Impact of a plant extract on the viability of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in nonfat yogurt

The effect of a plant extract (prepared from olive, garlic, onion and citrus with sodium acetate as a carrier) on the viability of yogurt starter cultures was studied. Nonfat yogurt was prepared with various levels of supplements: plant extract (0, 0.5 or 1.0%, w/v) or l-cysteine HCl (0.014 or 0.028%, w/w). Microbial and physicochemical analyses were conducted weekly for 50 days. Fermentation time increased for supplemented yogurts compared with the non-supplemented yogurt. Lactobacillus bulgaricus counts in supplemented yogurts were >6 log cfu mL^?1 for a longer time (7–21 days) compared with the non-supplemented yogurt. Streptococcus thermophilus counts in all yogurts were > 6 log cfu mL^?1 throughout the storage. Overall, redox potential and titratable acidity of yogurts on day 50 were greater compared with day 1, but pH and syneresis were less. Plant extract at 0.5% enhanced L. bulgaricus viability in nonfat yogurt while least affecting the physicochemical characteristics.     

The critical role of urease in yogurt fermentation with various combinations of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus

The protocooperation between Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus relies on metabolite exchanges that accelerate acidification during yogurt fermentation. Conflicting results have been obtained in terms of the effect of the Strep. thermophilus urease and the NH₃ and CO₂ that it generates on the rate of acidification in yogurt fermentation. It is difficult to perform a systematic study of the effects of urease on protocooperation because it is necessary to distinguish among the direct, indirect, and strain-specific effects resulting from the combination of the strains of both species. To evaluate the direct effects of urease on protocooperation, we generated 3 urease-deficient mutants (ΔureC) of fast- and slow-acidifying Strep. thermophilus strains and observed the effects of NH₃ or CO₂ supplementation on acidification by the ΔureC strains. Further, we examined 5 combinations of 3 urease-deficient ΔureC strains with 2 CO₂-responsive or CO₂-unresponsive strains of L. bulgaricus. Urease deficiency induced a shortage of ammonia nitrogen and CO₂ for the fast- and slow-acidifying Strep. thermophilus and for the CO₂-responsive L. bulgaricus, respectively. Notably, the shortage of ammonia nitrogen had more severe effects than that of CO₂ on yogurt fermentation, even if coculture with L. bulgaricus masked the effect of urease deficiency. Our work established (1) that urease deficiency inhibits the fermentative acceleration of protocooperation regardless of the Strep. thermophilus and L. bulgaricus strain combinations, and (2) that urease is an essential factor for effective yogurt acidification.     

Exopolysaccharide production from whey lactose by fermentation with Lactobacillus delbrueckii ssp. bulgaricus

Ropy Lactobacillus delbrueckii ssp. bulgaricus (strain RR) was used for production of exopolysaccharide in sweet whey and simulated whey permeate (SWP) supplemented with combinations of lactose, KH₂PO₄, NH₄Cl, casamino acids, and mineral salts. Media were incubated at 32, 37, and 44°C for 72h. Periodic adjustment of pH to ～6.2 increased viscosity and lactose utilization, and the free galactose and lactic acid in the media. The effect of pH adjustment was greater than that of supplementation with nutrients or minerals. Fermentation of supplemented SWP generally produced lower viscosities than did fermentation of supplemented sweet whey. After 24h fermentation, viscosity decreased in pH adjusted media. Viscosity of media was highest when incubation was at 32°C and lowest with incubation at 44°C.     

Effect of lactose hydrolysis on the milk-fermenting properties of Lactobacillus delbrueckii ssp. bulgaricus 2038 and Streptococcus thermophilus 1131

Yogurt is a well-known nutritious and probiotic food and is traditionally fermented from milk using the symbiotic starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. However, yogurt consumption may cause health problems in lactose-intolerant individuals, and the demand for lactose-free yogurt has been increasing. The standard method to prepare lactose-free yogurt is to hydrolyze milk by lactase; however, this process has been reported to influence the fermentation properties of starter strains. This study aimed to investigate the fermentation properties of an industrial starter culture of L. bulgaricus 2038 and S. thermophilus 1131 in lactose-hydrolyzed milk and to examine the metabolic changes induced by glucose utilization. We found that the cell number of L. bulgaricus 2038, exopolysaccharide concentration, and viscosity in the coculture of L. bulgaricus 2038 and S. thermophilus 1131 was significantly increased in lactose-hydrolyzed milk compared with that in unhydrolyzed milk. Although the cell number of S. thermophilus 1131 showed no difference, production of formic acid and reduction of dissolved oxygen were enhanced in lactose-hydrolyzed milk. Further, in lactose-hydrolyzed milk, S. thermophilus 1131 was found to have increased the expression of NADH oxidase, which is responsible for oxygen reduction. These results indicated that glucose utilization promoted S. thermophilus 1131 to rapidly reduce the dissolved oxygen amount and produce a high concentration of formic acid, presumably resulting in the increased cell number of L. bulgaricus 2038 in the coculture. Our study provides basic information on the metabolic changes in starter strains in lactose-hydrolyzed milk, and demonstrates that lactose-free yogurt with increased cell number of L. bulgaricus can be prepared without delay in fermentation and decrease in the cell number of S. thermophilus.     

Productions and monomer compositions of exopolysaccharides by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains isolated from traditional home-made yoghurts and raw milk

In this study, a total of forty‐five strains of lactobacilli and streptococci were determined exopolysaccharide (EPS) production in skim milk and Man Rogosa and Sharpe (MRS)/M17 medium, viscosity and proteolytic activity. The exopolysaccharide production by lactobacilli strains during growth in MRS medium was twenty‐one to 211 mg L^?1, while in skim milk was to thirty‐six to 315 mg L^?1. The EPS production by streptococci strains during growth in M17 medium was sixteen to 114 mg L^?1, while in skim milk was to twenty‐four to 140 mg L^?1. The EPS production of strains was lower in MRS/M17 medium than skim milk. Results showed that it was not clear correlation between the viscosity and EPS production of some strains. All strains were shown proteolytic activity. Positive correlations between exopolysaccharide production and proteolytic activity in skim milk were found some strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. These results indicated that the high exocellular protease‐producing strains can produce high EPS in skim milk. The monomer compositions of the EPSs formed by selected five strains were analysed. Mannose dominated (99–100%) on the EPS produced by L. delbrueckii subsp. bulgaricus and S. thermophilusstrains (except L. delbrueckii subsp. bulgaricus 22) in skim milk and MRS/M17 medium. Besides, the EPSs of strains in skim milk contained small amount of lactose.     

酸乳中乳杆菌和链球菌的实时荧光定量分析

采用实时荧光定量分析了酸乳中的德氏乳杆菌L2和嗜热链球菌S1的数量,德氏乳杆菌L2的活菌数与细胞循环数的相互关系曲线为Y=-2.936logX+34.72(R2=0.998),采用平板计数法获得的样品中L2浓度为8.82×10smL-1,荧光定量的结果是8.78×108mL-1;嗜热链球菌S1活菌数与细胞循环教的相互关系曲线为Y=-3.03910gX+35.45(R2=0.995),采用平板计数法,获得的样品中嗜热链球菌S1浓度1.08×108mL-1,荧光定量的结果是1.06×108mL-1.实时荧光定量的方法适合于酸乳中乳酸菌的快速计数.     

保加利亚乳杆菌和嗜热链球菌相互作用的研究

探讨了嗜热链球菌和保加利亚乳杆菌之间的相互作用(拮抗和共生效果)，指出这两个菌种的许多菌株在代谢过程中会产生过氧化氢、细菌素等抑菌性物质，导致嗜热链球菌与保加利亚乳杆菌间表现出非协同生长的现象。在此基础上，建立了评价发酵剂之间相互作用的新的指标体系，旨在方便优良酸奶发酵剂菌种的筛选工作。     

Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus,Streptococcus thermophilus,Lactobacillus acidophilus,bifidobacteria, Lactobacillus casei,Lactobacillus rhamnosus,and propionibacteria

Nineteen bacteriological media were evaluated to assess their suitability to selectively enumerate Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, bifidobacteria, and propionibacteria. Bacteriological media evaluated included Streptococcus thermophilus agar, pH modified MRS agar, MRS-vancomycine agar, MRS-bile agar, MRS-NaCl agar, MRS-lithium chloride agar, MRS-NNLP (nalidixic acid, neomycin sulfate, lithium chloride and paramomycine sulfate) agar, reinforced clostridial agar, sugar-based (such as maltose, galactose, sorbitol, manitol, esculin) media, sodium lactate agar, arabinose agar, raffinose agar, xylose agar, and L. casei agar. Incubations were carried out under aerobic and anaerobic conditions at 27, 30, 37, 43, and 45 degrees C for 24, 72 h, and 7 to 9 d. S. thermophilus agar and aerobic incubation at 37 degrees C for 24 h were suitable for S. thermophilus. L. delbrueckii ssp. bulgaricus could be enumerated using MRS agar (pH 4.58 or pH 5.20) and under anaerobic incubation at 45 degrees C for 72 h. MRS-vancomycine agar and anaerobic incubation at 43 degrees C for 72 h were suitable to enumerate L. rhamnosus. MRS-vancomycine agar and anaerobic incubation at 37 degrees C for 72 h were selective for L. casei. To estimate the counts of L. casei by subtraction method, counts of L. rhamnosus on MRS-vancomycine agar at 43 degrees C for 72 h under anaerobic incubation could be subtracted from total counts of L. casei and L. rhamnosus enumerated on MRS-vancomycine agar at 37 degrees C for 72 h under anaerobic incubation. L. acidophilus could be enumerated using MRS-agar at 43 degrees C for 72 h or Basal agar-maltose agar at 43 degrees C for 72 h or BA-sorbitol agar at 37 degrees C for 72 h, under anaerobic incubation. Bifidobacteria could be enumerated on MRS-NNLP agar under anaerobic incubation at 37 degrees C for 72 h. Propionibacteria could be enumerated on sodium lactate agar under anaerobic incubation at 30 degrees C for 7 to 9 d. A subtraction method was most suitable for counting propionibacteria in the presence of other lactic acid bacteria from a product. For this method, counts of lactic bacteria at d 3 on sodium lactate agar under anaerobic incubation at 30 degrees C were subtracted from counts at d 7 of lactic bacteria and propionibacteria.     

Differentiation of Enterococcus faecium from Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains by PCR and dot-blot hybridisation

Variations in length and sequence of the 16S/23S spacer region of Enterococcus faecium provided the basis for development of simple PCR and dot-blot hybridisation assays that enabled the differentiation of potentially probiotic Enterococcus faecium strains from Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Such assays may be useful for differentiation of yoghurt starter cultures and enterococcal strains when they are simultaneously present in probiotic food products.     

保加利亚乳杆菌与嗜热链球菌共生机理的研究进展

乳酸菌在乳中的共生机制是非常复杂的网络体系。通过对蛋白质代谢、核苷酸碱基代谢、氧化性应激以及碳源物质代谢等方面的研究,对保加利亚乳杆菌与嗜热链球菌共生机理的研究进展进行详细的阐述。旨在为发酵剂菌体的筛选及应用提供参考。     

The differential enumeration of Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus salivarius subspecies thermophilus in yogurt and labneh using an improved whey medium

Bromocresol green whey agar (BGWA), an elective differential medium for yogurt bacteria, was prepared by mixing 2 parts of whey obtained by autoclaving (121°C/15 min) reconstituted non-fat dry milk (NFDM) (15% w/w; pH 5.7 by 1 N HCl) with 1 part of a sterile agar solution (115°C/15 min) containing 3% yeast extract, 1.2% K₂HPO₄, 0.004% bromocresol green and 4% agar. Lactobacillus delbrueckii subsp bulgaricus colonies in BGWA pour plates were light in colour and in the form of an irregular mass with twisted filament projections, while Streptococcus salivarius subsp thermophilus colonies were green lenticular with entire edges. BGWA performed generally better than deMan-Rogosa-Sharpe agar and M17 agar when L bulgaricus and S thermophilus respectively were enumerated in samples of commercial yogurt and labneh (yogurt concentrated by removing part of its whey) and in single cultures of yogurt bacteria in NFDM. Immediately after processing, the counts of yogurt bacteria in labneh samples were similar to those of yogurt samples, indicating a loss of these bacteria with whey during processing. The decrease in the numbers of yogurt bacteria in labneh samples at the end of the shelf-life (14 days at 7°C) was slight, indicating a relative resistance of these bacteria to acidity and refrigeration.     

Rheological and physical characteristics of non-fat set yogurt prepared with EPS-producing Streptococcus thermophilus and an H+-ATPase-defective mutant Lactobacillus delbrueckii subsp. bulgaricus

The rheological and physical characteristics of non-fat set yogurt produced using co-cultures of ropy EPS-producing Streptococcus thermophilus S3.3 and Lactobacillus delbrueckii subsp. bulgaricus LTM and of Streptococcus thermophilus S3.3 and Lactobacillus delbrueckii subsp. bulgaricus H+-ATPase-defective mutant L6 under different conditions were studied. Yogurts produced with mutant L6 had a higher pH and water-holding capacity and better textural properties compared to those produced with the LTM strain. The highest storage modulus (G′) and thixotropic loops were found for yogurt made with L6 at 42°C on day 21 of cold storage. This yogurt contained a network of thick, continuous protein aggregates and large void spaces.     

摄入酸奶对辐照小鼠体重和免疫细胞的影响

辐射前后5d用含保加利亚乳杆菌和嗜热链球菌活菌的酸奶添加于饲料中饲喂小鼠，比较接受2Gy辐射小鼠的体重、胸腺细胞重量、腹膜巨噬细胞吞噬活性和脾淋巴细胞计数等指标，发现摄入添加酸奶饲料的处理小鼠与普通饲料对照组有显著的差异，证明饲喂酸奶对辐射后小鼠产生了保护作用，减轻其体重下降、胸腺重下降和淋巴细胞计数，提高其巨噬细胞吞噬活性。     

Influence of casein hydrolysates on the growth and lactic acid production of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus

The growth performance of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus (S. thermophilus) was determined in the presence of casein hydrolysates produced by the action of five proteolytic enzymes (Alcalase, Flavorzyme, Neutrase, Papain and Trypsin) with various degrees of hydrolysis (DH). In addition, these five kinds of casein hydrolysates were fractionated by ultrafiltration and the influence of the amino acid composition of the peptides on the growth and lactic acid yield of yoghurt lactic acid bacteria (LAB) was studied. The results showed that the ultrafiltered fraction (<3000 Da) was the determinant stimulator in crude hydrolysates. Furthermore, the hydrophilic amino acid residua including His, Lys, Glu and Ser were beneficial for bacterial growth. Compared with control, the cell growth and lactic acid yield of yoghurt LAB were increased with the supplementation of the peptides fraction (<3000 Da) produced with papain by 65.1% and 49.6%, respectively.     

菌株性能及球杆菌比例对酸奶品质的影响

分析测定了从某直投式发酵剂中分离的两株嗜热链球菌（Streptococcus thermophilus）S.t S1、S.t R1和一株保加利亚乳杆菌（Lactobacillus delbrueckii subsp.bulgaricus）L.bL1的发酵性能,并将球菌与杆菌以不同比例组合发酵,比较酸奶发酵时间、粘度、乳清析出、后酸化和感官评价差异。结果表明,嗜热链球菌S.tS1生长速度略慢于S.tR1,酸化速度相当,但S.tS1的胞外多糖产量和发酵乳粘度明显高于S.tR1,保加利亚乳杆菌L.bL1的上述性能均比两株球菌差。S.tS1与L.bL1菌株以100：1的比例进行组合发酵时,粘度达0.549Pa·s;乳清析出仅2.5mL/10g;4℃冷藏保存15d后,酸度上升12°T,酸奶产品各项指标优于其他菌株组合。菌株性能和组合方式对酸奶产品的品质具有显著的影响。     

Conjugated linoleic acid production by immobilized cells of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus acidophilus

Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) and L. acidophilus (CCRC14079), immobilized with chitosan and polyacrylamide, were tested for CLA production. A 10-ml aliquot of L. delbrueckii ssp. bulgaricus cell suspension (3.59 × 10⁷ CFU/ml) was adsorbed to 0.5 g chitosan and polyacrylamide, mixed with 0.2 ml linoleic acid (0.9 g/ml), and incubated at 37 °C for 24 h at pH 5, 6, 7, and 8 for CLA production. CLA levels, produced by immobilized cells of L. delbrueckii ssp. bulgaricus and L. acidophilus with increasing cell counts to 1.08 and 1.28 × 10¹⁰ CFU/ml, respectively, at optimal reaction pHs were evaluated. More CLA was formed at pH 8 of chitosan and pH 7 of polyacrylamide-immobilized L. delbrueckii ssp. bulgaricus cell treatments. Increase in cell count resulted in higher CLA production. The adsorption of L. delbrueckii ssp. bulgaricus cells onto polyacrylamide at pH 7 showed significant improvement in total CLA level. Results demonstrated a potential for enhancing CLA production through immobilization.     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.     

Factors influencing autoaggregation and aggregation of Lactobacillus delbrueckii subsp. bulgaricus isolated from handmade yogurt

Of 26 Lactobacillus delbrueckii subsp. bulgaricus strains isolated from yogurt, strains B2 and 22, which produce low levels (28 and 21 mg liter(-1), respectively) of extracellular polysaccharides (EPSs), and strains B3 and G12, which produce high EPS levels (211 and 175 mg liter(-1), respectively), were selected for further study. The two high EPS-producing strains showed a significant autoaggregation and coaggregation ability with Escherichia coli ATCC 11230 (P < 0.05). Moreover, the effect of bile was evaluated on autoaggregation and hydrophobicity. Autoaggregation and hydrophobicity of these L. delbrueckii subsp. bulgaricus strains decreased after treatment with bile. Only the high EPS-producing L. delbrueckii subsp. bulgaricus strain B3 showed greater autoaggregation (80%) and hydrophobicity (86%) than the other strains after bile treatment. When these strains were assessed for the inhibition of E. coli ATCC 11230 in coculture, L. delbrueckii subsp. bulgaricus B3 completely inhibited E. coli during 24 and 48 h of incubation. This investigation showed that a high EPS production and coaggregation ability may be important in the selection of probiotic strains.     

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.     

德氏乳杆菌保加利亚亚种自发突变抗噬菌体菌株的选育及其特性

利用德氏乳杆菌L.bulgaricus KLDS08021为宿主菌,采用次级感染的方法筛选自发突变的抗噬菌体菌株,将筛选出的有抗性的7株菌株进行RAPD-PCR鉴定,然后进行EOP、遗传稳定性、溶源性检测,并对其中的2株抗性菌BIM1、BIM7及野生株进行发酵性能测定。结果显示,7株菌RAPD-PCR电泳图谱一致,均来自宿主菌,是遗传性稳定的非溶源性菌;无论噬菌体是否存在2株抗性菌BIM1、BIM7其发酵活菌数、产酸能力都比野生株高,且制作的发酵乳黏度与硬度也比野生菌株强,适于作为酸奶发酵菌株。