A Study On The Influence Of Millet Dietary Fibre Fractions As A Main Carbon Source On The Growth Of Probioticbacteria And Enhancement Of Short Chain Fatty Acid Production During Fermentation

Millet is the staple food in many developing countries of Asia and Africa. This study wasaimed to evaluate, the potential of millet dietary fibre fractions as a main carbon source of probioticfermentation and effect on growth, SCFA production and adhesion mechanism of probiotic bacteria. Infirst phase of study, Dietary fbres were extracted from four commercially available millet varietiesPearl millet (pennisetum glaucum), Foxtail millet (Setaria italica), Proso millet (Panicum miliacum),Finger Millet (Eleusine coracana) and separated into individual fractions of total dietary fbre (TDF),insoluble dietary fbre (IDF) and soluble dietary fbre (SDF). The current study focused on the “wholegrain”. The dietary fibres of millet varied between0.55-13.70g per100g.TDF varied significantlyamong tested millet varieties. Pear millet (PM) variety gave a highest yield of TDF while the fingermillet (FM) produced the least. The relative proportions of IDF in the different millet varieties were thesame as for TDF (Pearl millet (PM)<Foxtail millet (FxM)<Proso millet (PrM)<Finger millet (FM)).However, the relative proportion of SDF was found different, highest amount of SDF was found in PM(13.13%) while other millet varieties FxM, PrM, and FM contained SDF at11.11%,9.57%and10.18%respectively. In second step, faecal microfora was collected from four volunteers, before andafter3months of given millet based diet and analyzed for anaerobic fermentation of the individualmillet fbres. Millet diet did not increase significantly the total number of human gut microbes,measured in faecal samples before and after three months of given millet rich diet. However, in vitrofermentation of millet dietary fibre fractions by faecal inocula recorded increase growth oflactobacillus species by9-12%and bifidobacterium species by3-5%. Millet dietary fibre fractions ata concentration of3%(v/v) were found sufficient to enhance the growth of gut microbiota after24hof fermentation.TDF fraction fermentation yielded a slight increase in the total number ofBifidobacterium and total Lactobacillus species as compared to SDF and IDF fractions but notsignificant. A significant change in pH (p<0.05) was noted but no significant differences in opticaldensity (OD540nm) and viable cell counts among the human faecal inocula. Individual millet fibrefractions were incubated anaerobically with the faecal microflora obtained from human volunteersbefore and after being fed the millet diet. Short chain fatty acid (SCFA) production was analyzed bygas liquid chromatography. SCFA produced by millet fibre fermentations using faecal microflora takenfrom subjects fed after3month millet diet, showed a significant increase compared with fermentationsusing faecal inocula taken before the millet diet. The fibre fractions from PM gave the highest yield ofSCFA while the variety FM gave the least. The highest SCFA was obtained between6and24hfermentation. Among the fibre fractions, TDF produced the most SCFA irrespective of the substrate.PM variety found more preferable as a dietary fibre source when compared with the other milletvarieties tested. Based on the result of our first phase of study, we selected two millet varieties (PMand FxM) and four probiotic bacteria (Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium longum and Bifidobacterium bifidus) to enhance the in vitro synthesis of short chainfatty acids through millet dietary fibre fermentation. Both bifidobacterium and lactobacillus speciesexhibited different pattern in the fermentation experiments, with Bifidobacterium species indicatinglonger slow and constant growth compared with lactobacillus species. Lactobacillus species showedthe highest growth irrespective of the substrate up to24h whereas Bifidobacterium speciesdemonstrated the highest growth at both24and48h.1.5%(v/v) concentration of millet dietary fibrein the medium enhanced probiotics growth. SCFA production in the fibre fractions followed the patternTDF>SDF>IDF irrespective of millet variety, indicating that TDF is the best possible dietary fibre forSCFA production. Lactobacillus and Bifdobacterium spp. digested60–80%and75–85%of the milletfbre fractions from both millet samples respectively. The quantity of different SCFAs produced was inthe order acetate>propionate>butyrate. The fermentation of millet dietary fibre using nine co-culturesof four probiotics was also investigated. Interestingly, co-cultures of different genus showed more cellcount than combinations from same genus. However, maximum co-cultures showed more preferencefor glucose than fibre as a substrate. Co-cultures prepared of more than one genus reached a lower pHthan those made up of bacteria from the same genus, providing more evidence for the synergisticassociation in these co-cultures. Co-culture BB+BL+LR showed growth on all substrates tested (TDF,SDF, IDF and Glucose). Millet dietary fibre fractions fermentation with most of the co-culturessignificantly increased (p<0.05) the production of SCFA when compared to pure cultures results in ourprevious study. During all co-culture fermentation with fibre fractions, acetate was found the mostpredominant SCFA and contributed to more than90%of the total SCFA formation at each hour. Purebifidobacteria and lactobacilli adhered to dietary fibre fractions within20to40minutes. A higherpercentage of Bifidobacteria almost40-55%adhered to millet fibre while lactobacilli percentage wasless than40%. Adhesion was not influenced by NaCl or Tween80but, was decreased bypolysaccharides. The optimum temperature for adhesion was found37°C and live cells found essentialfor adhesion. Adhesion was significantly inhibited by protease and by low pH (pH3-4.15). Whileadhesion of probiotic combinations to fibre was influenced by the temperature, pH, and proteindigestive enzymes, but not affected by bile and NaCl. The probiotic co-cultures appear to have similarmechanisms of adhesion as the individual probiotics. Colonization of millet fibre by bacterial cells wasinfluenced by the temperature, with adhesion observed higher at37°C. This study observed enhancedadhesion for BB+BL and LA+LR when these combinations were together as BB+BL+LA+LR. Thisstudy involved low oxygen and highly reducing experimental conditions which were similar to humangut distal colon. Millet fibre was found a suitable substrate for probiotic microflora.