Survival of Lactobacillus acidophilus and Bifidobacterium animalis ssp. lactis in stirred fruit yogurts

The effect of commercial fruit preparations (mango, mixed berry, passion fruit and strawberry) on the viability of probiotic bacteria, Lactobacillus acidophilus LAFTI^® L10 and Bifidobacterium animalis ssp. lactis LAFTI^® B94 in stirred yogurts during storage (35 days) at refrigerated temperature (4 °C) was evaluated. The results showed that addition of either 5 or 10 g/100 g fruit preparations had no significant (p>0.05) effect on the viability of the two probiotic strains except on L. acidophilus LAFTI L10 yogurt with 10 g/100 g passion fruit or mixed berry. After the addition of fruit preparation, 96% of the yogurts incorporated with fruit preparation did not exhibit a greater loss in the viability of probiotic bacteria compared to plain yogurt during the storage period. A correlation between the post-storage pH in yogurts and the survival of probiotic bacteria was observed. All the yogurts, however, contained the recommended levels of (10⁶-10⁷ cfu/g) probiotic bacteria at the end of 35-day shelf life.     

Temporal gene expression and probiotic attributes of Lactobacillus acidophilus during growth in milk

Lactic acid bacteria have been used as starter strains in the production of fermented dairy products for centuries. Lactobacillus acidophilus is a widely recognized probiotic bacteria commonly added to yogurt and used in dietary supplements. In this study, a whole genome microarray was employed to monitor gene expression of L. acidophilus NCFM cells propagated in 11% skim milk during early, mid and late logarithmic phase, and stationary phase. Approximately 21% of 1,864 open reading frames were differentially expressed at least in one time point. Genes differentially expressed in skim milk included several members of the proteolytic enzyme system. Expression of prtP (proteinase precursor) and prtM (maturase) increased over time as well as several peptidases and transport systems. Expression of Opp1 (oligopeptide transport system 1) was highest at 4 h, whereas gene expression of Opp2 increased over time reaching its highest level at 12 h, suggesting that the 2 systems have different specificities. Expression of a 2-component regulatory system, previously shown to regulate acid tolerance and proteolytic activity, also increased during the early log and early stationary phases of growth. Expression of the genes involved in lactose utilization increased immediately (5 min) upon exposure to milk. The acidification activity, survival under storage conditions, and adhesion to mucin and Caco-2 tissue culture cells of selected mutants containing insertionally inactivated genes differentially expressed in the wild-type strain during growth in milk were examined for any potential links between probiotic properties and bacterial growth and survival in milk. Some of the most interesting genes found to be expressed in milk were correlated with signaling (autoinducer-2) and adherence to mucin and intestinal epithelial cells, in vitro.     

Effect of microencapsulation on viability and other characteristics in Lactobacillus acidophilus ATCC 43121

Lactobacillus acidophilus ATCC 43121 were microencapsulated with sodium alginate by dropping method. The effects of microencapsulation on the changes in survival rate of the L. acidophilus ATCC 43121 during exposure to artificial gastrointestinal and on the change in heat susceptibility of L. acidophilus ATCC 43121 during the heat treatment were studied. In addition, cholesterol assimilation and intestinal adhesion of non-encapsulated and encapsulated L. acidophilus ATCC 43121 were also investigated to explore the effect of microencapsulation on health beneficial effect of lactic acid bacteria. Non-encapsulated cells were completely destroyed when exposed to artificial gastric juice (AGJ) of pH 1.2 and 1.5, while the treatment declined the viable count of encapsulated samples only by 3 log. Encapsulated cells exhibited a significantly higher resistance to artificial intestinal juice (AIJ) and heat treatment than non-encapsulated samples. The assimilative reductions of cholesterol by non-encapsulated and encapsulated L. acidophilus ATCC 43121 were 35.98% and 32.84%, respectively. However, encapsulation did not significantly (P>0.05) affect the adherence of L. acidophilus ATCC 43121 onto the human intestinal epithelial cell lines HT-29. The microencapsulation effectively protected the microorganisms from heat and acid treatment in delivering the viable cells to intestine without any significant adverse effect on their functionalities.     

The effect of probiotics (Lactobacillus rhamnosus HN001, Lactobacillus paracasei LPC-37, and Lactobacillus acidophilus NCFM) on the availability of minerals from Dutch-type cheese

The use of probiotic cultures in the production of Dutch-type cheeses did not lead to significant changes in their chemical composition but it lowered their acidity. The availability of calcium and magnesium analyzed by in vitro enzymatic hydrolysis was 19 and 35%, respectively; the availability of phosphorus was significantly higher, at >90%. The use of probiotic cultures significantly increased the availability of calcium (~2.5%), phosphorus (~6%), and magnesium (~18%). The in vitro method supports accurate determination of the effect of the Lactobacillus spp. cultures on the availability of mineral compounds ingested with Dutch-type cheese.     

An excessively high Lactobacillus acidophilus inoculation level in yogurt lowers product quality during storage

The effect of manufacturing yogurt with a wide variation in Lactobacillus acidophilus inoculation level while holding the yogurt culture inoculation level constant on the properties of the resulting yogurt was determined to find out if any problems can occur if an excessively high level of L. acidophilus is used in yogurt production. Four batches of plain, set-style yogurt were manufactured with skim milk, nonfat dry milk, yogurt cultures, and with or without L. acidophilus (0, 0.0239, 0.238, or 2.33 g/100 g). After homogenization, pasteurization, and cooling, yogurt mixes were inoculated, poured into containers, incubated to pH 4.5, and cooled. Lactobacilli and L. acidophilus counts, pH, amount of syneresis, color, apparent viscosities, and sensory scores were determined during storage. The yogurt inoculated with 0.238 g/100 g L. acidophilus had the highest L. acidophilus counts from 4 to 7 wk. Yogurts inoculated with 2.33 g/100 g L. acidophilus generally had lower lactobacilli counts, L* values, apparent viscosities, and sensory scores but more syneresis and higher a* and b* values than the remaining yogurts. An excessively high inoculated level of L. acidophilus (2.33 g/100 g) resulted in an inferior quality yogurt.     

Elaboration of a probiotic oblea from whey fermented using Lactobacillus acidophilus or Bifidobacterium infantis

A novel probiotic product was developed, which was formulated as an oblea (wafer-type dehydrated traditional Mexican dessert) using goat sweet whey fermented with Bifidobacterium infantis or Lactobacillus acidophilus. To obtain the probiotic oblea, the fermented whey was formulated with prebiotic carbohydrates (inulin and resistant starch) and gelatin, and the preparation was poured onto a polytetrafluoroethylene-coated nonstick baking pan, dried in a convection oven, and finally dehydrated at a low relative humidity and room temperature (23 ± 2°C). The amounts of prebiotic carbohydrates and gelatin to be used in the formulation were determined by a factorial experimental design. An untrained sensory panel evaluated 3 quality characteristics (film formation, homogeneity, and smoothness) in the final product. Three different drying temperatures were tested, namely, 40, 55, and 70°C. Bacterial survival at each temperature was determined by viable plate-counting. The best formulation, based on the quality characteristics tested, consisted of 58.33% (vol/vol) of fermented whey, 8.33% (vol/vol) of 6% (wt/vol) resistant starch dispersion, 16.66% (vol/vol) of 15% (wt/vol) inulin solution, and 16.66% (vol/vol) of a 10% (wt/vol) gelatin solution. Drying at 55 ± 2°C for 2.66 ± 0.22 h allowed for concentrations of probiotic bacteria above 9 log₁₀ cfu/g, which is above the minimum concentration required in a probiotic product.     

Microencapsulation of Lactobacillus acidophilus La-5 by spray-drying using sweet whey and skim milk as encapsulating materials

The aim of this study was to evaluate the effect of encapsulating material on encapsulation yield, resistance to passage through simulated gastrointestinal conditions, and viability of Lactobacillus acidophilus La-5 during storage. Microparticles were produced from reconstituted sweet whey or skim milk (30% total solids) inoculated with a suspension of L. acidophilus La-5 (1% vol/vol) and subjected to spray-drying at inlet and outlet temperatures of 180°C and 85 to 95°C, respectively. The samples were packed, vacuum-sealed, and stored at 4°C and 25°C. Encapsulation yield, moisture content, and resistance of microencapsulated L. acidophilus La-5 compared with free cells (control) during exposure to in vitro gastrointestinal conditions (pH 2.0 and 7.0) were evaluated. Viability was assessed after 0, 7, 15, 30, 45, 60, and 90 d of storage. The experiments were repeated 3 times and data were analyzed by ANOVA and Tukey test for the comparison between means. The encapsulating material did not significantly affect encapsulation yield, average diameter, or moisture of the particles, which averaged 76.58 ± 4.72%, 12.94 ± 0.78 μm, and 4.53 ± 0.32%, respectively. Both microparticle types were effective in protecting the probiotic during gastrointestinal simulation, and the skim milk microparticles favored an increase in viability of L. acidophilus La-5. Regardless of the encapsulating material and temperature of storage, viability of the microencapsulated L. acidophilus La-5 decreased on average 0.43 log cfu/g at the end of 90 d of storage, remaining higher than 10⁶ cfu/g.     

Evaluation of culture media for enumeration of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium animalis in the presence of Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus

The study compared the growth capability of probiotic (Lactobacillus acidophilus La05, Lactobacillus casei Lc01 and Bifidobacterium animalis Bb12) and non-probiotic (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus) cultures on twenty-one culture media grouped according to selectivity: non-selective agars, selective agars without antibiotics and MRS agars containing different combinations of lithium chloride, cystein, bile salts and antibiotics. Four of these media were selected for quantitative enumeration of L. acidophilus La05, L. casei Lc01, and B. animalis Bb12. The best culture media and incubation conditions for enumeration of the probiotic cultures were: B. animalis: MRS agar with dicloxacillin, 37 °C or 42 °C, anaerobiosis; L. acidophilus: MRS agar with bile salts, 37 °C or 42 °C, aerobiosis; L. casei: MRS agar with lithium chloride and sodium propionate, 37 °C or 42 °C, aerobiosis or anaerobiosis. Plating on MRS with glucose replaced by maltose, 37 °C or 42 °C, anaerobiosis, will distinguish probiotic from non-probiotic cultures. For enumeration of each probiotic in a mixed culture, the following media and incubation conditions were recommended: B. animalis: 4ABC-MRS, 42 °C, anaerobiosis, L. acidophilus: LC medium, 42 °C, aerobiosis or anaerobiosis and L. casei: LP-MRS, 42 °C, aerobiosis or anaerobiosis. In all experiments, differences in counts using pour plating or surface plating were not significant (P ≤ 0.05).     

Isolation and partial characterization of the surface protein of Lactobacillus acidophilus strains

A surface protein layer was found to be present in some Lactobacillus acidophilus strains. it could be visualized with electron microscopy and extracted with sodium dodecyl sulfate or guanidine hydrochloride. The protein was resistant to several proteolytic enzymes, but sensitive to pronase. Amino acid composition analysis revealed about 30% hydrophobic amino acids. Hydrophobicity study with a hexadecane partition assay demonstrated that the cells were hydrophobic and removal of the surface layer with pronase destroyed the hydrophobic nature of the cells. Several growth conditions studies were found to have little effect on synthesis of the surface protein.     

Quality attributes of yogurt with Lactobacillus casei and various prebiotics

The objective was to determine the effect of chain length of inulins on the characteristics of fat-free plain yogurt manufactured with Lactobacillus casei. Probiotic fat-free plain yogurts were manufactured using Streptococcus thermophilus, Lactobacillus bulgaricus and L. casei. The treatments were inulins of short (P95), medium (GR) and long (HP) chain lengths. The inulins were incorporated at a concentration of 1.5 g/100 g yogurt mix. Inulins of various chain lengths did not affect viscosity, L*, a*, b* and appearance of yogurts manufactured with L. casei. Yogurt with HP had less syneresis compared to the control, while yogurt with P95 had syneresis comparable to the control. Yogurt with P95 had a significantly lower pH than the control, while the pH of the yogurts with other treatments was not different from the control. Flavor scores of the control were comparable to yogurt with P95. The flavor scores for yogurts with P95 were significantly higher than for yogurts with HP. The yogurts with HP had better body and texture compared to the control and P95. Chain length of prebiotics affected some characteristics of the yogurts.     

Lactobacillus casei MYL01 modulates the proinflammatory state induced by ethanol in an in vitro model

Accumulating studies have suggested that probiotics have beneficial effects on liver injury but the underlying mechanism has remained unclear. Toll-like receptors (TLR) expressed on immune cells and hepatocytes recognize bacterial components that are translocated from the gut into the portal vein. To date, it has been demonstrated that ethanol alone, without microbial components, is able to activate TLR, leading to promotion of proinflammatory cytokine production. Because the enhanced signaling of TLR triggers persistent inflammation, we hypothesized that development of hepatocyte TLR tolerance to repetitive stimulation plays an important role in protecting the liver from hypergeneration of proinflammatory cytokines. In this study, we showed that Lactobacillus casei MYL01 modulated the proinflammatory state induced by ethanol and investigated in detail the mechanism underlying the observation that L. casei MYL01 gave rise to TLR tolerance toward ethanol stimulation. The effects of L. casei MYL01 in the attenuation of ethanol-induced liver damage were due to enhancement of IL-10 production, which limited the proinflammatory process. Furthermore, better defense of hepatocytes against ethanol challenge by treatment of L. casei MYL01 was attributed to previous induction of toll interacting protein (TOLLIP) and suppressor of cytokine signaling (SOCS)1 and SOCS3 expression via activation of TLR1, TLR2, TLR6, and TLR9, an action that cross-regulated ethanol–TLR4–nuclear factor κB signal transduction events. This finding might help establish an in vitro platform for selecting hepatoprotective probiotic strains in terms of ethanol-induced liver damage.     

The substitution of a traditional starter culture in mutton fermented sausages by Lactobacillus acidophilus and Bifidobacterium animalis

Common starter cultures used in fermented mutton sausages were substituted by probiotic strains of Lactobacillus acidophilus CCDM 476 and Bifidobacterium animalis 241a. Technological properties of the traditional and the probiotic sausages were compared. The potential probiotic effect was evaluated by enumeration of bifidobacteria and lactobacilli in stool samples of 15 volunteers before and after a 14-day consumption period. The numbers of lactobacilli (10⁷ cfu/g) and bifidobacteria (10³ cfu/g) in the final product did not affect the technological properties. The use of L. acidophilus as a starter culture was found more beneficial than the use of B. animalis. Even after 60 days of storage, high counts of L. acidophilus (10⁶ cfu/g) were detected; on the other hand, the counts of B. animalis were under the detection limit. Regarding sensory properties, the probiotic products showed better texture, and, curiously, a reduction of the typical smell of mutton. The numbers of lactobacilli in stool samples increased significantly after the consumption of the probiotic sausages.     

Short communication: Effect of milk and milk containing Lactobacillus casei on the intestinal microbiota of mice

BALB/c mice were fed milk or Lactobacillus casei BL23 in milk for 14 d and fecal samples were collected at d 0, 4, and 7 as well as 1 and 8 d after the last administration. According to high-throughput DNA sequencing of the 16S rRNA genes extracted from the fecal microbiota, the bacterial diversity in the fecal samples of all mice increased over time. After 14 d of administration, the consumption of milk and milk containing L. casei BL23 resulted in distinct effects on the microbial composition in the intestine. Specifically, the proportions of bacteria in the Lactobacillaceae, Porphyromonadaceae, and Comamonadaceae were significantly higher in mice fed the L. casei BL23-milk culture compared with one or more of the other groups of mice. The relative amounts of Lachnospiraceae were higher and Streptococcaceae were lower in mice fed milk alone. The changes were not found at d 4 and 7 during milk and L. casei feeding and were no longer detected 8 d after administration was stopped. This study shows that consumption of milk or probiotic L. casei-containing milk results in non-overlapping, taxa-specific effects on the bacteria in the distal murine intestine.     

Effect of probiotic yogurt containing Lactobacillus acidophilus and Bifidobacterium lactis on lipid profile in individuals with type 2 diabetes mellitus

The purpose of this study was to investigate the effects of probiotic and conventional yogurt on the lipid profile in type 2 diabetic people. In a randomized double-blind controlled trial, 60 people (23 males and 37 females) with type 2 diabetes and low-density lipoprotein cholesterol (LDL-C) greater than 2.6 mmol/L were assigned to 2 groups. Participants consumed daily 300 g of probiotic yogurt containing Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 or 300 g of conventional yogurt for 6 wk. Fasting blood samples, anthropometric measurements and 3-d, 24-h dietary recalls were collected at the baseline and at the end of the trial. Probiotic yogurt consumption caused a 4.54% decrease in total cholesterol and a 7.45% decrease in LDL-C compared with the control group. No significant changes from baseline were shown in triglyceride and high-density lipoprotein cholesterol (HDL-C) in the probiotic group. The total cholesterol:HDL-C ratio and LDL-C:HDL-C ratio as atherogenic indices significantly decreased in the probiotic group compared with the control group. Probiotic yogurt improved total cholesterol and LDL-C concentrations in type 2 diabetic people and may contribute to the improvement of cardiovascular disease risk factors.     

A synbiotic containing Lactobacillus acidophilus CHO-220 and inulin improves irregularity of red blood cells

This randomized, double-blind, placebo-controlled, and parallel-design study was conducted to investigate the effect of a synbiotic product containing Lactobacillus acidophilus CHO-220 and inulin on the irregularity in shape of red blood cells (RBC) in hypercholesterolemic subjects. The subjects (n = 32) were randomly allocated to 2 groups, a treatment group (synbiotic product) and a control group (placebo), and received 4 capsules of either synbiotic or placebo daily for 12 wk. Morphological representation via scanning electron microscopy showed that the occurrence of spur RBC was improved upon supplementation of the synbiotic. In addition, the supplementation of synbiotic reduced the cholesterol:phospholipids ratio of the RBC membrane by 47.02% over 12 wk, whereas the control showed insignificant changes. Our present study also showed that supplementation of the synbiotic reduced the concentration of saturated fatty acids (SFA), increased unsaturated fatty acids (UFA), and increased the ratio of UFA:SFA over 12 wk, whereas the control showed inconspicuous changes. The alteration of RBC membrane was assessed using fluorescence anisotropy (FAn) and fluorescence probes with different affinities for varying sections of the membrane phospholipid bilayer. A noticeable decrease in FAn of three fluorescent probes was observed in the synbiotic group compared with the control over 12 wk, indicative of increased membrane fluidity and reduced cholesterol enrichment in the RBC membrane.     

Preparation of Bifidobacterium breve encapsulated in low methoxyl pectin beads and its effects on yogurt quality

Yogurt is a popular product worldwide partly because of the health-promoting effects of the probiotics that it contains. Probiotics with high survivability constitute a promising direction for fortified yogurt products. This study aimed to prepare Bifidobacterium breve–loaded yogurt with the bacteria surviving transit to the lower part of small intestine or colon. Bifidobacterium breve beads were prepared through an ion-crosslinking method using low methoxyl pectin as the encapsulating material. Features such as encapsulation efficiency and stability during storage and passage through the simulated gastrointestinal tract were studied in vitro. A commercial starter was used for yogurt fermentation, and B. breve with or without encapsulation was added as a probiotic supplement with the starter or 3 to 4 h after fermentation. The effects of B. breve beads on yogurt characteristics were evaluated after different fermentation processes: BC, milk fermented with marketed yogurt starter; UBFF, unencapsulated B. breve added to fresh milk and then fermented; EBFF, encapsulated B. breve added to fresh milk and then fermented; UBAF, unencapsulated B. breve added after fermentation with the starter; and EBAF, encapsulated B. breve beads added 3 to 4 h after fermentation with the starter. Evaluation was based on texture, electronic nose, and electronic tongue analyses. The particle size analysis of B. breve beads showed that they were uniform, mostly spherical, 1 to 1.5 mm in diameter with encapsulating efficiency higher than 99%. Following treatment with the simulated gastrointestinal tract conditions, the number of B. breve decreased by 1.76 and 4.82 log cfu/g for B. breve beads and unencapsulated B. breve, respectively. The EBAF group showed the lowest viscosity (2,235.67 cP) at d 0, and the lower postfermentation degree was reflected by the slow increase in yogurt viscosity. All groups kept a relatively stable pH during storage. The cohesiveness values of the EBAF and UBAF groups were significantly higher than those of the other groups. The trends in texture changes within the BC, UBFF, and EBFF groups were similar, and the UBAF and EBAF groups showed similar trends. In conclusion, B. breve beads showed good stability in vitro and improved yogurt characteristics by increasing the survival rate of the encapsulated cells. Good compatibility of low methoxyl pectin beads with yogurt was also observed.     

酸奶中嗜酸乳杆菌和植物乳杆菌检测方法的研究

根据嗜酸乳杆菌、植物乳杆菌、嗜热链球菌和保加利亚乳杆菌对不同碳水化合物的选择性利用，设计了适合上述4种乳酸菌的选择性计数培养基，并对其效果进行了验证。结果表明，利用MRS培养基、麦芽糖-MRS培养基和山梨醇-MRS培养基(两种培养基共同使用)能满足选择性计数的要求。以所设计的选择性培养基，研究了含有4种乳酸菌的酸奶中不同乳酸菌在产品货架期内活菌数的变化规律。     

酸奶中游离氨基酸含量及乳清蛋白组成分析

用反相高效液相色谱法（RP-HPLC）测定了5种原味发酵型酸奶中游离氨基酸含量，并用十二烷基硫酸钠-聚丙稀酰胺凝胶电泳（SDS-PAGE）分析了酸奶乳清蛋白组成。结果表明：被测酸奶中乳清蛋白含量的范围在5.66-10．45g／L，其主要成分为α-乳清蛋白、β-乳球蛋白及少量酪蛋白。用HPLC在酸奶中检测到17种氨基酸，总游离氨基酸质量浓度范围285．90-1116．21mg／L，其中4个品牌酸奶中精氨酸（她）质量浓度最高，1个品牌酸奶中天冬氨酸（Asp）质量浓度最高。