In this study, microbial succession and metabo-lite changes during 7 days of traditional Vietnamese alcoholic fermentation were monitored.. Keywords: Traditional alcoholic fermentation A
Trang 1New insight into microbial diversity and functions in traditional
Vietnamese alcoholic fermentation
a
Food Industries Research Institute, 301 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam
b
Hanoi University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 20 January 2016
Received in revised form 13 April 2016
Accepted 20 May 2016
Available online 24 May 2016
The roles of microorganisms in traditional alcoholic fermentation are often assumed based on abundance in the starter and activity in pure culture There is a serious lack of hard evidence on the behavior and activity of indi-vidual microbial species during the actual fermentation process In this study, microbial succession and metabo-lite changes during 7 days of traditional Vietnamese alcoholic fermentation were monitored Special attention was devoted to starch degradation In total, 22 microbial species, including 6 species offilamentous fungi (Rhizo-pus microsporus, Rhizo(Rhizo-pus arrhizus, Mucor indicus, Mucor circinelloides, Cunninghamella elegans, Aspergillus niger),
1 yeast-like fungus (Saccharomycopsisfibuligera), 7 yeasts (Saccharomyces cerevisiae, Clavispora lusitaniae, Wickerhamomyces anomalus, Lindnera fabianii, Pichia kudriavzevii, Candida rugosa, Candida tropicalis), and 8 bac-teria (Stenotrophomonas maltophilia, Lactobacillus brevis, Lactobacillus helveticus, Acinetobacter baumannii, Staph-ylococcus hominis, Bacillus megaterium, Enterobacter asburiae, Pediococcus pentosaceus) were identified Despite the presence of a complex microbiota in the starter, the fermentation process is consistent and involves a limited number of functional species Rapid change in microbial composition of fermentation mash was observed and it was correlated with ethanol content Microbial biomass reached maximum duringfirst 2 days of solid state fer-mentation Acidification of the medium took place in day 1, starch degradation in days 2, 3, 4, and alcohol accu-mulation from day 3 Although Sm.fibuligera dominated by cell count amongst potential starch degraders, zymography indicated that it did not produce amylase in the fermentation mash In mixed culture with Rhizopus, amylase production by Sm.fibuligera is regulated by the moisture content of the substrate Rhizopus was identi-fied as the main starch degrader and S cerevisiae as the main ethanol producer Bacterial load was high but un-stable in species composition and dominated by acid producers M indicus, Sm.fibuligera, W anomalus and bacteria were regarded as satellite microorganisms Their possible influence on organoleptic quality of fermenta-tion product was discussed
© 2016 Elsevier B.V All rights reserved
Keywords:
Traditional alcoholic fermentation
Amylolytic starter
Microbial succession
Rhizopus
Saccharomycopsis fibuligera
Amylase
1 Introduction
In Vietnam, traditional alcoholic fermentation of rice has centuries of
history Before the introduction of beer, this had been the main way for
obtaining alcoholic drinks (Peters, 2012) Even now, traditional
fermen-tation of alcohol from rice still makes up 80% of the total national
dis-tilled liquor production Alcohol is being produced by a vast network
of small breweries across the country (Da, 2009) Being one of the
coun-tries where rice cultivation originated, Vietnam possesses a rich
assort-ment of rice varieties and so the rice liquors produced from (Da, 2009)
Also, with 54 distinct ethnic groups, each with its own language,
life-style, and cultural heritage, there is high variation in methods for
alco-hol production Despite of the differences, breweries share the same
production principle where starter (banh men) containing a relatively
stable microbial community is used Banh men provides microorgan-isms necessary for breaking down of starch and subsequent fermenta-tion of the released monomers to ethanol (Lee and Fujio, 1999; Aidoo
et al., 2006; Thanh et al., 2008) Banh men is made from uncooked dough of rice, water, a variety of oriental herbs, and a small amount of the starter from previous batch By loopback inoculation, banh men is passed from generation to generation and is regarded as technological and cultural heritage
Due to economic and cultural importance, traditional alcoholic fer-mentation receives strong interest from scientific and technological communities Studies on traditional Vietnamese alcoholic fermentation could be categorized into two main groups: (i) microbiological charac-terization of banh men; and (ii) selection of active strains from banh men for process improvement Microbial composition of banh men is rel-atively well understood Typically, each gram of the starter contains
103–106CFU of mold, 106–107CFU of yeast and 103–106CFU of lactic acid bacteria (Lee and Fujio, 1999; Dung et al., 2007) Banh men contains
⁎ Corresponding author.
http://dx.doi.org/10.1016/j.ijfoodmicro.2016.05.024
Contents lists available atScienceDirect International Journal of Food Microbiology
j o u r n a l h o m e p a g e :w w w e l s e v i e r c o m / l o c a t e / i j f o o d m i c r o
Trang 2a complex and relatively stable microbiota, including Amylomyces rouxii,
Rhizopus microsporus, Mucor indicus, Mucor circinelloides,
Saccharomycopsis fibuligera, Hyphopichia burtonii, Saccharomyces
cerevisiae, Issatchenkia orientalis, Pichia anomala, Candida tropicalis,
Pichia ranongensis, Clavispora lusitaniae, Pediococcus pentosaceus,
Lacto-bacillus plantarum, LactoLacto-bacillus brevis, Weissella confusa, and Weissella
paramesenteroides (Lee and Fujio, 1999; Dung et al., 2007; Thanh et al.,
2008) Strains of A rouxii and S cerevisiae have been selected for the
production of defined granulated starters for controlled production of
rice wine (Dung et al., 2005) There is also a number of other attempts
to improve fermentation process that has been published locally in
the forms of technological reports
Until now, the implementation of modified processes has proven to
be difficult and commercial production of rice alcohol in Vietnam still
relies on the starter produced in the traditional way There is a major
gap in understanding of the role and behavior of individual species in
actual fermentation process Most often, strains are selected from banh
men based on amylase activity and alcohol production capability
How-ever, banh men production and alcoholic fermentation using banh men
are two independent processes and the populations thrived from the
first process may not be functioning in the second one In this article
we describe microbial succession and metabolite changes during the
course of rice fermentation using banh men Special attention was
de-voted to starch degradation and the group of amylase producers
2 Materials and methods
2.1 Rice fermentation
Rice fermentation was carried out in the laboratory using traditional
procedures Three fermentation experiments were conducted
separate-ly using one of the three starter samples collected at My Duc, Phuc Tho
and Son Tay districts of Hanoi, Vietnam Khang Dan rice variety was
cooked using a kitchen rice cooker For each fermentation experiment,
3 kg of cooked rice was sprinkled over with 60 g of pulverized banh
men, mixed well and dispensed on a sterile tray with a layer of ca
5 cm The tray was covered with a sterile cotton blanket and incubated
at 28 °C After 2 day of solid state fermentation, the fermentation mass
was transferred into a cylindrical ceramic jar and 3 litter of sterile
water was added The jar was closed with a ceramic lid and alcoholic
fermentation was carried out for 5 days at 28 °C Samples were collected
at different intervals (seeFig 2) for microbiological and biochemical
analyses In order to ensure uniformity, samples were collected at 3
dif-ferent positions and mixed before analysis For solid state fermentation
samples, an equal amount (by weight) of sterile distilled water was
added Data were presented as mean values obtained from three
experiments
2.2 Microbial isolation and enumeration
For microbial enumeration, serial dilutions of samples were made
using sterile physiological solution containing 0.9% NaCl For isolation
of yeast and fungi, the diluted samples were plated on malt-glucose
agar (malt, 10 g/L; glucose, 10 g/L; agar, 15 g/L; chloramphenicol,
100 mg/L) The plates were incubated at 28 °C for 5 days Fast growing
mucoraceous fungi were counted after 1–2 days, and yeast after
5 days For isolation of bacteria, plate count agar with bromocresol
pur-ple (yeast extract, 2.5 g/L; peptone, 5.0 g/L; glucose, 1.0 g/L; L-cysteine,
0.1 g/L; Tween-80, 1.0 g/L; bromocresol purple, 0.04 g/L; agar, 15 g/L)
was used The plates were incubated at 37 °C in a candle extinction jar
for 24 h Acidification of the medium was indicated by the formation
of yellow halos around colonies Microbial colonies with distinctive
ap-pearances were picked from each day of fermentation (8 points) and
with one representative per fermentation experiment (3 batches) The
isolates were purified and maintained in cultures
2.3 Identification of microorganisms For identification of microorganisms, the isolates were initially di-vided into genetic groups by microsatellite PCRfingerprinting and then the representatives from each groups were identified by rRNA gene sequencing The identification was extrapolated to the rest of the isolates of the same genetic group For DNA extraction, one loop-full of cells was transferred to a micro-tube containing 1 mL 2× SSC (15 mM sodium citrate, 150 mM NaCl, pH 7.0) and heated at 99 °C for 10 min using a PHMT Thermo-Shaker dry heater block (Grant-bio, England) Cells were collected by centrifugation at 10,000 g for 1 min To the cell pellet, ca 100μL glass beads (0.2–0.5 mm in diameter) (Roth, Germa-ny), 100μL phenol/chloroform and 150 μL water were added Cells were disrupted using a Mini-Beadbeater-8 (Biospec, USA) for 45 s The tube was centrifuged at 14,000 g for 10 min and the upper layer was transferred to a new micro-tube The DNA solution was further purified using a Silica Bead DNA Gel Extraction kit (Thermo Scientific) according
to the manufacturer's instructions
For microsatellite PCRfingerprinting, primer (GAC)5was used PCR was carried out in a C1000 Touch Thermal Cycler (BioRad, USA) using following thermal program: 94 °C for 3 min; 30 cycles of 94 °C for
40 s, 52 °C for 40 s and 72 °C for 1 min 30 s; 72 °C for 10 min PCR prod-ucts were separated in 1% agarose gel in ×0.5 TAE (20 mM Tris, 10 mM acetic acid, 0.5 mM EDTA) For identification of yeast, 26S rRNA gene (D1/D2) was amplified and sequenced using primer pair NL1
′-GGTCCGTGTTTCAAGACGG-3′) (Kurtzman and Robnett, 1998) For fungi, the ITS region was amplified and sequenced using primer pair
′-TCCTCCGCTTATTGATATGC-3′) (Esteve-Zarzoso et al., 1999) For identi-fication of bacteria, the 16S rRNA gene was amplified and sequenced using the universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) (Lane, 1991) The PCR conditions were as follows: 94 °C for 3 min; 30 cycles of 94 °C for
30 s, 52 °C for 40 s and 72 °C for 60 s; 72 °C for 10 min PCR products were sequenced using the service provided by Macrogen Inc (Korea) Species identification was based on the similarity of the obtained se-quences with the reference sese-quences at EzTaxon (http://www ezbiocloud.net/eztaxon) (Kim et al., 2012) and GenBank (http://www ncbi.nlm.nih.gov/nuccore/) databases
2.4 Chemical and biochemical analyses The pH of fermentation mash was measured using a SevenEasy bench-top pH meter (Mettler-Toledo, Austria) For determination of ethanol content, samples were centrifuged at 10,000 g for 10 min at
4 °C and solid content discarded Ethanol concentration was measured based on boiling temperature using a traditional ebulliometer (Dujardin-Salleron, France) For amylase extraction, to the sample an equal amount (by weight) of 100 mM sodium acetate buffer, pH 5.0 was added The mixture was shaken at 200 rpm in an orbital shaker for 1 h at 30 °C and then enzyme extract was obtained by centrifugation
at 10,000 g for 10 min at 4 °C For amylase assay, soluble starch (Sigma) was used as the substrate Enzymatic hydrolysis was carried out in
50 mM sodium acetate buffer, pH 5.0 at 50 °C for 20 min The amount
of reducing sugar produced was measured using Nelson-Somogyi method (Nelson, 1944; Somogyi, 1952) One unit of amylase was de-fined as the amount of the enzyme that produced 1 μM of reducing sugar (calculated as glucose) per minute
2.5 Zymography of enzyme extracts Zymography was performed based on conventional SDS-PAGE (Sambrook et al., 1989) using 10% polyacrylamide gel containing 1% starch Samples were loaded without the heat-treatment step After electrophoresis, the gel was rinsed twice in 2% Triton X-100 for
Trang 330 min at 4 °C Then, it was rinsed twice in 100 mM sodium acetate
buff-er, pH 5.0 for 15 min at 4 °C To perform starch hydrolysis reaction, the
gel was incubated in 100 mM sodium acetate buffer, pH 5.0 at 50 °C for
30 min For visualization, the gel was stained in 100 mL Lugol's solution
(I2, 5 g/L; KI, 10 g/L) Active amylase fractions appeared as transparent
bands on the dark-blue background
2.6 Influence of moisture content on amylase production
For preparation of rice substrates with different moisture contents,
rice (100 g) was soaked with different amounts (from 20 to 150 mL)
of distilled water in 1000 mL Erlenmeyerflasks for 2 h The flasks
were autoclaved (without post-autoclave drying) at 121 °C for 15 min
After cooling, pure or mixed cultures of Sm.fibuligera BMQ908 and R
microsporus BMF40 were inoculated The initial moisture contents
were determined using a KERN DBS (Kern & Sohn GmbH, Germany)
infra-red moisture meter After 48 h of solid state fermentation at 28 °C,
amylase was extracted and analyzed as described above
3 Results and discussion
3.1 Microorganisms associated with traditional alcoholic fermentation
In total, 130 microbial strains were isolated from different stages of
rice fermentation Amongst them, 41 were fungi, predominantly of
Mucoraceae, 16 yeast-like fungus Sm.fibuligera, 29 yeasts, mainly of S
cerevisiae, and 44 bacteria (seeTable 1) Grouping and identification a
larger number of strains based on morphological and physiological
characteristics are often difficult and time consuming Different species
may share similar phenotypic characteristics and strains of the same
species may show high variation depending on the culture age and
sex-ual activity By using microsatellite primed PCR we could successfully
differentiate a large number of strains A typical grouping of fungi
based on microsatellite PCR is presented inFig 1 The primer (GAC)5
also provided good resolution for yeasts and bacteria
The most frequent fungus was R microsporus, which accounted for
17 strains amongst 41 fungal strains identified, and followed by R
arrhizus (9 strains), M indicus (7 strains), M circinelloides (3 strains),
Cn elegans (3 strains) (seeTable 1) The fermentation mash nearly lacked of Aspergillus, Trichoderma, and Penicillum, the most common saprotrophic fungi in indoor environments (Haleem Khan and Mohan Karuppayil, 2012) There was a single strain of A niger isolated on the first day and most likely by chance Traditional Vietnamese alcoholic fermentation substantially differs from the fermentation of Japanese sake (Kitamoto, 2015) and Chinese maotai (Chen et al., 2014), where As-pergillus spp are the predominant This specificity might be due to high moisture content of the substrates used in Vietnamese alcoholic fer-mentation We found that the initial moisture contents of dough for starter preparation and cooked rice for alcoholic fermentation were in the range from 50% to 55% The condition would facilitate the growth
of mucoraceous fungi which are well adapted to the substrates with high moisture contents (Richardson, 2009) and are capable of growing
in microaerophilic and anaerobic environments (Hesseltine et al.,
1985)
With 6 species identified, highest fungal diversity was observed at the beginning of solid state fermentation and the diversity reduced as the fermentation progressed On the second day, only three species, namely R microsporus, R arrhizus, and M indicus were detected Strains
of R microsporus and R arrhizus are well known as strong amylase pro-ducers (Peixoto et al., 2003; Dung et al., 2006) M indicus is a dimorphic fungus that exhibits bothfilamentous and yeast growth M indicus is an
efficient ethanol producer and can accumulate up to 5% of ethanol when growing in glucose containing medium (Karimi and Zamani, 2013) M indicus was the only fungus detected in the fermentation broth till day
6 of 7 days of fermentation Strains of M indicus obtained in this study showed very low or no amylase activity when growing on rice substrate Although occupied second position in terms of the number of strains obtained in this study, R arrhizus is the best known fungus in banh men
R arrhizus (previously known as Amylomyces rouxii) wasfirst described
by Calmette in his comprehensive study on banh men dated back to
1892 (Calmette, 1892) Calmette considered the fungus as the major agent causing liquefaction of rice starch and developed a technology for alcohol production based on A rouxii and Saccharomyces strains (Calmette, 1895) The technology was utilized by the alcohol monopoly,
Table 1
Microorganisms isolated on different days during fermentation of rice using banh men starter.
Microbial species Number of isolates Day of isolation
Rhizopus microsporus (=R oligosporus) 17 + + + +
Rhizopus arrhizus (=Amylomyces rouxii, =R oryzae) 9 + +
Wickerhamomyces anomalus (=Pichia anomala) 2 + +
Trang 4the Société Des Distilleries de L'indochine for a large scale industrial
pro-duction in colonial Vietnam from 1897 to 1945 (Peters, 2012) A rouxii
was also utilized for upgrading of banh men for production of rice wine
from purple glutinous rice (Dung et al., 2006) The name A rouxiii
rep-resents a special phenotype of R arrhizus that widely occurs in Asian
amylolytic starters It is characterized by rapid growth, intensive
forma-tion of large chlamydospores, and the lack of black pigmented sporangia
(Hesseltine, 1983) A rouxiii itself is a heterogeneous group A rouxiii
strains isolated from banh men produce malic acid but not lactic acid
while strains from look-pang and ragi-tape produce majorly lactic acid
(Kito et al., 2009) The separation of the two groups is supported by
RFLP and DNA sequencing data Two varieties, R arrhizus var delemar
for lactic acid negative strains from banh men and R arrhizus var
arrhizus for lactic acid producing strains from look-pang and ragi-tape,
have been proposed (Kito et al., 2009; Dolatabadi et al., 2014a) In this
study, we have found two genetic groups of R arrhizus (seeFig 1)
Similar to the case of R arrhizus, the domesticated form of R
microsporus having reduced sporulation activity was treated as a
sepa-rate species R oligosporus or a variety (R microsporus var oligosporus)
However, this variety along with others (azygosporus, chinensis,
oligosporus, microsporus, rhizopodiformis and tuberosus) are not
support-ed genetically by matting compatibility and sequencing data of ITS, ACT,
1-α (TEF) genes (Dolatabadi et al., 2014b) Strains of R microsporus can
produce rhizoxins, a potent antimitotic macrocyclic polyketide toxins It
was revealed that the fungus cannot produce toxin by itself and the
toxin is produced by bacteria of the genus Burkholderia that live inside
the fungal cells (Partida-Martinez et al., 2007) Alarming was the fact
that the bacteria have been found in R microsporus CBS 111563, a strain
originally isolated from banh men in Vietnam (Lackner et al., 2009) When the strain was tested for sufu and tempe production, considerable amounts of rhizoxins could be detected in thefinal products (Rohm et al., 2010) It is not clear about the extent of toxigenic phenotype amongst R microsporus populations associated with traditional Viet-namese alcoholic fermentation
Beside amylolytic mucoraceous fungi, Sm.fibuligera, an active amy-lase producer was also found in high density (up to 8.2 log CFU/g) dur-ing traditional Vietnamese alcoholic fermentation (seeTable 1andFig
2) Sm.fibuligera produces extracellular α-amylase, glucoamylase and raw starch digesting glucoamylase (Chi et al., 2009) In culture medium,
Sm.fibuligera produces extracellular C14-C182-D-hydroxy fatty acids in the form of needle shaped crystals (Kurtzman et al., 1973) Sm.fibuligera strains obtained in this study also exhibited similar phenotype There is
no information on the possible effect of these compounds on microbial community and organoleptic quality of the fermentation products, al-though it seems quite obvious.Kurtzman et al (1973)informed that the compounds had little or no antibacterial activity However, in the study, the target microorganisms were restricted to common enteric pathogens (Kurtzman et al., 1973)
Amongst the fermentative yeasts isolated during rice fermentation,
S cerevisiae was the most prevalent The yeast occurred in all samples from beginning to the end of fermentation On the last day of fermenta-tion, beside bacteria, S cerevisiae existed practically as a pure culture (seeTable 1andFig 2) The presence of S cerevisiae populations in Asian amylolytic fermentation is not a result of spontaneous introduc-tion from environment but a consequence of long term domesticaintroduc-tion Recent phylogenetic analysis based on multi-gene sequencing data
Fig 1 Typical fingerprints generated by micro-satellite PCR of fungi associated with traditional alcoholic fermentation The lanes were manually rearranged Number in boxes indicated strains identified by DNA sequencing M - 1 kb DNA ladder (Fermentas).
Fig 2 Microbiological (A) and biochemical (B) parameters during the course of rice fermentation using banh men starter Error bar represents differences between thee independent
Trang 5indicated that S cerevisiae strains obtained from Japanese sake, Chinese
rice wine and Indonesian ragi represent a well-supported clade and
dis-tant from natural populations (Wang et al., 2012; Li et al., 2014)
Yeasts W anomalus (= P anomala), Pichia kudriavzevii
(=Issatchenkia orientalis), C lusitaniae were isolated sporadically at
dif-ferent days of fermentation (Table 1) They have also been reported to
occur in banh men (Lee and Fujio, 1999; Dung et al., 2007; Thanh et
al., 2008) W anomalus is a regular component in several types of
Asian alcoholic fermentation starters (Haard et al., 1999; Limtong et
al., 2002) The yeast can accumulate up to 5% (w/v) ethanol in broth
cul-ture (Limtong et al., 2002) W anomalus strains are known to produce
killer toxin, a glycoprotein that inhibits the growth of other yeasts
(Schneider et al., 2012) W anomalus produces a spectrum of small
vol-atile compounds, such as ethyl acetate, ethyl propanoate, phenyl
etha-nol and 2-phenylethyl acetate (Passoth et al., 2006) These volatile
compounds might contribute to the aroma of rice liquor
Bacteria of banh men received little attention and were often
regarded as contaminant and unwanted (Haard et al., 1999) In this
study, a rather high count (up to 8.8 log CFU/g) of bacteria was observed
in the fermentation mash (seeFig 2) Following species were identified:
Stenotrophomonas maltophilia, L brevis, L helveticus, Acinetobacter
baumannii, Staphylococcus hominis, Bacillus megaterium, Enterobacter
asburiae, and P pentosaceus (seeTable 1)
Fungal and yeast composition was consistent and all three
fermenta-tion batches shared the same dominating species (R microsporus, R
arrhizus, M indicus, Sm.fibuligera, and S cerevisiae) Meanwhile, the
bac-terial composition was inconsistent Sten maltophilia dominated in one
fermentation batch but was not detected in the other two Similar
find-ing was reported for the microbial composition of banh men (Thanh et
al., 2008) Taken that bacteria present in high cell density but with
un-stable species composition, controlling of bacterial microbiota might
help in improving quality consistency of traditional Vietnamese
alcohol-ic fermentation product
3.2 Microbial succession during traditional alcoholic fermentation
Microbial succession and changes in biochemical parameters during
the course of traditional alcoholic fermentation is presented inFig 2
During thefirst 24 h, the pH of the fermentation mash rapidly dropped
from 6.5 to 3.5 and this coincided with the growth of acid producing
bacteria and mucoraceous fungi After 24 h of fermentation, the
bacteri-al count reached 8.7 log CFU/g, meanwhile the count for mucoraceous fungi reached 5.6 log CFU/g Beside lactic acid bacteria (L brevis, L helveticus, Staph hominis, Pd pentosaceus), other bacterial species de-tected (Sten maltophilia, A baumannii, and E asburiae) are also known
to produce acid when growing on sugar substrates (Brenner et al., 1986; Juhnke and des Jardin, 1989; Constantiniu et al., 2004) R microsporus and R arrhizus, the major mucoraceous fungi during the onset of fermentation can produce fumaric, malic and lactic acids (Kitpreechavanich et al., 2008; Kito et al., 2009) It should be noted that quantification of mucoraceous fungi based on colony forming unit may be bias From our observation, in the fermentation mash, Rhizopus formed extensivefilamentous growth with hyphae penetrating deep in-side the rice grains This pattern of growth would be difficult to quantify The comparative contributions of bacteria and mucoraceous fungi in the acidification of fermentation mash, thus remained unclear Acidic condi-tion would inhibit the growth of spoilage bacteria and facilitate the growth of yeast and fungi
Rapid changes in microbial composition observed during fermenta-tion correlated with ethanol accumulafermenta-tion (Fig 2) On thefirst day of solid state fermentation, non-Saccharomyces dominated amongst yeast populations Yeast growth peaked at 60 h, both in terms of cell density (8.9 log CFU/g) and diversity At that point, ethanol concentration was 2.2% (v/v) Soon after, the diversity rapidly decreased and S cerevisiae thrived to be the dominant At 78 h, ethanol concentration reached 4.9% (v/v) only two types of yeasts were detected At 120 h, when eth-anol concentration was 7.6% (v/v), S cerevisiae existed practically as a pure culture
Starting with 6 fungal species, mainly the ones associated with banh men, fungal growth reached maximum after 2 days and only 3 species (R microsporus, R arrhizus, and M indicus) thrived Upon addition of water, fermentation turned into alcohol production phase, species of Rhizopus died out but M indicus could still grow until the near end of fermentation (144 h, 8.9% ethanol) Similarly, Sm.fibuligera achieved maximum density during solid state fermentation and died out after
96 h (6.9% ethanol) Bacteria reached maximal density and diversity after 24 h of fermentation, roughly a day earlier than yeast and fungi Al-though reduced in diversity, bacterial populations maintained high cell density until the end of fermentation (seeFig 2,Table 1)
During thefirst 24 h of solid state fermentation, amylase activity and concentration of free sugar were low (seeFig 2) At this stage, microbial growth perhaps was supported by the ready available nutrients present
in cooked rice During the next 24 h, rapid increase in amylase activity was observed The rate of starch degradation surpassed sugar consump-tion and led to the accumulaconsump-tion of free sugars which peaked at 7.5% at the end of solid state fermentation At this moment, ethanol fermenta-tion still did not start although yeast density reached maximum Upon
Fig 4 Zymography of amylases extracted from pure culture of R arrhizus BMF4, Sm fibuligera BMQ908, banh men starter, and fermentation mash samples collected at Fig 3 Mobility in starch containing gel of amylases extracted from pure cultures of
mucoraceous fungi and Sm fibuligera grown on rice.
Trang 6addition of water, ethanol fermentation took place, the concentration of
free sugar decreased rapidly although amylase activity was still on the
rise for another 12 h During 5 days of ethanol fermentation, yeast
remained in high density but shifted from multispecies to practically
pure culture of S cerevisiae Amylase activity decreased after 72 h of
fer-mentation and stabilized at low level At the end of ferfer-mentation,
etha-nol concentration reached an average value of 9.3% (v/v) and the mash
exhausted in free sugar
3.3 Zymography and the origin of amylases in fermentation mash
The correlation of amylase activity in the fermentation mash with
the growth of mucoraceous fungi and Sm.fibuligera (seeFig 2) has
prompted the question concerning the contribution of each group in
starch degradation There are compelling opinions regarding the role
of Sm fibuligera and Rhizopus spp in saccharification of starch
(Uchimura et al., 1990; Limtong et al., 2002; Dung et al., 2006) We
de-cided to trace the origin of amylase in fermentation mash by comparing
the electrophoretic mobility of the amylase extracted from
fermenta-tion mash with the ones produced by pure cultures R microsporus, R
arrhizus, M indicus, M circinelloides, Cn elegans, and Sm.fibuligera
strains were grown in cooked rice Except M indicus, all tested strains
demonstrated amylase activity at different extents Amylases produced
by mucoraceous fungi had low electrophoretic mobility and could be
differentiated from the ones produced by Sm.fibuligera (seeFig 3)
When comparing the amylase extracted from the starter (banh men)
with amylases produced by pure cultures, it become clear that banh
men contained both amylases from mucoraceous fungi and Sm
fibuligera (Fig 4) It was surprising that although Sm.fibuligera
dominat-ed amongst amylase producers (0.8 to 1.9 log CFU higher) in the
fer-mentation mash (see Fig 2), amylase of Sm fibuligera was not
detected throughout the fermentation process (seeFig 4) Instead,
am-ylase with mobility similar to that of Rhizopus was the main component
Light bands presumably of Cn elegans were also observed In order to
verify the consistency of the phenomenon, fermentation test was
re-peated using 8 additional starters Also, 9 fermentation samples from a
traditional alcohol producer (Van village, Bac Ninh province) were
col-lected and analyzed The results were similar, i.e amylase of Sm
fibuligera was detected in the starters but not in fermentation mashes
(data not shown) Thus, it could be concluded that, although present
in high density during solid state fermentation, M indicus and Sm
fibuligera play little or no role in starch degradation and Rhizopus is
the main amylase producer Due to similar electrophoretic mobility, it
was not possible to discriminate between amylases produced by R microsporus and R arrhizus
3.4 Substrate moisture content as regulating factor for amylase production
in co-culture The fact that amylase of Sm.fibuligera presents in the starter but not
in the fermentation mash has prompted the question concerning the regulation of amylase production in Sm.fibuligera Since banh men is rel-atively drier comparing with the fermentation mash, it was hypothe-sized that moisture content could play a regulating role Sm.fibuligera BMQ908 and R microsporus BMF40 were grown in pure and mixed cul-tures on cooked rice with different moisture contents (seeFig 5) and amylases were extracted It was shown that Sm.fibuligera BMQ908 and R microsporus BMF40 produced amylase at all tested moisture con-tents but most optimum at 29–38% (lanes 2, 3, 4) In mixed culture, while R microsporus BMF40 maintained the same amylase production pattern, Sm.fibuligera BMQ908 ceased to produce amylase when the moisture content was equal or above 34% Taken that cooked rice used
in traditional Vietnamese alcoholic fermentation has the moisture con-tent in the range from 50% to 55%, the absence of Sm.fibuligera's amylase
in fermentation mash is thus understandable It is most likely that free sugar released by the action of Rhizopus amylase repressed amylase pro-duction in Sm.fibuligera, and such interaction is facilitated in the sub-strates with high moisture contents
Thus, despite the presence of a rather complex microbiota in the starter, traditional Vietnamese alcoholic fermentation is a stable process that involves a limited number of functional species The role of satellite microorganisms, however should not be overlooked since they may contribute to the organoleptic quality offinal product Surprising behav-ior of Sm.fibuligera has highlighted the need for understanding the sys-tem in interaction Since banh men is similar to Chinese yeast ball, murcha (India, Nepal), bubod (Philippines), loog-pang (Thailand), ragi (Indonesia, Malaysia) in terms of microbiological composition and method of application (Hesseltine et al., 1988), what described here for traditional Vietnamese alcoholic fermentation might also be relevant
to other processes
Acknowledgements
We thank Truong A Tai and Tran Thi Thom for valuable assistance This study was supported by Grant from The National Foundation for Science and Technology Development (NAFOSTED) #106.03.146.09
Fig 5 Influence of moisture content on the production of amylases by pure and mixed cultures of Sm fibuligera BMQ908 and R microsporus BMF40 grown on rice substrate Amylase was extracted after 48 h of fermentation Lanes 1 to 7 are substrates with increasing moisture contents: 1–23%; 2–29%; 3–34%; 4–38%; 5–48%; 6–55%; 7–60%.
Trang 7Appendix A Supplementary data
Supplementary data to this article can be found online athttp://dx
doi.org/10.1016/j.ijfoodmicro.2016.05.024
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