How to select a probiotic? A review and update of methods and criteria

International competition within the dairy market and increasing public awareness about the importance of functional food consumption are providing new challenges for innovation in the probiotic sector. In this context, countless references are currently dedicated to the selection and characterization of new species and more specific strains of probiotic bacteria. In general, these studies adopt basic selection criteria established by the World Health Organization (WHO), including host-associated stress resistance, epithelium adhesion ability, and antimicrobial activity. These aspects are applied to ensure that the candidate probiotic could withstand the stressful conditions of the human digestive system and exert functional proprieties. However, it cannot be assumed that these novel microbial strains are capable of offering several biological benefits attributed to probiotics. Additionally, safety-associated selection criteria, such as plasmid-associated antibiotic resistance spreading and enterotoxin production, are often neglected. This article reviews the recent developments in the processes, strategies, and methods, such as anticarcinogenic, antidepression, antianxiety, antiobesity, antidiabetic, immunostimulatory, and cholesterol-lowering assessments, to select probiotic strains with the ultimate objective of assisting future probiotic microbe evaluation studies.     

The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus

Human intestinal glycoproteins extracted from faeces were used as a model for intestinal mucus to investigate adhesion of pathogenic Escherichia coli and Salmonella strains, and the effect of probiotics on this adhesion. S-fimbriated E. coli expressed relatively high adhesion in the mucus model, but the other tested pathogens adhered less effectively. Probiotic strains Lactobacillus GG and L. rhamnosus LC-705 as well as a L. rhamnosus isolated from human faeces were able to slightly reduce S-fimbria-mediated adhesion. Adhesion of S. typhimurium was significantly inhibited by probiotic L. johnsonii LJ1 and L. casei Shirota. Lactobacillus GG and L. rhamnosus (human isolate) increased the adhesion of S. typhimurium suggesting that the pathogen interacts with the probiotic.     

Diversity and evolutionary aspects of mucin binding (MucBP) domain repeats among Lactobacillus plantarum group strains through comparative genetic analysis

Mucin binding protein (Mub) of Lactobacillus plantarum- group (LPG) is considered as a potential marker gene due to its diversity and adherence property to host intestinal layer. We recently observed strain specific diversity in mub gene of LPG strains (n = 8) and grouped them into Type I, II and III based on the insertion and/or deletion of MucBP domain repeats (Devi et al., 2016). The mub gene expression pattern noted by real-time PCR revealed 67.64 increased fold change in MTCC 9483 with four MucBP repeats. Whereas, the isolates with 3 and 2 MucBP repeats showed ∼4 and 2 enhanced expression, respectively. Detection of integrase (int) and transposases (tra) in the flanking regions of mub, suggested variation in amplicon sizes and revealed strain diversity. The IS finder program identified tra nucleotide sequence similarity towards IS256 and IS5 family insertion elements that are present among other lactic acid bacteria. Phylogenetic dendrogram suggested the evolution of D₇–D₁₀ domains of L. paraplantarum MTCC 9483 from Lactobacillus namurensis, Lactobacillus hammesii and Lactobacillus spicheri. These results collectively gave information on the genetic diversity, functional MucBP domains and also facilitated in the selection of an efficient novel LPG strain with good adherence ability.     

Potentials of Exopolysaccharides from Lactic Acid Bacteria

Recent research in the area of importance of microbes has revealed the immense industrial potential of exopolysaccharides and their derivative oligosaccharides from lactic acid bacteria. However, due to lack of adequate technological knowledge, the exopolysaccharides have remained largely under exploited. In the present review, the enormous potentials of different types of exopolysaccharides from lactic acid bacteria are described. This also summarizes the recent advances in the applications of exopolysaccharides, certain problems associated with their commercial production and the remedies.     

The influence of polyunsaturated fatty acids on probiotic growth and adhesion

The establishment of the intestinal microflora, and probiotic bacteria, may control the inflammatory conditions in the gut. As polyunsaturated fatty acids (PUFA) possess antimicrobial activities, they may deter the action of probiotics. We assessed whether free linoleic, gamma-linolenic, arachidonic, alpha-linolenic and docosahexaenoic acids at physiological concentrations in the growth media would influence the growth and adhesion of Lactobacillus GG (probiotic), Lactobacillus casei Shirota (probiotic) and Lactobacillus bulgaricus (dairy strain). Higher concentrations of PUFA (10-40 microg PUFA ml(-1)) inhibited growth and mucus adhesion of all tested bacterial strains, whilst growth and mucus adhesion of L. casei Shirota was promoted by low concentrations of gamma-linolenic acid and arachidonic acid (at 5 microg ml(-1)), respectively. PUFA also altered bacterial adhesion sites on Caco-2 cells. Caco-2 cells grown in the presence of arachidonic acid were less adhered to by all three bacterial strains. Yet, L. casei Shirota adhered better on Caco-2 cells grown in the presence of alpha-linolenic acid. As the adhesion to mucosal surfaces is pivotal in health promoting effects by probiotics, our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA.     

Evaluation of selenite bioremoval from liquid culture by Enterococcus species

The genus Enterococcus belong to the genera of bacteria that produce lactic acid and can confer health benefits to living organisms. Selenium (Se) is an essential micronutrient for humans and animals. Thirty-six Enterococcus species isolated from dairy products were screened for Se(IV) sorption capacity for use as a probiotics in animal nutrition. Several isolates grew luxuriantly and significantly removed Se(IV) from Se(IV) amended medium. Two isolates, LAB 14 and LAB 18, identified by 16S rRNA gene sequence analysis as Enterococcus faecalis (98% nucleotide sequence similarity) and Enterococcus faecium (97% nucleotide sequence similarity), respectively, were selected for further studies. The two isolates grew optimally and removed selenium at initial pH 7.0. Optimum removal of Se(IV) from the medium was recorded at 25 °C. Time course studies showed that after 8 h of incubation LAB 14 and LAB 18 cultures displayed the highest biomass production and Se(IV) bioremoval and most selenite in culture depleted in 24 h. At initial concentrations of 10 mg L⁻¹ and 60 mg L⁻¹, E. faecium (LAB 18) removed 9.91 mg L⁻¹ and 59.70 mg L⁻¹, respectively after 24 h. Similar Se(IV) bioremoval capacity was recorded with E. faecalis (LAB 14). Substantial amount of Se was detected in biomass of E. faecium (0.4599 mg g⁻¹ of dry weight) and E. faecalis (0.4759 mg g⁻¹ of dry weight). The significant uptake and transformation of Se(IV) by the Enterococcus species observed in this study suggest that they can be used to deliver dietary Se through feed augmentation with Se(IV)-enriched Enterococcus biomass.     

Use of a continuous culture fermentation system to investigate the effect of GanedenBC30 (Bacillus coagulans GBI-30, 6086) supplementation on pathogen survival in the human gut microbiota

Single-stage continuous fermentation systems were employed to examine the effects of GanedenBC(30) supplementation on the human gastrointestinal microbiota in relation to pathogen challenge in vitro. Denaturing gradient gel electrophoresis analysis demonstrated that GanedenBC(30) supplementation modified the microbial profiles in the fermentation systems compared with controls, with profiles clustering according to treatment. Overall, GanedenBC(30) supplementation did not elicit major changes in bacterial population counts in vitro, although notably higher Bcoa191 counts were seen following probiotic supplementation (compared to the controls). Pathogen challenge did not elicit significant modification of the microbial counts in vitro, although notably higher Clit135 counts were seen in the control system post-Clostridium difficile challenge than in the corresponding GanedenBC(30)-supplemented systems. Sporulation appears to be associated with the anti-microbial activity of GanedenBC(30), suggesting that a bi-modal lifecycle of GanedenBC(30)in vivo may lead to anti-microbial activity in distal regions of the gastrointestinal tract.     

Modulation of virulence and antibiotic susceptibility of enteropathogenic Escherichia coli strains by Enterococcus faecium probiotic strain culture fractions

The increasing rate of antimicrobial resistance drastically reduced the efficiency of conventional antibiotics and led to the reconsideration of the interspecies interactions in influencing bacterial virulence and response to therapy. The aim of the study was the investigation of the influence of the soluble and cellular fractions of Enterococcus (E.) faecium CMGB16 probiotic culture on the virulence and antibiotic resistance markers expression in clinical enteropathogenic Escherichia (E.) coli strains.The 7 clinical enteropathogenic E. coli strains, one standard E. coli ATCC 25,922 and one Bacillus (B.) cereus strains were cultivated in nutrient broth, aerobically at 37 °C, for 24 h. The E. faecium CMGB16 probiotic strain was cultivated in anaerobic conditions, at 37 °C in MRS (Man Rogosa Sharpe) broth, and co-cultivated with two pathogenic strains (B. cereus and E. coli O28) culture fractions (supernatant, washed sediment and heat-inactivated culture) for 6 h, at 37 °C. After co-cultivation, the soluble and cellular fractions of the probiotic strain cultivated in the presence of two pathogenic strains were separated by centrifugation (6000 rpm, 10 min), heat-inactivated (15 min, 100 °C) and co-cultivated with the clinical enteropathogenic E. coli strains in McConkey broth, for 24 h, at 37 °C, in order to investigate the influence of the probiotic fractions on the adherence capacity and antibiotic susceptibility. All tested probiotic combinations influenced the adherence pattern of E. coli tested strains. The enteropathogenic E. coli strains susceptibility to aminoglycosides, beta-lactams and quinolones was increased by all probiotic combinations and decreased for amoxicillin-clavulanic acid. This study demonstrates that the plurifactorial anti-infective action of probiotics is also due to the modulation of virulence factors and antibiotic susceptibility expression in E. coli pathogenic strains.     

Impact of the probiotic bacteria Enterococcus faecium NCIMB 10415 (SF68) and Bacillus cereus var. toyoi NCIMB 40112 on the development of serum IgG and faecal IgA of sows and their piglets

Abstract

To examine the influence of two different probiotic bacteria on the humoral immune system of swine, two animal studies were carried out with sows and their litters. The sows' feed was supplemented with either Enterococcus faecium NCIMB 10415 (SF68) or Bacillus cereus var. toyoi NCIMB 40112 beginning early in pregnancy. The total IgA content in the faeces as well as the total IgG concentration in the blood of the sows was recorded before and after weaning. The same parameters were determined in the blood and faeces of the piglets. In sows, only feed supplementation with B. cereus led to a clear increase in faecal IgA. Serum IgG levels were not significantly affected by any probiotic feeding in sows. In piglets, the group that was fed B. cereus showed significantly higher faecal IgA levels shortly before weaning, whereas in the E. faecium group, a significant decrease in IgA levels was observed one week after weaning. In both probiotic fed groups the post-weaning IgG levels were significantly decreased compared to the respective control groups. We conclude that B. cereus var. toyoi feed supplementation led to an increased intestinal IgA secretion both in sows and piglets. This effect could be related to a more successful mucosal defence which in turn led to a lower level in systemic IgG production in piglets after weaning.     

微生态制剂辅助治疗溃疡性结肠炎的疗效与安全性研究

目的观察微生态制剂(思连康)辅助治疗活动期溃疡性结肠炎(UC)的疗效和安全性,并进一步探讨微生态制剂对肠黏膜局部细胞因子的影响。方法活动期UC患者86例,随机分为2组,对照组(40例)口服美沙拉嗪,同时用琥珀酸氢化考的松及庆大霉素、蒙脱石散保留灌肠,重度患者加静脉用激素。治疗组(46例)在上述治疗基础上,口服思连康1.5 g,每天3次。治疗6个月后评价2组疗效,主要症状和结肠镜变化情况,免疫组化法检测患者肠黏膜局部细胞因子IL-10、IL-6和IL-18的变化。结果治疗后2组临床表现均有明显好转,且治疗组优于对照组,2组比较差异有统计学意义(P<0.05)。2组治疗后结肠镜表现均有明显改善,治疗组优于对照组;疾病活动指数(DAI)降低(P<0.05),降低幅度治疗组优于对照组(P<0.05)。所有患者均未见明显的不良反应。免疫组化结果显示治疗后肠黏膜细胞因子IL-10的水平升高,治疗组优于对照组,IL-6和IL-18均有降低,IL-6下降治疗组优于对照组,而IL-18的降低2组间差异无统计学意义。结论微生态制剂口服辅助治疗UC疗效明显,并能降低UC的复发,且无不良反应。其机制可能与上调局部肠黏膜抗炎性细胞因子,下调炎性细胞因子有关。