Study on Antioxidant and Antimicrobial Activities of Peptides Derived from Fermented Foxtail Millet

Abstract

Millets are a major food source in arid and semi-arid parts of the world. Millets are good sources of energy. They provide protein, fatty acids, minerals, vitamins, dietary fibre and polyphenols. Typical millet protein contains high quantity of essential amino acids especially the sulphur containing amino acids (methionine and cysteine). Processing millet by milling removes the bran and germ layers that are rich in fibre and phytochemicals, causing significant loss. The millets are source of antioxidants, such as phenolic acids and glycated flavonoids. Millet foods are characterized to be potential prebiotic and can enhance the viability or functionality of probiotics with significant health benefits. Traditionally fermented foods and beverages obtained from millet or millet mixed with other cereals (corn and sorghum) include koko (millet porridge), fura, mangishi, jandh, uji, burukutu, kunu-zaki, ogi and bushera while dambu, masvusvu and roti are representatives of unfermented millet based products. Solid-state bioprocessing of foxtail millet by Lactobacillus paracasei Fn032 is a biotechnological strategy to produce fermented foxtail millet meal with more beneficial components. The effect of L. paracasei Fn032 fermentation and heat-moisture treatment (HMT) on the physicochemical properties of foxtail millet (Setaria italica) flour was investigated. The results obtained showed a significant (P < 0.05) increase in proximate chemical composition in general after fermentation and HMT. Differential scanning calorimetry analysis showed high decomposition temperature (Td) trend of 180.59 and 189.82o C after HMT. However, there was significant (P < 0.05) enthalpy (∆H) decrease. Flour digestion resulted in variation of slow digestible starch and resistant starch count from 6.83 to 18.42% and 7.61 to 22.68% respectively, after fermentation and HTM. Following this observation, it was ascertained that in X-ray diffraction; pasting viscosity and fluorescence spectrophotometry show greater HMT influenced on the flour components. Findings from the scanning electron microscopy analysis showed microstructure differences of the flours samples. Evaluation of antioxidant, antimicrobial properties and nutritional values of water extracts from fermented foxtail millet flour and its bran with and without protease by L. paracasei Fn032 was done. Fermented foxtail millet flour with added protease extract showed higher scavenging ability on DPPH radicals as well as reducing power than Jiangnan University ABSTRACT PhD Thesis xi fermented foxtail millet flour and fermented foxtail millet bran extracts. Both extracts, fermented foxtail millet flour and fermented foxtail millet flour with protease, showed significant (P < 0.05) effectiveness inhibition abilities on microbial growth when compared with fermented foxtail millet bran extracts. Amino acid profile revealed that fermented foxtail millet flour with protease, with relatively strongest antioxidant, antimicrobial activity, also had the highest total hydrophobic amino acids content (51.39%) and hydrophobic index (8.47 Kj/moL amino acid residue). Moreover, fermented foxtail millet flour with protease revealed the highest protein content, predicted protein efficiency ratio, and protein digestibility. Molecular weight of the whole extracts varied from 180– 5000 Da. Purification by RP-HPLC and amino acid sequencing by LC-MS of peptides derived from foxtail millet (Setaria italica) meal fermented by L. paracasei Fn032 were carried out. The purified foxtail millet peptide fractions (FFMp) of Tyrosine/Leucine-rich (FFMp4=756.84, FFMp6=678.74 and FFMp10=678.87 Da) showed significant (P < 0.05) scavenging activities for DPPH, and superoxide anion radicals. FFMp peptides (synthesized) have shown fairly inhibition of the Escherichia coli ATCC 8099 growth. Furthermore, FFMp4 and FFMp6 peptides showed resistance to trypsin proteolysis while FFMp10 appeared to have partial hydrolysis. The effect of chemically synthesized short peptides (6 residues), previously identified from a fermented foxtail millet meal fraction (FFMp10) on the cell surface hydrophobicity (CSH), biomass growth, DNA binding ability of Escherichia coli ATCC 8099 was verified. The proteolytic responses of these peptides were also tested using the high performance liquid chromatography. Changes made in the FFMp10 sequence were: Mp1, Mp2, Mp3 and Mp4 peptides sequences by Arg and Lys amino acids substitutions. The results revealed that all the peptides resisted to pepsin treatment for 1 h. However, Mp2, Mp3 and Mp4 showed no significant HPLC elution profile changes after the trypsin treatment. The values of CSH were significantly higher (P < 0.05) with FFMp10, Mp1 and Mp2 2 h treatment on bacteria, whereas, lowered with Mp3 and Mp4 treatment. Overall peptides treatment lowered the rate of bacterial biomass growth when compared to control. Slight E. coli ATCC 8099 DNA bindings were observed in lane 2, 3 and lane 3 respectively for Mp1 and Mp2 incubation. Jiangnan University ABSTRACT PhD Thesis xii Fermentation and heat moisture treatment methods present a possible way of changing or improving the physicochemical properties and add nutritional value to foxtail millet meal. Fermented foxtail millet flour extracts were relatively effective in the antioxidant, antimicrobial properties assayed. Indeed, it is feasible to derive natural antioxidants peptides along with antimicrobial activity and resistance to enzyme from fermented foxtail millet meal