一株嗜酸乳杆菌产共轭亚油酸条件的优化

共轭亚油酸(conjugated linoleic acid,CLA)是具有抗癌、减肥等许多生理功能的天然脂肪酸.生物法合成共轭亚油酸无毒且异构体单一,但目前产量较低.为提高CLA产量,以嗜酸乳杆菌(Lactobacillus acidophilus)为出发菌株,优化其培养条件.结果表明:发酵液初始pH为6.5,红花油添加量为0.15%,接种量5%,发酵温度为37°C,发酵时间为60h时最有利于共轭亚油酸的合成,此时CLA的合成最为94.7μg/mL.     

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.     

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.     

Augmentation of macrophage functions by an extracellular phosphopolysaccharide from Lactobacillus delbrueckii ssp. bulgaricus

The effect of an extracellular neutral- and phosphopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus OLL 1073R-1 on macrophage functions was examined in short-term studies. The total number of peritoneal macrophages elicited by an intraperitoneal injection of 100 mg kg⁻¹of the polysaccharide was three fold greater than that by PBS. The macrophage phagocytosis was augmented by the phosphopolysaccharide in vivo and in vitro, but not or less by the neutral polysaccharide. The cytostatic activity of thioglycolate-induced macrophages against tumour cell lines (Sarcoma-180 or P388) was significantly augmented by a 6-h treatment of the phosphopolysaccharide at a concentration of 10–100 μg ml⁻¹, while only at 100 μg ml⁻¹of the neutral polysaccharide. The phosphopolysaccharide purified by high performance liquid chromatography also had a substantial effect on macrophage cytostatic activity. The partial hydrolysis by trifluoroacetic acid treatment and dephosphorylation by hydrofluoric acid degradation of phosphopolysaccharides reduced the cytostatic activity in macrophages. The phosphopolysaccharide produced byLactobacillus delbrueckii ssp. bulgaricus 1073R-1 is a potent enhancer of macrophage functions in which the conformational structure or phosphate group may play an important role.     

Improvement of the resistance of Lactobacillus delbrueckii ssp. bulgaricus to freezing by natural selection

Lactic acid bacteria are often produced as frozen or freeze-dried cultures that can be used for the direct inoculation of milk in cheese and fermented milk production processes. The objective of this study was to investigate whether the resistance of Lactobacillus delbrueckii ssp. bulgaricus to freezing could be improved by natural selection. Three parallel cultures of strain CFL1 were propagated for 30 cycles in which each cycle involved three serial transfers through milk, one freezing step, and one thawing step. The concentration in viable cells after thawing as well as the acidifying activity of the thawed cultures increased dramatically throughout the experiment. This may be explained by the random appearance of better-adapted mutants that can outcompete the other genotypes. However, after 30 cycles of subcultivation, freezing, and thawing, all the cultures contained subpopulations having different survival rates to freezing. Our results show that serial transfer culture experiments may be used to improve technological properties of lactic acid bacteria. Furthermore, investigation of the mutations that are responsible for an increased cryotolerance may help to define new targets for improving the resistance of lactic acid bacteria to several stresses.     

Immunomodulatory effects of polysaccharides produced by Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1

The extracellular polysaccharides (EPS) produced by lactic acid bacteria (LAB) are associated with the rheology, texture, and mouthfeel of fermented milk products, including yogurt. This study investigated the immunomodulatory effects of EPS purified from the culture supernatant of Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) OLL1073R-1. The crude EPS were prepared from the culture supernatant of L. bulgaricus OLL1073R-1 by standard chromatographic methods, and were fractionated into neutral EPS and acidic EPS (APS). Acidic EPS were further fractionated into high molecular weight APS (H-APS) and low molecular weight APS (L-APS). High molecular weight APS were shown to be phosphopolysaccharides containing D-glucose, D-galactose, and phosphorus. Stimulation of mouse splenocytes by H-APS significantly increased interferon-γ production, and, moreover, orally administered H-APS augmented natural killer cell activity. Oral administration of yogurt fermented with L. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059 to mice showed a similar level of immunomodulation as H-APS. However, these effects were not detected following administration of yogurt fermented with the starter combination of L. bulgaricus OLL1256 and S. thermophilus OLS3295. We conclude from these findings that yogurt fermented with L. bulgaricus OLL1073R-1, containing immunostimulative EPS, would have an immunomodulatory effect on the human body.     

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.     

Impedimetric method for estimating the residual activity of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus

The residual activity of Lactobacillus delbrueckii ssp. bulgaricus cultures was analysed using pH and various impedimetric methods (impedance detection time (IDT), conductance and capacitance) to quantify the loss in activity following freeze-drying. The large variation recorded in IDT values for similar levels of activity suggests that IDT is not an adequate parameter for estimating the culture's fermentative activity. Comparison of the impedance signals generated revealed that capacitance yields values that are more reproducible than those of conductance, and also gives a better correlation with pH. Statistical analysis (p<0.05) indicated that there are no significant differences between the capacitance and the pH method when attempting to estimate residual activity.     

Increase of viability of entrapped cells of Lactobacillus delbrueckii ssp. bulgaricus in artificial sesame oil emulsions

A technique was developed to protect lactic acid bacteria (Lactobacillus delbrueckii ssp. bulgaricus) against simulated gastrointestinal conditions by encapsulation of bacterial cells within artificial sesame oil emulsions. Purified sesame oil bodies consisting of approximately 99% oil, 0.5% phospholipid, and 0.5% protein were decomposed by heating at 70 degrees C for 1 h. The bacteria cultured in nonfat milk were encapsulated in artificial oil emulsions constituted with decomposed sesame oil bodies and excess sesame or vegetable cooking oil. Viability of bacteria in storage at 4 degrees C for 16 d was substantially elevated from 0.023 to 5.45% after encapsulation. Compared with free cells, the entrapped bacteria demonstrated a significant increase (approximately 10(4) times) in survival rate when subjected to simulated high acid gastric or bile salt conditions. The results indicate that artificial sesame oil emulsion may serve as an effective biocapsule for encapsulation of bacteria in dairy products.     

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus ND02 derived from 2 propagation procedures was determined. The subpopulations consisted of 3 categories (physiological states): viable cells capable of forming colonies on agar plates (VC+), viable cells incapable of forming colonies on agar plates (VC?), widely referred to as viable but nonculturable (VBNC) cells, and nonviable or dead cells (NVC). Counts of VC+ were recorded using a conventional plate count procedure. A fluorescent vital staining procedure that discriminates between viable (VC+ and VC?) and NVC cells was used to determine the number of viable and nonviable cells. Both propagation procedures had 2 variables: in procedure (P)1, the propagation medium was rich in yeast extract (4.0%) and the pH was maintained at 5.7; in P2, the medium was devoid of yeast extract and the pH was maintained at 5.1. The results showed that post-propagation operations—concentration of cells by centrifugation and subsequent freezing or lyophilization of cell concentrate—induced different degrees of transience from VC+ to VC? states in cells derived from P1 and P2. Compared with cells derived from P2, cells from P1 were more labile to stress associated with centrifugation, freezing, and lyophilization, as revealed by differential counting.     

Exploring the industrial potential of Lactobacillus delbrueckii ssp. bulgaricus by population genomics and genome-wide association study analysis

Lactobacillus delbrueckii ssp. bulgaricus is one of the most commonly used starter cultures for yogurt production. However, how its genetic background affects acid production capacity is unclear. This study investigated the industrial potential of L. delbrueckii ssp. bulgaricus using population genomics and GWAS analysis. To meet our goal, population genetics and functional genomics analyses were performed on 188 newly sequenced L. delbrueckii ssp. bulgaricus strains isolated from naturally fermented dairy products together with 19 genome sequences retrieved from the NCBI database. Four distinct clusters were identified, and they were correlated with the geographical sites where the samples were collected. The GWAS analysis about acidification fermentation results with sucrose-fortified reconstituted skim milk revealed a significant association between l-lactate dehydrogenase (lldD; Ldb2036) and the bacterial acid production rate. Our study has broadened the understanding of the population structure and genetic diversity of L. delbrueckii ssp. bulgaricus by identifying potential targets for further research, development, and use of lactic acid bacteria in the dairy industry.     

Immunostimulatory oligonucleotide,CpG-like motif exists in Lactobacillus delbrueckii ssp. bulgaricus NIAI B6

The present study was conducted to find an immunostimulatory oligonucleotide derived from yogurt starter cultures. The chromosomal DNA was purified from nine strains of Lactobacillus delbrueckii ssp. bulgaricus and six strains of Streptococcus thermophilus. An immunostimulatory ability of the DNA was examined in a proliferation of peyer's patch and splenic B cells. Only the DNA from L. bulgaricus NIAI B6 induced a significant proliferation of both cells. When the DNA was cloned and amplified using PCR, the mitogenic activities to B cells were significantly increased by 13 of 135 DNA clones. Ten homologous nucleotide sequences were found as possible oligonucleotide sequences of mitogens, and were then chemically synthesized (sOL-LB1 to sOL-LB10). One CpG-like motif (sOL-LB7; 5'-CGGCACGCTCACGATTCTTG-3') was identified as an immunostimulatory oligonucleotide, but it did not contain palindromic CpG structure known as a B cell-specific mitogen. The sOL-LB7 substantially bound to B cells and increased the CD69 positive cells in peyer's patch cells. This study demonstrated that L. bulgaricus NIAI B6 was a good candidate of a starter culture for the production of new functional foods, "Bio-Defense Foods".     

德氏乳杆菌保加利亚亚种耐酸机制

德氏乳杆菌保加利亚亚种是最具经济价值的乳酸菌之一,其耐酸能力是影响其发酵性能和发挥益生功效的关键因素。本文就该菌的耐酸能力调节进行阐述。     

The impact of alternative nitrogen sources on the growth and viability of Lactobacillus delbrueckii ssp. bulgaricus

In this study, we developed and optimized a growth medium using various nitrogen sources for the cultivation of Lactobacillus delbrueckii ssp. bulgaricus, a probiotic and essential dairy starter culture. The composition of de Man, Rogosa, and Sharpe (MRS) culture medium was modified, and the nitrogen content was replaced by alternative nitrogen sources X-Seed Nucleo Max, X-Seed KAT, and X-Seed Carbo Max (Ohly GmbH) in various blends of 5 and 10 g/L. Results showed that bacterial growth was significantly higher when the nitrogen source blend of 10 g/L of KAT and 10 g/L of Carbo Max [KCMax (10/10)] was used. The optical densities of the Lb. bulgaricus strains were significantly higher in the KCMax (10/10) medium than in the MRS medium. There was no significance in bacterial counts for both the MRS and the KCMax (10/10) medium, and all bacterial counts were estimated at 8 log cfu/mL. The buffering capacity of the KCMax (10/10) medium was also tested and supplemented with l-histidine and was significantly higher than that of the MRS control medium. KCMax (10/10) also supported the freeze-stability and viability of the Lb. bulgaricus cells during freezing and freeze-drying operations. Our results suggest that the alternative nitrogen sources X-Seed Nucleo Max, X-Seed KAT and X-Seed Carbo Max can substantially support the growth of lactic acid bacteria as demonstrated with Lb. bulgaricus. These alternative nitrogen sources could thus be recommended for lactic acid bacteria fermentation and for the cultivation of dairy starter cultures.     

保加利亚乳杆菌胞外多糖合成条件的优化及其在发酵乳中的应用研究

研究了不同碳源、氮源、碳氮比及发酵条件等因素对保加利亚乳杆菌Y5菌株产胞外多糖的影响。结果表明,以果糖为碳源时,胞外多糖的产量是55．46mg／L。显著高于用半乳糖作为碳源时的产量（29．59mg／L）;以鱼蛋白胨作为氮源为时,胞外多糖产量可达到46．89mg／L,明显高于用胰蛋白胨作为氮源时的产量（P〈0．01）;当碳氮比为2：1时,Y5菌株产生的胞外多糖最多,可这50．10g／L,显著高于碳氮比为1：2时的产量（P〈0．01）。培养温度为40℃,发酵时间为16h,初始pH值为6．0,葡萄糖添加量为2％的发酵条件是Y-5菌株产胞外多糖的最佳工艺条件,其胞外多糖水平可达105．36g／L。与采用ST和LB菌株的发酵乳相比,应用Y5菌株生产的发酵乳,其乳清析出量仅为7mL（P〈0．01）：冷藏30d后,Y5菌株制备的发酵乳仍能维持10^7mL^-1以上的活菌数,明显高于对照菌株的存活数量（P〈0．01）。     

Effect of 'ropy' strains of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus on rheology of stirred yogurt

The TA-TX2 Texture Analyser and the Brookfield RVT Viscometer have been used to investigate the contribution of ropiness to the texture of stirred yogurts made using ropy strains of bacteria. Back extrusion and texture profile analysis, not commonly used to quantify rheological properties of semi-solid foods, have been found useful in distinguishing the contribution of exopolysaccharides to different texture attributes (Toba et al ., 1990). Thus ropiness, a characteristic which is imparted to the product as a result of fermentation with particular polysaccharide-producing strains, contributes to 'adhesiveness', while 'firmness' and 'elasticity' are likely to be influenced more by the protein matrix of the yogurt than by secretion of the polysaccharide by the ropy strains. Effects on viscosity and ability to recover viscosity after disruption were apparent, although the contribution of ropiness was not always positive. Ropy strains increased viscosity of stirred yogurts when compared to yogurt made with non-ropy cultures. But, whilst a ropy Lactobacillus delbrueckii ssp. bulgaricus (Lb r⁺) combined with a non-ropy Streptococcus thermophilus (St r⁻) produced a viscous product which recovered its viscosity well, a yogurt made by combining both ropy strains did not recover its viscosity as well as yogurt made by combining two non-ropy cultures and lost its structure more rapidly during the destructive testing. These results show therefore that inclusion of a ropy strain will not always lead to improved texture attributes, that while ropy strains may increase viscosity they may not influence 'firmness' and lend support to the view that this latter attribute is more influenced by protein–protein interactions.     

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.