Profiling of dynamic changes in the microbial community during the soy sauce fermentation process

Soy sauce is a traditional condiment manufactured by natural inoculation and mixed culture fermentation. As is well known, it is the microbial community that plays an important role in the formation of its flavors. However, to date, its dynamic changes during the long period of fermentation process are still unclear, intensively constraining the improvement and control of the soy sauce quality. In this work, we revealed the dynamic changes of the microbial community by combining a cultured dependent method and a cultured independent method of polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis. Results indicated that the two methods verified and complemented each other in profiling microbial community, and that significant dynamics of the microbial community existed during the fermentation process, especially the strong inhibition of the growth of most of the microbes when entering into the mash stage from the koji stage. In the analysis of bacterial community, Staphylococcus and Bacillus were found to be the dominant bacteria and detected in the whole fermentation process. Kurthia and Klebsiella began to appear in the koji stage and then fade away in the early stage of the mash fermentation. In the analysis of fungal community, Aspergillus sojae and Zygosaccharomyces rouxii were found to be the dominant fungi in the koji and mash fermentation, respectively. It was clearly shown that when A. sojae decreased and disappeared in the middle stage of the mash fermentation, Z. rouxii appeared and increased at the meantime. Aspergillus parasiticus, Trichosporon ovoides and Trichosporon asahii also appeared in the koji and the early period of the mash fermentation and disappeared thereafter. Similar to Z. rouxii, Millerozyma farinosa and Peronospora farinosa were also found spontaneously which appeared in the mid-late period of the mash fermentation. The principal component analysis suggested that the microbial community underwent significant changes in the early period of the fermentation and, thereafter, tended to the stabilization in the mid-late periods. This study gave us important clues to understand the fermentation process and can serve as a foundation for improving the quality of soy sauce in the future.     

Isolation and Analysis of Molds from Soy Sauce Koji in Thailand

Five different isolates of Aspergillus and one of Mucor were compared with a Japanese commercial strain of Aspergillus oryzae for proteolytic activity on wheat bran substrate. One isolate of Aspergillus with superior protease production, identified as Aspergillus flavus var. columnaris, showed no detectable aflatoxin production on glutinous rice or soybean substrate. Preliminary tests using this fungus as a koji mold in a traditionally operated factory resulted in a soy sauce superior in quality to that usually produced.     

New potent antioxidative o-dihydroxyisoflavones in fermented Japanese soybean products.

A potent antioxidative 6-hydroxydaidzein (6-OHD) was newly isolated from soybean koji fermented with Aspergillus oryzae. 6-OHD, in addition to 8-hydroxydaidzein and 8-hydroxygenistein, were found to be present in various fermented soybean products, including their koji. Considering that these o-dihydroxyisoflavones had strong antioxidative activities, they may contribute to protecting from oxidative deterioration during the processing of fermented soybean products.     

Growth of nisin-producing lactococci in cooked rice supplemented with soybean extract and its application to inhibition of Bacillus subtilis in rice miso

Lactic acid fermentation of cooked rice and rice koji by supplementation with soybean extract (SBE) and its application to rice miso fermentation were investigated. By supplementing the cooked rice with SBE, lactic acid bacteria (LAB) grew well without any unfavorable effects on the rice such as off-flavor or coloration. Lactococcus lactis subsp. lactis IFO12007 (Lc. lactis, a producer of the bacteriocin nisin) proliferated at 10(8 to approximately 9) cells/g after 24 h of incubation and produced high activity of nisin. The fermented rice with Lc. lactis strongly inhibited not only Bacillus subtilis ATCC19659 but also the other Bacillus strains. While some strains of LAB markedly inhibited the growth of Asp. oryzae, resulting in failure of koji fermentation, Lc. lactis did not affect the growth of these molds. When Lc. lactis was used for rice miso fermentation as a lactic acid starter culture, Lc. lactis rapidly proliferated and produced high nisin activity of 6,400 IU/g, in the steamed rice, resulting in complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation. The rice miso after 12 weeks of aging had a suitable pH, and favorable taste and color. Furthermore, hyposalting of rice miso could be done without difficulty by lactic acid fermentation of both rice and soybeans.     

传统豆酱发酵过程中细菌多样性动态

细菌在豆酱发酵过程中起到非常重要的作用,并与豆酱的风味和质量密切相关,因此研究豆酱中细菌的多样性具有重要意义。以自然发酵的豆酱样品为研究对象,采用细菌16S rDNA的部分可变区的PCR-DGGE技术对自然发酵豆酱样品的细菌群落组成和优势菌群进行研究。结果表明,传统豆酱发酵过程细菌群体中既有原始种群的减少和增长,也有次级种群的增多和演变。在整个发酵过程中,初期和末期以不可培养细菌为主,初期细菌群体快速演替,细菌种群多样性指数在发酵42 d和56 d达到两次高峰。     

Complete Growth Inhibition of Bacillus subtilis by Nisin-Producing Lactococci in Fermented Soybeans

The growth inhibition by nisin-producing lactococci against Bacillus subtilis and its application to soybean miso fermentation were investigated. Lactococcus lactis subsp. lactis IFO12007 (nisin-producing, salt-intolerant) rapidly proliferated to more than 10⁹ cells/g in cooked soybeans without any excessive pH decrease. In spite of the mild decrease in pH, the growth of B. subtilis was completely inhibited; no living cells were detected in a soybean sample inoculated with 10⁶ cells/g and incubated for 24 to 72 h. This Lc. lactis was applied to soybean miso fermentation as a starter culture. It produced high nisin activity (1.28×10⁵ AU/g) in cooked soybean, resulting in the complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation, throughout the process of miso production. Over-acidification, which is undesirable for miso quality, was successfully prevented simply by adding salt which killed the salt-intolerant Lc. lactis. Furthermore, the nisin activity in miso disappeared with aging.     

Metabolomics-based optimal koji fermentation for tyrosinase inhibition supplemented with Astragalus radix

The present study was focused on improving the quality of rice koji by fermentation with a selected Aspergillus oryzae strain and a plant Astragalus radix. A. oryzae KCCM 60345 was used as main inoculant and the Astragalus radix was added as supplement in rice koji preparation. LC-MS based metabolite analysis and tyrosinase inhibitory activities were studied for different time periods. A. oryzae KCCM 60345 fermented rice koji supplemented with Astragalus showed higher tyrosinase inhibition activity at 4 d of fermentation and metabolite analysis with PCA and PLS-DA indicated differences in kojic acid, calycosin-7-O-β-D-glucoside, ononin, calycosin, and formononetin as compared with other forms of rice koji fermentation. By correlation analysis between metabolites and tyrosinase inhibitory activity, calycosin and kojic acid were identified as major tyrosinase inhibitors. Based on these results, we concluded that A. oryzae KCCM 60345 supplemented with Astragalus radix is useful for whitening effects, and we identified optimal conditions for rice koji preparation.     

雅致放射毛霉液体培养工艺及其多糖的免疫调节作用研究

对雅致放射毛霉液体培养研究表明,其适宜碳源为饴糖和可溶性淀粉,适宜氮源为黄豆粉和酵母膏,最适培养基含黄豆粉3%、饴糖0.5%、磷酸二氢钾0.2%、硫酸镁0.2%、酵母膏0.1%。适宜液体培养条件是温度28℃,接种量5%,发酵前期通风量1∶0.8,之后将风量调至1∶1.5~1∶2.0,全程不搅拌培养至18h~20h,生物量为每100mL发酵液中菌丝鲜重达30g以上。菌丝体氨基酸含量为46.1mg/100mL。免疫调节作用检验表明,雅致放射毛霉多糖具有一定的增强免疫力作用。     

Production of a New Type of Acid Carboxypeptidase of Molds of the Aspergillus niger Group

The ability of 88 fungi, which had been obtained as high-potency strains for acid proteinase production, to produce a new type of acid carboxypeptidase (having on optimal pH of about 3 for hydrolysis of benzyloxycarbonyl-glutamyltyrosine) in surface koji culture was determined. Among the aspergilli, substantial amounts of this new acid carboxypeptidase were produced by Aspergillus saitoi, A. usamii, A. awamori, A. inuii, and A. niger. Maximum yields of acid carboxypeptidase per gram of substrate were obtained by submerged culture in a medium containing 0.9% defatted soybean and 0.6% wheat bran. However, the maximum enzyme concentration per milliliter was obtained with a medium containing 3% defatted soybean and 2% wheat bran. The terminal pH could be controlled by varying the concentrations of soybean oil meal and wheat bran. The maximum enzyme production was reached after 4 days or more at 30 C.     

Indigenous Bacteria and Fungi Drive Traditional Kimoto Sake Fermentations

Sake (Japanese rice wine) production is a complex, multistage process in which fermentation is performed by a succession of mixed fungi and bacteria. This study employed high-throughput rRNA marker gene sequencing, quantitative PCR, and terminal restriction fragment length polymorphism to characterize the bacterial and fungal communities of spontaneous sake production from koji to product as well as brewery equipment surfaces. Results demonstrate a dynamic microbial succession, with koji and early moto fermentations dominated by Bacillus, Staphylococcus, and Aspergillus flavus var. oryzae, succeeded by Lactobacillus spp. and Saccharomyces cerevisiae later in the fermentations. The microbiota driving these fermentations were also prevalent in the production environment, illustrating the reservoirs and routes for microbial contact in this traditional food fermentation. Interrogating the microbial consortia of production environments in parallel with food products is a valuable approach for understanding the complete ecology of food production systems and can be applied to any food system, leading to enlightened perspectives for process control and food safety.     

内生真菌发酵提取物和植物生长调节剂对大豆根际细菌多样性的影响

为了研究内生真菌发酵提取物和植物生长调节剂对大豆根际细菌多样性的影响,采用PCR-DGGE技术分析了各处理中不同发育期的大豆根际细菌群落变化。结果发现发酵提取液和植物生长调节剂能增加部分优势菌群的数量,但对根际细菌类群结构影响并不明显;生育周期也是影响根际细菌数量的重要因素。另外割胶测序发现优势菌群主要是Proteobacteria(变形菌门)、Acidobacteria(酸杆菌纲)、Nitrospira(硝化螺旋菌属)、Bradyrhizobium(慢生根瘤菌属)等,这些也都是大豆根际比较常见的细菌类群。     

Production of red mold rice using a modified Nagata type koji maker

In this research, a commercial koji maker with a rotary perforated bed of 5-m diameter was modified for red mold rice production. Monascus purpureus BCRC 31499 was selected for its high production capacities of monacolin K and red pigment. The selected strain was first cultivated in a 120-l submerged type fermentor at 34°C and 2 vvm aeration rate with 60 rpm agitation for 5 days using 20% liquefied rice porridge as carbon source. The high concentration red mold rice broth (>3.5 g/ml) was harvested for inocula and well mixed with cooked rice to an initial concentration of 2% v/w. The inoculated cooked rice then was directed into the modified koji maker, in which temperature and humidity profiles were kept at varied levels at different stages, respectively. Air was circulated to remove fermentation heat while the perforated bed rotated slowly for providing mild agitation. Lag phase of the Monascus sp. in the modified koji maker was determined to be 16 h by the time the koji temperature raised rapidly. Water was added into the koji bed by a water curtain at the 36th hour to keep the moisture content of the rice koji at 50% or above. At the final stage, temperature was adjusted to 34°C to direct red pigment production. After 7 days, 1,200-kg high quality red mold rice was harvested per batch. Labor costs, space, and fermentation time were reduced tremendously compared with those made by traditional methods.     

Dual Role for the Tyrosine Decarboxylation Pathway in Enterococcus faecium E17: Response to an Acid Challenge and Generation of a Proton Motive Force

In this work we investigated the role of the tyrosine decarboxylation pathway in the response of Enterococcus faecium E17 cells to an acid challenge. It was found that 91% of the cells were able to remain viable in the presence of tyrosine when they were incubated for 3 h in a complex medium at pH 2.5. This effect was shown to be related to the tyrosine decarboxylation pathway. Therefore, the role of tyrosine decarboxylation in pH homeostasis was studied. The membrane potential and pH gradient, the parameters that compose the proton motive force (PMF), were measured at different pHs (pH 4.5 to 7). We obtained evidence showing that the tyrosine decarboxylation pathway generates a PMF composed of a pH gradient formed due to proton consumption in the decarboxylation reaction and by a membrane potential which results from electrogenic transport of tyrosine in exchange for the corresponding biogenic amine tyramine. The properties of the tyrosine transporter were also studied in this work by using whole cells and right-side-out vesicles. The results showed that the transporter catalyzes homologous tyrosine/tyrosine antiport, as well as electrogenic heterologous tyrosine-tyramine exchange. The tyrosine transporter had properties of a typical precursor-product exchanger operating in a proton motive decarboxylation pathway. Therefore, the tyrosine decarboxylation pathway contributes to an acid response mechanism in E. faecium E17. This decarboxylation pathway gives the strain a competitive advantage in nutrient-depleted conditions, as well as in harsh acidic environments, and a better chance of survival, which contributes to higher cell counts in food fermentation products.     

豆粕营养成分对蝙蝠蛾拟青霉发酵产物的影响

为探索培养基中的豆粕以及制备豆粕的原材料大豆对蝙蝠蛾拟青霉发酵产物的影响,我们比较了不同品种大豆和用其制成的豆粕的主要营养成分含量,包括粗蛋白、粗脂肪、6种微量元素（钙、镁、铜、锌、铁、锰）和总异黄酮（大豆苷、黄豆黄苷、染料木苷、大豆苷元、黄豆黄素、染料木素）,以及豆粕作为培养基对蝙蝠蛾拟青霉发酵产物的生物量和有效成分含量包括腺苷、腺嘌呤、虫草素和麦角甾醇的影响,结果表明大豆中钙和大豆苷与发酵产物中腺苷含量正相关。豆粕中钙、大豆苷、大豆苷元、染料木素与发酵产物中腺苷含量正相关;豆粕中钙、镁、大豆苷、大豆苷元、染料木素、总异黄酮与发酵产物中腺嘌呤含量负相关;豆粕中铁与菌丝体干重值正相关。结果表明,培养基中的豆粕和原料大豆主要营养成分含量对蝙蝠蛾拟青霉发酵产物品质有显著影响。     

豆粕发酵制备大豆肽菌株的筛选

微生物发酵豆粕可以降解大豆蛋白为大豆肽,试验以蛋白酶活力和水解度为指标,结合液体发酵和固体发酵工艺考察菌株的发酵能力,从而筛选优良的豆粕发酵菌株。结果表明,通过初筛和复筛获得3株能较好发酵豆粕的芽胞菌菌株,为豆粕发酵工艺的研究奠定了基础。     

Comparison of Metabolites Variation and Antiobesity Effects of Fermented versus Nonfermented Mixtures of Cudrania tricuspidata,Lonicera caerulea,and Soybean According to Fermentation In Vitro and In Vivo

We used ultra-performance-liquid-chromatography with quadrupole-time-of-flight mass spectrometry to study the changes in metabolites in the mixture of Cudrania tricuspidata, Lonicera caerulea, and soybean (CLM) during fermentation. Additionally, the antiobesity effects of CLM and fermented-CLM (FCLM) were studied based on the analysis of plasma from high-fat diet (HFD)-fed mice. The levels of cyanidin and the glycosides of luteolin, quercetin, and cyanidin derived from L. caerulea were decreased, whereas the levels of luteolin and quercetin were increased during fermentation. Isoflavone glycosides and soyasaponins originating from the soybean were decreased, whereas their aglycones such as daidzein, glycitein, and genistein were increased. As for prenylated flavonoids from C. tricuspidata, these metabolites were decreased at the early stage of fermentation, and were increased at end of the fermentation. In terms of the functional food product, various metabolites derived from diverse natural products in CLM had complementary effects and demonstrated higher antioxidant and pancreatic lipase inhibition activities after fermentation; these activities were closely related to flavonoid aglycones including genistein, daidzein, glycitein, luteolin, and quercetin. In an in vivo experiment, several clinical parameters affected by HFD were improved by the administration of either CLM or FCLM, but there was a difference in the antiobesity effects. The levels of lysoPCs with C20:4, C16:0, and C22:6 were significantly attenuated by CLM administration, while the attenuated levels of lysoPCs with C20:4 and C18:2 were significantly restored by FCLM administration. These metabolites may explain the above-mentioned differences in antiobesity effects. Although only the changes in plasma lysophospholipids could not fully explain antiobesity effects between non-fermented and fermented plant mixtures from our results, we suggest that metabolomics approach could provide a way to reveal the metabolite alterations in the complex fermentation process and understand the differences or changes in bioactivity according to fermentation.     

Genomic Features and Niche-Adaptation of Enterococcus faecium Strains from Korean Soybean-Fermented Foods

Certain strains of Enterococcus faecium contribute beneficially to human health and food fermentation. However, other E. faecium strains are opportunistic pathogens due to the acquisition of virulence factors and antibiotic resistance determinants. To characterize E. faecium from soybean fermentation, we sequenced the genomes of 10 E. faecium strains from Korean soybean-fermented foods and analyzed their genomes by comparing them with 51 clinical and 52 non-clinical strains of different origins. Hierarchical clustering based on 13,820 orthologous genes from all E. faecium genomes showed that the 10 strains are distinguished from most of the clinical strains. Like non-clinical strains, their genomes are significantly smaller than clinical strains due to fewer accessory genes associated with antibiotic resistance, virulence, and mobile genetic elements. Moreover, we identified niche-associated gene gain and loss from the soybean strains. Thus, we conclude that soybean E. faecium strains might have evolved to have distinctive genomic features that may contribute to its ability to thrive during soybean fermentation.     

Characterization of liposomes coated with S-layer proteins from lactobacilli

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein-lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.     

Influence of prebrining treatments on microbial and biochemical changes during the baceman stage in Indonesian kecap (soy sauce) production

Prior to the baceman stage or brine fermentation in Indonesian kecap (soy sauce) production, the soybeans undergo a number of treatments, such as soaking, cooking and a fungal solid state fermentation (bungkil stage). The influence of these prebrining steps on the baceman stage was investigated. Acidification during soaking inhibited bacterial growth during the solid state fermentation, causing lower initial numbers of bacteria in and a slower acidification of the baceman. Cooking time influenced the softness of the beans and thus fungal growth during the bungkil stage. Increasing the cooking time from 1 to 3 h resulted in a better solubility of macromolecules and gave higher final concentrations of fermentation products and formol nitrogen in baceman. When cooking time was increased from 3 to 5 h only slight differences could be observed. Length of the bungkil stage had some minor influences; the amount of fermentation products and residual monosaccharides decreased by 16% when the length was increased from 2 to 7 d. The highest amounts of formol nitrogen were found with 5 d of bungkil fermentation. The addition of rice flour showed that the absence of yeast fermentation in baceman is related to the lack of sugars after the previous lactic acid fermentation. In addition to yeast fermentation, the growth of fungi during the bungkil stage was also positively influenced by the addition of rice flour. Sun drying and crumbling bungkil caused lower enzyme activities and resulted in lower amounts of formol nitrogen and glycerol. Growth of Pediococcus halophilus during the baceman stage was faster when bungkil was sun dried, although the final concentrations of fermentation products were lower due to a lower content of amino acids which resulted in less buffering. Increasing the amount of bungkil added to salty water caused a similar increase in formol nitrogen and fermentation products.