传统乳制品中潜在益生植物乳杆菌的体外筛选

对从内蒙古牧区乳制品中分离到的29株植物乳杆菌的耐酸性（pH=3．0）、耐胆汁性（质量浓度3g／L牛胆酸钠）、抗菌活性进行了系统性研究。结果表明，有10株菌具有较好的耐酸耐胆盐特性，结合抗菌特性及黏附性试验结果，菌株WZ47-1，WZ32-2-1和WH13-2有可能成为潜在益生菌菌株。     

In vitro assessment of the gastrointestinal transit tolerance of taxonomic reference strains from human origin and probiotic product isolates of Bifidobacterium

Next to health promoting effects, the functional aspect of probiotic strains also involves their capacity to reach the colon as viable metabolically active cells. The present study aimed to assess the potential of 24 probiotic product isolates and 42 human reference strains of Bifidobacterium to survive gastrointestinal transit under in vitro conditions. The survival capacity of exponential and stationary phase cultures upon exposure to gastric and small intestinal juices was determined using a recently developed microplate-based assay in combination with the LIVE/DEAD BacLight Bacterial Viability kit. All 66 strains tested displayed a considerable loss in viability during exposure to an acidic pepsin containing solution (pH 2.0). Among the 10 taxa tested, cultures of B. animalis ssp. lactis appeared to be most capable to survive gastric transit. Although to a lesser extent, the presence of bile salts also affected the viability of most of the strains tested. Except for 3 strains, all 66 strains showed bile salt hydrolase activity using an agar-based assay. In contrast, the bifidobacterial strains used in this study appeared to possess a natural ability to survive the presence of pancreatin (pH 8.0). Although the effect was not significant, a slightly enhanced tolerance to gastrointestinal transit was observed when cells were in the stationary phase, especially when exposed to acid, compared with cells being in the exponential phase. Survival in the gastrointestinal tract appeared to be largely strain-dependent and hence implies that different strains will likely display a different behavior in functionality. The assay used in this study allows an initial assessment of strains for use as probiotic cultures prior to selecting potential candidate strains for further investigation in vivo.     

In vitro evaluation of the suitability potential probiotic of lactobacilli isolates from the gastrointestinal tract of chicken

Probiotic lactobacilli could be used to decrease the colonization of pathogenic bacteria in chicken and therefore decrease the risk of foodborne illness to consumers. The present study was conducted to select appropriate microbial strains for the development of potential probiotic. In experiment 1, 18 strains of lactobacilli isolated from the gastrointestinal tract of chicken were evaluated. The strains were demonstrated for their lactic acid, hydrogen peroxide, and exopolysaccharide productions. For experiment 2, the strains were tested for their acid, bile, antimicrobial activity, and antibiotic resistance levels. Among them, Lactobacillus delbrueckii ssp. delbrueckii BAZ32, Lactobacillus acidophilus BAZ29, BAZ36, BAZ43, and BAZ63 which produced high EPS were selected to aggregation ability. It is concluded that L. delbrueckii ssp. delbrueckii BAZ32, L. acidophilus BAZ29 confer high tolerance to acid, bile, antibiotic resistance, high antimicrobial activity, aggregation ability, and EPS production. These strains may be functional feed additives as potential probiotic in chicken.     

In vitro tolerance to digestive stresses of propionibacteria: influence of food matrices

This study investigated Propionibacterium freudenreichii tolerance to stresses encountered during food technology or in the digestive tract. The impact of food matrices on stress survival was evaluated. The ability of P. freudenreichii strains SI 41 and CNRZ 81 to survive both acid and bile salts stresses was studied in vitro. Stress tolerance was examined after inclusion of bacteria in different food matrices (alginate beads, xanthan–gellan beads, fermented milk) and compared to the stress tolerance of the same strains in pure cultures (either in exponential growth phase, in stationary phase, or after freeze-drying). Fermented milk was determined as the best probiotic vector to protect propionibacteria from this trial. This in vitro investigation gave promising results. Indeed, some food matrices can significantly improve protection of bacterial cells from stress injury. These data, which should be confirmed in an in vivo study, will be taken into consideration for the improvement of technological processes and for the choice of an adequate probiotic vector.     

In vitro assessment of probiotic bacteria: From survival to functionality

Selection by means of in vitro tests of new probiotic bacteria presents a challenge for scientists as well as for dairy companies. Presently, most probiotics are assessed using assays focused on their ability to survive in, and subsequently colonise, the gastrointestinal environment. This approach has led to selection of strains with confirmed in vivo probioticity, but the science underlying these assays still needs to be refined. As a consequence, we see a different approach emerging that uses functional properties and/or genomic evaluation to select appropriate probiotic strains.     

益生性植物乳杆菌的体外抗氧化活性研究

对从内蒙古奶豆腐及新生儿肠道中分离的5株植物乳杆菌的抗氧化活性进行分析.将保存的5株植物乳杆菌用MRS液体培养基活化培养后,对其耐酸特性、耐胆盐特性、疏水性和抗氧化能力进行分析.结果表明,5株植物乳杆菌均具有一定的耐酸和耐胆盐特性,其中植物乳杆菌MGSHC4302能够耐受pH=2.0的酸性环境,植物乳杆菌CRFA4608、MGSHC4301和MGSHC4302能够耐受1.0％的胆盐:5株植物乳杆对二甲苯和三氯甲烷的疏水性明显好于LGG,表明这些菌株具有较好的黏附性.实验结果表明,植物乳杆菌CRFA4608和MGSHC4302对羟自由基的清除能力高于对照菌株鼠李糖乳杆菌LGG,除植物乳杆菌MGSHC4303外其他4株茵对DPPH的清除能力均高于鼠李糖乳杆菌LGG或与之相近,而植物乳杆菌MGSHC4302、MGSHC4303具有较高的超氧阴离子清除能力.综上所述,植物乳杆菌MGSHC4302可以作为今后开发具有益生性产品的潜在菌株.     

In vitro testing of commercial and potential probiotic lactic acid bacteria

Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host. The objective of this study was to investigate the diversity of selected commercial and potential probiotic lactic acid bacteria using common in vitro screening assays such as transit tolerance in the upper human gastrointestinal tract, adhesion capacity to human intestinal cell lines and effect on epithelial barrier function. The selected bacteria include strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus sakei, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri and Pediococcus pentosaceus. Viable counts after simulated gastric transit tolerance showed that L. reuteri strains and P. pentosaceus tolerate gastric juice well, with no reduction of viability, whereas L. pentosus, L. farciminis and L. sakei strains lost viability over 180min. All strains tested tolerate the simulated small intestinal juice well. The bacterial adhesion capacity to human intestinal cells revealed major species and strain differences. Overall, L. plantarum MF1298 and three L. reuteri strains had a significant higher adhesion capacity compared to the other strains tested. All strains, both living and UV-inactivated, had little effect on the epithelial barrier function. However, living L. reuteri strains revealed a tendency to increase the transepithelial electrical resistance (TER) from 6 to 24h. This work demonstrates the diversity of 18 potential probiotic bacteria, with major species and strain specific effects in the in vitro screening assays applied. Overall, L. reuteri strains reveal some interesting characteristics compared to the other strains investigated.     

In vitro evaluation of the probiotic potential of bacteriocin producer Lactobacillus sakei 1

Lactobacillus sakei 1 is a food isolate that produces a heat-stable antimicrobial peptide (sakacin 1, a class IIa bacteriocin) inhibitory to the opportunistic pathogen Listeria monocytogenes. Bacterial isolates with antimicrobial activity may be useful for food biopreservation and also for developing probiotics. To evaluate the probiotic potential of L. sakei 1, it was tested for (i) in vitro gastric resistance (with synthetic gastric juice adjusted to pH 2.0, 2.5, or 3.0); (ii) survival and bacteriocin production in the presence of bile salts and commercial prebiotics (inulin and oligofructose); (iii) adhesion to Caco-2 cells; and (iv) effect on the adhesion of L. monocytogenes to Caco-2 cells and invasion of these cells by the organism. The results showed that L. sakei 1 survival in gastric environment varied according to pH, with the maximum survival achieved at pH 3.0, despite a 4-log reduction of the population after 3 h. Regarding the bile salt tolerance and influence of prebiotics, it was observed that L. sakei 1 survival rates were similar (P > 0.05) for all de Man Rogosa Sharpe (MRS) broth formulations when tests were done after 4 h of incubation. However, after incubation for 24 h, the survival of L. sakei 1 in MRS broth was reduced by 1.8 log (P < 0.001), when glucose was replaced by either inulin or oligofructose (without Oxgall). L. sakei 1 was unable to deconjugate bile salts, and there was a significant decrease (1.4 log) of the L. sakei 1 population in regular MRS broth plus Oxgall (P < 0.05). In spite of this, tolerance levels of L. sakei 1 to bile salts were similar in regular MRS broth and in MRS broth with oligofructose. Lower bacteriocin production was observed in MRS broth when inulin (3,200 AU/ml) or oligofructose (2,400 AU/ml) was used instead of glucose (6,400 AU/ml). L. sakei 1 adhered to Caco-2 cells, and its cell-free pH-neutralized supernatant containing sakacin 1 led to a significant reduction of in vitro listerial invasion of human intestinal Caco-2 cells.     

In vitro evaluation of probiotic lactic acid bacteria isolated from dairy and non-dairy environments

Four strains and 2 strains of lactic acid bacteria (LAB) were isolated from the commercial yogurt and kimchi products in Korea, respectively. Based on the 16S rRNA sequencing data, strain A from a drink-type yogurt manufactured by dairy company S, was a Gram-positive, rod-shaped Lactobacillus helveticus, and both strain B (company N) and D (company H) were identified as L. casei ssp. casei, and strain C (company L) as L. paracasei. None of yogurt strain B and D was recovered from the samples exposed to the simulated gastric juice, pH 2.0 for 1.5 hr. Of the 6 isolates tested, strain YS93 from kimchi was the most resistant to acidic condition using the simulated gastric juice, pH 2.0. Moreover, it was shown that 2 kimchi isolates and yogurt strain D produced antibacterial substances, probably bacteriocin-like peptide, which was inhibitory against Staphylococcus aureus as an indicator. In an adhesion assay using a Caco-2 cell, the adherence activity of kimchi strains YS29 and YS93 was significantly higher than those of 4 yogurt starter strains tested.     

Survival of probiotic lactobacilli in the upper gastrointestinal tract using an in vitro gastric model of digestion

The aim of this study was to investigate survival of three commercial probiotic strains (Lactobacillus casei subsp. shirota, L. casei subsp. immunitas, Lactobacillus acidophilus subsp. johnsonii) in the human upper gastrointestinal (GI) tract using a dynamic gastric model (DGM) of digestion followed by incubation under duodenal conditions. Water and milk were used as food matrices and survival was evaluated in both logarithmic and stationary phase. The % of recovery in logarithmic phase ranged from 1.0% to 43.8% in water for all tested strains, and from 80.5% to 197% in milk. Higher survival was observed in stationary phase for all strains. L. acidophilus subsp. johnsonii showed the highest survival rate in both water (93.9%) and milk (202.4%). Lactic acid production was higher in stationary phase, L. casei subsp. shirota producing the highest concentration (98.2 mM) after in vitro gastric plus duodenal digestion.     

In vitro comparison of probiotic properties of Lactobacillus casei Zhang, a potential new probiotic, with selected probiotic strains

Since 2004, our research group has isolated 240 Lactobacillus strains from Koumiss, a traditional fermented alcoholic beverage prepared from mare's milk in Inner Mongolia, Xinjiang of China and Mongolia. Among these Lactobacillus strains a novel strain with potential probiotic properties, Lactobacillus casei Zhang, was screened out and studied extensively for its probiotic properties, health-promoting effects and fermentation characteristics. In vitro tests indicated that L. casei Zhang had high tolerance to simulated gastric, intestine juices and bile salts, similar to commercial probiotic strains such as Lactobacillus acidophilus NCFM, Lactobacillus rhamnosus GG, L. casei Shirota and Bifidobacterium animalis Bb12. Higher acid-production activity and proteolytic activity was observed in the fermented milk inoculated with L. casei Zhang during the refrigerated storage than in the samples inoculated with the selected commercial probiotics. The yogurt samples fermented with L. casei Zhang exhibited similarer viable count (1.0 × 10⁹ cfu/mL) as the other samples after 28 d of refrigerated storage. The results suggested that L. casei Zhang showed good potential for application in functional foods and health-related products.     

Adhesion of dairy propionibacteria to intestinal epithelial tissue in vitro and in vivo

Adhesion to the intestinal mucosa is a desirable property for probiotic microorganisms and has been related to many of their health benefits. In the present study, 24 dairy Propionibacterium strains were assessed with regard to their hydrophobic characteristics and their autoaggregation and hemagglutination abilities, since these traits have been shown to be indicative of adherence in other microorganisms. Six strains were further tested for their capacity to adhere to ileal epithelial cells in vitro and in vivo. The results of the study showed that propionibacteria were highly hydrophilic, and hemagglutination and autoaggregation were properties not commonly found among these microorganisms. No relationship was found between surface characteristics and adhesion ability, since hemagglutinating, autoaggregating, and nonautoaggregating bacteria were able to adhere to intestinal cells both in vitro and in vivo. Microscopic examination revealed that autoaggregating cells adhered in clusters, with adhesion being mediated by only a few bacteria, whereas the hemagglutinating and nonautoaggregating strains adhered individually or in small groups making contact with each epithelial cell with the entire bacterial surface. The in vitro assessment of adhesion was a good indication of the in vivo association of propionibacteria with the intestinal epithelium. Therefore, the in vitro method presented here should be valuable in screening routinely adhesive properties of propionibacteria for probiotic purposes. The adhesion ability of dairy propionibacteria would prolong their maintenance in the gut and increase the duration of their provision of beneficial effects in the host, supporting the potential of Propionibacterium in the development of new probiotic products.     

Relationship between gastrointestinal tolerance and exopolysaccharide production of propionibacteria strains under different pH and bile conditions

In this study, we report the effects of exposure to simulated gastric and intestinal juices on the survival of six strains of dairy propionibacteria. Gastrointestinal tolerance of propionibacteria strains was highly variable, depending on the strain and pH (P < 0.05). Exopolysaccharides (EPS) production of six propionibacteria strains under different pH conditions and bile concentrations was also studied. A positive correlation was obtained between the EPS production quantity of the strains and tolerance to simulated gastric juices at pH 2.0, 3.0 (P < 0.05). This investigation showed that high EPS production may be important in the selection of probiotic strains for resistance to upper gastrointestinal system conditions.     

Survival of potentially probiotic enterococci in dairy matrices and in the human gastrointestinal tract

The aim of this study was to investigate the stability of Enterococcus strains isolated from a traditional Portuguese cheese and previously proved to be safe, in dairy matrices, and to assess survival of the best strains in the human gastrointestinal (GI) tract. Enterococcus faecium 32 and Enterococcus durans 37 were added to yoghurt that was ingested by 4 healthy adults. Detection of the enterococcal strains was performed with RAPD-PCR. The intervention trial showed transient colonisation with both strains, via presence in faeces during the ingestion period and disappearance by 10 d post-ingestion. Viable numbers of enterococci increased during the consumption period by 1.8–4.4 log-values, and returned to baseline level during the follow-up period. Based on data of the dairy matrix stability trials and human intervention study involving yoghurt ingestion, E. faecium 32 survived well both in the food matrix and in the human GI tract, thus showing probiotic potential.     

In vitro evaluation of physiological probiotic properties of different lactic acid bacteria strains of dairy and human origin

Eleven lactic acid bacteria strains of importance to the dairy industry were subjected to in vitro analyses to determine their probiotic potential. Seven strains were isolated from ewe’s and cow’s milk (Enterococcus faecalis – five –, Lactococcus lactis and Lactobacillus paracasei). Four were obtained from American Type Culture Collection (ATCC), isolated from cheese (Lactobacillus casei 393), human feces (L. paracasei 27092 and Lactobacillus rhamnosus 53103) and used in cheese making (L. lactis 54104). Although none of the strains was able to degrade mucin, all E. faecalis showed, at least, one transferable antibiotic resistance, which excluded them as candidates for addition to foods. Of the remaining six safe strains, L. lactis strains were more tolerant to low pH than Lactobacillus spp.; all were tolerant to pancreatin and bile salts and showed antibacterial activity. The highest level of adhesion to Caco-2 cells was observed with L. lactis 660, even higher than L. rhamnosus ATCC 53103 (recognized probiotic and used as control). The physiological probiotic properties of these strains, mainly isolated from dairy sources, are interesting in view of their use in cheese productions as starter and non starter cultures. The five LAB safe strains studied may have potential as novel probiotics in the dairy foods.     

In vitro characterisation of probiotic properties of Enterococcus faecium and Enterococcus durans strains isolated from raw milk and traditional dairy products

In this study, some probiotic characteristics such as antimicrobial activity, vancomycin resistance, growth ability at pH, resistance to bile salts, bile salt deconjugation and hydrophobicity of 30 Enterococcus faecium and Enterococcus durans strains isolated and identified from raw milk and various dairy products were investigated. According to the study results, antimicrobial activity profiling, pH and bile salt resistance and bile salt deconjugation ability of Enterococcus strains varied depending on the species and strains and all the strains showed resistance to the tested bile salt concentrations. It was concluded that the E. faecium and E. durans strains tested showed probiotic characteristics and have the potential to be used in food production.