Improvement of the quality and shelf life of traditionally produced sorghum juice by addition of ashes, dried powered leaves and stem obtained from combretum spp

Thesis (Ph.D.(Microbiology)) -- University of Limpopo, 2022

Traditional sorghum juice is produced in many African countries for human consumption. The juice is very rich in calories, B-group vitamins including thiamine, folic acid, riboflavin, nicotinic acid, and essential amino acids such as lysine. Low earning income women at village level produce sorghum juice for home consumption and sale. The short shelf life (2 to 3 days) of sorghum juice is a major problem for both the brewers and consumers of this drink. The aim of the study was to use 12 Combretum plants to improve the microbiological quality and shelf life of sorghum juice. Fresh stems and leaves of C. caffrum, C. vendae, C. erythrophyllum, C. elaegnoides, C. apiculatum, C. imberbe, C. adenogdium, C. padoides, C. bracteosum, C. kraussii, C. mkuzense and C. zeyherii were collected at Nelspruit, National Botanical Gardens, Mpumalanga, South Africa. Voucher specimens and tree labels were used to verify the identity of the plants. The stems and bark collected were cut into pieces and air dried for 30 days. When dried, the plant material was ground to a fine powder and stored in paper bags at room temperature. The wood was burnt in an open fire; fuel was not used to minimise contamination. The qualitative phytochemical composition of both the leaves and stems of Combretum plants analysed in this study revealed the presence of saponins, tannins, terpenoids, steroids, cardiac glycosides and flavonoids. The following phytoconstituents were lost in the ashes; tannins with the exception of C. mkuzense and C. padoides; cardiac glycosides and flavonoids. The quantitative phytochemical analyses revealed that both the leaves, stems and some ashes such as C. apiculatum and C. vendae contained appreciable levels of phenolic compounds, tannins and flavonoids. Quantitative analysis of antioxidant activity, the 2, 2, diphenyl-1-picrylhydrazyl (DPPH) assay was used as a screen test for the radical scavenging ability of the compounds present in the different 36 70% acetone extracts. DPPH screening method indicated great scavenging activity with the 70% acetone leaf extracts of C. kraussii, C. zeyherii and C. mkuzense. The leaf and stem extracts showed substantial great antioxidant activity in a concentration-dependent manner. There was a significant decrease in the antioxidant activity in the ashes (p=001), when compared to both the leaves and the stems. The proximate and nutritional analysis of the 70% acetone extracts were performed by AOAC and ICPE protocols, respectively. The results indicated that all the extracts had substantial amounts of ash, moisture, protein and energy. Mineral content of the plant parts was analysed as well, calcium had the highest concentration, while zinc was lowest in concentration. The mineral content decreased significantly in the stems (p ≤ 0.05) when compared to the leaves. There was a further decrease in mineral content with regard to the ashes with the exception of calcium. Based on these findings, the leaves and ashes of C. adenogonium and C. apiculatum could provide a good source of calcium in the diet, while C. adenogonium, C. bracteosum and C. apiculatum had high levels of sodium. A serial micro-dilution assay was used to determine the minimum inhibitory concentration (MIC) values for 70% plant extracts using tetrazolium violet reduction as an indicator of growth. Two Gram-positive (Stapylococcus aureus ATCC 29213 and Enterobacter faecalis ATCC 29212) and two Gram-negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) bacterial strains were used in this study. The leaves had good antibacterial properties with the lowest MIC value being 0.04 mg/ml against E. coli and S. aureus. E. faecalis was found to be resistant against all the leaves with the exception of C. imberbe. The stem extracts of Combretum spp. tested in the study showed antimicrobial properties with the lowest MIC value being 0.04 mg/ml against E. coli shown by C. bracteosum. However, E. faecalis was resistant against all the 12 plants tested. All the test microorganisms showed resistance to the ashes, with the exception of S. aureus, which was found to be susceptible to 75% of the test ash extracts with the lowest MIC value of 0.16 mg/ml. Cytotoxicity and anticancer activity of the acetone extracts of the 12 Combretum plants were evaluated using tetrazolium-based colorimetric assay (MTT assay) on A549 lung carcinoma cells. The assays revealed that 50% of the leaf extracts of tested plants showed cytotoxicity and cell proliferation inhibition in A549 lung carcinoma cells in a concentration-dependent manner. The A549 cells were more sensitive to the following plants: C. elaegnoides, C. erythrophyllum C. imberbe, C. kraussii and C. mkuzense. The following stems extract, C. adenogdium and C. caffrum did not have any anticancer activity, whereas C. apiculatum and C. bracteosum were only able to reduce cell viability to less than 60%. C. mkuzense, C. padoides, C. vendae and C. zeyherii acted in a concentration-depended manner with the greatest activity seen at the highest concentration (1000 µg/ml). The plants had activity at concentrations between 31.25 and 1000 µg/ml allowing only 20% and 50%, respectively, of the cells to remain viable. Ashes from C. mkuzense showed good anti-cancer activity at the highest concentration (1000 µg/ml) reducing cell viability to around 10%. Enterobacteriaceae, total coliform, S. aureus, B. cereus, E. coli and lactic acid bacteria viability were studied during the four weeks storage period of prepared sorghum juices. Juice samples were collected after preparation; the samples were serially diluted using peptone water. Tempo instrument (Biomereiux) was used to enumerate total coliform, total aerobic count, E. coli, S. aureus, lactic acid bacteria, enteric bacteria, yeast and mould using the most probable number following the manufacturers instruction. C. mkuzense and C. padoides plants were able to inhibit the growth of B. cereus, lactic acid bacteria and S. aureus during the first three weeks of storage. E. coli was not present throughout the four weeks storage time. Vitek 2 Compact (Biomereiux) was used for the characterisation and identification of the dominant bacterial isolates using biochemical reactions. The isolates were characterised by morphological differences. Sixty five percent of the isolates were the Enterobacter genus that are commonly found in soil, water, and sewage. The nutritional composition and sensory properties of the prepared sorghum juices treated with Combretum plants were investigated. All the juices had appreciable amounts of protein, ash and energy. Sorghum juice treated with ashes of C. caffrum, C. erythrophyllum and C. kraussii had the highest levels of proteins when compared with other treated sorghum juices. The sorghum juices prepared in the study had varying levels of trace element or minerals with potassium -(3, 55 – 104 mg/l) and calcium (3.2-148 mg/l). Similarly, cobalt (-1.22), coppr (-0.99), iron(-0.962), magnesium (0.004), sodium (-0.145), nickel (-2.7)) and zinc (-1.2)are present in very low amounts. The juices treated with ashes had relatively higher levels of calcium, potassium, magnesium and sodium. Sorghum juices treated with the ashes had better sensory and organoleptic properties when compared with those treated with the leaves. The juices treated with the ashes of C. caffrum and C. bracteseum were more accepted by most of the panellist when compared with other treated juices. Overall, this study presents valuable information on the phytochemical composition, nutritional composition and antioxidant properties of some Combretum species in South Africa. It recommended its use as food and in pharmaceutical preparations for the local industries. In addition, Combretum plants showing the effects tested in this study may be explored further for development into drugs. functional food as food preservatives and nutraceutical applications, beside their traditional use

University of Limpopo and Foodbev SETA