Isolation and characterization of Lactobacillus species having potential for use as probiotic cultures for dogs

ABSTRACT:  The need to control pathogenic microorganisms in the intestinal tract of dogs is a growing concern. There is interest in using probiotics such as species of Lactobacillus to help control canine intestinal infections. For successful use as a probiotic, the bacterial species should be of canine intestinal origin since these species exhibit host specificity. Serial dilutions of freshly voided dog feces were plated on Lactobacillus selection (LBS) agar to isolate the cultures. Isolates were identified based on Gram stain, catalase test, and fermentation patterns using API 50 CH kits. All potential isolates were compared for bile resistance based on relative ability to grow in broth containing 0.3% Oxgall, the ability to inhibit Salmonella Typhimurium in associative broth cultures, and the production of reuterin. Of the lactobacilli isolated, Lactobacillus reuteri was the dominant species. However, some cultures of L. acidophilus also were isolated. We found variations among the isolates of L. reuteri and L. acidophilus with respect to bile tolerance. In general, isolates of L. reuteri appeared to be more bile resistant than were isolates of L. acidophilus . There were also variations in the ability to inhibit growth of S. Typhimurium. Some isolates of L. reuteri produced reuterin while others did not.     

Shelf life and quality of probiotic yogurt produced with Lactobacillus acidophilus and Gobdin

This study evaluated the effect of dry white mulberry and walnut paste (Gobdin, a traditional Turkish food) in probiotic yogurt on the survival of Lactobacillus acidophilus and yogurt properties. Six different yogurts were produced with 0%, 5% and 10% Gobdin using Lactobacillus bulgaricus + Streptococcus thermophilus and with 0%, 5% and 10% Gobdin using L. bulgaricus + S. thermophilus + L. acidophilus. The physical, chemical, microbiological and sensorial properties of the yogurts were evaluated based on storage at 4 ± 1 °C. Probiotic shelf life and the most suitable combinations were determined. The highest L. acidophilus count (8.65 log cfu g^?1) was found in the 5% Gobdin‐supplemented yogurt on the 7th day of storage, while the lowest count (8.11 log cfu g^?1) was found in the probiotic control yogurt on the 21st day. Although the L. acidophilus counts in the probiotic yogurts declined during storage, all values found throughout the 21‐day storage period were >8 log cfu g^?1. This is above the level necessary to provide the desired therapeutic effect in probiotic products (10⁶–10⁷ cfu g^?1). The highest overall acceptability score was obtained on the first day from the yogurt with 5% Gobdin. However, all yogurt samples had general acceptability scores between 7 and 8 points from a 9‐point maximum. Thus, this study determined that a new functional yogurt can be produced using L. acidophilus with 5% Gobdin.     

Antioxidant activity of prebiotic ginseng polysaccharides combined with potential probiotic Lactobacillus plantarum C88

The potential prebiotic properties of Panax ginseng polysaccharides were studied using ten strains of Lactobacillus plantarum isolated from traditional Chinese fermented foods. These probiotics showed different growth characteristics depending on the extract and strain specificity. L. plantarum C88 showed higher cell densities and growth rate when cultured on P. ginseng polysaccharides. In the in vitro antioxidant assay, P. ginseng polysaccharides combined with L. plantarum C88 were found to possess significant DPPH, ABTS and superoxide anion radicals scavenging activities, and acidic polysaccharides showed better antioxidant activity than neutral polysaccharides. Furthermore, we evaluated the antioxidant effect of acidic P. ginseng polysaccharide combined with L. plantarum C88 strain in natural ageing mice in vivo. Acidic P. ginseng polysaccharide and L. plantarum C88 together inhibited the formation of malondialdehyde (MDA) and increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), catalase (CAT) and total antioxidant capacities (T‐AOC) in a dose‐dependent manner.     

Quality characteristics of kefir as a carrier for probiotic Lactobacillus rhamnosus GG

Kefir was prepared using kefir grains of different origins (US, Australia and India) and starter culture. The sensory scores for kefir made using starter culture and US kefir grains were significantly higher than for the other samples (P < 0.05). The index of viscosity was significantly higher (P < 0.05) in kefir prepared using Indian kefir grains. Shear stress–shear rate plots indicated yield stress in all kefirs. All samples exhibited shear‐thinning behaviour. The average count of Lactobacillus rhamnosus GG in kefir after incubation was 7.09 log CFU/g. No significant difference (P > 0.05) was observed in quality characteristics in kefir after the addition of LGG, indicating that it could be a potential probiotic carrier.     

Viability of Lactobacillus acidophilus in rice bran‐enriched stirred yoghurt and the physicochemical and sensory characteristics of product during refrigerated storage

This study was aimed at investigating the fortification of probiotic yoghurt with rice bran to increase nutritional properties of the product. The different levels of rice bran (0.3%, 0.6%, 0.9% and 1.2%) were incorporated into milk. The yoghurt samples were produced after pasteurisation, addition of starter culture and 1% Lactobacillus acidophilus suspension (6 × 10⁸ CFU mL^?1) and incubation. During sample storage in refrigerator, the viability of L. acidophilus, viscosity and physicochemical and sensory properties of product were investigated. Rice bran significantly increased the viability of L. acidophilus (P < 0.05). In addition, all probiotic yoghurts incorporating rice bran indicated higher viscosity and acidity and lower pH and syneresis compared to plain yoghurts. Furthermore, increments in rice bran incorporation levels resulted in a reduction in consumers' sample preferences. In general, the addition of rice bran at a suitable level could increase L. acidophilus viability and improve quality attributes of yoghurt.     

Effect of simulated acidic and salty fermentation conditions on kinetic growth parameters and probiotic potential of Lactobacillus acidipiscis and Lactobacillus pentosus

The technological stress during food processing may alter the beneficial interactions of food-associated Lactobacillus on the host. The effect of initial combined acidic (pH 5.0) and osmotic stress (NaCl 5 %, w/v with or without CaCl₂ 0.02 %, w/v) typically present in vegetable fermentations and cheesemaking were investigated. The growth kinetics prediction, hydrophobicity, auto-aggregation, in vitro survival and adhesion were determined in Lactobacillus acidipiscis and Lactobacillus pentosus isolated from a Mexican salty cheese. The growth was modelled with the modified Gompertz equation (R > 0.99; RMSE < 0.01). The lag phase increased (>10 h), and auto-aggregation was reduced. Hydrophobicity and S-layer total proteins varied. Gut survival reduced >4 log 10 units compared to the initial intake. Nevertheless, adhesion remained the same. Hence, combined stress affected the technological and physiological properties of both strains negatively. Further research is needed to identify the effect of the combined stress on the potential bioactivity of the strains.     

Comparative evaluation of yogurt and low-fat cheddar cheese as delivery media for probiotic Lactobacillus casei

This study used Lactobacillus casei 334e, an erythromycin-resistant derivative of ATCC 334, as a model to evaluate viability and acid resistance of probiotic L. casei in low-fat Cheddar cheese and yogurt. Cheese and yogurt were made by standard methods and the probiotic L. casei adjunct was added at approximately 10(7) CFU/g with the starter cultures. Low-fat cheese and yogurt samples were stored at 8 and 2 degrees C, respectively, and numbers of the L. casei adjunct were periodically determined by plating on MRS agar that contained 5 microg/mL of erythromycin. L. casei 334e counts in cheese and yogurt remained at 10(7) CFU/g over 3 mo and 3 wk, respectively, indicating good survival in both products. Acid challenge studies in 8.7 mM phosphoric acid (pH 2) at 37 degrees C showed numbers of L. casei 334e in yogurt dropped from 10(7) CFU/g to less than 10(1) CFU/g after 30 min, while counts in cheese samples dropped from 10(7) CFU/g to about 10(5) after 30 min, and remained near 10(4) CFU/g after 120 min. As a whole, these data showed that low-fat Cheddar cheese is a viable delivery food for probiotic L. casei because it allowed for good survival during storage and helped protect cells against the very low pH that will be encountered during stomach transit.     

The potential of biodetoxification activity as a probiotic property of Lactobacillus reuteri

Previous work on the metabolism of Lactobacillus reuteri ATCC 55730 anticipated a variability in the use of organic electron acceptors as a means to relieve metabolic redox problems. Therefore, investigations focusing on this unique metabolism of L. reuteri may reveal a basis for new probiotic properties. For instance, L. reuteri may use reactive aldehydes and ketones as electron acceptors to balance their redox metabolism, which opens the possibility to exploit this bacterium for in vivo bioreduction of deleterious compounds in the gastrointestinal tract (GIT). Herein we demonstrate that L. reuteri ATCC 55730 cultures on glucose are able to use furfural (1 g/L), and hydroxymethylfurfural (HMF) (0.5 g/L), as electron acceptors. The former enhances the growth rate by about 25% and biomass yield by 15%, whereas the latter is inhibitory. Furfural is stoichiometrically reduced to furfuryl alcohol by the culture. The conversion of furfural had no effect on the flux distribution between the simultaneously operating phosphoketolase and Embden-Meyerhof pathways, but initiated a flux to acetate production. In addition to furfural and HMF, cellular extracts showed potential to reoxidize NADH and/or NADPH with acrolein, crotonaldehyde, and diacetyl, indicating that conversion reactions take place intracellularly, however, utilization mechanisms for the latter compounds may not be present in this strain. The strain did not reduce other GIT-related reactive compounds, including acrylamide, glyoxal, and furan.     

The probiotic role of Lactobacillus plantarum in reducing risks associated with cardiovascular disease

A novel strain of Lactobacillus plantarum DMDL 9010 (CGMCC No. 5172) was isolated from naturally fermented mustard. The potential cholesterol reduction effects of this strain were investigated using an in vivo model. The results showed that L. plantarum DMDL 9010 at a dose of 10⁹ cells per day significantly reduced (P ≤ 0.05) serum total cholesterol (TC), low‐density lipoprotein cholesterol content (LDL‐C) levels and atherosclerosis index (AI) by 23.03%, 28.00% and 34.03%, respectively, while L. plantarum DMDL 9010 did not exhibit a significant effect on reducing serum triglycerides (TG) and increasing the serum high‐density lipoprotein cholesterol content (HDL‐C) in experimental rats (P > 0.05). The morphological and pathological changes in the liver illustrated that L. plantarum DMDL 9010 protected the rats against hepatocyte steatosis. Additionally, a high dose of L. plantarum DMDL 9010 was shown to exhibit a positive cholesterol‐lowering effect through decreasing the liver cholesterol (?33.20%) and triglyceride (?40.86%) levels, and increasing significantly (P ≤ 0.05) faecal cholesterol (+31.07%) and bile acid excretion (+70.18%). The results demonstrated that L. plantarum DMDL 9010 acted in a dose‐dependent way to decrease serum and total hepatic cholesterol and triglyceride and enhance faecal excretion of bile acids. In conclusion, these findings suggest that L. plantarum DMDL 9010 has potential to be explored as a probiotic for hypercholesterolaemic preventive and therapeutic.     

The effects of ratio of cow's milk to soymilk,probiotic strain and fruit concentrate on qualitative aspects of probiotic flavoured fermented drinks

The effects of proportion of cow's milk to soymilk (100:0, 75:25, 50:50, 25:75, 0:100), probiotic bacteria (Lactobacillus acidophilus LA‐5 or Lactobacillus casei L‐01) and natural fruit concentrates (strawberry, apricot, peach and pear) on quality characteristics of soy‐based probiotic drink were investigated. The parameters were analysed at the end of fermentation and during 21 days of storage at 5 ^°C. The highest viability was observed when the equal proportion of cow's milk and soymilk and L. casei was used (50:50‐CY). During chilled storage, the flavouring apricot had the highest stimulatory effect on the survival of L. casei in 50:50 treatment. In general, the treatment 50:50‐CY was realised as the best one overall.     

Potential for prevention of non‐O157 Shiga toxin‐producing Escherichia coli contamination in traditionally fermented African maize gruel by fermentative probiotic Lactobacillus plantarum

Non‐O157 Shiga toxin‐producing Escherichia coli (STEC) are a frequent cause of STEC‐related infections such as diarrhoea. Fermentation by presumptive probiotic Lactobacillus plantarum strain B411 isolated from cereal fermentation was investigated to prevent the growth of acid‐adapted (AA) and non‐acid‐adapted (NAA) non‐O157 STEC in traditionally fermented maize gruel, a widely used complementary food in Africa. L. plantarum strain B411 possessed probiotic characteristics and antimicrobial activity against selected pathogenic bacteria. Growth of AA and NAA non‐O157 STEC strains was substantially inhibited by 3.6 and 4.8 log reductions, respectively, in the maize gruel fermented with the L. plantarum B411, while their growth was only inhibited by 1.0 and 1.2 log reductions, respectively, by traditional fermentation alone. Inclusion of fermentative strains of L. plantarum exhibiting probiotic activity is a feasible method to ensure safety of traditionally fermented African cereal porridges through inhibition of non‐O157 STEC.     

Nonfermented ice cream as a carrier for Lactobacillus acidophilus and Lactobacillus rhamnosus

Efficiency of a nonfermented ice cream for delivering Lactobacillus acidophilus and Lactobacillus rhamnosus to consumers was evaluated. Both of the microorganisms survived at the populations of greater than 10⁷ CFU g^?1 during 12 weeks of storage at ?19 °C. Addition of the microorganisms had no significant effect on the overrun, viscosity, firmness and melting behaviour; it changed the acidity, pH and sensory properties of the finished product. Resistance to acid and sensitivity to bile of both bacteria were tested separately on fresh harvested cells before inoculation to ice cream and then on the frozen‐thawed cells after 12 weeks of cold storage in ice cream. Ice cream processing followed by cold storage reduced acid resistance of both bacteria at pH 2.5. Resistance to bile in L. rhamnosus was not affected in frozen‐thawed ice cream when compared to fresh cell, whereas resistance to bile in L. acidophilus appeared to be more susceptible to the process and cold storage.     

Lactobacillus amylovorus DSM 19280 as a novel food-grade antifungal agent for bakery products

Mould spoilage is the main cause of substantial economic loss in bakery industry and might also cause public health problems due to the production of mycotoxins. The reduction of mould growth in bakery products is thus of crucial importance and there is great interest to develop safe and efficient strategies for this purpose. In this study Lactobacillus amylovorus DSM19280 has been shown to produce a wide spectrum of antifungal compounds active against common bread spoilage fungi. Among the indicator moulds, Aspergillus fumigatus and Fusarium culmorum were the most sensitive organisms. Several antifungal compounds were found to be present in synthetic medium inoculated with L. amylovorus DSM19280 strain, some of them being reported here for the first time. Wheat doughs fermented with L. amylovorus DSM19280 had good rheological properties and the breads thereof were of high quality as shown by rheofermentometer and texture analyser measurements. The results were compared with those obtained with a control non-antifungal L. amylovorus DSM20531^T strain, a non-acidified and a chemically acidified dough. The quality of sourdough and bread fermented with L. amylovorus DSM 19280 was comparable to that obtained by using L. amylovorus DSM20531 ^T. Additionally, breads were evaluated for the ability to retard the growth of Fusarium culmorum FST 4.05, Aspergillus niger FST4.21, Penicillium expansum FST 4.22, Penicillium roqueforti FST 4.11 and fungal flora from the bakery environment. The biological preservation of bread with L. amylovorus DSM 19280 was also compared to the most commonly used antifungal agent Calcium propionate. Breads containing sourdough fermented with L. amylovorus DSM 19280 were more effective in extending the shelf life of bread than the calcium propionate.     

Encapsulation of probiotic Lactobacillus acidophilus by ionic gelation with electrostatic extrusion for enhancement of survival under simulated gastric conditions and during refrigerated storage

The probiotic Lactobacillus acidophilus was encapsulated in biodegradable and biocompatible capsules prepared by ionic gelation between phytic acid (PA) and chitosan (CS) with an electrostatic extrusion method. Calcium carbonate (CaCO₃) and starch were used as co‐encapsulants for improvement of capsule stability. Capsules were characterised and evaluated for survival of encapsulated L. acidophilus cells in simulated gastric fluid (SGF) and during refrigerated storage. Loading capacity values of PA‐CS capsules, PA‐CS‐starch capsules and PA‐CS‐CaCO₃ capsules were 8.20, 8.12 and 7.81 log CFU g^?1 of wet capsule, respectively. Capsules showed particle sizes of 1.3–1.5 mm and a uniform spherical shape. PA‐CS‐CaCO₃ capsules were the most stable vehicles for the protection of probiotic cells against acidic damage, particularly at pH 1.5 and pH 2. L. acidophilus cells from PA‐CS‐CaCO₃ capsules showed only a 0.64 log CFU reduction in numbers after 2 h in pH 1.5 SGF conditions. The numbers of L. acidophilus encapsulated in PA‐CS‐CaCO₃ capsules were decreased by only 0.69 log CFU g^?1, while PA‐CS capsules and PA‐CS‐starch capsule numbers were reduced by more than 1.45 log CFU g^?1 after 4 weeks at 4 °C. Addition of calcium carbonate to PA‐CS capsules provided protection against acid injury via antacid and buffering effects for encapsulation of L. acidophilus.     

Effects of Mentha longifolia L. essential oil on viability and cellular ultrastructure of Lactobacillus casei during ripening of probiotic Feta cheese

The effects of Mentha longifolia L. essential oil during ripening and storage probiotic Feta cheese were studied, in relation to viability and cellular ultrastructure of Lactobacillus casei. The addition of the essential oil in the concentrations from 0.0 to 0.03% was trialled: the 0.03% treatment resulted in the highest viability of L. casei and the lowest pH value compared with other treatments (P < 0.05). Electron microscopy showed that essential oil caused no harm to L. casei. This study demonstrated the favourable effects of M. longifolia on optimal maintenance of L. casei at the end of cheese storage period.     

Discrimination of probiotic Lactobacillus strains for poultry by repetitive sequenced‐based PCR fingerprinting

BACKGROUND: Four repetitive element sequence‐based polymerase chain reaction (rep‐PCR) methods, namely repetitive extragenic palindromic PCR (REP‐PCR), enterobacterial repetitive intergenic consensus PCR (ERIC‐PCR), polytrinucleotide (GTG)₅‐PCR and BOX‐PCR, were evaluated for the molecular differentiation of 12 probiotic Lactobacillus strains previously isolated from the gastrointestinal tract of chickens and used as a multistrain probiotic. This study represents the first analysis of the comparative efficacy of these four rep‐PCR methods and their combination (composite rep‐PCR) in the molecular typing of Lactobacillus strains based on a discriminatory index (D). RESULTS: Species‐specific and strain‐specific profiles were observed from rep‐PCR. From the numerical analysis of composite rep‐PCR, BOX‐PCR, (GTG)₅‐PCR, REP‐PCR and ERIC‐PCR, D values of 0.9118, 0.9044, 0.8897, 0.8750 and 0.8529 respectively were obtained. Composite rep‐PCR analysis was the most discriminative method, with eight Lactobacillus strains, namely L. brevis ATCC 14869^T, L. reuteri C 10, L. reuteri ATCC 23272^T, L. gallinarum ATCC 33199^T, L. salivarius ATCC 11741^T, L. salivarius I 24, L. panis JCM 11053^T and L. panis C 17, being differentiated at the strain level. CONCLUSION: Composite rep‐PCR analysis is potentially a useful fingerprinting method to discriminate probiotic Lactobacillus strains isolated from the gastrointestinal tract of chickens. Copyright © 2011 Society of Chemical Industry     

Optimisation of probiotic yoghurt production enriched with phytosterols

To optimise the formulation of phytosterol‐enriched probiotic yoghurt, the effect of different concentrations of phytosterols, fat and the probiotic inoculation rate on the survival of Lactobacillus acidophilus LA‐5, Bifidobacterium lactis BB‐12 and physicochemical indices over 14 days of storage was explored. Data analysis showed that the phytosterol concentration and fat content were two crucial factors affecting probiotic viability. Addition of phytosterols had no adverse effect on the overall scores for sensory attributes. Regarding maximisation of viability and storage time, the optimum conditions were found to be fat = 8.07% (w/v), phytosterol = 18 g/L, probiotic inoculation rate = 71.42 mg/L and storage time = 12 days.     

Effect of different levels of fat and inulin on the microbial growth and metabolites in probiotic yogurt containing nonviable bacteria

Effects of different levels of fat and inulin on bacterial cell counts, degree of proteolysis and concentrations of organic acids in the yogurt containing inactivated cells of probiotic strains Bifidobacterium animalis and Lactobacillus acidophilus were investigated. Results showed that both L. acidophilus and B. animalis grew well in the yogurt samples reaching cell counts higher than 10⁶ CFU mL^?1 at the final pH of 4.5. Inulin at the concentration of 1% had no significant effects on the production of organic acids and cell counts of L. acidophilus, but promoted the growth of B. animalis with a reduction in the degree of proteolysis. Generally, different fat levels showed significant effects on the production of organic acids and nonsignificant effects on the cell counts of probiotic bacteria and degree of proteolysis. In case of lactic acid, the ratio of L‐ (+)to D‐ (?) isomer ranged from 50/50 to 80/20 in yogurt samples.     

Evaluation of in vitro probiotic potential of phytase‐producing bacterial strain as a new probiotic candidate

Sixty‐three phytase‐producing bacterial strains were isolated from natural sources, and their probiotic potential was evaluated. Of these, only fifteen strains were selected on the basis of confirmatory plate assay. Among these, five phytase‐producing strains exhibiting potent probiotic properties were identified as Bacillus cereus P1, Bacillus subtilis P6, B. subtilis P7, Pseudomonas aeruginosa P12 and P. aeruginosa P15 on the basis of morphological, biochemical and molecular characteristics. Maximum phytase activity (2.74 EU mL^?1) with potential probiotic properties, that is more than 70% survivability at pH 2.0, up to 2.0% bile salt tolerance, sporulation efficiency of more than 80% and survival in anaerobic condition (94.31%), was revealed by B. subtilis P6 as compared to the well‐established commercial probiotic strains Lactobacillus sporogenes and Lactobacillus casei. Thus, phytase‐producing B. subtilis P6 with promising probiotic features can be used in food and animal feed applications for betterment of mankind after further validation.     

Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses

Ninety-eight Lactobacillus plantarum strains isolated from Italian and Argentinean cheeses were evaluated for probiotic potential. After a preliminary subtractive screening based on the presence of msa and bsh genes, 27 strains were characterized. In general, the selected strains showed high resistance to lysozyme, good adaptation to simulated gastric juice, and a moderate to low bile tolerance. The capacity to agglutinate yeast cells in a mannose-specific manner, as well as the cell surface hydrophobicity was found to be variable among strains. Very high β-galactosidase activity was shown by a considerable number of the tested strains, whereas variable prebiotic utilization ability was observed. Only tetracycline resistance was observed in two highly resistant strains which harbored the tetM gene, whereas none of the strains showed β-glucuronidase activity or was capable of inhibiting pathogens. Three strains (Lp790, Lp813, and Lp998) were tested by in vivo trials. A considerable heterogeneity was found among a number of L. plantarum strains screened in this study, leading to the design of multiple cultures to cooperatively link strains showing the widest range of useful traits. Among the selected strains, Lp790, Lp813, and Lp998 showed the best probiotic potential and would be promising candidates for inclusion as starter cultures for the manufacture of probiotic fermented foods.