Isolation and Screening for Non-Conventional Yeast with Bakery Potentials

Isolation and Screening for Non-Conventional Yeast with Bakery Potentials

Eleanya, Lilian Chinyere*¹, Igboanugo, Edwin Uchechukwu², Dibua, Nwamaka Anthonia¹, Anagor, Ifeoma Sandra³, Ezeumeh, Eucharia Nkiruka¹, Chukwura, E. Nnamdi¹ and Okpalla, Jude¹

¹Chukwuemeka Odumegwu Ojukwu University, Uli, Anambra State Nigeria

²Legacy University, Okija, Anambra State, Nigeria

³Nnamdi Azikiwe University, Nnewi Campus, Nigeria

DOI: https://doi.org/10.51584/IJRIAS.2025.10020012

Received: 27 January 2025; Accepted: 01 February 2025; Published: 05 March 2025

ABSTRACT

The aim of this work was to isolate and screen for non-conventional yeast with bakery potentials. Thirty five isolates obtained from palm wine were screened. They were tested for sugar utilization, hydrogen sulphide production, urea hydrolysis and flocculation ability. Selected isolates were characterized using molecular methods. The isolate was subjected further to study its optimum growth conditions as well as for stress tolerance; Sodium chloride, ethanol and hyperosmotic tolerance. Result shows that the best among the selected isolates which was Yarrowia phangngensis (YP) utililized all the sugars, produced no hydrogen sulphide and exhibited high flocculation. Optimum growth temperature, pH and time were 30⁰C, pH 5 and 72h respectively. YP adapted to the various stress conditions. This study shows that Yarrowia phangngensis which was isolated from palm wine could be considered for use in the bakeries because it exhibited good bakery characteristics.

INTRODUCTION

Bakery products are good source of minerals, protein, and carbohydrates in addition to energy. Because of its extensive use and appeal, research on baked foods is of interest (Ibrahim et al., 2019). Numerous studies have been done on the optimisation of bakery goods, which entails enhancing their functionality, flavour, and quality. Functional food can be created by combining cutting-edge food technology with nutritional sciences. Foods with extra elements that support, function, and preserve human health are referred to as fuctional foods. Around the world, bread is a popular food (Ibrahim et al., 2019). It is a common component of many different international cuisines. The quality of bread is influenced by the type of flour and yeast used for its production (Manuel et al., 2021).

Centuries ago, bakery industry boosted with the discovery of incorporating yeast to leaven bread. Those year’s bakers used brewer’s yeast, though the performance was poor. Later, special strains of Saccharomyces cerevisiae with sort bakery potentials were isolated and it brought positive change (Olowonibii, 2017). Fermented beverages are well known to harbor heterogeneous microfloras that invariably include yeasts. In Nigeria, especially the Southern part, palm wine, the fermented sap of oil palm tree (Elaeis guineensis) is a popular beverage. The microbiology has been well studied and review by various researchers (Olowonibii, 2017).

Saccharomycescerevisiae, the conventional baker’s yeast has been in use for ages in the industry. This yeast increases dough by fermentation of simple sugars and CO₂   production which is its primary role in baking. Other secondary roles of a baker’s yeast, such as formation higher alcohols, aromatic esters, fatty acids, and other organic compounds which gives off aromatic and sensorial properties of bread are satisfied (Zhou et al., 2021). Yeasts encounter lots of stress during preparation of biomass and fermentation such as osmotic stress, thermal stress, salinity stress, oxidative stress, air-drying stress, freezing and thawing stress, ethanol stress, and others. However, the conventional baker’s yeast is sensitive to many of these baking-associated stresses; it has a streamlined carbon substrate utilization base, and has poor aromatic profiles as its major drawbacks (Zhou et al., 2021).

Several researches have shown that some of the non-conventional yeasts also known as wild yeast produces better results than the conventional baker’s yeast. Aslankooli et al. (2016) tested ten non-conventional yeasts for bread fermentation, including two Saccharomyces species that are not currently used in bread making and 8 non-Saccharomyces strains. The results show that Torulaspora delbrueckii and Saccharomyces bayanuspossessed good properties. Aslankooli et al. (2016) and many others have been able to prove the usefulness of non-covetional yeasts in the bakery industry. Reports of many researchers (Aslankoohi et al., 2016; Olowonibii, 2017; Tika et al., 2017; Umeh et al., 2019: Umeh et al., 2022) are also evidences that these yeasts are obtainable from local beverages and fruits could be potential sources leavening agents for bakery industry. The aim of this work was to isolate and screen for non-conventional yeast with bakery potentials.

MATERIALS AND METHODS

Sources and Collection of Samples

Fresh wine samples from raffia palm and oil palm (Elaensis guineensis) were bought from palmwine tappers. Five palm wine samples were purchased from each of the three senatorial zones in Anambra State, Nigeria: Mgbakwu and Ugbenu (Anambra central), Atani and Igbariam (Anambra north), and Ihiala and Umunze (Anambra south). Within three hours of collection, the palm wine samples were transported in an ice-packed container to the Chukwuemeka Odumegwu University Microbiology laboratory in Uli for analysis.

Isolation of Yeasts

Following a series of dilutions of the palm wine samples, an aliquot (0.1 mL) of the sample was spread uniformly using a sterile glass rod onto yeast extract peptone dextrose agar plates (YEPDA) enriched with chloramphenicol. Moreover, plates were incubated for 30 hours according to Nnodim et al.’s (2021) instructions. A variety of colonies were selected at random and sub-cultured on YEPDA plates to obtain pure culture and as well stored on agar slant for further analysis.

Screening for Bakery Yeast Potentials

The potential of each selected isolate was examined for its ability to produce ferment hydrogen sulphide, ferment sugar, hydrolyse urea, and degree of flocculation were tested.

Sugar fermentation test

The methodology for this experiment was the same as that which Sonia et al. (2019) outlined. The ability of the isolates to ferment lactose, glucose, sucrose, maltose, and fructose was assessed. Test tubes with an inverted Durham tube, peptone water, one gramme of sugar, and a drop of bromocresol purple indicator were used to inoculate the isolates. They were incubated for 48 hours at 30⁰C. The indicator’s colour changed from purple to yellow, or not at all, and the gas trapped in the Durham tube was released and trapped. The presence of gas was interpreted as proof of a reasonably high rate of fermentative activity.

Urea hydrolysis 

This test was conducted using the methodology outlined by Olowoniibi (2017). 250 millilitres of distilled water were combined with 9.68 grammes of urea broth (difco), and the mixture was filtered and sterilised to create urea broth. Test tubes that were sterile and empty were carefully filled with 0.5 ml of the solution aseptically. After being flame-sterilized, each isolate was placed into its allotted tube and incubated for twenty-four hours at 37 degrees Celsius. We looked for a change in colour from yellow to a bright pink in the soup.

Hydrogen sulfide production test

According to Tseganye et al. (2018), Bacto bismuth sulfite agar dehydrated – BSA (difco) was used to select the H₂S generating yeast isolates. Using a hot air oven, every glassware was adequately sterilised for one hour at 160 ⁰C. A mixture of 500 millilitres of distilled water and twenty grammes of BSA were dissolved. To dissolve the agar, the medium was gradually heated and stirred often until it began to boil and simmer for 30 seconds. After 15 minutes of sterilisation at 121°C, the medium was cooled to 50°C and then transferred into plates. The medium was allowed to harden by partially covering the dish. After being added, the yeast was kept at 30⁰C for 48 hours in order to proliferate. While the H₂S producing strains produced colonies that ranged in colour from light brown to black, contingent on production intensity, the non-sulfide producing bacteria produced white colonies.

Flocculation test

The test was conducted in accordance with Kumari et al.’s (2019) instructions. For 72 hours, the yeast isolates were incubated at 30 degrees Celsius in 5 millilitres of sterile yeast broth. To measure the amount of flocculation, the tubes were agitated after incubation. Following agitation, the yeast sediment’s adherence to the test tube was visually inspected and recorded, and the culture supernatant was carefully separated.

Characterization of Selected Isolates

Two of the best-performing isolates were selected for characterisation based on the results of the baking yeast screening tests. They were characterized using molecular method.

Molecular characterization

Genomic DNA was extracted from the samples using the Quick-DNA^TM bacterial/fungal kit  (Zymo Research, Catalogue No. D6005). The ITS target region was amplified using OneTaq Quick load  2X  Master Mix (NEB, CATALOGUE No. M0486) with the primers presented in Table 1. The PCR product were run on a gel and EXOSAP method was used for enzymatic clean up. The extracted fragments were sequenced in the forward and reverse direction (Nimagen, BrilliantDye^TM Terminator Cycle Sequencing kit V3.I, BRD3-100/1000) and purified fragments were analyzed on the ABI 3500xl Genetic analyzer (Applied Biosystems, ThermoFisher Scientific) for each reaction for every sample, as listed in section 1. DNASTAR was used to analyze the ab1 files generated by the ABI 3500XL Genetic Analyzer and results were obtained by a BLAST search (NCBI).

Optimum growth conditions of selected isolates

It was projected that the isolates (5 and 12) that were chosen were Yarrowia phangngensis. Isolate 5 was selected to have its culture conditions optimised based on the % ID of the isolates.

Optimum time

For this investigation, the approach outlined by Tika et al. (2017) was used. By plating the yeast isolates onto YEPD broth and incubating them at 300C for 24, 48, 72, and 96 hours, the ability of the yeast to proliferate at different time intervals was investigated. Using a spectrophotometer, growth was assessed at optical density 600. The culture media served as a placeholder.

Optimum temperature

For this investigation, the approach outlined by Tika et al. (2017) was used. By inoculating the yeast isolates onto YEPD broth and growing them at four distinct temperatures (24, 28, 30, and 370C) for 72 hours, the ability of the yeast to adapt to varying temperatures was investigated. The outcome was read as previously stated.

Optimum pH

For this investigation, the approach outlined by Tika et al. (2017) was used. By inoculating the yeast isolates onto YEPD broth and incubating at four various pH values (3, 5, 7, and 10) at 30 0C for 72 hours, the capacity of the yeast to grow at varied pH values was investigated. The outcome was read as previously stated.

Examination of the Selected Yeast Isolate’s Tolerance to Stress

In contrast to commercial baker’s yeast, the yeast isolate chosen based on its baking potentials underwent the following stress tolerance tests:

Test for ethanol tolerance

It was shown that the yeast could grow at greater ethanol concentrations, as reported by Tseganye et al. (2018). The yeast isolates were cultured on YEPD broth with varying ethanol concentrations (9%, 11%, and 15% (v/v), respectively, and were incubated for 72 hours at 30°C. The outcome was read as previously stated.

Test for hyper-osmotic tolerance

The test was conducted in accordance with Tika et al. (2017) instructions. For 72 hours, the yeast isolates were incubated at 30°C on YEPD broth that contained 25, 35, and 45% dextrose. It was measured how the isolates’ cell density responded to the concentration of dextrose. The outcome was read as previously stated.

Tolerance test for sodium chloride (Nacl)

The sodium chloride tolerance test was conducted using the Liu et al. (2023) method. The isolates of yeast were cultivated on YEPD broth that contained varying amounts of sodium chloride (2, 3, and 5%) and were incubated for seventy-two hours at 30°C. It was determined how the isolates’ cell density responded to the salt concentration. The outcome was read as previously stated.

Data Analysis

The statistical methods applied in this work are correlation analysis performed using SPSS version 22.

RESULTS

Screening for baking yeast potentials

All fungi isolates were screened for baking yeast potentials. They include; sugar fermentation test, H₂S production, degree of flocculation and urea hydrolysis. The isolates produced varying results as expressed in Table 1. Isolates 5 and 12 utilized almost all the sugars, produced no H₂S within the period of 48 h incubation and show reasonable degree of flocculation.

Characterization of selected isolate

Isolates 5 and 12 were chosen for further analysis. Molecular characteristics of the selected isolates are shown in Table 2. Both were identified as Yarrowiaphangngensis.

Table 1: Sugar fermentation test, H₂S production, Degree of flocculation and Urea hydrolysis

– = Not utilized; += low; ++=moderate ; +++=intense ++++= more intense

Table 2: Molecular identification of  selected yeast isolates

Optimization of growth conditions

After molecular characterization it was observed that isolates 5 and 12 were same organism (Yarrowia phangngaensis). Further analysis was carried out using isolate 5 against control (commercial yeast). The Commercial yeast is denoted as CY while Yarrowia phangngaensisis denoted as YP. The optimum growth temperature of the test organisms is 28⁰C and 30⁰C for CY and YP respectively as shown in figure 3. Statistically, there was weak negative correlation between growth density and temperature. As the temperature increased growth density decreased.

Figure 1 show the optimum growth time of the isolates. Both organisms grew optimally after 72 h. The optical density of the broth culture for both isolates rose 24 h to 72 h and a decline was observed after 72 h through 96 h. There was weak positive correlation between the two variables. Increase in growth density was proportional with increase in time.

Figure 2 shows the optimum pH for growth of the isolates. Both organisms grew optimally at pH 5. CY adapted better to pH 5 than YP with optical densities of 0.9 and 0.7 respectively. Statistically, there was weak positive correlation between the variables.

Figure 3 shows the optimum time for growth of the isolates. Optimum growth was observed in both isolates after 72 h.

Figure 1: Optimum growth temperature of YP and CY

Figure 2: Optimum growth pH of YP and CY

Figure 3: Optimum growth time of YP and CY

Stress tolerance of selected isolate

Figure 4 shows the Sodium chloride tolerance of CY and YP. Both tolerated all the salt concentrations they were exposed to, but grew optimally at the least salt concentration of 2%. Their tolerance decreased with increase in salt concentration. YP showed higher tolerance to salt than CY. Statistically, there is very strong negative correlation between salt concentration and growth density.

Figure 5 shows the ethanol tolerance of CY and YP. Both tolerated all the ethanol concentrations they were exposed to, but optimally growth was observed at 10% concentration. Their tolerance increased from 5% to 10% and fall in optical density was observed at 15% concentration. YP has higher tolerance to ethanol than CY. Statistically, there was no correlation between the variables, this result showed there was no correlation between ethanol concentration and growth density.

Figure 6 shows the hyper-osmotic tolerance of CY and YP. Both tolerated all the glucose concentrations they were exposed to. YP showed optimum growth at 45% glucose concentration, optical density increased with increase in glucose concentration. CY showed optimum growth at 45% glucose concentration, optical density increased from 25% to 45% glucose concentration. YP tolerated higher glucose concentration than CY. CY showed weak correlation while YP showed very strong positive correlation. Growth tends to increase with increase in glucose concentration.

Figure 4: Nacl tolerance of YP and CY

Figure 5: Ethanol tolerance of YP and CY

Figure 6: Hyper-osmotic tolerance of YP and CY

DISCUSSION

The sugary juice of palm trees is fermented by the impetuous yeast-lactic acid combination to create palm wine, a whitish, sparkling alcoholic beverage. When the juice of Arecaceae tropical plants is fermented, palm wine is created. It is widely produced and consumed in the southeast region of Nigeria. It is full of vitamins, proteins, carbohydrates, and amino acids—all nutritionally necessary components. The native flora of palm wine is primarily influenced by the palm tree species and its geographical location (Djeni et al., 2020). These factors make this wine an authentic medium for the growth of a group of microbes, whose growth causes a change in the physicochemical conditions of the wine, resulting in rivalry and successions of organisms (Oti et al., 2021). Das and Tamang (2021) state that one of the variables that may affect the microbial community in palm wine is the sugar profile.

Samples of palm wine were checked for baker’s yeast in the process. They were plated on YEPD agar for 48 hours after being serially diluted. Yeast colonies were seen on the agar following incubation. The potentials of each isolate for bakery yeast, such as sugar fermentation, urease hydrolysis, H₂S generation, and flocculation ability, were screened for. In this investigation, 35 isolates were evaluated for their potential as baking yeast; Table 4 presents the varied degrees of capacity for each isolate.

Palm wine has a high (10–12%) sugar content. The yeasts grow more easily at this high sugar content. The test isolate’s fermentative ability was assessed using the yeast fermentative capacity test. When there is a sugar source and a suitable environment, yeasts initiate fermentation. The primary purpose of sugar is to nourish yeast. Because the different sugars utilised in the tests are the same sugars found in dough, the yeast’s capacity to ferment them will demonstrate that it has the different enzymes needed to ferment these sugars. The milling process created the amylase enzyme, which is present in the flour’s starch. The enzyme in flour is triggered when it comes into contact with water, converting starch to maltose. Then the yeasts’ enzymes start to work. Maltase, for example, breaks down maltose into simple sugars like glucose, which is then processed by zymase to produce carbon dioxide and alcohol. However, if table sugar (sucrose) is added to the dough, the yeast produces the enzyme invertase, which breaks the sugar down into glucose and fructose, and then into carbon dioxide and alcohol (Ezemba et al., 2022). Table 4 illustrates the high fermentative ability and gas production of many of the isolates obtained in this experiment. Isolates 5, 12, and KP4 were selected in light of this. They created a respectable amount of gas and were able to use all of the sugar, as evidenced by a shift in colour from purple to yellow. Within the test tube, the gas was contained in an inverted Durham tube.

After 48 hours, every isolate aside from 5 and 12 generated H₂S, although KP4 only produced modest amounts. The commercial yeast obtained from the market, used as a control, also produced H2S in a positive manner. KP4 displayed minimal dark colouration, however isolates 5 and 12 did not. The top two of these three isolates were chosen for comparison with commercial yeast due to their distinct qualities that set them apart from the rest. According to findings from prior investigations, they were distinct from ordinary organisms. The complexity and variety of the isolated yeast species may be attributed to their origins from several sources, including the tapping environment and the tapping container (which may hold onto some yeasts) (Ezemba et al., 2022).

The isolates’ capacity for flocculation was also examined, as indicated in Table 1. The ability of yeast cells to flocculate due to the cell adhesion mechanism was chosen. Yeast cells that adhere to one another during flocculation facilitate simple separation from the broth medium. This phenomena can lower the energy cost associated with biomass centrifugation, which has an economic impact on the production of yeast biomass. Furthermore, yeast’s ability to flocculate ensures a high cell density and a sizable volume of retrieved cells. It can also increase the productivity of ethanol during fermentation. When a cell stops dividing due to a nitrogen shortage, the flocculation ability of the cell is initiated. Cell division and the start of flocculence were both affected by changes in the limiting nutrient’s concentration (Olowonibi 2017). There was a reasonable amount of flocculence seen in isolates 5, 12, and KP4.

As shown in Table 1, nearly every isolate recovered from our investigation generated hydrogen suphide (H₂S) to varied degrees. While some isolates produced low, moderate, or high H₂S, three isolates produced no H₂S at all. Commercial yeast was used as the control, and it produced H₂S only sporadically. According to Umeh et al. (2019), yeasts that produce a lot of hydrogen sulphide are not good for manufacturing bread because they give the bread a taste and flavour that lowers its quality. This work’s H₂S production report contrasts with Umeh et al.’s (2019) and Okafor et al.’s (2022) reports. Gebreslassie et al. (2019) found low H₂S producers as a control, but their controls were non-H₂S producers. The findings of this study and Gebreslassie’s demonstrate that certain commercial yeasts create hydrogen sulphide, which may have a negative impact on baked bread. Isolates 5 and 12 were carefully chosen for additional analysis.

Molecular analysis of isolates 5 and 12 was carried out (Table 2). The results differed from those of Bolaniran et al. (2017), Olowonibi (2017), and Ezemba et al. (2022), whose primary isolates were Sacharomyces cescerevisae. It is aligns with the findings of Nwagu et al. (2021), who isolated yeast from fruit, soil, and wine samples and tested it for the ability to produce lipase, cellulase, xylanase, pectinase, amylase, and protease precursors. Yarrowia phangngaensis XB3 was the isolate that worked best for cassava-wheat composite bread among the others.Isolate 5 was chosen for additional examination based on the percentage IDs of 98.73% and 98.08 % for isolates 5 and 12, respectively, displayed in Table 2.

Numerous variables, including temperature, pH, incubation time, inoculum size, and genetic background, affect how efficiently cells metabolise and produce. (Gebreslassie et al., 2019). To determine which growth environment best supports its growth, the isolate Yarrowia phangngensisis (YP) was exposed to a variety of growth conditions (figures 1-3). At 30 ⁰C, pH 5, and 72 hours of incubation, it displayed a greater biomass, while the commercial yeast (control) exhibited similar traits in this study. The growth of the isolated yeasts in this investigation is comparable to that of the commercial yeast strain, as demonstrated by this finding. Nwagu et al. (2019), in contrast, discovered that although the yeast could tolerate temperatures as high as 28 and 37⁰C for 48 hours of incubation, it flourished most at 24⁰C. Figure 3 illustrates that the strain of yeast used in this investigation could withstand temperatures as high as 40°C. Given that yeast can handle high temperatures, it is possible to use the isolate to carry out fermentation at a variety of temperature ranges since they are able to tolerate the excess heat generated during the fermentation process. According to Gebreslassie et al. (2019), Umeh et al. (2019), and Ezemba et al. (2022), yeasts can thrive at high temperatures of 40⁰C, though with difficulty. The optimum temperature is 30^oC. These findings are consistent with the findings of this study.

The organism’s ability to withstand stress was evaluated by comparing it to commercial yeast, which acted as a control. To find out if the chosen strain could adjust to the conditions needed to make bread, a stress tolerance test was conducted. The ability of baker’s yeast to survive under different stress situations may reveal important details about how well the yeast can proliferate and carry out fermentation when it is compromised. When damaged yeast is subjected to many challenges, such as ethanol and osmotic stress, its growth during fermentation may not be at its best (Olowonibi, 2017). Figures 1-3 in this study show that the yeast grew in adverse situations (Olowonibi, 2017).

Figure 3 from the current study shows that the maximum biomass was reached after 72 hours of incubation; however, as the incubation duration increased, the biomass dropped. This is corroborated by scientific data, which shows that the stationary phase of yeast growth is a non-growing phase during which metabolism slows and cell division is halted as a result of high temperatures, poisonous metabolites, and food shortages, which causes cells to autolyze and die. Mamun-Rashid et al. (2013) reported that the maximum biomass was reached after 144 hours of incubation, which is at odds with the findings of the current investigation. The genetic makeup of their cells and the environment under which they were grown may have contributed to the discrepancy in these results.

Figure 4 illustrates how YP and CY tolerate different salt concentrations. Every strain of yeast demonstrates tolerance to the different salt concentrations (2%, 3%, and 5%). The yeast demonstrated tolerance to varying salt concentrations, a behaviour that was consistent with findings from studies by Olowonibi (2017) and Eshet et al. (2022). As the salt content increased its adaptability declined. The significance of salt tolerance resides in the fact that in Nigeria, experience suggests that some bakeries use salt instead of sugar because the former is cheaper and preferred by those with some health condition such as diabetes. Salt has numerous purposes in baked goods. It affects taste, boosts crust color and influences the pace of yeast fermentation and enzyme activity. Salt also strengthens gluten, making it more cohesive and less sticky. Because salt increases the amount of water and carbon dioxide that gluten can store, the dough can expand without breaking (Olowonibi 2017).

Ethanol stress experiments were carried out to examine the yeast cells’ resistance to ethanol as they create ethanol as a secondary metabolite during the bread-making process. To get the desired flavour when preparing bread, the alcohol content must be at an appropriate level. All of the yeast strains were able to grow in a medium with 5%, 10%, and 15% (v/v) ethanol, as Figure 5 illustrates. For both test species, the optical density significantly decreased at 15% (v/v) of ethanol. This could be due to toxic effect of high concentration of alcohol on yeast, which restricts the cells development owing to the degradation of the cell membrane as described by Olowonibi (2017). Those strains which were capable of growing in same concentration were believed to have ability to generate similar quality of bread as commercial strain.

The yeasts were tested for hyper-osmotic tolerance (Figure 6). Optimum growth was recorded at 35% while the least growth was recorded at 45% glucose concentrations.  Adaptability of yeasts in hyper-osmotic environments is important in bread making and their output is dependent on how they respond to this stress. The increase of osmotic pressure which can be induced by the addition of stressful concentrations of sugars or salts is important in prolonging the shelf life of food (Ribeiro et al., 2022).

CONCLUSION

This work has shown that there could be more yeast strains in palm wine with little or no attention for industrial use. Yarrowia phangngaensis which was one of the yeasts isolated from palm wine was chosen as strain of choice in this work. It has been neglected with all attention directed at the conventional yeast Saccharomyces cerevisae. It has proven as observed here to be of good prospect for use in the bakery industry on the basis of its bakery yeast potentials.

RECOMMENDATION

It is recommended that yeast (Yarrowia phangngaensis), could be tested for toxicity, dough rising capability and keeping quality since it is not a common yeast in the food industry.

Conflict of Interest

There is no conflict of interest among the authors.

REFERENCES

1. Altschul, F. Stephen, Madden, L. Thomas, Schaumlffer, A. Alejandro, Zhang Jinghui, Zhang Zheng, Miller Webb and Lipman, J. David (1997). “Gapped Blast and PSI-BLAST: a new generation of protein database search programs”. Nucleic Acids Res. 25:3389-3402.
2. Aslankoohi E, Herrera-Malaver B, Rezaei MN, Steensels J, Courtin CM, Verstrepen KJ (2016) Non-Conventional Yeast Strains Increase the Aroma Complexity of Bread. PLoS ONE 11(10): e0165126. https://doi.org/10.1371/journal.pone.0165126
3. Bitrus, J., Amadi, O., Nwagu, T., Nnamchi, C. and Moneke, A. (2020) Application of wild yeast (Saccharomyces cerevisiae) isolates from palm wine and honey in baking of cassava/wheat composite bread. Food and Nutrition Sciences 11: 695-711. doi: 4236/fns.2020.117050.
4. Bolaniran, T., Arotupin, D. J., Afolami, O. I. and Fasoranti, O. F.  (2017). Sensory Evaluation Assessment of Bread Produced with Composite Flour Fermented by Baker’s Yeast in Akure, Nigeria. Asian Journal of Advances in Agricultural Research 4(1): 1-9, 2017; Article no.AJAAR.36899 ISSN: 2456-8864
5. Das, S. and Tamang, J.P. (2021). Changes in microbial communities and their predictive functionalities during fermentation of toddy, an alcoholic beverage of India. Microbiol Research 248:126769. doi: 10.1016/j.micres.2021.126769
6. Ribeiro, R.A., Bourbon-Melo, N. and Sá-Correia, I. (2022). The cell wall and the response and tolerance to stresses of biotechnological relevance in yeasts. Frontiers of Microbiology, 13:953479. doi: 10.3389/fmicb.2022.953479
7. Gebreslassie, E.B., Tefera, A.T., Muleta, D., Fantaye, S.K. and Wessel, G.M. (2019). Optimization of the cultivation conditions of indigenous wild yeasts and evaluation of their leavening capacity. International Journal of Sciences 8(6) DOI: 10.18483/ijSci.2043
8. Jiya, M. J., Damisa, D., Abdullahi, I.O. and Chukwu, V.C. (2020) Comparative studies of the dough raising capacity of local yeast strains isolated from different sources. Anchor University Journal of Science and Technology 1(1): 90 – 95
9. Oti , V. O., Okwulehie, I. C., NwankwoB. J. and  Ogoke, H. I. (2021). Assessment of the Physico-Chemical Properties of Oil Palm Wine (Elaeis guineensis) and Raphia Palm Wine (Raphia vinifera). Nigeria Agricultural Journal, 52(3) 
10. Olowonibi, O.O. (2017). Isolation and characterization of palm wine strains of Saccharomyces cerevisiae potentially useful as bakery yeasts. European Experimental Biolology 7:11. doi:10.21767/2248-9215.100011
11. Liu, T., Li, Y., Sadiq, F. A., Yang, H., Gu, J., Lee, Y. K., and He, G. (2018). Predominant yeasts in Chinese traditional sourdough and their influence on aroma formation in Chinese steamed bread. Food Chemistry 242: 404–411. doi: 10.1016/j.foodchem. 2017.09.081
12. Karki, T.B. and Timilsina, P.M., Yadav, A., Pandey, G.R., Joshi, Y., Bhujel, S., Adhikari, R. and Neupane, K. (2017). Selection and Characterization of Potential Baker’s Yeast from Indigenous Resources of Nepal. Biotechnology Research International. Volume 2017 https://doi.org/10.1155/2017/1925820
13. Kumari, S., K.Jha, A. and Singh, A.K. (2019) Isolation and characterization of temperature and ethanol tolerant strain of Saccharomyces cerevisiae strains from naturally fermented juices. Biosciences Biotechnology Research Asia 16(1): 97-103. http://dx.doi.org/10.13005/bbra/27266
14. Ezemba, C. C., Onyemalu, V. T., Nwanya, A. C., Ogbonna, E., Ezemba, A. S., Anakwenze, V. N., Anaukwu, G. C., Okoye, P. O. and Odibo, F. J. C. (2022). Isolation and characterization of palm wine yeasts and a comparison of their dough-raising activity with baker’s yeast. Asian Journal of Plant and Soil Sciences 7(1): 66-72, 2022
15. Nwagu, T.N. T., Osilo, Arinze, M.N., Okpala, G.N., Amandi, O.C., Ndubuisi, I.A. and Agu, R. (2021). A novel strain of Yarrowia phangngaensis producing a multienzyme complex; a source of enzyme additives for baking high cassava-wheat composite bread. Food Biotechnology 35(2) 158-177 https:// doi.org/ 10.1080/08905436.2021.1910520
16. Umeh, S. O., Okpalla, J. and Okafor, J. N. C. (2019). Novel Sources of Saccharomyces Species as Leavening Agent in Bread Making. International Journal of Trend in Scientific Research and Development (IJTSRD) 3(2) 827-832
17. Zhou, N., Semumu, T. and Gamero, A. (2021). Non-Conventional Yeasts as Alternatives in Modern Baking for Improved Performance and Aroma Enhancement. Fermentation 7(3) 102; https://doi.org/10.3390/fermentation7030102