Abstract
Archaeological evidence from the Matobo Hills shows that marula fruits have been used to brew traditional marula fruit beer for over 10,000 years. Indeed, marula fruit beer is recognised as an essential source of nutrients, particularly during food scarcity. Different types of marula fruit beers are high in dietary fibre, sugars, organic acids, polyphenols and flavonoids, amino acids, vitamin C, and minerals. Despite its nutritional value, marula fruit beer remains a relatively artisanal product in South Africa. This is a consequence of several processing constraints, including laborious juice extraction processes, high juice viscosity, lack of standardised production processes, susceptibility to stuck fermentations, and a short shelf life. As a result, this study attempted to address some of these challenges by developing a standard production process, controlling the fermentation process by using selected non-Saccharomyces yeasts, and extending the shelf life of the beer. To this end, a cold-contact fermentation (CCF) process was developed and optimised to produce low alcohol marula fruit beer using Metschnikowia pulcherrima, Pichia fermentans, and P. kluyveri. Fermentation at temperatures below 10 °C produced low alcohol marula fruit beer (0.00 – 0.20% v/v) with an apparent attenuation of above 80%. Cold-contact fermentation by non-Saccharomyces was shown to be an effective biological method to produce low alcohol marula fruit beer. However, the low alcohol marula fruit beer lacked the microbial inhibitory potential typically associated with higher alcohol content, increasing the beer’s susceptibility to microbial spoilage. To address this challenge, edible medicinal plant powders (EMPPs) of Devil’s Claw plant root and Moringa oleifera plant leaf were added to enhance the beer’s storage stability and reduce the growth of spoilage microorganisms. This is because EMPPs contain a wide variety of bioactive compounds that can inhibit spoilage microorganisms. However, to ensure that EMPPs didn’t also negatively impact the inoculum—M. pulcherrima, its physiological responses were determined. Results showed that the EMPPs positively impacted the cell viability of M. pulcherrima under CCF conditions. Compared to the control sample, the EMPPs-treated marula fruit beers showed higher overall cell viability, with Moringa oleifera plant leaf powder treatment 1 (MO1) and Devil’s Claw plant root powder treatment 2 (DC2) showing better cell viability after 312 h. The addition of EMPPs also improved overall carbon source utilisation (94.27%) and nitrogen source utilisation (71.47%). The addition of EMPPs increased the total flavonoid content and total phenolic content before fermentation and enhanced the antioxidant activity (AA). Specifically, the AA in MO2 and DC1 showed AA of 74% DPPH inhibition between 24 h and 312 h. Thereafter, the physiological response of M. pulcherrima under stress
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conditions was determined to ensure the yeast maintained a high fermentative activity to assert its dominance in the fermentation media. The stress response protein histidine kinase/HSP90-like ATPase domain-containing protein was upregulated in the marula fruit juice and down-regulated in the marula fruit beer. This suggests that EMPPs inhibited the expression of this protein. M. pulcherrima possibly uses histidine kinase to adapt to the fermentation medium to ensure cell viability throughout the exponential growth phase. Pyruvate decarboxylase and alcohol dehydrogenase were both upregulated in EMPPs-treated marula fruit beers. These enzymes are highly regulated in M. pulcherrima to limit the conversion of pyruvate to acetaldehyde to reduce the substrate available for alcohol production. Acids made up 18.50% of volatile organic compounds (VOCs), followed by alcohols (14.80%), aldehydes (13.10%), and ketones (8.50%). The major acid, acetic acid, lowered the pH and imparted a sour taste to the beer. Glycerol (9.2%) contributed to the beer’s sweetness and mouthfeel. Aldehydes benzaldehyde and 4-hydroxybenzaldehyde, imparted the pineapple-like or almond, and vanillic or nutty aromas in the marula fruit beer. Overall, EMPPs demonstrated the ability to inhibit specific spoilage microorganisms, induce stress-related proteins, and stabilise volatile organic compounds (VOCs). As a result, Devil’s Claw plant root powder and M. oleifera plant leaf powder influenced consumer acceptance of low alcohol marula fruit beer and were effective in extending the microbiological safety for at least 21 days. While the control showed the highest overall rating (5.32), the DC-treated marula fruit beer showed the highest overall rating (4.12) among the EMPPs-treated marula fruit beers. The DC-treated marula fruit beer showed a good balance in appearance, aroma, and mouthfeel, which enhanced consumer acceptance. In contrast, the MO-treated beer scored lowest in most attributes due to its oily, bitter, and medicinal characteristics. Slight changes in pH and alcohol content indicated microbial activity during storage. This was confirmed by the presence of lactic acid bacteria (LAB) from day 14 in all samples. No growth of aerobic spore-forming bacteria or moulds was observed during the storage period. Yeast colonies observed from day 14 on all samples were suspected and confirmed to be M. pulcherrima. Using an iron chelation reaction, M. pulcherrima was confirmed to produce pulcherrimin, which exhibits antibacterial and antifungal activity. Overall, this study has been able to develop and use a standardised CCF process in combination with M. pulcherrima to produce a storage-stable, consumer-acceptable, low alcohol marula fruit beer that has industrial upscaling and commercialisation potential.