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System design for production of biopreservatives from yeasts for reduction of fruit and beverage spoilage organisms
Ngongang, Maxwell Mewa
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The agro-processing industry is currently facing losses due to microbial spoilage of agricultural produce and associated value-added products such as beverages. Decay and undesired fermentation of fruit and beverages by fungal, yeast and bacterial spoilage organisms are among the major contributors of product losses in the food industry. When looking at the different level of food spoilage, it is common to find different spoilage organisms occurring in the same food item; which usually requires food producers to utilise a mixture of synthetic preservatives for spoilage organism control. Some of the synthetic chemical compounds with growth inhibition properties that have been used in food preservation are sulphur dioxide, benzoic, lactic, sorbic and acetic acid. These compounds act against a variety of spoilage microorganisms. In post-harvest control of fungi, triazoles, hydroanilide fenhexamid, dicarboximides and succinate dehydrogenase are also being used. Some spoilage organisms have been found to be resistant to the use of synthetic chemical preservatives which usually favour the use of higher dosage of preservatives in food. The use of synthetic chemicals as preservative and as postharvest control agents has been found to present serious health risks such as cardiovascular diseases, muscles and stomach pains, eyesight and skin damages and impairment of brain functions. The problem posed by the current use of synthetic chemicals in food put pressure on food producers and exporters to seek alternatives that will allow for the eradication of the use of synthetic chemicals as preservative in beverages and as postharvest control agents on fruits. Yeasts have been found to have the ability to grow at a faster rate on cheap media and to colonise dried surfaces rapidly. It has also been found that yeasts produce extracellular compounds of proteinaceous and volatile organic nature with growth inhibition properties against spoilage organisms. The current findings lack some engineering concept that could assist in the design of a production system for high scale production of biopreservation compounds from yeasts. The availability of a cost effective production media, the growth and production kinetics data using a cheaply available nutrient sources as well as the biological thermodynamic data are some of the gaps in biopreservation bioprospecting. Although several yeasts have already been studied to have great inhibition properties against fruit fungal pathogens, it was still unclear what was the minimum inoculum dose to be able to have a fungistatic and fungicidal effect on the growth of fruit spoilage organisms. The concept of combination of biopreservatives and the interaction effect of their biopreservation activity against consortia of spoilage organisms has also been lacking. As an attempt to seek alternatives to the use of synthetic chemicals as preservatives or postharvest control agents, Candida pyralidae Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 strains were assessed for antimicrobial activity against spoilage yeasts (Dekkera bruxellensis, Dekkera anomala, Zygosaccharomyces bailii) and spoilage fungi (Botrytis cinerea, Colletotrichum acutatum and Rhizopus stolonifer). As alternative to refined media, a cost effective approach was explored whereby the use of agro-waste, i.e. grape pomace extracts (GPE), as production medium for biopreservation compounds, was studied. Production kinetics using modified existing models, subsequent to optimization using response surface methodology (RSM) for biopreservation compounds production was studied for the three biocontrol yeasts using GPE broth as the fermentation medium. The evaluation of the interaction study between mixtures of crude biopreservatives against consortia of common spoilage organisms present in beverages was also conducted by producing the crude biopreservation compounds separately from yeasts and then formulating growth inhibition combinations (GICs); GIC 1 (Candida pyralidae Y1117 and Pichia kluyveri Y1125); GIC 2 (C. pyralidae Y1117 and P. kluyveri Y1164), GIC 3 (P. kluyveri Y1125 and Pichia kluyveri Y1164); GIC 4 (C. pyralidae, P. kluyveri Y1125 and P. kluyveri Y1164). The spoilage organism consortia combinations, i.e. SC1, D. anomala and D. bruxellensis; SC2 (D. anomala and Z. bailii); SC3 (D. bruxellensis and Z. bailii) and SC4 (D. anomala, D. bruxellensis and Z. bailii) were also prepared. This study also investigated the effect of varying inoculum dose (ID) of Candida pyralidae strain Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 on the biocontrol of Botrytis cinerea by contaminating the headspace of the growth medium with a fungal plug subsequent to biotreatment with different initial inoculum dose of the respective biocontrol yeasts. Finally, grape pomace extracts was used as fermentation medium to study the biological thermodynamics of biopreservation compound production from the three biocontrol yeasts. The results obtained demonstrated some interesting results. The antagonistic properties of C. pyralidae and P. kluyveri were observed on cheap solidified medium (grape pomace extracts) as well as on fruits (grapes and apples). These yeasts produced extracellular volatile organic compounds (VOCs) that could be responsible for yeast and fungal growth inhibition. Twenty-five VOCs in the category of alcohols, organic acids and esters were identified by GC-MS. The results of the kinetic study showed that the highest volumetric zone of inhibition (VZI) was 1.24 L contaminated solidified media (CSM) per mL biopreservation compounds used (BCU) when Candida pyralidae Y1117 was inoculated in a pH 3-diluted GPE broth (150 g L−1) incubated at 25 °C for 24 h. Similar conditions were applied for Pichia kluyveri Y1125 and P. kluyveri Y1164, albeit under slightly elongated fermentation periods (up to 28 h), prior to the attainment of a maximum VZI of only 0.72 and 0.76 L CSM mL−1 ACU, respectively. The potential biopreservation compounds produced were identified to be isoamyl acetate, isoamyl alcohol, 2-phenyl ethylacetate and 2-phenyl ethanol. The growth inhibition interaction study showed a variation in growth inhibition proficiency depending on the spoilage organisms or the consortia of spoilage organisms being deactivated. It was then suggested that, a food environment contaminated with a consortium of spoilage organisms can be controlled by employing either the crude biopreservation compounds from individual yeast or those of the following yeast combinations, GIC1-4, which showed a better growth inhibition proficiency against SC1-3. The fungistatic and fungicidal effects on the fungal pathogen were dose dependent. The fungistatic characteristics against Botrytis cinerea were displayed after 7 days when 102-105 cells mL-1 of Candida pyralidae Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 were independently used in-vitro and in-vivo. However, 106-108 cells mL-1 inoculum doses displayed fungicidal characteristics. Additionally, the fungicidal property of yeasts studied was also confirmed on table grape (in vivo studies) using closed jar method. The biological thermodynamic study showed that, dried biomass molecular weight of 28.9 g/C-mol, 29.163 g/C-mol, and 27.176 g/C-mol were obtained for Candida pyralidae strain Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 respectively. The results obtained successfully established useful biological thermodynamic data applicable to the design of adequate biopreservatives production system from yeasts using cheaply available nutrients source.