Luận văn β d fructofuranosidase production and application to the manufacture of frutooligosaccharides

Dependence of rate of hydrolysis of immobilized 1 and free FFase from yeast on the pH of the medium - The effect of temperature The same as the effect of temperature, immobilized enzyme

characteristics, many of these sugar possess properties that ar

beneficial to the heath of

consumers These include non-cariogenicity, a low calorific value andthe ability to

stimulate the growth of beneficial bacteria in the colon Both the production and the

applications of food-grade oligosaccharides are increasing rapidly Major uses are in

beverages, infant milk powders, confectionery, bakery products, yoghurts and dairy

desserts Research continues into the development of new

oligosaccharides with a range

of physiological properties and applications in the food industry

FOS has been attracted attention of many researchers with its prebiotical property

recently In industrial scale, immobilized fungal β-fructofuranosidase

frucose from sucrose, some microbial β-D-fructofuranosidase may

catalyse the synthesis

of short-chain fructooligosaccharides (FOS), in which one to three

fructosyl moieties are

linked to sucrose by different glycosidic bonds depend on the enzyme source (Sangeetha

et al., 2005) This enzyme has been used in food industry to produce inverted sugar and

mostly used for the preparation of jams, candies and soft-centered chocolates (Aranda C,

2006) FFase has been found in many different plants and

microorganisms FFase from

different sources differs in optinum pH of activity (which may be neutral, acid or

alkaline) (Winter H, 2000), optinum temperature of activity,

and thiol groups Reddy and Maley also indicated that carboxylic

groups from Asp-23

and Glu-204 play an important role in the catalytic process

Nevertheless, amino acid that

participate in the catalytic process of FFase from Arabidopsis thaliane’cell wall

(Arabidopsis thaliane is a small flowering plant native to Europe, Asia, and northwestern

Africa) are Asp-23 and Glu-203 (M Verhaest, 2006)

The catalytic sucrose process of FFase is divided into

three steeps:

-First, FFase links with sucrose to form enzyme-substrate

complex at the

immobilized FFase may be a consequence of the selective

binding of the more

neutral forms of the enzyme with the modified support in the immobilization

process

Fig.1 Dependence of rate of hydrolysis of immobilized (1) and free FFase

from yeast on the pH of the medium

- The effect of temperature

The same as the effect of temperature, immobilized enzyme are more stable

with the effect of temperature than free enzyme The optimal pH

of FFase from

2

Fig.2 Determination of the pH optima for immobilized (1) and free (2) FFase

from yeastThe thermostability cureves are shown in Fig.3 As can be seen, the free

enzyme was inactivated completely at 60-700C for 0.5-1h

- The effect of substrate and product concentration

It has been shown experimentally that if the amount of the enzyme is kept

constant and the substrate concentration is then gradually increased, the

reaction velocity will increase until it reaches a maximum After this point,

increases in substrate concentration will not increase the velocity

(Worthington, Biochemical corporation, 1972) This is represented

native enzyme was inactivated after 15 min

Fig.5 Kinetics of the formation of the products of the enzymatic hydrolysis of

sucrose: 1)immobilized enzyme; 2) native enzyme

4

1.2 Fructooligosacharides

(FOS)In response to an increasing demand from the customer for

healthier and

calorie-controlled foods, a number of so-called alternative sweeteners such

similar to that of sucrose, a traditional sweetener

Various fructans of higher molecular weight have been produced

2,1 position of sucrose (GF), respectively, which should be

distinguished from other

kinds of fructose oligomers (Hidaka H Eida, 1986 and Hayash, 1989)

The production yield of FOS using enzymes originated from plants is low and

mass production of enzyme is quite limited by seasonal conditions; therefore, industrial

production depends chiefly on fungal enzymes from either Aureobasidium sp

1-fructosyltransferase, 6”- 1-fructosyltransferase, and lF

-fructosyltransferase They further

purified and characterized the individual fructosyltransferases It was found that the

general properties resembled those of the Jerusalem artichoke, but its substrate specificity

differed Satyanarayana, 1976 described the biosynthesis of

oligosaccharides and fructans

from agave He isolated various oligosaccharides, (DP 3-15),

synthesized them in vitro,

5

and proposed a reaction mechanism Unlike most enzymes, this agave enzyme is capable

of synthesizing inulotriose from inulobiose The naturally occurring oligosaccharides in

agave consists of l-kestose, neokestose, 6-kestose, and their derivatives These

oligosaccharides arise not only by transfructosylation reactions but

enzymes from plants that were discover by some workers in the past

Table 1: Fructooligosaccharide-synthetic enzymes

1-glucose, respectively (they seem to be 1-kestose and nystose,

according to Jong Won

Yun, 1996) The action of C.purpurea enzyme on sucrose also gives rise to

Enzymes with the potential for achiving a high yield of FOS production were

found in the late 1980s and early 1990s Hidaka et al, 1988 studied A

immobilized in gluten One gram of mycelia-immobilized particials

having a cell content

of 20% (w/w) was incubated with 100ml sucrose solution with an initial content of

-1

400g.L After a reaction period of 5h, the FOS yield was 61% of the totalsugars When

Aspergillus oryzae was used, the cultural conditions and reaction

parameters have been

standardized to get FOS yield of 58% (Sangeetha, Ramesh and Prapulla, 2002)

Besides fungi, bacterial strain have been reported to

fructooligosaccharides The final yield of FOS was reported to be 33% when 50%

sucrose was used as substrate (Park and Oh, 2001) Lactobacillus reutri

represented by formula GFn as depicted in Figure 6

Fig 6 Chemical structure of fructooligosaccharides

A research group of Meiji Seika Co, the first commercial

producer of FOS,

introduced the chemical structure of FOS produced from A niger

fructosyltransferase

8

Until now, FOSs are widely known that oligosaccharides contain

1-kestose, nystose and

1f - fructofuranosyl nystose However, this definition is not completely true FOSs are not

only contain these sugar but also others sugar with higher

together whereas a single enzyme works in most other

microorganisms For instance,

fructosan metabolism in Jerusalem artichoke is established by the combination of two

enzyme: sucrose:sucrose l-fructosyltransferase (SST) and β (2->1) fructan:β(2->1)

fructan l-fructosyltransferase (FFT) In the first instance, SST

converts sucrose into

glucose and an oligosaccharide but unable to promote

polymerization above the

trisaccharide level; further higher polymers are consecutively

synthesized by FFT The

overall reaction mechanism was expressed as follows:

where GF is a sucrosyl group and n is the number of extrasucrosyl fructose residues

Agave enzyme catalyzed a stepwise transfructosylation reaction to give rise to higher

FOS formation where synthesis of FOSs from sucrose takes place as follows: GF + fructosyltransferase -> F-

fructosyltransferase +GF-fructosyltransferase + GF -> GF2 + frucotsyltransferase

Here, it is notable that glucose, not fructose, acts as the acceptor

Fig 7.Network of the reaction mechanism for the

properties of ome kestosides such as 1-kestose, 6-kestose, and

neokestose The specific

rotation ([α]D20 ) and melting temperature of 1-kestose are 28.5 and 199-200°C

respectively It forms fine white crystals fairly rapidly The relative sweetness of 1-

kestose, nystose, and 1F-fructofuranosyl nystose to 10% sucrose

solution are 31, 22, and

16%, respectively Indeed, FOSs have a nice, clean sweet taste typically 0.3–0.6 times as

sweet as sucrose depending on the chain length – sweetness

decreases with increasing

chain length.FOSs are highly hygroscopic; it is difficult to keep the lyophilized

There have been few published studies comparing the

2005),…Among of them, S.cerevisiae is considered as the organism of

choice for FFase

production because of its hight sucrose fermentability (Rouwen horst

consequently contribute to increased productivity

The production level of FFase depends to a great extent on the microorganism,

basal substrate and microbial production process Moreover, the

fermentation operation

mode also influences the efficency of the process Submerged

fermentation has been

preferred over the solid-state for FFase production as it is

with immobilized cells is a convenient manner to reduce the

fermentation time during

repeated batch fermentation due to the elimination of the time

needed for cell growth

(Yang X, 2005) S.I.Mussatto, 2009 also studied a system by using

by using submerged fermentation with some new methods to

increase the yield of

product

11

2.1 Material

Material for the production of FFas are microorganism

and nutrient.Saccharomyces cerevisiae: is oftens isolated from different soil samples

Subsequently, mutant S.cerevisiae is cultured in YPSA medium,

harvested during the 6

exponential phase growth (about 1.6.10 cells/ml), wash with distilled water and plated

on suitable medium before fermentation Medium for the production

Table 3: Microorganism and medium for the

production of FFase

Sch.occidentalis YEPD (1%, w/v, yeast extract, 2%, w/v, peptone, Miguel

A´

lvaro-%, w/v, glucose) or Lactose Medium (0.3lvaro-%, Benito, 2007

2w/v, yeast extract from Difco, 0.35%, w/v,

bactopeptone, 0.5%, w/v, KH PO , 0.1%, w/v,

0.10 2007

ul-Haq,

As japonicus (% w/v) sucrose 20.0, yeast extract 2.75, NaNO3 S I

Mussatto,0

02

.2, K HPO 0.5, MgSO 7H2O 0.05, and KCl 2009.05

0% sucrose, 2% yeast extract (Difco), 2% Chang Chen,

inoculator at 30°C for 48 h The agitation rate is often kept at 200

revolutions per minute

On the other hand, in large scale operation, the fermentation process istaken place in a

dadecated fermentor with contains drive motor, heaters, pumps, gascontrol, vessel,

intrumenstation and sensors These base components combine to

perform some important

functions such as: maintain a specific temperature, provide adequate mixing and aeration,

allow monitoring and/or control of dissolved oxygen, allow feeding of nutrient solutions

and reagents,… The production medium is sterilized by heating it to 121ºC at a pressure2

of 1.2 Kgf/cm and maintaining those conditions for 30 minutes Heat

is supplied by

circulating steam through the fermenter jacket Air is filtered by passing it through

polypropylene filter Cold water is then circulated through the

fermenter’s jacket and the

broth is cooled to about 30 ºC The production line of FFase production is shown below:

13

SterilizeCool

InoculateInoculum

Ferment

Purify and concentrate

Fructofuranosidase

β-D-Fig 8: β-D-Fructofuranosidase production line

The process is monitored continuously through periodic

measurement of the

following parameters: temperature, pH, activity,… When the peak

activity is reached, the

batch (crude enzyme) is harverted The crude enzyme is purified by different methods

such as: ultrafiltration, gel filtration, ion-exchange chromatography,…Ultrafiltration is

used to separate the biomass from the culture fluid, which is later used as a source of

fructosyltransferase for the production of FOS For commercial FFase, purified FFase is

dried by spray drier or freeze drier to obtain powder product

To determine the highest yield of FFas in the process, the

experiments, FFase activity is measured by exame the amount of

glucose released in the

14

whole time of the reaction The mount of glucose is measured by determining color

intensity by a UV/Vis spectrophoto meter after glucose reacts with DNS reagent

microfilter

waste

Gel filter

Fig 9: FFase production

diagram

15

growth stage of a

culture is a critical

factor for the optimal

enzyme production

.IKram ul.Hag, 2008

studied

S.cerevisiae

improved

mutanttotheproduction of FFase

by submergedfermentation

Timecourse profiles for

FFase production by

wildstyle S.cerevisiae

IS-14 and mutan

S.cerevisiae UMF are

shown in fig 10 As

the result, maximumFFase production by

mutant(34.72±2.6U/ml with

7.05±1.2 g/L sugar

S.cerevisiae

1consumptionand 7.85±1.8 g/L dry

cellobserved

8 h after the onsetof

mass) was4

incubation

Thereforethe

rate of volumetric

productivit

y improvedwasapproximately

31-fold

parental strain Longer

decrease in available nitrogen, the age of the cells, inhibitors produced

by yeast itself and

protease production Other workers have reported maximum FFase production by S

cerevisiae incubated for 48 (Barlikova et al., 1991; Gomez et al., 2000) Not only

S.cerevisiae but also a mould, aspergillus japonicus can produce FFase

fermentation of sucrese by As.japonicus immobilized in vegetal fiber As

can se, in the

subsequence seven cycle, enzyme production remain almost satble at 40.6U/ml and this

value decreased (22%) only at the end of the eighth cycle This is an interesting result

because demonstrates an important increase in the productivity of the process to obtain a

higer yield of FFase

Fig 11 β-Fructofuranosidase (FFase) activity

during repeated batch fermentation of sucrose by

Aspergillus japonicus immobilized in vegetal

As.japonicus immobilized on lignocellulosic material, the pH of media

was set at 7.0

before inoculation and was not controlled during the experiment, being gradually

decrease during the cultivation (Fig 13)

Fig12 Effect of initial pH on the FFase production in

submerged culture by the mutant Saccharomycescerevisiae

UME-2 Incubation period 48 h, sucrose concentration 5.0 g/L,

temperature 30 °C, agitation rate 200 revolutions per minute Y-

error bars indicate standard deviation among three parallel

replicates

As can see in Fig13, the final pH of the fermented media was just around 5.5 This fac

could explain the hight activity (48.81U/ml) obtained at the

fermentation’s end Similar

result was obtained by R.C.FErnadez, 2007; L.L.Hocine, 2000 who also investigated the

FFas production by using cells immobilized on corn cobs and the final

Fig13 Kinetic behavior of pH during the sucrose

fermentation by A japonicus immo-or not in different

As.japonicus 3028 6.5 Ikram ul-Haq, 2006S.I Mussatto, 2009

Wen chang chen, 1997A.K.Balasubramaniem,2001

Quang D Nguyen, 2004Carolina Janer, 2004

5.0 (initial pH)

5.5 (initial pH)

5.0 (initial pH)

6.5 (initial pH)

6.4 (initial pH)

6.0 (initial pH)

Beside time, pH and temperature, subtrate concentration is one of themost important

factors that affects the enzyme production In FFase production the effect of sucrose

concentration must be observed Depending on the microorganic strain,suitable amount

of sucrose should be used According to Ikram ul – Haq 2006, maximumFFase activity

produced by S.cerevisiae obtained at a sucrose concentration of

5.0g/l Sucrose

concentrations higher than 5.0g/l caused an increase in sugar

consumption and cells

19