New potentially antihypertensive peptides liberated in milk during fermentation with selected lactic acid bacteria and kombucha cultures

Compounds with the ability to inhibit angiotensin-converting enzyme (ACE) are used medically to treat human hypertension. The presence of such compounds naturally in food is potentially useful for treating the disease state. The goal of this study was to screen lactic acid bacteria, including species commonly used as dairy starter cultures, for the ability to produce new potent ACE-inhibiting peptides during milk fermentation. Strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus, Lactobacillus paracasei, Lactococcus lactis, Leuconostoc mesenteroides, and Pediococcus acidilactici were tested in this study. Additionally, a symbiotic consortium of yeast and bacteria, used commercially to produce kombucha tea, was tested. Commercially sterile milk was inoculated with lactic acid bacteria strains and kombucha culture and incubated at 37°C for up to 72 h, and the liberation of ACE-inhibiting compounds during fermentation was monitored. Fermented milk was centrifuged and the supernatant (crude extract) was subjected to ultrafiltration using 3- and 10-kDa cut-off filters. Crude and ultrafiltered extracts were tested for ACE-inhibitory activity. The 10-kDa filtrate resulting from L. casei ATCC 7469 and kombucha culture fermentations (72 h) showed the highest ACE-inhibitory activity. Two-step purification of these filtrates was done using HPLC equipped with a reverse-phase column. Analysis of HPLC-purified fractions by liquid chromatography–mass spectrometry/mass spectrometry identified several new peptides with potent ACE-inhibitory activities. Some of these peptides were synthesized, and their ACE-inhibitory activities were confirmed. Use of organisms producing these unique peptides in food fermentations could contribute positively to human health.     

Antimicrobial interactions of microbial species involved in the fermentation of cassava dough into agbelima with particular reference to the inhibitory effect of lactic acid bacteria on enteric pathogens

Lactic acid bacteria, Bacillus species and yeasts are involved in the fermentation of cassava dough into agbelima. Microbial interactions within and between these groups of microorganisms were investigated in addition to the survival of five enteric pathogens inoculated into agbelima under various conditions. Nine out of 10 cultures of lactic acid bacteria isolated at the end of agbelima fermentation showed inhibitory effect against 10 cultures of lactic acid bacteria isolated at the start of fermentation. Only 3 out of 10 isolates of Bacillus subtilis were inhibited by 10 isolates of lactic acid bacteria tested. No interactions were observed between yeasts and the lactic acid bacteria, whereas three of the Bacillus isolates showed inhibitory effects against the yeasts. Twelve isolates of Lactobacillus plantarum tested inhibited the growth of an isolate each of Lactobacillus fermentum and Lactobacillus brevis but none tested positive for bacteriocin production. The antimicrobial effect of the lactic acid bacteria was attributed to acid production. In fermenting cassava dough, enteric pathogens survived to different extents depending on pH and their sensitivity to acids. Vibrio cholerae C-230, Salmonella typhimurium 9 and Salmonella enteritidis 226 were not detectable in 10 g of sample after 4 h when inoculated into the 48-h fermented product, agbelima, whereas Shigella dysenteriae 2357T and Escherichia coli D2188 were detectable up to 8 h in the product.     

Lactic acid starter and probiotic bacteria: a comparative “in vitro” study of probiotic characteristics and biological barrier resistance

Probiotic characteristics (deconjugation of bile salts, hydrophobicity and β-galactosidase activity) and the resistance to biological barriers (gastric juice and bile salts) of 24 strains of lactic acid starter bacteria (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactococcus lactis) and 24 strains of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus and bifidobacteria) were compared. Among the probiotic bacteria tested, Lactobacillus acidophilus was the most interesting species since it showed high values of resistance to gastric juice and bile, hydrophobicity and β-galactosidase and bile salts deconjugation activities. Bifidobacterium bifidum strains showed the same behavior, although the values of the parameters investigated were slightly lower than those obtained for Lactobacillus acidophilus. On the other hand, it was demonstrated that Lactobacillus delbrueckii subsp. bulgaricus was the lactic acid starter species with the best probiotic characteristics among the starter species assessed. It was resistant to gastric juice and bile, and showed high values for β-galactosidase activity. On the other hand, lactic acid starter bacteria showed hydrophobicity values similar to or higher than those obtained for the strains of the Lactobacillus casei . According to the results found, the total probiotic value of a fermented dairy product should take into account not only the intestinal probiotic cultures used in the formulation but also the probiotic contribution of the lactic acid starter microflora.     

Antagonistic action of lactic cultures toward spoilage and pathogenic microorganisms in food

The antibacterial properties of cell-free filtrate from lactic cultures were assessed against 10 bacterial cultures. All the five species of lactic culture examined showed antimicrobial activity against tested bacteria. S. aureus was least sensitive of the tested bacteria, followed by E. coli and S. typhosa. E. aerogenes was the most sensitive one. L. casei had the greatest antimicrobial activity. Leu. mesenteroides weakly inhibited the growth of the tested bacteria. In general, inoculum density had little effect on inhibition.     

Interactions among lactic acid starter and probiotic bacteria used for fermented dairy products

Interactions among lactic acid starter and probiotic bacteria were investigated to establish adequate combinations of strains to manufacture probiotic dairy products. For this aim, a total of 48 strains of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium spp. (eight of each) were used. The detection of bacterial interactions was carried out using the well-diffusion agar assay, and the interactions found were further characterized by growth kinetics. A variety of interactions was demonstrated. Lb. delbrueckii subsp. bulgaricus was found to be able to inhibit S. thermophilus strains. Among probiotic cultures, Lb. acidophilus was the sole species that was inhibited by the others (Lb. casei and Bifidobacterium). In general, probiotic bacteria proved to be more inhibitory towards lactic acid bacteria than vice versa since the latter did not exert any effect on the growth of the former, with some exceptions. The study of interactions by growth kinetics allowed the setting of four different kinds of behaviors between species of lactic acid starter and probiotic bacteria (stimulation, delay, complete inhibition of growth, and no effects among them). The possible interactions among the strains selected to manufacture a probiotic fermented dairy product should be taken into account when choosing the best combination/s to optimize their performance in the process and their survival in the products during cold storage.     

乳酸菌菌粉定量计数方法研究

目的:以9株乳杆菌、6株双歧杆菌、3株球菌、1株凝结芽孢杆菌菌粉为研究对象,在国标GB 4789.35-2016的基础上,对稀释倍数、稀释液成分、培养基成分进行比较研究,考察对乳酸菌菌粉计数活菌数的影响。方法:采用稀释平板计数的方法,对不同的乳酸菌菌粉进行活菌计数。结果:初始乳酸菌菌粉样品稀释倍数对最终计数活菌数无明显影响,ISO稀释液对部分乳杆菌、双歧杆菌菌粉的活菌计数结果有显著提高(P<0.05);双歧杆菌菌粉采用TOS琼脂培养基的计数结果显著优于国标培养基(P<0.05);凝结芽孢杆菌菌粉采用改良芽孢计数培养基计数结果优于PCA和NA计数培养基。结论:平板计数方法中,稀释液中含有酪蛋白胨能提升双歧杆菌菌粉的活菌计数数量;TOS琼脂培养基更有利于双歧杆菌的增殖培养;改良芽孢计数培养基更有利于凝结芽孢杆菌的芽孢萌发增殖。     

Lactic acid bacteria profiles and tyramine and tryptamine contents of Turkish tulum cheeses

Tulum cheese, commonly produced in Turkey, is a traditional Turkish dairy product made from raw milk. In this research, 20 samples of tulum cheese were purchased from different markets in Isparta and Ankara (Turkey) and the populations of lactic acid bacteria (LAB) and contents of biogenic amines of Turkish tulum cheeses were investigated. The mean counts of LAB (as log10 cfu/g) were 7.22±0.67 on MRS agar, 6.93±1.77 on M17 agar, 7.28±0.72 on APT agar and 5.98±2.20 on ADA agar. Isolates of LAB obtained from 20 tulum cheeses were characterised as lactobacilli (48.5%), enterococci (32.7%), lactococci (15.8%) and streptococci (3.0%). Lactobacillus paracasei ssp. paracasei, Lactobacillus bifermentans and Enterococcus faecium predominated among the LAB in the samples of tulum cheeses; these organisms represented 30.2% of the isolates. As amino acids, tyrosine and tryptophan were determined in tulum cheeses. Tyramine and tryptamine, biogenic amines, were found at 0.00–329.00 and 0.32–40.44 mg/kg, respectively.     

酸和盐胁迫对乳酸菌活性的影响

该研究以13株食品常见乳酸菌为研究对象,考察酸和盐胁迫对其活性的影响,探究这13株乳酸菌的最适酸、盐生长条件及耐酸、耐盐能力。结果表明,适当的低盐浓度（1%、2%）对部分乳酸菌的生长有促进作用,植物乳杆菌和副干酪乳杆菌的最适盐浓度为1%,保加利亚乳杆菌和嗜酸乳杆菌的最适盐浓度为2%;盐浓度进一步增大,13株乳酸菌的生长受到抑制;盐浓度为10%时,除戊糖片球菌、乳酸乳球菌乳酸亚种、嗜热链球菌和乳酸片球菌外,其余9株菌的生长抑制率都＞95%。当pH值为5～7时,13株乳酸菌都生长良好,pH=6时,7株乳酸菌活性均最高;随着酸胁迫的增强,13株乳酸菌生长受抑制程度增大;pH为1～3时,13株乳酸菌的生长抑制率都＞90%,高盐及高酸对9株杆菌的抑制作用强于4株球菌。     

Probiotic culture survival and implications in fermented frozen yogurt characteristics

Low-fat ice cream mix was fermented with probiotic-supplemented and traditional starter culture systems and evaluated for culture survival, composition, and sensory characteristics of frozen product. Fermentations were stopped when the titratable acidity reached 0.15% greater than the initial titratable acidity (end point 1) or when the pH reached 5.6 (end point 2). Mix was frozen and stored for 11 wk at -20 degrees C. The traditional yogurt culture system contained the strains Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. The probiotic-supplemented system contained the traditional cultures as well as Bifidobacterium longum and Lactobacillus acidophilus. We compared recovery of Bifodobacterium by three methods, a repair-detection system with roll-tubes and plates on modified bifid glucose medium and plates with maltose + galactose reinforced clostridial medium. Culture bacteria in both systems did not decrease in the yogurt during frozen storage. The roll-tube method with modified bifid glucose agar and repair detection system provided at least one-half log10 cfu/ml higher recovery of B. longum compared with recoveries using modified bifid glucose agar or maltose + galactose reinforced clostridial agar on petri plates. No change in concentrations of lactose or protein for products fermented with either culture system occurred during storage. Acid flavor was more intense when product was fermented to pH 5.6, but yogurt flavor was not intensified. The presence of probiotic bacteria in the supplemented system seemed to cause no differences in protein and lactose concentration and sensory characteristics.     

Binding of free bile acids by cells of yogurt starter culture bacteria

Several strains of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus, which produced exocellular polysaccharides (EPS), varied in the amount produced. The streptococci tended to produce the most EPS per milliliter of culture; however, when compared on the basis of amounts per 10(7) cfu, the lactobacilli produced the most. Lactobacillus delbrueckii ssp. bulgaricus strains Lb-18 and Lb-10442 and S. thermophilus St-143 produced significantly larger amounts per 107 cfu than did other strains tested. These three cultures plus two strains of the streptococci that produced the greatest amounts of EPS per ml of culture were tested for the ability to bind bile acids from laboratory media. The two cultures of L. delbrueckii ssp. bulgaricus (Lb-18 and Lb-10442) bound significantly higher amounts of cholic acid than did the three strains of streptococci. These two cultures of lactobacilli bound up to 15.3% of the cholic acid present in laboratory media, up to 452 microg/mg of EPS and 2.9 microg/10(7) cfu. None of the cultures tested in this study were able to bind the conjugated bile acid, glycocholic acid.     

A selective medium for the enumeration and differentiation of Lactobacillus delbrueckii ssp. bulgaricus

Modified reinforced clostridial medium (mRCM) was developed and evaluated for the differential enumeration of Lactobacillus delbrueckii ssp. bulgaricus. Lactobacillus bulgaricus, an important species of lactic acid bacteria with health benefits, is used in the production of yogurt and other fermented foods. Our results showed that supplementing reinforced clostridial medium with 0.025% CaCl₂, 0.01% uracil, and 0.2% Tween 80 (mRCM) significantly enhanced the growth rate of L. bulgaricus RR and ATCC 11842 strains as measured by the optical densities of these strains after 12 h of incubation at 42°C. The bacterial populations (plate count) of the RR and ATCC 11842 strains were 0.76 and 0.77 log cfu/g higher in mRCM than in de Man, Rogosa, and Sharpe and reinforced clostridial medium media, respectively. Conversely, the population counts for other bacterial species (Bifidobacterium, Lactobacillus rhamnosus, and Lactobacillus reuteri) were significantly inhibited in the mRCM medium. The addition of aniline blue dye to mRCM (mRCM-blue) improved the selectivity of L. bulgaricus in mixed lactic bacterial cultures compared with de Man, Rogosa, and Sharpe medium and lactic agar with regard to colony appearance and morphology. The mRCM-blue performed better than the conventional medium in culturing, enumerating, and differentiating L. bulgaricus. Therefore, mRCM-blue could be used as a selective medium to enhance the growth and differentiation of L. bulgaricus in order to meet the increasing demand for this beneficial species of bacteria.     

清香型白酒酒醅发酵菌株分离鉴定及细菌群落结构分析

采用纯培养技术对清香型白酒酒醅中的乳酸菌和芽孢杆菌进行分离鉴定,同时通过Illumina MiSeq高通量测序技术解析清香型白酒酒醅细菌群落结构。结果表明,所有酒醅样品共分离得到13株乳酸菌,分别鉴定为乳酸片球菌（Pediococcus acidilactici）4株、副干酪乳杆菌（Lactobacillus paracasei）3株、短乳杆菌（Lactobacillus brevis）3株、粪肠球菌（Enterococcus faecium）2株以及希尔氏乳杆菌（Lactobacillus hilgardii）1株。从酒醅中分离得到的17株芽孢杆菌菌株分别鉴定为地衣芽孢杆菌（Bacillus licheniformis）9株、枯草芽孢杆菌（Bacillus subtilis）2株、短小芽胞杆菌（Bacillus pumilus）2株、澳洲芽孢杆菌（Bacillus australimaris）2株、解淀粉芽孢杆菌（Bacillus amyloliquefaciens）1株以及蜡样芽孢杆菌（Bacillus cereus）1株。Illumina MiSeq高通量测序技术结果显示,酒醅中优势菌门分别为厚壁菌门（Firmicutes）（97.96%）、放线菌门（Actinobacteria）（1.24%）和变形菌门（Proteobacteria）（0.76%）;优势细菌属为乳酸杆菌属（Lactobacillus）（97.42%）、栖热嗜油菌属（Thermoleophilum）（1.22%）和假单胞菌属（Pseudomonas）（0.72%）。     

开菲尔粒中乳酸菌的分离方法研究

本文分别对开菲尔滤液和洗液中乳酸菌的分离方法进行了研究。结果表明:使用添加了0.5%山梨酸钾的乳清改良培养基,倾注法(划线法)厌氧培养乳酸菌分离效果较好,并且滤液中的菌株比洗液中的长势更好,活力更高。     

Lactic acid bacteria with cholesterol-lowering properties for dairy applications: In vitro and in situ activity

Cholesterol-lowering activity is one of the most promising properties of lactic acid bacteria with probiotic characteristics. In the present study, 58 potentially probiotic lactic acid bacteria were tested for their ability to survive in vitro digestion and reduce cholesterol in a medium containing cholesterol and bile acids. The best-performing strains (Lactobacillus casei VC199, Lactobacillus paracasei ssp. paracasei SE160 and VC213, Lactobacillus plantarum VS166 and VS513, Enterococcus faecium VC223, and Enterococcus lactis BT161) resulted in a 42 to 55% reduction of the cholesterol level in broth and were further tested in cheese manufacture. The cholesterol content in all the cheeses decreased with ripening. All the strains were present in the cheese at levels higher than 10⁷ cfu/g until 60 d of ripening, the highest reductions (up to 23%) being obtained when Lb. paracasei ssp. paracasei VC213 and E. lactis BT161 were added during the cheese-making. The adjunct cultures had no negative effect on the sensory characteristics of the cheese. Thus, these strains with proven in vitro properties are good candidates for novel probiotic-containing formulations and could be used to functionalize foods such as dairy fermented products.     

眉山泡菜中乳酸菌的分离鉴定

目的：分析眉山泡菜的基本数据和乳酸菌的菌种构成。方法：从眉山市采集泡菜12 份,测定pH值、总酸度、盐度、乳酸菌计数,用16S rRNA序列分析法鉴定菌种。结果：样品pH值的范围是3.37～3.89,总酸度的范围是0.65～0.92 g/100 g（以乳酸计）,盐度的范围是4.16%～5.28%（以NaCl计）,MRS和M17乳酸菌计数分别是1.71～6.33、1.33～5.78（lg（CFU/g））。总酸度对乳酸菌计数影响最大。共分离鉴定出16 株乳酸菌：Lactobacillus fermentum（2 株）、Lactobacillus plantarum（3 株）、Lactobacillus brevis（2 株）、Lactobacillusacidophilus（1 株）、Lactobacillus casei（1 株）、Lactobacillus pentosus（1 株）、Lactobacillus delbrueckii（1 株）、Lactococcus lactis（3 株）、Pediococcus pentosaceus（2 株）。结论：眉山泡菜中乳酸菌存在多样性,实验结果为工业发酵生产泡菜积累了菌株。     

Evaluation of isolated starter lactic acid bacteria in Ras cheese ripening and flavour development

Eighteen cultures of starter lactic acid bacteria with or without added adjunct cultures, isolated from Egyptian dairy products, were evaluated in experimental Ras cheese for flavour development. Chemical composition of experimental cheeses was within the legal limit for Ras cheese in Egypt. All cultures used in this study had no effect on chemical composition of Ras cheese. Very significant variations in free amino acids, free fatty acids and sensory evaluation have been found among the cultures used in Ras cheesemaking. The levels of free amino acids and free fatty acids were correlated well with flavour development in Ras cheese. Seven of the tested cultures produced acceptable flavour and texture of Ras cheese. The highest overall score of flavour intensity, flavour and texture acceptability were in cheese made using YY47 lactic culture in addition to adjunct culture of Lactobacillus helveticus, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. lactis and Enterococcus faecium. This culture can be recommended for Ras cheese manufacture using pasteurized milk.     

直投式发酵剂制备中乳酸菌增殖条件的探讨

为获得高浓度的乳酸菌细胞,对化学中和法培养乳酸菌的增殖条件进行了研究。主要采用菌落计数和显微镜检的方法,确定乳酸菌增殖的最佳温度、pH值及菌种中球菌与杆菌的比例。结果表明,在优化条件下,即32℃、pH5.8及球菌与杆菌比例为1∶2的培养条件下,乳酸菌活菌数达3.2×1010cfu/mL。     

Microbiological characteristics of "androlla", a Spanish traditional pork sausage

Counts of total aerobic mesophilic microflora, lactic acid bacteria, salt-tolerant microflora, Enterobacteriaceae, enterococci, moulds and yeasts, and staphylococci, and some physico-chemical parameters (total solids, NaCl and nitrate contents and pH and aw values) were determined in 20 units of "androlla", a traditional dry-fermented sausage made in the NW of Spain. In general, high counts of all the investigated microbial groups were observed, with average values of 8.99 +/- 0.46 log cfu/g for the total aerobic mesophilic microflora, 9.11 +/- 0.16 log cfu/g for the lactic acid bacteria, 6.87 +/- 0.68 log cfu/g for the salt-tolerant microflora, 2.80+/-1.85 log cfu/g for the Enterobacteriaceae, 3.25 +/- 1.86 log cfu/g for the enterococci, 4.30 +/- 1.73 log cfu/g for the moulds and yeasts, and 3.62 +/- 0.60 log cfu/g for the staphylococci. From MRS agar, SPC agar + 7.5% NaCl, VRBG agar, and KAA agar, 10 colonies were randomly taken from each androlla unit and from each culture medium. A total of 200 strains per culture medium were then identified using the classical methods. Among the isolates from MRS agar, Lactobacillus sakei predominated, followed by Lactobacillus curvatus, Lactobacillus alimentarius and Lactobacillus plantarum. Of the 200 isolates obtained from SPC agar + 7.5% NaCl, only 56 strains belonged to the Staphylococcaceae or Micrococcaceae families. Among the Staphylococcaceae, Staphylococcus xylosus was the main species, followed by Staph. epidermidis; Staph. equorum, Staph. capitis and Staph. saprophyticus were isolated in very low proportions. Among the Micrococcaceae, Micrococcus luteus predominated, followed by Micrococcus lylae, Kocuria varians and Kocuria kristinae. Of the 150 isolates obtained from VRBG agar, Hafnia alvei was the main species, followed by Serratia liquefaciens and Enterobacter amnigenus; six isolates were identified as Salmonella. Among the 190 isolates obtained from KAA agar, 122 were considered enterococci; 20 isolates were identified as Enterococcus faecium, one as Enterococcus faecalis and 101 as Enterococcus inter faecalis-faecium.     

乳酸菌富硒及产胞外多糖条件的研究

试验以实验室保藏的6株乳酸菌为研究对象,筛选出最佳试验条件下富硒率和产胞外多糖含量最优的菌株,以期为生产富硒调味品提供一定的理论依据.通过测定其生长曲线确定各菌的加硒时间,对比6株乳酸菌在不同亚硒酸钠质量浓度下的颜色变化、富硒率变化和胞外多糖含量变化,选定鼠李糖乳杆菌作为最佳试验菌株;通过测定鼠李糖乳杆菌在不同培养时间...     

Microbial and Physicochemical Characterization of the Horse Meat in Fermented Sausage

In this study, 150 lactic acid bacterial strains, originating from horse meat, were subjected to a step-by-step screening and characterization to search for potential protective cultures to be used in the meat industry. Strains were first tested for their homofermentation and salt tolerance. Second, the antibacterial capacities towards Listeria monocytogenes were determined in an agar spot test. In total, 50% of the tested strains were inhibitory towards Listeria monocytogenes. However, only 12 strains produced a bacteriocin. Finally, three strains with strong bacteriocin activity were competitively evaluated by comparing their growth rate, acidifying character and lactic acid production at 15°C under anaerobic conditions in a liquid broth. All three strains had a fast growth rate with rapid acidification caused by the production of high levels of lactic acid. Based on this Lactobacillus sakei was used as starter culture for producing sausage horse meat. In this study, fermentations were followed analyzing the microbiological and physicochemical aspects of this product. The sausages were characterized based on microbial activity of lactic acid bacteria that resulted in a product with a final pH of about 4.56. No Listeria monocytogenes, Salmonella spp. nor Sulfite reducing clostridia were ever isolated from the raw materials or the fermented sausages during the maturation, reflecting the safety of this product. The final water activity of the product was 0.85. Lactobacillus sakei was efficient in reducing amine production since this strain caused a quick pH drop during sausage fermentation.