Supportive Role of Probiotic Strains in Protecting Rats from Ovariectomy-Induced Cortical Bone Loss

Osteoporosis is a major health problem that occurs as a result of an imbalance between bone formation and bone resorption. Different approaches have been established for treating osteoporosis. Recently, because of their health benefits and also low adverse reaction, probiotics have been receiving considerable attention. In this study, we compared the effectiveness of five probiotic strains, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei, Bifidobacterium longum, and Bacillus coagulans, in protecting rats from ovariectomized (OVX)-induced bone loss. Forty-nine adult female Sprague-Dawley rats were allocated into seven groups as follows: group 1, control; group 2, OVX; group 3, OVX + Lactobacillus acidophilus; group 4, OVX + Lactobacillus casei; group 5, OVX + Bacillus coagulans; group 6, OVX + Bifidobacterium longum; and group 7, OVX + Lactobacillus reuteri. Probiotics were fed to OVX groups at the concentration of (1 × 10⁹ CFU/ml/day) for 4 weeks. Then, biochemical parameters, including vitamin D, calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP), were assessed. Dual-energy X-ray absorptiometry (DEXA) scans were used that assess bone mineral density (BMD), bone marrow concentration (BMC), and area of global, femur, spine, and tibia. Lactobacillus acidophilus and Lactobacillus casei significantly increased Ca and ALP and decreased P in treated groups. Lactobacillus casei, Lactobacillus reuteri, and Bifidobacterium longum increased vitamin D significantly. Lactobacillus acidophilus and Lactobacillus casei indicated the most effects on BMD. In terms of BMC, and bone area, Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus casei demonstrated the significant enhancement in OVX groups treated with. Among the probiotics used in this study, Lactobacillus acidophilus and Lactobacillus casei showed the most effects in terms of BMD, BMC, bone area, and biochemical parameters. It seems that probiotics effects on bone health are strain dependent, but further studies should be done to prove these findings.

     

More Protection of Lactobacillus acidophilus Than Bifidobacterium bifidum Probiotics on Azoxymethane-Induced Mouse Colon Cancer

Based on the ability of the probiotics in the gut microflora modification, they can have the beneficial effects on diseases in the short and/or the long term. In previous study, we revealed that unlike Bifidobacterium bifidum, the amount of Lactobacillus acidophilus remained almost unchanged in mice gut microflora in the long term, indicating more stability of L. acidophilus than B. bifidum which can be used to prevent some incurable diseases such as cancer. Thirty-eight male BALB/c mice were divided into four groups, control, azoxymethane (AOM), L. acidophilus, and B. bifidum probiotics, to evaluate the protective effects of the probiotics on AOM-induced mouse colon cancer. Except for the control group, the rest of the animals were weekly given AOM (15 mg/kg, s.c) in three consecutive weeks. Colon lesion incidence was 74% in the AOM group in comparison with the control (0%) (P < 0.05). The lesions were varied from mild to severe dysplasia and colonic adenocarcinoma. Administration of the probiotics inhibited the incidence of colonic lesions by about 57% in L. acidophilus (P < 0.05) and 27% in B. bifidum (P > 0.05) compared to the AOM group. The serum levels of CEA and CA19-9 tumor markers were significantly decreased in L. acidophilus in comparison with the AOM group (P < 0.05). Moreover, the serum levels of IFN-γ and IL-10 and the number of CD4⁺ and CD8⁺ cells were significantly increased in L. acidophilus compared to AOM (P < 0.05). Our study highlighted the more potential effects of L. acidophilus probiotic than B. bifidum on mouse colon cancer.

     

Effects of Dietary Lactiplantibacillus plantarum subsp. plantarum L7, Alone or in Combination with Limosilactobacillus reuteri P16, on Growth,Mucosal Immune Responses,and Disease Resistance of Cyprinus carpio

Skin mucosal lymphoid tissues of fish are the first line of defence against pathogen invasion. We investigated the effects of Lactiplantibacillus plantarum subsp. plantarum L7, singularly or in combination with Limosilactobacillus reuteri P16, on mucosal immunity and diseases resistance of carp Cyprinus carpio. C. carpio (average weight: 26.28 ± 1.02 g) were divided into five experimental groups. Fish in each group were fed with one of the following potential probiotic-supplemented diets: control (0 – basal diet), D1 (10⁷ CFU/g L7), D2 (10⁸ CFU/g L7), D3 (10⁹ CFU/g L7), and D4 (10⁸ CFU/g L7 + 10⁸ CFU/g P16). Eight weeks post-feeding, growth performance was higher in D4, with a final weight gain of 67.18 ± 1.47 g. Results showed a significantly higher skin mucosal lysozyme, alkaline phosphatase, mucus protein level, superoxide dismutase, and catalase activities in D2 and D4 compared to the control. However, potential probiotics had no significant effect on skin mucosal immunoglobulin level. Skin mucus of D4 exhibited stronger inhibition zones against pathogenic bacterial strains. Moreover, digestive enzyme activities (protease, lipase) were highest in D4. Intesinal lactic acid bacterial counts of fish fed combind probiotics (i.e. D4) was significantly higher than the control. Further, supplementation of potential probiotics altered the expression of IL-1β, TNF-α, and IL-10 cytokines. Fish from D4 exhibited significantly higher relative post-challenge survival (69.7%) against Aeromonas hydrophila, followed by D2 (66.67%). Therefore, the inclusion of L. plantarum subsp. plantarum L7 at 10⁸ CFU/g or in combination with L. reuteri P16 could enhance the growth performance, mucosal immune responses, and disease resistance of C. carpio.

     

Low-Dose Electron-Beam Irradiation for the Improvement of Biofilm Formation by Probiotic Lactobacilli

The effects of 50–150 gray electron-beam irradiation on the biofilm-formation ability and cell surface hydrophobicity of the commercial strain, Lactobacillus acidophilus DDS®-1, from Lacto-G (a marketed synbiotic formulation) and the putative probiotic, L. rhamnosus Vahe, were evaluated. No significant changes in cell surface hydrophobicity were found after irradiation, while increases in biofilm-formation abilities were documented for both investigated microorganisms 0.22 ± 0.03 vs. 0.149 ± 0.02 (L. rhamnosus Vahe, 150 Gy) and 0.218 ± 0.021 vs. 0.17 ± 0.012 (L. acidophilus DDS®-1, 150 Gy). Given this, the use of electron-beam irradiation (50–100 Gy) for the treatment of L. rhamnosus Vahe and L. acidophilus DDS®-1 cells may be considered in product sterilization, quality improvement, and packaging practices.

     

Cell Wall Contents of Probiotics (Lactobacillus species) Protect Against Lipopolysaccharide (LPS)-Induced Murine Colitis by Limiting Immuno-inflammation and Oxidative Stress

Currently, there are no effective therapeutic agents to limit intestinal mucosal damage associated with inflammatory bowel disease (IBD). Based on several clinical studies, probiotics have emerged as a possible novel therapeutic strategy for IBD; however, their possible mechanisms are still poorly understood. Although probiotics in murine and human improve disease severity, very little is known about the specific contribution of cell wall contents of probiotics in IBD. Herein, we investigated the protective effects of cell wall contents of three Lactobacillus species in lipopolysaccharide (LPS)-induced colitis rats. LPS-sensitized rats were rendered colitic by colonic instillation of LPS (500 µg/rat) for 14 consecutive days. Concurrently, cell wall contents isolated from 10⁶ CFU of L. casei (LC), L. acidophilus (LA), and L. rhamnosus (LA) was given subcutaneously for 21 days, considering sulfasalazine (100 mg/kg, p.o.) as standard. The severity of colitis was assessed by body weight loss, food intake, stool consistency, rectal bleeding, colon weight/length, spleen weight, and histological analysis. Colonic inflammatory markers (myeloperoxidase activity, C-reactive protein, and pro-inflammatory cytokines) and oxidative stress markers (malondialdehyde, reduced glutathione, and nitric oxide) were also assayed. Cell wall contents of LC, LA, and LR significantly ameliorated the severity of colitis by reducing body weight loss and diarrhea and bleeding incidence, improving food intake, colon weight/length, spleen weight, and microscopic damage to the colonic mucosa. The treatment also reduced levels of inflammatory and oxidative stress markers and boosted anti-oxidant molecule. In conclusion, cell wall contents of LC, LA, and LR attenuate LPS-induced colitis by modulating immuno-inflammation and oxidative stress.

     

E. coli Nissle microencapsulation in alginate-chitosan nanoparticles and its effect on Campylobacter jejuni in vitro

Microencapsulation enhances the oral delivery of probiotic bacteria. In this study, the probiotic Escherichia coli Nissle 1917 (EcN) was microencapsulated using alginate and chitosan nanoparticles. The result showed 90% encapsulation yield of EcN, and the encapsulated EcN displayed significantly (P < 0.05) increased survival in low pH (1.5), high bile salt concentration (4%), and high temperature (70 °C). The most effective cryopreservatives of EcN during freezing and thawing was skim milk and sucrose. Exposure to microencapsulated EcN significantly (P < 0.05) reduced the Campylobacter jejuni growth by 2 log CFU. The rate of EcN release from microcapsule was 9.2 × 10⁵ cell min⁻¹, and the appropriate model to describe its release kinetics was zero order. Importantly, the entrapment of EcN inside the microcapsule did not eliminate the exterior diffusion of EcN produced antioxidant compounds. In addition, the EcN microcapsule efficiently adhered to intestinal HT-29 cells and the pre-treatment of HT-29 cells with EcN-microcapsule for 4 h significantly (P < 0.05) reduced the invasion (1.9 log) of C. jejuni; whereas, completely abolished the intracellular survival. Furthermore, HT-29 cells pre-treated with encapsulated EcN in PCR array showed decreased expression (> 1.5-fold) of genes encoding chemokines, toll-like receptors, interleukins, and tumor necrosis factors. In conclusion, the alginate-chitosan microcapsule can provide effectual platform to deliver probiotic EcN and thereby can reduce the Campylobacter infection in chickens and humans.

     

Measurement of particle diameter of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> microcapsule by spray drying and analysis on its microstructure

Lactobacillus acidophilus, as a probiotic, is widely used in many functional food products. Microencapsulation not only increases the survival rate of L. acidophilus during storage and extends the shelf-life of its products, but also optimal size microcapsule makes L. acidophilus have an excellent dispersability in final products. In this paper, L. acidophilus was microencapsulated using spray drying (inlet air temperature of 170°C; outlet air temperature of 85–90°C). The wall materials used in this study were β-cyclodextrin and acacia gum in the proportion of 9:1 (w/w), and microcapsules were prepared at four levels of wall materials (15, 20, 25 and 30% [w/v]) with a core material concentration of 6% (v/v). The microcapsule diameters were measured by Malvern’s Mastersizer-2000 particle size analyzer. The results showed that the particle diameters of microcapsule were mostly within 6.607 μm and 60.256 μm and varied with 2.884–120.226 μm (the standard smaller microcapsule designated as <350 μm). Through comparison of microcapsule size and uniformity with different concentration of wall materials, we concluded that the optimal concentration of wall material was 20% (w/v), which gave microcapsule with a relatively uniform size (averaging 22.153 μm), and the number of surviving encapsulated L. acidophilus was 1.50 × 10⁹ c.f.u./ml. After 8 weeks storage at 4°C, the live bacterial number was above 10⁷ c.f.u./ml, compared with unencapsulated L. acidophilus, 10⁴–10⁵ c.f.u./ml. Through the observation of scanning electron microscopy, we found that the shapes of microcapsule were round and oval, and L. acidophilus cells located in the centre of microcapsule.     

Fermentation of coconut water by probiotic strains Lactobacillus acidophilus L10 and Lactobacillus casei L26

Coconut water is becoming an increasingly popular beverage and sports drink in tropical countries due to its high mineral content. Probiotic fermentation of coconut water would provide consumers with a novel probiotic beverage which can provide both hydration and probiotic benefits. The aim of this study was to assess the growth, survival and fermentation performance of two probiotic bacteria in coconut water. Lactobacillus acidophilus L10 and L. casei L26 grew well in coconut water and showed similar growth patterns. The viable cell count of the two probiotic cultures reached approximately 10⁸ CFU/ml after 2 days fermentation at 37 °C and maintained approximately10⁷–10⁸ CFU/ml after 26 days at 4 °C. Changes in total soluble solids (^oBrix), pH, sugars, organic acids and minerals were similar between the two probiotic cultures, except for fructose, glucose, copper, phosphorus and lactic, acetic and malic acids. There were significant variations between the two cultures in their ability to produce and consume these compounds. L. acidophilus produced higher amounts of 2-heptanone, 2-nonanone, benzaldehyde, 2-heptanol, 2-nonanol, δ-octalactone and δ-dodecalactone, whereas L. casei produced higher amounts of acetic acid, diacetyl, acetoin, δ-decalactone, 3-methyl-3-buten-1-ol, linalool, 1-octanol, p-tolualdehyde and ethyl 2-hydroxypropanoate. There was no substantial change in mineral content. These results suggest the feasibility of fermenting coconut water into a probiotic beverage, especially for sports nutrition, with the dual benefits of electrolytes and probiotics.     

The Effects of Probiotic Honey Consumption on Metabolic Status in Patients with Diabetic Nephropathy: a Randomized,Double-Blind,Controlled Trial

To the best of our knowledge, this study is the first evaluating the effects of probiotic honey intake on glycemic control, lipid profiles, biomarkers of inflammation, and oxidative stress in patients with diabetic nephropathy (DN). This investigation was conducted to evaluate the effects of probiotic honey intake on metabolic status in patients with DN. This randomized, double-blind, controlled clinical trial was performed among 60 patients with DN. Patients were randomly allocated into two groups to receive either 25 g/day probiotic honey containing a viable and heat-resistant probiotic Bacillus coagulans T11 (IBRC-M10791) (10⁸ CFU/g) or 25 g/day control honey (n = 30 each group) for 12 weeks. Fasting blood samples were taken at baseline and 12 weeks after supplementation to quantify glycemic status, lipid concentrations, biomarkers of inflammation, and oxidative stress. After 12 weeks of intervention, patients who received probiotic honey compared with the control honey had significantly decreased serum insulin levels (− 1.2 ± 1.8 vs. − 0.1 ± 1.3 μIU/mL, P = 0.004) and homeostasis model of assessment-estimated insulin resistance (− 0.5 ± 0.6 vs. 0.003 ± 0.4, P = 0.002) and significantly improved quantitative insulin sensitivity check index (+ 0.005 ± 0.009 vs. − 0.0007 ± 0.005, P = 0.004). Additionally, compared with the control honey, probiotic honey intake has resulted in a significant reduction in total-/HDL-cholesterol (− 0.2 ± 0.5 vs. + 0.1 ± 0.1, P = 0.04). Probiotic honey intake significantly reduced serum high-sensitivity C-reactive protein (hs-CRP) (− 1.9 ± 2.4 vs. − 0.2 ± 2.7 mg/L, P = 0.01) and plasma malondialdehyde (MDA) levels (− 0.1 ± 0.6 vs. + 0.6 ± 1.0 μmol/L, P = 0.002) compared with the control honey. Probiotic honey intake had no significant effects on other metabolic profiles compared with the control honey. Overall, findings from the current study demonstrated that probiotic honey consumption for 12 weeks among DN patients had beneficial effects on insulin metabolism, total-/HDL-cholesterol, serum hs-CRP, and plasma MDA levels, but did not affect other metabolic profiles. http://www.irct.ir: IRCT201705035623N115.

     

Enterocin M-Producing Enterococcus faecium CCM 8558 Demonstrating Probiotic Properties in Horses

The effects of non-authochtonous Enterococcus faecium AL41 = CCM 8558, enterocin M-producing and probiotic strain were tested on the microbiota, phagocytic activity, hydrolytic enzymes, biochemical parameters and dry matter in horses based on its previous benefits demonstrated in other animals. E. faecium CCM 8558 sufficiently colonized the digestive tract of horses. At day 14, its counts reached 2.35 ± 0.70 CFU/g (log 10) on average. The identity of CCM 8558 was confirmed by means of PCR after its re-isolation from horse faeces. The inhibition activity of CCM 8558 was demonstrated against Gram-negative aeromonads, counts of which were significantly reduced (P < 0.001). After 14 days application of CCM 8558, a tendency towards increased phagocytic activity (PA) was measured; PA value was 73.13% ± 8.55 on average at day 0/1; at day 14, it was 75.11 ± 8.66%. Cellulolytic, xylanolytic and pectinolytic activity in horse faeces was significantly increased (P < 0.001) at day 14 (after CCM 8558 application) and amylolytic activity as well (P < 0.01) compared to day 0/1. Inulolytic activity increased with mathematical difference 1.378. Dry matter value reached 20.81 ± 2.29% on average at day 0/1; at day 14, it was 20.77 ± 2.59% (P = 0.9725). Biochemical parameters were influenced mostly in the physiological range. These results achieved after application of CCM 8558 in horses are original, giving us further opportunity to continue these studies, to measure additional parameters and to show the benefits of CCM 8558 application in horses.

     

The Effect of Lactococcus lactis subsp. lactis PTCC 1403 on the Growth Performance,Digestive Enzymes Activity,Antioxidative Status,Immune Response,and Disease Resistance of Rainbow Trout (Oncorhynchus mykiss)

The effect of Lactococcus lactis subsp. lactis strain PTCC 1403 as a potential probiotic was investigated on the growth, hematobiochemical, immune responses, and resistance to Yersinia ruckeri infection in rainbow trout. A total of 240 fish were distributed into 12 fiberglass tanks representing four groups (× 3 replicates). Each tank was stocked with 20 fish (average initial weight: 11.81 ± 0.32 g) and fed L. lactis subsp. lactis PTCC 1403 at 0 (control, T0), 1 × 10⁹ (T1), 2 × 10⁹ (T2), and 3 × 10⁹ (T3) CFU/g feed for 8 weeks. The results showed enhanced protein efficiency ratio and reduced feed conversion ratio in the fish-fed T2 diet. Further, fish-fed T2 and T3 diets showed a significantly higher survival rate than the control (p < 0.05). Trypsin, lipase, and protease activities were increased in fish-fed L. lactis subsp. lactis PTCC 1403 compared to the control (p < 0.05). Fish fed with a T2 diet showed significantly (p < 0.05) lower glucose content than other groups. The blood lysozyme activity and IgM showed significantly (p < 0.05) higher values in fish-fed T2 and T3 diets than in other groups. The antioxidative responses were increased in fish-fed T2 and T3 diets (p < 0.05). After 7 days post-Y. ruckeri challenge, the cumulative mortality rate showed the lowest value in fish fed with T1 and T2 diets, while the highest value was recorded in the control group. In conclusion, the results revealed beneficial effects of L. lactis subsp. lactis PTCC 1403 on the feed efficiency, immune response, and resistance to Y. ruckeri infection in rainbow trout.

     

Storage stability and sourdough acidification kinetic of freeze-dried Lactobacillus curvatus N19 under optimized cryoprotectant formulation

In this study, the response surface methodology was used to optimize the cryoprotective agent (skimmed milk powder, lactose and sucrose) formulation for enhancing the viability of Lactobacillus curvatus N19 during freeze-drying and storage stability of cells freeze-dried by using optimum formulation was evaluated. Our results showed that the most significant cryoprotective agent influencing the viability of L. curvatus N19 to freezing and freeze-drying was sucrose and skim milk, respectively. The optimal formulation of cryoprotective agents was 20 g/100 mL skim milk, 3.57 g/100 mL lactose and 10 g/100 mL sucrose. Using the optimum formulation during freeze-drying, the cell survival was found more than 98%. Under the optimal conditions, although only storage of the cells at 4 °C for 6 month retained the maximum stability (8.85 log cfu/g), the employed protectant matrix showed promising results at 25 °C (7.89 log cfu/g). The storage stability of cells under optimized conditions was predicted by accelerated storage test, which was demonstrated that the inactivation rate constant of the freeze-dried L. curvatus N19 powder was 9.74 × 10⁻⁶ 1/d for 4 °C and 2.08 × 10⁻³ 1/d for 25 °C. The loss of specific acidification activity after the storage at 4 and 25 °C was determined.     

Survival of spray-dried <Emphasis Type="Italic">Lactobacillus kefir</Emphasis> is affected by different protectants and storage conditions

Survival of two Lactobacillus kefir strains after spray drying in reconstituted skim milk with or without the addition of 12.5 g monosodium glutamate/l, 20 g sucrose/l, or 20 g fructo-oligosaccharides (FOS)/l and during subsequent storage under different conditions of temperature (20 and 30°C) and relative humidity (RH) (0, 11 and 23%) was evaluated. After being dried, L. kefir 8321 and L. kefir 8348 had a decrease in viability of 0.29 and 0.70 log cfu/ml respectively, while the addition of different protectants improved the survival of both strains significantly. During storage, bacterial survival was significantly higher under lower conditions of RH (0–11%), and monosodium glutamate and FOS proved to be the best protectants.     

Using banana to generate lactic acid through batch process fermentation

We evaluated the usefulness of waste banana for generating lactic acid through batch fermentation, using Lactobacillus casei under three treatments. Two treatments consisted of substrates of diluted banana purée, one of which was enriched with salts and amino acids. The control treatment comprised a substrate suitable for L. casei growth. When fermentation was evaluated over time, significant differences (P<0.05) were found in the three treatments for each of five variables analyzed (generation and productivity of lactic acid, and consumption of glucose, fructose, and sucrose). Maximum productivity was (in g l^–1 h^–1) 0.13 for the regular banana treatment, 1.49 for the enriched banana, and 1.48 for the control, with no significant differences found between the latter two treatments. Glucose consumption curves showed that L. casei made greater use of the substrate in the enriched banana and control treatments than in the regular banana treatment. For fructose intake, the enriched banana treatment showed significantly better (P<0.05) results than the regular one. Sucrose consumption was insignificant (P<0.05), probably because fermentation time was too short. Even when enriched, diluted banana purée is an ineffective substrate for L. casei, probably because it lacks nutrients.     

The Effects of Probiotic Supplementation on Genetic and Metabolic Profiles in Patients with Gestational Diabetes Mellitus: a Randomized,Double-Blind,Placebo-Controlled Trial

This study was carried out to evaluate the effects of probiotic supplementation on genetic and metabolic profiles in patients with gestational diabetes mellitus (GDM) who were not on oral hypoglycemic agents. This randomized, double-blind, placebo-controlled clinical trial was conducted in 48 patients with GDM. Participants were randomly divided into two groups to intake either probiotic capsule containing Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum, Lactobacillus fermentum (2 × 10⁹ CFU/g each) (n = 24) or placebo (n = 24) for 6 weeks. Probiotic intake upregulated peroxisome proliferator-activated receptor gamma (P = 0.01), transforming growth factor beta (P = 0.002) and vascular endothelial growth factor (P = 0.006), and downregulated gene expression of tumor necrosis factor alpha (P = 0.03) in peripheral blood mononuclear cells of subjects with GDM. In addition, probiotic supplementation significantly decreased fasting plasma glucose (β, − 3.43 mg/dL; 95% CI, − 6.48, − 0.38; P = 0.02), serum insulin levels (β, − 2.29 μIU/mL; 95% CI, − 3.60, − 0.99; P = 0.001), and insulin resistance (β, − 0.67; 95% CI, − 1.05, − 0.29; P = 0.001) and significantly increased insulin sensitivity (β, 0.009; 95% CI, 0.004, 0.01; P = 0.001) compared with the placebo. Additionally, consuming probiotic significantly decreased triglycerides (P = 0.02), VLDL-cholesterol (P = 0.02), and total-/HDL-cholesterol ratio (P = 0.006) and significantly increased HDL-cholesterol levels (P = 0.03) compared with the placebo. Finally, probiotic administration led to a significant reduction in plasma malondialdehyde (P < 0.001), and a significant elevation in plasma nitric oxide (P = 0.01) and total antioxidant capacity (P = 0.01) was observed compared with the placebo. Overall, probiotic supplementation for 6 weeks to patients with GDM had beneficial effects on gene expression related to insulin and inflammation, glycemic control, few lipid profiles, inflammatory markers, and oxidative stress.

     

Assessment of the Safety of Lactobacillus casei IMV B-7280 Probiotic Strain on a Mouse Model

Probiotics, in particular Lactobacillus (lactic acid bacteria, LAB) strains, are widely used in clinical practice. Despite that these probiotics have GRAS (generally regarded as safe) and qualified presumption of safety (QPS) statuses, the safety of particular strains still needs to be thoroughly studied. The aim of the study was to evaluate the safety of Lact. casei IMV B-7280 strain by investigating toxicity and the effects on gut microbiota in experimental animal model. Male BALB/c mice (7–8 weeks, weight 20–24 g) were treated with amounts of Lact. casei IMV B-7280 strain: 5 × 10⁶, 5 × 10⁸, or 5 × 10⁹ CFU/animal once per day during 7 days, or in the amount of 1 × 10¹⁰ CFU/animal once per day during 3 days (most of the proposed probiotic doses for humans—from 10⁸ to 10⁹ CFU) and monitored during 14 days. Blood tests and serum biochemistry were conducted; the cecal content from mice of the experimental and control groups were freshly collected and analyzed. At the end of the experiments (15th day), the presence of LAB in the heart, liver, kidney, and mesenteric lymph nodes and peripheral blood was determined; histology of the brain, liver, heart, fragments of the small and large intestine, and mesenteric lymph nodes was conducted. Survival rate of BALB/c mice treated with Lact. casei IMV B-7280 strain in different concentrations in toxicity experiments during 14 days was 100%. We observed no signs of toxicity as changes in gait, lethargy, sleep, somatomotor activity as well as changes in fur, eyes, skin and mucous membranes, tremors, behavior pattern, convulsions, salivation, diarrhea, and local injuries in mice from all experimental groups. After administration of probiotic strain, the number of opportunistic bacteria in cecal contents, such as Staphylococcus spp., Candida spp., Pseudomonas spp., and total aerobic and optionally anaerobic bacteria decreased compared to controls; the population of beneficial bacteria such as lactobacilli increased in cecal contents of these mice. LAB were not detected in the peripheral blood, heart, liver, kidneys, and mesenteric lymph nodes after administration of this strain to intact mice. Lact. casei IMV B-7280 strain is safe at dose up to 10¹⁰ CFU/animal during 3- and 7-day oral administration to mice and has a positive effect on the gut microbiota composition; it could be potentially considered as safe probiotic for humans.

     

Reduction of fecal shedding of enterohemorrhagic Escherichia coli O157:H7 in lambs by feeding microbial feed supplement

Enterohemorrhagic Escherichia coli O157:H7, an emerging food-borne pathogen, has been implicated in several outbreaks in the US. Ruminants, including cattle, sheep and deer are reservoirs of E. coli O157:H7 and fecal shedding of the pathogen forms the vehicle of entry into the human food chain. We studied the efficacy of Lactobacillus acidophilus, Streptococcus faecium, a mixture of L. acidophilus and S. faecium and a mixture of L. acidophilus, S. faecium, Lactobacillus casei, Lactobacillus fermentum and Lactobacillus plantarum in reducing fecal shedding of E. coli O157:H7 by sheep experimentally infected with the pathogen prior to administration with the microbials. Following oral inoculation with 10¹⁰ CFU of E. coli O157:H7, 30 Suffolk ram lambs were blocked by body weight (six blocks of five lambs each) and lambs within the block randomly assigned to five groups. The lamb groups were fed daily for 7 weeks a basal diet without microbial supplement (control) or the basal diet with L. acidophilus or with S. faecium or with a mixture of L. acidophilus and S. faecium or with a mixture of L. acidophilus, S. faecium, L. casei, L. fermentum and L. plantarum. The microbial supplements contained stabilized live naturally occurring bacteria and were mixed with the diet at the rate of 6.0×10⁶ CFU per kilogram of diet. Fecal samples were collected weekly and analyzed for E. coli O157:H7 using modified tryptic soy broth with novobiocin as a pre-enrichement broth and cefixim-tellurite sorbitol MacConkey agar (CT-SMAC) as a selective media. E. coli O157:H7 was confirmed by its reaction with O157 and H7 antisera. E. coli O157:H7 was shed continuously and in varying numbers in the feces throughout the 7-week experimental period by all five groups. However, lambs administered a mixture of L. acidophilus, S. faecium, L. casei, L. fermentum and L. plantarum shed significantly lower (P=0.0211) average number of E. coli O157:H7 (2.3 log₁₀ CFU per gram of feces per week) than the other lamb groups over the entire experimental period. S. faecium supplemented lambs were comparable (P=0.0884) to lambs fed a mixture of L. acidophulus and S. faecium in fecal shedding of the pathogen (3.5 versus 4.4 log₁₀ CFU per gram of feces) but significantly lower (P=0.0001) than the control lambs (5.6 log₁₀ CFU per gram of feces) and those supplemented with L. acidophilus (5.5 log₁₀ CFU per gram of feces). Average daily gain (ADG) and gain to feed ratio (G:F) were significantly improved (P=0.0145) by the mixed culture microbials (163.0 g and 0.33 for the control, 186.4 g and 0.37 for L. acidophulus, 168.2 g and 0.36 for S. faecium, 213.6 g and 0.46 for L. acidophulus and S. faecium, and 219.1 g and 0.44, respectively for L. acidophilus, S. faecium, L. casei, L. fermentum and L. plantarum supplemented lambs. The study indicates that supplementing lambs infected with E. coli O157:H7 with S. faecium or a mixture of S. faecium, L. acidophilus, L. casei, L. fermentum and L. plantarum in the diet can reduce total number of E. coli O157:H7 shed in the feces and improve animal meat production performance as well.     

Probiotic Clostridium butyricum Improves the Growth Performance,Immune Function,and Gut Microbiota of Weaning Rex Rabbits

Probiotics could promote animal growth and enhance immune function. This study investigated the effects of Clostridium butyricum (CB) on the growth performance, intestinal immune, and gut microbiota of weaning rex rabbits. A total of 60 healthy female rabbits (5-month-old) were divided equally into four groups and mated on the same day: control group (CTRL, fed with basal feed), low-dose group (LDG, fed with basal feed + 1.0 × 10³ CFU/g CB), middle-dose group (MDG, fed with basal feed + 1.0 × 10⁴ CFU/g CB), and high-dose group (HDG, fed with basal feed + 1.0 × 10⁵ CFU/g CB). Then, 30 weaning rex rabbits (35-day-old) were collected from each group for this experiment, and they were offered the same feeds as their mother. The results demonstrated that high-dose CB treatment significantly increased average daily weight gain of weaning rex rabbits. Further studies suggested that CB enhanced small intestinal digestive enzyme activity and improved mucosal morphology and antioxidant status. Supplemented with CB, small intestinal barrier function was maintained with the upregulation of mRNA levels of ZO-1, claudin, and occludin as well as the increase of sIgA production. Moreover, the relative expressions of MyD88, TLR2, and TLR4 were elevated in HDG; simultaneously, pro-inflammatory cytokines including IL-6, INF-γ, and TNF-α were decreased after CB administration. In addition, CB showed beneficial effects in improving weaning rex rabbit intestinal microflora via increasing the abundance of beneficial bacteria. Therefore, our results indicated CB can promote rex rabbit growth, which is likely to the enhancement of immune function and the improvement of intestinal microbiota.

     

Functional Identification of a Putative β-Galactosidase Gene in the Special lac Gene Cluster of Lactobacillus acidophilus

The putative β-galactosidase gene (lacZ) of Lactobacillus acidophilus has a very low degree of homology to the Escherichia coli β-galactosidase gene (lacZ) and locates in a special lac gene cluster which contains two β-galactosidase genes. No functional characteristic of the putative β-galactosidase has been described so far. In this study, the lacZ gene of L. acidophilus was hetero-expressed in E. coli and the recombinant protein was purified by a three-step procedure. The product of the lacZ gene was also extracted from L. acidophilus ATCC 4356 and active staining was carried out. The enzymatic properties of the purified recombinant LacZ were assayed. The results of hetero-expression showed the recombinant LacZ without tag had β-galactosidase activity. The purified recombinant LacZ had a specific activity of 43.2 U/mg protein. The result of active staining showed that the functional product of the lacZ gene did exist in L. acidophilus. The L. acidophilus β-galactosidase (LacZ) had an optimal pH of 6, an optimal temperature of 37°C and could hydrolyze 73% of lactose in milk in 30 h at 10°C. The L. acidophilus β-galactosidase (LacZ) was identified as cold-adapted β-galactosidase in this study for the first time, and may be useful for lactose removal from dairy products at low temperatures.     

Fructooligosaccharide production by <Emphasis Type="Italic">Penicillium expansum</Emphasis>

Fructooligosaccharides (FOS) production by Penicillium expansum was evaluated. In a first stage, the best conditions for P. expansum growth and sporulation were established with potato/dextrose/agar being the most suitable medium at between 22 and 25°C, giving good growth and good sporulation. The inocula from this medium were used for FOS production using shake-flask cultures, and yielded 0.58 g FOS/g sucrose (3.25 g FOS/l.h), demonstrating the potential of this strain for sucrose conversion to FOS.