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GROWTH RESPONSES AND TOLERANCE OF OSMOPHILIC AND HALOTOLERANT YEASTS ISOLATED FROM LOCAL HONEY TO ENVIRONMENTAL STRESSORS

CHAPTER ONE

INTRODUCTION

1.1 Background to the Study

Yeasts are eukaryotic microorganisms that are of immense biotechnological, industrial and ecological importance. In the yeast kingdom, osmophilic and halotolerant species form a physiologically unique group due to their outstanding survival, growth, and metabolism under conditions of high sugar concentrations, high salt concentrations, low water activity (aw), and other adverse physicochemical factors (Hohmann, 2022; Magan and Aldred, 2022). Osmophilic yeasts characterized by the ability to grow on water activity values as low as 0.61 0.70 (i.e., sugar content above 60 w/v) are of specific interest to fermentation, spoilage and biotransformation of high-sugared food systems including honey, fruit concentrates, jam, syrups, and dried fruits (Ndegwa et al.,)

Honey is a highly complex high-sugar matrix of fructose (38%), glucose (31%) and sucrose, and a mixture of oligosaccharides dissolved in limited proportions of water (usually 1420%). It is an inhospitable environment to the majority of microorganisms because of its natural antimicrobial activity due to hydrogen peroxide, defensin-1, low pH (3.24.5), and volatile phenols (Alvarez-Suarez et al., 2023). Nevertheless, osmophilic and halotolerant yeasts have developed several particular molecular adaptations to endure and take advantage of such conditions, such as the build-up of compatible solutes (especially glycerol), activating osmotic stress response systems, altering membrane lipid constituents, and the expression of particular transporters and stress-response factors (Hohmann, 2022; Liti, 2022).

The local (artisanal) Nigerian and other sub-Saharan African honey is especially of relevance because it is a source of osmophilic yeast diversity, as it is usually produced under non-commercial conditions and subjected to little to no heat treatment, filtration, or quality control, and often kept at ambient temperatures, which are more conducive to yeast survival and fermentative activity (Igwe et al Other African studies have also reported the availability of osmophilic yeasts such as Zygosaccharomyces rouxii, Candida species, Torulaspora delbrueckii, Debaryomyces hansenii in local honey (Nweze et al., 2022; Ayansola et al., 2021). These yeasts are known to cause honey fermentation a type of spoilage, which produces alcoholic off-flavours and decreases the shelf life leading to huge economic losses by honey producers and traders.

In addition to acting as agents of spoilage, osmophilic and halotolerant honey yeasts are a valuable genetic source in biotechnological practices. They are capable of withering osmotic stress, low water activity, high sugar concentrations, and thermal stress, which makes them suitable to be utilized in high-sugar fermentation processes, the generation of bioethanol out of lignocellulosic hydrolysates, synthesis of osmolytes and compatible solutes, and as platforms to generate value-added metabolites under industrial stress conditions (Liti, 20 Knowledge of how these yeasts respond to certain environmental stressors such as different temperature, pH, salt, sugar, UV radiation and hydrogen peroxide is essential to their utilization in industries and to their manipulation in food preservation effects.

Although the ecological and economic potential of osmophilic and halotolerant yeasts in Nigerian honey is broad, little to no systematic research has been done to investigate the stress tolerance physiology, growth dynamics of the yeasts in the presence of various environmental stress factors, and comparative tolerance of the locally isolated strains, respectively. The growth response and tolerance of these yeasts to selected environmental stressors will yield information that is pertinent to honey management, local food preservation, microbiological biotechnology and the basic microbiology of osmotic stress responses in sub-Saharan African cultures.

1.2 Statement of the Problem

The following issue is stated in the paper:

The issue of Honey fermentation and spoilage by osmophilic yeasts is also a major challenge to honey producers and traders in Nigeria and causes economic loss and low product quality. Although it has been reported that such yeasts may be present in some local honey, the physiological characteristics of their stress tolerance (especially growth responses to temperature and pH) and osmotic pressure, salt, hydrogen peroxide, and UV stress are not characterised in detail by Nigerian isolates (Ayansola et al., 2021; Nweze et al., 2022). This gap constrains the elaboration of local honey production specific preservation plans.

Moreover, the biotechnological application of the osmotolerant yeasts in Nigeria such as production of bioethanol, osmolyte, and fermentation of high sugar content has not been studied with regard to the stress physiology. Their applicability in industry cannot be rationally evaluated or optimized without the knowledge of their tolerance limits and growth under environmental stressors and their mode of growth kinetics. This work thus solves a food quality/safety issue and a biotechnology opportunity, by systematically describing the profiles of the stress tolerance of osmophilic and halotolerant yeast, local to the honey source in Nigeria.

1.3 Objectives of the Study

The aim of this study is to evaluate the growth responses and tolerance of osmophilic and halotolerant yeasts isolated from local honey samples to selected environmental stressors.

The specific objectives are to:

1. Isolate and identify osmophilic and halotolerant yeasts from locally sourced honey samples using morphological, biochemical, and molecular (ITS rDNA sequencing) methods.
2. Determine the growth kinetics (specific growth rate, generation time, and maximum cell density) of isolates at varying sugar concentrations (20–70% w/v sucrose).
3. Evaluate the tolerance of isolates to temperature (15–50°C), pH (2.0–8.0), and NaCl concentrations (0–20% w/v).
4. Assess the sensitivity of isolates to oxidative stress (hydrogen peroxide) and UV radiation.
5. Compare the stress tolerance profiles of isolates and determine the species with the broadest stress tolerance for potential biotechnological applications.

1.4 Research Questions

1. What osmophilic and halotolerant yeast species are present in locally sourced honey samples?
2. How do growth kinetic parameters of the isolates vary with increasing sugar concentration?
3. What are the optimal and maximum tolerance ranges of isolates for temperature, pH, and salt concentration?
4. How do isolates respond to oxidative (H₂O₂) and UV radiation stress?
5. Which isolate exhibits the broadest overall stress tolerance, and what are its potential biotechnological attributes?

1.5 Research Hypotheses

Ho1: There is no significant difference in growth rates of osmophilic yeast isolates across varying sugar concentrations.

Ho2: Temperature, pH, and salt concentration have no significant effect on the growth of halotolerant yeasts isolated from local honey.

Ho3: There is no significant difference in the oxidative and UV stress tolerance among the yeast isolates.

1.6 Significance of the Study

The results of the research will offer practical information on honey producers about identity and stress tolerance pattern of yeasts that cause honey spoilage to assist in designing suitable storage and preservation configuration. To biotechnologists, the definition of stress-tolerant strains will tell them about potential yeasts to be exploited in industrial fermentation under the pressure of osmotic and thermal stress. The research will also add to the microbiological characterization of Nigerian artisanal honey, which will contribute to the relative lack of information on local fermentative microflora of food matrices in Africa.

1.7 Scope of the study 

The analysis will involve local honey samples which will be acquired in selected arteries that produce honey and markets among a chosen number of states. The laboratory will be used to carry out yeast isolation, identification and stress tolerance experiments. It will be followed by molecular confirmation of the species identity of representative isolates with the help of ITS1/ITS4 rDNA sequencing. As the key environmental factors to be tested in stress tolerance experiments, the sugar concentration, temperature, pH, NaCl, hydrogen peroxide, and UV radiation will be listed.

1.8 Operational Definition of Terms

Osmophilic yeast: A yeast capable of growth at high solute (sugar) concentrations, typically at water activity values below 0.85.

Halotolerant yeast: A yeast that can tolerate high salt (NaCl) concentrations and maintain growth under elevated ionic stress conditions.

Water activity (aw): A measure of the availability of free water in a substrate, defined as the ratio of the vapour pressure of water in the solution to that of pure water at the same temperature; pure water has aw = 1.0.

Growth kinetics: The quantitative description of microbial growth over time, characterized by parameters including specific growth rate, lag phase duration, and maximum cell yield.

Environmental stressor: A physical, chemical, or biological factor that induces stress responses in an organism and may inhibit growth or reduce viability.

 

References

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Ayansola, A. A., Babatunde, S. K., & Oke, M. A. (2021). Osmophilic yeast diversity in Nigerian honey and their biochemical characteristics. Journal of Applied Biosciences, 159, 16400–16411. https://doi.org/10.35759/JABs.v159.5

Fleet, G. H. (2023). Yeasts in foods and beverages: Impact on product quality and safety. Current Opinion in Biotechnology, 18(2), 170–175. https://doi.org/10.1016/j.copbio.2007.01.010

Hohmann, S. (2022). Osmotic adaptation in yeast: Control of the yeast osmoadaptation pathway. Science Signaling, 15(724), re1. https://doi.org/10.1126/scisignal.abl8088

Igwe, O. U., Onwu, F. K., & Nweze, E. I. (2022). Microbial evaluation of honey samples sold in markets in Enugu State, Nigeria. International Journal of Food Microbiology, 2022, 7843921.

Liti, G. (2023). The fascinating and secret wild life of the budding yeast S. cerevisiae. eLife, 4, e05835. https://doi.org/10.7554/eLife.05835

Magan, N., & Aldred, D. (2022). Environmental fluxes and fungal interactions: Maintaining the ménage à trois of water activity, temperature and pH. In N. Magan & M. Olsen (Eds.), Mycotoxins in Food: Detection and Control (pp. 19–35). Woodhead Publishing.

Ndegwa, E. N., Irungu, J. W., & Raina, S. K. (2021). Yeasts associated with honey bees (Apis mellifera) and their products in Kenya. Journal of Apicultural Research, 51(1), 65–78. https://doi.org/10.3896/IBRA.1.51.1.07

Nweze, E. I., Nweze, J. A., & Onoja, U. S. (2022). Occurrence of spoilage yeasts in Nigerian honey and their antibiotic susceptibility pattern. African Journal of Biotechnology, 21(1), 1–9. https://doi.org/10.5897/AJB2021.17349

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