Detection and Specific Enumeration of Multi-Strain Probiotics in the Lumen Contents and Mucus Layers of the Rat Intestine After Oral Administration

Although the detection of viable probiotic bacteria following their ingestion and passage through the gastrointestinal tract (GIT) has been well documented, their mucosal attachment in vivo is more difficult to assess. In this study, we investigated the survival and mucosal attachment of multi-strain probiotics transiting the rat GIT. Rats were administered a commercial mixture of the intestinal probiotics Lactobacillus acidophilus LA742, Lactobacillus rhamnosus L2H and Bifidobacterium lactis HN019 and the oral probiotic Streptococcus salivarius K12 every 12 h for 3 days. Intestinal contents, mucus and faeces were tested 6 h, 3 days and 7 days after the last dose by strain-specific enumeration on selective media and by denaturing gradient gel electrophoresis. At 6 h, viable cells and DNA corresponding to all four probiotics were detected in the faeces and in both the lumen contents and mucus layers of the ileum and colon. Viable probiotic cells of B. lactis and L. rhamnosus were detected for 7 days and L. acidophilus for 3 days after the last dose. B. lactis and L. rhamnosus persisted in the ileal mucus and colon contents, whereas the retention of L. acidophilus appeared to be relatively higher in colonic mucus. No viable cells of S. salivarius K12 were detected in any of the samples at either day 3 or 7. The study demonstrates that probiotic strains of intestinal origin but not of oral origin exhibit temporary colonisation of the rat GIT and that these strains may have differing relative affinities for colonic and ileal mucosa.     

Supercritical CO2 interpolymer complex encapsulation improves heat stability of probiotic bifidobacteria

The probiotic industry faces the challenge of retention of probiotic culture viability as numbers of these cells within their products inevitably decrease over time. In order to retain probiotic viability levels above the therapeutic minimum over the duration of the product’s shelf life, various methods have been employed, among which encapsulation has received much interest. In line with exploitation of encapsulation for protection of probiotics against adverse conditions, we have previously encapsulated bifidobacteria in poly-(vinylpyrrolidone)-poly-(vinylacetate-co-crotonic acid) (PVP:PVAc-CA) interpolymer complex microparticles under supercritical conditions. The microparticles produced had suitable characteristics for food applications and also protected the bacteria in simulated gastrointestinal fluids. The current study reports on accelerated shelf life studies of PVP:PVAc-CA encapsulated Bifidobacterium lactis Bb12 and Bifidobacterium longum Bb46. Samples were stored as free powders in glass vials at 30 °C for 12 weeks and then analysed for viable counts and water activity levels weekly or fortnightly. Water activities of the samples were within the range of 0.25–0.43, with an average a _w  = 0.34, throughout the storage period. PVP:PVAc-CA interpolymer complex encapsulation retained viable levels above the recommended minimum for 10 and 12 weeks, for B. longum Bb46 and B. lactis Bb12, respectively, thereby extending their shelf lives under high storage temperature by between 4 and 7 weeks. These results reveal the possibility for manufacture of encapsulated probiotic powders with increased stability at ambient temperatures. This would potentially allow the supply of a stable probiotic formulation to impoverished communities without proper storage facilities recommended for most of the currently available commercial probiotic products.     

Relationship between the cell surface hydrophobicity and survival of bacteria <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> after exposures to ethanol,freezing or freeze-drying

An inverse linear relationship (P < 0.01) was detected between the cell surface hydrophobicity (CSH) and survival of ethanologenic bacteria Zymomonas mobilis 113S exposed to elevated (2.55 M) ethanol concentration. In the same way, viable cell counts of relatively hydrophobic Z. mobilis were less diminished by growing (0.85-3.40 M) ethanol concentrations as compared to more hydrophobic bacteria. Very similar inverse relationships (P < 0.01) were observed between the CSH of intact Z. mobilis and survival of cells subjected to subsequent freeze-drying or freezing/thawing cycles thereby affinity substantially lowered ability of hydrophobic bacteria to survive under adverse environments. Observed relationships were supported by significant correlations between independent analytical data of the carbohydrate content within fractions of lipopolysaccharide and surface proteins extracted from cells of varied hydrophobicity. The results suggest that the CSH could be of value to predict the ability of intact bacteria to endure stress conditions and should be monitored towards lower values during cultivation in order to reduce subsequent unwanted structural and physiological disturbances provoked by multiple stress factors.     

Influence of chicory inulin on the survival of microbiota of a probiotic fermented milk during refrigerated storage

Inulin-supplemented and control probiotic fermented milks were produced using a dairy starter culture containingsStreptococcus thermophilus CHCC 742/2130,Lactobacillus acidophilus La-5 andBifidobacterium lactis Bb-12, and were then stored at 4°C for 6 weeks. Microbiological analyses were performed at weekly intervals. The results showed that the presence of inulin at 1 t o 5% (wt/v) did not influence significantly (P>0.05) the survival rates of eitherS. thermophilus orL. acidophilus. However, the addition of inulin at 5% (wt/v) had a significant beneficial effect (P<0.05) on the viability of bifidobacteria after 28 days of refrigerated storage.     

Immunoprotective Effect of Probiotic Dahi Containing Lactobacillus acidophilus and Bifidobacterium bifidum on Dextran Sodium Sulfate-Induced Ulcerative Colitis in Mice

In the present study, probiotic Dahi (LaBb Dahi) containing Lactobacillus acidophilus LaVK2 and Bifidobacterium bifidum BbVK3 was selected as a probiotic therapy to investigate its protective effect on dextran sodium sulfate (DSS)-induced ulcerative colitis model in mice that mimics the picture in human. LaBb Dahi was prepared by co-culturing Dahi bacteria (Lactococcus lactis ssp. cremoris NCDC-86 and Lactococcus lactis ssp. lactis biovar diacetylactis NCDC-60) along with selected strain of L. acidophilus LaVK2 and B. bifidum BbVK3 in buffalo milk (3% fat). Four groups of swiss albino male mice (12 each) were fed buffalo milk (3% fat), buffalo milk (3% fat) plus DSS, Dahi plus DSS, and LaBb Dahi plus DSS, respectively, for 17?days with basal diet. The myeloperoxidase (MPO) activity, levels of tumor necrosis factor-?? (TNF-??), interleukin-6 (IL-6) and interferon (IFN-??) were assessed as inflammatory markers, and the histopathological picture of the colon of mice was studied. DSS-induced colitis appeared to induce significant increase in MPO activity, levels of TNF-??, IL-6 and IFN-??. Feeding with LaBb Dahi offered significant reduction in MPO activity, levels of TNF-??, IL-6 and IFN-?? when compared to either buffalo milk group or group III (Dahi). The present study suggests that LaBb probiotic Dahi can be used to combat DSS-induced biochemical and histological changes and to achieve more effective treatment for ulcerative colitis.     

Antiproliferative Effects of Homogenates Derived from Five Strains of Candidate Probiotic Bacteria

Unheated and heat-treated homogenates were separately prepared from candidate probiotic bacteria, including Lactobacillus rhamnosus GG, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Streptococcus thermophilus. We compared the phytohemagglutinin-induced proliferation of mononuclear cells in the presence of homogenates and in the presence of a control containing no homogenate by assessing thymidine incorporation in cell cultures. All homogenates suppressed proliferation, whether the enzymatic activity was inactivated or not inactivated by heating. When the proliferation assays were repeated with cytoplasmic and cell wall extracts derived from the homogenate of L. rhamnosus GG, the cytoplasmic extract but not the cell wall extract was suppressive. These findings indicate that candidate probiotic bacteria possess a heat-stable antiproliferative component(s). These bacteria may be used to generate microbiologically nonviable yet immunologically active probiotic food products that are easier to store and have a longer shelf life.     

Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms'' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions.     

The possible causal relationship between fragmentation of genomic DNA and formation of viable,but non‐culturable probiotic bacteria upon storage in dry state

In this study, the aim was to establish if loss of DNA integrity is a cause of loss of culturability for probiotic bacteria during storage in dry state. The number of colony forming units (CFU), number of metabolically active cells, and DNA integrity during dry storage of probiotic strains, B. animalis subsp. lactis BB‐12 and L. acidophilus LA‐5, were investigated. The probiotic strains were freeze‐dried and stored at 20°C, with and without oxygen present, and at water activity levels 0.22 or 0.32. Dry storage resulted in a decrease in CFU during the entire storage period. The number of metabolically active cells was unchanged during storage of B. animalis subsp. lactis BB‐12, but did decrease during the first week of storage of L. acidophilus LA‐5. Loss of DNA integrity was evident for both strains during storage and correlated well with the loss of CFU. Both loss of CFU and loss of DNA integrity were significantly greater for both strains when oxygen was present and when a_w was increased. Statistical analysis indicates a possible causal relationship between DNA degradation and loss of culturability and this idea is consistent with the function of DNA at cell division. The study contributes with new knowledge of the cause for loss of CFU during dry storage of probiotic bacteria, which possibly can aid in the improvement of preservation techniques. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:231–242, 2018     

Isolation of Lactic Acid Bacteria from Kuruma Shrimp (Marsupenaeus japonicus) Intestine and Assessment of Immunomodulatory Role of a Selected Strain as Probiotic

Fifty-one lactic acid bacteria (LAB) strains were isolated and identified based on 16S ribosomal DNA sequence from the intestinal tracts of 142 kuruma shrimps (Marsupenaeus japonicus) collected from Kanmon Strait, Fukuoka and Tachibana Bay, Nagasaki, Japan. Cellular immunomodulatory function of 51 isolated LAB strains was assessed by measuring the level of interferon (IFN)-γ induction in mouse spleen cell culture. The strain Lactococcus lactis D1813 exhibited the highest amount of IFN-γ production and also bactericidal activity and was selected for testing its immunomodulatory role as a probiotic in kuruma shrimp. We also assessed the effect of dietary incorporation of this probiotic on resistance to Vibrio penaeicida infection in the kuruma shrimp. Our results demonstrate that probiotic L. lactis D1813-containing diet-fed (10⁵ cfu g^?1) shrimps displayed a significant up-regulation of lysozyme gene expressions in the intestine and hepatopancreas. However, insignificantly higher expression of anti-lipopolysaccharide factor, super oxide dismutase, prophenoloxidase, and toll-like receptor 1 was recorded in the intestine of shrimps fed the probiotic diet. Moreover, significantly increased (P?<?0.01) resistance to the bacterial pathogen in term of better post-infection survival (61.7 %) was observed in the shrimps fed with the probiotic-incorporated diet compared with the control diet-fed group (28.3 %). The present study indicates the immunomodulatory role of the LAB L. lactis D1813 on the kuruma shrimp immune system and supports its potential use as an effective probiotic in shrimp aquaculture.     

An In Vitro Study on Bacterial Growth Interactions and Intestinal Epithelial Cell Adhesion Characteristics of Probiotic Combinations

The aims of this study were to examine long-term growth interactions of five probiotic strains (Lactobacillus casei 01, Lactobacillus plantarum HA8, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC 55730 and Bifidobacterium lactis Bb12) either alone or in combination with Propionibacterium jensenii 702 in a co-culture system and to determine their adhesion ability to human colon adenocarcinoma cell line Caco-2. Growth patterns of probiotic Lactobacillus strains were not considerably affected by the presence of P. jensenii 702, whereas lactobacilli exerted a strong antagonistic action against P. jensenii 702. In the co-culture of Bif. lactis Bb12 and P. jensenii 702, a significant synergistic influence on growth of both bacteria was observed (P < 0.05). The results of adhesion assay showed that when probiotic strains were tested in combination, there was evidence of an associated effect on percentage adherence. However, in most cases these differences were not statistically significant (P < 0.05). Adhesion percentage of Lb. casei 01 and Lb. rhamnosus GG both decreased significantly in the presence of P. jensenii 702 compared to their adhesion levels when alone (P < 0.05). These results show that the survival and percentage adhesion of some probiotic strains may be influenced by the presence of other strains and this should be considered when formulating in the probiotic products.     

Using Goat’s Milk,Barley Flour,Honey, and Probiotic to Manufacture of Functional Dairy Product

Stirred yogurt manufactured using probiotic culture which usually called Rayeb milk in the Middle East region is one of the most important functional fermented milk products. To increase the health and functionality properties to this product, some ingredients like fruits, cereal, and whey protein are used in production. This study was carried out to prepare functional Rayeb milk from goat’s milk, barley flour (15%) and honey (4%) mixtures using ABT culture. Also, vanilla and cocoa powder were used as flavorings. Adding barley flour and honey to goat’s milk increased curd tension and water-holding capacity and decreased coagulation time and susceptibility to syneresis. The values of carbohydrate, total solids, dietary fiber, ash, total protein, water soluble nitrogen, total volatile fatty acids, unsaturated fatty acids, oleic, linoleic, α-linolenic acids, and antioxidant activity were higher in Rayeb milk supplemented with barley flour and honey than control. The viabilities of Lactobacillus acidophilus and Bifidobacterium lactis Bb12 (Chr. Hansen’s Lab A/S) increased in fortified Rayeb milk. The recommended level of 10⁷ cfu g^?1 of bifidobacteria as a probiotic was exceeded for these samples. Addition of vanilla (0.1%) or cocoa powder (0.5%) improved the sensory properties of fortified Rayeb milk.     

The Effect of Dietary Prebiotics and Probiotics on Body Weight,Large Intestine Indices,and Fecal Bile Acid Profile in Wild Type and IL10?/? Mice

Previous studies have suggested roles of probiotics and prebiotics on body weight management and intestinal function. Here, the effects of a dietary prebiotic, inulin (50 mg/g diet), and probiotic, Bfidobacterium animalis subsp. lactis (Bb12) (final dose verified at 10⁵ colony forming unit (cfu)/g diet, comparable to human consumption), were determined separately and in combination in mice using cellulose-based AIN-93G diets under conditions allowed for the growth of commensal bacteria. Continuous consumption of Bb12 and/or inulin did not affect food intake or body, liver, and spleen weights of young and adult mice. Fecal bile acid profiles were determined by nanoESI-MS/MS tandem mass spectrometry. In the presence of inulin, more bacterial deconjugation of taurine from primary bile acids was observed along with an increased cecal weight. Consumption of inulin in the absence or presence of Bb12 also increased the villus cell height in the proximal colon along with a trend of higher bile acid sulfation by intestinal cells. Feeding Bb12 alone at the physiological dose did not affect bile acid deconjugation and had little effect on other intestinal indices. Although interleukin (IL)10-null mice are susceptible to enterocolitis, they maintained the same body weight as the wild type mice under our specific pathogen-free housing condition and showed no signs of inflammation. Nevertheless, they had smaller cecum suggesting a mildly compromised intestinal development even before the disease manifestation. Our results are consistent with the notion that dietary factors such as prebiotics play important roles in the growth of intestinal microbiota and may impact on the intestinal health. In addition, fecal bile acid profiling could potentially be a non-invasive tool in monitoring the intestinal environment.     

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.     

Adaptation of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> to Thermal Stress Yields a Thermotolerant Variant Which Also Exhibits Improved Survival at pH 2

Loss in probiotic viability upon exposure to stressful storage and transport conditions has plagued the probiotic market worldwide. Lactobacillus acidophilus is an important probiotic that is added to various functional foods. It is known to be fairly labile and susceptible to temperature variations that it encounters during processing and storage which increases production cost. It has been repeatedly demonstrated that pre-exposure to sub-lethal doses of stress, particularly, temperature and pH, leads to improved survival of various probiotics when they subsequently encounter the same stress of a much greater magnitude. Attempts to adapt L. acidophilus to temperatures as high as 65 °C to arrive at a thermotolerant variant have not been reported previously. To improve viability at elevated temperatures, we gradually adapted the L. acidophilus NCFM strain to survival at 65 °C for 40 min. Following adaptation, the variant showed a 2-log greater survival compared to wild-type at 65 °C. Interestingly, this thermotolerant variant also demonstrated a 2-log greater stability compared to wild-type at pH 2.0. The improved pH and temperature stress tolerance exhibited by this variant remained unaltered even when the strain was lyophilized. Moreover, the thermotolerant variant demonstrated improved stability compared to wild-type when stored for up to a week at 37 and 42 °C. Probiotic properties of the variant such as adherence to epithelial cells and antibacterial activity remained unaltered. This strain can potentially help address the issue of significant loss in viable cell counts of L. acidophilus which is typically encountered during probiotic manufacture and storage.     

Probiotic Strawberry Yogurts: Microbiological,Chemical and Sensory Properties

This study was performed to determine the viability of Lactobacillus acidophilus and Bifidobacterium bifidum in yogurt made with strawberry marmalade (SM) and to examine the quality properties of probiotic yogurt. Acidity, pH, bacterial counts and sensory analysis of the yogurt samples were investigated on days 1, 3, 5, 7, 10 and 14 during storage at 4 °C. The survival rate of L. acidophilus was greater than that of B. bifidum. The viability of L. acidophilus decreased during the storage period, but B. bifidum numbers remained stable during the storage period. The highest L. acidophilus count (7.20 log cfu/g) was found in L. acidophilus + B. bifidum SM yogurt on day 1. The highest B. bifidum count (6.13 log cfu/g) was detected in yogurt containing L. acidophilus + B. bifidum SM yogurt on day 7. Yeast and mould counts of all yogurts increased during the storage period. Coliform bacteria and Staphylococcus aureus were not detected in the yogurt samples. The highest overall acceptance sensory score was observed in yogurts containing L. acidophilus. Considering the sensory and probiotic characteristics of all yogurt samples, this study suggested that strawberry yogurt with a suitable 5–7 day storage period can be produced with single L. acidophilus addition or single B. bifidum addition.     

Development of Yoghurt with Juçara Pulp (<Emphasis Type="Italic">Euterpe edulis</Emphasis> M.) and the Probiotic <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> La5

Yoghurts are dairy products consumed worldwide and can be supplemented with substances that provide extra health benefits as well as probiotic strains. In this context, the present study aimed to prepare a yoghurt added of juçara (Euterpe edulis M.) pulp and the commercial probiotic strain Lactobacillus acidophilus La5. Moreover, the probiotic survival during storage and after in vitro exposure to simulated gastric and enteric conditions was evaluated. Four formulations of yoghurt were prepared: (a) natural yoghurt, (b) yoghurt added of probiotic, (c) yoghurt added of juçara pulp, and (d) yoghurt added of probiotic culture and juçara pulp. The preparations were evaluated for survival of probiotic strain during storage and its tolerance to gastric and enteric conditions in vitro. The probiotic population in yoghurt remained unchanged during 28 days of storage. In addition, juçara pulp increased the probiotic resistance to simulated gastric and enteric conditions in the first day of storage. These data indicate that juçara pulp is a potential ingredient for the production of probiotic yoghurts.     

Hydrophobicity of bacteria Zymomonas mobilis under varied environmental conditions

Changes in the cell surface hydrophobicity (CSH) of bacteria Zymomonas mobilis 113S were examined in response to varied environmental conditions (temperature and phase of growth, concentration or type of carbon source, the presence of amphiphilic compounds). The values of CSH were elevated with a decreased growth rate over the time of cultivation up to 20–22% at the stationary phase. CSH values increased proportionally with the growth of cultivation temperature and concentration of carbon source (glucose or sucrose) or amphiphilic compound (aliphatic alcohols, Tween80) in the medium. Replacement of sucrose by glucose and the presence of Tween20 in the growth medium resulted in reduced values of CSH. An inverse relationship was detected between the number of attached cells to the hydrophilic glass surfaces and the CSH values of Z. mobilis whereas direct linear relationship was observed for hydrophobic surfaces. Permeation rates of the fluorescent probe (NPN) into the cells were directly proportional to the concentration of extracellular protein in the medium and to the values of CSH indicating the impaired barrier function for more hydrophobic cells. The multiple correlation between the CSH values and absorption indices of FT-IR spectra at the fingerprint region (866–1088 cm⁻¹) suggests the possible contribution of carbohydrates and/or lipopolysaccharides in observed changes of Z. mobilis hydrophobicity.     

Characterization of the Properties of Human- and Dairy-Derived Probiotics for Prevention of Infectious Diseases in Fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

Probiotic Dahi containing Lactobacillus acidophilus and Bifidobacterium bifidum alleviates age-inflicted oxidative stress and improves expression of biomarkers of ageing in mice

The potential benefiting effects of probiotic Dahi on age-inflicted accumulation of oxidation products, antioxidant enzymes and expression of biomarkers of ageing were evaluated in mice. Probiotic Dahi were prepared by co-culturing in buffalo milk (3% fat) Dahi bacteria (Lactococcus lactis ssp. cremoris NCDC-86 and Lactococcus lactis ssp. lactis biovar diacetylactis NCDC-60) along with selected strain of Lactobacillus acidophilus LaVK2 (La-Dahi) or combined L. acidophilus and Bifidobacterium bifidum BbVK3 (LaBb-Dahi). Four groups of 12 months old mice (6 each) were fed for 4 months supplements (5 g/day) of buffalo milk (3% fat), Dahi, La-Dahi and LaBb-Dahi, respectively, with basal diet. The activities of catalase (CAT) and glutathione peroxidase (GPx) declined and the contents of oxidation products, thiobarbituric acid reactive substances (TBARS) and protein carbonyls, increased in red blood corpuscles (RBCs), liver, kidney and heart tissues and superoxide dismutase (SOD) activity increased in RBCs and hepatic tissues during ageing of mice. Feeding ageing mice with La-Dahi or LaBb-Dahi increased CAT activity in all the four tissues, and GPx activity in RBCs and hepatic tissue, and a significant decline in TBARS in plasma, kidney and hepatic tissues and protein carbonyls in plasma. Feeding mice with probiotic Dahi also reversed age related decline in expression of biomarkers of ageing, peroxisome proliferators activated receptor-α, senescence marker protein-30 (SMP-30) and klotho in hepatic and kidney tissues. The present study suggests that probiotic Dahi containing selected strains of bacteria can be used as a potential nutraceutical intervention to combat oxidative stress and molecular alterations associated with ageing.     

Specific Identification and Targeted Characterization of Bifidobacterium lactis from Different Environmental Isolates by a Combined Multiplex-PCR Approach

The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of B. lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.