Evaluation of bacterial flora during the ripening of Kedong sufu, a typical Chinese traditional bacteria-fermented soybean product

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Research ArticleReceived: 20 May 2012 Revised: 31 July 2012 Accepted article published: 11 September 2012 Published online in Wiley Online Library: 15 October 2012

(wileyonlinelibrary.com) DOI 10.1002/jsfa.5918

Evaluation of bacterial flora duringthe ripening of Kedong sufu, a typical Chinesetraditional bacteria-fermented soybeanproductZhen Feng,a∗ Wei Gao,b Dan Ren,a Xi Chena and Juan-juan Lia

Abstract

BACKGROUND: Kedong sufu is a typical bacteria-fermented sufu in China. Isolation and identification of the autochthonousbacteria involved would allow the design of specific starters for this speciality. The purpose of the present study was to evaluatethe bacterial flora during the ripening of Kedong sufu using polymerase chain reaction denaturing gradient gel electrophoresis(PCR-DGGE) and culturing.

RESULTS: In terms of bacterial diversity, 22 strains were isolated and identified and 27 strains were detected by DGGE. Regardingbacterial dynamics, the results of culturing and PCR-DGGE exhibited a similar trend towards dominant strains. Throughout thefermentation of sufu, Enterococcus avium, Enterococcus faecalis and Staphylococcus carnosus were the dominant microflora,while the secondary microflora comprised Leuconostoc mesenteroides, Staphylococcus saprophyticus, Streptococcus lutetiensis,Kocuria rosea, Kocuria kristinae, Bacillus pumilus, Bacillus cereus and Bacillus subtilis.

CONCLUSION: This study is the first to reveal the bacterial flora during the ripening of Kedong sufu using both culture-dependentand culture-independent methods. This information will help in the design of autochthonous starter cultures for the productionof Kedong sufu with desirable characteristic sensory profiles and shorter ripening times.c© 2012 Society of Chemical Industry

Keywords: Kedong sufu; denaturing gradient gel electrophoresis (DGGE); bacteria; flora

INTRODUCTIONSufu is a traditional fermented soybean product in China. It is asoft, creamy, cheese-like product made from cubes of soybeancurd (tofu) by microbial action. Sufu can be used in the same wayas cheese.1 This fermented product has a characteristic flavourand texture and has been widely consumed by Chinese people asan appetiser for many centuries. There are many different types ofsufu, which are produced by various processes in different localitiesin China. On the basis of colour and flavour, sufu can be classifiedinto four types, i.e. red sufu, white sufu, grey sufu and other types,which are mainly attributable to different ingredients of dressingmixtures during ripening. On the basis of the starter culture used,sufu can be classified into three types, i.e. bacteria-fermentedsufu (Bacillus spp. or Micrococcus spp.), mould-fermented sufu(Actinomucor elegans, Mucor flavus or Mucor racemosus) and othertypes (moulds, bacteria and yeasts). Kedong sufu is a typicalbacteria-fermented sufu in China.2 The ripening stage of Kedongsufu takes more than 6 months including pre-fermentation andpost-fermentation.

Investigation of the microbial typing of fermented foods may

aid in the design of specific starter and/or adjunct cultures.3–5

Compared with traditional spontaneous fermentation, the useof starter cultures for fermented food production is becomingincreasingly necessary in order to standardise the product’s

properties to obtain a consistent flavour and texture, a standardcolour and a shorter ripening time.6,7 A better understanding ofthese characteristics can be obtained by studying the diversityand dynamics of the microbial community involved in thefermentation process. Most previous studies have focused on

WangZhiHe sufu (a typical mould-fermented sufu).8–11 Moreover,to the best of our knowledge, the microbial flora of Kedong sufu ispoorly understood and no evaluation of the microbial flora duringripening has been reported. The lack of information about Kedongsufu microecology has become a bottleneck in the developmentof specific starter and/or adjunct cultures to obtain a consistentflavour and texture, a standard colour and a shorter ripening time.

The traditional plate count method is useful for monitoringthe viable count of only known and cultured micro-organismsin a sample. On the other hand, the denaturing gradient gelelectrophoresis (DGGE) method is able to monitor the population

∗ Corresponding author: Zhen Feng, Fax: +86-451-55190577, Tel: +86-451-55190459 E-mail address: [email protected]

a College of Food Science, Northeast Agricultural University, 59 Mucai Road,150030, Harbin, Heilongjiang, China

b College of Food Science and Technology, Harbin Institute of Technology, 202Haihe Road, 150090, Harbin, Heilongjiang, China

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www.soci.org Z Feng et al.

Figure 1. Schematic diagram of sufu production process: A, tofu curd; B, sufu samples of pre-fermentation; C, sufu samples of post-fermentation.

dynamics of constituents of a microbiota including unculturedor/and unknown micro-organisms. The two methods have beenused as a complementary approach for the ecological assessmentof fermented foods.12 In order to obtain a detailed overviewof the microbial populations involved in ripening, combinationsof culture-dependent and culture-independent approaches are

typically applied.13–15

The purpose of this study was to evaluate the microbial floraassociated with the process of Kedong sufu fermentation usinga combination of conventional culturing and polymerase chainreaction (PCR) DGGE methods. The overall aim was to lay afoundation for the selection of autochthonous starter cultures. Tothe best of our knowledge, this is the first report to investigateextensively the bacterial flora in Kedong sufu by both culture-dependent and culture-independent methods.

MATERIALS AND METHODSKedong sufu samplingTraditional fermentation technology was used to produce sufuaccording to the methods of Wang et al.2 at Kedong Sufu Co. Ltd(Kedong, Heilongjiang, China). A schematic diagram of the sufuproduction process is shown in Fig. 1. Seven pre-fermentationsamples (collected after culture for 1, 2, 3, 4, 5, 6 and 7 days) and 19post-fermentation samples (collected after culture for 10, 15, 20,25, 30, 37, 44, 51, 58, 68, 78, 88, 98, 108, 118, 128, 143, 158 and 173days) were used in the study. Nine samples from three differentbatches were taken at the corresponding times of each stage ofripening.

Physicochemical analysesThe pH of sufu was monitored with a Beckman 72 pH meter(Beckman Instruments Inc., Fullerton, CA, USA). Salt (NaCl) con-centration was monitored with a Corning 926 chloride analyser

(Corning, Halstead, UK). Titratable acidity was determined bytitration with 0.1 moL L−1 NaOH. Water activity was measured witha Pawkit water activity meter (Decagon Devices, Pullman, WA,USA). The concentration of total free amino acids was monitoredwith a Beckman 6300 high-performance amino acid analyser(Beckman Instruments Ltd, High Wycombe, UK).

Isolation and enumeration of bacteriaSamples (25 g) were transferred to individual sterile plastic bagsand homogenised in 225 mL of sterile saline solution. Appropriatedecimal dilutions of the homogenates were spread over plates oftryptic soy broth (TSB) agar (Becton, Dickinson and Co., Sparks,MD, USA) and incubated at 30 ◦C for 48–72 h. After enumeration,colonies of different morphology were selected at random from theTSB plates according to the Harrison disc method.16 The isolateswere phenotyped based on colony morphology and colour usinga SZ-ST stereo zoom microscope (Olympus, Tokyo, Japan), cellmorphology using a BX61 phase contrast microscope (Olympus),and Gram and catalase reactions. The isolates were purified bystreaking on TSB agar.

The Harrison disc method was adopted from Harrigan.17 Thismethod was used to determine the prevalent microbes thatdeveloped at each dilution and to select representative coloniesfrom each plate in a random manner for further purification andidentification. The Harrison disc method is able to calculate thedistribution of various micro-organisms present in a sample.

Identification of pure culturesFor the extraction of DNA from pure cultures, each culture (2 mL)was collected by centrifugation (12 000 × g, 3 min, 4 ◦C). DNAwas extracted from the bacterial pellet using a TIANamp BacteriaDNA Kit (Tiangen Biotech Co., Beijing, China) according to themanufacturer’s instructions. The isolated DNA was resuspended

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Evaluation of bacterial flora in Kedong sufu www.soci.org

in sterile distilled and deionised water (dd-H2O) and stored at −20◦C. The identity of strains was confirmed with 16S rDNA sequencedata. The 16S rDNA gene was amplified using the forward primer27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and the reverse primer1512R (5′-ACGGCTACCTTGTTACGACT-3′). PCR amplification wasperformed using a GenAmp PCR System 9700 (Perkin-Elmer,Foster City, CA, USA). PCR was carried out in a total reactionvolume of 50 µL containing 25 µL of 2× Taq PCR MasterMix(Tiangen Biotech Co.), 0.5 µL of each primer, 3 µL of templateDNA and 21 µL of sterile dd-H2O. The PCR protocol was inaccordance with conventional PCR conditions. Sequencing ofthe 16S rDNA fragment was performed by BGI (Beijing, China).Sequence similarity was analysed with a BLAST search of theGenBank database (http://www.ncbi.nlm.nih.gov/BLAST/).

Extraction of total DNA from Kedong sufu samplesSamples (25 g) were transferred to individual sterile plastic bagsand homogenised in 225 mL of sterile saline solution. To digestthe protein aggregates, 3 mL of 100 g L−1 pronase solution(Sigma Chemical Co., St Louis, MO, USA) and 300 µL of β-mercaptoethanol were added and samples were incubated at37 ◦C for 3 h. Cells were separated by centrifugation (14 000 × g,5 min) and washed twice with buffer (10 mmol L−1 Tris–HCl, 10mmol L−1 ethylenediaminetetraacetic acid, 50 mmol L−1 NaCl, pH8). A QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) wasused to extract bacterial DNA from 700 mg of the bacterial pelletaccording to the manufacturer’s protocol. The yield and qualityof DNA were analysed electrophoretically (Mupid-21, Advance,Tokyo, Japan) on 10 g L−1 agarose gel. The extracted DNA wasused as a template for PCR to amplify the V3 region of the 16SrRNA gene.

PCR-DGGE analysisFor the DGGE analysis of bacterial communities, theprimer set 338F (5′-ACTCCTACGGGAGGCAGCAG-3′)/518R(5′-ATTACCGCGGCTGCTGG-3′) was used to amplify the V3 regionof the 16S rDNA. The GC-338F primer has a GC clamp (5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGAGGGG-3′)attached to the 5′ end of the primer 338F. PCR was performed ina total reaction volume of 50 µL containing 0.5 µL of each primer,3 µL of template DNA, 21 µL of dd-H2O and 25 µL of 2× Taq PCRMasterMix (Tiangen Biotech Co.). Touchdown PCR was carried outwith the GC-338F/518R primer set to increase the specificity of theamplification and reduce the formation of spurious by-products.The PCR products were generated using an initial denaturationstep of 5 min at 94 ◦C, followed by denaturation at 94 ◦C for 30s. The annealing temperature of 65 ◦C for 30 s was decreasedby 1 ◦C during each of the successive cycles until a touchdowntemperature of 55 ◦C was reached, and the remaining 25 cycleswere accomplished at 55 ◦C for 1 min. The elongation step wasconducted at 72 ◦C for 3 min. A final chain extension at 72 ◦Cfor 10 min was done. PCR amplification was performed usinga GenAmp PCR System 9700 (Perkin-Elmer). Amplified productswere run on 20 g L−1 agarose gel, stained with ethidium bromide(Fluka-Riedel-de Haen, Basel, Switzerland) and visualised underUV light to assess the quality of the amplification.

DGGE analysis of PCR amplicons was performed on a DcodeSystem apparatus (BioRad, Hercules, CA, USA) using the methodof Muyzer and Smalla.18 The gel was stained with AgNO3 anddeveloped after completion of electrophoresis. Different DGGEbands were punched out from the acrylamide gels. The DNA

fragments were purified using an E.Z.N.A. Cycle-Pure Kit (OmegaBio-Tek, Doraville, GA, USA) according to the manufacturer’srecommendations. The purified products were used directlyfor reamplification of the PCR products with 338F and 518R.Sequencing was performed by BGI. Sequence identity wasdetermined by a BLAST nucleotide search of the GenBankdatabase (http://www.ncbi.nlm.nih.gov/BLAST/). Sequences with97% or higher identity were considered to represent the samespecies.

RESULTSPhysicochemical parametersChanges in physicochemical parameters during fermentation ofKedong sufu are reported in Table 1. Salt concentration wasmoderately high, between 64 and 78 g kg−1. Water activity wasin the range 0.88–0.77. pH increased from 6.59 to 7.54 duringpre-fermentation and decreased from 7.29 to 6.09 during post-fermentation. Titratable acidity decreased from 1.86 to 1.47 g kg−1

during pre-fermentation and increased from 1.58 to 6.25 g kg−1

during post-fermentation. Total free amino acids showed a signifi-cant increase throughout fermentation, from 4.77 to 108.97 g kg−1.

Analysis of bacterial flora by culture methodThe TSB agar used in this study isolated fermenting microbes andenabled visualisation of distinct colony morphologies among theisolates. A total of 132 isolates associated with the fermentationprocess were selected according to colony colour and morphologyand cell morphology, most of which were Gram-positive andcatalase-positive. These isolates were subjected to 16S rRNAsequence-based identification, which revealed that 22 isolates(P1–P22) were different. Identification results of the 16S rDNAsequences of the 22 isolates are presented in Table 2. The colonymorphology of the 22 isolates is shown in Fig. 2. Samples of Kedongsufu from different stages of ripening showed huge diversityin colony morphology. The diversity of colony morphologyprovides an advantageous condition for analysis of the microbialdynamics of sufu by the Harrison disc and colony-countingmethods.

The populations of total bacteria in sufu were enumeratedusing TSB agar media. The initial number of bacteria was 6.63 logcolony-forming units (CFU) g−1, which increased to 7.97 log CFUg−1 after 7 days of ripening. However, the number of bacteriapresent decreased significantly to 5.83 log CFU g−1 after 15 daysof ripening. The total bacterial count decreased to 3.96 log CFUg−1 after 173 days of ripening (data not shown). The Harrison discmethod used for random statistical selection of representativecolonies allowed for the calculation of the percentage distributionof the bacteria found in sufu samples. Figure 3 depicts thepercentage of the prevalent bacterial population present atdifferent stages during sufu ripening. The sufu pre-fermentation(1–7 days) was governed by the distinct population dynamics ofsix strains, namely Enterococcus faecium, Streptococcus lutetiensis,Staphylococcus saprophyticus, Kocuria kristinae, Kocuria rosea andLeuconostoc mesenteroides. Enterococcus faecium accounted for18% of the total isolates, making it the most dominant bacterialspecies, followed by K. kristinae (17%), S. saprophyticus (15%), L.mesenteroides (13%), S. lutetiensis (13%) and K. rosea (11%). Amongthem, increases and decreases in the percentage of bacterialcounts were not obvious. The sufu post-fermentation (10–173days) was governed by the distinct population dynamics of

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Table 1. Physicochemical changes during fermentation of Kedong sufu

Fermentation time

(days) Water activity

Titratable acidity

(g kg−1) pH

Salt concentration

(g kg−1)

Total free amino acids

(g kg−1)

1 0.86 1.86 6.59 68 4.77

3 0.87 1.77 6.76 65 11.57

5 0.88 1.59 7.15 64 18.97

7 0.88 1.47 7.54 66 25.46

10 0.78 1.58 7.29 72 22.85

20 0.78 1.76 6.98 74 38.92

30 0.79 2.29 6.32 73 47.35

44 0.80 3.66 6.03 75 59.62

58 0.81 4.87 6.09 76 72.99

68 0.82 5.28 5.96 73 73.87

78 0.81 4.97 6.24 75 74.15

88 0.79 5.12 6.18 77 74.20

98 0.78 5.18 6.11 77 74.76

108 0.79 5.22 6.07 76 75.97

118 0.77 5.29 6.12 75 76.90

128 0.79 5.31 6.14 77 82.60

143 0.80 5.86 6.27 78 87.34

158 0.81 6.14 6.35 76 94.93

173 0.81 6.25 6.09 74 108.97

Table 2. Molecular similarity and identity of 16S rDNA sequences of bacterial strains isolated from Kedong sufu by culturing

Representative isolate Type strain Similarity (%) Identification

P1 Enterococcus faecium LMG 11423T (AJ301830) 99.6 Enterococcus faecium

P2 Enterococcus durans DSM 20633T (AJ276354) 99.5 Enterococcus durans

P3 Enterococcus canintestini LMG 13590T (AJ888906) 100 Enterococcus canintestini

P4 Streptococcus lutetiensis NEM 782T (AJ297215) 99.9 Streptococcus lutetiensis

P5 Staphylococcus gallinarum ATCC 35539T (D83366) 100 Staphylococcus gallinarum

P6 Staphylococcus sciuri DSM 20345T (AJ421446) 100 Staphylococcus sciuri

P7 Staphylococcus carnosus ATCC 51365T (AB009934) 99.7 Staphylococcus carnosus

P8 Staphylococcus saprophyticus ATCC 15305T (AP008934) 99.9 Staphylococcus saprophyticus

P9 Kocuria kristinae DSM 20032T (X80749) 99.6 Kocuria kristinae

P10 Kocuria rosea DSM 20447T (X87756) 99.5 Kocuria rosea

P11 Leuconostoc mesenteroides ATCC 8293T (CP000414) 100 Leuconostoc mesenteroides

P12 Marinilactibacillus psychrotolerans M13-2T (AB083406) 100 Marinilactibacillus psychrotolerans

P13 Aerococcus viridans ATCC 11563T (M58797) 99.9 Aerococcus viridans

P14 Ornithinibacillus bavariensis WSBC 24001T (Y13066) 99.0 Ornithinibacillus bavariensis

P15 Corynebacterium variabile DSM 20132T (AJ222815) 99.9 Corynebacterium variabile

P16 Corynebacterium stationis ATCC 14403T (FJ172667) 99.4 Corynebacterium stationis

P17 Bacillus cereus ATCC 14579T (AE016877) 99.4 Bacillus cereus

P18 Bacillus amyloliquefaciens DSM 7T (FN597644) 99.4 Bacillus amyloliquefaciens

P19 Bacillus subtilis DSM 10T (AJ276351) 99.9 Bacillus subtilis

P20 Bacillus megaterium IAM 13418T (D16273) 98.2 Bacillus megaterium

P21 Bacillus tequilensis 10bT (HQ223107) 98.5 Bacillus tequilensis

P22 Proteus mirabilis NCTC 11938T (DQ885256) 99.8 Proteus mirabilis

five strains, namely Enterococcus durans, Staphylococcus carnosus,Bacillus cereus, Bacillus subtilis and Proteus mirabilis. Among them,increases and decreases in the percentage of bacterial countswere obvious. Staphylococcus carnosus (12%) and P. mirabilis(10%) dominated in the initial stages of post-fermentation, butthe proportions of E. durans, B. cereus and B. subtilis increased asfermentation progressed. After 51 days, E. durans (12%), B. cereus

(10%) and B. subtilis (13%) also became predominant bacterialgroups in the microbial community.

Analysis of bacterial flora by DGGE methodThe DGGE separation patterns of PCR-amplified V3-16S rDNAsegments derived from Kedong sufu samples cultured for 1–173days are shown in Fig. 4. The identity of selected DGGE bands from


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Figure 2. Morphology of different colonies of bacterial groups associated with Kedong sufu fermentation.

0%10%20%30%40%50%60%70%80%90%

100%

1 2 3 4 5 6 7 10 15 20 25 30 37 44 51 58 68 78 88 98 108 118 128 143 158 173

Days of fermentation

% I

sola

tion

P2 P7 P17 P19 P22 P1 P 4 P8 P9 P10 P11 Other strains

Figure 3. Dynamic distribution results for micro-organisms isolated from TSB agar during different stages of Kedong sufu production. The results arerepresentative of a typical experiment of three different batches samples.

the bacterial DGGE fingerprint is shown in Table 3. High microbialdiversity from the beginning to the end of the fermentationprocess was observed, which was illustrated by the presence ofmultiple bands (Figs 4A and 4B). During the pre-fermentationstage, which ranged from day 1 to day 7 of the ripening process(Fig. 4A, lanes 1–7), 22 bands were present in DGGE. During thepost-fermentation of Kedong sufu, which ranged from day 10 today 173 of the ripening process (Figs 4A and 4B, lanes 10–173), 25bands were present in DGGE. A total of 27 strains were detectedby DGGE throughout fermentation (Table 3).

Variability in the dominance of microbial species, which wasassessed by the intensity of bands in DGGE, was detected. Thebands A11, A12, A18, A27 and A28 showed high intensity duringpre-fermentation. This indicated that S. saprophyticus, S. lutetiensis,L. mesenteroides, K. rosea and K. kristinae were dominant bacterialspecies during pre-fermentation. The bands A4 (or B4), A8 (orB8), A14 (or B16), A15 (or B17) and B14 showed high intensityduring post-fermentation. This indicated that E. durans, B. pumilus,

S. carnosus, B. subtilis and B. cereus were dominant bacterial speciesduring post-fermentation. The bands A4 (or B4), A5 (or B5) andA14 (or B16) were detected almost throughout fermentation andshowed different intensities at different stages. This indicatedthat E. durans, E. faecalis and S. carnosus were dominant bacterialspecies throughout fermentation. The other bands were detectedonly for short periods during the ripening of Kedong sufu.

The profiles obtained by the culture-independent DGGE methodgenerally agreed with the results obtained by the culture-dependent method in terms of dominant strains. In general, duringpre-fermentation of sufu the dominant strains were E. faecalis,L. mesenteroides, S. saprophyticus, S. lutetiensis, K. rosea andK. kristinae. During post-fermentation of sufu the dominant strainswere E. durans, B. pumilus, B. cereus, B. subtilis and S. carnosus.Throughout fermentation of sufu the dominant strains wereE. durans, E. faecalis and S. carnosus and the subdominant strainswere L. mesenteroides, S. saprophyticus, S. lutetiensis, K. rosea,K. kristinae, B. pumilus, B. cereus and B. subtilis.


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Figure 4. DGGE profiles of bacterial V3-16S rDNA gene fragments amplifiedfrom Kedong sufu samples at different stages. Lane numbers denotetime points (days) of the fermentation process. Numbered bands wereexcised and, after reamplification, subjected to sequencing. The results arerepresentative of a typical experiment of three different batches samples.

DISCUSSIONThe bacterial flora of Kedong sufu was assessed using culture-independent and culture-dependent approaches. Some strainsin the sufu samples remained undetected using PCR-DGGEbut were isolated in cultures. The culture-dependent approachallowed isolation and identification of some strains, and theircorresponding V3-16S rDNA fragments were represented by afragment in the PCR-DGGE profiles. In contrast, some strains werenot isolated in the sufu samples but were represented by afragment in the DGGE profiles (Tables 2 and 3). The comparisonof culture-independent and culture-dependent approaches usedin the current study highlights the limitations of each approach.Thus culture-independent and culture-dependent approaches arecomplementary for microbial analysis of fermented food products.

Staphylococcus carnosus, S. saprophyticus, K. rosea and K. kristinaewere the dominant and subdominant coagulase-negative coccifound in naturally fermented Kedong sufu. Staphylococcus sapro-phyticus is dominant in Taiwanese naturally fermented ham andsome Greek and Italian sausages.7,19,20 Staphylococcus carnosus isthe dominant species in Spanish sausages.21 Kocuria kristinae is thedominant bacterium in cheese and fermented seafood.22,23 The

coagulase-negative cocci are important because these bacteriahave nitrite and nitrate reductase activity, promote the desiredred colour development and help stabilisation, which limits lipidoxidation and prevents rancidity.24,25 They also contribute toflavour through their proteolytic and lipolytic activities.26 ThusS. saprophyticus, S. carnosus, K. rosea and K. kristinae could besuitable candidates for starter cultures for Kedong sufu.

Enterococcus faecalis was the dominant strain during thefermentation of Kedong sufu. It is also the most commonenterococcal species in cheese.27,28 Han et al.10 detectedEnterobacteriaceae in mould-fermented sufu. Enterococci area common component of Corsican cheese microflora.29 Somestrains of E. faecalis may also produce bacteriocins that are activeagainst various food-borne pathogens, which makes them suitablecandidates for use in the control of emerging pathogens duringfood fermentation.30,31 However, the presence of enterococci infoods is highly controversial. Although some authors considerthem undesirable and indicators of faecal contamination, othershave reported that they have an important role in flavourdevelopment and bioprotection.30,32 In this study, Escherichiacoli was never detected in any sample, indicating that E. faecalisdid not arise from faecal contamination. Thus enterococci seem tobe a common component of sufu microflora.

Bacillus subtilis and B. cereus were subdominant strains duringthe fermentation of Kedong sufu. Baruzzi et al.33 reported thatB. subtilis was dominant in southern Italian sausages. Bacillussubtilis and B. cereus were found to be the predominant micro-organisms in ‘Hawaijar’.34 Han et al.10 detected B. cereus and B.subtilis in mould-fermented sufu. Some Bacillus strains show highproteolytic and lipolytic activities. These species could play a rolein the development of texture and organoleptic characteristicsof fermented foods. In addition, consumption of Bacillus strainsincluding B. subtilis and B. cereus has been shown to have abeneficial health impact on humans.35 Therefore these speciescould be used in specific starter cultures for Kedong sufu.

One of the broadly recognised advantages of subjectingfood materials to lactic acid bacteria (LAB)-driven fermentationprocesses is the inhibitory effect this has on the growth ofother micro-organisms, especially food-borne pathogens.36,37 Inthe present study, L. mesenteroides, Tetragenococcus halophilus,Marinilactibacillus psychrotolerans, Weissella confusa, Weissellaparamesenteroides, E. faecium and E. durans belong to the LABgroup and thus are candidates for use in the control of emergingpathogens during food fermentation.

Throughout fermentation, total free amino acids obviouslyincreased, because E. faecalis, K. kristinae, S. carnosus andB. subtilis may be involved in proteolysis and express otherenzymatic activities.26,30,32,35 During pre-fermentation, titratableacidity decreased and pH increased in the sufu owing to thepresence of higher levels of basic amino acids (Arg, Lys and His)and ammonia compared with those of acidic amino acids (Aspand Glu) and free fatty acids. However, during post-fermentation,titratable acidity increased and pH decreased in the sufu becauseof increased production of acidic amino acids and free fatty acids(data not shown). A relatively high NaCl content (above 60 g kg−1)inhibits the acidification of LAB.29 In this study the LAB detectedwere not those typical of acidification (e.g. Lactobacillus acidipiscis,Lactobacillus plantarum and Lactobacillus casei). Therefore the pHdid not drop substantially.

In the current study the presence of bacterial speciesrepresenting Enterococcus, Staphylococcus, Proteus and Bacillusduring fermentation might be due to the presence of


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Table 3. Sequencing results of selected DGGE bands from bacterial DGGE fingerprint illustrated in Fig. 4

Band(s)a Closest relative Identity(%)b Accession no.c Band(s)a Closest relative Identity(%)b Accession no.c

A1 Myroides pelagicus 100 AB176662.1 A22, B22 Uncultured bacterium clone 100 EU703029.1

A2 Uncultured bacterium clone 99 EU797159.1 A23 Virgibacillus sp. 99 GU826620.1

A3, B3 Enterococcus casseliflavus 98 AB671565.1 A24 Oceanobacillus picturae 99 JN049914.1

A4, B4 Enterococcus durans 100 AJ276354 A25 Corynebacterium variabile 100 AB116137.1

A5, B5 Enterococcus faecalis 100 JN644614.1 A27 Kocuria rosea 99 FJ745378.1

A6 Enterococcus gallinarum 98 JN020631.1 A28 Kocuria kristinae 100 EU379300.1

A8, B8 Bacillus pumilus 99 FJ973535.1 A7, A16, A21, A26, B20 Unidentified

A9, B9 Proteus mirabilis 100 EU703029.1 B1 Aerococcus viridans 99 JN377813.1

A10, B11 Tetragenococcus halophilus 100 JF909577.1 B2 Weissella confusa 98 HQ711354.1

A11 Staphylococcus saprophyticus 99 JF909593.1 B6, B7 Weissella paramesenteroides 98 HQ721255.1

A12 Streptococcus lutetiensis 100 HQ293091.1 B10 Uncultured bacterium clone 97 JF680802.1

A13 Uncultured bacterium 100 FJ679491.1 B12 Lysinibacillus sphaericus 97 HQ829965.2

A14, B16 Staphylococcus carnosus 98 EU727183.1 B13 Proteus vulgaris 100 JF970208.1

A15, B17 Bacillus subtilis 100 EU862320.1 B14 Bacillus cereus 100 JF907013.1

A17, B18 Uncultured bacterium clone 98 JN162422.1 B15 Uncultured bacterium 96 GU586697.1

A18 Leuconostoc mesenteroides 100 JN673550.1 B19 Shewanella sp. 91 EU563345.1

A19 Gamma proteobacterium 100 HQ663044.1 B21 Aeromonas veronii 88 JF496559.1

A20 Uncultured bacterium 96 AY241953.1

a Bands are numbered as indicated on the DGGE gels shown in Fig. 4.b Percentage of identical nucleotides in the sequence obtained from the DGGE band and the sequence of the closest relative found in the GenBankdatabase.c Accession number of the sequence of the closest relative found by BLAST search.

microenvironments in the food matrix that support the growthof these bacteria.38,39 The strains that were dominant during pre-fermentation, such as S. saprophyticus, K. rosea and K. kristinae,were absent during post-fermentation. During the processing ofKedong sufu, there is a drying process between pre-fermentationand post-fermentation, which may lead to the death of heat-labile micro-organisms. In addition, pre-fermentation is aerobicfermentation, while post-fermentation is anaerobic fermentation.Overall, the increase, decrease or disappearance of some strainsduring fermentation is likely due to an unfavourable environmentbeing created for specific strains by the inhibitory effects of growth,environmental factors or secondary metabolites.

CONCLUSIONCulture-dependent and culture-independent methods were usedfor the first time to reveal the microbial diversity and dynamics innaturally fermented Kedong sufu. This is an important step that willhelp in the selection of starter cultures, particularly strains that arewell adapted to the particular production technology used for thistypical bacteria-fermented sufu in China. Our research suggestedthat a combination of culture-independent and culture-dependentmethods is a useful approach to effectively describe the microbialcommunity in a complex environment. The totality of this informa-tion will help the design of autochthonous starter cultures for theproduction of Kedong sufu with desirable characteristic sensoryprofiles and shorter ripening times. To select autochthonousstarter cultures for Kedong sufu, we are now evaluating thetechnological properties of such potential starter cultures.

ACKNOWLEDGEMENTSThis research was supported by the National Natural ScienceFoundation of China (31000808), the Special Fund of the National

Postdoctoral Science Foundation of China (No. 4, 201104409),the Postdoctoral Science Foundation of Heilongjiang Province(LBH-Z10233) and the Doctor Scientific Research Start-up Fund ofNortheast Agricultural University (2010RCB60).

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Evaluation of bacterial flora during the ripening of Kedong sufu, a typical Chinese traditional bacteria-fermented soybean product