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Identification of yeast isolates using ITS/5.8S ribosomal DNA and their accession numbers obtained from GenBank.
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This study is about the isolation of yeast from fermented dairy and non-dairy products as well as the characterization of their survival in in vitro digestion conditions and tolerance to bile salts. Promising strains were selected to further investigate their probiotic properties, including cell surface properties (autoaggregation, hydrophobicity a...
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Citations
... Survival in the GIT conditions is promising, but a probiotic is also expected to be able to colonize the GIT, through its adhesive properties, which are influenced by surface hydrophobicity and autoaggregation tendencies [56]. These parameters are considered indirect indicators of the adhesion abilities of the probiotics candidate strains [21]. It is worth noting that studies have considered strains as hydrophobic, when their level of hydrophobicity was found more than 40% [57]. ...
... of the colonic mucosa, and allows prevention of pathogenic infections [21]. Here, percentages above 80% were obtained from the autoaggregation assay, demonstrating that the autoaggregation properties are time-dependent and consistent with those obtained by Fernandez-Pacheco, Arévalo-Villena, Bevilacqua, et al. [58] and Tchamani Piame et al. [56]. ...
Interest in Saccharomyces and non-Saccharomyces yeasts as biotechnological agents is growing worldwide. Here, Kluyveromyces marxianus GBC2 and two Saccharomyces cerevisiae strains FBZ4 and FBK9 were isolated from pomegranate (Punica granatum) and fig (Ficus carica), respectively, and extensively characterized for their probiotic attributes and health benefits. Overall, these strains were found to be γ-hemolytic, non-cytotoxic against Caco-2 cells, and sensitive to therapeutic antifungals. In terms of probiotic characterization, the strains were able to survive at pH 2 and in 1% bile and had high hydrophobicity and self-aggregation properties, which could explain their ability to form biofilm on a polystyrene and adhere to Caco-2 cells. Adhesion rates of 23.52%, 14.05%, and 9.44% were recorded at 37 °C for K. marxianus GBC2, S. cerevisiae FBK9, and S. cerevisiae FBZ4, respectively. Furthermore, biological screening showed a cholesterol assimilation of 54.32% for K. marxianus GBC2 and almost 33% for both Saccharomyces, more than 73% α-amylase inhibition, and good antioxidant potential for all strains; however, only K. marxianus GBC2 showed antibacterial activity against Staphylococcus aureus ATCC 25923. In light of these findings, the strains could be potential candidates for the development of novel functional foods and for probiotic applications.
... The isolates demonstrating both acid and bile tolerance were subjected to a cell-surface hydrophobicity assay, following the method described by Alkalbani et al. [47]. In this experiment, hydrocarbons such as n-hexadecane (RM2238, HiMedia, Mumbai, India) and xylene (61772105001730, Merck, Mumbai, India) were used as solvents. ...
... More than 80% hydrophobicity was considered the threshold for the high hydrophobic nature of the yeast isolates [47]. ...
... The antimicrobial activity was performed following the protocol described by Alkalbani et al. [47], with slight modifications. The agar-well diffusion method was used, and the target pathogens used were E. coli, S. enterica, S. aureus, and B. cereus. ...
Introduction
In the present study, we focused on the screening of the potential probiotic yeasts isolated from two Indian fermented cereal-based foods, viz., idli and selroti. A total of 260 yeast isolates were isolated from the batters of idli (140 isolates) and selroti (120 isolates).
Method
Preliminary screening of basic probiotic traits such as tolerance to low pH, bile, and cell surface attachment was checked for the selection of potential probiotic yeasts from total isolates. Finally, 8 yeast isolates were selected for further in-depth assessment by in vitro and genetic screening, which included Clavispora lusitaniae AIY-4, Wickerhamomyces anomalus MIY-30, Pichia kudriavzevii BIY-8 (from idli), Yarrowia lipolytica SGLY-15, Y. lipolytica SGLY-21, Candida parapsilosis SPRY-17, C. parapsilosis SBRY-12, and Kodamaea ohmeri SBRY-25 (from selroti).
Results
A principal component analysis (PCA) biplot was designed to evaluate the differences and similarities amongst the yeast strains, and two clusters were formed using the paired group (UPGMA) algorithm and Euclidean similarity index. Cluster one was comprised of Cl. lusitaniae AIY- 4, W. anomalus MIY-30, C. parapsilosis SBRY-12, and P. kudriavzevii BIY-8, and another cluster included C. parapsilosis SBRY-12 and Y. lipolytica SGLY-21.
Conclusion
Hence, based on statistical analysis for probiotic in vitro and genetic screening, Wickerhamomyces anomalus MIY-30 (idli) and Kodamaea ohmeri SBRY-25 (selroti) were selected as the most potential probiotic strains.
... Though hydrophobicity was lower for Pichia kudraivzevii, auto aggregation and coaggregation were higher than for Kluyveromyces marxianus. Cell surface hydrophobicity values in similar range has been reported for Pichia kudraivzevii isolated from traditional fermented dairy product, Shanklish by Alkalbani et al. (2022) [4] . Auto aggregation and CSH values obtained for Pichia kudraivzevii is in agreement with Pious et al. (2024) [21] . ...
... Though hydrophobicity was lower for Pichia kudraivzevii, auto aggregation and coaggregation were higher than for Kluyveromyces marxianus. Cell surface hydrophobicity values in similar range has been reported for Pichia kudraivzevii isolated from traditional fermented dairy product, Shanklish by Alkalbani et al. (2022) [4] . Auto aggregation and CSH values obtained for Pichia kudraivzevii is in agreement with Pious et al. (2024) [21] . ...
... Several studies have shown that novel yeasts can withstand gastrointestinal conditions, tolerate osmotic stress, adhere to epithelial cells and exhibit inhibitory effects against pathogens [35,40,45]. Consequently, yeast probiotics have gained importance in enhancing nutrition and health, originally used as a feed additive, due to their richness in proteins, organic acids, fibre and vitamin B [4]. Probiotic yeasts have found numerous applications in various fields [4,7,53]. ...
... Consequently, yeast probiotics have gained importance in enhancing nutrition and health, originally used as a feed additive, due to their richness in proteins, organic acids, fibre and vitamin B [4]. Probiotic yeasts have found numerous applications in various fields [4,7,53]. ...
Though numerous bacteria have been used as probiotics by industries, at present, Saccharomyces boulardii and Saccharomyces cerevesiae are the only yeast probiotics which are industrially exploited. In view of this, yeast probiotics were isolated from traditional fermented foods and products collected from different parts of Karnataka, India. In this work, we have studied the probiotic attributes of ten yeast isolates isolated from different traditionally fermented foods and products. About 73 yeast isolates were initially isolated by serially diluting the samples and plating on the Potato Dextrose Agar (PDA) plates. The spot assay was performed to screen the yeast isolates against test pathogens. Ten isolates were selected based on their significant antimicrobial activity. These isolates were subjected to biochemical characterization and then assessed for probiotic properties. The ability of probiotics to endure at pH 2.0 and tolerate bile conditions (0.3%) are crucial attributes for the survival in the gastrointestinal tract (GIT). The yeast isolates were also assessed for cell surface hydrophobicity and autoaggregation capabilities. All the ten isolates showed endurance in GIT tract and > 40% of adhesion. The study further examined cholesterol assimilation, antioxidant and antagonistic properties of the yeasts. Subsequently, the molecular characterization was performed by isolating the DNA of yeast isolates by phenol–chloroform method and identified molecularly through sequencing of D1/D2 regions. The isolates tested negative for gelatinase and DNase and were non-haemolytic indicating they are safe for consumption. Among ten isolates, Meyerozyma guillermondii (MYSY23), Meyerozyma caribbica (MYSY22) and Meyerozyma guillermondii (MYSY19) showed significant results for all probiotic and functional characteristics with greater than 65% survivability in GIT tract and > 50% of antagonistic activity against test pathogens and also proved non-cytotoxic and safe. These findings suggest that yeasts with significant probiotic attributes could be recommended for various probiotic application.
... The toxin from P. kudriavzevii RY55 demonstrated antimicrobial activity against common pathogens such as Escherichia coli, Enterococcus faecalis, Klebsiella sp., Staphylococcus aureus, Pseudomonas aeruginosa, and Pseudomonas alcaligenes, showing promise as a metabiotic for treating various intestinal diseases. Consequently, attention has turned towards yeast of the genus Pichia in probiotics [23,36,37]. ...
... The study by Alkalbani et al. [36] aimed to isolate yeast from fermented milk and nondairy products. Twelve yeast isolates demonstrated robust survival under acidic conditions, cholesterol reduction abilities, bile salt hydrolysis, heat stability, hydrophobicity, strong coaggregation, autoaggregation after 24 h, and potent antimicrobial activity. ...
The emergence of antibiotic-resistant pathogens in clinical settings has intensified the search for new probiotic strains with both health benefits and technological utility. This study aims to identify and characterize promising antimicrobial cultures derived from milk and dairy products, capable of inhibiting opportunistic pathogens. The samples of dairy products were collected from various markets across Kazakhstan. Microorganisms isolated from these samples underwent identification through 16S rRNA and ITS gene sequencing, using the BLAST algorithm. Their antimicrobial activity was assessed using the delayed antagonism method against pathogenic microorganisms including E. coli, S. aureus, Pseudomonas sp., Candida sp., and B. subtilis. Additionally, the isolates were evaluated for resistance to environmental stress factors such as temperature, pH, salt, ethanol, glucose, and peroxide. From 24 distinct samples, 33 isolates were purified, with 15 demonstrating high viability (108–109 CFU/mL) and stress resistance. Notably, Lacticaseibacillus casei AK and Enterococcus faecium KS exhibited resistance to all tested stress conditions. Antimicrobial screening revealed strong activity by strains LP, LB, and S-2 against multiple pathogens. Genotyping and carbohydrate fermentation tests identified these effective isolates as belonging to the genera Lactobacillus, Lactococcus, Enterococcus, Lactiplantibacillus, Streptococcus, and the yeast genus Pichia. This study underscores the industrial and health potential of the identified microorganisms. Prominent among the strains, Lactiplantibacillus pentosus LP, Lacticaseibacillus casei AK, Lactiplantibacillus argentoratensis LB, Lactiplantibacillus plantarum S-2, and Enterococcus faecium KS have been recognized as potent probiotics. These strains exhibit broad-spectrum antimicrobial activity coupled with robust stress tolerance, making them suitable candidates for probiotic applications.
... Traditionally, probiotic research has primarily focused on bacteria, such as Lactobacillus and Bifidobacterium species. However, recent studies have highlighted the diverse capabilities and benefits offered by yeast as probiotics such as Saccharomyces, Kluyveromyces, Candida and Pitchia (Alkalbani et al. 2022;Azhar and Munaim 2023;Staniszewski and Kordowska-Wiater 2021). Yeast strains possess distinct characteristics, including robust resistance to harsh environmental conditions, the ability to survive in the gastrointestinal tract, and the capacity to interact with the gut microbiota. ...
... The yeast species Pichia kudriavzevii, Candida spp., and Diutina mesorugosa have been the subject of extensive research due to their potential probiotic attributes and their presence in traditional fermented foods. Pichia kudriavzevii, in particular, has been studied for its probiotic potential and its ability to thrive in low pH conditions similar to the human gut (Alkalbani et al. 2022;Chelliah et al. 2016). Similarly, Candida species have also displayed probiotic attributes and are being investigated for commercial applications (Kunyeit, Anu-Appaiah, and Rao 2020). ...
Probiotic microorganisms, offering health benefits when consumed in sufficient quantities, are gaining recognition for their potential role in promoting wellness. This study focuses on isolating and characterising potential probiotic yeast strains sourced from fermented food products. This research evaluates the gastrointestinal tolerance and antimicrobial activity of isolated yeast strains, with the potential application in probiotic supplements and functional foods. Yeast strains were isolated from fermented food sources and identified using morphological analysis, PCR, gene sequencing, and genetic identification. Gastrointestinal tolerance was assessed through simulated gastric fluid (SGF) exposure, and antimicrobial activity was tested against foodborne pathogens. Six yeast strains (Diutina mesorugosa, Pichia kudriavzevii, Candida mesorugosa, Candida sp) were identified. They exhibited varying resistance to low pH in SGF, suggesting survivability in the stomach. Some strains selectively inhibited specific Gram-negative pathogens like Pseudomonas aeruginosa and Salmonella sp. These findings suggest the isolated yeast strains may serve as probiotics, promoting digestive health and food safety. They are potentially used in probiotic supplements and functional foods, promising improved overall well-being.
... In this research, studies showed that T. delbrueckii could survive in acid and bile environments, allowing yeast cells to be calculated in a numerical value in virtual environments. In the same line with Alkalbani et al. who examined various yeast isolates including T. delbrueckii, which was capable to breaking down bile salts and dramatically lowering cholesterol [45]. Besides, Diguță et al. [46] illustrated the capability of yeast to tolerate various temperatures, sodium chloride levels, and various pH levels. ...
Purpose Yeasts are gaining attention as potential emerging tools for enhancing the dietary benefits of food attributes and for preventing food spoilage because of their anti-microbial properties.
Methods In this study, both Torulaspora delbrueckii (T. delbrueckii) as a prospective probiotic and bananas as a prebiotic were used to investigate their potential roles in modulating the lipid content and bacterial number in the feces of examined rats.
Results Milk has been used to isolate a yeast stain that has been identified using conventional and molecular tools as T. delbrueckii. The isolated yeast showed promising results upon testing for acid and bile tolerance. T. delbrueckii also had the capacity to thrive on simulating stomach and intestinal fluids. According to an animal feeding experiment, rats fed T. delbrueckii developed and acquired mass in a regular manner. Consuming T. delbrueckii also dramatically lowers LDL, cholesterol, and triglyceride levels while dramatically raising HDL levels. Consuming both T. delbrueckii and bananas along with regular animals’ diet considerably reduced the amount of coliforms and Staphylococcus sp. in the rats’ excrement. Conclusions These findings suggested a potential function for T. delbrueckii in treating hypocholesteremia and controlling the bacterial flora of the intestine, which can then be used widely after more confirmation of the outcomes.
... Adhesion to HT-29, chicken crop epithelial cells and human buccal epithelial According to the findings of Jaiswal et al. (2022) a strain of Pediococcus produces considerable levels of butyrate along with other short-chain fatty acids and has an anti-proliferative effect on colonic cancer cells HT-29 and SW-480 (Joishy et al., 2019). In a study of Alkalbani et al. (2022) the yeasts collected from fermented dairy products and non-dairy products could attach to the HT-29 cell line with an average of 6.3 Log10 CFU/mL after 2 h. According to the research by Hidalgo et al. there were strain-dependent differences in the amount of adhesion to chicken crop epithelial cells: L. crispatus CRL 1453 demonstrated the highest levels of adhesion (>19%), while Lig. ...
The current study aims to evaluate and characterize the probiotic andantidiabetic properties of lactic acid bacteria (LAB) obtained from milk and other dairy-based products. The strains were tested physiologically, biochemically, and molecularly. Based on biochemical tests and 16S rRNA gene amplification and sequencing, all three isolates RAMULAB18, RAMULAB19, and RAMULAB53 were identified as Lacticaseibacillus paracasei with homology similarity of more than 98%. The inhibitory potential of each isolate against carbohydrate hydrolysis enzymes (α-amylase and α-glucosidase) was assessed using three different preparations of RAMULAB (RL) isolates: the supernatant (RL-CS), intact cells (RL-IC), and cell-free extraction (RL-CE). Additionally, the isolate was evaluated for its antioxidant activity against free radicals (DPPH and ABTS). The strain’s RL-CS, RL-CE, and RL-IC inhibited α-amylase (17.25 to 55.42%), α-glucosidase (15.08–59.55%), DPPH (56.42–87.45%), and ABTS (46.35–78.45%) enzymes differently. With the highest survival rate (>98%) toward tolerance to gastrointestinal conditions, hydrophobicity (>42.18%), aggregation (>74.21%), as well as attachment to an individual’s colorectal cancer cell line (HT-29) (>64.98%), human buccal and chicken crop epithelial cells, all three isolates exhibited extensive results. All three isolates exhibited high resistance toward antibiotics (methicillin, kanamycin, cefixime, and vancomycin), and other assays such as antibacterial, DNase, hemolytic, and gelatinase were performed for safety assessment. Results suggest that the LAB described are valuable candidates for their significant health benefits and that they can also be utilized as a beginning or bio-preservative tradition in the food, agriculture, and pharmaceutical sectors. The LAB isolates are excellent in vitro probiotic applicants and yet additional in vivo testing is required.
... [38] In order to live in bile salt stress environments, probiotic yeasts employ a variety of strategies, including the production of organic molecules such as glycerol and trehalose. [39] In the current investigation, isolates' hydrophobicity percentages of chloroform, ethyl acetate, and xylene ranged from 33% to 51%, 42% to 82%, and 16% to 41%, respectively (Figure 2a). The isolates with ethyl acetate had a percentage of hydrophobicity that fulfilled the minimum standard requirement for using probiotic sources. ...
... [42] The solvent with the lowest percentage of hydrophobicity among the others was xylene (Figure 2a). This result was consistent with the finding of Alkalbani et al. [39] According to Pereira et al., [30] xylene was the solvent where isolates had the least adhesion ability. The degree to which yeast and lactic acid bacteria isolates adhered to the solvent varied. ...
... Increasing the incubation time resulted in yeast and LAB isolates being more capable of self-aggregation (Figure 2b). This result was consistent with the finding of Alkalbani et al., [39] who reported that yeasts' capacity for auto-aggregation increased as incubation time rose. ...
Ersho is an undefined starter culture used for Ethiopian injera production, and it is a good source of yeasts and lactic acid bacteria (LAB). However, few researchers have been done on the characterization of yeasts and LAB from ersho samples as a source of probiotics. The objective of the current work was to isolate and screen yeasts and LAB from ersho samples and in vitro characterization of their probiotic properties. An experimental research design was used. A total of 55 ersho samples were collected. Based on the ersho samples, 220 yeast colonies and 220 LAB colonies were isolated and purified. For the probiotic characterization, three yeasts (4A, 14D, 54A) and three LAB (1A, 13E, and 55A) were chosen. The isolates 4A, 14D, 54A, 1A, 13E, and 55A were identified as Saccharomyces cerevisiae KRSAN1, Saccharomyces cerevisiae JYC2577, Kazachstania humilis, Enterococcus lactis, Lactobacillus paracasei, and Lactobacillus species, respectively. All the isolates showed positive probiotic characteristics. They had very good acid resistance (percentage survival > 80%), high bile salt resistance (percentage survival > 140%), high cell surface hydrophobicity (>40%), high levels of auto-aggregation (>40%), variable co-aggregation with pathogens (ranged between 42% and 72%), variable antimicrobial activity (clear halo zone ranged from 4.0 to 31 mm), and variable antibiotic resistance responses. The results indicated that the ersho samples can be used as yeast and LAB sources for developing food and probiotic starter cultures.
... Saccharomyces cerevisiae and Pichia kudriavzevii, with accession numbers OK441070 and OK441073, respectively, were previously isolated from traditional fermented food sources and identified as potential probiotic yeasts [32]. Both yeasts were subcultured in yeast extract-peptone-dextrose (YPD) broth and stored in glycerol stocks (50% v/v) at −80 • C. YPD broth (Himedia Laboratories Pvt ltd, Nashik, India) was used to culture S. cerevisiae and P. kudriavzevii, separately, before being used for fermentation. ...
Utilizing agricultural byproducts, such as date pulp residue (DPR), can contribute significantly towards achieving sustainable food production. This paper aimed to investigate the physicochemical properties of DPR and evaluate the health benefits of nonfermented and fermented DPR samples both pre- and post-digestion (bioaccessible portions). Additionally, it aimed to analyze the carbohydrates and untargeted metabolites in the bioaccessible portions. Fermentation of DPR by Saccharomyces cerevisiae and Pichia kudriavzevii resulted in an abundance of malic acid (over 2400 mg/100 g) and the identification of 42 volatiles, with different degrees of predominance observed in the samples. Twenty phenolics were determined by UPLC in fermented DPR, with (-)-epicatechin, tyrosol, and gallic acid being the most abundant. Bioaccessibility studies revealed that fermented DPR samples retained at least ~44% of α-glucosidase inhibition and exhibited improved α-amylase inhibition compared to nonfermented and undigested samples. In vitro cytotoxicity assays showed a more potent inhibitory effect of fermented DPR against MCF-7 and Caco2 cell lines (average inhibition of 55% and 74.4% for the two types of fermented samples) compared to nonfermented DPR. The untargeted metabolomics analysis identified C5-branched dibasic acid metabolism as the most prominent pathway, with four metabolites identified. Furthermore, the analysis of bioaccessible carbohydrate metabolites in the fermented DPR using LC-QTOF showed the presence of a group of phytochemicals, including three terpenoid metabolites.
