Protection of Lactobacillus acidophilus from the low pH of a model gastric juice by incorporation in a W/O/W emulsion

The protective effect of a W/O/W emulsion for Lactobacillus acidophilus from a model gastric juice was investigated in order to develop a method for utilizing the advantages of the probiotics. The bacteria were included in the inner-phase solution of the W/O/W emulsion, and a method for counting the viable-bacteria included in the W/O/W emulsion was developed. The relative viability of the bacteria included in the W/O/W emulsion was 49% at 2 h in the model gastric juice, whereas the viability of the bacteria directly dispersed in the juice declined to 1.3% even at 0.67 h. The relative viabilities of the encapsulated bacteria in the model gastric juice at 2 h were 0.12 and 1.10 for the emulsions having the median diameters of 11.9 and 25.4 μm, prepared with inner-phase volume ratios at 0.03 and 0.45, respectively. The relative viabilities of the bacteria in the W/O/W emulsions with the median diameters of 11 and 27 μm, prepared at the homogenization speed of 2.2×10⁴ and 9.8×10³ rpm, were 0.4 and 0.8, respectively. These results suggested that both the inner-phase volume ratio and the median diameter of oil droplet affected the relative viability of the included bacteria.     

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.     

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.     

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.     

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.     

Quantitative profiling of bacteriocins present in dairy-free probiotic preparations of Lactobacillus acidophilus by nanoliquid chromatography-tandem mass spectrometry

Bacteriocins are a heterogeneous group of ribosomally synthesized peptides or proteins with antimicrobial activity, produced predominantly by lactic acid bacteria, with potential applications as biopreservatives and probiotics. We describe here a novel strategy based on a bottom-up, shotgun proteomic approach using nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) with multiple fragmentation techniques for the quantitative profiling of bacteriocins present in the probiotic preparations of Lactobacillus acidophilus. A direct LC-MS/MS analysis with alternate collision-induced dissociation, high-energy collision dissociation, and electron-transfer dissociation fragmentation following a filter-assisted size-exclusion sample prefractionation has resulted in the identification of peptides belonging to 37 bacteriocins or related proteins. Peptides from lactacin F, helveticin J, lysin, avicin A, acidocin M, curvaticin FS47, and carocin D were predominant. The process of freeze drying under vacuum was observed to affect both the diversity and abundance of bacteriocins. Data acquisition using alternating complementary peptide fragmentation modes, especially electron-transfer dissociation, has significantly enhanced the peptide sequence coverage and number of bacteriocin peptides identified. Multi-enzyme proteolytic digestion was observed to increase the sample complexity and dynamic range, lowering the chances of detection of low-abundant bacteriocin peptides by LC-MS/MS. An analytical platform integrating size exclusion prefractionation, nanoLC-MS/MS analysis with multiple fragmentation techniques, and data-dependent decision tree-driven bioinformatic data analysis is novel in bacteriocin research and suitable for the comprehensive bioanalysis of diverse, low-abundant bacteriocins in complex samples.     

Identification of seven species of the Lactobacillus acidophilus group by FT-IR spectroscopy

Fourier transform infrared (FT-IR) spectra of 102 strains of the seven species of the Lactobacillus acidophilus group were collected and investigated for their potential use in classification and identification on species level. The database built contains more than 370 spectra. Various procedures of pre-processing and classification methods have been compared with respect to their predictive ability. The most encouraging results were achieved with linear discriminant analysis (LDA) of the absorbance values of normalized spectra at selected wavenumbers. The rate of correct species assignment in cross-validation (Jackknife procedure with one spectrum left out for model building) were 95%, 95%, 69%, 100%, 88%, 100%, and 91% for L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, L. helveticus, and L. johnsonii, respectively. Very distinct grouping was found for L. gallinarum and L. helveticus, the most difficult differentiation in LDA was between the pairs L. crispatus/L. amylovorus and L. gasseri/L. johnsonii.     

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.     

Evaluation of proteolytic and ACE-inhibitory activity of Lactobacillus acidophilus in soy whey growth medium via response surface methodology

Soy whey is a rich by-product of tofu-manufacturing industries. We have previously optimized the growth of Lactobacillus acidophilus FTCC 0291 in soy whey upon supplementation of meat extract, vegetable extract and peptone using response surface methodology (RSM). The present study evaluated the proteolytic and angiotensin-I converting enzyme (ACE)-inhibitory activities of L. acidophilus FTCC 0291 in the optimized soy whey medium. The probiotic-fermented soy whey exhibited growth-associated proteolysis and ACE-inhibitory activity. Proteolysis was highly correlated with ACE-inhibitory activity, indicating that peptides liberated via fermentation may have exerted in vitro antihypertensive properties. Of the three nitrogen sources studied, peptone was found to have the highest influence on growth performance and ACE-inhibitory activity. Our results strongly indicated that probiotic-fermented soy whey produced in vitro antihypertensive bioactivity, and hence could be further developed into a carrier for probiotics with enhanced functional properties.     

Propionic acid production by cofermentation of Lactobacillus buchneri and Lactobacillus diolivorans in sourdough

Cooperative metabolism of lactobacilli in silage fermentation converts lactate to propionate. This study aimed to determine whether propionate production by Lactobacillus buchneri and Lactobacillus diolivorans can be applied for bread preservation. Propionate formation was observed in cofermentation with L. buchneri and L. diolivorans in modified MRS broth as well as sourdough with low, medium and high ash contents. 48 mM of propionate was formed in sourdough with medium ash content, but only 9 and 28 mM propionate were formed in sourdoughs prepared from white wheat flour or whole wheat flour, respectively. Acetate levels were comparable in all three sourdoughs and ranged from 160 to 175 mM. Sourdough fermented with L. buchneri and L. diolivorans was used in breadmaking and its effect on fungal spoilage was compared to traditional sourdough or propionate addition to straight doughs. Bread slices were inoculated with Aspergillus clavatus, Cladosporium spp., Mortierella spp. or Penicillium roquefortii. The use of 20% experimental sourdough inhibited growth of three of the four moulds for more than 12 days. The use of 10% experimental sourdough deferred growth of two moulds by one day. Bread from traditional sourdough with added acetate had less effect in inhibiting mould growth.     

Viability of Lactobacillus acidophilus La-5 added solely or in co-culture with a yoghurt starter culture and implications on physico-chemical and related properties of Minas fresh cheese during storage

The effect of a probiotic culture of Lactobacillus acidophilus (La-5), added solely or in co-culture with a starter culture of Streptococcus thermophilus, on texture, proteolysis and related properties of Minas fresh cheese during storage at 5 °C was investigated. Three cheese-making trials were prepared and produced with no addition of cultures (T1 - control), supplemented with La-5 (T2), and with La-5 + S. thermophilus (T3). Viable counts of La-5 remained above 6.00 log cfu g⁻¹ during the whole storage for T2, reaching 7.00 log cfu g⁻¹ on the 14th day. For T3, the counts of La-5 remained above 6.00 log cfu g⁻¹ after 7 days of storage. Due to the presence of S. thermophilus, T3 presented the highest proteolytic index increase and titratable acidity values. Nevertheless, these results and S. thermophilus addition had no influence on viability of La-5 which presented satisfactory populations for a probiotic food. Moreover, the use of a yoghurt culture for the production of Minas fresh cheese T3 supplemented with La-5 resulted in a good quality product, with a small rate of post-acidification, indicating that traditional yoghurt culture could be employed in co-culture with La-5 to improve the quality of this cheese.     

Probiotic beverage from cashew apple juice fermented with Lactobacillus casei

This study optimized the conditions of Lactobacillus casei NRRL B-442 cultivation in cashew apple juice, as well as, determined the proper inoculum amount and fermentation time. Moreover, it was investigated the survivability ability of L. casei in cashew apple juice during refrigerated storage (4 °C) for 42 days. The optimum conditions for probiotic cashew apple juice production were initial pH 6.4, fermentation temperature of 30 °C, inoculation level of 7.48 Log CFU/mL (L. casei) and 16 h of fermentation process. It was observed that the L. casei grew during the refrigerated storage. Viable cell counts were higher than 8.00 Log CFU/mL throughout the storage period (42 days). The values of lightness, yellowness and total color change increased and the values of redness reduced along the fermentation and refrigerated storage periods. The fermented juice with L. casei is a good and healthy alternative functional food containing probiotics. Cashew apple juice showed to be as efficient as dairy products for L. casei growth.     

基于熊果酸的W/O/W型Pickering乳液制备方法

目的:提高熊果酸的应用范围和生物利用率。方法:以熊果酸为乳化剂,果胶为外水相,采用两步法制备W/O/W型Pickering乳液。通过单因素试验优化W/O乳液制备条件,研究果胶质量浓度对W/O/W型Pickering乳液理化性质、微观结构、流变学特性及长期稳定性的影响。结果:W/O乳液的最佳制备工艺为熊果酸质量浓度4 g/100 mL、水油质量比2∶3、均质转速9 000 r/min、均质时间5 min;以果胶为外水相制备的双层乳液粒径均在30～40μm,果胶质量浓度的增加使乳液的zeta电位绝对值、流变性能以及4℃贮藏稳定性有着显著性的提高。结论:果胶质量浓度为0.3,0.4,0.5 g/100 mL时能够制备稳定的Pickering双层乳液。     

Effect of calcium alginate and resistant starch microencapsulation on the survival rate of Lactobacillus acidophilus La5 and sensory properties in Iranian white brined cheese

Two types of probiotic cheese, with free and microencapsulated bacteria, were manufactured in triplicate under the same conditions. The number of viable cells during 182 days of storage in refrigerated conditions was evaluated. The number of viable cells of Lactobacillus acidophilus was reduced significantly from day 28 to day 182 of storage period in both types of cheese, but reduction in the cheese containing free cells (5.1 ± 0.67 log cfu g⁻¹) was significantly p < 0.05 higher than the cheese containing microencapsulated cells (11.00 ± 0.58 log cfu g⁻¹). The results showed that, microencapsulation in calcium alginate gel and resistant starch was able to increase the survival rate of L. acidophilus La5 in Iranian white brined cheese after 6 months of storage.     

O/W emulsion and W/O/W multiple emulsion: physical characterization and skin pharmacokinetic comparison in the delivery process of caffeine

The aim of this study was to compare the characteristics of an O/W emulsion and a W/O/W emulsion, formulated with similar ingredients. The physical properties of these two types of vehicles were characterized first. Using three types of in vitro models, human skin biopsies, nitrate acetate cellulose membranes and human reconstituted epidermis, the delivery potential of each vehicle for one hydrophilic drug, caffeine, was compared. The assessment of physical parameters, such as particle size, conductivity and rheological behavior enabled the nature of the test emulsions to be clearly identified. Clear differences were observed in the ability of each type of emulsion to deliver caffeine. Whatever the nature of the membrane used for the pharmacokinetic study, the absorption of caffeine was roughly two-fold lower (2.6 for human skin) when the W/O/W multiple emulsion was used as the vehicle. The concomitant determination of physical and kinetic properties of these two test emulsions allowed the W/O/W multiple emulsion and a simple O/W emulsion to be clearly differentiated.     

Evolution of aroma volatiles during storage of sourdough breads made by mixed cultures of Kluyveromyces marxianus and Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus helveticus

Two mixed starter cultures were used for sourdough bread making to evaluate their ability to improve quality and increase bread shelf-life: Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus helveticus mixed with the lactose fermenting yeast Kluyveromyces marxianus as alternative baker’s yeast. Control sourdough breads (K. marxianus) without the addition of bacteria, were also prepared. The changes on the headspace aroma volatiles during storage were assessed using solid-phase microextraction (SPME) GC–MS analysis. The effect of these changes on bread flavour was evaluated by consumer preference evaluations and the results were co-evaluated with those from the GC–MS analysis. The obtained results showed differences in the volatile composition of the different types of breads examined, as well as dramatic decreases of the number and the amount of volatiles after five days of storage. The sourdough breads made with K. marxianus and L. bulgaricus, had a more complex aroma profile, longer shelf-life and achieved the highest scores in the sensory tests.     

Effect of microencapsulation on survival of Lactobacillus plantarum in simulated gastrointestinal conditions, refrigeration, and yogurt

In the present research the survival of free and microencapsulated cells of a new strain of Lactobacillus plantarum BL011 under stress conditions was tested in sodium alginate or pectin, coated with sodium alginate or chitosan. Results for the simulated gastrointestinal medium (SGT) showed no change in viability of cells in relation to the control. However, the simulated gastric medium (GM) drastically reduced the viability under the tested conditions, with no significant differences between free and immobilized cells. Under refrigerated storage viability of immobilized cells were greatly enhanced compared to the free microorganisms, and the treatments showing the lowest loss of viability were those of 4% (w/v) pectin, 3% (w/v) sodium alginate coated with chitosan and a mixture of 2% (w/v) sodium alginate and 2% (w/v) pectin, respectively. Loss of viability of immobilized L. plantarum in 3% alginate coated with chitosan in yogurt was of 0.55 log cycles during 38 days of storage. The results of this study suggest the efficiency of immobilization techniques to increase the survival of lactobacilli in yogurt under refrigerated storage.     

Effects of a probiotic fermented milk beverage containing Lactobacillus casei strain Shirota on defecation frequency,intestinal microbiota,and the intestinal environment of healthy individuals with soft stools

The effects of drinking a fermented milk beverage that contains Lactobacillus casei strain Shirota (LcS) at 40 billion bacterial cells/bottle for 4 weeks (probiotics, 1 bottle/day) on defecation frequency, intestinal microbiota and the intestinal environment of healthy individuals with soft stools were evaluated. Thirty-four healthy adults who had soft stools were randomised into 2 groups, and the effects of a regular 4-week intake of probiotics were evaluated by a placebo-controlled, double-blind, parallel-group comparative design. Defecation frequency significantly decreased after the 4-week intake period compared with before the probiotic treatment. The stool quality significantly improved (hardened) compared to the placebo. Also, the water content of the stools was lower in the probiotic group than in the placebo group. Live LcS was recovered at 6.9 ± 1.3 and 7.2 ± 0.8 log₁₀ CFU per 1 g of stool after 2 and 4 weeks, respectively, of probiotic treatment. The number of bifidobacteria in the stools also increased significantly compared with the level before starting the probiotics. The organic acid levels (total, acetic acid, propionic acid, and butyric acid) significantly increased compared with the level before intake in both the probiotic and placebo groups, but they returned to the original levels after the end of the intake period. These results suggest that probiotic fermented milk beverage has an intestine-conditioning effect by improving the frequency of defecation and stool quality and increasing the intrinsic bifidobacteria in healthy individuals with soft stool.