Survival of Lactobacillus rhamnosus strains in the upper gastrointestinal tract

In the present study six probiotic Lactobacillus rhamnosus strains were investigated for their ability to survive in the human upper gastrointestinal tract through a dynamic gastric model of digestion. MRS broth was used as delivery vehicle and survival was investigated during in vitro gastric and gastric plus duodenal digestion. Results highlighted that all tested strains showed good survival rate during both gastric and duodenal digestion. In particular, three strains exhibited a great survival showing a recovery percentage in the range between 117 and 276%. In agreement with survival data, high lactic acid production was detected for all strains, confirming their metabolic activity during digestion.     

Lactobacillus rhamnosus alleviates intestinal barrier dysfunction in part by increasing expression of zonula occludens-1 and myosin light-chain kinase in vivo

The effects of lactobacilli on impaired intestinal barrier function and paracellular permeability were evaluated in human epithelial Caco-2 cells treated with tumor necrosis factor-α and in mice with colitis induced by dextran sodium sulfate (DSS). Filter-grown Caco-2 monolayers were used as the intestinal epithelial model. Among the 4 lactobacilli studied, Lactobacillus rhamnosus OLL2838 most effectively suppressed barrier impairment and increased IL-8 secretion induced by tumor necrosis factor-α in Caco-2 cells; however, the conditioned medium from OLL2838 did not show any effect on barrier functions. The in vivo effects of OLL2838 on intestinal epithelial barrier function and colonic inflammation were assessed in DSS-induced colitis of BALB/c mice. Oral treatment with both live and heat-killed OLL2838 suppressed weight loss and recovered colon length. Additionally, barrier function was restored by the administration of live and heat-killed OLL2838 to the DSS-treated animals, which conferred protection against the increase in mucosal permeability associated with DSS-induced colitis. This may at least partially be because of the increased expression of zonula occludens-1 (4.8-fold) and myosin light-chain kinase (3.1-fold) in intestinal epithelial cells isolated from mice of the heat-killed OLL2838 group. Therefore, L. rhamnosus OLL2838 would be useful in the treatment of gastrointestinal diseases such as inflammatory bowel disease.     

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.     

Antialcoholic liver activity of whey fermented by Lactobacillus casei isolated from koumiss

Whey fermented liquid (WFL) was studied for its hepatoprotective effects by using chronic alcohol-induced mice. Whey fermented liquid, prepared by inoculating whey with 4% (vol/vol) Lactobacillus casei and then incubating at 41°C for 8 h, was used to orally treat alcohol-induced mice at 3 dosages for 5 wk. Ethanol consumption significantly reduced the activity of superoxide dismutase and glutathione peroxidase, while lowering glutathione content and increasing levels of aspartate aminotransferase, alanine aminotransferase, total triglyceride, malondialdehyde, and cytochrome P450 2E1. Treatment with WFL significantly attenuated the increased levels of alanine aminotransferase, aspartate aminotransferase, triglyceride, and cytochrome P450 2E1, while decreasing superoxide dismutase, glutathione peroxidase, malondialdehyde, and glutathione levels. Pathological changes in the livers of mice who had ingested alcohol were improved by the administration of WFL. These results suggest that WFL may exert a protective effect against alcoholic liver disease by increasing antioxidant activity, which supports the use of WFL as an antialcoholic liver disease treatment.     

Antioxidant activity of Lactobacillus plantarum strains isolated from traditional Chinese fermented foods

Eleven Lactobacillus plantarum strains isolated from traditional Chinese fermented foods were investigated for their in vitro scavenging activity against hydroxyl and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals, and their resistance to hydrogen peroxide. L. plantarum C88 at a dose of 10(10) CFU/ml showed the highest hydroxyl radical and DPPH scavenging activities, with inhibition rates of 44.31% and 53.05%, respectively. Resistance of intact cells to hydrogen peroxide was also found in all strains. L. plantarum C88 was the most resistant strain against hydrogen peroxide. When L. plantarum C88 was administered to senescent mice suffering oxidative stress induced by d-galactose, the serum superoxide dismutase activity, the glutathione peroxidase activity and the total antioxidant capacity in liver increased significantly, while the level of malondialdehyde in liver decreased significantly. L. plantarum C88 isolated from traditional Chinese fermented dairy tofu could be considered as a potential antioxidant to be applied in functional foods.     

Proteolysis development in enzyme-modified Cheddar cheese using natural and recombinant enzymes of Lactobacillus rhamnosus S93

Proteolysis in enzyme-modified cheese was investigated with natural crude enzyme or recombinant aminopeptidase, both derived from Lactobacillus rhamnosus S93 in the presence of a commercial proteinase, Neutrase. For production of enzyme-modified cheeses, a cheese slurry was produced and pre-incubated with Neutrase. Natural enzyme or recombinant aminopeptidase (50 units 200 g⁻¹ slurry) was added alone or in combination to the cheese slurries, which were then incubated anaerobically under vacuum at 37 °C for 1, 3 and 6 d. The greatest levels of phosphotungstic acid soluble nitrogen and free amino acids were observed in the enzyme-modified cheese containing natural enzyme followed by the one treated with a combination of the natural enzyme and recombinant aminopeptidase. The enzyme-modified cheese containing the recombinant aminopeptidase alone resulted in the complete disappearance of proline after 1 d of maturation time.     

Aglycone production by Lactobacillus rhamnosus CRL981 during soymilk fermentation

Lactobacillus rhamnosus CRL981 showed the highest levels of β-glucosidase and was selected to characterize this enzyme system, among 63 strains of different Lactobacillus species. The maximum activity was obtained at pH 6.4 and 42 °C. The enzyme showed weak resistance to thermal inactivation maintaining only 20% of the initial activity when it was exposed at 50 °C for 5 min. It also, showed stability when stored at 4 °C for 60 days. Afterwards, L. rhamnosus was evaluated for hydrolysis of isoflavones to aglycones, cell population, residual sugars and organic acid produced during fermentation on soymilk (37 °C for 24 h). Higher viable counts were obtained after 12 h of fermentation (8.85 log CFU ml⁻¹) followed by a drop of pH and an increase of acidity during fermentation due the production of organic acids. L. rhamnosus CRL981 was able to proliferate in soymilk and produce a high β-glucosidase activity achieving a complete hydrolysis of glucoside isoflavones after 12 h of fermentation. The present study indicates that L. rhamnosus CRL981 could be used in the development of different aglycone-rich functional soy beverages.     

Temperate and virulent Lactobacillus delbrueckii bacteriophages: Comparison of their thermal and chemical resistance

The aim of this work was to study the efficiency of diverse chemical and thermal treatments usually used in dairy industries to control the number of virulent and temperate Lactobacillus delbrueckii bacteriophages. Two temperate (Cb1/204 and Cb1/342) and three virulent (BYM, YAB and Ib3) phages were studied. The thermal treatments applied were: 63 °C for 30 min (low temperature – long time, LTLT), 72 °C for 15 s (high temperature – short time, HTST), 82 °C for 5 min (milk destined to yogurt elaboration) and 90 °C for 15 min (FIL-IDF). The chemical agents studied were: sodium hypochlorite, ethanol, isopropanol, peracetic acid, biocides A (quaternary ammonium chloride), B (hydrogen peroxide, peracetic acid and peroctanoic acid), C (alkaline chloride foam), D (p-toluensulfonchloroamide, sodium salt) and E (ethoxylated nonylphenol and phosphoric acid). The kinetics of inactivation were drew and T₉₉ (time necessary to eliminate the 99% of phage particles) calculated. Results obtained showed that temperate phages revealed lower resistance than the virulent ones to the treatment temperatures. Biocides A, C, E and peracetic acid showed a notable efficiency to inactivate high concentrations of temperate and virulent L. delbrueckii phages. Biocide B evidenced, in general, a good capacity to eliminate the phage particles. Particularly for this biocide virulent phage Ib3 showed the highest resistance in comparison to the rest of temperate and virulent ones. On the contrary, biocide D and isopropanol presented a very low capacity to inactivate all phages studied. The efficiency of ethanol and hypochlorite was variable depending to the phages considered. These results allow a better knowledge and give useful information to outline more effective treatments to reduce the phage infections in dairy plants.     

Evaluation of stored lamb bio-preserved using a three-strain cocktail of Lactobacillus sakei

Commercially prepared lamb was stored at −1.5 °C after inoculation with a combination of three Lactobacillus sakei strains previously shown to inhibit spoilage and pathogenic bacteria of importance to the meat industry. Between 6 and 14 weeks storage samples were evaluated for growth of inoculated strains, production of fermentation end-products and sensory acceptance of the cooked product. All three L. sakei strains flourished during storage, formed consistently dominant populations and were associated with lower surface pH and increased levels of lactic and acetic acids. Inoculated samples were determined to be as equally acceptable for smell, acidity, rancidity and overall liking as un-inoculated controls.     

Volatile compounds produced by the probiotic strain Lactobacillus plantarum NCIMB 8826 in cereal-based substrates

The production of volatile compounds by the probiotic strain, Lactobacillus plantarum NCIMB 8826, in cereal-based media (oat, wheat, barley and malt) was investigated. Sixty compounds, including fatty acids and their esters, amides, alcohols, aldehydes, aromatic hydrocarbons, furans, ketones, peroxides and pyrans, were identified. L. plantarum significantly changed the aroma profile of the four cereal broths, and each substrate showed a specific volatiles profile. Oat and barley media were the substrates more influenced by the fermentation process. The most abundant volatiles detected in oat, wheat, barley and malt were oleic acid, linoleic acid, acetic acid, and 5-hydroxymethylfurfural, respectively. Analysis of these products confirmed the heterofermentative pathway of L. plantarum. Maillard compounds were not detected during sterilisation and fermentation. This study is the first to report the volatile composition of probiotic drinks produce with non-supplemented cereal-based media and the results obtained could contribute to the development of new non-dairy probiotic formulations.     

Characterization of glucan-producing Leuconostoc strains isolated from sourdough

Sourdough was previously demonstrated to be a fruitful biotope for isolation of lactic acid bacteria producing exopolysaccharides and more accurately diverse glycan polymers which have interesting applications as texturing agents or prebiotics. Characterization of polymers by ¹H and ¹³C NMR spectroscopy analysis demonstrated that these strains could synthesize glucans of high structural variety and containing different amounts of α-(1 → 2), α-(1 → 3) and α-(1 → 6) linkages. In this study, fifteen glucan-producing Leuconostoc mesenteroides and L. citreum strains from sourdoughs were characterized according to carbohydrate fermentation, rep-PCR fingerprinting using (GTG)₅ primers and glycansucrase activity (soluble or cell-associated). Enzyme characterization using SDS-PAGE and in situ polymer production after incubation with sucrose correlated with synthesis of classical or α-(1 → 2) branched dextrans, alternan and levan. In addition, the presence of genes coding for alternansucrase was detected by PCR and partially characterized by sequence analysis. We thus provide new information on the biodiversity of glucan production by sourdough Leuconostoc strains.     

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.     

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.     

Characterization of bacteriocins produced by two strains of Lactobacillus plantarum isolated from Beloura and Chouriço, traditional pork products from Portugal

Bacteriocins bacST202Ch and bacST216Ch, produced by Lactobacillus plantarum strains isolated from Beloura and Chouriço, respectively, inhibited the growth of a number of Gram-positive and Gram-negative meat spoilage bacteria. According to trycine–SDS–PAGE, bacST202Ch and bacST216Ch are approximately 3.5 and 10.0 kDa in size, respectively. Maximal activity of bacST202Ch (25,600 AU/ml) was recorded after 27 h of and bacST216Ch (102,400 AU/ml) after 22 h of growth. The mode of activity, as determined against Enterococcus faecium HKLHS, is bactericidal. Both peptides adsorb to the surface of the producer cells, but at very low concentrations. Both peptides remained active after 120 min at 100 ^oC and after 2 h of incubation at pH 2.0–12.0. Treatment for 120 min at 121 ^oC did not affect bacST216Ch activity. Activity of bacST202Ch and bacST216Ch was not affected by 1% Triton X-100, Tween 80, Tween 20, SDS, NaCl, urea and EDTA. Bacteriocin ST216Ch was deactivated in the presence of 1% Triton X-114. The nucleotide sequence of a 1044 bp DNA fragment amplified from L. plantarum ST202Ch is identical to the structural gene encoding pediocin PA-1, suggesting that the two bacteriocins are identical. Based on the broad spectrum of antibacterial activity, strains ST202Ch and ST216Ch may be used as starter cultures in the fermentation of meat products.     

Ability of Lactobacillus brevis strains to degrade food phenolic acids

The potential to degrade 15 food phenolic acids was investigated for several Lactobacillus brevis strains isolated from different sources. All the strains analysed in this study showed a similar metabolism on phenolic acids. Among the cinnamic acids assayed, only p-coumaric, ferulic and caffeic acids were metabolized by the L. brevis strains. These acids were decarboxylated to produce their corresponding vinyl derivatives. Contrarily to the results previously reported on Lactobacillus plantarum, the L. brevis strains analysed in this study were unable to subsequently reduce or metabolize these vinyl derivatives. In L. brevis, vinyl phenol, vinyl catechol, and vinyl guaiacol were the final metabolic products from p-coumaric, caffeic or ferulic acids, respectively. From the benzoic acids analysed, and similarly to L. plantarum strains, only gallic and protocatechuic acids were modified by L. brevis strains. Both acids were decarboxylated to pyrogallol and catechol, respectively. Currently, the enzymes involved in the metabolism of phenolic acids in L. brevis remain uncharacterized.     

Both stereo-isomers of glucose enhance the survival rate of microencapsulated Lactobacillus rhamnosus GG during storage in the dry state

This study investigates the protective effects of a metabolizable and a non-metabolizable stereoisomer of glucose on encapsulated Lactobacillus rhamnosus GG (LGG) bacteria in the dry state. Separate experiments confirmed that the probiotic strain metabolizes only d-glucose. Encapsulated LGG formulations (2 wt.% LGG, dry basis) were prepared by freeze drying a mixture of the freshly cultured bacteria within various whey protein–carbohydrate matrices. The encapsulant matrices used were (i) a 1:2 mixture of protein:maltodextrin, (ii) a 1:1:1 mixture of protein:maltodextrin:d-glucose and (iii) a 1:1:1 mixture of protein:maltodextrin:l-glucose. The partial substitution of maltodextrin with either d-glucose or l-glucose in the encapsulant matrix significantly enhanced the survival of LGG powders stored at a relative humidity of 33% RH and 25 °C over 35 days. In comparison, there was a protective effect afforded by partial substitution of maltodextrin with either stereoisomer of glucose during storage of LGG powders at relative humidity of 70% RH and 25 °C over 7 days only. However, on longer term storage at relative humidity of 70% RH and 25 °C over 14–35 days, the viability of LGG was similar for all formulations. The results suggest that the protection afforded to LGG by the incorporation of d-glucose or l-glucose in the encapsulant matrix was primarily due to the physico-chemical effect of the glucose molecules rather than d-glucose being a metabolizable sugar. These experiments allowed discrimination between the physico-chemical and nutritional roles of glucose on LGG viability.     

Comparison of inactivation pathways of thermal or high pressure inactivated Lactobacillus rhamnosus ATCC 53103 by flow cytometry analysis

The effect of thermal and pressure treatments on Lactobacillus rhamnosus ATCC 53103 was evaluated by flow cytometric analysis in conjunction to standard cultivation techniques. A double staining technique with fluorochromes carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) revealed that depending on temperature regime used heat-killed cells had different fluorescence behaviors. Cells killed at 60 °C were not stained at all whereas heat treatment at 75 °C resulted in a single population entirely labelled by PI. These findings indicated that thermal-induced cell death was achievable with or without membrane degradation. Hydrostatic pressures beyond 400 MPa inactivated L. rhamnosus ATCC 53103 in a different way. It was observed that the irreversible damage of the membrane-bound transport systems could be largely accounted for the cause of high pressure-induced cell death.     

Bacteriocinogenic Lactobacillus plantarum ST16Pa isolated from papaya (Carica papaya) — From isolation to application: Characterization of a bacteriocin

Strain ST16PA, isolated from papaya was identified as Lactobacillus plantarum based on biochemical tests, PCR with species-specific primers and 16S rDNA sequencing. L. plantarum ST16PA produces a 6.5 kDa bacteriocin, active against different species from genera Enterobacter, Enterococcus, Lactobacillus, Pseudomonas, Streptococcus and Staphylococcus and different serotypes of Listeria spp. The peptide is inactivated by proteolytic enzymes, but not when treated with ??-amylase, catalase, lipase, Triton X-100, SDS, Tween 20, Tween 80, urea, NaCl and EDTA. However, presence of 1% Triton X-114 deactivates the bacteriocin. No change in activity was recorded after 2 h at pH values between 2.0 and 12.0, and after treatment at 100 °C for 120 min or 121 °C for 20 min. The mode of activity against Lactobacillus sakei ATCC 15521, Enterococcus faecalis ATCC 19443 and Listeria innocua 2030C was bactericidal, resulting in cell lysis and enzyme-leakage. No significant differences in cell growth and bacteriocin production were observed when strain ST16Pa was cultured in MRS broth at 26 °C and 30 °C for 24 h (25 600 AU/ml). However, even though strain ST16PA grows well in MRS broth at 15 °C and 37 °C, a reduction of bacteriocin production was observed (400 AU/ml and 1600 AU/ml, respectively). In addition, effect of MRS medium components, different initial pH and additions of glycerol or vitamins to the media on bacteriocin ST16Pa production was studied.Peptide ST16PA adsorbs (400 AU/ml) to producer cells. However, bacteriocin ST16Pa was adsorbed at 50% to cells of L. innocua 2030C and at 75% to L. sakei ATCC 15521 and E. faecalis ATCC 19433 when experiments were conducted at 30 °C and pH 6.5. Adsorption of bacteriocin ST16Pa to target cells at different temperatures, pH and in presence of potassium sorbate, sodium nitrate, sodium chloride, ascorbic acid, Tween 80 and Tween 20 were also studied. To the best of our knowledge, this is the first report on detection of L. plantarum in papaya.     

Effect of different fermentation parameters on l-lactic acid production from liquid distillery stillage

Expansion of lactic acid applications, predominantly for the preparation of biodegradable polymers increased the research interest for new, economically favourable production processes. Liquid stillage from bioethanol production can be an inexpensive, valuable source of nutrients for growth of lactic acid bacteria. Utilisation of residual biomass with spent fermentation media as a functional animal feed can greatly influence the process value and its ecological aspect. In this paper, the kinetics of lactic acid and biomass production on liquid stillage by Lactobacillus rhamnosus ATCC 7469 was studied. In addition, the impact of temperature, inoculum concentration, shaking and pH control by addition of CaCO₃ was evaluated. Maximal lactic acid yield of 73.4%, as well as high biomass production (3 × 10⁸ CFU ml⁻¹) were achieved under selected conditions (41 °C, 5% (v/v) of inoculum, 1% (w/v) of CaCO₃, initial pH of 6.5 and shaking rate of 90 rpm). These results were achieved without supplementation of the stillage with nitrogen or mineral sources.     

Intragastric administration of a superoxide dismutase-producing recombinant Lactobacillus casei BL23 strain attenuates DSS colitis in mice

Human immune cells release large amounts of reactive oxygen species (ROS) such as superoxide radical and hydrogen peroxide via respiratory burst. In inflammatory bowel diseases, a sustained and abnormal activation of the immune response results in oxidative stress of the digestive tract and in a loss of intestinal homeostasis. We previously reported that heterologous production of the Lactobacillus plantarum manganese catalase (MnKat) enhances the survival of Lb. casei BL23 when exposed to oxidative stress. Anti-inflammatory effects were observed after Lb. casei BL23 oral administrations in moderate murine dextran sodium sulfate (DSS)-induced colitis, without added effects of the MnKat production. Here, we evaluated the protective effects obtained by an improved antioxidative strategy. The Lactococcus lactis sodA gene was expressed in Lb. casei BL23 which acquired an efficient manganese superoxide dismutase (MnSOD) activity. The effects of Lb. casei MnSOD alone or in combination with Lb. casei MnKat were compared first in eukaryotic cell PMA-induced oxidative stress model and then in severe murine DSS-induced colitis. Based on ROS production assays as well as colonic histological scores, a significant reduction of both oxidative stress and inflammation was observed with Lb. casei MnSOD either alone or in combination with Lb. casei MnKat. No added effect of the presence of Lb. casei MnKat was observed. These results suggest that Lb. casei BL23 MnSOD could have anti-inflammatory effects on gut inflammation.