Starter Culture Selection for Making Chinese Sesame-Flavored Liquor Based on Microbial Metabolic Activity in Mixed-Culture Fermentation

Selection of a starter culture with excellent viability and metabolic activity is important for inoculated fermentation of traditional food. To obtain a suitable starter culture for making Chinese sesame-flavored liquor, the yeast and bacterium community structures were investigated during spontaneous and solid-state fermentations of this type of liquor. Five dominant species in spontaneous fermentation were identified: Saccharomyces cerevisiae, Pichia membranaefaciens, Issatchenkia orientalis, Bacillus licheniformis, and Bacillus amyloliquefaciens. The metabolic activity of each species in mixed and inoculated fermentations of liquor was investigated in 14 different cocultures that used different combinations of these species. The relationships between the microbial species and volatile metabolites were analyzed by partial least-squares (PLS) regression analysis. We found that S. cerevisiae was positively correlated to nonanal, and B. licheniformis was positively associated with 2,3-butanediol, isobutyric acid, guaiacol, and 4-vinyl guaiacol, while I. orientalis was positively correlated to butyric acid, isovaleric acid, hexanoic acid, and 2,3-butanediol. These three species are excellent flavor producers for Chinese liquor. Although P. membranaefaciens and B. amyloliquefaciens were not efficient flavor producers, the addition of them alleviated competition among the other three species and altered their growth rates and flavor production. As a result, the coculture of all five dominant species produced the largest amount of flavor compounds. The result indicates that flavor producers and microbial interaction regulators are important for inoculated fermentation of Chinese sesame-flavored liquor.     

Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens

Aims: To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product‐derived Bacillus amyloliquefaciens. Methods and Results: An unknown bacterial species cultured from the Yogu Farm? probiotic dairy beverage was identified through 16S ribosomal RNA analysis as B. amyloliquefaciens, a phylogenetically close relative of Bacillus subtilis. The cell‐free supernatant (CFS) of overnight cultures was active against Listeria monocytogenes and also against clinical isolates of Gardnerella vaginalis and Streptococcus agalactiae. At the same time, several isolates of vaginal probiotic Lactobacilli were resistant to the CFS. The nature of the compound causing inhibitory activity was confirmed as proteinaceous by enzymatic digestion. The protein was isolated using ammonium sulfate precipitation, and further purified via column chromatography. PCR analysis was conducted to determine relatedness to other bacteriocins produced by Bacillus spp. Conclusion: The antimicrobial protein isolated from B. amyloliquefaciens was shown to be subtilosin, a bacteriocin previously reported as produced only by B. subtilis. Significance and Impact of the Study: This is the first report of intra‐species horizontal gene transfer for subtilosin and the first fully characterized bacteriocin isolated from B. amyloliquefaciens. Finally, this is the first report on subtilosin’s activity against bacterial vaginosis‐associated pathogens.     

接种发酵和自然发酵中酿酒酵母菌株多样性比较

[目的]探究自然发酵和接种发酵两种发酵方式,对霞多丽葡萄发酵中酵母菌种多样性和酿酒酵母菌株遗传多样性的影响.[方法]以霞多丽葡萄为原料,分别进行自然发酵和接种不同酿酒酵母菌株(NXU 17-26、UCD522和UCD2610)的发酵,利用26S rDNA D1/D2区序列分析和Interdelta指纹图谱技术分别进行酵...     

解淀粉芽孢杆菌诱变育种及其突变株在降低酱油中氨基甲酸乙酯的应用

【目的】通过诱变育种提高解淀粉芽孢杆菌JY06利用精氨酸的能力,并将其用于降低酱油中的氨基甲酸乙酯及前体,从而提高酿造酱油的安全性。【方法】采用等离子诱变和紫外诱变两种诱变育种方法对解淀粉芽孢杆菌JY06进行突变,应用高通量筛选手段获得具有高精氨酸利用能力的突变株,验证突变株降低酱油中氨基甲酸乙酯的能力。【结果】获得了12株精氨酸利用能力提高的突变株,与出发菌株JY06相比,突变株C12和E6可使酱油中瓜氨酸含量分别降低了15.6%和14.7%,EC的含量分别降低了19.3%和13.1%。【结论】通过等离子诱变和紫外诱变进一步提高了解淀粉芽孢杆菌JY06降低酱油中EC及其前体瓜氨酸的能力,具有控制或减少酱油中生物危害物的应用潜力。     

Prediction of problematic wine fermentations using artificial neural networks

Artificial neural networks (ANNs) have been used for the recognition of non-linear patterns, a characteristic of bioprocesses like wine production. In this work, ANNs were tested to predict problems of wine fermentation. A database of about 20,000 data from industrial fermentations of Cabernet Sauvignon and 33 variables was used. Two different ways of inputting data into the model were studied, by points and by fermentation. Additionally, different sub-cases were studied by varying the predictor variables (total sugar, alcohol, glycerol, density, organic acids and nitrogen compounds) and the time of fermentation (72, 96 and 256 h). The input of data by fermentations gave better results than the input of data by points. In fact, it was possible to predict 100% of normal and problematic fermentations using three predictor variables: sugars, density and alcohol at 72 h (3 days). Overall, ANNs were capable of obtaining 80% of prediction using only one predictor variable at 72 h; however, it is recommended to add more fermentations to confirm this promising result.     

A model of xylitol production by the yeast Candida mogii

Production of xylitol from xylose in batch fermentations of Candida mogii ATCC 18364 is discussed in the presence of glucose as the cosubstrate. Various initial ratios of glucose and xylose concentrations are assessed for their impact on yield and rate of production of xylitol. Supplementation with glucose at the beginning of the fermentation increased the specific growth rate, biomass yield and volumetric productivity of xylitol compared with fermentation that used xylose as the sole carbon source. A mathematical model is developed for eventual use in predicting the product formation rate and yield. The model parameters were estimated from experimental observations, using a genetic algorithm. Batch fermentations, which were carried out with xylose alone and a mixture of xylose and glucose, were used to validate the model. The model fitted well with the experimental data of cell growth, substrate consumption and xylitol production.     

The unconventional adverse effects of fungal pretreatment on iturin A fermentation by Bacillus amyloliquefaciens CX-20

Fungal pretreatment is the most common strategy for improving the conversion of rapeseed meal (RSM) into value-added microbial products. It was demonstrated that Bacillus amyloliquefaciens CX-20 could directly use RSM as the sole source of all nutrients except the carbon source for iturin A fermentation with high productivity. However, whether fungal pretreatment has an impact on iturin A production is still unknown. In this study, the effects of fungal pretreatment and direct bio-utilization of RSM for iturin A fermentation were comparatively analysed through screening suitable fungal species, and evaluating the relationships between iturin A production and the composition of solid fermented RSM and liquid hydrolysates. Three main unconventional adverse effects were identified. (1) Solid-state fermentation by fungi resulted in a decrease of the total nitrogen for B. amyloliquefaciens CX-20 growth and metabolism, which caused nitrogen waste from RSM. (2) The released free ammonium nitrogen in liquid hydrolysates by fungal pretreatment led to the reduction of iturin A. (3) The insoluble precipitates of hydrolysates, which were mostly ignored and wasted in previous studies, were found to have beneficial effects on producing iturin A. In conclusion, our study verifies the unconventional adverse effects of fungal pretreatment on iturin A production by B. amyloliquefaciens CX-20 compared with direct bio-utilization of RSM.     

Diversity of the total bacterial community associated with Ghanaian and Brazilian cocoa bean fermentation samples as revealed by a 16 S rRNA gene clone library

Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, starting with yeasts, followed by lactic acid bacteria and acetic acid bacteria. So far, all microbiological studies about cocoa bean fermentation were based on culture-dependent (isolation, cultivation, and identification), or, more recently, culture-independent (PCR-DGGE, or polymerase chain reaction denaturing gradient gel electrophoresis) methods. Using a metagenomic approach, total DNA was extracted from heap and box fermentations at different time points and from different locations (Ghana and Brazil, respectively) to generate a 16 S rDNA clone library that was sequenced. The sequencing data revealed a low bacterial diversity in the fermentation samples and were in accordance with the results obtained through culture-dependent and a second, culture-independent analysis (PCR-DGGE), suggesting that almost all bacteria involved in the fermentation process are cultivable. One exception was the identification by 16 S rDNA library sequencing of Gluconacetobacter species of acetic acid bacteria that were not detected by the two other approaches. The presence of Enterobacteriaceae related to Erwinia/Pantoea/Tatumella, as revealed by 16 S rDNA library sequencing, suggests an impact of these bacteria on fermentation.     

Bacterial biodiversity and dynamics during malolactic fermentation of Tempranillo wines as determined by a culture-independent method (PCR-DGGE)

The bacterial population during malolactic fermentation of Tempranillo wine was studied using the polymerase chain reaction-denaturing gradient gel electrophoresis, a culture-independent method successfully used for identification and monitoring of bacterial population in different habitats included food fermentations. The results showed that Oenococcus oeni was the predominant species in the malolactic fermentation of Tempranillo wines, although the presence of Gluconobacter oxydans, Asaia siamensis, Serratia sp., and Enterobacter sp. was also observed. These results were partly coincidental with those obtained from a culture-dependent method, using a selective medium. Therefore, it may be concluded that for a more complete knowledge of the bacterial community present during malolactic fermentation of Tempranillo wine, an approach that combines a culture-independent method and a culture-dependent method would be advisable.     

生防解淀粉芽胞杆菌的研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)因能抑制植物病害和促进植物生长而被广泛研究。随着测序技术的不断发展,解淀粉芽胞杆菌菌株的全基因组序列陆续被测定,综述了其拮抗作用相关的功能基因、生防机制及植物-病原物-生防菌的相互作用,并对后续研究趋势进行了展望,为解淀粉芽胞杆菌的深入研究及其更好的应用提供理论参考     

养猪微生物发酵床芽胞杆菌空间分布多样性

了解微生物发酵床大栏养猪垫料中的芽胞杆菌多样性和空间分布规律,为微生物发酵床管理、芽胞杆菌新资源挖掘及菌剂开发奠定基础。将发酵床划分为32个方格(4行×8列),采用五点取样法获得每个方格的样品。采用可培养法从32份样品中分离芽胞杆菌菌株,利用16S rRNA基因序列初步鉴定所分离获得的芽胞杆菌种类。利用聚集度指标和回归分析法,分析芽胞杆菌的样方空间分布型。通过Shannon-Wiener多样性指数、Simpson优势度指数、Hill指数及丰富度指数分析,揭示微生物发酵床中芽胞杆菌的空间分布多样性。从32份样品中共获得芽胞杆菌452株,16S rRNA基因鉴定结果表明它们分别隶属于芽胞杆菌纲的2个科、8个属、48个种。其中,种类最多的为芽胞杆菌属(Bacillus),30种;赖氨酸芽胞杆菌属(Lysinibacillus),6种;类芽胞杆菌属(Paenibacillus),5种;短芽胞杆菌属(Brevibacillus),3种;鸟氨酸芽胞杆菌属(Ornithinibacillus)、大洋芽胞杆菌属(Oceanibacillus)、少盐芽胞杆菌属(Paucisalibacillus)和纤细芽胞杆菌属(Gracilibacillus)各1个种。芽胞杆菌种类在发酵床空间分布差异很大,根据其空间出现频次,可分为广分布种类,如地衣芽胞杆菌(Bacillus licheniformis);寡分布种类,如根际芽胞杆菌(B.rhizosphaerae);少分布种类,如弯曲芽胞杆菌(B.flexus)。依据其数量,可分为高含量组优势种群,如地衣芽胞杆菌(B.licheniformis);中含量组常见种群,耐盐赖氨酸芽胞杆菌(Lysinibacillus halotolerans);寡含量组寡见种群,如根际芽胞杆菌(B.rhizosphaerae);低含量组偶见种群,如土地芽胞杆菌(B.humi)。空间分布型聚集度和回归分析测定表明,芽胞杆菌在微生物发酵床的分布类型为聚集分布。微生物发酵床垫料中芽胞杆菌种类总含量高达4.41×108个/g,其种类含量范围为0.01—94.1×106个/g(均值为8.96×106个/g),丰富度指数(D)、优势度指数(λ)、Shannon-Wiener指数(H')和均匀度指数(J')分别为0.4928、0.2634、1.3589和0.9803,其中香农指数最大的单个芽胞杆菌种类为地衣芽胞杆菌(B.licheniformis)。根据芽胞杆菌种类多样性指数聚类分析,当欧式距离λ=17时,可分为高丰富度高含量和低丰富度低含量类型。微生物发酵床的芽胞杆菌种类丰富、数量高,是一个天然的菌剂"发酵罐",有望直接作为微生物菌剂,应用于土壤改良、作物病害防控、污染治理等领域。     

Dynamics of bacterial communities during solid-state fermentation using agro-industrial wastes to produce poly-γ-glutamic acid,revealed by real-time PCR and denaturing gradient gel electrophoresis (DGGE)

The dynamics of bacterial communities play an important role in solid-state fermentation (SSF). Poly-γ-glutamic acid (γ-PGA) was produced by Bacillus amyloliquefaciens C1 in SSF using dairy manure compost and monosodium glutamate production residuals as basic substrates. The production of γ-PGA reached a maximum of 0.6% after 20 days fermentation. Real-time polymerase chain reaction showed the amount of total bacteria reached 3.95 × 10⁹ 16S rDNA copies/g sample after 30 days, which was in good accordance with the 4.80 × 10⁹ CFU/g obtained by plate counting. Denaturing gradient gel electrophoresis profile showed a reduction of microbial diversity during fermentation, while the inoculum, B. amyloliquefaciens C1, was detected as the dominant organism through the whole process. In the mesophilic phase of SSF, Proteobacteria was the dominant microbial, which was replaced by Firmicutes and Actinobacteria in the thermophilic phase. The molecular analysis of the bacterial diversity has significant potential for instructing the maturing process of SSF to produce γ-PGA at a large-scale level, which could be a benefit in the production of high quality and stable SSF products.     

Patagonian wines: implantation of an indigenous strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in fermentations conducted in traditional and modern cellars

In this work we evaluate the implantation capacity of the selected S. cerevisiae indigenous strain MMf9 and the quality of the produced wines in a traditional (T) and a modern (M) cellar with different ecological and technological characteristics in North Patagonia (Argentina). Red musts were fermented in 10,000 l vats using the indigenous strain MMf9 as well as the respective controls: a fermentation conducted with a foreign starter culture (BC strain) in M cellar and a natural fermentation in T cellar. Since commercial S. cerevisiae starters are always used for winemaking in M cellar and in order to compare the results, natural fermentations and fermentations conducted by the indigenous strain MMf9 were performed at pilot (200 l) scale in this cellar, concomitantly. Thirty indigenous yeasts were isolated at three stages of fermentation: initial, middle and end. The identification of the yeast biota associated to vinifications was carried out using ITS1-5.8S-ITS2 PCR-RFLP. The intra-specific variability of the S. cerevisiae populations was evaluated using mtDNA-RFLP analysis. Wines obtained from all fermentations were evaluated for their chemical and volatile composition and for their sensory characteristics. A higher capacity of implantation of the indigenous MMf9 strain was evidenced in the fermentation carried out in M cellar (80% at end stage) than the one carried out in T cellar (40%). This behaviour could indicate that each cellar differs in the diversity of S. cerevisiae strains associated to wine fermentations. Moreover a higher capacity of implantation of the native starter MMf9 with regard to the foreign (BC) one was also found in M cellar. The selected indigenous strain MMf9 was able to compete with the yeast biota naturally present in the must. Additionally, a higher rate of sugar consumption and a lower fermentation temperature were observed in vinifications conducted by MMf9 strain with regard to control fermentations, producing wines with favourable characteristics. Even when its implantation in T fermentation was lower than that observed in M one, we can conclude that the wine features from MMf9 fermentations were better than those from their respective controls. Therefore, MMf9 selected indigenous strain could be an interesting yeast starter culture in North Patagonian wines.     

Pilot-scale production of carboxymethylcellulase from rice hull by Bacillus amyloliquefaciens DL-3

Optimal conditions for pilot-scale production of the carboxymethylcellulase (CMCase) by Bacillus amyloliquefaciens DL-3 were investigated. The best carbon and nitrogen sources for the production of CMCase by B. amyloliquefaciens DL-3 were found to be rice hull and peptone and their optimal concentrations were 5.0 and 0.20% (w/v), respectively. Optimal temperature and initial pH for the production of CMCase were 37°C and 6.8. Optimal agitation speed and aeration rate for the production of CMCase were 300 rpm and 1.0 vvm in a 7 L bioreactor, which were different from those for the cell growth of B. amyloliquefaciens DL-3. The highest productions of CMCase by B. amyloliquefaciens DL-3 from 5.0% (w/v) rice hull as a carbon source under optimal conditions in a 7 or 100 L bioreactor were 220 and 367 U/mL, respectively.     

芽胞杆菌BJ-6的鉴定及对甜瓜细菌性果斑病的防治

芽胞杆菌是目前植物病害生物防治研究最多的一类微生物,其在自然界中分布广泛,开发潜力大。【目的】为了探究从杏树根际土壤分离的芽胞杆菌BJ-6的分类地位及其防病促生作用。【方法】本研究测定了芽胞杆菌BJ-6的形态和生理生化特征,通过PCR扩增了该菌的16S rRNA、gyrA和gyrB基因并进行了序列测定,通过多基因聚类分析确定其分类地位,平板对峙法测定抗菌谱,盆栽幼苗实验验证其对甜瓜细菌性果斑病的防治效果和对甜瓜的促生作用。【结果】结合形态特征、生理生化特性及多基因序列分析建立的系统进化树,确定菌株BJ-6为解淀粉芽胞杆菌(B. amyloliquefaciens),抑菌实验发现该菌株对15种植物病原菌均有不同程度的抑菌活性,盆栽实验结果发现该菌株发酵液对甜瓜细菌性果斑病有很好的防治效果,并对甜瓜苗有很好的促生作用。【结论】BJ-6属于解淀粉芽胞杆菌,抑菌谱广,且具有防病促生作用,具有进一步开发为生防制剂的前景。     

Isolation and characterization of antagonistic bacteria against bacterial leaf spot of Euphorbia pulcherrima

Aims: To investigate the inhibition potential of leaf‐associated bacteria against the pathogen of bacterial leaf spot of Euphorbia pulcherrima. Methods and Results: Seven out of 200 bacterial strains were effective antagonists by in vitro screening and the two strains PAB241 and PAB242 significantly reduced the disease incidence and severity as foliar treatments of E. pulcherrima. The two effective strains, PAB241 and PAB242, were both identified as Bacillus amyloliquefaciens by a polyphasic approach including phenotypic feature, carbon source utilization profile, fatty acid methyl esters and analysis of 16S rRNA gene sequence. In addition, the suspensions of B. amyloliquefaciens PAB241 and PAB242 showed antibacterial activities against the pathogen of bacterial leaf spot of E. pulcherrima under different treatments. Conclusions: The leaf‐associated bacteria, B. amyloliquefaciens PAB241 and PAB242, markedly inhibited the growth of X. axonopodis pv. poinsettiicola under different treatments and protected E. pulcherrima from pathogen infection in growth chamber conditions. Significance and Impact of the Study: This is the first study that showed B. amyloliquefaciens from plant leaves was a potential bactericide against bacterial leaf spot of E. pulcherrima.     

Effect of ragi and Lxxx-lactate-producing cultures on enteric pathogens in a rice-based weaning food

The use of the Southeast-Asian starter culture ragi in enhancing the safety of a rice-based model weaning food is described and compared with the use of diastatic malt extract. Ragi was shown to be an effective saccharifying agent convenient for use in weaning-food preparation on a domestic scale. Saccharification and fermentation with ragi alone produced some antimicrobial effect against the three enteric bacterial pathogens tested but this was much improved when ragi was used in conjunction with the Lxxx-lactate-producing Lactococcus lactis or Lactobacillus bavaricus. The latter showed the greatest inhibition of pathogens, reducing viable numbers by more than a factor of 10⁴ within 4 h. The antibacterial effects observed correlated with the total acid produced (ragi alone giving 0.2%; ragi with Lc. lactis giving 0.3% and ragi with Lb. bavaricus giving 0.4%). The proportion of the physiological Lxxx-lactate isomer was highest in Lc. lactis fermentations (>99% compared with 80% with Lb. bavaricus). There was no evidence of any pronounced antimicrobial effect due to the nisin produced during fermentation by Lc. lactis (150 IU/g). Whereas bacteriocin production may play little role in pathogen control, it may be desirable as a way of preventing fermentations conducted non-aseptically from becoming dominated by lactic acid bacteria producing unacceptable amounts of Dxxx-lactate.R.M. Yusof is with the Department of Nutrition and Community Health, Faculty of Human Ecology, University Pertanian Malaysia, 43400 UPM, Serdang, Selangor, Malaysia. T.A. Baker, J.B. Morgan and M.R. Adams are with the School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK.     

Assessment of the Role of Chemotaxis and Biofilm Formation as Requirements for Colonization of Roots and Seeds of Soybean Plants by <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> BNM339

This article correlates colonization with parameters, such as chemotaxis, biofilm formation, and bacterial growth, that are believed to be connected. We show here, by using two varieties of soybean plants that seeds axenically produced exudates, induced a chemotactic response in Bacillus amyloliquefaciens, whereas root exudates did not, even when the exudates, also collected under axenic conditions, were concentrated up to 200-fold. Root exudates did not support bacterial cell division, whereas seed exudates contain compounds that support active cell division and high cell biomass at stationary phase. Seed exudates of the two soybean varieties also induced biofilm formation. B. amyloliquefaciens colonized both seeds and roots, and plant variety significantly affected bacterial root colonization, whereas it did not affect seed colonization. Colonization of roots in B. amyloliquefaciens occurred despite the lack of chemotaxis and growth stimulation by root exudates. The data presented in this article suggest that soybean seed colonization, but not root colonization, by B. amyloliquefaciens is influenced by chemotaxis, growth, and biofilm formation and that this may be caused by qualitative changes of the composition of root exudates.     

Cell-recycle batch fermentation using immobilized cells of flocculent Saccharomyces cerevisiae wine strains

Five, highly flocculeng strains of Saccharomyces cerevisiae, isolated from wine, were immobilized in calcium alginate beads to optimize primary must fermentation. Three cell-recycle batch fermentations (CRBF) of grape musts were performed with the biocatalyst and the results compared with those obtained with free cells. During the CRBF process, the entrapped strains showed some variability in the formation of secondary products of fermentation, particularly acetic acid and acetaldehyde. Recycling beads of immobilized flocculent cells is a good approach in the development and application of the CRBF system in the wine industry.     

Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

A Bacillus amyloliquefaciens neutral protease gene was cloned and expressed in Bacillus subtilis.The chromosomal DNA of B. amyloliquefaciens strain F was partially digested with restriction endonuclease Sau3AI, and 2 to 9 kb fragments isolated were ligated into the BamHI site of plasmid pUB110. Then, B. subtilis strain 1A289 was transformed with the hybrid plasmids by the method of protoplast transformation and kanamycin-resistant transformants were screened for the formation of large halo on a casein plate. A transformant that produced a large amount of an extracellular neutral protease harbored a plasmid, designated as pNP150, which contained a 1.7 kb insert.The secreted neutral protease of the transformant was found to be indistinguishable from that of DNA donor strain B. amyloliquefaciens by double immunodiffusion test and SDS-polyacrylamide gel electrophoresis.The amount of the neutral protease activity excreted into culture medium by the B. subtilis transformed with pNP150 was about 50-fold higher than that secreted by B. amyloliquefaciens. The production of the neutral protease in the transformant was partially repressed by addition of glucose to the medium.