Bei-Zhong Han

Complex microbiota of a Chinese “Fen” liquor fermentation starter (Fen-Daqu), revealed by culture-dependent and culture-independent methods

Daqu is a traditional fermentation starter that is used for Chinese liquor production. Although partly mechanized, its manufacturing process has remained traditional. We investigated the microbial diversity of Fen-Daqu, a starter for light-flavour liquor, using combined culture-dependent and culture-independent approaches (PCR-DGGE). A total of 190 microbial strains, comprising 109 bacteria and 81 yeasts and moulds, were isolated and identified on the basis of the sequences of their 16S rDNA (bacteria) and 26S rDNA and ITS regions (fungi). DGGE of DNA extracted from Daqu was used to complement the culture-dependent method in order to include non-culturable microbes. Both approaches revealed that Bacillus licheniformis was an abundant bacterial species, and Saccharomycopsis fibuligera, Wickerhamomyces anomalus, and Pichia kudriavzevii were the most common yeasts encountered in Fen-Daqu. Six genera of moulds (Absidia, Aspergillus, Mucor, Rhizopus, Rhizomucor and Penicillium) were found. The potential function of these microorganisms in starters for alcoholic fermentation is discussed. In general the culture-based findings overlapped with those obtained by DGGE by a large extent. However, Weissella cibaria, Weissella confusa, Staphylococcus saprophyticus, Enterobacter aerogenes, Lactobacillus sanfranciscensis, Lactobacillus lactis, and Bacillus megaterium were only revealed by DGGE.

Effects of soaking, germination and fermentation on phytic acid, total and in vitro soluble zinc in brown rice

Rice is an important staple food in Asian countries. In rural areas it is also a major source of micronutrients. Unfortunately, the bioavailability of minerals, e.g. zinc from rice, is low because it is present as an insoluble complex with food components such as phytic acid. We investigated the effects of soaking, germination and fermentation with an aim to reduce the content of phytic acid, while maintaining sufficient levels of zinc, in the expectation of increasing its bioavailability. Fermentation treatments were most effective in decreasing phytic acid (56-96% removal), followed by soaking at 10°C after preheating (42-59%). Steeping of intact kernels for 24h at 25°C had the least effect on phytic acid removal (<20%). With increased germination periods at 30°C, phytic acid removal progressed from 4% to 60%. Most wet processing procedures, except soaking after wet preheating, caused a loss of dry mass and zinc (1-20%). In vitro solubility, as a percentage of total zinc in soaked rice, was significantly higher than in untreated brown rice while, in steeped brown rice, it was lower (p<0.05). Fermentation and germination did not have significant effects on the solubility of zinc. The expected improvement due to lower phytic acid levels was not confirmed by increasing levels of in vitro soluble zinc. This may result from zinc complexation to other food components.

Microbial composition during Chinese soy sauce koji-making based on culture dependent and independent methods

Koji-making is a key process for production of high quality soy sauce. The microbial composition during koji-making was investigated by culture-dependent and culture-independent methods to determine predominant bacterial and fungal populations. The culture-dependent methods used were direct culture and colony morphology observation, and PCR amplification of 16S/26S rDNA fragments followed by sequencing analysis. The culture-independent method was based on the analysis of 16S/26S rDNA clone libraries. There were differences between the results obtained by different methods. However, sufficient overlap existed between the different methods to identify potentially significant microbial groups. 16 and 20 different bacterial species were identified using culture-dependent and culture-independent methods, respectively. 7 species could be identified by both methods. The most predominant bacterial genera were Weissella and Staphylococcus. Both 6 different fungal species were identified using culture-dependent and culture-independent methods, respectively. Only 3 species could be identified by both sets of methods. The most predominant fungi were Aspergillus and Candida species. This work illustrated the importance of a comprehensive polyphasic approach in the analysis of microbial composition during soy sauce koji-making, the knowledge of which will enable further optimization of microbial composition and quality control of koji to upgrade Chinese traditional soy sauce product.

Microbiota dynamics related to environmental conditions during the fermentative production of Fen-Daqu, a Chinese industrial fermentation starter

Chinese Daqu is used as a starter for liquor and vinegar fermentations. It is produced by solid state fermentation of cereal-pulse mixtures. A succession of fungi, lactic acid bacteria and Bacillus spp. was observed during the production of Daqu. Mesophilic bacteria followed by fungi, dominated the first phase of fermentation. Next, lactic acid bacteria increased in relative abundance, resulting in an increase of the acidity of Daqu. At the final stages of fermentation, Bacillus spp. and thermophilic fungi became the dominant groups, possibly due to their tolerance to low water activity and high temperature. Both culture-dependent and culture-independent analyses confirmed that Bacillus spp. were ubiquitous throughout the process. Yeast species such as Wickerhamomyces anomalus, Saccharomycopsis fibuligera and Pichia kudriavzevii were present throughout almost the entire fermentation process, but the zygomycetous fungus Lichtheimia corymbifera proliferated only during the final stages of fermentation. Canonical correspondence analysis (CCA) revealed the significance of acidity, moisture content and temperature in correlation with the composition of the microbial communities at different stages.

In vitro solubility of calcium, iron and zinc in relation to phytic acid levels in rice-based consumer products in China

Abstract In vitro solubility of calcium, iron and zinc in relation to phytic acid (PA) levels in 30 commercial rice-based foods from China was studied. Solubility of minerals and molar ratios of PA to minerals varied with degrees of processing. In primary products, [PA]/[Ca] values were less than 5 and [PA]/[Fe] and [PA]/[Zn] similarly ranged between 5 and 74, with most values between 20 and 30. [PA]/[mineral] molar ratios in intensively processed products were lower. Solubility of calcium ranged from 0% to 87%, with the lowest in brown rice (12%) and the highest in infant foods (50%). Iron solubility in two-thirds of samples was lower than 30%, and that of zinc narrowly ranged from 6% to 30%. Solubility of minerals was not significantly affected by [PA]/[mineral]. At present, neither primary nor intensively processed rice-based products are good dietary sources of minerals. Improvements should be attempted by dephytinization, mineral fortification or, preferably, combination of both.

Lactic acid bacteria diversity of fresh rice noodles during the fermentation process, revealed by culture-dependent and culture-independent methods

Rice noodles, a traditional Chinese food, are made using natural fermentation. The fermentation process plays an important role for the texture and flavour, which are due to the metabolism of the microorganisms involved, especially lactic acid bacteria (LAB). However, the fermentation of rice noodles is a non-controlled and spontaneous process; the quality of products is not stable. In order to improve and stabilize the quality of the production, the LAB functions on rice noodles during fermentation need to be elucidated. In this study, the community composition and dynamics of LAB associated with the fermented rice were investigated by using a culture-dependent approach in combination with a culture-independent approach, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE). Sixty-four strains were isolated by culture-dependent analysis in different rice noodles factories and mainly 10 species in total were found. In addition, PCR-DGGE detected 17 species (including uncultured species) in total. Seven Lactobacillus species were detected by both methods, namely Lb. delbrueckii, Lb. salivarius, Lb. helveticus, Lb. reuteri, Lb. fermentum, Lb. amylovorus and Lb. oris, and the rest were detected by different methods separately. The potential role of these predominant LAB in the fermentation process is discussed. In general, the two techniques complement each other to give a better understanding on the LAB composition, which is necessary for the isolation of functional strains and improvement of the quality and consistency of rice noodles.

Effect of the environment microbiota on the flavour of light-flavour Baijiu during spontaneous fermentation

Light-flavour Baijiu is a type of Chinese liquor with a pure and mild flavour produced by traditional spontaneous solid-state fermentation. The flavour of this liquor has been found to vary in the different periods of annual production. To explore the factors affecting flavour, the microbiota of the surrounding environment, starter and fermentation process in different periods were investigated. Results showed that the ester content and acidity of light-flavour Baijiu were significantly lower when annual production was resumed after a summer break. HCA plot of volatile flavour profile and bacterial PCoA results indicated that the differences occurred at later stages, mainly due to different structures of Lactobacillus. Correlation analysis by O2PLS indicated that Lactobacillus positively correlated with esters. Species-level analysis showed that the lack of L. acetotolerans on the surface of the jar might cause a lag in fermentation and lower ester content. Thereafter, L. acetotolerans was revived during fermentation and enriched on the surface of the jar, which promoted ester formation. As important sources of L. acetotolerans, the air and fermentation jars played a critical role during fermentation. Therefore, this systematic study on environmental microbial ecology is valuable for quality control and to explore environmental microbiota functions during spontaneous fermentation.

Biochemical characterisation and dominance of different hydrolases in different types of Daqu – a Chinese industrial fermentation starter

BACKGROUND Daqu is a fermentative saccharification agent that is used to initiate fermentation in the production of Chinese liquor and vinegar. This study investigated the differences of amylase, protease and esterase in dominance of different types of Daqu, which can be useful for quality control and flavor improvement of Daqu production by enzyme technology. RESULTS Hydrolase activities in different Daqu samples were compared by principal component analysis (PCA). Based on protein electrophoresis and 1 H NMR spectroscopy, the protein patterns and metabolites in Daqu were further analysed. The results indicated that the highest amylase activities and diversities were found in low/medium-temperature of Daqu which had light-flavour and strong-flavour. Proteases play a significant role in determining the quality of high-temperature Daqu samples which had a sauce-flavour. Furthermore, the contributions of esterase to both strong and sauce flavour development in high-temperature Daqu are similar. CONCLUSION Results from the present work showed that differences in amylase, protease and esterase play a leading role in different types of Daqu, which can be useful for quality control and technology development of Daqu.

Microbial diversity and dynamics of microbial communities during back-slop soaking of soybeans as determined by PCR–DGGE and molecular cloning

Tempe is a traditional fermented food in Indonesia. The manufacture process is quite complex, which comprises two stages, preparatory soaking of soybeans and fungal solid state fermentation. Daily addition of previous soak water (back-slopping) during the soybean soaking step is considered to be crucial in the manufacture of high quality tempe. The microbial diversity and dynamics of the microbial communities evolving during back-slop soaking of soybeans for tempe making was investigated by culture-independent PCR–DGGE and molecular cloning. Both DNA and total RNA were isolated and included in this study, to obtain a view on the succession of total and viable bacteria in the complex microbiota. DGGE profiles indicated that Enterobacter sp., Enterococcus sp., Pseudomonas putida, Leuconostoc fallax, Pediococcus pentosaceus, and Weissella cibaria, were the predominant bacteria. Their occurrence shifted dramatically during the back-slop soaking procedure. This study combined with previous culture-dependent studies could gain a better understanding of the complex microbiota of traditional fermented food and give useful information for its quality control.