In Vitro Probiotic Evaluation of Yeasts from Coconut and Raffia Juices

. Eukaryotic probiotics currently attract a lot of scientific attention, with Saccharomyces cerevisiae and Saccharomyces


Introduction
Probiotics have a long history, dating back over 10 000 years.Fermented foods, such as yogurt, are high in probiotics and are widely consumed worldwide.These days, probiotics are part of various healthy diets, and supplements with probiotic microbes have long established themselves in commercial production [1,2].
Criteria for in vitro assessment of potential probiotics.In 2002, the Food and Agriculture Organization of the United Nations published guidelines for evaluating probiotics in a variety of foods.Currently, the criteria for evaluating probiotic candidates include: low pH tolerance, bile salt tolerance, osmotolerance, phenotypic and genotypic stability, carbohydrate tolerance, etc.
Although the exact mechanism through which probiotics exert their positive benefits remains uncertain, a range of beneficial outcomes derived from probiotics is well documented [5].
Sources of probiotics.Previously, most probiotics ingested by humans came from fermented foods, e.g., dairy products.Eventually, the human body itself became the predominant source, with faces and breast milk serving as the primary providers.Probiotics isolated from human breast milk are mainly of the Lactobacillus genus, while those isolated from feces of healthy human adults and breastfed infants belong to Lactobacilli and Bifidobacterium [6].
Fermented foods with potential probiotics can be of plant or animal origin.All other probiotic organisms are bacterial species, except for Saccharomyces cerevisiae (bakery and brewing), Saccharomyces bayanus (winemaking), and Saccharomyces boulardii.These yeasts have been isolated from a variety of sources, including soy paste, and used as a probiotic in medicine [7,8].
In Middle Eastern countries, fermented foods are abundant sources of lactic acid bacteria.The list includes parboiled dried wheat, garlic, parsley and olives among many others.Non-germinated cereals, such as sorghum and millet grains, are known for their functional properties [9].Traditional non-dairy fermented beverages are also high in probiotics.They are made from millets, legumes, fruits, and vegetables [10,11].Probiotic features of lactic acid bacteria include resistance to pH 3 and 3% bile, as well as antibacterial activity against S. aureus, E. coli, Pseudomonas aeruginosa, and Enterococcus faecalis.Such new developments as paraprobiotics and postbiotics go beyond the current trend of consuming live bacteria in food or as supplements: they imply that bacterial viability alone may not be required for health benefits.This discovery presents a potential opportunity for functional food producers [12].
Coconut and raffia palm juice are widely consumed around the world.In tropical countries such as Nigeria, they are natural refreshing beverages used to quench thirst.In addition to minerals, these fruit drinks contain several local microorganisms.For instance, coconut juice contains Pichia kudriavzevii and Kluyveromyces marxianus while raffia palm juice contains Schizosaccharomyces pombe and Wickherhamomyces anomalus.Raphia rinfera, Raphia hookeri, and Elaeis guineensis are the most common sources of palm wine in Nigeria.Fresh palm wine is widely regarded as a healthy beverage that aids lactation, heals conjunctivitis, and even improves vision [13,14].This delicious drink is popular in southeastern Nigeria, as well as in many tropical countries all over the world, including Asia and South America.
This study featured four yeasts.P. kudriavzevii and K. marxianus were isolated from coconut juice; S. pombe and W. anomalus came from raffia palm juice.They were tested for potential use as probiotics.

Study objects and methods
Yeast strains.Pichia kudriavzevii and Kluyveromyces marxianus (coconut juice) and Schizosaccharomyces pombe and Wickherhamomyces anomalus (raffia palm juice) were obtained from the Microbiology Department of Landmark University, Omu-Aran, Nigeria.They were subjected to four in vitro tests: survival in low and alkaline pH, survival in 30% simulated gastric juice, survival in 1, 2, and 3% bile, and survival in 5, 10, 15, 20, 25, and 30% sugar (glucose) to check osmotolerance.
Culture conditions.This study employed yeast extract peptone dextrose broth and agar as culture media.After purification, the yeasts were counted to obtain 10 9 CFU/mL in sterile phosphate buffered saline, as proposed by Moradi et al. [15].The tests took place within 60 min after the count.
Gastric juice tolerance test.To test the capacity of the yeasts to survive in simulated gastric juice, we modified the procedure described by Lohith & Anu Appaiah and Ragavan & Das [16,17].In brief, the simulated gastric juice was prepared by dispensing 10 mL of phosphate buffered saline (0.9% w/v) into sterile universal tubes (n = 4) and adjusting pH to 2.0 with HCl.After that, we added 0.03 g pepsin into the solution to achieve a concentration of 3 mg/mL.Subsequently, we put 20 µL of overnight cultures (~ 10 9 CFU/mL) of each yeast into the simulated gastric juice to inoculate and incubate them at 37°C for 90 min.Following the incubation, 10 µL simulated gastric juice with yeast cultures was added to 10 mL yeast extract peptone dextrose broth.Each test was performed in triplicates.The optical density (absorbance) values made it possible to determine the viability of the yeasts spectrophotometrically.The test involved the use of a UV/VIS Spectrophotometer, Model AE S80-2S (A&E Lab, UK).After 0, 24, 48, 72, and 96 h of incubation, we measured the absorbance at 660 nm.All the tests were carried out in triplicates, and each value was a mean calculated from all three.
Survival in acid and alkaline environments.The study used the methods developed by Lohith & Anu Appaiah and Ragavan & Das with minimal modifications [16,17].The pH of the yeast extract peptone dextrose broth was adjusted with 1N HCL to 2.0, 3.0, and 5.0 for acidic conditions.For alkaline conditions, the adjustment was carried out using 1N NaOH to bring pH up to 7.5 and 8.0.The samples of pH-adjusted yeast extract peptone dextrose broth (9.9 mL) were dispensed into clean universal bottles.After that, we inoculated 0.1 mL (~ 10 9 CFU/mL) yeasts purified in phosphate buffered saline into 9.9 mL of pH-adjusted broths.The obtained mixes were swirled to homogenate.The absor-bance of each inoculated broth at 660 nm was measured before incubation and repeated the procedure every 24 h for a total of 96 h.All the tests were carried out in triplicates, and the growth and survival of the yeasts were measured from the mean absorbance values recorded for each yeast organism.
Bile tolerance test.We prepared 1, 2, and 3% bile in the yeast extract peptone dextrose broth.Then, we dispensed 0.2 mL (~ 10 9 CFU/mL) of overnight culture in phosphate buffered saline into the broth and mixed.The absorbance of the broth cultures was measured at 660 nm before the incubation and 24, 48, 72, and 96 h after the incubation.All the tests were carried out in triplicates, and each value was a mean calculated from all three.
Osmotolerance test.Glucose concentrations of 5, 10, 15, 20, 25, and 30% (w/v) were prepared in the yeast extract peptone dextrose broth.From each of these stock solutions, we dispensed 19.8 mL into sterile universal tubes, to which was added 0.2 mL (~ 10 9 CFU/mL) of each yeast (in phosphate buffered saline) for subsequent testing.The test included three replicates per yeast.Before incubation, the absorbance of the broth cultures was read at 660 nm.During the incubation, measurement of the absorbance was repeated every 24 h for a total of 96 h and the mean values calculated.

Results and discussion
Gastric juice tolerance test.Three of the four yeasts showed remarkable ability to thrive in the simulated 30% gastric juice at pH 2.0 and 37°C for 96 h.Schizosaccharomyces pombe demonstrated the best results with absorbance value of 2.704 at 96 h.Its concentration exceeded that of Kluyveromyces marxianus by 13% and that of Wickerhamomyces anomalus by 97.7% (p < 0.05).W. anomalus appeared to be the least viable yeast in the 30% gastric juice environment.The samples showed no significant difference (p > 0.05) in viability within the first 24 h.Pichia kudriavzevii, with an absorbance value of 1.984 at 96 h, showed no significant difference (p > 0.05) from S. pombe (2.704) and K. marxianus (2.352) after 96 h.However, its difference from W. anomalus for the same period was significant (p < 0.05) (Fig. 1).
Acid and alkaline tolerance test.All four yeasts exhibited acidic tolerance, with P. kudriavzevii showing evidence of remarkable survival at pH 2.0 and 3.0 for up to 96 h when compared to S. pombe, W. anomalus, and K. marxianus.Notwithstanding, all the yeasts fared better at pH 3.0 than at pH 2.0 with an approximately 89% (1.695/0.185mean OD values) higher growth in pH 3.0 and 2.0, respectively, after 96 h (Figs.2a and b).Similarly, all four yeasts grew better at pH 5.0 than at pH 3.0 and 2.0.For pH 5.0, we recorded a mean increase of 14.05% for all four yeasts after 96 h.K. marxianus and P. kudriavzevii showed better survival results (Fig. 2c).At 96 h, S. pombe and W. anomalus survived best in the alkaline medium, with no discernible difference between growth and survival results in acidic pH 5.0 and alkaline pH 7.5 and 8.0.(Figs.2c, d, e, and f).
Bile tolerance test.S. pombe maintained stable concentration in all three bile solutions for 96 h while other yeasts showed variable concentrations after 48 h (Fig. 3).
Each of the assay periods demonstrated minimal but not appreciable differences in yeast concentrations between 1 and 2% bile and between 2 and 3% bile.
P. kudriavzevii was consistently the most viable species in 1, 2, and 3% bile medium, especially at 48 h.
Osmotolerance test.All four yeasts demonstrated signs of survival (5-30%) in the glucose solutions (Figs.4a-f).P. kudriavzevii, K. marxianus, and W. anomalus reduced in concentration as the glucose concentration increased from 5 to 30%.However, their concentrations increased as the incubation time proceeded from 0 to 96 h.
S. pombe followed the same pattern as the other three yeasts but had a slightly lower concentration as the incubation time increased from 0 to 96 h.
Overall, as the concentration of glucose increased from 5 to 30% after 48 h of incubation, we detected a variable degree of reduction in yeast concentrations, with P. kudriavzevii being the least affected (Fig. 4g).
Such yeast strains as Saccharomyces boulardi are popular in healthcare and food industry for their well documented therapeutic properties, e.g., alleviation of digestive issues.K. marxianus is another highly researched probiotic yeast with a set of established methods of screening and assessing probiotic potential [15].Because low pH is one of the most basic criteria, most in vitro studies recommend selecting probiotic yeast strains that can grow at extremely low pH.All the yeasts tested in this work showed resilience to low pH of 2, 3, and 5 for up to 96 h.P. kudriavzevii performed remarkably well: its ability to thrive in low pH exceeded that of the other three yeasts.In addition, its pH corresponded to the pH range of human stomach, which is 1.5-3.5.This fact qualified P. kudriavzevii as a protic candidate, provided the strain meets other, untested criteria.The pH of human intestine ranges between 6 and 7, and all the yeasts in this study were able to thrive in a comparable environment, which also indicates their probiotic potential.
In a previous study, Moradi et al. compared Saccharomyces cerevisiae with K. marxianus and different strains of P. kudriavzevii [15].They reported that the other yeasts thrived better in acid environments than S. cerevisiae.Our findings imply that P. kudriavzevii and K. marxianus from coconut juice, as well as S. pombe and W. anomalus from raffia juice, may have probiotic properties.
If these yeasts meet all other probiotic criteria that were not investigated in this study, their ability to thrive in low pH environments, gastric juice, bile, and 5-30%   [15].In this respect, our findings are consistent with those mentioned above.
The small intestine and colon contain relatively large quantities of bile salts, which are poisonous to living cells.As a result, the ability of bacteria and yeasts to tolerate bile is now an important criterion for probiotic organisms [19].In the human digestive environment, the optimal bile content ranges from 0.30 to 0.60%.
In this research, all four yeasts isolated from plant sources were able to grow in simulated bile salt concentrations of 1, 2, and 3%, all of which exceeded the optimal concentration in human intestine.Their ability to pass the bile tolerance test suggests that they could be effective as probiotics.
Yeast can use a wide range of carbohydrates, including glucose, to fuel its growth.S. pombe, W. anomalus, K. marxianus, and P. kudriavzevii survived well in all glucose concentrations (5-30%).Other studies identified P. kudriavzevii as an osmotolerant yeast species to be used in bioethanol production [20].In our research, P. kudriavzevii and K. marxianus remained the most stable yeasts in the varied glucose concentrations, which makes them excellent candidates for probiotics.

Conclusion
Bacterial species, such as Lactobacillus and Bifidobacterium, are universally accepted probiotic organisms.Currently, the only probiotic yeast in use is Saccharomyces boulardii.However, yeasts with therapeutic benefits can be found in a variety of fruits and dairy products that people consume on a regular basis in fermented drinks and yogurts.
As observed in this study, Pichia kudriavzevii isolated from coconut juice survived in both acidic and alkaline environments, concentrated gastric juice, 30% pepsin, 1-3% bile, and 5-30% glucose medium.Its survival properties exceeded those demonstrated by Schizosaccharomyces pombe, Wickerhamomyces anomalus, and Kluyveromyces marxianus.P. kudriavzevii showed acid and osmotolerance survival which corresponds with some earlier reports of its usefulness as an ethanologenic yeast strain [21].However, the other three yeasts also exhibited reasonable probiotic potential, particularly S. pombe, which thrived in the bile medium.
Probiotics' microbial viability and metabolic activity must be maintained throughout the production process, i.e., fermentation, which demands further in vitro and in vivo studies [22].In this study, S. pombe, W. anomalus, K. marxianus, and P. kudriavzevii all proved viable in each of the four conditions studied.Presumably, other plants used in national cuisines can offer new sources of eukaryotic probiotic organisms with potential commercial use as part of functional foods.