Evaluation of probiotic characteristics of newly isolated Lactobacillus spp.: immune modulation and longevity

In the current study, the probiotic potential of approximately 350 strains of lactic acid bacteria isolated from Korean infant feces and Kimchi was investigated. Common probiotic properties of the bacterial strains, such as acid tolerance, bile tolerance and adhesion to human intestinal epithelial cells (HT-29 cells), were examined. Some strains were found to have immune modulatory and antimicrobial properties. Antagonistic activity against a panel of pathogenic bacteria was found to be strain dependent. To evaluate the immune modulatory activity of the strains, lymphocyte interferon (IFN)-γ secretion was determined in conjunction with cell proliferation. Some strains of Lactobacillus gasseri, L. fermentum and L. plantarum exhibited increased IFN-γ levels and lymphocyte proliferation. To evaluate the effects of these immune modulating lactobacilli on host life span, Caenorhabditis elegans was used as an in vivo model. Nematodes that were supplied heat-killed lactobacilli as a food source exhibited obvious differences in life span compared with those fed Escherichia coli OP50. The mean life span (determined as mean percent survival) of worms fed L. plantarum CJLP133 and L. fermentum LA12 was 13.89% and 13.69% greater, respectively, than that of control nematodes after 21 days (P = 0.036 and 0.043, respectively). In addition, some of safety profiles, including hemolytic type, gelatin hydration and degradation of urea, were found to be positive. These newly identified lactobacilli hold promise for use as probiotic agents, feed additives and/or in food applications.     

Cellular injuries of spray-dried Lactobacillus spp. isolated from kefir and their impact on probiotic properties

The injuries caused by spray drying (SD) of three potential probiotic lactobacilli isolated from kefir grains and the impact on some probiotic properties, were evaluated. Results demonstrated that Lactobacillus plantarum 83114 and L. kefir 8321 showed a slight reduction of viability (0.11 and 0.29 log CFU/ml respectively) after SD process, and L. kefir 8348 was found to be more sensitive to the process with a reduction in viability of 0.70 log CFU/ml. Neither membrane damage, evaluated by increased sensitivity to NaCl, lysozyme, bile salt and penicillin G, nor changes in acidifying activity in MRS and milk by lactobacilli were detected after SD. L. plantarum 83114 and L. kefir 8321 after SD did not lose their capacity to adhere to intestinal cells. Nevertheless, L. kefir 8348 showed a significant loss of adhesion capacity after SD. In addition, rehydrated spray-dried L. kefir 8321 retained the ability to protect against Salmonella invasion of intestinal cells. This effect was observed when L. kefir is co-incubated with Salmonella before invasion assay.This work shows that the membrane integrity evaluated by indirect methods and some probiotic properties of lactobacilli isolated from kefir did not change significantly after SD, and these powders could be used in functional foods applications.     

Physicochemical analysis of non-fermented probiotic milk with probiotic Lactobacillus plantarum Ln1 isolated from Korea traditional fermented food

Food Science and Biotechnology - The objective of this study was to develop a non-fermented probiotic milk that maintains its physicochemical properties, microbial properties, antioxidant activity,...     

食源假单胞菌16S rRNA鉴定及其食品腐败特性的研究

从市售鱼中分离的三株革兰氏阴性菌FML05-1、FML05-2、FML05-3,经16s rRNA鉴定为假单胞菌属。该菌是一种导致食品腐败的重要腐败细菌。选取代表菌株的16S rRNA序列,利用phylip软件网上构建系统进化树,同时对其蛋白水解活性、嗜铁素产生和生物膜形成等与食品腐败相关的特性进行检测,发现FML05-2具有很强的蛋白水解活性和生物膜的形成能力,并且在脱脂牛奶中生长还产生大量的嗜铁素,菌株FML05-1具有很强的生物膜形成能力,但蛋白水解活性较弱,在LB及脱脂牛奶中生长均不产生嗜铁素,而菌株FML05-3不具有上述三种特性。     

Growth characteristics of motile Aeromonas spp. isolated from different environments

The growth of 80 strains of motile Aeromonas spp. derived from environments with temperatures above 25°C and below 15°C, respectively, were examined at five temperatures (5°C, 10°C, 25°C, 37°C and 44°C) and four salt levels (0.05%, 2%, 4% and 6% NaCl). Sixty-one strains were further examined at two pH levels (pH 7.3 and pH 5.3). All strains grew at 25°C and 10°C with the majority of the isolates proliferating from approx. 10² to approx. 10⁷ cfu/ml within 1 and 3 days, respectively. In contrast, there were significant differences in the proportion of isolates able to grow at 5°C and 37°C depending on the temperature of their source of isolation. The ecological background of the organisms thus influences their thermal growth range and their ability to proliferate at body temperature, a highly significant factor in infective disease. At 25°C and pH 7.3, all strains grew in 0.05% NaCl, 96% grew in 2% NaCl, 96% grew in 2% NaCl while few grew in broth containing 4% or 6% NaCl. Lowering the pH to 5.3 with lactic acid caused a marked increase in the lag phase at 25°C and prevented growth of a large number of isolates at suboptimal conditions. Thus, none of the isolates from warm environments and only 8% of the isolates from cold environments grew at this pH at 5°C. The observed differences in growth optima between strains from different environments are discussed in relation to food- or waterborne infection.     

Mechanisms of erythromycin resistance of Campylobacter spp. isolated from food, animals and humans

Macrolides are regarded as drugs of choice for treatment of human campylobacteriosis. The use of antimicrobials for this purpose as well as in food animal production could result in macrolide resistance in Campylobacter species. Campylobacter isolates exhibit two different phenotypes with regard to erythromycin resistance: high-level resistance (HLR) and low-level resistance (LLR). Thirty-six food/animal and human isolates of Campylobacter jejuni and C. coli were examined for their mechanisms of resistance to erythromycin. The data presented here confirm the previous findings that the A2075G mutation in the 23S rRNA gene is the most frequently reported mechanism of high-level erythromycin resistance in Campylobacter isolates. The efflux pump inhibitor PAbetaN increased susceptibility to erythromycin for at least 16-32-fold in all examined HLR isolates, suggesting that the efflux mechanism acts in synergy with the 23S rRNA mutation to confer high-level erythromycin resistance. This was also confirmed in the isolates with sequence variation in the efflux pump cmeB gene. Additionally, the PAbetaN restored the susceptibility of LLR strains to the level of minimal inhibitory concentrations (MICs) of the susceptible strains and also reduced the MICs of the susceptible C. jejuni and C. coli isolates. The data suggest that active efflux contributes to the intrinsic resistance to erythromycin in Campylobacter and also contribute to high-level resistance.     

Molecular characterization of the probiotic strain Bacillus cereus var. toyoi NCIMB 40112 and differentiation from food poisoning strains

Bacillus cereus var. toyoi strain NCIMB 40112 (Toyocerin), a probiotic authorized in the European Union as feed additive for swine, bovines, poultry, and rabbits, was characterized by DNA fingerprinting applying pulsed-field gel electrophoresis and multilocus sequence typing and was compared with reference strains (of clinical and environmental origins). The probiotic strain was clearly characterized by pulsed-field gel electrophoresis using the restriction enzymes Apa I and Sma I resulting in unique DNA patterns. The comparison to the clinical reference strain B. cereus DSM 4312 was done with the same restriction enzymes, and again a clear differentiation of the two strains was possible by the resulting DNA patterns. The use of the restriction enzymes Apa I and Sma I is recommended for further studies. Furthermore, multilocus sequence typing analysis revealed a sequence type (ST 111) that was different from all known STs of B. cereus strains from food poisoning incidents. Thus, a strain characterization and differentiation from food poisoning strains for the probiotic strain was possible.     

The probiotic characteristics and GABA production of Lactobacillus plantarum K154 isolated from kimchi

In order to identify strains with a high GABA production ability and glutamate decarboxylase activity, 273 bacteria were isolated from kimchi. K154 produced 154.86 μg/mL of GABA in an MRS broth containing 1% MSG, 170.42 μg/mL of GABA in an MRS broth containing 2% MSG, and 201.78 μg/mL of GABA in an MRS broth containing 3% MSG. K154 was identified as Lactobacillus plantarum based on API carbohydrate fermentation pattern testing. The 16s rDNA sequence was investigated in order to determine physiological characteristics. The optimum growth temperature of K154 was 37°C. K154 was more sensitive to novobiocin and bacitracin than to other antibiotics, and exhibited greater resistance to polymyxin B and vancomycin. K154 was comparatively tolerant to bile juice and acid, and displayed resistance to Escherichia coli, Salmonella Typhimurium, and Staphylococcus aureus at rates of 19.0, 18.9, and 13.6% respectively.     

Growth and toxin profiles of Bacillus cereus isolated from different food sources

Eleven strains of Bacillus cereus isolated from milk and meat products have been used to study growth and sporulation profiles in detail. Polymerase chain reaction (PCR) using primers detecting cold shock protein A gene signatures (cspA), showed that none of the strains were the newly suggested species in the B. cereus group, B. weihenstephanensis, comprising psychrotolerant cereus strains, although one of the strains grew at 4 degrees C, two at 6 degrees C and seven grew at 7 degrees C. One of the two strains that grew at 6 degrees C had a maximum growth temperature of 42 degrees C, while the remaining 10 strains all grew at temperature of 43 degrees C or higher. Only three strains grew at 48 degrees C. At 42 degrees C, the generation time varied between 11 and 34 min. Spore germination was much faster for the two strains that grew at 6 degrees C than for the other nine strains in milk at 7 degrees C and 10 degrees C. All strains were cytotoxic and contained the non-haemolytic enterotoxin gene (nhe), 10 strains contained the enterotoxin T gene (bceT), and only six had the gene (hbl) encoding haemolytic enterotoxin. Two strains showed some microheterogeneity in the nhe operon. but contained all three genes. We can conclude that true B. cereus strains can have growth profiles as expected for B. weihenstephanensis, and that nhe and bceT were not correlated with growth profiles. However, the two psychrotolerant strains with minimal growth temperature of 4 degrees C and 6 degrees C did not contain hbl, as judged from our PCR results.     

Incidence, diversity and toxin gene characteristics of Bacillus cereus group strains isolated from food products marketed in Belgium

The major objectives of this study were to determine the incidence, diversity and characteristics of Bacillus cereus group spp. isolated from food products marketed in Belgium. The food products investigated in this study included cooked pasta, lasagna, béchamel sauce, bolognaise sauce, fresh minced beef, fresh-cut vegetables and raw basmati rice. B. cereus group spp. were detected in 56.3% (324 of 575) of the samples giving rise to 380 strains. The highest incidence (100%) occurred in the raw basmati rice. Although only 10 (2.6%) of the 380 isolates were determined to be psychrotolerant (able to grow at ≤ 7 °C), 25 (6.2%), 189 (49.7%) and 334 (87.9%) isolates were able to grow at mild temperature abuse conditions of 8 °C, 9 °C and 10 °C, respectively. The large diversity of the isolates obtained (overall and between isolates obtained from the same product type) was highlighted by the results of the (GTG)₅ PCR fingerprinting of 80 selected isolates. Sixty-one of these 80 isolates belonged to 15 distinct clusters (≥ 85% Pearson correlation) whereas the remaining 19 were each clustered separately. Further diversity was also found in the distribution of toxin genes as 16 different profiles were observed in the 80 selected isolates. Whilst none of 80 selected strains harboured the ces gene required for the production of the emetic toxin cereulide, 42 strains (52.5%) carried all seven genes required for the production of the diarrhoeal enterotoxins: haemolytic BL, non-haemolytic enterotoxin and cytotoxin K. The results of this study highlight not only the omnipresence but also the highly diverse ecology of B. cereus spp. within and across several food product types available on the retail market in Belgium. They should also provide the impetus for more studies to enable detailed risk assessment studies to be performed.     

益生菌食品的开发

<正>在健康成人肠道内栖息的细菌种类约有100多种,达100兆个以上,如把这些细菌换算成重量,大约有1,000克重。     

Prevalence and characteristics of Shiga toxin-producing Escherichia coli, Salmonella spp. and Campylobacter spp. isolated from slaughtered sheep in Switzerland

Caecum samples collected from 653 slaughtered sheep from two Swiss abattoirs were examined. The aim of this study was: (i) to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC), Salmonella spp. and Campylobacter spp.; (ii) to further characterize isolated strains; and (iii) to discuss the results obtained with their relevance to food safety. The percentage of samples testing positive for STEC by a polymerase chain reaction was 29.9%. The prevalence of positive Salmonella spp. samples was 11.0% and of Campylobacter spp. 17.5%. In 55.3% of the 76 isolated non-O157 STEC strains, stx2 variants (mostly stx2d) were detected. Additional virulence factors were harbored by 55.3% of the STEC strains, 10.5% of them being eae positive, 55.3% ehxA positive and 2.6% astA positive. All isolated salmonella were identified as Salmonella enterica subsp. diarizonae serovar 61:k:1,5,(7). Pulsed-field gel electrophoresis (PFGE) was performed for genotyping and 22 different restriction endonuclease digestion profiles were found among these strains for the different farms of origin. Of the 114 isolated Campylobacter spp. strains, 64.9% were shown to be Campylobacter jejuni and 35.1% Campylobacter coli, nine strains showed resistance against tetracycline, ciprofloxacin/nalidixic acid or streptomycin. In conclusion, sheep are a reservoir for the pathogens of latent zoonoses as non-O157 STEC, S. enterica subsp. diarizonae and Campylobacter spp. The maintenance of slaughter hygiene is consequently of crucial importance. It can be measured in daily practice by "slaughter-process-controls" and regular microbiological monitoring of carcasses. These are valuable tools for verifying slaughter hygiene according to hazard analysis critical control point principles.     

Discrimination of Bacillus cereus from Selected Bacillus spp. by Pyrolysis Gas-Liquid Chromatography

Pyrolysis gas-liquid chromatography (PGLC) and statistical analysis employing stepwise discriminant analysis (SDA), were used to classify five species and varieties in the genus Bacillus. Nine strains were harvested, lyophilized, and pyrolyzed under replicate conditions. The combination of PGLC and SDA enabled classification of these selected varieties and species of Bacillus at 96% accuracy. Stepwise discriminant analysis of the same PGLC data discriminated (100%) B. cereus, a foodborne pathogen, from the remaining selected nonpathogenic bacilli when the two groups were compared. One particular elution peak resulting from the PGLC-SDA analysis was prominent in both discriminations reported. The PGLC and SDA method offers potential as an accurate, objective procedure for the discrimination of these selected bacilli.     

Prevalence and characterization of toxigenic Bacillus cereus group isolated from low-moisture food products

Food Science and Biotechnology - The present study was conducted to determine the occurrence of B. cereus group members in low-moisture food products by phenotypic and genetic assessment and to...     

Antimicrobial properties of probiotic strains isolated from breast-fed infants

The protective and probiotic characteristics of intestinal lactobacilli, bifidobacteria and enterococci isolated from breast-fed infant faeces were investigated. Fifty-two selected isolates were identified at the species level and further characterized for their antimicrobial and safety features. Resistance to simulated gastric digestion and adherence to Caco-2 cells were also studied in the selected strains Lactobacillus paracasei INIA P272, Lactobacillus rhamnosus INIA P344, L. rhamnosus INIA P426, Lactobacillus mucosae INIA P459, Bifidobacterium pseudolongum INIA P2, Bifidobacterium breve INIA P18, Enterococcus faecium INIA P445 and Enterococcus faecalis INIA 127 due to their tolerance to GIT (gastrointestinal tract) conditions and interesting antimicrobial properties compared to the other strains investigated. A considerable diversity in the probiotic and antimicrobial properties among breast-fed infant lactic acid bacteria and bifidobacteria isolates was recorded. Some of the selected strains could be promising candidates to be exploited in the development of infant formulas or novel food products.     

Metabolic activities of Lactobacillus spp. strains isolated from kefir

A total of 21 strains of Lactobacillus species were isolated from Turkish kefir samples, in order to select the most suitable strains according to their metabolic activities including probiotic properties. As a result of the identification tests, 21 Lactobacillus isolates were identified as L. acidophilus (4%), L. helveticus (9%), L. brevis (9%), L. bulgaricus (14%), L. plantarum (14%), L. casei (19%) and L. lactis (28%). The amount of produced lactic acid, hydrogen peroxide, proteolytic activity, and acetaldehyde productions of Lactobacillus spp. were determined. Different amounts of lactic acid were produced by strains studies; however, lactic acid levels were 1.7-11.4 mg/mL. All strains produced hydrogen peroxide. L. bulgaricus Z14L strain showed no proteolytic activity, L. casei Z6L strain produced the maximum amount (0.16 mg/mL) of proteolytic activity. Acetaldehyde concentration produced in Lactobacillus strains ranged between 0.88-3.52 microg/mL.     

Importance of food in probiotic efficacy

Foods are carriers for the delivery of probiotics to the human body. In addition, foods help to buffer the probiotic through the gastrointestinal tract, regulate their colonization and contain other functional ingredients, such as bioactive components, which may interact with probiotics to alter their functionality and efficacy. The growth and survival of probiotics during gastric transit is affected by the physico-chemical properties of food carriers. Gastric acid, juices and bile tolerance, adherence to gastrointestinal epithelium and the acid production of probiotics are also affected by the food ingredients used in probiotic delivery. Same probiotic strains could vary in functional and technological properties in the presence of different food ingredients. Prebiotic food ingredients encourage the growth of probiotic bacteria. The appropriate combination of prebiotics and probiotics manifest higher potential for a synergistic effect. Originally, probiotic delivery was consistently associated with foods, particularly dairy foods. But nowadays, there is an increasing trend toward using probiotics in different food systems despite its original sources and even as nutraceuticals, such as in capsules. This changing trend in delivering probiotics may lead to a reduction in functional efficacy due to the exclusion of the potential synergistic effect of the food. Thus, selection of suitable food systems to deliver probiotics is a vital factor that should be considered in developing functional probiotic foods. This review focuses on information related to the effect of processed food products on functional efficacy of probiotics.     

Lactobacillus spp. with in vitro probiotic properties from human faeces and traditional fermented products

Lactobacillus strains from traditional African fermented milk products, as well as human intestinal isolates were identified and investigated in vitro for their technological and functional characteristics as potential new probiotic strains. To test survival under gastrointestinal conditions, first the protective effect of milk and the effects of medium composition, lysozyme, pepsin, and pH of the medium on bacterial viability were assessed in vitro using the Plackett-Burman statistical model and the commercially used L. johnsonii LA1 probiotic strain. The use of either an artificial gastric electrolyte solution or MRS did not play a significant role in the viability of the cultures, while lysozyme, acidic conditions (pH 2.5), pepsin and the presence of milk significantly influenced the survival of the strain. Therefore, these parameters were selected as important test variables in a model stomach passage survival trial. Five strains identified as L. plantarum and two identified as L. johnsonii showed good survival under simulated gastrointestinal conditions. These selected strains also showed antimicrobial activity, probably due to production of organic acids. All strains exhibited bile salt hydrolase activity, while only the L. plantarum strains showed beta-galactosidase activity.     

Putative virulence factors of the Aeromonas spp. isolated from food and environment in Abu Dhabi, United Arab Emirates

Thirty randomly selected Aeromonas isolates from food and the environment in Abu Dhabi, United Arab Emirates, were characterized for putative virulence determinants, such as production of cytotoxin, cytotonic toxin, and hemolysin and their capacity to adhere to and invade Henle 407 cells in vitro. Seventy percent of the tested isolates were cytotoxin producers, and 80% were hemolytic. Cytotoxin was produced by 6 of 7 A. hydrophila strains, 6 of 13 A. caviae strains, and 6 of 7 A. veronii bv. sobria strains, mostly from food sources. A. schubertii, A. jandaei, and A. trota also produced both cytotoxin and hemolysin. All of the 30 isolates tested adhered to Henle 407 cells, but none were able to invade the cells, as determined with the in vitro assay. However, no significant correlation of the presence of these putative virulence factors was found among these aeromonad food isolates.     

Genotypic characterization of Listeria spp. isolated from fresh water fish

A total of 200 samples (muscles and viscera, 100 of each) of fresh water fish, walking catfish (Clarias batrachus) were screened for Listeria spp. All the samples were subjected to a two-step enrichment followed by plating on selective media. Confirmation of the isolates was on the basis of biochemical characters, haemolysis on blood agar and Christie, Atkins, Munch Petersen test. A total of 39 isolates of Listeria spp. were recovered. Of these 26 (67%), 8 (21%), 3 (8%) and 2 (5%) were Listeria monocytogenes, Listeria seeligeri, Listeria grayi and Listeria welshimeri, respectively. The isolates were subjected to a PCR assay for detection of the virulence-associated genes individually or together. The plcA, actA, hlyA and iap genes were detected in six strains, three genes (actA, hlyA and iap) in nine strains, the plcA, hlyA and iap in our strain, the hlyA and iap were in three strains, actA and hlyA in four strains, plcA and hlyA in our strain and hlyA in two strains. The hlyA and iap were also detected in L. seeligeri.