Traditional Fermentation Starter Research Articles

The incidence and distribution of ochratoxin A in Daqu, a Chinese traditional fermentation starter

Daqu, a traditional starter culture mainly used to produce Chinese liquor and vinegar, is spontaneously fermented by diverse bacteria, yeasts and filamentous fungi under thermophilic condition. Therefore, mycotoxins may exist in Daqu, resulting in the contamination of end-foods. Ochratoxin A (OTA), a mycotoxin produced by certain species of Aspergillus and Penicillium, is not known whether existing in Daqu. However, specific method to detect OTA as well as OTA occurrence in Daqu has not been reported so far. With this in mind, a new method was developed to detect OTA in Daqu by the combination of ultrasound-assisted solid-liquid extraction (USLE), solid phase extraction (SPE) cleanup and UPLC-MS/MS. The USLE conditions of OTA from Daqu were optimized using Plackett-Burman (PB) design coupled with Box-Behnken (BB) design. Under the optimized conditions, no matrix effects were found, and the external standard method can be used to determine OTA in Daqu. The recoveries for spiked samples were 87–106% with the relative standard deviations (RSD) < 15%. The limits of detection and quantification were respectively 0.33 and 0.41 ppb. This approach was then applied to analyze 133 Daqu samples from different geographical regions in China, including 26 low temperature-, 33 medium temperature- and 74 high temperature-type Daqu. The results showed that OTA was detected in 66 samples with a maximum concentration of 28.87 ppb in low temperature Daqu, and the OTA incidence was on increase in the order of high temperature-, medium temperature- and low temperature-type Daqu. This implied that fermentation temperature is the key factor influencing OTA occurrence in Daqu. Moreover, there may be some fungi possessing the biosynthesis ability of OTA under high temperature environment (more than 45 °C).

Restoration of Traditional Korean Nuruk and Analysis of the Brewing Characteristics.

In this study, a total of 58 different kinds of nuruk (a traditional Korean fermentation starter) were prepared, including 46 kinds of restored nuruk from ancient documents. Each nuruk was evaluated by analysis of its saccharification power, and the enzyme activities of glucoamylase, α-amylase, β-amylase, protease, and β-glucanase. The range of saccharification power (sp) of the restored nuruk ranged between 85 and 565 sp. The diastatic enzymes, α-amylase, β-amylase, and glucoamylase, were significantly correlated to the saccharification power value; conversely, β-glucanase and protease did not have a correlative relationship with saccarification power. In addition, their brewing properties on chemical and organoleptic aspects of traditional alcoholic beverage production were compared. Each raw and supplementary material contained in nuruk showed its own unique characteristics on Korean alcoholic beverage brewing. For the first time, in this study, the traditional Korean nuruk types mentioned in ancient documents were restored using modernized production methods, and also characterized based on their brewing properties. Our results could be utilized as a basis for further study of traditional alcoholic beverages and their valuable microorganisms.

Attraction of Coffee Bean Weevil, Araecerus fasciculatus, to Volatiles from the Industrial Yeast Kluyveromyces lactis.

The coffee bean weevil (CBW), Araecerus fasciculatus (De Geer, 1775) (Coleoptera: Anthribidae) is an important pest of stored products such as grains, coffee beans, cassava, and traditional Chinese medicine materials. In China, CBW causes large losses of Daqu, a traditional Chinese liquor fermentation starter, and, unfortunately, the use of conventional insecticides against CBW is not suitable in Daqu storage. We found CBW to be highly attracted to fermenting yeast cultures, such as Kluyveromyces lactis. Eight volatile compounds, produced by fermenting cultures and not by sterile samples, were identified by gas chromatography coupled with mass spectrometry. Five of these substances elicited significant responses in Y-tube behavioral bioassays. Field trapping experiments revealed 2-phenylethanol and 2-phenylethyl acetate to be crucial for attraction of CBW. Results show that yeast volatiles play an important role in host location, and that 2-phenylethanol and 2-phenylethyl acetate could be utilized as potential attractants in monitoring and control systems against this important pest.

Effects of initial moisture content of Korean traditional wheat-based fermentation starter nuruk on microbial abundance and diversity.

The brewing of makgeolli, one of Korea's most popular alcoholic beverages that is gaining popularity globally, is facilitated by nuruk, a traditional Korean cereal starter. The nuruk microbiome greatly influences the fermentation process as well as the nutritional, hygienic, and aromatic qualities of the product. This study is a continuation of our efforts to examine nuruk biodiversity at a depth previously unattainable. In this study, microfloral dynamics in wheat-based nuruk C, composed of traditional ingredients such as barley, green gram, and wheat and fermented under various internal moisture contents of 20% (C20), 26% (C26), and 30% (C30), was evaluated using 454 pyrosequencing during the 30-day fermentation process. Rarefaction analysis and alpha diversity parameters indicated adequate sampling. C20 showed the greatest fungal richness and diversity, C20 and C26 exhibited similar bacterial richness and diversity, while C30 had low fungal and bacterial richness. Fungal taxonomic assignments revealed that the initial moisture content caused selective enrichment of Aspergillus candidus with a decreasing trend during fermentation, whereas Saccharomycetales sp. exhibited increasing relative abundance with increasing moisture content from day 6 of the fermentation process. Depending on initial moisture level, changes in bacterial communities were also observed in the genera Streptomyces, Bacillus, and Staphylococcus, with decreasing trends whereas Saccharopolyspora exhibited a sigmoidal trend with the highest abundance in C26. These findings demonstrate the possible impact of initial moisture content of nuruk on microfloral richness, diversity, and dynamics; this study is thus a step toward our ultimate goal of enhancing the quality of nuruk.

Effect of bioaugmented inoculation on microbiota dynamics during solid-state fermentation of Daqu starter using autochthonous of Bacillus, Pediococcus, Wickerhamomyces and Saccharomycopsis

Daqu, a traditional fermentation starter that is used for Chinese liquor and vinegar production, is still manufactured through a traditional spontaneous solid-state fermentation process with no selected microorganisms are intentionally inoculated. The aim of this work was to analyze the microbiota dynamics during the solid-state fermentation process of Daqu using a traditional and bioaugmented inoculation with autochthonous of Bacillus, Pediococcus, Saccharomycopsis and Wickerhamomyces at an industrial scale. Highly similar dynamics of physicochemical parameters, enzymatic activities and microbial communities were observed during the traditional and bioaugmented solid-state fermentation processes. Both in the two cases, groups of Streptophyta, Rickettsiales and Xanthomonadales only dominated the first two days, but Bacillales and Eurotiales became predominant members after 2 and 10 days fermentation, respectively. Phylotypes of Enterobacteriales, Lactobacillales, Saccharomycetales and Mucorales dominated the whole fermentation process. No significant difference (P > 0.05) in microbial structure was observed between the traditional and bioaugmented fermentation processes. However, slightly higher microbial richness was found during the bioaugmented fermentation process after 10 days fermentation. Our results reinforced the microbiota dynamic stability during the solid-state fermentation process of Daqu, and might aid in controlling the traditional Daqu manufacturing process.

Taxonomic Characterization, Evaluation of Toxigenicity, and Saccharification Capability of Aspergillus Section Flavi Isolates from Korean Traditional Wheat-Based Fermentation Starter Nuruk

The most economically important species used in a wide range of fermentation industries throughout Asia belong to Aspergillus section Flavi, which are morphologically and phylogenetically indistinguishable, with a few being toxigenic and therefore a major concern. They are frequently isolated from Korean fermentation starters, such as nuruk and meju. The growing popularity of traditional Korean alcoholic beverages has led to a demand for their quality enhancement, therefore requiring selection of efficient non-toxigenic strains to assist effective fermentation. This study was performed to classify the most efficient strains of Aspergillus section Flavi isolated from various types of traditional wheat nuruk, based on a polyphasic approach involving molecular and biochemical evaluation. A total of 69 strains were isolated based on colony morphology and identified as Aspergillus oryzae/flavus based on internal transcribed spacer and calmodulin gene sequencing. Interestingly, none were toxigenic based on PCR amplification of intergenic regions of the aflatoxin cluster genes norB-cypA and the absence of aflatoxin in the culture supernatants by thin-layer chromatography analysis. Saccharification capability of the isolates, assessed through α-amylase and glucoamylase activities, revealed that two isolates, TNA24 and TNA15, showed the highest levels of activity. Although the degrees of variation in α-amylase and glucoamylase activities among the isolates were higher, there were only slight differences in acid protease activity among the isolates with two, TNA28 and TNA36, showing the highest activities. Furthermore, statistical analyses showed that α-amylase activity was positively correlated with glucoamylase activity (p < 0.001), and therefore screening for either was sufficient to predict the saccharifying capacity of the Aspergillus strain.

Draft genome sequence of alcohol-tolerant bacteria Pediococcus acidilactici strain K3.

Pediococcus acidilactici strain K3 is an alcohol-tolerant lactic acid bacterium isolated from nuruk, which is a traditional Korean fermentation starter for makgeolli brewing. Draft genome of this strain was approximately 1,991,399 bp (G+C content, 42.1%) with 1525 protein-coding sequences (CDS), of which 44% were assigned to recognized functional genes. This draft genome sequence data of the strain K3 will provide insights into the genetic basis of its alcohol-tolerance.

Metagenomic analysis of fungal diversity in Korean traditional wheat-based fermentation starter nuruk

Nuruk, a traditional natural starter, is extensively used in the brewing of Makgeolli, one of Korea’s most popular alcoholic beverages that has been recently gaining global popularity. Thus, the quality of traditional nuruk needs to be enhanced. The nuruk mycobiome greatly influences both fermentation process as well as palatability enhancement. Limitations of culture-dependent identification restrict an accurate analysis of fungal diversity and distribution in nuruks. 454 pyrosequencing of two traditional wheat-based nuruks, prepared at two representative temperature conditions revealed a total of 153 and 53 OTUs for nuruks A and B, respectively, from a total of 33,157 ITS sequences. Phylogenetic assignments indicated that nuruk A mycobiota was dominated by the genera Aspergillus and Mucorales, whereas nuruk B by Rhizomucor. Species-level identification indicated that Mucorales sp., Aspergillus candidus, and Aspergillus cibarius predominated in nuruk A mycoflora whereas Rhizomucor pusillus, Mucorales sp., and Thermoascus crustaceus in nuruk B. The alpha diversity indices suggest nuruk A mycobiota to be more diverse than that of nuruk B at almost all time points of fermentation. Resemblances of patterns of predominant species composition and succession between culture-dependent and -independent phylogenetic analysis creates the potential to reconstruct the nuruk mycobiome in vitro, which allows the establishment of a standard inoculum for scientific comparison.

Microbiological characterization of traditional dough fermentation starter (Jiaozi) for steamed bread making by culture-dependent and culture-independent methods

In this study, the microbial composition of two types of Jiaozi (a dough fermentation starter in making steamed bread) was investigated using both culture-dependent and culture-independent (PCR-DGGE) methods. The numbers of the cultivable bacteria on MRS at 30°C and yeast on YPD at 28°C in the maize flour Jiaozi (MFJ) were 9.21±0.16 Log CFU/g and 9.18±0.05 Log CFU/g, respectively, which were higher than that in the rice flour Jiaozi (RFJ) (P<0.05). A total of 140 bacteria and 124 yeasts were isolated and identified on the basis of the sequences of their 16S rRNA gene and ITS region. The culture-dependent method showed that Acetobacter tropicalis and Enterococcus durans were the predominant bacteria strains in MFJ, and accounted for 45.7% and 25.7% of the bacteria, and Lactobacillus plantarum and Pediococcus pentosaceus represented 12.8% and 8.6%. In the RFJ sample, the most prominent isolate was P. pentosaceus (38.6%), followed by L. plantarum (24.3%), A. tropicalis (22.8%), and E. durans (5.7%). P. pentosaceus and L. plantarum were also detected in both starters by PCR-DGGE, while some bacteria species such as A. tropicalis and E. durans, recovered as pure cultures, were not detected by direct PCR-DGGE. On the other hand, Lactobacillus brevis, Weissella sp. and Lactobacillus alimentarius detected by PCR-DGGE were not recovered in any of the media and conditions used. In the MFJ sample, the isolated main yeast species were identified as Wickerhamomyces anomalus (67.2%), Saccharomyces cerevisiae (27.9%) and Torulaspora delbrueckii (4.9%). In addition to S. cerevisiae (42.9%), W. anomalus (27.0%) and T. delbrueckii (7.9%), Saccharomycopsis fibuligera was also identified as the predominant isolate in RFJ samples and accounted for 22.2%. PCR-DGGE also indicated the presence of W. anomalus and S. cerevisiae in both MFJ and RFJ starters and S. fibuligera was also detected in RFJ, but T. delbrueckii was not detected in both samples.

Characterization of the dominant bacterial communities of traditional fermentation starters for Hong Qu glutinous rice wine by means of MALDI-TOF mass spectrometry fingerprinting, 16S rRNA gene sequencing and species-specific PCRs

The main goal of this work was to investigate the bacterial flora present in the traditional fermentation starters using matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry fingerprinting combined with 16S rRNA gene sequencing and species-specific PCRs. A total of 1314 bacterial strains were obtained from ten kinds of representative wine fermentation starters (5 Hong Qu and 5 Yao Qu) and classified by MALDI-TOF mass spectrometry. Sixteen different bacterial species belonging to six different genera (Bacillus, Staphylococcus, Leuconostoc, Pediococcus, Lactobacillus and Lactococcus) were identified with high accuracy by comparing them with generated spectra of MALDI Biotyper database. Comparison of 16S rRNA gene phylogeny and MALDI-TOF mass spectrometry analysis showed that MALDI-TOF mass spectrometry fingerprinting was a good complementary approach to 16S rRNA sequencing and even a more powerful tool in the accurate differentiation and classification of Bacillus species, especially for differentiating between Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus methylotrophicus. MALDI-TOF mass spectrometry was also found to provide valuable information at species levels for the genus of Lactobacillus. The identification revealed that most strains isolated from traditional fermentation starters were B. amyloliquefaciens (31.81%), followed by Bacillus megaterium (16.89%), Lc. lactis subsp. lactis (8.60%) and B. subtilis (8.14%). In the culture-dependent quantitative analysis, 69.01% of the detected isolates were Bacillus species, and 30.44% of the detected isolates belonged to the class of lactic acid bacteria (LAB), including Leu. mesenteroides, Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus brevis, Pediococcus acidilactici, Lactobacillus coryniformis and Lc. lactis subsp. lactis. This study provides detailed evaluation of bacterial communities in the traditional fermentation starters for Hong Qu glutinous rice wine using a fast and cost-effective technique—MALDI-TOF mass spectrometry fingerprinting. Information provided by this study is fundamental and useful to develop effective strategies for the selection of beneficial bacterial strains and improve of quality of Hong Qu glutinous rice wine.

Utilization of Oil Palm Empty Fruit Bunch Hydrolysate for Ethanol Production by Baker's Yeast and Loog-Pang

Oil palm empty fruit bunch is a potential source of glucose which can serve as a promising raw material for production of ethanol. Ethanol production was investigated by using separation hydrolysis and fermentation process (SHF), in which yeast from loog-pang (a Thai traditional fermentation starter culture) and baker's yeast were used as biocatalyst. After alkali pretreatment with 15% w/v NaOH for 40min at 130°C, Cellulose was analyzed to be 61.94% (on dry weight basis). Glucose was 33.45g/L after cellulose was hydrolyzed by 7% v/v H2SO4 at 119°C for 110min. The resulting aqueous phase rich in glucose was fermented by yeast from baker's yeast to produce ethanol with the concentration of 8.49g/L with is 56 times higher than loog-pang as yeast. Therefore, implementation of oil palm empty fruit bunch for the development of alternative source of energy could be an effective approach and key enable to the billion-ton biofuel vision.

국내 전통주 주박의 이용과 유용생리활성

The lees of Korean traditional wine called as Jubak or Sul-jigemi in Korea is byproduct from alcohol fermentation industry, which is remnant of fermentation broth after filtration, centrifugation, distillation, or sedimentation during aging. Since, Korean traditional wines are produced from edible plant sources such as rice, foxtail millet, fruits and medicinal herbs with nuruk (a traditional fermentation starter and starch degrader), the Jubak from rice wine (takju), medicinal herb wine (yakju) and fruits wine are considered as safe byproduct and have various useful bioactivity. Considering the recent rapid increased production of Jubak from takju industry, and the reinforcement of dispose of Jubak as waste material in worldwide, the development of efficient reuse process for Jubak is necessary in traditional wine industry. In this review, the status of current industry, research and patent trends in relation with Jubak production, treatment, utilization and renewal was analyzed and different bioactive compounds including phenolic acids from Jubak were provided. Jubak is not any more waste material, and is the source of bioactive functional materials for food, cosmetics and medicinal industry. To develop the efficient and economic renewal technology including recovery process for bioactive substances from Jubak, systematic collaboration and research among the industry, academy and government is necessary.

1H NMR-based metabolomics approach for understanding the fermentation behaviour ofBacillus licheniformis

Bacillus licheniformis has been found to be one of the persistent dominant microorganisms in Daqu, which is a traditional fermentation starter, and it has been used to intensify certain strains. To understand the impact of B. licheniformis on Daqu, the fermentation behaviour of B. licheniformis was studied using 1H NMR-based non-targeted analysis and principal component analysis. During the fermentation, 53 compounds were identified. Among them, seven compounds were largely consumed and 17 metabolites were largely accumulated. The macromolecular starch and cellulose were degraded by B. licheniformis, and then utilized to produce acetic acid, lactic acid, propionic acid, succinate acid, etc. Principal component analysis was carried out to study the variations of analytes during the fermentation. Samples collected at each time point could be clearly discriminated and the biomarkers of each time point were defined. A variety of biochemical compounds (such as acetate, ethanol, lactate, pyruvate, malate, maltose and sucrose) were changed during the fermentation of B. licheniformis. The results are useful to reveal how and why B. licheniformis becomes dominant in Daqu, and to reveal its impact on the aroma formation of Daqu and its derived products. Copyright © 2015 The Institute of Brewing & Distilling

Ethyl carbamate formation regulated by Saccharomyces cerevisiae ZJU in the processing of Chinese yellow rice wine

SummaryEthyl carbamate (EC) is a probable carcinogen existing in most fermented foods. Throughout traditional fermentation processes, the Chinese fermentation starter plays an important role, but it contains varieties of microorganisms which make inhibiting EC efficiently become a challenge. Therefore, the traditional fermentation starter is substituted with a single yeast strain (Saccharomyces cerevisiae ZJU) to regulate EC catabolism. In this work, S. cerevisiae ZJU can reduce EC formation and the data of EC concentration show that there is 85.6% reduction of EC at most using S. cerevisiae ZJU instead of the traditional fermentation starter. Extracellular urea and citrulline were the leading precursors of EC. The content of amino acids and volatile flavour compounds in the experimental group has no significant influence compared to the natural fermentation. The findings in this work suggest that EC can be regulated by means of the fermentation starters variation.

시판 전통누룩의 젖산균 분리동정과 제조단양주의 품질특성

Five takju prepared using four types of nuruk (a traditional Korean fermentation starter made of malted wheat; non-cooked, naturally inoculated) labeled SH, SS, JJ, and SJ, and one type of koji (cooked, inoculated with an inoculum) labeled MN, were compared. Titratable acidity, pH, sugars, ethanol, amino acids, organic acids, and microbial changes in samples were measured, and the sensory properties were evaluated. Titratable acidity, alcohol, and organic acid content increased as sugar contents decreased. The overall ethanol concentration of all takju increased over time, reaching a maximum of 13.08-14.96% (w/v) at 7-21 days. The total amino acid contents of takju prepared with nuruk, except for one (SJ), were higher than the takju prepared with koji (MN). Lactic acid bacteria were also isolated from the starters. Sequence analysis of 16S rRNA genes (500 - 600 bp) of 223 isolates revealed that the major strains were in the genera of Leuconostoc, Weissella, Pediococcus, and Lactobacillus.

과일과 전통발효제로부터 추출한 단백질분해 조효소의 비교 특성

본 연구에서는 과일(무화과, 키위, 파인애플)에서 추출한 단백질분해 조효소와 전통발효제(개량누룩, 전통누룩, 메주, 입국)에서 추출한 단백질분해 조효소의 특징을 비교하였다. 전통발효제에서 추출한 단백질분해 조효소는 과일에서 추출한 단백질분해조효소보다 높은 온도(70°C), 높은 염 내성(1~3%), 그리고 낮은 pH(3~6)에서 활성을 보였다. 같은 조건에서 활성을 비교하였을 때, 바이오 누룩>입국>전통누룩>무화과>파인애플>메주>키위 순으로 활성이 높았다. 이러한 결과는 과일에서 추출한 단백질분해 조효소보다 전통발효제에서 추출한 단백질분해 조효소의 활성이 우수하고 고온, 산성, 그리고 염의 조건에서도 활성이 저하되지 않으므로, 소시지나 치즈 제조 등의 다양한 식품산업에 이용될 수 있는 가능성을 보여준다.

Bacterial community dynamics during the traditional brewing of Wuyi Hong Qu glutinous rice wine as determined by culture-independent methods

Wuyi Hong Qu (black-skin-red-koji) glutinous rice wine, as one of the most typical representatives of Hong Qu glutinous rice wine in Fujian province of China, is brewed under non-sterile and uncontrolled fermentation condition based on empirical knowledge, causing uncontrollability of fermentation process and instability of the final quality. The objective of this study was to investigate the bacterial dynamics during the traditional fermentation of Wuyi Hong Qu glutinous rice wine using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S ribosomal RNA (rRNA) gene clone libraries analysis.The DGGE profile indicated that the dominant bacterial species in the traditional wine fermentation starters were Pediococcus pentosaceus, Pediococcus acidilactici and Bacillus sp. (including Bacillus aryabhattai or Bacillus megaterium and Bacillus amyloliquefaciens). Bacterial dynamic obtained from the PCR-DGGE revealed the presence of Bacillus sp. and LAB (including Lactobacillus plantarum group, Lactobacillus brevis, P. acidilactici and P. pentosaceus) during the traditional fermentation process, but they varied in different brewing phases. The relative proportions of some bacterial species (such as Bacillus sp., P. acidilactici, L. brevis and P. pentosaceus) detected at early fermentation stage decreased as the fermentation progressed. While L. plantarum group was consistently detected with high light band intensity throughout the fermentation process. 16S rRNA gene clone libraries revealed that the two different molecular biological methods gave similar results, but clone library analysis was more representative of the bacterial community to some extent. For example, Leuconostoc mesenteroides was detected by 16S rRNA gene clone library but not discovered by bacterial DGGE profile throughout the whole fermentation process. Therefore, the combined approach of nested PCR-DGGE and 16S rRNA gene clone libraries would give a more comprehensive profile of the bacterial dynamics than either alone. Finally, species-specific multiplex PCR was also performed to confirm the L. plantarum group. Result showed that only L. plantarum can be detected from the total bacterial DNA extracted from samples of different fermentation phases.This is the first report to reveal the dynamics of bacterial species involved in Wuyi Hong Qu glutinous rice wine brewing process using culture-independent methods. It might be useful to control wine production systems and improve wine quality.

Monitoring the Ecology of Bacillus During Daqu Incubation, a Fermentation Starter, Using Culture-Dependent and Culture-Independent Methods

Daqu, a traditional fermentation starter, has been used to produce attractively flavored foods such as vinegar and Chinese liquor for thousands of years. Although Bacillus spp. are one of the dominant microorganisms in Daqu, more precise information is needed to reveal why and how Bacillus became dominant in Daqu, and next, to assess the impact of Bacillus sp. on Daqu and its derived products. We combined culture-dependent and culture-independent methods to study the ecology of Bacillus during Daqu incubation. Throughout the incubation, 67 presumptive Bacillus spp. isolates were obtained, 52 of which were confirmed by 16S rDNA sequencing. The identified organisms belonged to 8 Bacillus species: B. licheniformis, B. subtilis, B. amyloliquefaciens, B. cereus, B. circulans, B. megaterium, B. pumilus, and B. anthracis. A primer set specific for Bacillus and related genera was used in a selective PCR study, followed by a nested DGGE PCR targeting the V9 region of the 16S rDNA. Species identified from the PCR-DGGE fingerprints were related to B. licheniformis, B. subtilis, B. amyloliquefaciens, B. pumilus, B. benzoevorans, and B. foraminis. The predominant species was found to be B. licheniformis. Certain B. licheniformis strains exhibited potent antimicrobial activities. The greatest species diversity occurred at the Liangmei stage of Daqu incubation. To date, we lack sufficient knowledge of Bacillus distribution in Daqu. Elucidating the ecology of Bacillus during Daqu incubation would enable the impact of Bacillus on Daqu to be accessed, and the quality and stabilization of Daqu-derived products to be optimized.

Yeast diversity of traditional alcohol fermentation starters for Hong Qu glutinous rice wine brewing, revealed by culture-dependent and culture-independent methods

Hong Qu glutinous rice wine, one of the most popular traditional rice wines in China, is brewed from glutinous rice with the addition of two traditional fermentation starters – Hong Qu (hóng qū) and Yao Qu (yào qū). The aim of this study was to investigate the yeast flora present in these fermentation starters using a combination of culture-dependent and -independent molecular biological methods.Molecular identification of a total of 500 yeast isolate from 10 kinds of representative wine fermentation starters (5 Hong Qu and 5 Yao Qu) was achieved by a combination of polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis of the internal transcribed spacer region ITS1-5.8S-ITS2 and sequencing of the D1/D2 domain of the 26S rRNA gene. The PCR–RFLP method generated 13 different ITS/RFLP profiles. Sequencing identification allowed identifying 13 different ITS/RFLP profiles into 12 different species of yeast belong to eight different genera (Pichia, Saccharomyces, Candida, Cryptococcus, Rhodotorula, Sporobolomyces, Rhodosporidium and Saccharomycopsis). Species diversity was thus considerable: Saccharomycopsis fibuligera was the most commonly isolated specie, followed by Saccharomyces cerevisiae. On the other hand, the yeast diversity associated with wine starters for Hong Qu glutinous rice wine was also investigated through culture-independent method using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) patterns and sequencing of the DNA bands. The results revealed by culture-independent method were almost the same as that of culture-dependent methodology. However, several yeast species can not be detected by traditional microbiological procedures but can be detected using PCR–DGGE analysis. Similarly, some species could only be detected by culture-dependent method. For example, Sporobolomyces nylandii was detected existing in wine starter samples (Q7 and Q10) through culture-dependent method, but not detected in wine starters for Hong Qu glutinous rice wine through fungal PCR–DGGE assay. Therefore, it is recommended that polyphonic approaches based on culture-dependent and culture independent methods seem the best strategy to get a more comprehensive picture of the microbial diversity in traditional wine starters. These results enrich our knowledge of rice wine-related yeasts, and can be used to promote the development of the traditional brewing industry.

Evaluation of the Probiotic Potential of Bacillus polyfermenticus CJ6 Isolated from Meju, a Korean Soybean Fermentation Starter

To evaluate the probiotic potential of Bacillus polyfermenticus CJ6 isolated from meju, a Korean traditional soybean fermentation starter, its functionality and safety were investigated. B. polyfermenticus CJ6 was sensitive to all antibiotics listed by the European Food Safety Authority. The strain was also non-hemolytic, carried no emetic toxin or enterotoxin genes, and produced no enterotoxins. The resistance of B. polyfermenticus CJ6 vegetative cells and spores to simulated gastrointestinal conditions was high (60-100% survival rate). B. polyfermenticus CJ6 produced high amounts (0.36 g as a purified lyophilized form) of gamma-polyglutamic acid (PGA). We speculate that the improved cell viability and the production of gamma-PGA have a significant correlation. Adhesion of the strain to Caco-2 and HT-29 cells was weaker than that of the reference strain (Lb. rhamnosus GG), but it was comparable to or stronger than those of reported Bacillus spp. When B. polyfermenticus CJ6 spores were given orally to mice, the number of cells excreted in the feces was 4-fold higher than the original inocula. This suggests the inoculated spores propagated within the intestinal tract of the mice. This idea was confirmed by field emission scanning electron microscopy, which revealed directly that B. polyfermenticus CJ6 cells germinated and adhered within the gastrointestinal tract of mice. Taken together, these findings suggest that B. polyfermenticus CJ6 has probiotic potential for both human consumption and use in animal feeds.