Proteolysis of reconstituted goat whey fermented by Streptococcus thermophilus in co‐culture with commercial probiotic Lactobacillus strains

Reconstituted goat whey was fermented with the starter Streptococcus thermophilus TA‐40 in co‐culture with four probiotic adjuncts (independent treatments): Lactobacillus casei BGP93 (T1), Lactobacillus paracasei BGP1 (T2), Lb. paracasei LPC37 (T3) and Lactobacillus rhamnosus LR32 (T4). Lactobacillus populations were higher than 7 log cfu/mL after fermentation and storage. Proteolysis increased significantly (P < 0.05) during fermentation in all trials. Relative amount of low‐molecular‐weight protein fractions (<6.5 kDa) increased in goat whey trials with T1, T3 and T4 during fermentation and storage. The goat whey powder was considered a potential substrate for starter and probiotic cultures, which raised the opportunities to upgrade this by‐product into a functional food.     

益生菌Lactobacillus plantarum P8与Streptococcus thermophilus混合发酵对发酵乳品质的影响

将益生菌Lactobacillus plantarum P8以不同接种比例分别与Streptococcus thermophilus ND03、ND05和ND07复配发酵牛乳,并以S.thermophilus单菌发酵为对照.于0h、发酵结束(pH=4.5)及4℃冷藏24 h,分别测定发酵乳样品的发酵时间,及pH值、黏度、脱水收缩率和活菌数并进行感官鉴评.结果表明,L.plantarum P8与S.thermophilus ND03复配,发酵时间显著高于其他组,各组样品黏度和脱水收缩率差异不显著;L.plantarum P8与S.thermophilus ND05分别以5×106 mL-1接种1∶1复配时,样品中L.plantarum P8的活菌数为5.26×107mL-1,酸度适宜,无乳清析出,有良好的黏度,产品风味较好,将该组合应用于发酵乳生产,较可行.     

Effect of inulin on the growth and metabolism of a probiotic strain of Lactobacillus rhamnosus in co-culture with Streptococcus thermophilus

Metabolic studies are very important to improve quality of functional dairy products. For this purpose, the behaviors of pure cultures of Streptococcus thermophilus (St) and Lactobacillus rhamnosus (Lr) as well a co-culture of them (St–Lr) were investigated during skim milk fermentation, and the inulin effect as prebiotic was assessed. Lr was able to metabolize 6 g/100 g more galactose than St and St–Lr. Final lactic acid production by Lr was higher (9.8 g/L) compared to St (9.1 g/L) and St–Lr (9.1 g/L). Acetic acid concentration varied from 0.8 g/L (St–Lr) to 1.5 g/L (Lr) and that of ethanol from only 0.2 g/L (St–Lr) to 0.4 g/L (Lr), which suggests the occurrence in Lr of a NADH oxidase activity and citrate co-metabolization via pyruvate, both dissipating a part of the reducing power. Diacetyl and acetoin accumulated at the highest levels (18.4 and 0.8 mg/L, respectively) with St–Lr, which suggests possible synergistic interactions between these microorganisms as well as the Lr capability of co-metabolizing citrate in the presence of lactose. Inulin stimulated both biomass growth and levels of all end-products, as the likely result of fructose release from its partial hydrolysis and subsequent metabolization as an additional carbon and energy source.     

保加利亚乳杆菌和嗜热链球菌生物膜形成研究

采用置片法和菌落计数法对嗜热链球菌(Streptococcus thermophilus)和保加利亚乳杆菌(Lactobacillus bulgaricus)在不同载体表面(椰果粒、不锈钢网布、塑料片、陶瓷片和玻璃片)上形成生物膜的能力进行研究。结果表明：椰果粒和不锈钢网布是适宜乳酸菌单菌生物膜形成的载体,培养7d椰果粒和不锈钢网布上的单菌生物膜初始菌密度可达107CFU/cm2,并且在后续培养中能稳定在3.2×106CFU/cm2左右;混菌生物膜菌密度连续7d稳定在1×107CFU/cm2左右。利用扫描电镜对S. thermophilus和L. bulgaricus形成的混菌生物膜进行观察,结果表明在椰果粒和不锈钢网布表面上S. thermophilus和L. bulgaricus形成典型的混菌生物膜结构。     

Time-dependent persistence of enhanced immune response by a potential probiotic strain Lactobacillus paracasei subsp. paracasei NTU 101

The possible time-dependent role of lactic acid bacteria (LAB) in immunomodulation was investigated in BALB/c mice fed daily with Lactobacillus paracasei subsp. paracasei NTU 101 (10(8) colony forming units) for 3, 6, and 9 weeks, and following feeding with Lactobacillus-free food for a further 7 days. We observed up-regulation of the antigen-presenting ability of dendritic cells, and expression of natural killer group-2 D (NKG2D) molecules capable of trigger natural killer cell-mediated cytotoxicity. Lymphocyte proliferation and antibody production were also significantly increased in mice after treatment. Innate and adaptive immunity remained constant even at the most protracted feeding time, indicative of the time dependence of the bacterial-mediated enhanced immunity. To better correlate intestinal microflora with immunity, the intestinal contents of probiotics and harmful microorganisms were determined. Results showed an altered intestinal microflora, with increases in bifidobacteria and lactobacilli and a decreased content of Clostridium perfringens after feeding with L. paracasei subsp. paracasei NTU 101. It is possible that persistent activation of immunity might be induced by intestinal probiotics.     

Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses

Ninety-eight Lactobacillus plantarum strains isolated from Italian and Argentinean cheeses were evaluated for probiotic potential. After a preliminary subtractive screening based on the presence of msa and bsh genes, 27 strains were characterized. In general, the selected strains showed high resistance to lysozyme, good adaptation to simulated gastric juice, and a moderate to low bile tolerance. The capacity to agglutinate yeast cells in a mannose-specific manner, as well as the cell surface hydrophobicity was found to be variable among strains. Very high β-galactosidase activity was shown by a considerable number of the tested strains, whereas variable prebiotic utilization ability was observed. Only tetracycline resistance was observed in two highly resistant strains which harbored the tetM gene, whereas none of the strains showed β-glucuronidase activity or was capable of inhibiting pathogens. Three strains (Lp790, Lp813, and Lp998) were tested by in vivo trials. A considerable heterogeneity was found among a number of L. plantarum strains screened in this study, leading to the design of multiple cultures to cooperatively link strains showing the widest range of useful traits. Among the selected strains, Lp790, Lp813, and Lp998 showed the best probiotic potential and would be promising candidates for inclusion as starter cultures for the manufacture of probiotic fermented foods.     

Multiple interactions between Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii strongly affect their growth kinetics during the making of hard cooked cheeses

In hard cooked cheeses, any interactions between the thermophilic starters as they grow during the cheese-making are critical, since they modify bacterial growth kinetics and acidification kinetics, so affecting the ripening process and the final characteristics of the cheese. Twenty-four experimental hard cooked cheeses were made under controlled conditions, the milk being inoculated with various combinations of thermophilic strains of Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii. Over the first day of manufacturing we recorded a wide range of different growth kinetics for each starter species used, and a wide range of pH kinetics, depending on the starter combination. Most of the bacterial variability could be statistically explained by the nature, quantity, and/or presence or absence of the different strains inoculated. Four main interactions between the three species were evidenced during cheese-making. There was antagonism between L. helveticus and L. delbrueckii. The lactobacilli had a positive effect on S. thermophilus, which was reciprocal for L. helveticus. L. helveticus had a negative effect on S. thermophilus cultivability. And the combination of S. thermophilus inoculated in large quantities and L. helveticus strain H2 had a negative effect on the growth of the L. delbrueckii strain D2. While the positive effect of L. delbrueckii on S. thermophilus probably corresponds to interactions in milk that have already been described and published, the other interactions were hitherto unknown. These interactions are of major importance for the growth kinetics of streptococci and thermophilic lactobacilli during cheese-making.     

保加利亚乳杆菌和嗜热链球菌发酵紫红薯酸牛奶

研究以鲜牛奶和紫红薯为主要原料,保加利亚乳杆菌(Lactobacillus bulgaricus)与嗜热链球菌(Streptococcus thermophilus)为发酵剂,木糖醇为甜味剂,进行了发酵条件的平行和L9(34)正交试验.结果表明,保加利亚乳杆菌与嗜热链球菌混合量比1∶1(数量比),接种量5％,紫红薯添加量15％,木糖醇10％,果胶0.1％,明胶0.1％,CMC 0.1％,变性淀粉0.5％,45℃,发酵4h,制得的紫红薯酸牛奶不仅保留纯酸牛奶的芳香和口感,又具备紫红薯的营养和色泽,是新型紫红薯营养酸奶发酵的优化工艺.     

Inulin and probiotic concentration effects on fatty and linoleic conjugated acids in cream cheeses

The aim of this study was to evaluate the changes in the lipid profile, especially in conjugated linoleic acid (CLA), in 12 cream cheese batches with different prebiotic (inulin) and probiotic (Bifidobacterium animalis and Lactobacillus acidophilus) concentrations stored for 45 days at 8 ± 0.5 °C and analyzed every 15 days. A Central Rotational Composite Design was used to establish the prebiotic and probiotic concentrations. The effect of the pre- and probiotic concentrations on CLA contents (mg g⁻¹ of fat) was verified by plotting the response surface. The linear, quadratic, and interaction coefficients of the multiple linear regression for both prebiotic and probiotics were not significant (p > 0.05). No significant changes were observed neither in the lipid profile nor in the CLA content among the different batches, in which fatty acid composition and CLA concentrations were similar to the milk used in the preparation of the cream cheese and also to the control batch.     

Adhesive and chemokine stimulatory properties of potentially probiotic Lactobacillus strains

Five Lactobacillus plantarum strains and two Lactobacillus johnsonii strains, stemming either from African traditionally fermented milk products or children's feces, were investigated for probiotic properties in vitro. The relationship between the hydrophobic-hydrophilic cell surface and adhesion ability to HT29 intestinal epithelial cells was investigated, and results indicated that especially the L. johnsonii strains, which exhibited both hydrophobic and hydrophilic surface characteristics, adhered well to HT29 cells. Four L. plantarum and two L. johnsonii strains showed high adherence to HT29 cells, generally higher than that of the probiotic control strain Lactobacillus rhamnosus GG. Most strains with high adhesion ability also showed high autoaggregation ability. The two L. johnsonii strains coaggregated well with the intestinal pathogens Listeria monocytogenes Scott A, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Salmonella enterica serovar Typhimurium ATCC 14028. The L. plantarum BFE 1685 and L. johnsonii 6128 strains furthermore inhibited the adhesion of at least two of these intestinal pathogens in coculture with HT29 cells in a strain-dependent way. These two potential probiotic strains also significantly increased interleukin-8 (IL-8) chemokine production by HT29 cells, although modulation of other cytokines, such as IL-1, IL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNF-alpha), and transforming growth factor beta (TGF-beta), did not occur. Altogether, our results suggested that L. plantarum BFE 1685 and L. johnsonii BFE 6128 showed good adherence, coaggregated with pathogens, and stimulated chemokine production of intestinal epithelial cells, traits that may be considered promising for their development as probiotic strains.     

Enhancement of immune response in mice fed with Streptococcus thermophilus and Lactobacillus acidophilus

Swiss mice, fed for 8 consecutive d with 50 micrograms/d of viable cultures of Lactobacillus acidophilus and Streptococcus thermophilus, showed significant variation in their immune system. In order to study this phenomenon assays for macrophage and lymphocyte function were carried out. Both lactic acid bacteria enhanced significantly the enzymatic and phagocytic activity of peritoneal macrophages as checked against the controls and also accelerated the phagocytic function of the reticuloendothelial system as revealed by the carbon clearance test. On the 2nd d (100 micrograms), L. acidophilus reached a peak of K = .271, which remained high. Streptococcus thermophilus was effective only on the 2nd d and then decreased. The lymphocytic activity studied by immunoglobulin secreting cells was assayed by Jerne's method of plaque-forming cells (PFC). This activity also was increased by the two microorganisms. Streptococcus thermophilus proved more effective than L. acidophilus. Lactobacillus acidophilus and S. thermophilus activated macrophages and lymphocytes and produced the same increase in the immune response of mice whether administered orally or intraperitoneally.     

Lactobacillus delbrueckii subsp. bulgaricus和Streptococcus thermophilus接种比例对Bifidobacterium lactis益生菌发酵乳品质的影响

将Lactobacillus delbrueckii subsp.bulgaricus ND02(LB-ND02)和Streptococcus thermophilus ND03(ST-ND03)按1∶1、1∶10、1∶100、1∶1000接种于脱脂乳中,同时接入益生菌Bifidobacterium lactis V9(B.lactis V9,接种量为2.0×107g-1),于42℃进行发酵。通过对发酵及贮藏过程中发酵乳指标的测定,评价LB-ND02和ST-ND03的接种比例对发酵乳品质的影响。结果表明,随着LB-ND02接种比例减小,凝乳时间显著延长,B.lactis V9活菌数显著提高。4℃贮藏28 d后,随LB-ND02接种比例减小,B.lactis V9存活率差异显著,后酸化也显著减弱。研究发现,LB-ND02和ST-ND03的接种比例,显著影响发酵乳的发酵时间、B.lactis V9活菌数、后酸化及黏度。     

Effect of microencapsulation and resistant starch on the probiotic survival and sensory properties of synbiotic ice cream

Two types of synbiotic ice cream containing 1% of resistant starch with free and encapsulated Lactobacillus casei (Lc-01) and Bifidobacterium lactis (Bb-12) were manufactured. The survival of L. casei and B. lactis were monitored during the product’s storage for 180 days at −20 °C. The viable cell number of L. casei and B. lactis in the free state in prepared ice cream mixture was 5.1 × 10⁹ and 4.1 × 10⁹ CFU/mL at day one and after 180 days storage at −20 °C, these numbers were decreased to 4.2 × 10⁶ and 1.1 × 10⁷ CFU/mL, respectively. When we encapsulated the mentioned probiotic bacteria in calcium alginate beads, the probiotic survival raised at rate of 30% during the same period of storage at same temperature. In general, the results indicated that encapsulation can significantly increase the survival rate of probiotic bacteria in ice cream over an extended shelf-life. The addition of encapsulated probiotics had no significant effect on the sensory properties of non-fermented ice cream in which we used the resistant starch as prebiotic compound.     

全脂乳中2种乳酸菌的HPLC研究

研究了可应用于高效液相离子交换色谱分析的嗜热链球菌及保加利亚乳杆菌的培养方法,同时探讨了全脂乳中培养的乳酸菌HPLC上样菌悬液的制备方法。对全脂乳中培养不同时间的嗜热链球菌及保加利亚乳杆菌进行色谱表征,并首次发现保加利亚乳杆菌在全脂乳培养过程中菌体形态变化与色谱表征的一致性。     

Influence of process temperature and salting methods on starter and NSLAB growth and enzymatic activity during the ripening of cheeses produced with Streptococcus thermophilus and Lactobacillus helveticus

The effect of varying cook temperature (40 or 50 °C) and salting method (dry or brine salting) on bacterial viability, enzymatic activity and chemical composition in cheeses made using Streptococcus thermophilus and Lactobacillus helveticus were investigated. Dry salting resulted in decreased cell viability of L. helveticus, increased lactate dehydrogenase activity and increased free amino acid levels, compared with brine salted cheeses, irrespective of cook temperature. A cook temperature of 50 °C resulted in reduced primary proteolysis and increased pH in comparison with that in cheeses cooked to 40 °C. Salting method influenced moisture content with higher levels in brine salted cheeses; cook temperature was also influential with higher cook temperature resulting in lower moisture within each salting method. Variations in manufacture procedure can allow for the development of cheese varieties with novel flavour and texture profiles using existing Cheddar or Swiss-style manufacturing facilities.     

Selection, application and monitoring of Lactobacillus paracasei strains as adjunct cultures in the production of Gouda-type cheeses

Raw milk cheeses have more intense flavours than cheeses made from pasteurized milk and harbour strains with potential adjunct properties. Two Lactobacillus paracasei strains, R-40926 and R-40937, were selected as potential adjunct cultures from a total of 734 isolates from good quality artisan raw milk Gouda-type cheeses on the basis of their prevalence in different cheese types and/or over several production batches, safety and technological parameters. Conventional culturing, isolation and identification and a combined PCR-DGGE approach using total cheese DNA extracts and DNA extracts obtained from culturable fractions were employed to monitor viability of the introduced adjuncts and their effect on the cheese microbiota. The control cheese made without adjuncts was dominated by members of the starter, i.e. Lactococcus lactis and Leuconostoc pseudomesenteroides. In the cheeses containing either R-40926 or R-40937, the respective adjuncts increased in number as ripening progressed indicating that both strains are well adapted to the cheese environment and can survive in a competitive environment in the presence of a commercial starter culture. Principal component analysis of cheese volatiles determined by steam distillation-extraction and gas chromatography-mass spectrometry could differentiate cheeses made with different concentrations of adjunct R-40926 from the control cheese, and these differences could be correlated to the proteolytic and lipolytic properties of this strain. Collectively, results from microbiological and metabolic analyses indicate that the screening procedure followed throughout this study was successful in delivering potential adjunct candidates to enrich or extend the flavour palette of artisan Gouda-type cheeses under more controlled conditions.     

Selective enumeration and identification of mixed cultures of Streptococcus thermophilus,Lactobacillus delbrueckii subsp. bulgaricus,L. acidophilus,L. paracasei subsp. paracasei and Bifidobacterium lactis in fermented milk

This study describes selective plating methodologies for enumeration of mixed cultures of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. acidophilus, L. paracasei subsp. paracasei and Bifidobacterium lactis in fermented milk based on selective antibiotic-free media. Enumeration of S. thermophilus was performed using M17-lactose. MRS-fructose was suitable for enumeration of L. bulgaricus and MRS-maltose for differentiation between L. acidophilus and L. paracasei. The selective enumeration of B. lactis was obtained using MRS-raffinose containing 0.05% LiCl. The bacterial counts obtained using selective methods were equivalent to those under optimum culture conditions at a probability level of 95%. Performance of the methods was verified in fermented milk products where identification of the enumerated species was confirmed by species-specific polymerase chain reaction. This study shows that combination of species-specific polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis has great detection and identification potential for verification of accurate species labelling in fermented milk without prior isolation of the bacteria.     

嗜热链球菌与保加利亚乳杆菌麦芽复合汁增菌培养基的优化筛选

研究了在麦芽汁中添加不同的营养因子对嗜热链球菌S.t-3的细胞生长量的影响,进一步采用正交试验优化筛选出S.t-3的麦芽复合汁增菌培养基,并对保加利亚乳杆菌L.b-DR和L.b-S1进行了验证性增菌试验。结果表明,S.t-3、L.b-DR和L.b-S1在麦芽汁中能进行正常的产酸代谢,大豆蛋白胨、牛肉膏、酵母膏可显著促进S.t-3的细胞生长(P<0.05);K2HPO4不仅可以促进菌体增殖,而且还可缓冲基质pH值的变化,避免了低pH值对乳酸菌的伤害;利用L9(33)正交试验,筛选出S.t-3的麦芽复合汁增菌培养基最佳配比为:在10%的麦芽汁中添加0.5%大豆蛋白胨、0.5%酵母膏、1%牛肉膏、0.2%K2HPO4;在麦芽复合汁增菌培养基中,S.t-3、L.b-DR和L.b-S1,37℃恒温培养16 h,活菌数分别为2.42×109cfu/mL、2.85×109cfu/mL和4.81×109cfu/mL,与液体MRS培养基的活菌数相当,成本较MRS培养基降低1 000元人民币/t。