Probiotic properties of yeasts in traditional fermented foods and beverages

The interest in potentiality and functionality of probiotic yeasts from fermented foods has increased drastically over the years. In many fermented foods and beverages, lactic acid bacteria and yeasts exist synergistically by stimulating their growth and survival. Probiotic strains of lactic acid bacteria are more widely studied than potential probiotic yeasts. Saccharomyces cerevisiae variety boulardii is the only commercialized probiotic yeast, which are extensively studied. This review article provides information on the presence of potential probiotic yeasts in some traditional fermented foods and beverages.     

Yeasts from kefir grains: isolation, identification, and probiotic characterization

Kefir—a traditional beverage whose consumption has been associated with health benefits—is a logical natural product to investigate for new probiotic strains. The aim of the present work was to isolate and identify kefir yeasts and select those with acid and bile tolerance to study their adhesion to epithelial cells and their transit through mouse gut. From 4 milky and 3 sugary kefir grains, 34 yeast strains were isolated and identified by means of classical microbiological and molecular-genetic methods (whole-cell protein pattern, internal-transcribed-spacer amplification, and analysis of restriction-fragment–length polymorphisms). We identified 4 species belonging to 3 genera—Saccharomyces cerevisiae (15 strains), Saccharomyces unisporus (6 strains), Issatchenkia occidentalis (4 strains), and Kluyveromyces marxianus (9 strains)—and selected 13 strains on the basis of resistance to low pH and bile salts. Among the strains selected, Kluyveromyces marxianus CIDCA 8154 and Saccharomyces cerevisiae CIDCA 8112 were further studied. Both strains evidenced the capacity to adhere to epithelial intestine-derived cells in vitro and to survive passage through the gastrointestinal tract of BALB/c mice. The investigation of the potential probiotic features of these kefir-yeast strains should be useful for the development of novel functional foods.     

Growth kinetics on oligo- and polysaccharides and promising features of three antioxidative potential probiotic strains

Aims: To determine the antioxidative activity, glutathione production, acid and bile tolerance and carbohydrate preferences of Lactobacillus plantarum LP 1, Streptococcus thermophilus Z 57 and Bifidobacterium lactis B 933. Methods and Results: The intact bacteria exhibited antioxidative capacity against linolenic acid and ascorbate oxidation. The antioxidative activity of cell-free extracts was determined by chemiluminescent assay and agreed with total glutathione content. Superoxide dismutase was negligible in all the strains. Bile and gastric juice resistance was tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. Bifidobacterium lactis B 933 and L. plantarum LP 1 were more acid tolerant than S. thermophilus Z 57. All the strains were resistant to bile. Among 13 indigestible carbohydrates, galacto-oligosaccharides and fructo-oligosaccharides were utilized by all the strains and did not affect survival in human gastric juice. Conclusions: These potential probiotic strains exhibited antioxidative properties and good viability in gastric juice and bile may indicate tolerance to the transit through the upper gastrointestinal tract. Galacto-oligosaccharides and fructo-oligosaccharides are the most appropriate prebiotics to be used in effective synbiotic formulations. Significance and Impact of the Study: These results outline promising strains with antioxidative properties. Carbohydrate preferences can be exploited in order to develop synbiotic products.     

Probiotic Potential,Antioxidant Activity,and Phytase Production of Indigenous Yeasts Isolated from Indigenous Fermented Foods

While many bacteria have been used as probiotics by industries, only two yeasts, Saccharomyces cerevisiae var. boulardii and Kluyveromyces fragilis (B0399), have been used for this purpose. In the present work, a total of 116 yeasts isolated from Brazilian indigenous fermented food, cocoa fermentation, and kefir were in vitro characterized for probiotic attributes. From 116 isolates, 36 were tolerant to gastrointestinal conditions evaluated by tolerance to pH 2.0, bile salts (0.3% w/v), and 37 °C temperature. From those, 15 isolates showed a similar or higher percentage (P < 0.05) of hydrophobicity, autoaggregation, and coaggregation with E. coli than the reference strain S. boulardii. All these strains showed a high percentage of adhesion to Caco-2 cells (> 63%) and antioxidant activity (ranging from 18 to 62%). Phytate hydrolysis was evaluated for these yeasts and 13 strains showed positive results, which is important for nutrient availability in plant-based foods. These results are important insights for characterization of novel probiotic yeast strains as well as to aggregate functional value to these food products.

     

Immunostimulatory activity of potential probiotic yeast strains in the dorsal air pouch system and the gut mucosa

Aims: To determine the immunostimulatory activity of 15 presumptive probiotic yeast strains in the dorsal air pouch system in comparison with their activity in the gut mucosa. Methods and Results: Presumptive probiotic yeast strains previously isolated from human gastrointestinal tract and Feta cheese were further characterized genotypically and biochemically. The Saccharomyces cerevisiae 982, Saccharomyces boulardii KK1 and Kluyveromyces lactis 630 strains exhibited in the air pouch increased polymorphonuclear cell influx and phagocytic activity as well as cytokine production with similar potency as the probiotics Ultra levure S. boulardii and Lactobacillus acidophilus NCFB 1748. Oral administration of these strains in mice results in differential activation of small intestine immune responses concerning IgA and cytokine production as well as Toll‐like receptor expression. Conclusion: Besides the Saccharomyces strains 982 and KK1, the K. lactis 630 strain could also be considered as a candidate probiotic. Significance and Impact of the Study: The air pouch model may be used as an alternative and rapid method for the discrimination and selection of potential probiotic yeast strains.     

Differentiation of probiotic and environmental Saccharomyces cerevisiae strains in animal feed

Aims: To establish an identification system for probiotic Saccharomyces cerevisiae strains based on artificial neural network (ANN)–assisted Fourier‐transform infrared (FTIR) spectroscopy to improve quality control of animal feed. Methods and Results: The ANN‐based system for differentiating environmental from probiotic S. cerevisiae strains comprises five authorized feed additive strains plus environmental strains isolated from different habitats. A total of 108 isolates were used as reference strains to create the ANN. DHPLC analysis and δ‐PCR were used as reference methods to type probiotic yeast isolates. The performance of the FTIR‐ANN was tested in an internal validation using unknown spectra of each reference strain. This validation step yielded a classification rate of 99·1 %. For an external validation, a test data set comprising 965 spectra of 63 probiotic and environmental S. cerevisiae isolates unknown to the ANN was used, resulting in a classification rate of 98·2 %. Conclusions: Our results demonstrate that probiotic S. cerevisiae strains in feed can be differentiated successfully from environmental isolates using both genotypic approaches and ANN‐based FTIR spectroscopy. Significance and Impact of the Study: FTIR‐based artificial neural network analysis provides a rapid and inexpensive technique for yeast identification both at the species and at the strain level in routine diagnostic laboratories, using a single sample preparation.     

Characterization of Intestinal Lactobacillus reuteri Strains as Potential Probiotics

This study was conducted to evaluate the probiotic properties of Lactobacillus reuteri isolated from human infant feces (less than 3?months). Out of thirty-two representative L. reuteri strains isolated from the infant human feces, nine isolates (i.e. LR5, LR6, LR9, LR11, LR19, LR20, LR25, LR26 and LR34) showed survival in acid, bile and simulated stomach?Cduodenum passage conditions, indicating their high tolerance to gastric juice, duodenal juice and bile environments. The nine isolates did not show strong hydrophobic properties because the percentages of adhesion to the apolar solvent, n-hexadecane, did not exceed 40%, showing that their surfaces were rather hydrophilic. Functionality of these nine probiotic isolates was supported by their antagonistic activity and their ability to deconjugate bile salts. The safety of the nine indigenous L. reuteri isolates was supported by the absence of transferable antibiotic resistance determinants, DNase activity, gelatinase activity and hemolysis. The results obtained so far suggest that the nine strains are resistant to low pH, bile salts and duodenum juice, so they could survive when passing through the upper part of the gastrointestinal tract and fulfill their potential probiotic action in the host organism. According to these results, the L. reuteri strains isolated from human infant feces possess interesting probiotic properties that make them potentially good candidates for probiotics.     

Mitogenic response and probiotic characteristics of lactic acid bacteria isolated from indigenously pickled vegetables and fermented beverages

Lactic acid bacteria from indigenous pickled vegetables and fermented beverages (fermented rice and Madhuca longifolia flowers) were isolated and investigated for their functional characteristics in vitro as potential new probiotic strains. Four isolates (all Lactobacillus spp.) selected on the basis of high tolerance to bile (0.2%) were identified by standard and molecular methods (16S rDNA) as L. helveticus, L. casei, L. delbrueckii and L. bulgaricus from pickled vegetables and fermented beverages respectively. These selected strains had antibiotic resistance, tolerance to artificial gastric juice and phenol (0.4%), enzymatic profile, and antagonistic activity against enteric pathogens (Enterobacter sakazakii, Salmonella typhimurium, Shigella flexneri 2a, Listeria monocytogenes, Yersinia enterocolitica and Aeromonas hydrophila). All strains survived well in artificial gastric juice at low pH (3.0) values for 4 h, possessed bile salt hydrolase activity and were susceptible to most antibiotics including vancomycin. Additionally, the isolates exhibited high tolerance to phenol, high cell surface hydrophobicity (>60%) and induced proliferation of murine splenocytes. All the four strains of present study suppressed the Con A-stimulated proliferation of the mouse spleen cells, although L. casei had the strongest suppressive effect. The results of this study suggest a potential application of the strains (following human clinical trials), for developing probiotic foods.     

A possible application of ale brewery strains ofSaccharomyces cerevisiae in lager beer production

The physiological characteristics of two strains of brewery ale yeasts,Saccharomyces cerevisiae, with sedimentation abilities, were investigated to see if the strains were suitable for lager beer production. Compared with typical industrial ale strains ofS. cerevisiae and lager strains ofS. uvarum (nowS. cerevisiae), the investigated strains differ in fermentation dynamics, as well as in biological properties. The differences, however, particularly between the two strains and the lager brewing yeasts, were not significant.     

Microarray karyotyping of maltose‐fermenting Saccharomyces yeasts with differing maltotriose utilization profiles reveals copy number variation in genes involved in maltose and maltotriose utilization

Aims: We performed an analysis of maltotriose utilization by 52 Saccharomyces yeast strains able to ferment maltose efficiently and correlated the observed phenotypes with differences in the copy number of genes possibly involved in maltotriose utilization by yeast cells. Methods and Results: The analysis of maltose and maltotriose utilization by laboratory and industrial strains of the species Saccharomyces cerevisiae and Saccharomyces pastorianus (a natural S. cerevisiae/Saccharomyces bayanus hybrid) was carried out using microscale liquid cultivation, as well as in aerobic batch cultures. All strains utilize maltose efficiently as a carbon source, but three different phenotypes were observed for maltotriose utilization: efficient growth, slow/delayed growth and no growth. Through microarray karyotyping and pulsed‐field gel electrophoresis blots, we analysed the copy number and localization of several maltose‐related genes in selected S. cerevisiae strains. While most strains lacked the MPH2 and MPH3 transporter genes, almost all strains analysed had the AGT1 gene and increased copy number of MALx1 permeases. Conclusions: Our results showed that S. pastorianus yeast strains utilized maltotriose more efficiently than S. cerevisiae strains and highlighted the importance of the AGT1 gene for efficient maltotriose utilization by S. cerevisiae yeasts. Significance and Impact of the Study: Our results revealed new maltotriose utilization phenotypes, contributing to a better understanding of the metabolism of this carbon source for improved fermentation by Saccharomyces yeasts.     

模拟人体胃肠道环境筛选益生乳杆菌

【目的】筛选具有益生特性的乳杆菌作为保健型酸奶的候选菌株。【方法】从健康人肠道和奶豆腐中分离筛选出耐受人工胃液的乳杆菌,对其进行体外益生特性(人工胃肠液耐受性、胆盐耐受性、抑菌活性及胆固醇降解能力)研究。【结果】从在乳杆菌分离培养基上有溶钙圈的41株菌株中筛选出5株耐酸、耐人工胃液较强的菌株,经16S rR NA基因测序鉴定,其中3株为乳杆菌,分别命名为植物乳杆菌Lp MT-3、植物乳杆菌Lp MT-5和唾液乳杆菌LsA F-7。在人工胃液中3株菌的耐受力均强于商品化的对照菌株LGG(鼠李糖乳杆菌GG);转入肠液4 h后直至26 h,Lp MT-5存活率基本稳定在45%左右,仅次于LGG。胆盐浓度为0.10%时,3株乳杆菌的耐胆盐能力均强于LGG;胆盐浓度为0.20%时,Lp MT-3和LsA F-7仍能存活。3株乳杆菌均具有抑菌活性,对粪肠球菌的抑制最明显,其次是金黄色葡萄球菌,对大肠杆菌、沙门氏菌的抑制作用较差。3株乳杆菌对胆固醇的清除效力依次为Lp MT-3LpM T-5Ls AF-7;清除率依次为Ls AF-7Lp MT-3LpM T-5。【结论】筛选出3株适应人体胃肠液环境、耐胆盐、抑菌及降胆固醇活力强的乳杆菌,可作为进一步开发新的益生菌产品和保健型酸奶的菌株。     

Occurrence and Growth of Killer Yeasts during Wine Fermentation

Sixteen wine fermentations were examined for the presence of killer yeasts. Killer property and sensitivity to killer action were found in isolates of Saccharomyces cerevisiae but not in isolates of Kloeckera, Candida, Hansenula, and Torulaspora spp. Several killer and killer-sensitive strains of S. cerevisiae were differentiated by colony morphology, and this property was used to monitor their growth kinetics in mixed cultures in grape juice. Killer-sensitive strains died off within 24 to 48 h during mixed-strain fermentation. Killer action was demonstrated at pH 3.0 and pH 3.5 and over the range of 15 to 25°C but depended on the proportion of killer to killer-sensitive cells at the commencement of fermentation. The dominance of killer strains in mixed-strain fermentations was reflected in the production of ethanol, acetic acid, and glycerol.     

Probiotic potential of lactic acid bacteria isolated from chicken gastrointestinal digestive tract

This study was conducted in order to evaluate the probiotic properties of lactic acid bacteria (LAB) isolated from intestinal tract of broilers and Thai indigenous chickens. The major properties, including the gastric juice and bile salts tolerance, starch, protein and lipid digesting capabilities, and the inhibition on certain pathogenic bacteria were investigated. Three-hundred and twenty-two and 226 LAB strains were isolated from ten broilers and eight Thai indigenous chickens, respectively. The gastrointestinal transit tolerance of these 548 isolates was determined by exposing washed cell suspension at 41°C to simulated gastric juice (pH 2.5) containing pepsin (3 mg ml⁻¹), and to simulated small intestinal juice (pH 8.0) in the presence of pancreatin (1 mg ml⁻¹) and 7% fresh chicken bile, mimicking the gastrointestinal environment. The survival of 20 isolates was found after passing through the gastrointestinal conditions. The survival rates of six strains; KT3L20, KT2CR5, KT10L22, KT5S19, KT4S13 and PM1L12 from the sequential study were 43.68, 37.56, 33.84, 32.89, 31.37 and 27.19%, respectively. Twelve isolates exhibited protein digestion on agar plate but no isolates showed the ability to digest starch and lipid. All 20 LAB showed the antimicrobial activity against Salmonella sp., Staphylococcus aureus and Escherichia coli except one strain which did not show the inhibitory activity toward E. coli. Accordingly, five isolates of selected LAB (KT2L24, KT3L20, KT4S13, KT3CE27 and KT8S16) can be classified as the best probiotics and were identified as Enterococcus faecalis, Enterococcus durans, Enterococcus faecium, Pediococcus pentosaceus, and Enterococcus faecium, respectively. The survival rate of microencapsulation of E. durans KT3L20 under simulated small intestine juice after sequential of simulated gastric juice was also investigated. An extrusion technique exhibited a higher survival rate than emulsion technique and free cell, respectively.     

Resistance to Simulated Gastrointestinal Conditions and Adhesion to Mucus as Probiotic Criteria for Bifidobacterium longum Strains

Eight Bifidobacterium longum strains, including reported probiotic strains (commercial and noncommercial), collection strains, and laboratory isolates, were investigated for their ability to adhere to mucin as well as their ability to tolerate acid and bile. Strains could be discriminated based on their sensitivity at pH values of 2.0 to 2.5 and bile concentrations of 0.5% to 2.0%. B. longum NCC 2705, a strain known for its probiotic properties, showed the highest resistance to gastrointestinal conditions, whereas the commercial probiotic strains B. longum BB 536 and SP 07/3 were the least resistant. In parallel, the human isolate B. longum BIF 53 showed the highest adhesion to mucin, whereas the commercial probiotic strains B. longum W 11, BB 536, and SP 07/3 were the least adhesive. The bacterial adhesion to mucin of strains B. longum NCC 2705 and BIF 53 could be reduced by lysozyme, indicating that cell-wall components are involved in the adhesion process. These results showed that there is no obvious link between adhesion and resistance to gastrointestinal conditions and the probiotic status of the studied strains. This calls for a definition of conditions for in vitro tests that better predict the in vivo functionality of probiotic strains.     

In vitro assessment of probiotic properties of <Emphasis Type="Italic">Bacillus</Emphasis> isolated from naturally fermented congee from Inner Mongolia of China

A total of thirty-three strains of Bacillus were isolated from sixteen samples of naturally fermented congee in Inner Mongolia of China and identified by 16S rDNA sequence analysis. Probiotic properties including acid, bile tolerance and artificial gastrointestinal juice resistance as well as inhibition on pathogenic bacteria were used for screening of Bacillus. After the preliminary selection, four strains including Bacillus licheniformis IMAUB1002, Bacillus subtilis IMAUB1011, Bacillus amyloliquefaciens IMAUB1014 and Bacillus amyloliquefaciens IMAUB1034 showed high tolerance to simulated gastric juice at pH 2.0 for 3 h with survival rate all above 92%. And then through gastrointestinal transit, survival rates of these four strains were above 90%. Furthermore, Bacillus licheniformis IMAUB1002 performed well in tolerance to bile salt (0.6%) and inhibitory activity to five food-borne pathogens among four strains of Bacillus. The results suggested that Bacillus licheniformis IMAUB1002 should be considered as a potential probiotics. Further study will be focused on evaluation of these porbiotics properties in vivo and clarification of its other functional properties so as to use it in functional foods production in future.     

The probiotic properties of Lactobacillus buchneri P2

Aims: To isolate new lactobacilli strain with cholesterol‐lowering effect and analyse its probiotic properties and possible mechanisms of cholesterol removal. Methods and Results: The strain with cholesterol‐lowering effect was isolated from pickled juice. The acid and bile tolerance and antimicrobial activity were tested. The free cholalic acid, the cholesterol in supernatant fluid, washing buffer and cell extract, the cholesterol removed by growing, dead and resting cells were quantified. The isolated strain with high cholesterol‐reducing rate of 43·95% was identified as Lactobacillus buchneri (Lact. buchneri) P2. It had acid and bile tolerance and antimicrobial activity. Moreover, it could remove cholesterol via coprecipitating with deconjugated bile salts, assimilating and adsorbing by cells. And the assimilation was considered to be the main reason of cholesterol removal. Conclusions: The isolated Lact. buchneri P2 showed probiotic properties of cholesterol reduction, acid and bile tolerance and antimicrobial activity and could remove cholesterol via different ways. Significance and Impact of the Study: A new strain of Lact. buchneri P2 with efficient cholesterol‐reducing ability was isolated to provide species diversity of lactobacilli for functional dairy products. And the possible mechanism of cholesterol removal by Lact. buchneri was discussed.     

Transgenic wine yeast technology comes of age: is it time for transgenic wine?

Saccharomyces cerevisiae is the main yeast responsible for alcoholic fermentation of grape juice during wine making. This makes wine strains of this species perfect targets for the improvement of wine technology and quality. Progress in winemaking has been achieved through the use of selected yeast strains, as well as genetic improvement of wine yeast strains through the sexual and pararexual cycles, random mutagenesis and genetic engineering. Development of genetically engineered wine yeasts, their potential application, and factors affecting their commercial viability will be discussed in this review.     

Killer phenotype of indigenous yeasts isolated from Argentinian wine cellars and their potential starter cultures for winemaking

Of 31 yeasts, from different surfaces of two cellars from the northwest region of Argentina, 11 expressed killer activity against the sensitive strain Saccharomyces cerevisiae P351. Five of these killer yeasts were identified as S. cerevisiae by phenotypic tests and PCR-RFLP analysis. Two S. cerevisiae killer strains, Cf5 and Cf8, were selected based on their excellent kinetic and enological properties as potential autochthonous mixed starter cultures to be used during wine fermentation. They could dominate the natural microbiota in fermentation vats and keep the typical sensorial characteristics of the wine of this region.     

Identification of industrial yeast strains of Saccharomyces cerevisiae by fatty acid profiles

Summary The fatty acid composition of 20 Saccharomyces cerevisiae strains (laboratory and industrial strains, top and bottom brewery yeasts, distillery and wine yeasts) was determined. Fatty acids with chain lengths of 12 to 28 carbons were separated. It has been found that individual strain groups differ in their fatty acid profiles.     

The inhibitory activity of Lactobacillus spp. isolated from breast milk on gastrointestinal pathogenic bacteria of nosocomial origin

Milk acts as a mean for transporting many essential substances from the mother to the child. In human beings, milk includes several predominant bacteria, such as staphylococci, streptococci, micrococci, lactobacilli, enterococci, lactococci and bifidobacteria. Besides, its intake favors the predominance of bifidobacteria and lactobacilli in the child’s intestinal microbiota. The present work explores the isolation and selection of lactobacilli strains with probiotic potential, focusing in their degree of hydrophobicity and antagonism against important gastrointestinal nosocomial pathogens. 98 lactobacilli were isolated from 48 breast milk samples, with most strains belonging to the obligately homofermentative group (36.7%). 63% of the isolated strains showed a high degree of hydrophobicity when tested on three solvents and were selected for detecting antimicrobial activity against gastrointestinal pathogens, including Escherichia coli, Shigella spp, Pseudomonas spp and Salmonella spp strains. When applying the agar diffusion test, many isolated strains presented inhibitory activity against pathogenic strains. We observed that: Salmonella enteriditis was the most inhibited pathogen, and the strains with the most inhibitory power were AR2 and O1 (both highly hydrophobic lactic acid bacteria), which showed an opposing effect against all nosocomial pathogens tested. Although more in vitro, in vivo or clinical data would be needed before any conclusion on the probiotic properties of the strains can be drawn, our results demonstrate that some of the tested strains may have good probiotic potential for their inclusion in products targeting infants.