biodegrad-A wide range of biodegrad-AM films prepared from modified biodegradable materials have the potential to be used for packaging of various food products.. Keywords: active packag
Trang 2JFS: Vol 76, Number 3 Industrial Application Briefs Industrial Applications of Selected JFS Articles
A major point of interest is the determination of antioxidants
and the functionality of those compounds Of course, also of
interest are any short-cuts that can lead to safer products with
less hands-on inspection, as the budget for food safety appears to
be eyed by budget choppers Health is important—and concerns
about all aspects of health are being studied carefully Some papers
in this issue are particularly interesting; some may cut the amount
of time and expense regarding food products and their safety and
healthfulness
Reigning in Uncertainty of Foodborne Infections
If a person consumes a large number of infectious organisms,
will that person become ill or die? One can build simulations of
that probability, but usually, if the person knows they’ve gobbled
down piles of bad stuff they will perform a ritual—take a couple
of aspirins, drink a lot of water, have a bottle of Scotch—and
wait Depending on their immune system, maybe yes, maybe no
But building a probability of illness can be helpful, and that’s
what Micha Peleg, Mark D Normand, and Maria G Corradini
have done in the paper titled “Construction of Food and Water
Borne Pathogens’ Dose–Response Curves Using the Expanded
Fermi Solution.” The authors note that “The proposed Expanded
Fermi Solution is not intended to replace clinical or
experimen-tal methods to determine microbial dose–response curves but a
way to generate such curves in situations where information on
the infection either does not exist or is insufficient to develop an
accurate mechanistic model Uncertainties concerning infection
by food and waterborne pathogens stem from inherent variability
in their physiological state when ingested, the number of “units”
actually consumed, their fate during and after passing through the
digestive tract, and the state of the host’s immune system, among
others.” The uncertainty of calamity occurring from consuming
pathogens should not slow the progress toward better
understand-ing of the relationship between high numbers of pathogens and
the probability of sickness, nor should it be used as an excuse for
reducing vigilance in food safety.R82–89
Phenols and Phenolic Acids Trap Methylglyoxal
Methylglyoxal, a reactive aldehyde that is very toxic to cells,
is triggered by excessive intake of glucose by a cell It appears
to be trapped by certain benzenediols and di-hydroxyl benzoic
acids, and the compound hydroquinone is able to decrease MG by
about 13% It has been shown that an accumulation of AGEs
(ad-vanced glycation end products) over time in patients with diabetes
mellitus can causes micro- or macrovascular complications, such
as retinopathy, peripheral neuropathy, and arteriosclerosis
Fur-ther studies of the role of various phytochemicals is described in
the paper “Efficiency of Trapping Methylglyoxal by Phenols and
Phenolic Acids,” evaluating the activity of 20 different phenolic
compounds These results showed high reactivity of MG and
cer-tain polyphenols with specific chemical structural arrangements
The authors concluded that: “Computational chemistry analysis
can provide a potentially important screening tool for compounds
with favorable effects for the control of carbonyl stress.”H90–96
Stress Triggers Antioxidant Activity in Peanuts
Peanut roots produce a number of phytoalexins, which oftenhave antioxidant activity The development of these compoundsand their activities are thought to be a response to environmentalstress, such as the infestation of the roots by an actual fungal ormicrobial infection, or by UV light, heavy metal salts, physicaldamage, and other environmental stressors “ORAChromatog-raphy and Total Phenolics Content of Peanut Root Extracts”offers information about the oxygen-radical absorbing capacity(ORAC) value and total phenolic contents of peanut root extractsand peanut root extract fractions collected via HPLC The peanutsused in this experiment were from 4 varieties that are commer-cially important They were planted in similar conditions, andwere not intentionally stressed The 2 sets of results represented
by the total phenolics contents of the fractions and ORAC values
of the fractions do not appear to be particularly well correlated.The fractions exhibiting antioxidant activity may either containnonphenolic compounds that exhibit antioxidant activity or arenot accurately measured with the total phenolics method that wasused The goal of measuring the total phenolic content of thefractions was to normalize the ORAC data from the fractions.Combining the ORAC and total phenolic content data may per-mit better understanding of the antioxidant capacity of a fraction,and may supply information about commercial use of some ofthese compounds.C380–384
Brook Trout Fatty Acid Profiled Improved by Feeding Flax
The results of a feeding study that altered the fatty acid profile
of Brook Trout is published in “The Effect of a Flaxseed Enhanced Diet on the Product Quality of Farmed Brook Trout
Oil-(Salvelinus fontinalis) Fillets.” The feeding study indicated that
en-hancing feed for the fish with flaxseed increased the total omega-3fatty acids in the fish flesh, making it more valuable Since there is
a preference of the desaturation enzymes forω-3 fatty acids,
in-creasing the amount ofω-3 PUFA would likely interfere with the
desaturation and elongation of LA to metabolites that could tentially induce a prothrombotic and proaggregatory state Therewere concerns about the flavor profiles that would be produced
po-by increasing the less stable omega-3 fatty acids; however, the
re-searchers found that “Flax fillets were preferred (P < 0.05) over
CD [commercial feed, fish oil-supplemented] fillets Of the 53participants, 34 preferred the Flax fillets, while 19 preferred the
CD fillets Preference of Flax fillets over the CD fillets may havebeen due to the greater presence of fish odor and fishy flavor thatwas typical of the CD fillets Manipulating feed with Flax resulted
in an increase in the total amount ofω-3 PUFAs, yet a reduction in
the longer and less stable DHA and EPA.” This change may haveaffected sensory characteristics, with the impact being generallymore positive than negative.S192–197
Sesame Seed Oil Offers Flavor Variety
The variation in color and flavor of sesame seed oil runs from thenearly colorless and flavorless, to dark brown in color and redolent
Trang 3of chocolate and peach flavors – and many others In
“Identifica-tion of 2-Ethyl-4-methyl-3-thiazoline and
2-Isopropyl-4-methyl-3-thiazoline for the First Time in Nature by the Comprehensive
Analysis of Sesame Seed Oil”, a total of 87 components were
iden-tified and confirmed by GC and GC-MS methods Toasted sesame
seed oil, characteristic of Chinese cuisine, was extracted using
the SAFE (Solvent-Assisted Flavo Evaporation) technique The
major components were methylpyrazine; 2,5-dimethylpyrazine;
2,6-dimethylpyrazine; 2-ethyl-3,6-dimethylpyrazine; furfuryl
al-cohol; and guaiacol In addition, as part of this analysis,
2-ethyl-4-methyl-3-thiazoline and 2-isopropyl-4-2-ethyl-4-methyl-3-thiazoline were
confirmed as being present in a natural product for the first time
The oil derived from toasted sesame seed is used as cooking oil in
East-Asian countries, and as a flavor enhancer in baked goods and
confectionery products, especially in Japan and China Defining
the different flavors is interesting: “In water, the material tasted:
strong, chocolate, peach, liverwurst, gassy, coffee, roasted, brown,
dark, roasted coffee In sugar water, it tasted: cola, peach,
liv-erwurst, brown, and savory In sugar-acid solution, it tasted:
sa-vory, succulent, sulfurous, fried garlic, gassy, skunk, sulfur, strong,
roasted coffee In salt water, it tasted: peach, onion, pretzel, strong,
liverwurst, gassy, savory, brown, burnt, chocolate, coffee, roasted,
roasted vegetables In general, this material is more peach-like in
character at lower levels; at higher levels, the material is perceived
as being more chocolate-like.” One of the interesting attributes is
the wide range of flavors and colors, depending on how sesame is
processed into oil.C385–391
Examining Psicose
Psicose an epimer of d-fructose with a twist at the C03
po-sition, has some very interesting characteristics It’s a ketohexose
sugar that forms interesting Maillard reaction products with aminoacids such as l-lysine Psicose has been identified for awhile—atfirst it was thought to be a contaminant to fructose-containingproducts In the paper “Characteristics and Antioxidant Activity
of Maillard Reaction Products from Psicose-Lysine and Lysine Model Systems”, the difference between the Maillard re-action products produced via psicose and fructose are compared.Psicose enabled Maillard products appear to have some interestingantioxidant and textural qualities.C398–403
Fructose-A New Method for Estimating Weight of Whole Fish
A medical X-ray unit may be the new way to estimate theweight of a whole fish in the fast-paced environment of Nor-wegian fisheries By calculating the integral intensity of the totalnumber of pixels in each image, the resulting linear relationshipcorrelated with the actual weight of each fish The paper titled “A
Simple Method for Weight Estimation of Whole Herring pea harengus) Using Planar X-Ray Imaging” describes the method
(Clu-and the possibilities of adjusting equipment using the method toimprove speed, safety, and profitability According to the authors,fish weight is a key parameter for sorting ocean fish, and theweight affects price Processors depend on weight to help themadjust equipment Say the authors, “The industry is interested in
an automated weight grading of individual fish, as accurate ing into different weight classes would be economically beneficialfor the pelagic industry Development of any method for weightestimation should fulfill 2 most important requirements from theindustry: high degree of accuracy and on-line speed of operation.”
grad-E328–331
vii
Trang 4JFS: Vol 76, Number 3 Editorial
Reducing Page Count, and When Language
Inhibits Understanding the Science
The Scientific Editors, Associate Editors, and staff of theJournal
of Food Science have worked diligently to increase the quality of
the content and the impact factor of the journal Our successes
in both areas have resulted in a significant increase in submissions
from authors For publishing year 2007, there were 899
submis-sions and the rejection rate was 55.2% In comparison, in 2010
there were 1430 submissions and the rejection rate had increased
to 62.9%, a 59% increase in submissions and a 14% increase in
rejection rate The net result is that the size of the journal
in-creased from 2597 printed pages for 368 papers to 3513 printed
pages for 466 papers, an increase in average page count per
pa-per from 7.1 to 7.5, respectively Recently we have taken steps to
reduce paper length by requiring that manuscripts must be 5000
words or less for the research sections and 10000 words or less for
the Concise Review section The word count excludes tables and
figures
Another trend that is readily apparent is that the majority of
manuscripts now submitted to JFS are from authors whose
na-tive language is not English In 2010, authors from 2
English-language countries submitted 26.1% of the manuscripts (United
States 19.6% and India 6.5%) Of the 8 remaining top 10
submit-ting countries, China had far and away the most submissions with
15.0% Although the non-English-speaking authors are making a
conscientious effort to write in this non-native language, it is
ap-parent that in many manuscripts the poor language gets in the way
of interpreting the science in the study Although our copyeditors
can correct some language issues, it is the Scientific Editors,
As-sociate Editors, and reviewers who are in the difficult position of
interpreting the science Unfortunately the journal cannot offer
English-language assistance but there are 2 resources to which werefer authors for editing and English language help:
Wiley Author Services’ author resources page:
http://authorservices.wiley.com/bauthor/english_language.aspAmerican Journal Experts (recommended by ScholarOneManuscripts): http://www.journalexperts.com
In many cases, the authors can also avail themselves for assistancefrom within their respective universities When the language in-terferes with interpreting the science and its value, SEs, AEs, andreviewers have been advised to reject the manuscript with theadvice to get assistance with English language editing We arehopeful that these efforts will continue the trend of an improved
Journal of Food Science.
Daryl LundEditor-in-Chief
Trang 5Contamination by Taking Advantage
of the Egg’s Self-Defense System: A Review
Wei Zhang, Jiang-Xia Zheng, and Gui-Yun Xu
Abstract: Egg-associated salmonellosis is a major problem for food safety It can be caused by vertical transmission(transovarian transmission) in hens and horizontal transmission though penetration Despite a series of physical andchemical defense mechanisms naturally found in eggs, they cannot provide complete protection for them Environmentalhygiene, bacteria vectors such as birds, rodent, flies, and beetles along with feed and water contamination are the most
frequently reported causes of Salmonella colonization in hens, and finally to eggs In addition, inappropriate egg handling will cause eggs to lose their self-protection ability, thus resulting in the survival and multiplication of Salmonella in an egg’s contents, which contributes to the horizontal dissemination The routes of Salmonella contamination were discussed, and
the effectiveness and shortcomings of different decontamination methods were evaluated in this review Various studies
on egg storage indicated that the low-temperature storage without temperature fluctuation was beneficial for the control
of Salmonella This review, based on an understanding of the stages of Salmonella transmission and an egg’s self-protection mechanisms, highlights a comprehensive strategy toward Salmonella control in a process from egg production and handling
to human consumption
Keywords: egg safety, egg’s self-protection mechanism, Salmonella control, Salmonella transmission
Introduction
In the United States, 164044 Salmonella infections were reported
during 1998 to 2002 (Lynch and others 2006) In China, a total
of 4207 Salmonella Enteritidis infections from egg products were
reported from 1991 to 1996 (Liu 2008) The latest egg-associated
Salmonella outbreaks, which happened in the United States from
May to August in 2010, led to a nationwide recall of more than
half a billion eggs from 2 Iowa egg producers, and nearly 2752
illnesses were reported from May to September 2010 This caused
egg safety to come into the spotlight of the food industry once
again
Salmonella is a genus of rod-shaped, Gram-negative, nonspore
forming, thermolabile, predominantly motile enterobacteria with
diameters of approximately 0.7 to 1.5 μm, and lengths from 2
to 5 μm Most Salmonella have flagella with the exception of
Salmonella Gallinarum and Salmonella Pullorum Flagella and curli
fimbriae are vital for penetration through an egg’s physical
bar-riers where multiplication can occur in the nutrient-rich yolk
The most commonly isolated Salmonella serovars from poultry and
eggs were Salmonella enterica serovar Typhimurium and S enterica
serovar Enteritidis (Omwandho and Kubota 2010) However, S.
Enteritidis phage type (PT)4was more frequently reported than
other S Enteritidis phage types (Cogan and others 2004).
Salmonella is a food borne bacteria causing human
infec-tion, which often results from the consumption of uncooked
MS 20100955 Submitted 8/24/2010, Accepted 12/27/2010 Authors are with
Dept of Animal Genetics and Breeding, Natl Engineering Laboratory for Animal
Breeding, China Agricultural Univ., Beijing, China Direct inquiries to author Xu
(E-mail: ncppt@cau.edu.cn).
contaminated eggs or infected egg products A level of Salmonella
contamination of 10 to 20 CFU per egg was sufficient to cause man salmonellosis (Kapperud and others 1990; Vought and Tatini1998) Although humans, regardless of their age or sex, were vul-
hu-nerable to Salmonella contamination, the most vulhu-nerable were
children under the age of 5, the elderly, and those with impairedimmune systems Symptoms developed 8 h to 3 d after eatingcontaminated food and last 4 to 7 d with characteristics includingdiarrhea, fever, nausea, stomach cramps, vomiting, and headache(Lin and others 1997; FSIS 2005)
In artificially infected hens, the percentage of infected eggscould range from 0% to 27.5% (De Reu and others 2008) Innaturally infected situations, the detection rate varied accordingthe seriousness of contamination in flocks For example, in the
United Kingdom, there was a Salmonella isolation rate of 3.4% of
17000 eggs sampled between 2002 and 2004 (Little and others2007), while the estimation from the World Health Organization
showed that 0.03% of the eggs were contaminated with S
Enteri-tidis in infected flocks in Israel The average for more than 60% ofthe flocks was only 0.025% (Lublin and Sela 2008) Even though
the Salmonella detection rate is relatively low in most cases, the
problem is still serious if we take the whole shell egg tion amount into consideration According to the risk assessmentconducted by USDA-FSIS, 46.8 billion eggs were produced an-nually in the United States, and 2.3 million of them might contain
consump-Salmonella Those eggs contaminated with Salmonella pose a great
risk to public health without proper handling and processing
A better understanding of the interactions and mechanisms
be-tween Salmonella and chicken eggs is necessary to reduce outbreaks
of salmonellosis Considerable research has been reported on eggcomponents in terms of bacteria contamination examples includethe quality of the cuticle and eggshell, components of the egg
Trang 6white and their functions, and factors that affect the penetration
of Salmonella and its resistance abilities Additional research has
been reported on how to control Salmonella infection in table eggs
more effectively In this review, all items were summarized from
the whole picture
Two Routes for Eggs to Be Contaminated
There are 2 possible routes for egg contamination by Salmonella.
Eggs can be contaminated by Salmonella originating from the
in-fection of reproductive organs though direct contamination of the
yolk, albumen, eggshell membranes, or eggshells before
ovipo-sition Second, eggs can also be contaminated by penetration
through the eggshell from the colonized gut or from
contami-nated faeces during or after oviposition In this case, many
dif-ferent serotypes of the genus Salmonella can be involved (FEHD
2004) However, it is uncertain that route is most important for
egg contents to become contaminated by Salmonella Entertidis.
Vertical transmission
In the case of vertical transmission, S Enteritidis could be
intro-duced into the egg from infected ovaries or oviduct tissue before
oviposition (Gantois and others, 2009) When Salmonella are
di-rectly deposited inside the egg contents of an egg, they are able
to survive especially in egg yolk, which is a favorable
environ-ment for Salmonella multiplication Once the bacteria colonized
in the hen’s intestinal tract, it could invade and disseminate to
internal organs and maintain colonization throughout a whole
production period (Gast 1994; Gast and Holt 1998) Based on
the strain and the dose of the inoculum, flocks infected with S.
Enteritidis showed various clinical symptoms, such as depression,
anorexia, diarrhea, reduced egg production, and even mortality
as well as physiological effects like different levels of antibodies
in egg yolk and serum (Shivaprasad and others 1990; Gast 1994)
Infected flocks excreted S Enteritidis intermittently (Shivaprasad
and others 1990) Carrique-Mas and others (2008) revealed that
only 5% of the birds from the infected flocks actively excreted
Salmonella at the end of lay and lower in earlier stages Eggs from
those infected birds could be internally contaminated in
intermit-tent clusters (Humphrey and others 1989) Further experiments
showed that short environmental stress, such as water and food
deprivation, an infection with other pathogens, and molting, were
often the common catalysts leading to an increase of Salmonella
excretion in flocks (Barrow 1992; Nakamura and others 1994)
However, no certain correlation was found between persistence
of Salmonella in flocks and the likelihood of egg contamination
(Gast and others 2005a) And according to FSIS (2005), most
cases of foodborne salmonellosis in the United States were
asso-ciated with the consumption of shell eggs, and the predominant
Salmonella were transferred by vertical transmission.
Horizontal transmission
Environment hygiene is rather critical for the control of
Salmonella dissemination in horizontal transmission Carrique-Mas
and others (2009) revealed that Salmonella could exist in laying
houses over subsequent flock cycles therefore posing a persistent
threat to the poultry industry Salmonella existing in laying house
is able to penetrate though the eggshell and vitelline membrane
into egg contents and consequently infects humans
Penetration through eggshell. The eggshell is composed of
a cuticle and mammillary, cone, palisade, and vertical crystal layers
The eggshell has pores that allow the exchange of gas and water
between its contents and the external environment Therefore, thepores also provide a passageway for bacteria in the environment toenter the egg Accordingly, an understanding of the influence of
eggshell quality and Salmonella penetration is necessary for taking measures to prevent the introduction of Salmonella into eggs via
penetration of the eggshell Penetration is facilitated mainly bycondensation that occurs as the egg passes through the vagina andexperiences a temperature change from that of the hen to the out-side environment Eggshell characteristics changed with the age ofthe hen with its best overall qualities during the middle of the eggproduction (Messens and others 2005a) However, there was nocorrelation between eggshell characteristics, such as the number
of pores or thickness and Salmonella penetration (Kraft and others
1958; Williams and others 1968; Nascimento and others 1992;
Messens and others 2005a) And current evident showed that teria were checked in their movement by their structural modifi-cations in their mammillary layer (Messens and others 2005b) Onthe other hand, some extrinsic factors, such as strain of bacteria,temperature differential, moisture, number of organisms present,and storage conditions, were identified as being important to thetrans-shell contamination (Messens and others 2005b)
bac-Penetration through vitelline membrane. The vitellinemembrane surrounding the egg yolk is made up of 2 layers Theouter layer consists of the ovomucin, vitelline membrane outerlayer protein I (VMO I), VMO II, and lysozyme The inner layerconsists mainly of glycoprotein I(GP I), GP II, and GP III (Kidoand others 1975, 1992) Bacteria penetration through the vitellinemembrane can result in rapid and extensive multiplication in the
nutrient-rich yolk content In vitro experiments, which involved inoculating S Enteritidis and Salmonella Heidelberg onto the exte-
rior surface of egg yolk vitelline membranes, showed that bacteriamay sometimes reach the yolk (Gast and others 2005b)
Leleu’s study (2009) showed that vitelline membrane strength(VMS) decreased from the beginning of a production cycle to themiddle of it and then plateaued until the end of the productioncycle Comparisons of VMS during different periods of lay and
between the VMS and the moment of penetration by Salmonella
revealed little effect of the lay period and a slight but significantcorrelation between the VMS at the moment of penetration by
Salmonella Thus, it can be assumed that a higher VMS leads to
a longer resistance of the vitelline membrane against penetration
Interesting, however, was that lower yolk temperatures resulted inhigher VMS (Ngoka and others 1983) This partly explains why
low-temperature storage reduces the risk of Salmonella vitelline
membrane penetration Other studies showed that VMS decreasedwith storage (Kirunda and McKee 2000; Jones and Musgrove2005), which was partly a result of loss of structural integrity(Fromm 1966; Back and others 1982)
Self-Defense Mechanism of Chicken Eggs
An egg is a potential life form having its own protection nisms Thus, the intact egg is naturally equipped with both physicaland chemical defenses in order to protect the embryo from bac-teria invasion and physical harm This is shown in the followingaspects
mecha-Physical barriers
Cuticle. Oviposited eggs have a cuticle deposition, which ers the outer surface of the eggshell The cuticle is composed of aprotein carbohydrate complex and contains a small amount of thecrystal complex hydroxyapatit This layer is secreted in the shellgland pouch during the last hour of eggshell formation (EFSA
Trang 72009) The cuticle is the first line of defense to bacteria by closing
the eggshell pores and decreases with the age of the hen
Eggshell. The eggshell has many pores, which can vary in
number from 6000 to 10000 according to egg size and location
There are more pores on the blunt pole than at the apex and some
of the proteins related to the eggshell have antibacterial properties
The pore diameters range from 6 to 65μm and are wide enough
for the penetration of Salmonella.
Eggshell membrane. The inner and outer eggshell
mem-brane can act as a “filter” in the process of penetration from
external sources The eggshell membrane is composed of highly
cross-linked proteins that are structurally similar to a meshwork
of entangled threads and can obstruct the invading
microorgan-isms (Baker and Balch 1962) Ahlborn and others (2006)
re-ported that bacteria thermal resistance and/or their inactivated
cells were greatly reduced by exposing the selected Gram-positive
and Gram-negative bacterial pathogens to eggshell membranes
This process is associated with several antibacterial proteins, such
as ß-N-acetylglucosaminidase, lysozyme, and ovotransferrin
Chemical barriers
Bactericidal effects of albumen were factors that influence S
En-teritidis’s growth in egg albumen with the restriction of iron being
a major factor (Schade and Caroline 1944; Clay and Board 1991;
Baron and others 1997) A high concentration of ovotransferrin
in egg albumen could chelate iron and inhibit bacterial growth by
binding free iron and making it inaccessible to bacteria resulting
in a depletion of ferric and ferrous iron for Salmonella Moreover,
ovotransferrin and lysozyme could interact with the surface of S.
Enteritidis and form pores in the Salmonella cell wall, thus
prevent-ing its multiplication (Kang and others 2006) Also, egg albumen
could penetrate S Enteritidis and kill bacteria through nuclease
activity (Lu and others 2003)
Genes involved in cell wall structural and functional integrity,
as well as nucleic and amino acid metabolism were important for
S Enteritidis to persist in egg albumen (Clavijo and others 2006).
Therefore, damaging bacterial DNA became one of the
mecha-nisms that egg albumen uses to control bacteria growth This might
be because egg albumen nuclease could damage the chromosomal
DNA of Salmonella by entering bacterial cells through pores in
the cell wall formed by ovotransferrin and lysozyme In addition,
there were other factors that work against Salmonella such as
im-munoglobulins in egg albumen and yolk, the albumen’s alkaline
pH, protease inhibitors, proteins chelating essential elements, and
the viscosity of albumen that prevents Salmonella’s movement into
the egg’s yolk These bactericide effects not only play an
impor-tant role on horizontal transmission, but also limit the growth of
Salmonella in the eggs contents in the case of vertical transmission
(Keller and others 1995)
Controlling Salmonella Contamination
in Eggs and Egg Products
Reasons that eggs are vulnerable
toSalmonella contamination
Although an egg has many self-defense mechanisms against
bac-terial invasion, Salmonella also set up systems to avoid defenses by
the egg albumen For instance, yaf D and xthA were genes that play
an essential role in the repair of DNA damage cause by the
albu-men and hence facilitate the survival of S Enteritidis in chicken
eggs (Lu and others 2003; Gantois and others 2009) The survival
ability for Salmonella in egg contents also depends on the strain
of Salmonella and preservation temperature Many studies indicate that S Enteritidis can survive in egg content (Gast and Holt 2000;
Messens and others 2004; Murase and others 2005; Gurtler and
Conner 2009) However, there was no agreement about Salmonella
growth in albumen because it is difficult to compare various studiesdue to different inoculum size, strains, incubation temperatures,storage time, and age of the eggs Gast and Holt (2000) indicated
that extensive multiplication of S Enteritidis was less frequently
observed at lower inoculum doses (15 cells), shorter storage times(1 d), and lower temperatures (10 to 17.5 ◦C) (Gast and Holt
2000) Salmonella Enteritidis inoculated onto a vitelline
mem-brane could proliferate in albumen surrounding the yolk, possiblyresulting from the use of nutrient compounds emerging from theyolk though the vitelline membrane (Murase and others 2005).However, no such indication was found that nutrients or factorsleaking out from the yolk could impair the inhibitory properties of
the albumen And growth of Salmonella occurred more frequently
in the albumen of fresh eggs compared to eggs stored prior to ulation, which was partly due to the alkalinity pH environment inegg albumen since longer periods of storage results in higher albu-men pH (Scott and Silversides 2000; Messens and others 2004) In
inoc-Gurtler and Conner’s study (2009), survival of Salmonella in liquid
egg products was also related to storage temperature and egg
prod-uct composition Humphrey and Whitehead (1993) found that S.
Enteritidis can grow in the contents of naturally contaminatedeggs at room temperature Besides, the effectiveness of an egg’sself-protection abilities was affected in the case of inappropriate
preservation Salmonella can double every 20 min and a single
bac-terium can multiply into more than a million in 6 h if not properly
handled (Hasan and others 2009) Multiplication of Salmonella in
eggs could occur rapidly within a single day of storage at a warm
temperature Study showed that the number of S Enteritidis in
yolk can reach a mean level of 8.4 to 8.7 log units/mL at 2 d
in samples initially contaminated with doses of 15 and 150 CFU,respectively, while a mean level of 4.3 log units/mL (15 CFUdose) and 6.1 log units/mL (150 CFU dose) at 2 d in whole egg
No multiplication was found in albumen (Gast and Holt 2000).Therefore, concerns on how to reduce egg contamination to thegreatest extent through better treatment and handling of fresh eggs
is a rather important issue for public health
Comprehensive Measures toward
Salmonella Control in Flocks
The primary reason for eggs getting contaminated with
Salmonella is the infection of flocks To the best of our edge, numerous factors can result in the colonization of Salmonella
knowl-in hens such as feed, water, vectors, as well as general hygiene
con-dition According to FDA investigation, the Salmonella outbreak
that happened at the Wright County Egg farms in the UnitedStates was possibly due to feed contamination and other commonenvironmental risk factors such as equipment, walkways, and othersurfaces in and around the farm (CDC 2010) Given the serious-ness of this issue, it is urgent for egg producers to strictly implementpreventive measures throughout the whole egg production pro-cess First of all, a timely disposal of waste and dead birds limits thenumber of vectors on farms The hygiene procedure including athorough cleaning of flock housing followed by an effective disin-fection of the flock surface should be carried out consistently For
another, feeds as a common source of Salmonella for poultry flocks,
should always be decontaminated with heat treatment, and cessed in pellet form rather than in meal form to lower the flock’s
pro-risk of Salmonella contamination In some cases, when Salmonella
Trang 8infection becomes a serious problem, a special control strategy
such as the use of a vaccine is necessary to prevent Salmonella
colonization in the reproductive tract, as well as to reduce fecal
shedding and further contamination of eggs Two types of vaccines
are available for poultry at present and both of them can be used
throughout the life of birds except during the withdrawal period
before slaughter However, the use of a vaccine does not provide
100% protection in flocks And it has limited effects on
improv-ing animal health and welfare Therefore, its use depends on the
aim of the control program, type of poultry, stage of production,
true prevalence of Salmonella, serovars targeted, detection
meth-ods used, and cost-benefit analysis (EFSA 2004) In addition, an
integrated control program for Salmonella should include the
“all-in-all-out” principle and “test-and-removal of flock policy.” Since
it is unrealistic for the complete eradication of Salmonella,
com-prehensive bio-security measures should be adopted constantly
Egg handling and processing
Decontamination. Because egg decontamination effectively
reduces the bacteria load on an eggs surface and prevents rapid
penetration, it should be carried out as soon as eggs are
col-lected Ways to decontaminate eggs include different detergents in
wash water, such as free chlorine, new N-halamine compounds,
and an iodine-based disinfectant, (Worley and others 1992; Knape
and others 2001), electrolyzed oxidative water (Russell 2003),
microwave (Mudau 2007; Sivaramakrishnan 2007), ultrasonic in
combination with heat and/or pressure treatment (Piyasena and
others 2003; Cabeza and others 2004, 2005), ozone, and UV
(ul-traviolet radiation) (Rodr´ıguez Romo 2004) The use of egg
wash-ing is a continuous debate despite its broad commercial application
Current concerns focus on whether egg washing increases the
in-ternal microbial load Within the European Union, egg washing
is prohibited except in Sweden and parts of the Netherlands The
reason offered is that egg-washing procedures may damage the
quality of the cuticle enhancing the opportunity for bacterial
in-vasion (Peebles and Brake 1986; Bialka and others 2004; EFSA
2005) Factors related to cuticle damage caused by egg washing
include presence of water on the eggshell, presence of iron in the
wash water, physical brushing damage, and high pressure
(Com-mission of European Communities, 2003) These are the reasons
that class A eggs for human consumption are not eligible for the
practice of egg washing by European Union legislation and eggs
will be downgraded if any forms of disinfection are used However,
this reasoning is at odds with research that showed the washing
procedure did not appear to affect the incidence of open pores
and the overall cuticle quality Meanwhile, it was also indicated
that brown eggs in general were of better quality in terms of
their cuticle scores than the white eggs when 4 standards, such
as mechanical damage, debris, open pores, and cuticle coverage,
were considered (Messens 2009) And the use of egg washing is
yet authorized in Canada, America, Japan, Australia, Russia, and
Mexico for the reason that egg washing can reduce the total
mi-crobial load on the surface of sanitized eggs by approximately 2 to
above 5 log units (Hutchison and others 2004; Rodr´ıguez Romo
2004)
Given the controversy on the advantages and disadvantages of
egg washing, other procedures are being evaluated Hierro and
others (2009) used pulsed light (PL) as a method of egg
decon-tamination and the effects were more notable when the cuticle was
preserved intact In addition, this treatment is most effective when
applied as soon as eggs are laid And, it is limited by the motility
of Salmonella and low penetration depth of UV radiation Hot air
treatment for table eggs (2 shots for 8 s at 600◦C with an interval
of 30 s of cold air) reduced Salmonella load up to 1.9 log units
without significant changes for any of the egg’s quality traits, such
as the cuticle, breaking strength, and yolk index (Pasquali 2009)
Nonthermal atmospheric gas plasma device, a resistive barrier charge prototype able to generate an ionized gas containing freeelectrons and neutral reactive species such as atoms, molecules,and radicals at atmospheric conditions, which is able to reduce
dis-Salmonella load up to 4.5 log units per eggshell with a humidity
of 65% at 25◦C for 90 min of treatment, provides a nation choice for farmers and industries that need stock eggs for
decontami-a reldecontami-atively long period (Rdecontami-agni decontami-and others 2010) However, thetime and cost needed for this method may become a limitationfor its practical use in commercial production
In hot water immersion, heat is transferred from hot waterthrough the eggshell all the way to the inside egg contents untilthe center of the yolk reaches the desired temperature for suffi-cient time In light of early research findings, the use of hot waterimmersion at 57 ◦C for 25 min followed by hot air heating at
55◦C for 60 min resulted in a 7 log unit reduction of Salmonella
in shell eggs and produced acceptable changes on egg qualities atthe same time (Hou and others 1996) The patent of Davidsonand others (2004) indicated that the heated fluid bath with a tem-perature of between about 128 to 145 ◦F allows a reduction of
at least 4.6 log units of any Salmonella bacteria within the eggs.
This followed by antibacterial gas treatment and further wax covercould result in at least another 5 log units reduction of bacteriaand provide additional antibacterial barriers to egg contents And,this method is commercially used by Davidson’s pasteurized eggs
Nevertheless, pasteurization methods employing liquid sion or spray washing of shell eggs are prohibited under certainregulatory schemes in many European countries due to possibleundesirable effects to egg quality (Ball and others 2002) Schumanand others (1997) revealed that 50 to 57.5 min treatment with
immer-a bimmer-ath temperimmer-ature of 58◦C or 65 to 75 min treatment with atemperature of 57◦C increased Haugh unit values and had no in-fluence on albumen pH values and yolk index but that it affectedalbumen clarity and functionality
In addition to Salmonella disinfection, an effective measure for preventing Salmonella growth in egg contents is necessary The
rapid cooling was introduced to cool eggs from 40 or 45 ◦C to
7◦C in approximately 15 min or less, which may take 7 or 10 d inconventional conditions, to suppress the significant bacteria mul-tiplication (Keener and others 2000b) Moreover, rapid coolingwas also found to improve internal egg quality and increase shelflife (Sabliov and others 2002) Further study showed that rapidcooling with CO2 produces higher quality eggs with increasedshelf life than rapid air cooling but with no difference on Haughunits unless followed by subsequent storage in CO2(Keener andothers 2000a) Although rapid cooling might cause slight cracks
in eggshells, if well managed, it is a good way for controlling
Salmonella growth on the whole (Thompson and Knutson 2000).
Most importantly, though lots of work can be done for shelleggs, secondary pollution in the process of packing and palletiz-
ing may also introduce Salmonella to decontaminated eggs Thus,
decontamination should not only focus on the egg itself, but theequipment for egg storage as well
Storage. Storage conditions present issues in contaminationwith focus on duration, temperature, and environmental hygiene
Different countries have different regulations Storage limits fortable eggs in the United Kingdom were 3 wk at 8◦C (Kinder-lerer 1994), while in Israel 3 mo for refrigerated eggs and 16 d at
Trang 9room temperature (Lublin and Sela 2008) In many countries, eggs
are required to be stored at low temperatures to restrict microbial
growth In Germany, legislation required that egg cooling be
ap-plied at 5 to 8◦C for 18 d maximum post lay (EFSA 2009) And
in the United States, either shell eggs packed for consumers or
eggs that receive a treatment from egg producers were required to
be kept at 45◦F (7.2◦C) no later than 36 h after the eggs are laid
during storage and transportation (FDA 2010) In this scenario,
it is more advisable to apply low-temperature storage in order to
minimize the possibility that eggs infected with S Enteritidis are
transmitted to humans This recommendation is supported by the
study of Gast and Holt (2000), which showed that low
temper-atures were more effective for controlling S Enteritidis
multipli-cation in the yolk when high concentration of S Enteritidis was
artificially introduced into egg contents (Gast and Holt 2000)
On the other hand, low temperature can slow down the
pro-cess of penetration (Chousalkar and others2010) However, Kang
and others (2006) suggested that it is preferable to store eggs at
37◦C for a certain period of time first, instead of 4◦C directly, to
allow the endogenous bactericidal activity of egg albumen to kill
the contaminating S Enteritidis This reasoning is valid especially
when most eggs are infected through trans-shell contamination
While in the case of vertical transmission, this application awaits
more research Further studies show that, although low
preser-vation temperature for table eggs will limit the multiplication of
Salmonella, it does not reduce the existing Salmonella
concentra-tion It may indeed prolong the survival of Salmonella because
Salmonella may be increased by low storage temperature (Baker
and Balch 1962; Radkowski 2002; Messens and others 2006) and
reduced with higher temperature (Rizk and others 1996)
From the aspect of an egg’s structure, Humphrey and Whitehead
(1993) showed that storage had little direct impact on albumen
with respect to the growth of Salmonella, but rather, it influenced
the integrity of the vitelline membrane that might result in a
dif-ference in Salmonella multiplication in the albumen and yolk The
pH of the albumen rose along with the storage time, and finally led
to the dissipation of fibers contained on the vitelline membrane
of fresh eggs, as well as the decline in protein and hexosamine
content of the vitelline membrane (Fromm 1967) Consequently,
it is easier for the bacteria to either invade the yolk or obtain
nutri-ents from it Therefore, cooling practices should to be carried out
shortly after lay to keep eggs fresh and also to prevent Salmonella
multiplication in eggs Besides, the speed with which changes in
membrane integrity occur as during storage, it is also highly
tem-perature dependent No significant changes occurred over 3 wk
of storage at 20◦C, whereas apparent changes occurred only after
7 to 10 d of storage with temperatures fluctuating between 18
and 30◦C (Humphrey and Whitehead 1993) Thus, it is highly
recommended that eggs should be kept in a cooling environment
and temperature fluctuation be avoided during egg storage
Additional concern for egg processing. Eggshell is
espe-cially fragile so special care should be taken in the process of egg
handling The data by USDA showed that 177 million dozen shell
eggs were cracked during August 2010, 3% up from a year ago
(USDA 2010) A cracked egg loses part of its defense system,
thereby is in danger of Salmonella invasion Cross-contamination,
on the other hand, always happens in transportation and egg
pro-cessing Therefore, safety education for egg handlers is necessary
to mitigate Salmonella dissemination and egg products should be
pasteurized It was estimated that the annual number of illness
would be reduced from 5500 to 3200, if all liquid egg products
produced in the United States were pasteurized for a 6 log units
reduction of Salmonella (FSIS 2005) Outreach efforts should stress the importance of properly cooking eggs Salmonella are suscepti-
ble to heat treatment Temperatures above 55◦C for enough time
are sufficient to destroy Salmonella Uncooked and semi-cooked
eggs should be avoided for the public
Conclusion
An egg can be contaminated either though vertical transmission
or horizontal transmission A well-organized Salmonella control scenario is necessary in virtue of the seriousness of Salmonella
dissemination First of all, flocks are the primary contaminationresource for eggs Thereby, egg producers have to keep good en-vironmental hygiene, ensure feed and water safety, and implement
effective management strategies to guard hens against Salmonella
infection On the other hand, decontamination and storage tions turn out to be rather critical in the process of egg handling Anumber of decontamination methods were evaluated in the review.Egg handlers have to make a proper choice in light of their practicalvalues coupled with specific regulations and cost-benefit analysis.When it comes to storage condition, low-temperature storage isbasically more favorable and should be carried out as soon as pos-sible after eggs are laid In this scenario, rapid cooling can shorten
condi-the cooling time to prevent multiplication of Salmonella Moreover,
temperature fluctuation and long time storage should be avoided
to keep the integrity of the vitelline membrane thus retarding the
growth of Salmonella in egg contents Most importantly, eggs have
to be well cooked for enough time in case Salmonella recover from
pasteurization
Acknowledgments
We thank Prof F Chen for providing technical guidance and
Dr Paul Siegel for critical reading of the manuscript This work
is supported by the earmarked fund for Modern Agro-industryTechnology Research System of China (nycytx-41)
Baron F, Gautier M, Brule G 1997 Factors involved in the inhibition of growth of Salmonella
Enteritidis in liquid egg white J Food Prot 60:1318–23.
Barrow PA 1992 Further observations on the serological response to experimental Salmonella
Typhimurium in chicken measured by ELISA Epidemiol Infect 108:231–41.
Bialka KL, Demirci A, Knabel SJ, Patterson PH, Puri VM 2004 Processing and products efficacy of electrolyzed oxidizing water for the microbial safety and quality of eggs Poult Sci 83:2071–8.
Cabeza MC, Garc´ıa ML, Hoz Ldl, Cambero I, Ordo ˜nez JA 2005 Destruction of Salmonella
Senftenberg on the shells of intact eggs by thermoultrasonication J Food Protect 68:841–4 Cabeza MC, Ord ´o ˜nez JA, Cambero I, Hoz Ldl, Garc´ıa ML 2004 Effect of thermoultrasonication
on Salmonella enterica serovar in distilled water and intact eggs to achieve its elimination from
shell J Food Protect 67:1886–91.
Carrique-Mas JJ, Breslin M, Sayers AR, Mclaren L, Arnold M, Davies R 2008 Comparison of
environmental sampling methods for detecting Salmonella in commercial laying flocks in the
UK Lett Appl Microbiol 47:514–9.
Carrique-Mas JJ, Breslin M, Snow L, McLaren I, Sayers AR 2009 Persistence and clearance of
different Salmonella serovars in buildings housing laying hens Epidemiol Infect 137:837–46 CDC 2010 Investigation update: multistate outbreak of human Salmonella Enteritidis infections
associated with shell eggs Available from: -http://www.cdc.gov/salmonella/enteritidis/ cessed Dec 2, 2010.
Ac-Chousalkar KK, Flynn P, Sutherland M, Roberts JR, Cheetham BF 2010 Recovery of
Salmonella and Escherichia coli from commercial shell eggs and effect of translucency on bacterial
penetration in eggs Int J Food Microbial 142:207–213.
Clavijo RI, Loui C, Andersen GL, Riley LW, Lu S 2006 Identification of genes associated
with survival of Salmonella enterica serovar in chicken egg albumen Appl Environ Microbiol
72:1055–64.
Clay CE, Board RG 1991 Growth of Salmonella Enteritidis in artificially contaminated hens’
shell eggs Epidemiol Infect 106:271–81.
Trang 10Cogan TA, Jorgensen F, Lappin-Scott HM, Benson CE, Woodward MJ, Humphrey TJ 2004.
Flagella and curli fimbriae are important for the growth of Salmonella enterica serovars in hen
eggs Microbiology 150:1063–71.
Commission of the European Communities 2003 COM (2003) 479 final Report from
the commission to the council with regard to developments in consumption,
wash-ing and markwash-ing of eggs Available from: http://eur-lex.europa.eu/LexUriServ/site/en/
com/2003/com2003_0479en01.pdf Accessed Jun 8, 2003.
Davidson LJ, Wagner MA, inventors; Davidson Food Co., assignee 2004 Jul 11 Pasteurized
eggs U.S patent 20020090429 A1.
De Reu K, Messens W, Heyndrickx M, Rodenburg TB, Uyttendsele M, Herman L 2008.
Bacterial contamination of table eggs and the influence of housing systems World’s Poultry
Sci J 64:5–19.
EFSA 2004 Opinion of the scientific panel on biological hazards on the requests from the
commission related to the use of vaccines for the control of Salmonella in poultry EFSA J
114:1–74.
EFSA 2005 Opinion of the scientific panel on biological hazards on the request from the
commission related to the microbiological risks on washing of table eggs EFSA J 269:1–
39.
EFSA 2009 Scientific opinion of the panel on biological hazards on special measures to reduce
the risk of consumers through Salmonella in table eggs-e.g cooling of table eggs EFSA J
957:1–29.
FDA 2010 New FDA regulations for Salmonella testing in commercial layer Available from:
http://www.vetmed.wsu.edu/depts_waddl/announcements/FDATesting.pdf Accessed Jul 9,
2010.
FEHD 2004 Salmonella in eggs and egg products Hong Kong: Food and
Environ-mental Hygiene Dept p 20 Available from: http://www.cfs.gov.hk/english/programme/
programme_rafs/files/egg_e.pdf Accessed Dec 2004.
Fromm D 1966 The influence of ambient PH on the moisture content and yolk index of the
hens yolk Poult Sci 45:234–41.
Fromm D 1967 Some physical and chemical changes in the vitelline membrane of the hen’s
egg during storage J Food Sci 32:52–6.
FSIS 2005 Risk assessments of Salmonella in shell eggs and Salmonella spp in egg products.
Available from: http://www.fsis.usda.gov/oppde/rdad/FRPubs/04–034N/Introduction.pdf.
Accessed Oct 2005.
Gantois I, Ducatelle R, Pasmans F, Haesebrouck F, Gast R, Humphrey TJ, Immerseel FV 2009.
Mechanisms of egg contamination by Salmonella FEMS Micobiol Rev 33:718–38.
Gast RK 1994 Understanding Salmonella in laying chickens: the contributions of experimental
infections Int J Food Microbiol 21:107–16.
Gast RK, Holt PS 1998 Persistence of Salmonella Enteritidis from one day of age until maturity
in experimentally infected layer chickens Poult Sci 77:1759–62.
Gast RK, Holt PS 2000 Influence of the level and location of contamination on the
multipli-cation of Salmonella Enteritidis at different storage temperatures in experimentally inoculated
eggs Poult Sci 79:559–63.
Gast RK, Guard-Bouldin J, Holt PS 2005a The relationship between the duration of fecal
shedding and the production of contaminated eggs by laying hens infected with strains of
Salmonella and Salmonella Heidelberg Avian Dis 49:382–6.
Gast RK, Holt PS, Murase T 2005b Penetration of Salmonella Enteritidis and Salmonella
Hei-delberg into egg yolks in a vitro contamination model Poult Sci 84:621–5.
Gurtler JB, Conner DE 2009 Survival and growth of Salmonella Enteritidis in liquid egg products
varying by temperature, product composition and carbon dioxide concentration Foodborne
Pathog Dis 6:561–7.
Hasan MN, Ara N, Mamun SA, Rahman MM, Rahman MH 2009 Prevalence of Salmonella
spp in chicken egg from Khulna city J Innov Dev Strategy 3:1–6.
Hierro E, Manzano S, Ord ´o ˜nez JA, Hoz Ldl, Fern´andez M 2009 Inactivation of Salmonella
enterica serovar on shell eggs by pulsed light technology Int J Food Microbiol 135:125–
30.
Hou H, Singh RK, Muriana PM, Stadelman WJ 1996 Pasteurization of intact shell eggs Food
Microbiol 13:93–101.
Humphrey TJ, Baskerville A, Mawer S, Rowe B, Hopper S 1989 Salmonella Enteritidis phage
type 4 from the contents of intact eggs: a study involving naturally infected hens Epidemiol
Infect 103:415–23.
Humphrey TJ, Whitehead A 1993 Egg age and the growth of Salmonella Enteritidis PT4 in
egg contents Epidemiol Infect 111:209–19.
Hutchison ML, Gittins J, Walker A, Sparks N, Humphrey TJ, Burton C, Moore A 2004.
An assessment of the microbiological risks involved with egg washing under commercial
conditions J Food Prot 67:4–11.
Jones DR, Musgrove MT 2005 Effects of extended storage on egg quality factors Poult Sci
84:1774–7.
Kang H, Loui C, Clsvijo RI, Riley LW, Lu S 2006 Survival characteristics of Salmonella enterica
serovar in chicken egg albumen Epidemiol Infect 134:967–76.
Kapperud G, Gustavsen S, Hellesnes I, Hansen AH, Lassen J, Him J, Jahkola M, Montenegro
MA, Helmuth R 1990 Outbreak of Salmonella Typhimurium infection trace to contaminated
chocolate and caused by a strain lacking the 60-megadalton virulence plasmid J Clin Microbiol
28:2597–601.
Keener KM, LaCrosse JD, Curtis PA, Anderson KE, Farkas BE 2000a Subsequent storage in
air and carbon dioxide on shell egg quality Poult Sci 79:1067–71.
Keener KM, Lacrosse JD, Farkas BE, Curtis PA, Anderson KE 2000b Gas exchange into shell
eggs from cryogenic cooling Poult Sci 79:275–80.
Keller LH, Benson CE, Krotec K, Eckroade RJ 1995 Salmonella colonization of the reproductive
tract and forming and freshly laid eggs of chickens Infect Immun 63:2443–9.
Kido S, Janado M, Nunoura H 1975 Macromolecular components of the vetelline membrane
of hen’s egg I Menbrane structure and its deterioration with age J Biochem 78:261–
8.
Kido S, Morimoto A, Kim F, Doi Y 1992 Isolation of a novel protein from the outer layer of
the vitelline membrane Biochem J 286:17–22.
Kinderlerer JL 1994 Salmonella in eggs BNF Nutrition Bull 19:11–8.
Kirunda DFK, McKee SR 2000 Relating quality characteristics of aged and fresh eggs
to vitelline membrane strength as determined by a texture analyzer Poult Sci 79:1189–
93.
Knape KD, Carey JB, Ricke SC 2001 Response of foodborne Salmonella spp marker strains
inoculated on egg shell surfaces to disinfectants in a commercial egg washer J Environ Sci Health 36:219–27.
Kraft AA, Mcnally EH, Brant AW 1958 Shell quality and bacterial infection of shell eggs Poult Sci 37:638–44.
Leleu S 2009 Penetration of Salmonella Enteritidis through the vitelline membrane of hen’s eggs
as affected by its strength during the laying period XIII European Symposium on the Quality
of Eggs and Egg Products; 2009 June 21–25 Turku, Finland: WPSA.
Lin C-TJ, Morales RA, Ralston K 1997 Raw and undercooked eggs: a danger of salmonellosis.
Food Saf 20:27–32.
Little CL, Surman-Lee S, Greenwood M, Bolton FJ, Elson R, Mitchell RT, Nichols GN, Sagoo
SK, Threlfall EJ, Ward LR, Gillespie IA, O’Brien S 2007 Public health investigations of
Salmonella Enteritidis in catering raw shell eggs, 2002–2004 Lett Appl Microbiol 44:595–601.
Liu X 2008 Isolation and Identification of Salmonella from eggs and biological characteristics.
[MSc dissertation] Mongolia: Mongolia Agricultural Univ 45 p.
Lu S, Killoran PB, Riley LW 2003 Association of Salmonella enterica serovar YafD with resistance
to chicken egg albumen Infect Immun 71:6734–41.
Lublin A, Sela S 2008 The impact of temperature during the storage of table eggs on the
viability of Salmonella enterica serovars and Virchow in the eggs Poult Sci 87:2208–14.
Lynch M, Painter J, Woodruff R, Braden C 2006 Surveillance for foodborne-disease United States, 1998–2002 Surveill Summ 55(SS10):1–34.
outbreaks-Messens W 2009 Effect of egg washing on the cuticle of table eggs XIII European symposium
on the quality of eggs and egg products; 2009 June 21–25 Turku, Finland: WPSA.
Messens W, Duboccage L, Grijspeerdt K, Heyndrickx M, Herman L 2004 Growth of Salmonella
serovars in hens’ egg albumen as affected by storage prior to inoculation Food Microbiol 21:25–32.
Messens W, Grijspeerdt K, Herman L 2005a Eggshell characteristics and penetration by
Salmonella enterica serovar through the production period of a layer flock Br Poult Sci
46:694–700.
Messens W, Grijspeerdt K, Herman L 2005b Eggshell penetration by Salmonella: a review.
World’s Poultry Sci J 61:71–82.
Messens W, Grijspeerdt K, Herman L 2006 Eggshell penetration of hen’s eggs by Salmonella enterica serovar upon various storage conditions Br Poult Sci 47(5):554–60.
Mudau MS 2007 Functional properties of microwave pasteurized and oil coated whole shell eggs [MSc dissertation] South Africa: Univ of Pretoria 111 p.
Murase T, Holt P, Gast RK 2005 Growth of Salmonella enterica serovar Enteritidis in albumn
and yolk contents of eggs inoculated with this organism onto the vitelline membrane J Food Protect 68:718–21.
Nakamura M, Nagamine N, Takahashi T, Suzuki S, Kijima M, Tamura Y, Sato S 1994.
Horizontal transmission of Salmonella and effect of stress on shedding in laying hens Avian
Dis 38:282–8.
Nascimento VP, Cranstoun S, Solomon SE 1992 Relationship between shell structure and
movement of Salmonella Enteritidis across the eggshell wall Br Poult Sci 33:37–48.
Ngoka DA, Froning GW, Babji AS 1983 Effect of temperature on egg yolk characteristics of eggs from young and old laying hens Poult Sci 62:718–720.
Omwandho COA, Kubota T 2010 Salmonella enterica serovar: a mini-review of contamination
routes and limitations to effective control Jpn Agr Res Quart 44:7–16.
Pasquali F 2009 Hot air treatment for surface decontamination of table eggs Food Control 21:431–5.
Peebles ED, Brake JT 1986 The role of the cuticle in water vapor conductance by the eggshell
of broiler breeders Poult Sci 65:1034–9.
Piyasena P, Mohareb E, McKellar RC 2003 Inactivation of microbes using ultrasound: a review.
Int J Food Microbiol 87:207–16.
Radkowski M 2002 Effect of moisture and temperature on survival of Salmonella on shell eggs.
Arch Geflugelkd 66:119–23.
Ragni L, Berardinelli A, Vannini L, Montanari C, Sirri F, Guerzoni ME, Guarnieri A 2010.
Non-thermal atmospheric gas plasma device for surface decontamination of shell eggs J Food Eng 100:125–32.
Rizk SS, Ayres JC, Kraft AA 1996 Effect of holding condition on the development of Salmonella
in artificially inoculated hens’ eggs Poult Sci 45:825–9.
Rodr´ıguez Romo LA 2004 Control of Salmonella enterica serovar in shell eggs by ozone,
ultraviolet radiation, and heat [DPhil dissertation] Ohio: Ohio State Univ 185 p.
Russell SM 2003 The effect of electrolyzed oxidative water applied using electrostatic spraying
on pathogenic and indicator bacteria on the surface of eggs Poult Sci 82:158–62.
Sabliov CM, Farkas BE, Keener KM, Curtis PA 2002 Cooling of shell egg with cryogenic carbon dioxide: a finite element analysis of heat transfer Lebensm-Wiss Technol 35:568–74.
Schade AL, Caroline L 1944 Raw hen egg white and the role of iron in growth inhibition
of Shigella dysenteriae, Staphylococcus aureus, Escherichia coli and Saccharomyces cerevisiae Science
100(1944):14–5.
Schuman JD, Sheldon BW, Vandepopuliere JM, Ball Jr HR 1997 Immersion heat treatments
for inactivation of Salmonella with intact eggs J Appl Microbiol 83:438–44.
Scott TA, Silversides FG 2000 The effect of storage and strain of hen on egg quality Poult Sci 79:1725–9.
Shivaprasad HL, Timoney JF, Morales S, Lucio B, Baker RC 1990 Pathogenesis of Salmonella
Enteritidis infection in laying chickens I Studies on egg transmission, clinical signs, fecal shedding, and serologic responses Avian Dis 34:548–57.
Sivaramakrishnan SR 2007 Microwave pasteurization of shell eggs-a preclude [MSc tion] Quebec: McGill Univ 88 p.
disserta-Thompson JF, Knutson J 2000 Rapid cooling of shell eggs J Appl Poultry Res 9:258–68.
USDA 2010 USDA, AMS, Poultry market news and analysis weekly egg products report october 15, 2010 The “source” of poultry and egg market information Available from:
http://www.ams.usda.gov/mnreports/pyweggproducts.pdf Accessed Oct 15, 2010.
Vought KJ, Tatini SR 1998 Salmonella Enteritidis contamination of ice cream associated with a
1994 multistate outbreak J Food Prot 61:5–10.
Williams JE, Dillard LH, Hall GO 1968 The penetration patterns of Salmonella Typhimurium
through the outer structures of chicken eggs Avian Dis 12:445–66.
Worley BS, Wheatley WB, Lauten SD, Williams DE, Mora EC, Worley SD 1992 Inactivation of
Salmonella on shell eggs by novel N-halamine biocidal compounds J Ind Microbiol 11:37–42.
Trang 11Pathogens’ Dose–Response Curves Using
the Expanded Fermi Solution
Micha Peleg, Mark D Normand, and Maria G Corradini
Abstract: Theoretically, the relationship between the number of pathogens that cause acute infection if settling in the gut,
N , and that initially ingested, M, can be constructed from the survival probabilities at the different “stations” along the digestive tract These probabilities are rarely known exactly, but their ranges can be estimated If for a given N one generates estimates of M using random probabilities within these ranges, the estimates’ distribution will be approximately lognormal
and its cumulative (CDF) form will represent the pathogen’s dose–response curve The distribution’s logarithmic meanand standard deviation can be calculated from the ranges with a formula and used to plot the curve The method wasused to generate dose–response curves of hypothetical food and waterborne pathogens and calculate their infective dose(ID) at 5%, 50%, and 95% probability The curves were compatible with the Beta Poisson model and robust against minorperturbations in the underlying probabilities’ ranges The calculation and plotting procedure was automated and posted
on the Internet as a freely downloadable interactive Wolfram Demonstration It allows the user to generate, modify,examine, and compare dose–response curves, and to calculate their characteristics, by moving sliders on the screen.Keywords: dose–response, food poisoning, infectious dose, lethal dose, risk assessment
Introduction
In the context of gastrointestinal infection, a dose–response
curve usually refers to a plot of the relationship between the
number of pathogens’ units ingested (x-axis) and the
probabil-ity or frequency that it will have a clinical manifestation as illness
or death (y-axis) The pathogen can be a food or waterborne
infectious microorganism (including protozoa), a virus, and, less
frequently, a bacterial spore Dose–response curves have been an
important tool in microbial risk assessment (MRA) They have
been amply investigated and documented, as well as
mathemat-ically characterized (see Cassin and others 1998; Holcomb and
others 1999; Haas and others 2000; Buchanan and others 2000,
Kothary and Babu 2001; Strachan and others 2005, for example)
The most commonly used 2 parameter mathematical model used
to describe dose–response curves is the Beta Poisson (BP) model
where Pinf(dose) is the probability of infection after ingesting a
dose, the number of pathogen units, and α and β constants,
char-acteristic to the particular pathogen and affected by environmental
circumstances (for example, Holcomb and others 1999; Teunis and
others 1999; Haas and others 2000) Alternative models include
lognormal, log logistic, simple exponential, a “flexible”
expo-MS 20100958 Submitted 8/24/2010, Accepted 11/10/2010 Authors Peleg and
Normand are with Dept of Food Science, Univ of Massachusetts, Amherst, MA
01003, U.S.A Author Corradini is with Inst de Tecnolog´ıa, Facultad de Ingenier´ıa y
Ciencias Exactas, Univ Argentina de la Empresa, Cdad de Buenos Aires, Argentina.
Direct inquiries to author Peleg (E-mail: micha.peleg@foodsci.umass.edu).
nential, and Weibull–Gamma models (Holcomb and others 1999;Haas and others 2000), expanded versions of the BP and othermodels (for example, Bartrand and others 2008) and a version ofthe BP model into which the Time Post Incubation (TPI) hasbeen incorporated (Huang and Haas 2009) The mathematicalproperties of these functions, their fit to simulated and experi-mental data, and relation to the infectious mechanism have alsobeen amply studied and compared (for example, Teunis and oth-ers 1996; Holcomb and others 1999; Teunis and Havelaar 2000;Xie Yang and others 2000; Latimer and others 2001) In manycases, experimental dose–response data on a particular pathogenhave been limited for logistic, technical and ethical considerations,especially when human volunteers have been involved The prob-lem is further complicated by that the infectivity of pathogens canvary dramatically from a few units in some of the most virulent(Alam and Zurek 2006) to many thousands or even millions inthe less infectious (Teunis and others 1996) Thus while the ordi-nate of the dose–response curves’ plots is almost always linear, that
of the abscissa is frequently logarithmic Also, data obtained fromepidemiological and laboratory studies frequently do not coverthe entire dose–response curve and are notoriously scattered Forthese reasons, comparison of the fit of the various dose–responsemodels has not always rendered a clear winner
The scatter in dose–response data is not unexpected As hasbeen long recognized, the clinical manifestation of an infection,and sometimes even its reporting, is the result of a stochasticprocess, involving probabilities that can rarely be accurately as-sessed, let alone determined exactly—see subsequently There-fore, an element of uncertainty is inherent in the construction ofdose–response curve, regardless of the mathematical model usedfor its characterization and whether the independent variable isthe actual number of pathogens ingested or their logarithm Sinceinfection by a food or water borne pathogen is largely but not
C
2011 Institute of Food Technologists R
Trang 12exclusively affected by the conditions of the infective agent, the
particulars of the medium in which it has been transmitted, and
the immunological state of the humans that have ingested it, the
observed scatter in recorded and reported dose–response curves is
not at all surprising
Construction of a dose–response curve, in contrast with merely
fitting epidemiological or experimental laboratory data, is based
on certain underlying assumptions The most well known of these,
is the “single-hit” hypothesis and its relation to the Beta Poison
model, which was critically evaluated by Teunis and Havelaar
(2000) The “stochastic approach” is based on identifying the
chain of events that leads to infection or death, assigning them
with probabilities and multiplying these probabilities to obtain
the occurrence’s probability (Teunis and others 1996) The
con-cept has been the foundation of several risk assessment methods—
see Cassin and others 1998, for example The starting point of the
methods based on it is the tacit admission that the details of the
events leading to the infection or death are rarely, if ever, known
with certainty and hence the appeal to probabilities The “Fermi
Solution” is a general method of estimation based on the
multi-plication and division of several factors whose values, which are
unknown, can nevertheless be “reasonably assumed.” It is named
after the great physicist Enrico Fermi (1901 to 1954) who made it
a high art (von Baeyer 1993) We will explain the term
“reason-ably assumed” and describe the method and its expansion in the
following sections The objectives of this study were to use an
ex-panded version of the Fermi Solution, which has been originally
developed for microbial risk assessment (Peleg and others 2007),
to generate food and water pathogens’ dose–response curves when
hard information on what happens to them prior to and after their
ingestion is scant or nonexistent, and to develop a user-friendly
in-teractive program to do the calculations and post it on the Internet
as freely downloadable software
Methodology
The “Fermi Solution”
Suppose the number of pathogens “units” reaching viable or
set-tling in the human gut needed to cause acute infection is known
or can be assumed to be N , N ≥ 1 How many “units,” M,
M ≥ N, ought to be ingested in order that N of them will
survive the digestive tract’s hurdles to cause the infection? The
question can be answered by animal studies but this will leave
extrapolation to humans an open issue When the infective agent
is not lethal and does not cause permanent damage, the answer
can come from studies where it is used to infect human
volun-teers But in this case, although the number of ingested units,
M, can be controlled, the actual number of surviving pathogen
units that had produced the acute infection, N , might have varied
among the subjects Either way, an element of uncertainty will
remain and the relationship between M and N would have to be
estimated rather than accurately determined Moreover, the
sus-ceptibility of individual humans to the same infective agent also
can vary widely as a result of its origin and history, their
im-mune system’s state, the particular food with which it has been
ingested, and other factors Another way to approach the
ques-tion is to assume that the number of viable pathogen units, N ,
reaching the gut is the number of units ingested, M, multiplied
by a series of probabilities, P i’s, that they will survive the
se-ries of hurdles posed by the host’s digestive and immune systems
Identification and quantification of these hurdles, or “barriers”
(Teunis and Havelaar 2002), is a major issue in what is known
as “Key Events Dose–Response Framework” (Julien and others2009) But while listing the hurdles or barriers can be straightfor-ward, see Buchanan and others (2009) for example, quantification
of their roles is not This is for the same reasons already tioned, that is, the inherent variability in the response of individ-ual humans, the pathogen’s infectivity dependence on its origin,history, and the food with which it has been ingested (for exam-ple, Peterson and others 1989, Hofmann and Eckmann 2006), and
men-so on
For simplicity, let us suppose that P1 is the probability thatthe microbial pathogen survives the stomach’s acid and enzymes,
P2 that it survives the pancreatic juice, P3 that it survives the
bile, P4that it survives the competition from resident microbiota(“microflora”) in the gut, and so on If indeed these are all themajor factors that determine this pathogen’s survival and ability
to infect (although an expert would certainly add a few other
probabilities to the list), then if all these Pi’s could be “reasonably
estimated,” the expected number of pathogen units that will cause
acute infection, N , would be:
N = M ∗ P1∗ P2∗ P3∗ P k (2)
Hence M, the number of ingested pathogen units that will result
in N viable pathogen units, is:
P1· P2· P3· P k
(3)
For example, if 10 surviving pathogens in the gut are required
to cause acute infection, N = 10, and there are 5 main survival
probabilities: P1= 0.05, P2= 0.8, P3= 0.7, P4= 0.5, P5= 0.9,
then M = 10/(0.05 ∗ 0.8 ∗ 0.7 ∗ 0.5 ∗ 0.9) = 794 (rounded tothe nearest integer) This is the “Fermi Solution,” albeit with a
fairly small number of factors Notice that if one of the Pi’s value
is very close to 1, then the corresponding factor can be considered
unimportant and discarded At the other end, if one of the Pi’s
value is very small, 10−4 or 10−6 say, then the correspondinghurdle is overwhelming as far as the pathogen is concerned Insuch a case, a very large number of pathogen units would have
to be ingested to be infective This number’s order of magnitudewould be determined not only by that particular probability but
by the other probabilities too
A “reasonable estimate” of a parameter’s role, expressed as a vival probability here, is not a scientifically defined term How-ever, it is not the same as a wild guess, and experts in the fieldcan come up with estimates on the basis of experience and/orpublished clinical or laboratory data, for instance, which might
sur-be fairly accurate (Peleg and others 2007) The reason why themethod frequently works is that in a series of “reasonably es-timated” parameter values it is unlikely that all or the decisivemajority will be exaggerated in one direction It is more likelythat overestimation of some parameter values will be at least partlycompensated by underestimation of the values of other parameters,and vice versa Thus, although never guaranteed, the Fermi Solu-tion method often renders an estimate that is close to the correctvalue
The Expanded Fermi Solution
One of the shortcomings of the original Fermi Solutionmethod, especially if the number of factors (probabilities in our
Trang 13Pathogens dose–response curves
case) is small, is that a substantial error in 1 or 2 can skew the
final estimate Also, different experts may disagree on some or
all of the parameters values, which will result in a considerable
discrepancy between their estimates A way to avoid such
er-rors and reduce potential disagreement among experts is to
spec-ify the parameters magnitudes not as single values but as their
likely lower and upper bounds instead (Peleg and others 2007,
2011) Thus, experts who might vigorously disagree on a
partic-ular value are more likely to reach a consensus concerning the
range in which this value ought to be Once the key factors have
been identified and their corresponding probability ranges
de-cided, one can generate numerous Fermi Solution estimates using
combinations of random values of the Pi’s each within its assigned
range It can be shown, that the distribution of these estimates,
will be approximately lognormal, a manifestation of the central
limit theorem (Peleg and others 2007, 2011) The distribution’s
logarithmic mean,μ L, and standard deviation,σ L, can be used
to calculate the sought “Best Estimate” being the distribution’s
mode (ibid) This is the Expanded Fermi Solution’s Monte Carlo
version
When the random numbers used to pick the parameters values
within their respective ranges have a uniform distribution,
reflect-ing “maximum ignorance,” the lognormal distribution’s
logarith-mic mean,μ L, and standard deviation,σ L, can be also calculated
analytically (see J Horowitz’s appendix to Peleg and others 2007)
In our case, we want to estimate M for a given (fixed) N , which
where,μ LM andσ LM are the distribution’s logarithmic (base e)
mean and standard deviation, respectively, andμ LPiandσ LPithose
of the probabilities
When the lower and upper bounds of a probabilities i is specified
by P imin and P imax, respectively, the logarithmic mean (μLPi) and
standard deviation (σLPi) are calculated from
μ L P i = P i maxloge (P i max)− P i minloge (P i min)
whereμ LPiis calculated by Eq 6
Onceμ LM andσ LM have been calculated in this way, the
cu-mulative form (CDF) of the lognormal distribution can be used to
generate the relationship between the probability of N pathogens
settling in the gut and causing acute infection for any M of them
ingested A plot of this relationship on linear or log-linear
coordi-nates (see Figure 1) will be the estimated dose–response curve for
the pathogen in question whose infectivity and survivability are
specified by the assigned N and set of probability ranges,
us-N and/or the P i’s minimum and maximum values They can beentered as numbers or by moving sliders on the screen with themouse As soon as any of the parameters is altered, the curvecorresponding to the new setting will be displayed almost instan-taneously on the screen Above the plot, the new numerical values
of the logarithmic mean (μL) and standard deviation (σL) lated by Eqs 4 to 7 will also appear, accompanied by the 5%, 50%,and 95% quantities, that is, the infective doses, ID5%, ID50%, and
calcu-ID95% The Demonstration also allows the user to enter any
par-ticular value of M by typing its value, or moving its slider, and the
corresponding probability’s numerical value will also be displayed.The location of the selection is shown as a moving colored dot onthe displayed curve The curve itself can be plotted on linear orlog-linear coordinates, the choice being made by clicking on the
“x-axis” bar setter
The ease at which N and the probabilities are varied enables the
user to generate a large number of their combinations in a veryshort time and examine their potential effect on the dose–responsecurve and its characteristics, notably ID50% The Demonstration
also facilitates the examination of how N , the assumed number of
surviving pathogen units needed to cause an infection, affects therisk when different amounts of the pathogen are ingested Or al-ternatively, the Demonstration can be used to assess how errors oruncertainties in the probabilities’ lower and upper bounds mightaffect the dose–response curve’s shape and its characteristics TheDemonstration, therefore, can facilitate an expert team’s discussionand help them reach a consensus Or, if a disagreement among theexperts persists, the Demonstration would enable them to quantifythe differences in their assessments in clearly defined terms, that is,the corresponding ID5%, ID50%, and ID95% The large ranges of N ’s and M’s that the Demonstration allows would enable the assessors
to examine scenarios involving a single or very few pathogens, as
in cases involving extremely virulent ones, and scenarios ing up to 108pathogens, which would include most of if not allthe more “benign” ones In its present version, the Demonstra-tion only allows the user to enter up to 6 different probabilities
involv-To include more will require the program’s code modification involv-Touse fewer than 6 probabilities, all the user has to do is set thelower and upper limits of the unneeded probabilities to 1 Inthe current version of the Demonstration posted in the Internet,the probabilities’ ranges are all set to 0.001 to 1 To change any
of them or zoom on a particular probability interval, from 0.0001
to 0.0005 or from 0.5 to 0.7, say, will require replacement the
Trang 14Figure 1–Examples of simulated “monotonic”
and sigmoid dose–response curves generated
by the Expanded Fermi Solution plotted on linear and log-linear coordinates.
Figure 2–Screen display of the Wolfram Demonstration that generates dose–response curves using the Expanded Fermi Solution method Note thatN, M, and the P i’s are all in lower case.
Trang 15Pathogens dose–response curves
corresponding current values of Pmin and Pmax by these new
values
The method and program can be extended to probabilities
in-volving a pretreatment of the food or water (Peleg and others
2010) For example, P1 can be the probability that the pathogen
in question has survived refrigeration or washing of the food,
or the water disinfection, in which case M will be the
num-ber of pathogen units present prior to the treatment In a
sim-ilar manner, one can include in the analysis the probability of
mortality from the infection In such a case, P5 might be the
probability of the pathogen establishing itself in the gut and
P6 that if there would cause the victim’s (human or animal)
death
Comparison of the Lognormal and Beta Poisson Models
As already stated, the Beta Poisson equation is the most
commonly used mathematical dose–response model However, it
has been amply demonstrated that alternative 2 parameter models,
including the cumulative form of the lognormal distribution, can
have a comparative fit, especially when applied to highly scattered
data To demonstrate the point, we have generated numerous
smooth dose–response data with the Beta Poisson model, added to
it a random scatter of various amplitudes, and fitted the scattered
data with the lognormal model (Eqs 8 and 9—see subsequently)
The procedure to generate the data, choose the number of points
(5 to 300 in the current web version) and the scatter’s amplitude,
execute the nonlinear regression and display the comparison
between the resulting dose–response curves has been automated
It is available to the reader in the form of another freely
downloadable Wolfram Demonstration, open: http://demon
of α and β values, and M range, where the Beta Poisson and
lognormal models can be used interchangeably for all practicalpurposes Notice that published real dose–response curves rarelyhave more than 5 to 10 data points and, in many cases, a largerscatter than that shown in the figure and allowed by the program.The reason for including the much larger number of data pointsand a smaller scatter is to demonstrate that the lognormal model’sfit is not accidental or merely an artifact of the scatter
The Beta Poisson model is a simple algebraic equation while thecumulative form of the lognormal distribution is not—it containsthe Error Function (erf), which requires numerical integration forits values calculation, that is,
p[m ] := CDF[LogNormalDistribution[muL, sigmaL], m] (9)where CDF stands for the cumulative density function of the dis-tribution specified between the brackets The function p[m] in
Eq 9, representing P(M) in the text, is executed and plotted just
as fast as an algebraic function containing only power, exponential
or logarithmic terms, and so on (For this reason, the WolframDemonstration’s response to changes in the sliders appears in-stantaneous.) Also, p[m] as defined by the equation can be used
as a model for nonlinear regression to extract μ L andσ L fromexperimental dose–response data The lognormal distribution
Figure 3–Screen display of the Wolfram Demonstration that generates scattered dose–response data with the Beta Poisson model and fits them with the lognormal model.
Trang 16model’s parameters may have little intuitive meaning to many
prac-titioners But the same can be said on the Beta Poisson model’s
parametersα and β and on the parameters of other dose–response
models, perhaps with the exception of the exponential model
The model parameters’ lack of intuitive meaning becomes a moot
issue when one compares different dose–response curves in terms
of their ID5%, ID50%, and ID95%as in the shown Wolfram
Demon-stration, or alternative terms such as ID1%, ID10%, and ID99%, and
so on
Table 1– The generation and virulence (“pathogenicity”)
pa-rameters used to generate the dose–response curves shown in
Figure 5 and calculate the hypothetical pathogen’s Infective
The Results’ Robustness Against Small Perturbations
Examples of dose–response curves created with the model areshown in Figure 5 and 6 Each pair of curves was generated with
2 sets of slightly different probability ranges whose numericalvalues and the resulting dose–response parameters are given inTable 1 and 2 The figures and tables demonstrate that themethod is robust against small perturbations This was also ob-served in other applications of the methods (Peleg and others 2007,
Table 2– The generation and virulence (“pathogenicity”) rameters used to generate the dose–response curves shown in Figure 6 and calculate the hypothetical pathogen’s Infective Doses. a
Figure 5–A pair of dose–response curves generated with the Expanded Fermi Solution method with slightly different probability ranges and plotted on linear coordinates The corresponding generation, fit and “pathogenicity parameters” are listed in Table 1 Note the method’s robustness against minor perturbations in the underlying probability ranges.
Trang 17Pathogens dose–response curves
Figure 6–A pair of dose–response curves generated with the Expanded Fermi Solution method with slightly different probability ranges and plotted on log-linear coordinates The corresponding generation, fit and
“pathogenicity parameters” are listed in Table 2 Note the method’s robustness against minor perturbations in the underlying probability ranges.
2010) and is therefore not surprising It suggests that experts can
reach a consensus on the final result even if minor differences of
opinion concerning the magnitudes of one or more probabilities
remain unsettled But more importantly, the ease at which the
dose–response curves are generated enables the experts to
exam-ine immediately how their differing estimates of the ranges would
affect the final assessment
Again, the closer the probabilities estimates are to their
cor-rect values and the narrower their ranges, the more accurate and
reliable the dose–response curve so created will be Had all the
probabilities been known exactly, the Expanded Fermi Solution
would be reduced to the original Fermi Solution method But
encountering such a scenario, as previously explained, is very
un-likely because of the inherent uncertainties concerning the events
that lead to an infection or in severe cases death
Conclusions
The proposed Expanded Fermi Solution is not intended to
re-place clinical or experimental methods to determine microbial
dose–response curves but a way to generate such curves in
situa-tions where information on the infection either does not exist or is
insufficient to develop an accurate mechanistic model
Uncertain-ties concerning infection by food and waterborne pathogens stem
from inherent variability in their physiological state when ingested,
the number of “units” actually consumed, their fate during and
after passing through the digestive tract, and the state of the host’s
immune system among others In many cases, the virulence of a
pathogen is determined in an animal model Thus, extrapolation
of the results to humans introduces another kind of uncertainty
The same can be said about dose–response data obtained from
human volunteers Although the most reliable, the issue of how
representative a group of rather healthy volunteers is of the
popula-tion at large remains unresolved The extrapolapopula-tion issue becomes
even more troublesome when it comes to assessing the risk of
par-ticularly vulnerable groups or segments of the populations, such
as the elderly, chronically ill, malnourished children or infants,
and people with a compromised immune system In such cases,
the Expanded Fermi Solution offers a way to integrate expertise
and knowledge obtained from related cases or the literature to
construct a dose–response curves that could not be determined
directly To do that, one would only need to adjust the number
of surviving pathogens that are likely to cause an acute infection
or death, identify the probabilities that determine their survival
and infectivity once ingested, and set their most likely lower and
upper bounds Although the method does not guarantee an rate result, the dose–response curves that it generates might have abetter chance to be close to the correct ones than those based onarbitrary assumptions or guessed parameters values
accu-The Expanded Fermi Solution method can also be useful inestablishing a decision making protocol that can be evaluated aposteriori and if necessary revised This could be done by adding
or eliminating probabilities or adjusting their lower and upper
boundaries, for example, or by modifying the value of N when
new information becomes available The Wolfram Demonstrationthat generates and plots the dose–response curve and calculatesits parameters (ID5%, ID50%, and ID95% in the current version),enables quick assessment of the consequences of a change or vari-ations in the model’s parameters Through modification of theprobability ranges, the method also enables the user to comparethe survivability and virulence of different pathogens and to sim-ulate the effects of environmental factors, such as the food withwhich the pathogen is ingested, on the dose–response curve’s char-acteristics Yet, we should reiterate that the probabilistic approachhas only been developed for situations where hard information isscarce and for simulations It should not be construed as a reason
to reduce the efforts to collect relevant clinical and ical data These have been and will continue to be the basis ofpathogens’ dose–response curves construction Hopefully, futureresearch will show that the clinical and probabilistic approaches arecomplementary, the first creating the database and its mechanisticinterpretation and the second serving as a tool of risk quantifi-cation and attempts at its extrapolation to populations for whichdata are scarce or nonexistent
Bartrand TA, Weir MH, Haas CN 2008 Dose–response models for inhalation of Bacillus anthracis
spores: Interspecies comparisons Risk Anal 28:1115–24.
Buchanan RL, Smith JL, Long W 2000 Microbial risk assessment: dose–response relations and risk characterization Int J Food Microbiol 58:159–72.
Buchanan RL, Havelaar AH, Smith MA, Whiting RC, Julien E 2009 The key events dose–response framework: its potential for application to foodborne pathogenic microor- ganisms Crit Rev Food Sci Nutr 49:718–28.
Cassin MH, Paoli GM, Lammerding AM 1998 Simulation modeling for microbial risk ment J Food Prot 61:1560–6.
Trang 18Haas CN, Thayyar-Madabusi A, Rose JB, Gerba CP 2000 Development of a dose–response
relationship for Escherichia coli O157: H7 Int J Food Microbiol 56:153–9.
Hofmann AF, Eckmann L 2006 How bile acids confer gut mucosal protection against bacteria.
Proc Natl Acad Sci 103:4333–4.
Holcomb DL, Smith MA, Ware GO, Hung YC, Brackett RE, Doyle MP 1999 Comparison
of six dose–response models for use with food-borne pathogens Risk Anal 19:1091–100.
Huang Y, Haas CN 2009 Time-dose–response models for microbial risk assessment Risk Anal
29:648–61.
Julien E, Boobis AR, Olin SS 2009 The key events dose–response framework: a
cross-disciplinary mode-of-action based approach to examining dose–response and thresholds Crit
Rev Food Sci Nutr 49:682–9.
Kothary MH, Babu US 2001 Infective dose of foodborne pathogens in volunteers: a review.
J Food Safety 21:49–73.
Latimer HK, Jaykus LA, Morales RA, Cowen P, Crawford-Brown D 2001 A weighted
com-posite dose–response model for human salmonellosis Risk Anal 21:295–305.
Peleg M, Normand MD, Horowitz J, Corradini MG 2007 An expanded Fermi solution for
microbial risk assessment Int J Food Microbiol 113:92–101.
Peleg M, Normand MD, Horowitz J, Corradini MG 2011 Expanded Fermi solution for
estimating the survival of ingested pathogenic and probiotic cells and spores Appl Environ
Microbiol 77:312–9.
Peterson WL, Mackowiak PA, Barnett CC, Marling-Cason M, Haley, ML 1989 The human gastric bactericidal barrier: mechanisms of action, relative antibacterial activity, and dietary influences J Infect Dis 159:979–83.
Strachan NJC, Doyle MP, Kasuga F, Rotariu O, Ogdend ID 2005 Dose response modelling
of Escherichia coli O157 incorporating data from foodborne and environmental outbreaks Int
284550002 Bilthoven, The Netherlands: Natl Insti of Public Health and the Environment.
Teunis PFM, Nagelkerke NJD, Haas CN 1999 Dose–response models for infectious teritis Risk Anal 19:1251–60.
gastroen-von Baeyer HC 1993 The fermi solution: essays on science, Random House, New York, N.Y.
Xie Yang BSX, Karmali M, Lammerding AH 2000 A novel dose–response model for foodborne pathogens using neural networks IEEE 4:2551–6.
Trang 19Polysaccharide and Protein-Based Films
Containing Active Agents
Kuorwel K Kuorwel, Marlene J Cran, Kees Sonneveld, Joseph Miltz, and Stephen W Bigger
Abstract: Significant interest has emerged in the introduction of food packaging materials manufactured from able polymers that have the potential to reduce the environmental impacts associated with conventional packaging mate-rials Current technologies in active packaging enable effective antimicrobial (AM) packaging films to be prepared frombiodegradable materials that have been modified and/or blended with different compatible materials and/or plasticisers
biodegrad-A wide range of biodegrad-AM films prepared from modified biodegradable materials have the potential to be used for packaging
of various food products This review examines biodegradable polymers derived from polysaccharides and protein-basedmaterials for their potential use in packaging systems designed for the protection of food products from microbial con-tamination A comprehensive table that systematically analyses and categorizes much of the current literature in this area
is included in the review
Keywords: active packaging, antimicrobial agents, biodegradable film, food packaging
Introduction
Films and coatings prepared from biodegradable materials are
in-creasingly being used in the food packaging industry (Rodriguez
and others 2006) Biodegradable polymers can be produced from
natural, renewable resources (for example, starch), chemically
syn-thesized from natural sources (for example, poly[lactic acid]), or
made from microbiologically produced materials (for example,
hydroxybutyrate and hydroxyvalerate) (Petersen and others 1999;
Cagri and others 2004; Cha and Chinnan 2004; Perez-Gago and
Krochta 2005; Pommet and others 2005; Weber and others 2002)
These biopolymers can decompose more readily in the
environ-ment than their synthetic polymeric counterparts such as
polyethy-lene (PE), polypropypolyethy-lene (PP), and polystyrene (PS) that are
derived from crude oils (Guilbert 1986; Chick and Ustunol 1998;
Tharanathan 2003; Cutter 2006; Lopez-Rubio and others 2006;
Altsk¨ar and others 2008; Iovino and others 2008; Dias and
oth-ers 2010) Consumer demands for preservative-free, high-quality
food products, packaged in materials that create less environmental
impact have inspired research into the application of biopolymeric
materials In combination with antimicrobial (AM) packaging
sys-tems, biopolymer materials with AM properties are emerging as
one of the more promising forms of active packaging systems
(Krochta and De Mulder-Johnston 1997; Cha and Chinnan 2004;
Hernandez-Izquierdo and others 2008) Further development of
food packaging materials manufactured from biodegradable
poly-mers such as starch-based materials have the potential to reduce
MS 20101154 Submitted 10/11/2010, Accepted 1/26/2011 Authors Kuorwel
and Bigger are with School of Engineering and Science and author Cran is with
Inst for Sustainability and Innovation, Victoria Univ., PO Box 14428, Melbourne,
8001, Australia Author Sonneveld is with KS PackExpert & Assoc., PO Box 399,
Mansfield, 3724, Australia Author Miltz is with Dept of Biotechnology and Food
Engineering, Technion-Israel Inst of Technology, Haifa, 3200, Israel Direct inquiries
to author Bigger (E-mail: stephen.bigger@vu.edu.au).
environmental impacts thereby being advantageous over tional synthetic-based packaging systems (Vlieger 2003).Active packaging (AP) is a system in which the product, thepackage and the environment interact in a positive way to extendshelf life or improve microbial safety or sensory properties whilstmaintaining the quality of food products (Miltz and others 1995;Rooney 1995; Devlieghere and others 2000; Han 2000; Quin-tavalla and Vicini 2002; Suppakul and others 2003b) According
conven-to Rooney (1995) and Matche and others (2004), the additionalpreservation roles rendered by AP systems to the packaged foodproduct differentiates them from traditional packaging systems,which offer only protective functions against external influences
A polymeric film mixed with an AM agent can be vital in ling microbial growth on the surfaces of foods; hence leading to
control-an extension of the shelf life control-and/or improved microbial safety offood products (Padgett and others 1998; Ojagh and others 2010).Several researchers have published review articles in the area ofbio-based polymers with a detailed discussion of potential foodpackaging applications (Krochta and De Mulder-Johnston 1997;Petersen and others 1999; Weber 2000; Tharanathan 2003; Cagriand others 2004; Cutter 2006) as well as the general issues affecting
AM packaging (Olivas and Barbosa-Canovas 2009) Many of the
previous studies focus on key foodborne pathogens such as Listeria,
S aureus, E coli, and Salmonella (Maizura and others 2008; Ojagh
and others 2010; Shen and others 2010) The reasons for focusing
on foodborne pathogens in particular is clear but to food facturers the cost/benefit is a major consideration and extendingthe shelf life of real foods, by diminishing spoilage, is a primarygoal The number of published research studies with AM packagesfor real foods is, however, limited In spite of the importance ofthe cost/benefit ratio for food manufacturers, a detailed analysis
manu-of the cost effectiveness manu-of AM packaging systems developed frombio-polymeric materials is outside the scope of this review
In the present review, the concept of AM packaging systemswith respect to food packaging applications is considered with a
C
2011 Institute of Food Technologists R
Trang 20focus on biodegradable films, mainly polysaccharides and
protein-based materials This is followed by a detailed discussion of various
forms of films incorporated and/or coated with AM agents
Fi-nally, consideration is given to coating and immobilization of AM
agents onto films prepared from biodegradable materials
Polysaccharides and Protein-Based Materials
Interest has increased recently in the potential uses of films
and coatings manufactured from biodegradable polymers
par-ticularly polysaccharides and protein-based materials In the last
15 y or so and especially in recent years the interest in these
ma-terials has been primarily for use in food packaging (Krochta and
others 1994; Baldwin and others 1995; Krochta and De
Mulder-Johnston 1997) Polysaccharides and proteins-based films
demon-strate adequate gas barrier properties (Hernandez-Izquierdo and
Krochta 2008) Examples of polysaccharide-based polymers that
have a potential to be used in AM packaging systems or can
be used in conjunction with AM agents include starch, alginate,
cellulose, chitosan, and carageenan Examples of proteins-based
materials include whey protein, soya protein, corn zein, and/or
their derivatives (Krotcha and others 1997; Dawson and
oth-ers 2002; Krochta 2002; Cagri and othoth-ers 2004; Brody 2005;
Phan and others 2005; Rodriguez and others 2006)
Further-more, various forms of polysaccharides, protein-based polymers,
and/or other biodegradable polymers identified by Weber (2002)
have the potential to be developed into active packaging
terials for food packaging applications Many bio-based
ma-terials such as polysaccharides and protein-based polymers are
hydrophilic with a relatively high degree of crystallinity causing
processing and performance problems Therefore, AM packages
made from such biodegradable films demonstrate high moisture
sensitivity, poor water barrier, and poor mechanical properties
compared to those made from synthetic polymers (Weber and
others 2002)
Packaging materials with suitable physico-mechanical
proper-ties can nonetheless be prepared from biopolymers such as
starch-based materials when the biodegradable materials are modified by
physical, mechanical, and/or chemical techniques or by
blend-ing them with compatible plasticisers (Arvanitoyannis and
oth-ers 1998; Garc´ıa and othoth-ers 2000a; Tharanathan 2003; Fang and
others 2005; Pommet and others 2005; Davis and Song 2006)
Plasticizers are relatively low molecular weight compounds that
can be copolymerized with the polymer or added to the
poly-mer to reduce the intermolecular and intramolecular forces and
thereby increase the mobility of the polymeric chains (Garc´ıa and
others 2000b; Tharanathan 2003; Sothornvit and Krochta 2005)
Plasticizers are usually mixed with biopolymers to improve
pro-cessing, increase film flexibility, and lower the glass transition
tem-perature (Arvanitoyannis and Biliaderis 1999; Av´erous and others
2000; Krochta 2002; Brody 2005; Fang and others 2005; L ´opez
and others 2008; Zhang and Liu 2009) Examples of plasticizers
that are commonly used with biopolymers include polyols such as
glycerol, sorbitol, and mannitol; monosaccharides such as fructose,
glucose, and mannose, and poly(ethylene glycol) (Kester and
Fen-nema 1986; Brody 2005) Water is another important plasticiser
for biodegradable films although excess moisture may affect the
film properties (Van Soest and Essers 1997; Krochta 2002) Water
can be added to a starch-based film to break its native granular
structure and hydrogen bonding (Yang and Paulson 2000; Mali
and others 2002; Myll¨arinen and others 2002)
When a biopolymer is chemically, mechanically, or physically
modified, it is able to exhibit thermoplastic properties
(Arvani-toyannis and Biliaderis 1999) Modified biodegradable materialssuch as starch can thus be manufactured into a suitable packagingfilm using conventional plastic conversion processes like compres-sion molding, extrusion, and thermoforming (Carvalho and oth-ers 2005; Jin and Zhang 2008; Kristo and others 2008) Packagingfilms made from biodegradable polymers such as polysaccharidesexhibit low gas permeability, enabling the extension of shelf life offood products without creating anaerobic conditions (Baldwin andothers 1995) These biodegradable films or coatings can also beused to prolong the shelf life of foods such as muscle food products
by preventing dehydration, oxidative rancidity, and surface ing (Nisperos-Carriedo 1994) Recently, commercially developedstarch-based packaging materials like PlanticR, EverCornTM, andBio-PTM made by Plantic Technologies (Melbourne, Australia),Novamont (Italy), and Bioenvelope (Japan), respectively, becameavailable (Robertson 2008; Garc´ıa and others 2009) These ma-terials can be used in commercial applications to package foodproducts such as biscuits and snacks Biodegradable materials havealso found successful applications in the pharmaceutical industry
brown-as films or coatings to control drug relebrown-ase (Soppimath and ers 2001; Tuovinen and others 2003; Siepmann and others 2004;
oth-Arifin and others 2006)
Preparation of AM Films from Biodegradable Materials
The main processing techniques used for the preparation ofbiodegradable films are similar to those used in synthetic plasticsprocessing; these include wet and dry processing methods (Brody2005; Pommet and others 2005) The wet methods comprise sol-vent casting (which is the most commonly used laboratory-scaletechnique to prepare AM films from biopolymers) whereas thedry methods usually involve compression molding or extrusion ofthe biopolymers that have been modified to become thermoplas-tic (Van Soest and Essers 1997; Mehyar and Han 2004; Pommetand others 2005; Liu and others 2006; Thunwall and others 2006;
Nam and others 2007; Chal´eat and others 2008) The processingtechniques may significantly affect the properties of the resultant
AM film made from a biodegradable material (Altsk¨ar and others2008) Different factors affect the choice of the processing tech-niques when preparing an AM packaging film (Han 2005) Theseinclude the type and properties of the polymer, the properties ofthe AM agent (such as polarity and compatibility with the poly-mer), the heat stability of the latter during processing and the resid-ual AM activity after manufacturing (Han 2000) When a polar
AM agent is added to a nonpolar polymer to produce an AM film,the incorporated AM agent may affect the physical and mechanicalproperties of the resultant AM film (Han 2003) However, if the
AM agent is compatible with the polymer, a considerable amount
of it can be incorporated into the packaging material with minimalphysico-mechanical property deterioration (Han and Floros 1997;
Suppakul 2004; Han 2005; Rupika and others 2008) Therefore,the polymer and/or the AM agent may require modification prior
to film processing to increase the compatibility between the two(Cha and Chinnan 2004) During manufacturing of AM films, thetemperature and the shearing forces must be carefully considered(Han 2003) High processing temperatures may result in consid-erable losses of volatile AM agents (Han and Floros 1997; Han2000; Rupika and others 2005) Moreover, Cooksey (2005) sug-gested that the AM agent might partly or completely lose its AMactivity when incorporated into the film under harsh processingconditions For example, Nam and others (2007) reported up to48% recovery of the initial lysozyme activity in an extruded starch-based film upon increasing the extrusion temperature Therefore,
Trang 21AM activity of biodegradable films
to minimize the loss of AM agent during processing, as low as
possible temperatures should be applied as recommended by Han
and Floros (1998)
Antimicrobial Activity of Biodegradable Films
Numerous studies have identified migratory and nonmigratory
systems as the 2 main types of AM packaging systems A migratory
system contains an AM agent that can migrate into the headspace
of the package A nonmigratory system contains an AM agent
immobilized onto the packaging film In the latter case, the AM
film becomes effective against microbial growth when the food
and the packaging material are in direct contact (Appendini and
Hotchkiss 1997, 2002; Brody and others 2001; Vermeiren and
oth-ers 2002; Davidson and othoth-ers 2005; Han and Gennadios 2005)
These forms of AM packaging systems are designed primarily for
the purpose of protecting food products from deterioration and
spoilage by microorganisms The following subsections provide a
detailed overview of each of the different forms of AM packaging
systems by utilizing biodegradable films Table 1 shows that
signif-icant progress has been made by effectively integrating AM agents
into various biodegradable polymers, particularly polysaccharides
such as starch-based and protein-based films Such AM films have
demonstrated inhibitory activity against the growth of various
mi-croorganisms Understandably, the physico-mechanical properties
of the films are other important aspects to be considered when
designing the film for food packaging applications
Antimicrobial activity of biodegradable films
incorporated with AM agents
Impregnation of an AM agent into a packaging material is
a feasible means for achieving optimal AM activity of an AM
film (Weng and Hotchkiss 1993; Han 2003; Suppakul and others
2003a) This method enables a slow release of the agent onto the
food surfaces and the maintaining of an adequate concentration of
the agent to effectively inhibit microbial growth throughout the
product shelf life (Cooksey 2005; Salleh and others 2007) An AM
agent can be incorporated into a packaging material by blending
it with a base polymer before manufacturing (extrusion or
com-pression molding) of the film (Mistry 2006; Suppakul and others
2006; Rardniyom 2008; Rupika and others 2008) This method
enables the AM agent to be evenly distributed in the amorphous
region of the material (Suppakul 2004)
Antimicrobial activity of polysaccharide films
incorporated with AM agents
Biodegradable polysaccharides can be used for the production
of biodegradable films Polysaccharide-based films demonstrate
adequate film-forming properties, although they are sensitive to
moisture due to the hydrophilic groups in their structure (Krochta
and others 1994; Baldwin and others 1995; Han and Floros 1997)
Phan and others (2005) studied the functional properties of
agar-based and starch-agar-based films as well as their potential application
in food packaging They reported that films made from agar and
cassava starch demonstrated advanced functional properties
How-ever, these films exhibited poor moisture barrier properties
com-pared to low-density polyethylene (LDPE) films because of the
inherent hydrophilicity of the polysaccharides Dias and others
(2010) developed biodegradable films based on rice starches that
had improved mechanical properties
Amongst the polysaccharide-based polymers, the starch-based
ones are the most abundant and relatively inexpensive renewable
materials Starch is a natural polysaccharide primarily sourced from
cereal grains, potatoes, tapioca, and arrowroot (Baldwin and ers 1995; Cutter 2006; Zhang and Liu 2009) Starch consists ofamylose and amylopectin molecules present at different molecularratios Amylose is a linear molecule consisting of glucose unitsconnected by 1,4-glucosidic linkages and amylopectin is a highlybranched molecule consisting of short 1,4-glucose chains con-nected by 1,6-glucosidic linkages (Wu and others 1998; Parkerand Ring 2001; Rodriguez and others 2003; Maizura and oth-ers 2007) Starch is a semicrystalline, very hydrophilic material(Bicerano 2003) The amorphous and crystalline phases affect thephysical and chemical properties of starch-based films such as themechanical and gas barrier properties (Cha and Chinnan 2004;Liu 2005) Films manufactured from starch-based materials havebetter gas barrier properties than synthetic polymer films but theirmechanical properties are poorer A high amylose starch polymercan be formed into consistent, relatively strong and flexible filmsthat are highly impermeable to oxygen and carbon dioxide This
oth-is in contrast to high amylopectin starch polymers, that can only
be formed into noncontinuous and brittle films (Gennadios andothers 1997; Cha and Chinnan 2004) As expected, starch alonecannot be formed into films with adequate properties for foodpackaging (Arvanitoyannis and Biliaderis 1998; Phan and others2005) The intrinsic high level of hydrophilicity, poor mechanicalproperties, and difficulties in processing limit its applications infood packaging unless modified mechanically, physically, chemi-cally, or genetically (Arvanitoyannis and others 1998; Garc´ıa andothers 2000b; Marron and others 2000; Tharanathan 2003; Davisand Song 2006; Zhang and Liu 2009) Several studies have demon-strated that modified starch-based materials can be used in com-mercial applications to package dry and other solid food productssuch as biscuits, snacks, cereals, fresh produce, fruits, and vegeta-bles (Nisperos-Carriedo 1994; Wong and others 1994; Gennadiosand others 1997; Debeaufort and others 1998; Av´erous and others2001; Bravin and others 2006; Cutter 2006) and/or products withlow water activity (Olivas and Barbosa-Canovas 2009)
Table 1 demonstrates that many researchers have made siderable progress by successfully impregnating starch-based filmswith natural or synthetic AM agents Such AM starch-based filmshave shown inhibitory activity to the growth of various microor-
con-ganisms such as S enteritidis, L plantarum, B thermosphaceta B2, and L monocytogenes, E coli O157:H7, E coli, S aureus, and S typhimurium Durango and others (2006) developed an AM film
based on yam starch incorporated with chitosan at different centrations (1%, 3%, and 5% [w/v]) and reported a significant
con-reduction of S enteritidis in liquid culture by each of the films.
Nam and others (2007) incorporated 1% (w/w) lysozyme into a
pea starch film and demonstrated an AM activity against B sphaceta B2 Salleh and others (2007) studied the synergistic effects
thermo-of wheat starch films incorporated with lauric acid and chitosan
and found a significant AM activity of these films against B subtilis but not against E coli The researchers claimed that starch-based
films inhibited the growth of all tested microorganisms in liquidculture The latter observation may be unrealistic in terms of therelease of AM agent in the film because the starch-based filmpresumably dissolves in the liquid culture medium
Baron and Sumner (1993) showed that starch films impregnatedwith potassium sorbate and acidified with lactic acid reduced
the growth of S typhimurium by 4 log CFU mL−1 after 2 h at
37◦C The population count of E coli O157:H7 decreased by
2 log CFU mL−1after 3.5 h at 37◦C Furthermore, they foundthat corn-starch films impregnated with potassium sorbate inhib-
ited the growth of S typhimurium and E coli O157:H7 on poultry
Trang 26products stored at 7◦C for 12 d Maizura and others (2008)
investi-gated the antibacterial activity of starch-alginate film incorporated
with lemongrass oil The AM film inhibited the growth of E coli
O157:H7 and S enteritidis determined by the agar disc diffusion
assay but did not show any inhibitory effect on the growth of S.
aureus A recent study by Shen and others (2010) showed that sweet
potato starch film incorporated with 15% (w/w) potassium sorbate
or 5% (w/w) chitosan resulted in a significant reduction of E coli
on solid and semi-solid media compared to a control film
con-taining no potassium sorbate or chitosan that did not inhibit the
growth of E coli The sweet potato starch film incorporated with
10% (w/w) chitosan suppressed the growth of S aureus Corrales
and others (2009) showed that pea starch films impregnated with
grape seed extract inhibited the growth of B thermosphaceta B2 on
pork loin by 1.3 log CFU mL−1within the first 4 d of storage at
4◦C compared to the control film Pelissari and others (2009)
in-vestigated the AM activity of starch-based film incorporated with
oregano essential oil (EO) The use of the AM starch-based film
effectively inhibited the growth of E coli O157:H7, B cereus, and
S enteritidis in the agar disc diffusion assay.
Many of the abovementioned studies demonstrated AM activity
against various microorganisms using techniques involving
agar-based and liquid culture media Unfortunately, the question of the
moisture sensitivity of the starch-based materials and the
subse-quent usefulness of their films as commercial packaging systems has
not been adequately addressed in the literature to date Therefore,
further research is needed to show how to diminish the moisture
sensitivity and to enhance the physico-mechanical properties of
such starch-based materials so that these can be used for packaging
of moist food products Although, many starch-based materials
incorporated with various AM agents demonstrate AM activity,
an important aspect to be considered is the effect of increasing
the concentration of AM agent on the physico-mechanical
prop-erties of the resultant films Shen and others (2010) reported a
deterioration in the physico-mechanical properties of films upon
an increase in the potassium sorbate concentration Indeed, such
adverse effects could limit the prospects of applying such films in
food packaging applications
In many studies the AM activity of other polysaccharide-based
materials such as chitosan incorporated with AM agents has been
investigated Chitosan films have exhibited inhibitory activity on
the growth of various microorganisms, when impregnated with
AM agents For example, Ojagh and others (2010) developed
chi-tosan films containing 0.4% to 2% (v/v) of cinnamon EOs and
evaluated the AM efficacy of these films against L monocytogenes,
L plantarum, E coli, L sakei, and P fluorescens in the disc diffusion
assay They reported that chitosan films containing these
concen-trations of cinnamon EOs inhibited the growth of all the tested
bacteria on agar media Li and others (2006) demonstrated that
chitosan films incorporated with 463 international units (IU) of
nisin inhibited the growth of S aureus, L monocytogenes, and B.
cereus using the agar diffusion method However, nisin
incorpo-rated into chitosan film had no inhibitory effect against E coli.
The later observation is in agreement with the results of Pranoto
and others (2005) who studied the AM effect of chitosan films
im-pregnated with nisin at different concentrations against E coli The
impregnated chitosan films were also tested against food pathogens
including S aureus, S typhimurium, L monocytogenes, and B cereus.
In their findings, the AM chitosan film demonstrated inhibitory
effects on L monocytogenes, S aureus and B cereus Increasing the
concentration of nisin in the film formulation did not improve
the AM activity of the film Ouattara and others (2000b) found
that chitosan films containing several organic acids (acetic and
propionic) and cinnamaldehyde reduced the growth of teriaceae, Serratia liquefaciens, and Lactobacillus sakei on the surfaces of
Enterobac-vacuum-packed cured meat products (bologna, cooked ham, andpastrami) after a storage period of 21 d at 4◦C Duan and others(2008) reported that chitosan films containing lysozyme demon-
strated inhibitory activity against E coli and L monocytogenes A
significant release of lysozyme from the films was found The age conditions (time and temperature) did not affect the watervapor permeability of the film M ¨oller and others (2004) studiedthe AM effectiveness of chitosan-hydroxypropylmethyl cellulose(HPMC) films, chitosan-HPMC films containing stearic and cit-ric acids, and chemically modified chitosan-HPMC films Thechitosan-HPMC films, with and without stearic acid, significantly
stor-reduced the growth of L monocytogenes.
Table 1 shows that other studies have evaluated the AM tivity of AM agents incorporated into cellulose-based materialssuch as methylcellulose (MC) films The cellulose-based mate-rials are some of the naturally occurring polysaccharides withimproved film-forming properties Similarly to the starch-basedmaterials, cellulose-based materials are hydrophilic in nature andhave a crystalline structure and so they are not generally suitablefor the packaging of moist food products (Baldwin and others1995; Cutter 2002)
ac-Many of the cellulose-based materials and/or their derivativessuch as MC, HMPC, and cellulose acetate are already producedcommercially The latter is widely used in the packaging of bakedgoods and fresh food products (Weber 2000) Although, therehave been a limited number of studies conducted in the past us-ing MC-based materials and/or their derivatives, more recentlythere has been increased recognition of the potential use of suchmaterials in AM packaging systems for the preservation of foodproducts against microbial contaminations and for the extension
of the shelf life of the packaged products Several researchers haveinvestigated the potential use of cellulose-based materials in AMpackaging systems particularly in coating systems as discussed inthe next section For example, Ayana and Nazan (2009) studiedthe antibacterial effectiveness of olive leaf extract incorporated
into MC films against S aureus in an agar disc diffusion test and on
surfaces of Kasar cheese The MC films demonstrated inhibitory
activity against S aureus on the agar medium The films containing 1.5% (w/v) olive leaf extract decreased the population count of S.
aureus on the surface of Kasar cheese by 1.22 log cycles after 14 d
of storage Santiago-Silva and others (2009) investigated the AMactivity of a cellulose-based film incorporated with pediocin Us-
ing the challenge test on sliced ham inoculated with L innocua and Salmonella spp the AM cellulose-based film reduced the growth of
L innocua by 2 log cycles after 15 d of storage at 12◦C Similarly,the AM cellulose-based film effectively inhibited the growth of
Salmonella spp by 0.5 log cycles after 12 d of storage.
Table 1 shows the AM activity of AM agents incorporated intoother polysaccharide-based materials such as alginate, poly(lacticacid) (PLA), and pullulan-based films as determined by differentresearchers Marcos and others (2007) studied the effect of ente-rocins incorporated into a series of biodegradable films (alginate,zein, and poly[vinyl alcohol]) for the preservation of ready-to-eat
food products including sliced ham inoculated with L genes These biodegradable AM films successfully delayed and/or reduced the growth of L monocytogenes during storage at 6 ◦Cfor 29 d Recently, Jin and Zhang (2008) investigated a PLA filmincorporated with nisin They found that PLA containing nisin
monocyto-significantly inhibited the growth of L monocytogenes in liquid
Trang 27AM activity of biodegradable films
culture and on liquid egg white The PLA-nisin film was more
active against the growth of E coli O157:H7 in orange juice than
on liquid culture Rojas-Grau and others (2006) studied the
an-tibacterial effectiveness of apple puree-based films impregnated
with EOs (oregano, cinnamon and lemongrass) against E coli
O157:H7 All the evaluated films containing EOs were reported
to be effective against E coli O157:H7 with the antibacterial
ac-tivity of oregano oil notably higher than that of lemongrass and
cinnamon oils Kandemir and others (2005) investigated the AM
activity of pullulan-based films incorporated with partially
puri-fied lysozyme against the growth of E coli and L plantarum The
AM pullulan-based films were found to be effective against E coli
but did not show any AM activity against L plantarum Natrajan
and Sheldon (2000) evaluated the antibacterial effectiveness of
cal-cium alginate and agar-based films incorporated with nisin against
S Typhimurium on broiler skin Their results showed that the films
containing nisin reduced the population of S Typhimurium.
Antimicrobial activity of protein films
incorporated with AM agents
Proteins are biopolymeric materials that can be used for the
production of biodegradable AM films as they have good
film-forming properties Protein-based polymers have amino acids as
their monomer units Packaging films have been manufactured
from different proteins, such as corn zein, wheat gluten, soy
pro-tein, whey propro-tein, or their derivatives (Hernandez-Izquierdo and
others 2008) Packaging films made from protein-based polymers
possess adequate physico-mechanical properties (Krochta 2002)
Whey protein and corn zein incorporated with natural or
syn-thetic AM agents have been extensively tested in vitro and on
different food products against the growth of various
microorgan-isms A summary of the studies investigating the antibacterial effect
of AM protein-based films is also presented in Table 1 Although
these studies are not directly comparable in terms of the AM agents
tested or microorganisms tested, the results in general demonstrate
that whey protein isolate (WPI) films can be impregnated with
AM agents and have the potential to be used as AM food
pack-aging materials However, no information is readily available in
the current literature on the cost/effective benefits of WPI-based
films and therefore such information is needed before fabricating
AM films from WPI-based materials for commercial applications
Pintado and others (2010) investigated the inhibitory effects of
whey protein films incorporated with nisin, natamycin, and malic
acid against P aeruginosa, L monocytogenes, Y lipolytica, P
roque-forti, and P commune using the agar disc diffusion method They
reported that whey protein films incorporated with AM agents
demonstrated inhibitory effects against all tested microorganisms
Seydim and Sarikus (2006) tested the AM efficacy of WPI films
incorporated with oregano, rosemary, and garlic EOs against E.
coli O157:H7, S aureus, S enteriditis, L monocytogenes, and L
plan-tarum The AM whey protein films containing oregano EOs at
2% (w/w) level demonstrated a higher inhibitory effect against
the tested microorganisms than similar films containing garlic and
rosemary extracts Min and others (2005) investigated the AM
ef-fectiveness of whey protein films containing lactoperoxidase
eval-uated against L monocytogenes using liquid and agar media as well
as on smoked salmon These films reduced the population of L.
monocytogenes on smoked salmon by 3 log CFU g−1 after 35 d
of storage compared with the control film Gadang and others
(2008) evaluated the AM effectiveness of WPI films incorporated
with a combination of nisin, malic acid, grape seed extract, and
ethylenediaminetetraacetic acid (EDTA) against the growth of L.
monocytogenes, E coli O157:H7, and S typhimurium inoculated on
the surface of a turkey frankfurter It was found that all the WPIfilms incorporated with the combination of AM agents decreased
the population of L monocytogenes, E coli O157:H7, and S phimurium on the surface of the turkey frankfurter by 3.2, 4.2, and
ty-4.6 log CFU g−1 after 28 d of storage at 4◦C compared to thecontrol film
Cagri and others (2001) developed WPI films containing 0.5%
to 1.5% (w/w) of sorbic acid (SA) or p-aminobenzoic acid (PABA) and evaluated the AM efficacy of these AM WPI films against L monocytogenes, E coli O157:H7, and S typhimurium DT104 in a
disc diffusion assay They reported that WPI films containing 1.5%
(w/w) PABA or SA inhibited the growth of L monocytogenes, E coli O157:H7, and S typhimurium DT104 in that assay These
results were verified by Cagri and others (2002) who examinedthe AM effectiveness of WPI films incorporated with 0.5% to 1%
(w/w) PABA or SA against L monocytogenes, E coli O157:H7, and S enterica subsp Enterica serovar typhimurium DT104 inoculated
on sliced bologna and summer sausage Whey protein isolate films
containing 1.5% (w/w) PABA or SA reduced the L monocytogenes,
E coli, and S enterica population on both products after 21 d at 4
◦C Ko and others (2001) studied the AM activity of WPI, SPI, egg
albumin, and wheat gluten films incorporated with nisin against
L monocytogenes They found that all these AM protein-based films inhibited L monocytogenes.
Corn zein materials obtained from plant sources are an tional form of proteins that demonstrate good film-forming prop-erties with the potential of being impregnated with AM agents
addi-to preserve food products from microbial contamination Previousstudies showed that corn zein films containing AM agents demon-strated AM activity against the growth of various microorganisms
both in vitro and in various food products A detailed study by
Hoffman and others (2001) found that corn zein films rated with lauric acid, nisin, EDTA, and combinations of these 3
incorpo-compounds reduced L monocytogenes in liquid culture, although
there was no observed inhibitory effect in films incorporated withEDTA alone All the films were reported to be bacteriostatic when
a 104CFU mL−1S enteritidis initial inoculum was used Padgett
and others (1998) investigated the inhibitory effect of heat-pressedand cast corn zein films containing lysozyme and nisin and re-
ported significant inhibition zones for Lactobacillus plantarum by
the cast film compared to the heat-pressed films In another studyPadgett and others (2000) found an inhibitory activity of cornzein films incorporated with various levels of lauric acid and nisin
on the growth of L plantarum in liquid culture G ¨ucbilmez and
others (2007) developed AM films from corn zein incorporatedwith lysozyme and albumin proteins They reported that these
films demonstrated AM activity against the growth of E coli and
B subtilis.
The AM activity of other types of protein-based films havebeen studied and reported in the scientific literature by differentresearchers (see Table 1) Kristo and others (2008) investigated theeffectiveness of sodium caseinate (SC) incorporated with nisin,
potassium, or sodium lactate against L monocytogenes They found
that SC films containing nisin exhibit the highest inhibitory effects
on the growth of L monocytogenes followed by films impregnated
with potassium sorbate, whereas films containing sodium lactatewere only slightly effective Sivarooban and others (2008) evaluatedthe AM properties of soy protein isolate (SPI) films containing1% (w/w) of grape seed extract and nisin (1 × 103 IU g−1).The AM SPI films demonstrated the greatest inhibitory activity
against L monocytogenes compared with the other systems that
Trang 28were tested Oussalah and others (2004) developed a protein-based
edible film containing 1% (w/w) oregano and pimento EOs or a
mixture of both EOs and evaluated the AM effects of these films
on the preservation of whole beef muscle The results suggested an
effectiveness of the AM films against Pseudomonas spp and E coli
O157:H7 inoculated on the surface of the beef Their results also
suggested that films containing oregano EO were more effective
against the growth of both microorganisms compared to films
containing pimento
Antimicrobial activity of biodegradable films coated with
AM agents
In addition to the direct incorporation of AM agents into
pack-aging films discussed previously, AM agents can be coated on the
surface of packaging materials to provide a high concentration of
the agent in contact with the surface of food product
(Genna-dios and others 1997; An and others 2000) The application of an
AM agent on a packaging material can be achieved by using
var-ious coating techniques including immersion of the substrate or
by spraying the substrate with a coating/carrier solution For this
purpose, the AM agent is dissolved in an appropriate solvent such
as water, ethanol, or isopropanol before applying it to the
pack-aging material (Krochta 2002) Little has been reported on the
activity of AM agents coated on biodegradable polymers Some
of the relevant studies are given in Table 1
Miltz and others (2006) studied the effectiveness of a corn
starch-based film coated with the peptide dermaseptin S4
deriva-tive as an AM agent against moulds and aerobic bacteria on
cu-cumbers They reported that this system was very effective Coma
and others (2001) found that cellulose films coated with nisin
in-hibited L innocua and S aureus on laboratory media Chen and
others (1996) prepared AM films containing 2% or 4% (w/w)
of sodium benzoate and potassium sorbate by casting MC,
chi-tosan, and their mixtures They evaluated the antimycotic activity
of the AM films against Rhodotorula rubra and Penicillium notatum
and found that MC and MC/chitosan films containing 2% and
4% (w/w) sodium benzoate and potassium sorbate, respectively,
inhibited the growth of these microorganisms Ming and others
(1997) reported that a cellulose casing coated with pediocin
com-pletely inhibited the growth of L monocytogenes on ham, turkey
breast, and beef products compared to the control film after 12
wk of storage at 4◦C Janes and others (2002) investigated the
AM effect of corn zein films coated with nisin and/or 1% (w/w)
calcium propionate against L monocytogenes inoculated on
ready-to-eat chicken samples and found that the coated films inhibited
the growth of the microorganism Kim and others (2008) evaluated
the AM effectiveness of chitosan and WPI coated with lysozyme
against the growth of L monocytogenes and S enteritidis inoculated
on hard-boiled eggs The chitosan-lysozyme system controlled the
growth of S enteritidis on hard-boiled shell-on and on peeled eggs.
Siragusa and Dickinson (1992; 1993) found that calcium alginate
coatings and films containing organic acids effectively reduced the
population of L monocytogenes, S typhimurium and E coli O157:H7
on the surface of beef carcass
Antimicrobial activity of biodegradable films with
immobilized AM agents
Effective AM packaging systems can also be achieved by the
immobilization of an AM agent in a polymeric material
Accord-ing to Steven and Hotchkiss (2003), the AM agents that can be
immobilized include peptides, proteins, or enzymes These agents
can be synthesized on the surface or extracted separately and then
covalently linked to the polymer substrate An AM agent that iscovalently immobilized onto the packaging material is not re-leased but becomes effective in inhibiting microbial growth when
in contact with the surface of the packaged food product (Han2003) Different studies have been conducted focusing on im-mobilization of AM agents onto packaging materials Appendiniand Hotchkiss (1997) investigated the efficiency of lysozyme im-mobilized on polyvinyl alcohol (PVOH) beads, nylon 6,6 pellets,and cellulose triacetate (CTA) films They reported that the vi-
ability of Micrococcus lysodeikticus was reduced in the presence of
immobilized lysozyme on CTA film that was found to show thehighest AM activity amongst the studied structures Cutter andSiragusa (1997) assessed the potential decontamination of raw beef
by applying organic acids (lactic or acetic acid) immobilized ontocalcium alginate films They reported a considerable reduction
of L monocytogenes growth with the treated films compared to a
calcium alginate film without acid treatment Cutter and Siragusa(1996) studied the AM activity of nisin immobilized onto calcium
alginate films against Brochothrix thermosphacta on beef surfaces.
They found that calcium alginate films treated with nisin
sup-pressed the growth of B thermosphacta by 2.42 log CFU cm−2
after 7 d compared to an untreated film A greater and steady nisinactivity was found when the tissues were ground and stored underrefrigerated conditions in the AM immobilized film for up to 7 dcompared to the use of sprayed nisin only
Conclusions
Consumer demands and requirements by regulatory agencies
to use more environmentally-friendly and less polluting packageshave directed researchers to look at packaging materials that arederived from natural or made from renewable resources to replace,
at least some, of the synthetic polymers Biodegradable materialsderived from polysaccharides and proteins, when combined with
AM agents, have the potential to be manufactured into food aging films with effective AM properties Polysaccharide-basedmaterials with AM agents, particularly the starch-based ones, havebeen studied extensively with some commercial success in the foodpackaging industry Many of the studies were carried out to obtain
pack-a “proof of concept” by mepack-asuring the inhibition zones crepack-ated
by the diffusion of the AM agent in solid media Some fied biodegradable polymers such as starch-based materials can bemanufactured into films and used to package dry and/or solid foodproducts such as biscuits, snacks, cereals, fresh produce, fruits, andvegetables Developing commercial biodegradable films with im-proved physical and mechanical properties is still a challenge due
modi-to their hydrophilic nature that limits their application for ing of food products with a high water activity The biodegradableand bio-compostable materials are also, many times, more expen-sive, and more difficult to process, a fact that further increases theircost compared to synthetic polymers However, when consideringthe cost of a package, the total “cradle to grave” economic ap-proach should be evaluated Thus, the economic evaluation shouldinclude not only the cost of the packaging material and of process-ing the material into a package but also the cost of disposing of thefinal package namely, recycling, and/or incineration, and/or landfilling This is very important especially for the last option, tak-ing into consideration the decreasing number of land filling sites,and the diminishing space for garbage disposal in the developedcountries If such considerations are taken into account, the dif-ference between the cost of biodegradable/bio-compostable andsynthetic polymers becomes much smaller Antimicrobial pack-aging films with improved physical and mechanical properties
Trang 29AM activity of biodegradable films
could be prepared from biodegradable polymers that have been
modified and/or blended with other compatible materials
incor-porated or coated with AM agents However, additional research
and development work is required to reduce the moisture
sensi-tivity of these polymers, enhance their physical properties and
im-prove their process-ability These goals can be achieved by proper
blending with appropriate materials and/or by copolymerization
Biodegradable materials could also be successfully prepared and
applied in AM packaging systems by the incorporation of
appro-priate AM agents Taking into consideration that the public, as a
whole, is already conscious (and becomes even more so as times
go by) to the environment, it is conceivable that the future will
see more biodegradable and AM biodegradable polymers and/or
their derivatives in the packaging of food, agricultural, and other
products
References
Altsk¨ar A, Andersson R, Boldizar A, Koch K, Stading M, Rigdahl M, Thunwall M 2008 Some
effects of processing on the molecular structure and morphology of thermoplastic starch.
Carbohydr Polym 71(4):591–7.
An DS, Kim YM, Lee SB, Paik HD, Lee DS 2000 Antimicrobial low density polyethylene film
coated with bacteriocins in binder medium Food Sci Biotechnol 9:14–20.
Appendini P, Hotchkiss JH 1997 Immobilization of lysozyme on food contact polymers as
potential antimicrobial films Packag Technol Sci 10:271–9.
Appendini P, Hotchkiss JH 2002 Review of antimicrobial food packaging Food Sci Emerg
Technol 3:113–26.
Arifin DY, Lee LY, Wang C-H 2006 Mathematical modeling and simulation of drug
re-lease from microspheres: Implications to drug delivery systems Adv Drug Deliv Rev
58(12-13):1274–325.
Arvanitoyannis I, Biliaderis CG 1998 Physical properties of polyol-plasticized edible films made
from sodium caseinate and soluble starch blends Food Chem 62(3):333–42.
Arvanitoyannis I, Biliaderis CG 1999 Physical properties of polyol-plasticized edible blends
made of methyl cellulose and soluble starch Carbohydr Polym 38(1):47–58.
Arvanitoyannis I, Nakayama A, Aiba S 1998 Edible films made from hydroxypropyl starch and
gelatin and plasticized by polyols and water Carbohydr Polym 36(2–3):105–19.
Av´erous L, Fringant C, Moro L 2001 Starch-based biodegradable materials suitable for
ther-moforming packaging Starch/St¨arke 53(8):368–71.
Av´erous L, Moro L, Dole P, Fringant C 2000 Properties of thermoplastic blends:
starch-polycaprolactone Polymer 41(11):4157–67.
Ayana B, Turhan KN 2009 Use of antimicrobial methylcellulose films to control Staphylococcus
aureus during storage of Kasar cheese Packag Technol Sci 22(8):461–9.
Baldwin EA, Nisperos-Carriedo MO, Baker RA 1995 Use of edible coatings to preserve quality
of lightly and (slightly) processed products Crit Rev Food Sci Nutr 35:509–24.
Baron JK, Sumner SS 1993 Antimicrobial containing edible films as inhibitory system to control
microbial growth on meat products J Food Protect 56:916.
Bicerano J 2003 Glass transition In: Mark HF, editor Encyclopedia of polymer science and
technology 3rd ed New York: John Wiley and Sons.
Bravin B, Peressini D, Sensidoni A 2006 Development and application of polysaccharide-lipid
edible coating to extend shelf-life of dry bakery products J Food Engineer 76(3):280–90.
Brody AL 2005 Packaging Food Technol 59(2):65–66.
Brody AL, Strupinsky ER, Kline LR 2001 Antimicrobial packaging active packaging for food
applications Lancaster, Pa.: Technomic Publishing Co p 131–96.
Cagri A, Ustunol Z, Ryser ET 2001 Antimicrobial, mechanical, and moisture barrier properties
of low pH whey protein-based edible films containing p-aminobenzoic or sorbic acids J Food
Sci 66(6):865–70.
Cagri A, Ustunol Z, Ryser ET 2002 Inhibition of three pathogens on bologna and summer
sausage using antimicrobial edible films J Food Sci 67(6):2317–24.
Cagri A, Ustunol Z, Ryser ET 2004 Antimicrobial edible films and coatings J Food Protect
67(4):833–48.
Carvalho AJF, Zambon MD, da Silva Curvelo AA, Gandini A 2005 Thermoplastic starch
modification during melt processing: hydrolysis catalyzed by carboxylic acids Carbohydr
Polym 62(4):387–90.
Cha DS, Chinnan MS 2004 Biopolymer-based antimicrobial packaging: a review Crit Rev
Food Sci Nutri 44:223–37.
Chal´eat CM, Halley PJ, Truss RW 2008 Properties of a plasticised starch blend Part 1: influence
of moisture content on fracture properties Carbohydr Polym 71:535–43.
Chen MC, Yeh GHC, Chiang BH 1996 Antimicrobial and physicochemical properties
of methylcellulose and chitosan films containing a preservative J Food Process Preserv
20:279–390.
Chick J, Ustunol Z 1998 Mechanical and barrier properties of lactic acid and rennet precipitated
casein-based edible films J Food Sci 63(6):1024–7.
Coma V, Sebti I, Pardon P, Deschamps A, Pichavant FH 2001 Antimicrobial edible packaging
based on cellulosic ethers, fatty acids, and nisin incorporation to inhibit Listeria innocua and
Staphylococcus aureus J Food Protect 64(4):470–5.
Cooksey K 2005 Effectiveness of antimicrobial food packaging materials Food Addit Contamin
22(10):980–7.
Corrales M, Han HJ, Tauscher B 2009 Antimicrobial properties of grape seed extracts and their
effectiveness after incorporation into pea starch films Int J Food Sci Technol 44(2):425–33.
Cutter CN 2002 Microbial control by packaging: a review Crit Rev Food Sci Nutr 42:151–61.
Cutter CN 2006 Opportunities for bio-based packaging technologies to improve the quality
and safety of fresh and further processed Meat Sci 74(1):131–42.
Cutter CN, Siragusa GR 1996 Reduction of Brochothrix thermosphacta on beef surfaces following
immobilization of nisin in calcium alginate gels Lett Appl Microbiol 23:9–12.
Cutter CN, Siragusa GR 1997 Growth of Brochothrix thermosphacta in ground beef following
treatments with nisin in calcium alginate gels Food Microbiol 14:425–30.
Davidson PM, Sofos JN, Branen AL 2005 Antimicrobials in food 3rd ed Boca Raton, Fla.: CRC Press, Taylor & Francis Group.
Davis G, Song JH 2006 Biodegradable packaging based on raw materials from crops and their impact on waste management Indust Crops Prod 23:147–61.
Dawson PL, Aton JC, Ogal AA 2002 Biopolymer films and potential applications to meat and poultry products 55 th annual reciprocal meat conference East Lansing, Mich.: American Meat Science Assoc p 75–81.
Debeaufort F, Quezada-Gallo JA, Voiley A 1998 Edible films and coatings: tomorrow’s aging: a review Crit Rev Food Sci Nutri 38(4):299–313.
pack-Devlieghere F, Vermeiren L, Bockstal A, Debevere J 2000 Study on antimicrobial activity of a food packaging material containing potassium sorbate Acta Alimentaria 29(2):137–46 Dias AB, M ¨uller CMO, Larotonda FDS, Laurindo JB 2010 Biodegradable films based on rice starch and rice flour J Cereal Sci 51(2):213–19.
Duan J, Kim K, Daeschel MA, Zhao Y 2008 Storability of antimicrobial chitosan-lysozyme composite coating and film-forming solutions J Food Sci 73(6):M321–9.
Durango AM, Soares NFF, Benevides S, Teixeira J, Carvalho M, Wobeto C, Andrade NJ 2006 Development and evaluation of an edible antimicrobial film based on yam starch and chitosan Packag Technol Sci 19(1):55–9.
Fang JM, Fowler PA, Escrig C, Gonzalez R, Costa JA, Chamudis L 2005 Development
of biodegradable laminate films derived from naturally occurring carbohydrate polymers Carbohydr Polym 60(1):39–42.
Fishman ML, Coffin DR, Onwulata CI, Willett JL 2006 Two stage extrusion of plasticized pectin/poly(vinyl alcohol) blends Carbohydr Polym 65(4):421–9.
Gadang VP, Hettiarachchy NS, Johnson MG, Owens C 2008 Evaluation of antibacterial activity
of whey protein isolate coating incorporated with nisin, grape seed extract, malic acid, and EDTA on a turkey frankfurter system J Food Sci 73(8):M389–94.
Garc´ıa MA, Martino MN, Zaritzky NE 2000a Lipid Addition to Improve Barrier Properties
of Edible Starch-based Films and coatings J Food Sci 65(6):941–4.
Garc´ıa MA, Martino MN, Zaritzky NE 2000b Microstructural characterization of plasticized starch-based films Starch/St¨arke 52(4):118–24.
Garc´ıa MA, Pinotti A, Martino MN, Zaritzky NE 2009 Characterization of starch and posite edible films and coatings In: Huber KC, Embuscado ME, editors Edible films and coatings for food applications New York: Springer, New York p 169–209.
com-Gennadios A, Hanna MA, Kurth LB 1997 Application of edible coatings on meats, poultry and seafoods: a review Lebensm Wiss Technol 30:337–50.
G ¨uc¸bilmez C ¸ M, Yemenicioglu A, Arslanoglu A 2007 Antimicrobial and antioxidant activity of edible zein films incorporated with lysozyme, albumin proteins and disodium EDTA Food Res Int 40(1):80–91.
Guilbert S 1986 Technology and application of edible protective films In: Mathlouthi M, editor Food packaging and preservation: theory and practice New York: Elsevier Applied Science p 371–94.
Han JH 2000 Antimicrobial food packaging Food Technol 54(3):56–65.
Han JH 2003 Novel food packaging techniques In: Ahvenainen R, editor Antimicrobial food packaging Cambridge: Woodhead Publishing Ltd.
Han JH 2005 Antimicrobial packaging systems In: Han JH, editor Innovations in food aging San Diego, Calif.: Elsevier Academic Press p 92–108.
pack-Han JH, Floros JD 1997 Casting antimicrobial packaging films and measuring their physical properties and antimicrobial activity J Plastic Film Sheet 13:287–98.
Han JH, Floros JD 1998 Simulating diffusion model and determining diffusivity of potassium sorbate through plastics to develop antimicrobial packaging film J Food Process Preserv 22(2):107–22.
Han JH, Gennadios A 2005 Edible films and coatings: a review In: Han JH, editor Innovations
in food packaging San Diego, Calif.: Elserier Academic Press p 239–62.
Hernandez-Izquierdo VM, Krochta JM 2008 Thermoplastic processing of proteins for film formation: a review J Food Sci 73(2):R30–9.
Hernandez-Izquierdo VM, Reid DS, McHugh TH, Berrios JDJ, Krochta JM 2008 mal transitions and extrusion of glycerol-plasticized whey protein mixtures J Food Sci 73(4):E169–75.
Ther-Hoffman KL, Han IY, Dawson PL 2001 Antimicrobial effects of corn zein films impregnated with nisin, lauric acid, and EDTA J Food Protect 64(6):885–9.
Hotchkiss JH 1997 Food packaging interactions influencing quality and safety Food Addit Contam 14:601–7.
Iovino R, Zullo R, Rao MA, Cassar L, Gianfreda L 2008 Biodegradation of poly(lactic acid)/starch/coir biocomposites under controlled composting conditions Polym Degrad Stabil 93(1):147–57.
Janes ME, Kooshesh S, Johnson MG 2002 Control of Listeria monocytogenes on the surface
of refrigerated, ready-to-eat chicken coated with edible zein film coatings containing nisin and/or calcium propionate J Food Sci 67(7):2754–7.
Jin T, Zhang H 2008 Biodegradable polylactic acid polymer with nisin for use in antimicrobial food packaging J Food Sci 73(3):M129–34.
Kandemir N, Yemenicioglu A, Mecitoglu C ¸ , Elmaci ZS, Arslanoglu A, G ¨oksungur Y, Baysal
T 2005 Production of antimicrobial films by incorporation of partially purified lysozyme
into biodegradable films of crude exopolysaccharides obtained from Aureobasidium pullulans
fermentation Food Technol Biotechnol 43(4):343–50.
Kester JJ, Fennema OR 1986 Edible films and coatings: a review Food Technol 40(12):47–59 Kim K, Daeschel MA, Zhao Y 2008 Edible coating for enhancing microbial safety and extend- ing shelf life of hard-boiled eggs J Food Sci 73(5):M227–35.
Ko S, Janes ME, Hettiarachchy NS, Johnson MG 2001 Physical and chemical properties of edible
films containing nisin and their action against Listeria monocytogenes J Food Sci 66(7):1006–11.
Kristo E, Koutsoumanis KP, Biliaderis CG 2008 Thermal, mechanical and water vapor barrier properties of sodium caseinate films containing antimicrobials and their inhibitory action on
Listeria monocytogenes Food Hydrocolloids 22(3):373–86.
Krochta JM 2002 Proteins as raw materials for films and coatings: definitions, current status, and opportunities In: Gennadios A, editor Protein-based films and coatings Boca Raton, Fla.: CRC Press p 1–41.
Trang 30Krochta JM, Baldwin EA, Nisperos-Carriedo MO 1994 Edible coatings and films to improve
food quality Lancaster, Pa.: Technomic Publishing Co Inc.
Li B, Peng J, Yie X, Xie B 2006 Enhancing physical properties and antimicrobial activity
of konjac glucomannan edible films by incorporating chitosan and nisin J Food Sci 71(3):
C174–8.
Liu L, Kerry JF, Kerry JP 2006 Effect of food ingredients and selected lipids on the physical
properties of extruded edible films/casings Int J Food Sci Technol 41:295–302.
Liu Z 2005 Edible films and coatings from starches In: Han JH, editor Innovations in food
packaging San Diego, Calif.: Elsevier Academic Press p 331–2.
Lopez-Rubio A, Gavara R, Lagaron JM 2006 Bioactive packaging: turning foods into healthier
foods through biomaterials Trends Food Sci Technol 17(10):567–75.
L ´opez OV, Garc´ıa MA, Zaritzky NE 2008 Film forming capacity of chemically modified corn
starches Carbohydr Polym 73(4):573–81.
Maizura M, Fazilah A, Norziah MH, Karim AA 2007 Antibacterial activity and mechanical
properties of partially hydrolyzed sago starch-alginate edible film containing lemongrass oil J
Food Sci 72(6):C324–30.
Maizura M, Fazilah A, Norziah M, Karim A 2008 Antibacterial activity of modified sago
starch–alginate based edible film incorporated with lemongrass (Cymbopogon citratus) oil Int
Food Res J 15:233–6.
Mali S, Grossmann MVE, Garcia MA, Martino MN, Zaritzky NE 2002 Microstructural
characterization of yam starch films Carbohydr Polym 50(4):379–86.
Marcos B, Aymerich T, Monfort JM, Garriga M 2007 Use of antimicrobial biodegradable
packaging to control Listeria monocytogenes during storage of cooked ham Int J Food Microbiol
120(1-2):152–8.
Marron V, Saari L, Floridi G, Boelck C, Innocenti F 2000 The market of biobased packaging
materials In: Weber, CJ, editor Biobased packaging materials for the food industry-status
and perspectives Copenhagen, Denmark p 105–12.
Matche RS, Kulkarni G, Ray B 2004 Modification of ethylene acrylic acid film for antimicrobial
activity J Appl Polym Sci 100:3063–8.
Mehyar GF, Han JH 2004 Physical and mechanical properties of high-amylose rice and
pea starch films as affected by relative humidity and plasticizer J Food Sci 69(9):
E449–54.
Miltz J, Passy N, Manneheim CH 1995 Trends and applications of active packaging systems.
In: Ackermann P, Jagerstad M, Ohlsson T, editors Foods and packaging materials: chemical
interactions Cambridge, UK: The Royal Society of Chemistry p 201–10.
Miltz J, Rydlo T, Mor A, Polyakov V 2006 Potency evaluation of a dermaseptin S4 derivative
for antimicrobial food packaging applications Packag Technol Sci 19:345–454.
Min S, Harris LJ, Krochta JM 2005 Listeria monocytogenes inhibition by whey protein films and
coatings incorporating the lactoperoxidase system J Food Sci 70(7):M317–24.
Ming X, Weber GH, Ayres JW, Sandine WE 1997 Bacteriocins applied to food packaging
materials to inhibit Listeria monocytogenes J Food Sci 62(2):413–5.
Mistry Y 2006 Development of LDPE-based antimicrobial films for food packaging packaging
and polymer research unit Melbourne, Australia: Victoria Univ p 101.
M ¨oller H, Grelier S, Pardon P, Coma V 2004 Antimicrobial and physicochemical properties
of chitosan-HPMC-based films J Agric Food Chem 52(21):6585–91.
Myll¨arinen P, Partanen R, Sepp¨al¨a J, Forssell P 2002 Effect of glycerol on behaviour of amylose
and amylopectin films Carbohydr Polym 50(4):355–61.
Nam S, Scanlon MG, Han JH, Izydorczyk MS 2007 Extrusion of pea starch containing lysozyme
and determination of antimicrobial activity J Food Sci 72(9):E477–84.
Natrajan N, Sheldon BW 2000 Efficacy of nisin-coated polymer films to inactivate Salmonella
typhimurium on fresh broiler skin J Food Protect 63(9):1189–96.
Nisperos-Carriedo MO 1994 Edible coatings and films based on polysaccharides In: Krochta
JM, Baldwin EA, Nisperos-Carriedo MO, editors Edible coatings and films to improve food
quality Lancaster, Pa.: Technomic Publishing Co p 305–35.
Ojagh SM, Rezaei M, Razavi SH, Hosseini SMH 2010 Development and evaluation of a novel
biodegradable film made from chitosan and cinnamon essential oil with low affinity toward
water Food Chem 122(1):161–6.
Olivas G, Barbosa-Canovas G 2009 Edible films and coatings for fruits and vegetables In:
Embuscado M, Huber K, editors Edible films and coatings for food applications New York:
Springer p 211–38.
Ouattara B, Simard RE, Piette G, B´egin A, Holley RA 2000a Diffusion of acetic and propionic
acid from chitosan-based antimicrobial packaging films J Food Sci 65(5):768–73.
Ouattara B, Simard RE, Piette G, B´egin A, Holley RA 2000b Inhibition of surface spoilage
bacteria in processed meats by application of antimicrobial films prepared with chitosan Int J
Food Microbiol 62(1-2):139–48.
Oussalah M, Caillet S, Salmieri S, Saucier L, Lacroix M 2004 Antimicrobial and antioxidant
effects of milk protein-based film containing essential oils for the preservation of whole beef
muscle J Agric Food Chem 52:5598–605.
Padgett T, Han IY, Dawson PL 1998 Incorporation of food-grade antimicrobial compounds
into biogradable packaging films J Food Prot 61:1330–5.
Padgett T, Han Y, Dawson PL 2000 Effect of lauric acid addition on the antimicrobial
ef-ficacy and water permeability of corn zein films containing nisin J Food Process Preserv
24(5):423–32.
Parker R, Ring SG 2001 Aspects of the physical chemistry of starch J Cereal Sci 34(1):1–
17.
Pelissari FM, Grossmann MVE, Yamashita F, Pineda EAG 2009 Antimicrobial, mechanical,
and barrier properties of cassava starch and chitosan films incorporated with oregano essential
oil J Agric Food Chem 57(16):7499–504.
Perez-Gago MB, Krochta JM 2005 Emulsion and bi-layer edible films In: Han JH, editor.
Innovations in food packaging San Diego, Calif.: Elsevier Academic Press p 384–402.
Petersen K, Væggemose Nielsen P, Bertelsen G, Lawther M, Olsen MB, Nilsson NH, Mortensen
G 1999 Potential of biobased materials for food packaging Trends Food Sci Technol
10(2):52–68.
Phan TD, Debeaufort F, Luu D, Voiley A 2005 Functional properties of edible agar-based and
starch-based films for food quality preservation J Agric Food Chem 53:973–81.
Pintado CMBS, Ferreira MASS, Sousa I 2010 Control of pathogenic and spoilage
microorgan-isms from cheese surface by whey protein films containing malic acid, nisin and natamycin.
Food Control 21(3):240–6.
Pommet M, Redl A, Guilbert S, Morel MH 2005 Intrinsic influence of various plasticizers
on functional properties and reactivity of wheat gluten thermoplastic materials J Cereal Sci 42:81–91.
Pranoto Y, Salokhe V, Rakshit S 2005 Enhancing antimicrobial activity of chitosan films by incorporating garlic oil, potassium sorbate and nisin Lebensm Wiss Technol 38:859–65.
Quintavalla S, Vicini L 2002 Antimicrobial food packaging in the meat industry Meat Sci 62:373–80.
Rardniyom C 2008 Development of multi-layer films containing natural antimicrobial agents.
packaging and polymer research unit Melbourne, Australia: Victoria Univ p 145.
Robertson G 2008 State-of-the-art biobased food packaging materials In: Chiellini E, editor.
Environmentally-compatible food packaging Boca Raton, Fla.: Woodhead Publishing Ltd.
and CRC Press LLC p 24–28.
Rodriguez M, Oses J, Ziani K, Mate JI 2006 Combined effects of plasticizers and surfactants
on the physical properties of starch-based edible films Food Res Int 39:840–6.
Rodriguez MS, Ramos V, Agullo E 2003 Antimicrobial action of chitosan against spoilage organisms in precooked pizza J Food Sci 68(1):271–4.
Rojas-Grau MA, Avena-Bustillos RJ, Friedman M, Henika PR, Martin-Belloso O, McHugh
TH 2006 Mechanical, barrier, and antimicrobial properties of apple puree edible films containing plant essential oils J Agric Food Chem 54(24):9262–7.
Rooney ML 1995 Active food packaging Glasgow, UK: Blackie Academic, Professional.
Rupika LAS, Sonneveld K, Miltz J, Bigger SW 2005 Development and evaluation of density polyethylene-based antimicrobial food packaging polymers containing thymol and carvacrol 22 nd IAPRI Symposium on Packaging Campinas, Brazil.
low-Rupika LAS, Sonneveld K, Miltz J, Bigger SW 2008 Modelling microbial inactivation by films containing thymol or carvacrol 16 th IAPRI World Conference on Packaging Bangok, Thailand.
Salleh E, Muhamadi l, Khairuddinr N 2007 Inhibition of Bacillus subtilis and Escherichia coli
by antimicrobial starch-based film incorporated with lauric acid and chitosan Proceedings
of the 3 rd CIGR Section Vl International Symposium on Food and Agricultural Products:
Processing and Innovation Naples, ltaly.
Santiago-Silva P, Soares NFF, N ´obrega JE, J ´unior MAW, Barbosa KBF, Volp ACP, Zerdas ERMA, W ¨urlitzer NJ 2009 Antimicrobial efficiency of film incorporated with pediocin (ALTA R 2351) on preservation of sliced ham Food Control 20(1):85–89.
Seydim AC, Sarikus G 2006 Antimicrobial activity of whey protein-based edible films porated with oregano, rosemary and garlic essential oil Food Res Int 39:639–44.
incor-Shellhammer TH, Krochta JM 1997 Whey protein emulsion film performance as affected by lipid type and amount J Food Sci 62(2):390–4.
Shen XL, Wu JM, Chen Y, Zhao G 2010 Antimicrobial and physical properties of sweet potato starch films incorporated with potassium sorbate or chitosan Food Hydrocolloids 24(4):285–90.
Siepmann J, Faisant N, Akiki J, Richard J, Benoit JP 2004 Effect of the size of able microparticles on drug release: experiment and theory J Control Release 96(1):123–
biodegrad-34.
Siragusa GA, Dickson JS 1992 Inhibition of Listeria monocytogenes on beef tissue by application
of organic acids immobilized in a calcium alginate gel J Food Sci 57:293–6.
Siragusa GR, Dickson JS 1993 Inhibition of Listeria monocytogenes, Salmonella typhimurium and Escherichia coli O157:H7 on beef muscle tissue by lactic or acetic acid contained in calcium
alginate gels J Food Saf 13(2):147–58.
Sivarooban T, Hettiarachchy NS, Johnson MG 2008 Physical and antimicrobial properties of grape seed extract, nisin, and EDTA incorporated soy protein edible films Food Res Int 41(8):781–5.
Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE 2001 Biodegradable polymeric nanoparticles as drug delivery devices J Control Release 70(1-2):1–20.
Sothornvit R, Krochta JM 2005 Plasticisers in edible films and coatings In: Han JH, itor Innovations in food packaging San Diego, Calif.: Elsevier Academic Press p 403–
ed-33.
Steven MD, Hotchkiss JH 2003 Non-migratory bioactive polymers (NMBP) in food packaging.
In: Ahvenainen R, editor Novel food packaging techniques Cambridge, UK: Woodhead Publishing Ltd and CRC Press LLC p 71–102.
Suppakul P 2004 Study of antimicrobial polymeric packaging films containing basil extracts.
School of molecular sciences Melbourne, Australia: Victoria Univ p 265.
Suppakul P, Miltz J, Sonneveld K, Bigger SW 2003a Active packaging technologies with
an emphasis on antimicrobial packaging and its applications J Food Sci 68(2):408–
Tharanathan RN 2003 Biodegradable films and composite coatings: past, present and future.
Trends Food Sci Technol 14:71–8.
Thunwall M, Boldizar A, Rigdahl M 2006 Compression molding and tensile properties of thermoplastic potato starch materials Biomacromolecules 7:981–6.
Thunwall M, Kuthanov´a V, Boldizar A, Rigdahl M 2008 Film blowing of thermoplastic starch.
Weber CJ 2000 Biobased packaging materials for the Food Industry In: Weber CJ, editor.
Frederiksberg, C Denmark: The Royal Veterinary and Agricultural Univ.
Weber GH, Haugard V, Festersen R, Bertelsen 2002 Production and applications of biobased packaging materials for the food industry Food Addit Contamin 19(Supplement):
172–7.
Weng Y-M, Hotchkiss JH 1993 Anhydrides as antimycotic agents added to polyethylene films for food packaging Packag Technol Sci 6:123–8.
Trang 31AM activity of biodegradable films
Wong DWS, Camirand WM, Pavlath AE 1994 Development of edible coatings for minimally
processed fruits and vegetables In: Krochta JM, Baldwin EA, Nisperos-Carriedo MO, editors.
Edible coatings and films to improve food quality Lancaster, Pa.: Technomic Publishing Co.
p 65–88.
Wu Y, Wulfsohn D, Lan Y, Singh N 1998 Experimental studies of temperature and
mois-ture movement in water limited starch-based food systems Food Process Preserv 22:91–
105.
Wulansari R, Mitchell JR, Blanshard JMV 1999 Starch conversion during extrusion as affected
by added gelatin J Food Sci 64(6):1055–8.
Yang L, Paulson AT 2000 Mechanical and water vapour barrier properties of edible gellan films Food Res Int 33(7):563–70.
Zhang Y, Liu Z 2009 Starch-based edible films In: Chiellini E, editor tally compatible food packaging Cambridge, UK: Woodhead Publishing Ltd p 108– 36.
Trang 32Vitamin C and Sugar Levels as Simple Markers
for Discriminating Spanish Honey Sources
Virginia Le´on-Ruiz, Soledad Vera, Amelia V Gonz´alez-Porto, and Mar´ıa Paz San Andr´es
Abstract: In this work, 7 Spanish honeys with different botanical origins were studied The honey origins were rosemary,chestnut, lavender, echium, thyme, multifloral, and honeydew The chemical compounds determined were ascorbic acid(vitamin C), hydroxymethylfurfural, and major sugar contents (glucose and fructose) The physicochemical parameters,
pH, conductivity, moisture, free acidity, and color, were also measured Vitamin C is an important antioxidant in food,and the possibility to use it as discriminate parameter among different honeys was studied The determination of vitamin
C in honey samples was carried out by 2 different methods, volumetric and chromatographic comparing the results byboth statistically Vitamin C content was higher in thyme honeys than in the other types; however a wide dispersion in thevalues was found Through a linear discriminant analysis (LDA), conductivity, glucose, fructose, and vitamin C contentwere the most important discriminant parameters
Keywords: ascorbic acid, honey analysis, HPLC, linear discriminant analysis, physicochemical parameters, vitamin C
Practical Application: Vitamin C content in different honey sources has been determined by a simple and rapid matographic method (less than 3 min) in honeys from 6 botanical origins The results together with glucose and fructosecontent and some physicochemical parameters have been studied in order to discriminate the botanical origin of honeysand in the future certified their quality A statistical LDA was applied to the data, and differentiation of honey sourceswas possible with very good agreement The vitamin C content found in thymus honeys was significantly higher than
chro-in other types This fact makes vitamchro-in C a special marker for thymus honeys that have a higher antioxidant effect thanthe others giving it special properties The identification of honey sources is essential for beekeepers in order to certifyhoneys for consumers
Introduction
The majority of the honeys in the market contains significant
nectar or honeydew contributions from several plant species, and
are therefore called multifloral honeys The term unifloral honey
is used to describe a honey in which the major part of nectar
or honeydew is derived from single plant species (Persano-Oddo
and Piro 2004) In Europe more than 100 plant species originate
unifloral honeys For complete information on European
uni-floral honeys, the Intl Honey Commission (IHC) conducted a
comprehensive study through the collection of bibliographic data,
and through information provided directly by members of IHC
(Persano-Oddo and others 2004) In order to compare the results
obtained by IHC with other European studies, an extensive review
that includes all analytical data available in the literature, for the
same type of honey, was carried out (Piazza and Persano-Oddo
2004)
At present, there is an increasing commercial interest to
pro-duce unifloral honeys, due to the therapeutic uses of certain honey
MS 20100652 Submitted 6/11/2010, Accepted 11/22/2010 Authors Le´on-Ruiz,
Vera, and San Andr´es are with Dept de Qu´ımica Anal´ıtica e Ingenier´ıa Qu´ımica,
Facultad de Qu´ımica, Univ de Alcal´a Ctra Madrid-Barcelona, Km 33.6,
28871-Alcal´a de Henares (Madrid), Spain Authors Le´on-Ruiz and Gonz´alez-Porto are with
Centro Agrario de Marchamalo, C/ Extramuros, s/n., 19180-Marchamalo
(Guadala-jara), Spain Direct inquiries to author San Andr´es (E-mail: mpaz.sanandres@uah.es).
varieties Also, the consumer would be important to have tional information of honey on the label, which is regulated forall foodstuffs by Regulation (EC) No 1924/2006 and Regulation(EC) No 108/2008 (OJEU 2006; OJEU 2008), and for honey
nutri-by Regulation (EC) No 110/2001 (OJEU 2002) of the EuropeanParliament and the Council
Honey is a natural bee product and its quality and compositiondepends on the botanical and geographic origin and climatologicconditions Honey characterization is based on the determination
of its chemical, physical, or biological properties Pollen analysis
of honey, or melissopalynology, is of great importance for qualitycontrol (Von der Ohe and others 2004) Honey always includesnumerous pollen grains and honeydew elements that altogetherprovide a good fingerprint of the environment where the honeycomes from
In general, a honey is considered as coming predominantly from
a given botanical origin (unifloral honey) if the relative frequency
of the pollen of that taxon exceeds 45% This percentage is not
valid for over- or under-represented pollen plants; for example,rosemary, lavender, or citrus honeys are considered to be unifloralwhen only 10% to 20% of pollen from these species is present(Piazza and Persano-Oddo 2004)
Honey contains around 200 compounds (Tomoi-Sato and Miyata 2000), and it is basically 20% water, 38% fructose, 31%glucose, 8% disaccharides (more than 20 are identified), 2% inother sugars, and the rest is other various substances as organicacids, enzymes, aromatic compounds, flavonoids, and vitamins
Trang 33Vitamin C and sugar markers as discriminators for Spanish honeys
In the last compounds, the vitamins, the water-soluble vitamins,
and specially vitamin C by its antioxidant character, are the most
important ones
In the last ten years, very few references on the determination of
vitamin C in honey have found Vitamin C determination was
car-ried out traditionally by colorimetric method after derivatization
in the presence of oxidants and by volumetric method in the
pres-ence of 2,6-dichloroindophenol (AOAC method 967.12 2005;
Guler and others 2007; Ferreira and others 2009) Recently,
vi-tamin C has been determined by reverse phase high-performance
liquid chromatography (RP-HPLC) in isocratic mode, with a
mo-bile phase of methanol/water at pH 2.5 This method is fast, it
does not required sample treatment and the retention time for
vitamin C is about 5 min (Castro and others 2001)
A gradient chromatographic method for the determination of
vitamin C has been developed (Gheldof and others 2002), however
despite the high number of honeys, the authors did not detect the
presence of vitamin C in any of the samples
Spain has several major areas for honey production, and one of
the most important is the natural region from Castilla-La Mancha
(Central Spain) In this area, origin denomination “La Alcarria
honey” was registered and protected by the European Commission
in 1996 This honey must have specific parameters in a range values
to belong to this denomination (JCCLM 1996)
Characterization of Spanish honeys of different floral type (for
example, rosemary, lavender, willow, thyme) has also been done
by using pollen analysis, physicochemical parameters, mineral
con-tent, and/or volatile composition data (Mateo and others 1992;
P´erez-Arquillu´e and others 1995; Mateo and Bosch 1998;
So-ria and others 2004; Terrab and others 2004; Nozal and others
2005) Chemometric techniques appear to be the most powerful
tools for characterizing and classifying honeys by geographical and
botanical source In this way, linear discriminant analysis (LDA)
and principal component analysis are commonly employed to
dis-tinguish between honeys from different botanical sources (Mateo
and Bosch 1998; Terrab and others 2002; Terrab and others 2003;
Devillers and others 2004; Marini and others 2004; Soria and
others 2004; Terrab and others 2004; Gonz´alez-Miret and
oth-ers 2005; Nozal and othoth-ers 2005; Consonni and Cagliani 2008;
Etzold and Lichtenberg 2008)
The determination of vitamin C by RP-HPLC and by titration
with the 2,6-dichloroindophenol and the classification by LDA of
7 Castilla-La Mancha honeys according to their floral origin are
the purposes of this article All the predictor variables have been
chosen among the parameters routinely determined for the quality
control of this product, with the only exception of the vitamin C
data
Material and Methods
Honey samples
The present study was conducted using 82 honeys from the
region Castilla-La Mancha (central Spain) that were collected
di-rectly from beekeepers: chestnut (11), lavender (9), thyme (16),
rosemary (11), echium (11), multifloral (19), and honeydews (5)
Samples were initially characterized by melissopalynological and
sensorial analysis to determine the botanical source Pollen analysis
was carried out using the methods established by the Intl
Com-mission of Bee Botany (Louveaux and others 1978; Von der Ohe
and others 2004)
Determination of physicochemical parameters
Some physicochemical parameters were analyzed using TheHarmonised Methods of the Intl Honey Commission (Bogdanovand others 2004)
Moisture levels were determined by noting the refractive index
at 20◦C in an Abbe model NAR-1T refractometer (Tokyo, Japan).Measurements of pH were performed potentiometrically at20.0◦C in a 10% (w/v) solution of honey in freshly boiled distilledwater using an Eutech System model XS PC510 pH-meter Thefree acidity is obtained by plotting the neutralization curve with
a titrated sodium hydroxide solution and determining the pH ofthe equivalence point
Electrical conductivity was measured at 20.0◦C in a 20% (w/v)solution of honey (dry matter basis) in deionized water using aRadiometer CDM-83 conductimeter (Copenhagen, Denmark).The determination of the hydroxymethylfurfural (HMF) con-tent was based on the determination of UV absorbance of HMF
at 284 nm with a Hitachi model U-1100 spectrophotometer(Krefeld, Germany)
The determination of sugar content (glucose and fructose) wasdone by HPLC but with refractometric detection (Merck RI-71)
A mixture of acetonitrile:water (87:13,% v/v) was used as a mobilephase, with a flow rate of 1 mL/min Separation was carried outusing a Lichrospher 100 NH2 (5μm), from Merck (Darmstadt,
Germany) at 40◦C Honey samples were dissolved to 5% (w/v)
in water and filtered through a nylon syringe filter (0.45μm).
Determination of color
Color parameters were established in the CIELAB system byreflection method (measuring geometry d/8◦, illuminant D65,range 400 to 700 nm, observer 10◦) using a Hitachi model U-
1100 spectrophotometer
Determination of vitamin C
method for juices). The AOAC method involves a redox tion with 2,6-dichloroindophenol (AOAC International 2005).The honey samples were prepared by dissolving 5 g of honey in
titra-25 mL of 2% oxalic acid (Panreac, Barcelona, Spain) and withfolded filters filtration prior to the assessment
High-performance liquid chromatography, RP-HPLC method. The determination of vitamin C was performed by
an RP-HPLC method in isocratic mode, with a mobile phase of0.01% (v/v) H2SO4(Panreac), at pH 2.5, a flow of 0.9 mL/min,with UV detection at 245 nm, at 25± 1◦C The method used was
adapted from the work of V´azquez (V´azquez-Oderiz and others1994) The chromatographic system (HPLC, Hitachi) consists of
a model L-6200 pump, a thermostatic oven for column T-6300,
an automatic injection system AS-2000, a UV-Vis L-4250 modeldetector, and interface D-6000 The software used for acquisitionand data processing is the D-6000 Chromatography Data Station.The column used was a Lichrosorb RP-18 10μm 150 mm ×
4.0 mm, from Merck
Standard solutions of vitamin C (Fluka, Madrid, Spain) wereprepared by dissolving the corresponding amount in 0.05 M HPO3(Sigma-Aldrich, Madrid, Spain) All solutions were stored in thedark at 4◦C The determination of vitamin C has been carriedout by the external standard method The range of concentrations
of vitamin C used to perform the calibration line ranged between0.11 and 276.70 mg/L The method sensitivity was calculated
as the slope of the straight line, with its corresponding units The
Vol 76, Nr 3, 2011 rJournal of Food Science C357
Trang 34detection and quantification limits were obtained from the straight
line, as the amount of analyte that yields a signal equal to intercept
plus 3 or 10 times the statistic s y /x, which estimates the random
errors in the y-direction (Miller and Miller 2000) The robustness
was the relative standard deviation (RSD) of the slope of the
calibration curve for 10 calibration curves obtained in different
days
Samples were prepared by dissolving 5 g of honey in 25 mL
of 0.05 M HPO3, then filtered through a paper filter and prior
to injection in the chromatographic system were filtered again
through a 0.45μm cellulose acetate membrane filter.
The statistical calculations in the linear discriminant
multivari-ate analysis have been carried out using the statistical program
STATGRAPHICS for windows 5.1
Results and Discussion
Vitamin C analysis in honey samples by volumetric and
chromatographic methods
The analysis of vitamin C in honeys has not assigned any official
method For this reason, vitamin C concentrations were obtained
for all honeys by volumetric AOAC official method 967.21 for
juices (AOAC method 967.12, 2005) and the results were
com-pared with a proposed reverse phase chromatographic method
(RP-HPLC) The chromatographic method is simple and rapid,
consuming less than 4 min per sample The results obtained by
both methods were compared to validate the RP-HPLC method
Figure 1A shows the chromatogram of a vitamin C standard
in-jected into the chromatographic system with a 0.01% H2SO4
mobile phase, pH= 2.5, and T = 25◦C The retention time for
vitamin C was 2.7 min, this time was lower than the one obtained
in octadecyl silane column C18 (5μm) with other mobile phases
such as methanol: H2O (15:85% v/v) in metaphosphoric acid at
pH 2.5 (5.1 min) (Castro and others 2001) and H2SO4at pH 2.2
(9 min) (V´azquez-Oderiz and others 1994)
The calibration curve was obtained by injecting different
con-centrations of vitamin C standards and its analytical characteristics
determined The parameters calculated were the linear range,
sen-sitivity, robustness, detection limit, and quantification limit The
linear range was long enough (2.28 to 276.7 mg/L) to analyze
all honey samples, and the method has a good sensitivity with a
detection limit 0.68 mg/L and a 2.3 mg/L quantification limit
value In addition, an RSD of 2.5% between the slopes values
was observed to determine the robustness These characteristics
were very good to determine the vitamin C content in the honey
samples studied
After the method was well established, 82 different honey
sam-ples with 7 different floral origins were analyzed, 11 chestnut, 9
lavender, 16 thyme, 5 honeydews, 11 rosemary, and 11 echium,
including also 19 multifloral honeys As an example, Figure 1B
shows a chromatogram of a rosemary honey obtained in the same
experimental conditions as Figure 1A
As was stated above, in order to validate the chromatographic
method as a useful method for vitamin C analysis in honey, samples
analyzed by both methods (AOAC 967.21 and RP-HPLC) were
compared to know if they were equal statistically The paired
t-test was not a correct t-test to use because the differences were not
normally distributed Therefore, a regression curve was used for
comparing analytical methods If each sample yields an identical
result with both methods, the regression line will have a zero
intercept and a slope of 1 As this situation never occurs due to
random errors, even if systematic errors are entirely absent, an
intercept differing significantly from zero and a slope differingsignificantly from 1 are tested generally at the 95% significancelevel Figure 2 shows the representation of the vitamin C valuesobtained for total analyzed honey samples by both methods inmg/100 g The values of the intercept and slope obtained for thelinear regression with their confidence limits were 1.63± 2.2 and1.04± 0.05, respectively As can be seen, the zero intercept value
is included in the confidence range, and again confidence rangefor slope includes the ideal value of 1
It is important to note that the statistical method concludes thatboth methods have got the statistically same result in spite of thedeviation that can be seen in a few values between methods Thesedeviations are relative errors because some of them are higher byRP-HPLC method and other by volumetric method
Assuming that the HPLC method obtains the same values thanthe AOAC official method, the next studies were realized usingthe values obtained by HPLC which is a faster and simpler thanvolumetric method
Vitamin C content found in the different honey sources
The vitamin C values obtained for 82 honey samples cited abovewere compared to look for statistical differences between eachhoney source group This comparison of the vitamin C values can
be made using a parametric test as ANOVA upon the results of
Figure 1–Ascorbic acid chromatogram with a mobile phase H 2 SO 4 0.01%,
pH= 2.5, and T = 25 ◦C Detection: UV at 254 nm (A) Standard solution and (B) rosemary honey sample.
Trang 35Vitamin C and sugar markers as discriminators for Spanish honeys
each honey group In our case, we have studied the conditions
to apply this test but the results were not a normal distribution
for vitamin C values and also, there was not a statistically equal
variance between all honey groups In spite of this, we consider
to be very important the visual analysis of data distribution in the
different honey groups As can be seen in Figure 3, the vitamin C
content is higher in the thyme honey group, reaching values close
to 170 mg/100 g This fact, lead us to think that this honey has
a huge antioxidant effect and is different from the others in base
to other vitamin C levels However, this honey type shows a great
dispersion in the values and its confidence interval is significantly
higher than the others In the lavender group, there was a wide
confidence interval, but if we look at Figure 3A, a discrepant
value was observed which was much higher than the other in this
honey
From these results, it could be conclude that the vitamin C
values obtained for the different honey types are higher for thyme
honey than the others, but they have a high standard deviation
(SD) that implies a wide distribution of values in this honey
type
Figure 2–Linear regression between ascorbic acid values in mg/100 g in
honey samples by AOAC and RP-HPLC analysis method.
Multivariate discriminant study of differentphysicochemical variables as markers for honey types
The aim of this study was to look for markers of the ent Castilla-La Mancha honey sources, and their characterizationbased to different physicochemical parameters Sixty-three honeysamples were studied in order to fix these markers with all of them
differ-Figure 3–Distribution of ascorbic acid values by RP-HPLC analysis method
in mg/100 g in function of unifloral honey floral origin (A) Obtained values in each honey type and (B) averages and confidence intervals for each honey type.
Table 1– Average values and standard deviation obtained for the physicochemical parameters determined in each unifloral honey studied.
Free
Honey type mg/100 g a ∗ 10 b ∗ 10 Cab ∗ 10 L ∗ 10 Hab ∗ 10 Turbidity mg/kg ms/cm mg/kg (%) pH (%) (%) Chestnut 10.66 21.05 59.52 63.37 38.46 71.21 0.41 45.02 1.06 4.64 16.06 4.62 31.67 26.75
Trang 36unifloral type The 19 multifloral honeys were excluded due to its
mixed botanical source Table 1 shows the average data of these
parameters and the SD of the measures realized in each typeof
honey sample In addition, the number of samples analyzed in the
average for each honey type is indicated in the table
The experimental data for the physicochemical parameters,
ex-cept vitamin C, have been contrasted with those available in the
literature for Spanish unifloral honeys In general, the results found
are in good agreement with data for rosemary, lavender, and thyme
(Mateo and Bosch 1998; Terrab and others 2004; Nozal and others
2005)
An LDA was applied to classify the different honey types The
parameters introduced in the analysis were free acidity, moisture,
conductivity, pH, HMF, glucose, fructose, and vitamin C After
that the statistical method discriminates between the parameters,
Figure 4–Discriminant functions obtained for discrimination of unifloral
honeys from their different botanical origin.
Table 2– Discriminate functions obtained for honey type and
percentage of explanation of each one in the variability of data
for variables studied.N = 63; honey types = 6.
Discriminate function Eigenvalue Percentage Correlation
Table 3– Standardized coefficients of 2 principal discriminate
functions obtained for the different honey types.N = 64; honey
Figure 4 shows the collected values of the discriminant tions obtained for the different honey types This figure shows
a clearly marked honey classification with 2 discriminant tions These functions have only 4 influential parameters fromall physicochemical parameters studied The final chosen param-eters for these functions are sugar content (glucose and fructose),electrical conductivity, and vitamin C content Table 2 shows thediscriminant functions and their auto-values with the significantpercentage of each one in the classification of honeys These dis-criminant functions are obtained with the quantitative variablesthat statistically discriminate between honey sources In this table,
func-it can be observed that the 2 first functions classify 88% of thedata measured for the 6 different honeys Table 3 summarized thestandardized coefficients obtained for the 4 quantitative variables
in the 2 principal discriminant functions
Figure 4 shows the obtained values in the 2 main discriminatefunctions for the 6 honey sources studied As can be seen in thisfigure, the classification of honeys in the corresponding source isclearly differentiated and the groups are separated in function ofthe parameters cited above In fact, the results obtained for differentvariables in the honey sources studied have very different influence
in the honey classification Therefore, it is possible to consider theparameters independently, because in some of them a variation infunction of honey source was found, but anything can be used
to define the botanical source marker Particularly, vitamin C wasclearly higher in the thyme honeys than the others, but the valueshad a great variability into this honey source On the other hand,rosemary honeys had higher sugar content than thyme honey andhoneydew had the lowest sugar content The conductivity wasalso different in each source, and the highest conductivity valuescorresponded to chestnut honeys and the lower values to rosemaryand lavender
The real classification of the 64 unifloral honey samples, usingthe discriminant functions obtained in this study is shown in Ta-ble 4 The values are percentages corresponding to the number ofhoneys well classified in relation to the total honeys in each honeysource The percentage of accurate classification (100%) was ob-tained in the chestnut and honeydew honey, and a high percentage
is obtained in lavender (88.9%) and thyme (87.5%) In the case ofthe other 2 honey types, the majority of cases are assigned to thecorrect group (rosemary 54.5% and echium 50%) being in bothcases a 36.4% in rosemary and 25% in echium assigned to lavendergroup This behavior is logical if it is taking into account that theassignment of a specific source to 1 honey corresponds to a mini-mum percentage of its own pollen and each honey has a mixture
of different pollen types Thus, it can be said that discriminantanalysis is a very good tool for the differentiation and classification
of Castilla-La Mancha studied honeys
Table 4–Percentage of correct classification data by discrimination functions in each honey type.
Trang 37Vitamin C analysis in these honeys can be carried out by
RP-HPLC isocratic method in 3 min with very good correlation with
respect to the official method for juices, AOAC
dichloroindophe-nol method Vitamin C content, which gives high antioxidant
properties to honey is higher in thyme samples than in other
honey types, reaching in some cases quantities up to 170 mg/
100 g
In the experimental physicochemical variables measured in
Castilla-La Mancha honeys, only electrical conductivity, sugar,
and vitamin C content showed a discriminant capacity as
mark-ers of the different honey types Considering vitamin C as a new
marker of honey types, this parameter together with conductivity
and sugar content have allowed correct classification into 6 honey
groups as it was shown in Figure 4
The assignment of each honey sample to their correct botanical
source is highly correct for chestnut, honeydew, lavender, and
thyme For rosemary and echium, the higher percentage of samples
is assigned to their correct type
Acknowledgments
The authors are gratefully acknowledge to “Junta de
Comu-nidades de Castilla-La Mancha” for financial support with the
projects PAI09-0018-9267 and 05-299/IA-47 and to beekeepers
for contributing with the samples used in this study and laboratory
personal of “Centro Agrario de Marchamalo.”
References
AOAC International 2005 Method 967.12 Official methods of analysis 18th ed Gaithersburg,
Md.: AOAC Intl.
Bogdanov S, Ruoff K, Persano Oddo L 2004 Physico-chemical methods for the characterization
of unifloral honeys: a review Apidologie 35:S4–S17.
Castro RN, Azeredo LC, Azeredo MA, de Sampaio C 2001 S.T HPLC assay for determination
of ascorbic acid in honey samples J Liq Chromatogr & Rel Technol 24:1015–20.
Consejer´ıa de Agricultura de la Junta de Comunidades de Castilla-La Mancha 1996 Principales
caracter´ısticas de la miel de la Alcarria Marchamalo (Guadalajara).
Consonni R, Cagliani LR 2008 Geographical characterization of polyfloral and acacia honeys
by nuclear magnetic resonance and chemometrics J Agric Food Chem 56:6873–80.
Council Directive 2001/110/EC of 20 December 2001 2001 Relating to honey Official
Journal of the European Union L10:47–52.
Council Directive 2006/1924/EC of 20 December 2006 2006 Nutrition and health claims
made on foods Official Journal of the European Union L404:9–25.
Council Directive 2008/108/EC of 15 January 2008 2008 Amending regulation (EC) No
1925/2006 on the addition of vitamins and minerals and of certain other substances to foods.
Official Journal of the European Union L39:11–3.
Devillers J, Morlot M, Pham-Del`egue MH, Dor´e JC 2004 Classification of mono floral honeys based on their quality control data Food Chem 869:3053–312.
Etzold E, Lichtenberg-Kraag B 2008 Determination of the botanical origin of honey by Fourier-transformed infrared spectroscopy: an approach for routine analysis Eur Food Res Technol 227:579–86.
Ferreira I, Aires E, Barreira JCM, Estevinho LM 2009 Antioxidant activity of Portuguese honey samples: different contributions of the entire honey and phenolic extract Food Chem 114:1438–43.
Gheldof N, Wang X, Engeseth NJ, 2002 Identification and quantification of antioxidant ponents of honeys from various floral sources J Agric Food Chem 50:5870–77.
com-Gonz´alez-Miret ML, Terrab A, Hernanz D, Fern´andez-Racamales MA, Heredia FJ 2005 Multivariate correlation between color and mineral composition of honeys and their botanical origin J Agric Food Chem 53:2574–80.
Guler A, Bakan A, Nisbet C, Yavuz O 2007 Determination of important biochemical properties
of honey to discriminate pure and adulterated honey with sucrose Food Chem 105:1119– 25.
Louveaux J, Maurizio A, Vorwohl G 1978 International Commission for Bee Botany of IUBS Methods of melissopalynology Bee World 59:139–57.
Marini F, Magri AL, Balestrieri F, Fabretti F, Marini D 2004 Supervised pattern recognition applied to the discrimination of the floral origin of six types of Italian honey samples Anal Chim Acta 515:117–25.
Mateo R, Bosch F 1998 Classification of Spanish unifloral honeys by discriminant analysis of electrical conductivity, color, water content, sugars and pH J Agric Food Chem 46:393– 400.
Mateo R, Jimenez M, Bosch F 1992 Evaluation of the color of some Spanish unifloral honey types as a characterization parameter J AOAC Int 75:537–42.
Miller JN, Miller JC 2000 Statistics and chemometrics for analytical chemistry Dorchester, Dorset, England: Prentice Hall p 120.
Nozal MJ, Bernal JL, Diego JC, Mart´ın MT 2005 Classifying honeys from the Soria province
of Spain via multivariate analysis Anal Bioanal Chem 382:311–9.
P´erez-Arquillu´e C, Conchello P, Ari ˜no A, Juan T, Herrera A 1995 Physicochemical attributes and pollen spectrum of some unifloral Spanish honeys Food Chem 54:167–72.
Persano-Oddo L, Piro R 2004 Main European unifloral honeys: descriptive sheets Apidologie 35:S38–S81.
Persano-Oddo L, Piana L, Bogdanov S, Bentabol A, Gotsiou P, Kerkvliet J, Martin P, Morlot
M, Ortiz VA, Ruoff K, Von der Ohe K 2004 Botanical species giving unifloral honey in Europe Apidologie 35:S82–S93.
Piazza MG, Persano-Oddo L 2004 Bibliographical review of the main European unifloral honeys Apidologie 35:S94–S111.
Soria AC, Gonz´alez M, de Lorenzo C, Mart´ınez-Castro I, Sanz J 2004 Characterization of artisanal honeys from Madrid (Central Spain) on the basis of their melissopalynological, physicochemical and volatile composition data Food Chem 85:121–30.
Terrab A, D´ıez MJ, Heredia FJ 2002 Characterisation of Moroccan unifloral honeys by their physicochemical characteristics Food Chem 79:373–9.
Terrab A, Gonz´alez AG, D´ıez MJ, Heredia FJ 2003 Mineral content and electrical conductivity
of the honeys produced in Northwest Morocco and their contribution to the characterisation
of unifloral honeys J Sci Food Agric 83:637–43.
Terrab A, Recamales AF, Hernanz D, Heredia FJ 2004 Characterization of Spanish thyme honeys by their physicochemical characteristics and mineral contents Food Chem 88:537– 42.
Tomoi-Sato MD, Go-Miyata MD 2000 The nutraceutical benefit, Part III: honey Nutrition 16:468–9.
V´azquez-Od´eriz ML, V´azquez-Blanco ME, L ´opez-Hern´andez J, Simal-Lozano J, Rodriguez MA 1994 Simultaneous determination of organic acids and vitamin C in green beans by liquid chromatography J AOAC Int 77:1056–9.
Romero-Von der Ohe W, Persano-Oddo L, Piana ML, Morlot M, Martin P 2004 Harmonized methods
of melissopalynology Apidologie 34:S18–S25.
Vol 76, Nr 3, 2011 rJournal of Food Science C361
Trang 38Derivatives from Waxy Corn Starch
W Zhu, H.L Xie, X.Y Song, and H.T Ren
Abstract: Waxy corn starch was esterified with 2-octen-1-ylsuccinic anhydride (OSA) using response surface ology The molecular structure and paste properties were also investigated Results indicated that the optimum parametersfor esterification were as follows: reaction period 4 h, temperature 36.7◦C, pH of reaction system 8.3, concentration
method-of starch slurry 36.9%, and amount method-of OSA 3% The degree method-of substitution was 0.0187 and the reaction efficiencywas 80.6% The ester carbonyl group in OSA starch was characterized by Fourier Transform Infrared Spectroscopy at
1723 cm−1 Compared with native starch, OSA derivative had higher peak viscosity, better freeze–thaw stability, anddecreased gelatinization temperature and digestibility by porcine pancreatic alpha–amylase Scanning electron microscopyshowed that the gels from OSA starch had less and smaller pores; however, the native starch gels changed to sponge-likestructure after 4 freezing/thawing cycles The OSA modified waxy corn starch offered a potential to be used in frozenfoods
Keywords: 2-octen-1-ylsuccinic anhydride, frozen food, physicochemical properties, response surface methodology, waxycorn starch
Practical Application: (a) To optimize operating conditions to achieve OSA modified waxy corn starch with a highdegree of substitution; (b) to give a deeper insight into the physicochemical properties of OSA modified waxy cornstarch, which offered a potential to be used in frozen foods
Introduction
The systematic search for food products ready for consumption
brought about a significant increase in the range of frozen products
offered in the market (Takeiti and others 2007) If the frozen food
is not stored under sufficiently cold and steady temperatures, ice
crystals will grow, or recrystallize to large ice crystals, which may
cause damage to the food texture and make these frozen products
less homogenous, creamy, and smooth
Starch has been used in a wide range of frozen products To
maintain the final quality of the products, the starch must keep its
characteristics and present low syneresis However, native starch
has limited use in the frozen products due to its poor stability
under the conditions of temperature, shear, pH, and refrigeration
commonly applied to processed foods Therefore, native starch is
often modified to meet different industrial requirements One
ex-ample of modified starches is octenyl succinic anhydride (OSA)
starch, produced by esterification of native starch with OSA in
aqueous slurry systems OSA starch was first patented by Caldwell
and Wurzburg (1953) In the United States, the Food and Drug
Administration approved OSA starch for food use in 1972
Re-cently, OSA starch has been permitted for use as an additive for
formula and weaning foods for infants and young children in the
MS 20100917 Submitted 8/12/2010, Accepted 12/21/2010 Authors Zhu and
Xie are with Dept of Agronomy and authors Song and Ren are with Dept of Food
Sci-ence and Engineering, Henan Agricultural Univ., Zhengzhou 450002, P R China.
Direct inquiries to author Xiao-Yan Song (E-mail: songxiaoyan2737@163.com).
European Union (Drusch and Schwarz 2006) The maximum level
of OSA treatment allowed is 3% (degree of substitution is imately 0.02) After OSA modification, the starch was an effectiveemulsifier due to the addition of dual functional hydrophilic andhydrophobic groups (Tesch and others 2002)
approx-The earlier research on modication of starches by OSA wasdone with corn, wheat, rice, and potato starches (Bao and others2003; Shih and Daigle 2003; He and others 2006; Song and others2006; Bhosale and Singhal 2007; Ruan and others 2009) But onlyOSA starches from waxy corn are commercially available as gumarabic substitutes for food production It is therefore important toinvestigate the physicochemical properties and functions of OSAmodified waxy corn starch
In this study, OSA starches were prepared from waxy corn starch
in aqueous slurry systems The major factors affecting the cation, including reaction temperature, pH of the reaction system,and starch concentration were evaluated by means of response sur-face methodology The molecular structure and paste properties(for example, pasting properties, thermal properties, freeze–thawstability, structure of freeze–thaw gels, and digestibility) of OSAstarches with different degrees of substitution were also investi-gated by means of Fourier Transform Infrared Spectroscopy (Ft-Ir), rapid visco analyser (RVA), differential scanning calorimeter(DSC), and scanning electron microscopy (SEM) The objectives
esterifi-of this investigation were: (a) to optimize operating conditions toachieve OSA starch with a high degree of substitution, and (b) togive a deeper insight into the physicochemical changes in OSAmodification from waxy corn starch, which might be useful forthe food industry
Trang 39Production of OSA modified corn starch
Materials and Methods
Materials
Waxy corn starch (the approximate composition was as follows:
crude protein content 0.19%± 0.02%, crude fat content 0.23% ±
0.03%, moisture content 9.00% ± 0.16%) was purchased from
Dacheng Group (Changchun, China) OSA was purchased from
Sigma-Aldrich Chemical Co (97%; St Louis, Mo., U.S.A.) The
other chemicals used in the study were of analytical grade
Preparation of OSA starch
Waxy corn starch (30 g, dry weight) was suspended in distilled
water with agitation The pH of the suspension was adjusted
ac-cording to Table 1 throughout the reaction by adding 3% NaOH
solution The 3% OSA (in proportion to starch, w/w) was added
slowly in a period of 2 h The reaction continued totally for 4 h
After reaction, the pH of the mixture was adjusted to 6.5 with
3% HCl solution, then the mixture was centrifuged The
precipi-tate was washed 2 times with distilled water and 2 times with 70%
aqueous alcohol, oven-dried at 40◦C for 24 h, and passed through
a 180-mesh nylon sieve (90μm opening).
Determination of the degree of substitution
Degree of substitution (DS) is the average number of hydroxyl
groups substituted per glucose unit The DS of OSA starch was
determined using a titration method (Kweon and others 2001)
The OSA starch (5 g, dry weight) was accurately weighed and
dispersed in 25 mL of 2.5 mol/L HCl-isopropyl alcohol solution
by stirring for 30 min A total of 100 mL 90% isopropyl alcohol
solution (in proportion to water, v/v) was added to the suspension,
followed by stirring for an additional 10 min The suspension was
filtered through a glass-filter and the residue was washed with 90%
isopropyl alcohol solution until no Cl− was detected (using 0.1
mol/L AgNO3solution) The starch was re-dispersed in 300 mL
distilled water, followed by being cooked in a boiling water-bath
for 20 min The starch solution was titrated with 0.1 mol/L
stan-dard NaOH solution, using phenolphthalein as an indicator A
blank was simultaneously titrated with native starch as a control
Table 1– DS and RE for the OSA modified waxy corn starches
Starch Temperature concentration
The reaction efficiency (RE) was calculated as follows:
The theoretical DS was calculated assuming that all of the addedanhydride reacted with starch to form the ester derivative
Experimental design and statistical analysis
According to the previous studies, the most important ters for the OSA modification were pH, temperature, and starchconcentration (Song and others 2006) In this study, response sur-face methodology was used to evaluate the effects of reactiontemperature, pH of reaction system, and starch concentration on
parame-DS (Table 1) The central composite design scheme consisted of
20 treatments, and the central point was replicated 6 times for thecalculation of the experimental error
Statistical analysis was performed with SAS 8.0 software.The variables were coded according to the following equation(Montagomery 1991)
x i = (X i − X0)/x i (3)
where, x i is the coded value of an independent variable, X i is
the real value of an independent variable, X 0is the real value of
an independent variable at the center point, andx i is the step
change value The model proposed for the response (Y ) was:
Y = b0+b i x i+b i i x i i2+b i j x i x j (4)
where b o is the value of the fixed response at the central point
(0, 0) of the experiment; b i , b ii , and b ij are the linear, quadratic,and cross product coefficients, respectively
Fourier transform infrared spectroscopy
The change in chemical structure of the starch was tively analyzed using FT-IR (Nexus 670, Nicolet, Madison, Wis.,U.S.A.) Samples were prepared by grinding the finely powderedstarch with KBr The spectrum was recorded over the wavenumberrange between 400 and 4000 cm−1
qualita-Pasting properties
The pasting properties were determined using a super 4D rapidvisco analyser (Newport Scientific Instruments, Warriewood,Australia) Samples were prepared by mixing 3 g of starch and
25 mL of distilled water The analysis was based on the AACCapproved method 76–21.01 (2000) with some modifications(AACC 2000) The mixture was stirred manually for 1 min tofacilitate dispersion before testing (Shih and Daigle 2003) Theheating and cooling cycles were programmed in the followingmanner: The samples were held at 50◦C for 1 min, heated to
95◦C in 3.42 min, held at 95 ◦C for 2.7 min, cooled to 50◦C
in 3.88 min, and held at 50◦C for 2 min
Vol 76, Nr 3, 2011 rJournal of Food Science C363
Trang 40The thermal properties of native and OSA modified starches
were determined using a differential scanning calorimeter (DSC)
(Q200, TA Inc., Newcastle, Del., U.S.A.) Total of 3 mg of samples
were placed in an aluminum cup, 7 μL the distilled water was
added The cup was hermetically sealed and then heated from
30 to 110 ◦C at a rate of 10◦C /min The major parameters
of DSC profile were described as onset temperature (T O), peak
temperature (T P), enthalpy of gelatinization (H gel), and final
temperature (T C)
Freeze–thaw stability
Freeze–thaw stability was determined according to the
pre-vious methods (Zheng and Sosulski 1998; Bhosale and Singhal
2007) with a slight modification Starch (3 g, dry weight) was
transferred to screwcap tubes and suspended in distilled water
(100 mL) in a boiling water bath for 20 min After being cool
down, the paste was divided into 4 equal parts by weight and
then each part was transferred to a centrifuge tube The samples
were kept in refrigeration at−18◦C for 24 h, and then thawed at
room temperature for 6 h One of the tubes was taken out every
time, and the other samples were put back to refrigeration for
another cycle The tube taken out was centrifuged at 3000 rpm
(1700 × g) for 20 min The water layer was decanted and the
residual paste was weighed The percentage of water separated
after each freeze–thaw cycle was measured and expressed as the
following equation:
Syneresis= m2−m3
m2−m1
where, m1is the weight of centrifuge tubes (g), m2is the weight of
centrifuge tubes and starch paste (g), m3is the weight of centrifuge
tubes and starch paste after centrifuge (g)
Frozen structure by scanning electron microscopy
The frozen structure was determined according to Charoenrein
and others (2008) The freeze–thaw samples were cut and
gradu-ally dehydrated in 50%, 70%, 90%, and absolute ethanol at room
temperature for 2 h at each concentration and finally dehydrated
using a critical point dryer The cut samples were mounted on
the stub, coated with gold, and observed using SEM (S-3400NII,
Hitachi Inc., Japan) Micrographs of each starch sample were taken
at 300× magnifications
In vitro digestibility
The in vitro digestibility of the native and OSA starches were
de-termined using porcine pancreatic alpha-amylase (E.C.3.2.1.1, 10
to 30 units/mg solids at pH 6.9 at 20◦C, Sigma-Aldrich
Chem-ical Co.) according to Anderson and others (2002) and Yang and
others (2006) with some modifications Each sample (100 mg,
dry basis) was weighed in a 50-mL test tube and 10 mL of
dis-tilled water was added The tubes were tightly capped to prevent
the loss of moisture The contents were mixed by vortexing for
2 min Then test tubes were placed in a boiling water bath for
30 min to gelatinize the starch After cooking, the tubes were
removed and cooled to room temperature Total of 2 mL of
0.2 mol/L phosphate buffer (pH 6.9) was added to the test tubes,
followed by the addition of 500μL of porcine pancreatic
alpha-amylase solution (1 mg/mL) The test tubes were vortexed and
placed in a 37 ◦C water-bath shaker Aliquots (250 μL) of the
solution were taken from each flask every 30 min from 0 to 3 h
and placed into corresponding test tubes containing 2 mL DNS(3,5-dinitrosalicylic acid) reagent The test samples were heated at
100 ◦C for 10 min to deactivate enzyme and develop reactioncolor After cooling, distilled water was added up to 20 mL andmixed The absorption of the reaction product of maltose-DNSAwas measured at 540 nm The maltose was used to make the stan-dard curve The extent of hydrolysis was calculated as the degree
of hydrolysis (%, maltose equivalent) Each sample was determined
35◦C, pH of reaction system 8.5, concentration of starch slurry35% Table 1 also indicated RE was positively correlated with DS,and therefore the regression analysis was only done for DS as theresponse variable
Determination of the optimum operating conditions
The ANOVA indicated that reaction temperature, pH, andstarch concentration had significant influence on the OSA mod-
ification of waxy corn starch (P < 0.05) x1 , x2 , and x3 also
had significant influence on the esterification (P < 0.05) But the
effects of cross products were not significant (Table 2)
The application of response surface methodology yielded a gression equation of empirical relationship between the DS andthe variables in coded units (Eq 6)
re-DS = 0.0184 + 0.000319x1− 0.000407x2+ 0.000233x3
−0.000498x2− 0.00131x2− 0.000304x2 (6)
where, DS is the response and x 1 , x 2 , and x 3are coded values of
temperature, pH, and starch concentration, respectively R 2 was0.9779 (Table 2), suggesting that 97.79% of the variation could beattributed to the independent variables and only 2.21% of the total
variations could not be explained by the model The model’s
P-value of 0.0000 implies that the model was statistically significant
at 99% confidence level
Eq 6 showed that the DS reached its maximum value (DS=
0.0185) at a combination of coded level 0.35 (x 1), −0.17 (x 2)
and 0.38 (x 3) Effects of the reaction conditions of temperature36.7 ◦C, pH 8.3, starch concentration 36.9% on DS was exam-ined The data showed that the DS was 0.0187± 0.0003 and REwas 80.6%± 1.3% The previously mentioned results indicate that
Table 2– ANOVA for the model.
Quadratic 3 0.8352 126.07 <0.0001
Cross product 3 0.0069 1.04 0.4152
Total model 9 0.9779 49.20 <0.0001