Novel isolates of lactobacilli from fermented Portuguese olive as potential probiotics

The purpose of this work was to screen for and characterize the potential probiotic features of strains of lactic acid bacteria isolated from Galega cultivar fermented olives, to eventually develop an improved probiotic food from plant origin. From 156 isolated strains, 10 were acid – and bile salt tolerant, and exhibited survival rates up to 48%, following simulated digestion. All strains exhibited auto- (4–12%) and co-aggregation features (≥30%), as well as hydrophobicity (5–20%) and exopolysaccharide-producing abilities, while no strain possessed haemolytic capacity or ability to hydrolyse mucin. Antibiotic resistance, oleuropein degradation, proteolytic activity and antimicrobial activity were strain-dependent features. Overall, 10 strains – belonging to Lactobacillus plantarum and Lactobacillus paraplantarum, appear to possess a probiotic value.     

两株乳杆菌益生特性的体外研究

从健康个体口腔中分离得到了两株乳酸菌，经API细菌鉴定系统鉴定分别为植物乳杆菌Lactobacillus plantarum HO-69与Lactobacillus plantarum HO-160，并对两株植物乳杆菌的特性进行了研究。抑菌试验表明，两株菌的代谢产物具有较好的抑菌活性；两株菌对于人工消化液具有良好的耐受性，HO-69生长的最高胆汁质量分数为0．4％，HO-160为0.3％；两菌均具有较高的表面疏水性与酸碱电荷。     

Two strains of nonstarter lactobacilli increased the production of flavor compounds in soft cheeses

The contribution to flavor generation and secondary proteolysis of 2 strains of mesophilic lactobacilli isolated from cheese was studied. Miniature soft cheeses (200 g) were produced with or without the inclusion of a culture of Lactobacillus plantarum I91 or Lactobacillus casei I90 in the starter composed of Streptococcus thermophilus. During ripening, cheeses containing the added lactobacilli showed an increased content of total free amino acids, but this increase was only significant in cheeses with Lb. plantarum I91. In addition, free amino acid profiles were modified by selective increases of some amino acids, such as Asp, Ser, Arg, Leu, and Phe. Cheeses inoculated with Lb. plantarum I91 or Lb. casei I90 were also characterized by a significantly higher concentration of diacetyl, a key flavor compound, and an increased content of acetoin. Results suggest an increase in the catabolism of either citrate or aspartate, with the production of the derived aroma compounds. Overall, aspartate content increased in both lactobacilli-added cheeses, whereas citrate was more or less constant, suggesting that aspartate could be the source of increased diacetyl and acetoin. A triangle aroma test showed that the addition of the lactobacilli strains significantly changed the sensory attributes of cheeses. At least 11 of 12 panelists commented that the aroma of cheeses with adjuncts was more buttery than that of control cheeses, which is desirable in most soft cheeses. Both Lb. plantarum I91 and Lb. casei I90 performed well as adjunct cultures by influencing cheese aroma development and cheese proteolysis.     

二株嗜热链球菌益生特性的体外研究

对嗜热链球菌ST1和嗜热链球菌KS1的部分益生性及功能特性进行了研究。考察了ST1和KS1对酸和胆盐的耐受性,结果表明,ST1和KS1在pH值为3.0条件下培养3h的存活率分别为67.17%和67.49%,在pH值为4.0或质量分数为0.3%胆盐环境中仍可以生长,表现出较高的存活力。通过抑菌实验表明ST1和KS1可以抑制常见的致病菌金黄色葡萄球菌、大肠杆菌、鼠伤寒沙门氏菌和福氏志贺氏菌。在对抗生素敏感实验中,ST1对其中的3种抗生素有耐药性,而KS1对6种抗生素有耐药性。研究还发现ST1和KS1还具有较高的降胆固醇的能力,胆固醇的去除率分别为65.14%和74.49%。     

Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics

Probiotics have established their efficacy as dietary adjuncts providing benefits to consumers, but the selection of probiotics before incorporation in diet requires close scrutiny in the form of in vitro as well as in vivo tests. The present study was undertaken to check different in vitro characteristics of seven Lactobacillus casei strains. The characteristics studied include acid and bile tolerance, adhesion and cell surface hydrophobicity, antimicrobial effect on common pathogens and cholesterol reduction. All strains were able to resist pH 3 for 3 h, though resistance to pH 2 was exhibited by NCDC 17, C1 and Y strains only. NCDC 63 and VT strains were able to tolerate 1% and 2% bile concentrations for 12 h. There was wide variation in ability of strains to adhere to isolated rat epithelial cells. The index was highest for C1 at 66%. The electron microscopic adhesion studies on the stainless steel chips did not reveal any specific attachment to surfaces by any of strains. The hydrophobic character for octane was highest for strain C1 at 54.06% and lowest for strain C2 at 4.65%. The ability to antagonize common pathogens was observed in all strains but this activity was attributed to production of organic acids and no specific compound caused the inhibitory effect. The cholesterol reducing ability varied not only for strains but also for time of incubation. NCDC 17 showed maximum reduction in cholesterol level after 48 h of incubation with buffalo plasma as the source of cholesterol. Overall there existed variations in different strains with respect to different characters of significance to be a probiotic.     

Numbers and strains of lactobacilli in some probiotic products

The numbers and types of Lactobacillus strains in probiotic feed or food products for sale in Europe were compared with the information stated on the product labels. The labels of all 10 products gave information on the genus and species of Lactobacillus, and seven gave information on the numbers of a specified strain(s). Lactobacilli were recovered using a recently validated method, developed as part of the European Community Project SMT4 CT98-2235 for the official control of probiotic microorganisms used as feed additives. Polymerase chain reaction (PCR)-based methods were used to assess the accuracy of labelling with regard to genus and species, and pulsed-field gel electrophoresis (PFGE) was used to identify strains. Five products were mislabelled with respect to the numbers and three with respect to species of lactobacilli. In four cases, the specified strains were not detected. Four fermented milks sold under three trademarks contained the same strain, which was named differently on each label. As safety and functionality of probiotics are strain dependent, these results demonstrated the need to control lactobacilli present in commercially probiotic human food products, not only at the species but also at the strain level, to ensure their quality and protect the consumer. The usefulness of the official methods developed for animal feed additives to identify lactobacilli in probiotic foods for humans was demonstrated.     

Bacillus strains as human probiotics: characterization,safety, microbiome,and probiotic carrier

Food Science and Biotechnology - Both spore and vegetative forms of Bacillus species have been used as probiotics, and they have high stability to the surrounding atmospheric conditions such as...     

Acid and bile tolerance and cholesterol removal ability of lactobacilli strains

Eleven strains of lactobacilli were studied for their acid and bile tolerance. Possible mechanisms of cholesterol removal by strains of lactobacilli were examined. Cholesterol assimilation as determined by the difference in cholesterol content in the medium before and after the incubation period showed that all lactobacilli strains were able to assimilate cholesterol at varying levels ranging from 12.03 to 32.25 microg/mL. Cholesterol removal was associated with growth of cultures. Binding of cholesterol to lactobacilli cells was determined using growing, heat-killed, and resting cells in phosphate buffer. Cholesterol removed by dead and resting cells ranged from 0.79 to 3.82 mg/g of dry weight compared with growing cells, which ranged from 4.53 to 16.03 mg/g of dry weight. Fatty acid methyl esters, as quantified using gas chromatography, showed changes in lipid profiles in cells grown in the presence of cholesterol compared with those grown without cholesterol. Fatty acid profiles, especially of hexadecanoic, octadecanoic, total saturated, and unsaturated acids suggested that cholesterol from the medium was incorporated into the cellular membrane. These findings suggest that strains of lactobacilli could remove cholesterol via various mechanisms and may be promising candidates for use as a dietary adjunct to lower serum cholesterol in vivo.     

两株益生菌生物学特性的研究

研究了两株益生菌植物乳杆菌ST-Ⅲ和干酪乳杆菌BD-Ⅱ的生物学特性，特别是对其在模拟消化环境下的耐受性进行了研究。结果表明，ST-Ⅲ和BD-Ⅱ是性能优良的两株益生菌。     

两株具有潜在益生作用的人源链球菌的安全性评价

对分离于健康儿童口咽部的2株对金黄色葡萄球菌等常见致病菌具有广谱拮抗作用的甲型链球菌菌株1-4和4-2的安全性进行评价,为其作为呼吸道益生菌产品的研发和制备提供安全性依据.通过给予SPF级小鼠口服含有菌株1-4和4-2不同剂量的菌液的急性毒性实验以及耐药性的测定,观察动物是否出现死亡,急性毒性体征,细菌移位生长等情况....     

不同酿酒酵母发酵特性及抗氧化特性的比较

该文比较了不同来源的5株葡萄酒酿酒酵母(2株野生酿酒酵母CC17、SC5,2株实验室常用工程菌株BY4743、INVSC1,以及1株工业酿酒酵母菌株CICC1450)的生长曲线、泡沫产生能力、H2S产生能力、CO2产生能力,结果表明该实验室分离得到的野生酿酒酵母CC17具有良好的发酵特性.经不同浓度的氧化性物质H2O2和甲萘醌分别处理后得知,CC17在抗氧化性能上具有明显优势,说明该菌株在实际生产中具有开发利用的潜力.     

Influence on cheese proteolysis and sensory characteristics of non-starter lactobacilli strains with probiotic potential

The contribution to cheese proteolysis and sensory profile of four potentially probiotic non-starter lactobacilli strains was assessed in two different models: soft and semi-hard cheeses. All the strains were able to grow in both types of cheese, where they maintained high levels during ripening. Overall, adjunct cultures of Lactobacillus rhamnosus showed the strongest influence in peptidolysis, which was verified by changes in peptide profiles and increase of free amino acids concentration. Nevertheless, some cheeses treated with L. rhamnosus also showed post-acidification during ripening and decreased sensory characteristics compared to controls. Cheeses with adjunct cultures of Lactobacillus casei I90 and Lactobacillus plantarum I91 exhibited, in general, an intermediate level of peptidolysis between control and L. rhamnosus-added cheeses; their sensory characteristics were preserved or improved, and they did not show any defects. Adjunct cultures showed similar trends in both models, confirming L. casei I90 and L. plantarum I91 as the most performing adjunct cultures for cheese-making among the strains.     

Characterisation of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11 and its non-EPS producing derivative strains as potential probiotics

Traditional fermented foods are the best source for the isolation of strains with specific traits to act as functional starters and to keep the biodiversity of the culture collections. Besides, these strains could be used in the formulation of foods claimed to promote health benefits, i.e. those containing probiotic microorganisms. For the rational selection of strains acting as probiotics, several in vitro tests have been proposed. In the current study, we have characterized the probiotic potential of the strain Lactobacillus paraplantarum BGCG11, isolated from a Serbian soft, white, homemade cheese, which is able to produce a "ropy" exopolysaccharide (EPS). Three novobiocin derivative strains, which have lost the ropy phenotype, were characterized as well in order to determine the putative role of the EPS in the probiotic potential. Under chemically gastrointestinal conditions, all strains were able to survive around 1-2% (10(6)-10(7)cfu/ml cultivable bacteria) only when they were included in a food matrix (1% skimmed milk). The strains were more resistant to acid conditions than to bile salts and gastric or pancreatic enzymes, which could be due to a pre-adaptation of the parental strain to acidic conditions in the cheese habitat. The ropy EPS did not improve the survival of the producing strain. On the contrary, the presence of an EPS layer surrounding the strain BGCG11 hindered its adhesion to the three epithelial intestinal cell lines tested, since the adhesion of the three non-ropy derivatives was higher than the parental one and also than that of the reference strain Lactobacillus rhamnosus GG. Aiming to propose a potential target application of these strains as probiotics, the cytokine production of peripheral blood mononuclear cells (PBMC) was analyzed. The EPS-producing L. paraplantarum BGCG11 strain showed an anti-inflammatory or immunosuppressor profile whereas the non-ropy derivative strains induced higher pro-inflammatory response. In addition, when PBMC were stimulated with increasing concentrations of the purified ropy EPS (1, 10 and 100μg/ml) the cytokine profile was similar to that obtained with the EPS-producing lactobacilli, therefore pointing to a putative role of this biopolymer in its immune response.     

Survival and activity of 5 probiotic lactobacilli strains in 2 types of flavored fermented milk

The viability of 5 probiotic lactobacilli strains (Lactobacillus acidophilus LA-5, Lactobacillus casei L01, Lactobacillus casei LAFTI L26, Lactobacillus paracasei Lcp37, and Lactobacillus rhamnosus HN001) was assessed in 2 types of probiotic flavored drink based on fermented milk during 21 days of refrigerated storage (5°C). Also, changes in biochemical parameters (pH, titrable acidity, and redox potential) during fermentation as well as the sensory attributes of final product were determined. Among the probiotic strains, L. casei LAFTI L26 exhibited the highest retention of viability during refrigerated storage period, while L. acidophilus LA-5 showed the highest loss of viability during this period. The decline in cell count of probiotic bacteria in strawberry fermented milk was significantly greater compared to peach fermented milk. In an overall approach, peach fermented milk containing L. casei LAFTI L26 was selected as the optimal treatment in this study in both aspects of viability and sensory accpeptibility.     

Lactobacilli as multifaceted probiotics with poorly disclosed molecular mechanisms

Lactic acid bacteria and more particularly lactobacilli have been used for the production of fermented foods for centuries. Several lactobacilli have been recognized as probiotics due to their wide range of health-promoting effects in humans. However, little is known about the molecular mechanisms underpinning their probiotic functions. Here we reviewed the main beneficial effects of lactobacilli and discussed, when the information is available, the molecular machinery involved in their probiotic function.Among the beneficial effects, lactobacilli can improve digestion, absorption and availability of nutrients. As an example, some strains are able to degrade carbohydrates such as lactose or α-galactosides that may cause abdominal pain. Furthermore, they can hydrolyze compounds that limit the bioavailability of minerals like tannin and phytate due to tannin acylhydrolase and phytase activities. In addition, it was shown that some lactobacilli strains can improve mineral absorption in Caco-2 cells. Lactobacilli can also contribute to improve the nutritional status of the host by producing B group vitamins. More recently, the role of lactobacilli in energy homeostasis, particularly in obese patients, is the object of an increased interest.Lactobacilli are also involved in the prevention of diseases. They have potential to prevent carcinogenesis through the modulation of enzymes involved in the xenobiotic pathway, and may prevent cardiovascular diseases such as hypertension through the production of a bioactive peptide that may have angiotensin converting enzyme inhibitor activity. Lactobacilli are increasingly studied for the treatment of inflammatory bowel diseases and exhibit interesting potential in the reduction of pain perception. The ability of some strains to bind to intestinal cells, their pathogen-associated molecular patterns and the metabolites they produce confer interesting immunomodulatory effects. Finally, pathogenic fungi, virus or bacteria can be inhibited by probiotics. They can reinforce the intestinal barrier, simply occupying the ecological niche, or they can have an active role by synthesizing various metabolites inhibiting pathogen development.Lactobacilli have a long standing history with foods and humans but comparatively, their history as probiotics is recent. Their effects are investigated in in vitro and in vivo models leading sometimes to contradictory or controversial results that make necessary final demonstrations through clinical trials. Researches on molecular mechanisms involved in the beneficial effects reviewed here are necessary for a better understanding of these effects, but could also lead to the development of molecular tools to help the screening of the probiotic potential of lactobacilli that are common inhabitants of numerous fermented foods around the world.     

Enterococci as probiotics and their implications in food safety

Enterococci belong to the lactic acid bacteria (LAB) and they are of importance in foods due to their involvement in food spoilage and fermentations, as well as their utilisation as probiotics in humans and slaughter animals. However, they are also important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics and possess virulence factors such as adhesins, invasins, pili and haemolysin. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some can harbour virulence traits, but it is also thought that virulence is not the result of the presence of specific virulence determinants alone, but is rather a more intricate process. Specific genetic lineages of hospital-adapted strains have emerged, such as E. faecium clonal complex (CC) 17 and E. faecalis CC2, CC9, CC28 and CC40, which are high risk enterococcal clonal complexes. These are characterised by the presence of antibiotic resistance determinants and/or virulence factors, often located on pathogenicity islands or plasmids. Mobile genetic elements thus are considered to play a major role in the establishment of problematic lineages. Although enterococci occur in high numbers in certain types of fermented cheeses and sausages, they are not deliberately added as starter cultures. Some E. faecium and E. faecalis strains are used as probiotics and are ingested in high numbers, generally in the form of pharmaceutical preparations. Such probiotics are administered to treat diarrhoea, antibiotic-associated diarrhoea or irritable bowel syndrome, to lower cholesterol levels or to improve host immunity. In animals, enterococcal probiotics are mainly used to treat or prevent diarrhoea, for immune stimulation or to improve growth. From a food microbiological point of view, the safety of the bacteria used as probiotics must be assured, and data on the major strains in use so far indicate that they are safe. The advantage of use of probiotics in slaughter animals, from a food microbiological point of view, lies in the reduction of zoonotic pathogens in the gastrointestinal tract of animals which prevents the transmission of these pathogens via food. The use of enterococcal probiotics should, in view of the development of problematic lineages and the potential for gene transfer in the gastrointestinal tract of both humans and animals, be carefully monitored, and the advantages of using these and new strains should be considered in a well contemplated risk/benefit analysis.     

Spent coffee as a rich source of antioxidative compounds

Antioxidant activities and major antioxidants were investigated in the methanolic extracts of roasted and spent coffee to evaluate the feasibility of spent coffee as a source of functional ingredients. Phenolic compounds, such as gallic acid, protocatechuic acid, and chlorogenic acid, and nitrogenous compounds, including trigonelline and caffeine, were identified. Caffeine was the most abundant compound, followed by chlorogenic acid. Despite the significant reduction of antioxidants, 2,2,-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was retained in more than 95% of roasted coffee. The retentions of superoxide dismutase (SOD)-like activity and ferric reducing antioxidant power (FRAP) were 65–90 and 46–60%, respectively. Gallic acid had a positive correlation with SOD-like activity, whereas protocatechuic acid positively correlated with FRAP, suggesting that the major compounds contributing to each antioxidant activity are different. These results show that spent coffee can be used as an antioxidant source for functional foods and cosmetic products to improve antioxidant properties.     

Assessing and comparing antioxidant activities of lactobacilli strains by using different chemical and cellular antioxidant methods

Some lactobacilli strains had beneficial effects on human beings due to their antioxidant activities. In this study lactobacilli strains stored in our laboratory were screened for potential antioxidant activities by investigating their 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity, oxygen radical absorbance capacity, resistance to H₂O₂, and hydroxyl free radical scavenging activity; then the antioxidant activities of the screened strains were evaluated by cellular antioxidant assay and protection for HT-29 cells against H₂O₂ injury assay. The results showed that Lactobacillus plantarum Y44 could scavenge oxygen free radicals, inhibit the production of intracellular reactive oxygen species without creating obvious cytotoxic effects, and protect HT-29 cells against H₂O₂ injury evidenced by the significant decrease of the Bcl-2-associated X protein (Bax)/B-cell lymphoma 2 (Bcl-2) ratio and heat shock protein 70 expression, increase of superoxide dismutase and glutathione peroxidase activities, and decrease of malondialdehyde level of HT-29 cells damaged by H₂O₂. It was speculated that L. plantarum Y44 protect HT-29 cells against oxygen radical injury through scavenging reactive oxygen species and activating intracellular antioxidant enzymes. A significant correlation was observed among the results of the hydroxyl radical scavenging assay, protection assay for HT-29 cells against H₂O₂ injury, and the cellular antioxidant assay. The findings indicated that L. plantarum Y44 could be a probiotic candidate with antioxidant properties and combining several chemical antioxidant methods and antioxidant cellular models could be an effective procedure to screen lactobacilli strains with antioxidant activity.     

Lactobacilli isolated from chicken intestines: potential use as probiotics

Lactobacillus strains were tested for their in vitro probiotic properties. Cell surface hydrophobicity was found to be very high for Lactobacillus fermentum subsp. cellobiosus and Salmonella Gallinarum; high values could indicate a greater ability to adhere to epithelial cells. Studies on Lactobacillus animalis indicated relative cell surface hydrophobicities smaller than those of L. fermentum subsp. cellobiosus and L. fermentum. L. animalis and Enterococcus faecalis were able to coaggregate with L. fermentum subsp. cellobiosus and L. fermentum, respectively, but not with Salmonella Gallinarum. After mixed-culture studies for determining suitable growth behavior, the pair of strains L. animalis plus L. fermentum subsp. cellobiosus was selected for an attempted challenge against Salmonella Gallinarum. Double and triple mixed-culture studies indicated that selected lactobacillus strains were able to retain their beneficial characteristics in the presence of Salmonella Gallinarum such as presence of lectins, production of antimicrobial compounds, and ability to grow and compete. The selected microorganisms can be considered as potential ingredients for a chicken probiotic feed formulation intended to control salmonellosis and also improve poultry sanitation.     

Exopolysaccharides produced by probiotic strains modify the adhesion of probiotics and enteropathogens to human intestinal mucus

Exopolysaccharides (EPSs) are exocellular polymers present in the surface of many bacteria, including Lactobacillus and Bifidobacterium. The genome sequence of several strains revealed the presence of EPS-encoding genes. However, the physiological role that EPSs play in the bacterial ecology still remains uncertain. In this study, we have assessed the effect of EPSs produced by Lactobacillus rhamnosus GG, Bifidobacterium longum NB667, and Bifidobacterium animalis IPLA-R1 on the adhesion of probiotic and enteropathogen strains to human intestinal mucus. The EPS fraction GG had no significant effect on the adhesion of L. rhamnosus GG and B. animalis IPLA-R1. However, the EPS fractions NB667 and IPLA-R1 significantly reduced the adherence of both probiotic strains. In contrast, the three EPS fractions increased the adhesion of Enterobacter sakazakii ATCC 29544 and Escherichia coli NCTC 8603. Higher adherence of Salmonella enterica serovar Typhimurium ATCC 29631 and Clostridium difficile ATCC 9689 was detected in the presence of the EPS fractions GG and NB667. In general, these effects were obtained at EPS concentrations of up to 5 mg/ml, and they were EPS dose dependent. The competitive exclusion of probiotics in the presence of EPS could suggest the involvement of these biopolymers in the adhesion to mucus. The increase in the adherence of enteropathogens could be explained if components of the pathogen surface are able to bind to specific EPSs and the bound EPSs are able to adhere to mucus. To the best of our knowledge, this is the first work reporting the effect of EPSs from probiotics on bacterial adhesion properties.