NISIN: A BACTERIOCIN WITH A POTENTIAL USE IN BREWING

Depending on the amount used and the strain of bacteria involved, nisin either kills lactic acid bacteria or inhibits their growth. In medium inoculated with approximately 10⁵ cells ml^?1 of a sensitive strain of Lactobacillus (BSO 375) nisin, added at levels recommended for commercial use (100 International Units ml^?1, killed all the cells in less than 6 h. In the absence of nisin this inoculum grew to a concentration of 10¹⁰ cells ml^?1 in about 50 h. Lower nisin concentrations killed fewer cells but inhibited the growth of those still viable. For the more resistant strain Lactobacillus (BSO 343) growth was only inhibited at the higher nisin concentrations. Nisin maintained its activity against lactic acid bacteria in brewing fermentations. It had no effect on the growth and fermentative performance of the 9 brewing yeast strains tested, and, in a pilot brewery fermentation, had no deleterious affect on the taste of the beer produced. Nisin could be used either as a preventative measure by regular addition to fermentations, or as a remedial measure once contamination by lactic acid bacteria had been detected.     

NISIN AND BREWING

Nisin, an internationally accepted food preservative has been shown to inhibit the growth of almost all Gram-positive beer-spoilage bacteria investigated. The initial effects on these bacteria appear to be upon the cell membrane. Nisin has little effect on most Gram-negative bacteria and has no effect upon the growth and fermentation properties of brewing yeasts. Nisin activity survives kieselguhr filtration, fining and pasteurisation and has no effect on beer shelf life. Nisin has potential applications in preventing spoilage of worts or beers by Gram-positive bacteria (particularly lactic acid bacteria).     

高产Nisin和高产LAB EPS乳酸菌共生株筛查

通过对10株产Nisin乳酸菌株和10株产LAB EPS乳酸菌株进行培养、筛选和测定,筛选出3株高产Nisin的乳酸菌株和3株高产LABEPS的乳酸菌株,进而对3株高产LAB EPS乳酸菌株和3株高产Nisin乳酸菌株进行复合发酵试验,最终筛选出可共生的高产Nisin乳酸菌株和高产LAB EPS乳酸菌株各1株。     

溶氧(DO)对混菌发酵生产Nisin影响的研究

Nisin(乳酸链球菌素)是由乳酸菌产生的一种抑菌活性肽。其在生产过程中需要控制发酵体系的pH,以避免由于乳酸菌代谢积累的乳酸导致pH迅速降低抑制菌体生长。本文采用以能够代谢消耗乳酸的酵母菌为辅助发酵菌的新型混菌发酵方法,在小试条件下研究了在混菌发酵过程中对菌体特别是酵母菌代谢有显著影响的因素溶氧(DO)对混菌发酵生产过程中发酵体系的pH与乳酸含量的变化nisin产量的影响。结果表明,不同水平溶氧对混菌发酵体系中两种菌的生长及代谢产物生成有一定影响。DO 10%与DO 30%时的nisin效价的水平波动范围为24.35%;在分阶段控制发酵体系溶氧水平的条件下获得了较高的nisin效价,效价水平比恒定溶氧提高45.38%。说明分阶段控制发酵体系溶氧水平是较为理想的溶氧控制策略。     

Identification of microorganisms producing lactic acid during solid‐state fermentation of Maotai flavour liquor

Lactic acid is the main acid produced during the Maotai liquor brewing process, influencing the quality of the base liquor and fermentation process. However, the microorganisms responsible for lactic acid production have not been identified. In this work, the dynamic changes in bacterial community structure in the Zaosha round (second sorghum feeding and fermentation) of the brewing process were analysed by 16S rRNA high‐throughput sequencing. Results show that lactic acid bacteria (LAB) and Bacillus spp. are the dominant bacteria in the brewing process, where Bacillus spp. are found in the early stage, whilst LAB are found throughout the brewing process. Furthermore, 10 types of LAB and five Bacillus spp. were isolated from Zaopei (a mixture of fermented grains including sorghum and wheat) by a culture‐dependent method. Lactobacillus panis accounts for 68% of the LAB, and Bacillus amyloliquefaciens for 54% of Bacillus spp. Solid‐state fermentation experiments were performed with L. panis and B. amyloliquefaciens and lactic acid production was consistent with the accumulation of lactic acid in Zaosha. The results showed that L. panis was the main producer of lactic acid in pits, while B. amyloliquefaciens plays an important role in the production of lactic acid in the early stages of fermentation. The approach used in this study may facilitate the identification of key microorganisms with specific functionality involved in other food and beverage fermentation processes. © 2018 The Institute of Brewing & Distilling     

清香型白酒酒醅乳酸菌分离鉴定及其自溶特性研究

采用MRS-碳酸钙培养基从清香酒醅中筛选出具有明显溶钙圈的47株细菌,通过生理生化特征及16S rDNA序列比对分析鉴定,47株乳酸菌中有20株短乳杆菌(Lactobacillus brevis),16株植物乳杆菌(L.plantarum),7株布氏乳杆菌(L.buchneri)和4株戊糖乳杆菌(L.pentosus)。利用比浊法对47株乳酸菌自溶度进行测定,其自溶度为5.01%~29.27%,自溶度大小与菌种没有明显相关性,表现出自溶度菌株特异性。进一步考察了自溶度较高的9株乳酸菌的自溶特性,发现大多数菌株的自溶度随环境温度和pH的升高而增大。     

Microbiological Analyses of Dry and Slurry Yeasts for Brewing

Classical microbiological methods in association with molecular methods (DNA amplification, Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) were used. These methods, developed to rapidly analyze microbial communities on the basis of sequence‐specific separation of DNA amplicons, allowed the detection of DNA differences in the amplicons tested and the identification of the strains analyzed by the comparison of unknown sequences with sequences of known species. TGGE allowed the comparison of the different Saccharomyces cerevisiae strains used in brewing while DGGE allowed the identification of lactic acid bacteria (LAB) in beer. These methods are a reliable tool for fast comparison of strains of Saccharomyces cerevisiae collected from different craft breweries where they were used as starters to check the presence of possible yeast contaminants in the brewing process and for rapid LAB identification.     

Lactic acid bacteria in the brewing of traditional Daqu liquor

Lactic acid bacteria and their metabolites play important roles in defining Chinese liquor flavour. Currently, the role of lactic acid bacteria in liquor brewing remains unclear. This review discusses the role of lactic acid bacteria in fermentation of different types of Daqu and fermented grains. It focuses on the interaction between these bacteria and other microorganisms during liquor brewing, and analyses its effect on liquor flavour and quality. In addition, the roles of Lactobacillus in reducing the amount of unfavourable ingredients in liquor and the succession law of lactic acid bacteria during the fermentation of three Daqu liquors were also studied. Finally, the methods aimed at reducing lactic acid production and improving liquor quality are discussed. Overall, the review highlights the significance of lactic acid bacteria for brewing Daqu liquor. Nevertheless, further studies are required to clarify the role and mechanism of action of lactic acid bacteria during liquor fermentation. © 2019 The Institute of Brewing & Distilling     

乳酸链球菌素对乳酸菌抑菌作用的研究

目的：为了观察Nisin 对乳酸菌的抑菌效果,探讨Nisin 作为食品级载体筛选方法的最佳抑菌浓度和持续时间。方法：采用试管稀释法和纸片扩散法检测Nisin 对乳酸菌的抑菌浓度,并根据乳酸菌的OD600nm 作出生长曲线,以研究Nisin 对乳酸菌的抑菌效果。结果：Nisin 对乳品工业常用乳酸菌具有较强的抑菌作用,仅某些乳酸乳球菌对Nisin 表现出抗性;Nisin 对乳酸菌有较持久的抑菌效果,可达30～48h 以上。结论：该实验为Nisin 作为食品级载体筛选方法的最佳抑菌浓度和持续时间提供了依据。     

Stimulation of Cadaverine Production by Foodborne Pathogens in the Presence of Lactobacillus,Lactococcus, and Streptococcus spp.

Abstract: The effect of Lactobacillus plantarum (FI8595), Lactococcus lactis subsp. cremoris MG 1363), Lactococcus lactis subsp. lactis (IL 1403), and Streptococcus thermophilus on cadaverine and other biogenic amine production by foodborne pathogens was investigated lysine decarboxylase broth. Both of lactic acid bacteria and foodborne pathogens used (especially Staphylococcus aureus, E. coli, Lc. lactis subsp. lactis and Lb. plantarum) had an ability to convert aminoacids into biogenic amine. The conversion of lysine into cadaverine was the highest (167.11 mg/L) by Lactobacillus spp. Gram‐positive bacteria generally had a greater ability to produce cadaverine with corresponding value of 46.26, 53.76, and 154.54 mg/L for Enterococcus faecalis, S. aureus, and Listeria monocytogenes, respectively. Significant variations on biogenic amine production were observed in the presence of lactic acid bacteria strains (P < 0.05). The role of lactic acid bacteria on biogenic amine production by foodborne pathogens varied depending on strains and specific amine. Cadaverine accumulation by Enterobactericeae was increased in the presence of lactic acid bacteria strains except for St. thermophilus, which induced 2‐fold lower cadaverine production by S. Paratyphi A. Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris induced 10‐fold higher increases in histamine for E. coli and K. pneumoniae, respectively. Lactic acid bacteria resulted in strong increases in cadaverine production by P. aeruginosa, although remarkable decreases were observed for histamine, spermidine, dopamine, agmatine, and TMA in the presence of lactic acid bacteria in lysine decarboxylase broth . The result of the study showed that amine positive lactic acid bacteria strains in fermented food led to significant amine accumulation by contaminant bacteria and their accumulation in food product may be controlled by the use of proper starters with amine‐negative activity. Practical Application: Foodborne pathogens and certain lactic acid bacteria are particularly active in the production of biogenic amines. Most of the strains of bacteria possess more than 1 amino acid decarboxylase activity under lysine enrichment culture conditions. Lactic acid bacteria strains had a significant role on increase putrescine accumulation by foodborne pathogens. The increased production of biogenic amines in mixed culture is the result of presence of amine positive lactic acid bacteria strains. The addition of a proper selected starter culture with amine‐negative activity is advisable to produce safer fermented food with low contents of biogenic amines.     

Comparative study of spontaneously fermented sourdoughs originating from two regions of Greece: Peloponnesus and Thessaly

A total of 167 yeast and 136 lactic acid bacteria strains were isolated from spontaneously fermented wheat sourdoughs from two regions of Greece, namely Thessaly and Peloponnesus. Identification of the isolates exhibited dominance of Torulaspora delbrueckii with sporadic presence of Saccharomyces cerevisiae and, regarding the lactic acid bacteria, dominance of Lactobacillus sanfranciscensis in the sourdoughs from Thessaly and Lb. plantarum subsp. plantarum in the sourdoughs from Peloponnesus. The latter was accompanied by Pediococcus pentosaceus as secondary microbiota. None of the above mentioned strains exhibited amylolytic, lipolytic or proteolytic activities, and none of the lactic acid bacteria strains produced antimicrobial compounds.     

汾酒酒醅有机酸的研究及酿造工艺的优化

采用高压液相色谱法分析了夏季汾酒酒醅中有机酸成分,以平板划线法分离得到夏季汾酒正常酒醅和酸度过高酒醅中的产酸细菌。研究了影响酒醅总酸度的因素进而对汾酒酿造工艺进行了优化。结果表明:酒醅中有机酸以乙酸和乳酸为主,从正常酒醅中分离到23株产乙酸细菌,5株产乳酸细菌,从酸度过高酒醅中分离到22株产乙酸细菌,29株产乳酸细菌。酒醅酿造的环境温度、入缸水分和装料量对酒醅总酸度影响较大。在此基础上,通过响应面试验,得到了适宜酸度的汾酒酿造条件,即环境温度20℃、入缸水分51%、装料量0.83 kg,出缸酒醅的总酸度为2.12(0.1 mol NaOH mL/g酒醅),与模型的预测值2.07基本相符。上述结果为解决汾酒夏季生产酒醅酸度过高问题提供了科学依据。     

Diversity Analysis,Batch Fermentation and Characterization of Nisin in Identified Strains of Lactococcus lactis spp. lactis

Thirty-one strains of Lactococcus lactis spp. lactis were identified out of 89 isolates of lactic acid bacteria (LAB) from dairy and nondairy sources. Of the 31 strains, 24 (46.1%) were obtained from dairy and seven (18.9%) from non-dairy sources. The cluster analysis of rep-PCR showed that (GTG)₅-PCR followed by ERIC-PCR exhibited more discriminating potential than BOX-PCR. The obtained banding patterns characterized the polymorphism among strains. The strain level polymorphism was also obtained by the combined cluster analysis of (GTG)_5, ERIC and BOX-PCR which exhibited a level of heterogeneity among strains but not with the sources of isolation. Among 31 strains, 17 strains were able to produce zones of inhibition against Lactobacillus acidophilus NCDC 015 and therefore considered as nisin producers. Nisin production by strains was further confirmed by PCR amplification of nisA/Z of 174 bp size. The nisin activity and cell growth observed to be higher in pH controlled batch fermentation than in uncontrolled fermentation. The nisin activity, cell concentration, and acidity were high in immobilized cell system than free cell batch fermentation. The hot acid and chloroform extraction method was found to be the efficient way for the partial purification of nisin from fermented broth.     

Nisin‐producing microorganisms and their implementation in brewers' wort

Nisin is a bacteriocin, which is capable of eliminating more than 90% of all potential beer spoilage Gram‐positive bacteria. Hence, the implementation of nisin‐producing cultures into the brewing process needs to be evaluated systematically. In this work, the genetic relationships and properties, as well as the reactions of four strains of Lactococcus lactis ssp. lactis, known to be capable of producing nisin (NCIMB 8780, 8586, 701402 and 701403), and seven isolated strains of the same species found in the beverage environment (TUM 575, 8947, 8127, 8446, 8673, 8973 and 8872), were tested under typical brewery conditions. As in previous work, it was found, that all of the tested strains could be genetically differentiated via PCR‐(GTG)₅. In addition, the absence of the genes HorA, HorC and ORF5 indicated that all strains were sensitive to hop components. The agar diffusion assay test proved to be the most reliable method to precisely determine different nisin concentrations. Nisin formation, acid formation and the reproduction rates of the organisms were tested subsequently in various brewery relevant culture media such as MRS, first wort (unhopped) and wort (hopped). MRS provided the best environment for bacterial growth and hence acid and nisin production. The four NCIMB strains, which were the only ones capable of producing nisin under the named conditions, were chosen for study with regards to their tolerance to specific compounds found in the brewery environment. The strains NCIMB 8780 and 8586 produced comparatively higher amounts of nisin and were more tolerant to hop bitterness, ethanol, high gravity and low extract conditions. The growth rates, acid production and nisin production of all strains decreased with increasing bitter units and ethanol content. The optimum extract concentration was 5–10°P. Copyright © 2015 The Institute of Brewing & Distilling     

Plantaricin bacteriocins: As safe alternative antimicrobial peptides in food preservation—A review

Many peptides are excreted by gram-positive (+) and gram-negative (−) bacteria, possessing antimicrobial properties, called bacteriocins. Common bacterial species produce bacteriocins are called lactic acid bacteria. Nowadays, plantaricins are natural antimicrobial peptides produced by Lactobacillus plantarum strains have obtained special attention. The L. plantarum and their bacteriocins have got great importance in different areas as food biopreservatives and/or starters in dairy products, meat products, and fish products also, were used for treating diseases caused by pathogenic bacteria that is, reducing symptoms of irritable bowel syndrome disease (IBS) and decreasing the number of colonies of pathogenic bacteria in the wound-burning model in mice. Moreover, plantaricins have got a potent protective role against urinary tract infection (UTI). Although there are many studies on the types of bacteriocins and their purification and uses, such as Nisin and Pediocin, there have been no reports in the literature on the characterizations, production, and purification of plantaricins. The present review aims to describe plantaricins and some of their features and applications in general. Also mentioned are the most common methods of isolation and purification.     

Microbial community changes in Makgeolli during brewing

Makgeolli, a traditional Korean alcoholic beverage, is produced from the simultaneous saccharification and fermentation of glutinous rice by microbes introduced through nuruk (fermentation starter). The microbial community in Makgeolli is complex, and changes in the community are important for quality control and product development of Makgeolli. In this study, changes in the microbial community of Makgeolli during brewing were monitored by pyrosequencing. Pyrosequencing of microbes in Makgeolli illustrated dynamic changes in the populations and species of bacteria and fungi during brewing. Saccharomyces, a fungal genus, dominated the fungal community in Makgeolli throughout brewing, and produced alcohol after sufficient production of reducing sugars. As the bacterial genera Pediococcus, Weissella, Lactobacillus and Enterococcus, categorized as lactic acid bacteria, grew in Makgeolli, the pH decreased. The fungal community in Makgeolli was not significantly changed during brewing, but there were dynamic changes in the bacterial community, especially during the first 2 days of brewing. Copyright © 2015 The Institute of Brewing & Distilling     

山西老陈醋酿造过程中乳酸菌和醋酸菌的分离鉴定及高粱单宁对其生长的影响

利用稀释涂布法及透明圈法从山西老陈醋酿造过程中分离了36株乳酸菌和25株醋酸菌,利用形态观察、生理生化特性和16S rDNA序列测定对分到的菌株进行了鉴定;并研究了不同浓度的高粱单宁对这些菌株生长的影响。结果表明,36株乳酸菌分别为植物乳杆菌（Lactobacillus lantarum）,发酵乳酸杆菌（Lactobacillus fermentium）,干酪乳酸杆菌（Lactobacillus casei）,肠膜明串珠菌（Leuconostoc mesenteroides）,类食品乳杆菌（Lactobacillus paralimentarius）和短乳杆菌（Lactobacillus brevis）;25株醋酸菌分别为巴氏醋酸杆菌（Acetobacter pasteurianus）、醋化醋杆菌（Acetobacter aceti）,液化醋杆菌（Acetobacter liquefaciens）和恶臭醋酸杆菌（Acetobacter rancens）。当高粱单宁含量为0～1.5%时,所有乳酸菌和绝大部分醋酸菌的生长被促进;当单宁含量为2.0%时,对不同菌的生长影响不同（有一些菌株生长被促进,而另一些菌株生长被抑制）;当单宁含量为3.0%～6.0%时所有菌株的生长均被抑制。     

白酒窖泥中乳酸菌分离鉴定及其发酵产挥发性风味物质比较

从白酒窖泥中分离得到11株乳酸菌,经16S rDNA基因序列同源性分析,鉴定为1株短乳杆菌、1株鼠李糖乳杆菌、2株干酪乳杆菌和7株铅黄肠球菌。分析了11株菌MRS培养基发酵产乳酸性能,结果表明:乳酸产量与菌群生长呈正相关,鼠李糖乳杆菌L9、干酪乳杆菌L10与L11发酵液乳酸含量较高,分别为15.74、15.58、14.74 g/L。4株代表菌相同条件下发酵高粱培养液产挥发性风味组分,共有物质13种,包含了白酒中重要香气成分:乙酸、己酸、乙酸苯乙酯和苯乙醇,且乙酸、己酸相对含量较高。各菌产可挥发性组分差异明显,相对含量较高的化合物种类为:短乳杆菌L5产烷烃类化合物(27.91%),铅黄肠球菌L8产醇类化合物(43.14%),鼠李糖乳杆菌L9产酯类(7.35%)、酮类(3.61%)和吡嗪类(3.3%)化合物,干酪乳杆菌L11产酸类(27.98%)和芳香类(5.96%)化合物。仅有短乳杆菌L5产四甲基吡嗪,短乳杆菌L5和铅黄肠球菌L8可明显产乙醇,鼠李糖乳杆菌L9和干酪乳杆菌L11产3-羟基-2-丁酮。这些物质对白酒风味和口感有重要影响。研究显示,窖泥中各种乳酸菌特征不一,对白酒酿造有不同影响。本研究旨在为进一步挖掘白酒酿造功能微生物、拓展乳酸菌的应用提供理论依据。     

Emergence of [GAR+] cells in yeast from sake brewing affects the fermentation properties

In the traditional (kimoto) method of sake (Japanese rice wine) brewing, Saccharomyces cerevisiae yeast cells are exposed to lactate, which is produced by lactic acid bacteria in the seed mash. Lactate promotes the appearance of glucose-repression-resistant [GAR⁺] cells. Herein, we compared the resistance to glucose repression among kimoto, industrial, and laboratory yeast strains. We observed that the frequencies of the spontaneous emergence of [GAR⁺] cells among the kimoto strains were higher than those among the industrial and laboratory strains. The fermentation ability of a kimoto yeast (strain U44) was lower than that of an industrial strain (K701), as [GAR⁺] cells generally showed slower ethanol production. The addition of lactate decreased the fermentation abilities of the K701 strain by increasing the number of [GAR⁺] cells, but it did not affect those of the U44 strain. These results suggest that lactate controlled fermentation by promoting the appearance of [GAR⁺] cells in the industrial sake strains but not in the kimoto strains.     

High‐Throughput Isolation of Bacteriocin‐Producing Lactic Acid Bacteria,with Potential Application in the Brewing Industry

Lactic acid bacteria (LAB) were isolated from malted cereals by means of a high‐throughput screening approach and investigated for antimicrobial activity against a range of beer‐spoiling bacteria. Putative bacteriocin‐producing strains were identified by 16S rRNA analysis and the inhibitory compounds were partially characterized. Following determination of the inhibitory spectra of the strains, an unspeciated Lactobacillus sp. UCC128, with inhibitory activity against a range of beer‐spoiling strains was subjected to further characterization. A bacteriocin was purified from this strain and analyzed by mass spectrometry to determine the weight of the protein. The result indicated that the bacteriocin was highly similar to pediocin AcH/PA‐1 from Pediococcus acidilactici. The bacteriocin‐producers identified in this study have the potential to be used in the brewing industry to enhance the microbiological stability of beer in conjunction with hurdles already in place in the brewing process.