Application Of Leuconostoc Mesenteroides Strains Isolated From Chinese Food Sources To Produce Oligosaccharides To Be Used As Prebiotics

Leuconostoc mesemteroides,widely used in cabbage,butter and cheese fermentation, is able to use saccharides or maltose as acceptors to biosynthesize glucooligosaccharides via transglycosidation of dextransucrase.The glucooligosaccharides show strong resistance to various digestive enzymes existing in the gastro-intestinal system.They favor selectively the growth and proliferation of the beneficial microorganisms in intestinal tract due to not being digested and absorbed by body.These oligosaccharides,defined as non-digestible oligosaccharide,play as probiotics in functional foods.However,the oligosacchafides formed via starch hydrolysation contain generally large amount of monosaccharides,disaccharides or maltodextrin which are the carbohydrates liable to digestive enzymes.They only give energy to food,but do not promote the growth and activation of probiotic bacteria in intestines and thus have little functionality.Therefore,the objectives of this study were to:(1) examine the ability of L.mesemteroides strains 6055,DM1-2,PC13 and L4 isolated from traditional Chinese foods to produce oligosaccharides;(2) optimize medium components available for L.mesemteroides to synthesize oligosaccharides;(3) deal with the properties of key enzymes related to oligosaccharides synthesize;(4) investigate the change between the substrates and the end-products throughout glucooligosaccharides fermentation;and(5) establish a new way to produce functional oligosaccharides via L.mesemteroides.The following are main results:(1) 15 strains derived from different origins were proved to produce glucan and metabolize citrate.Additionally,8 strains isolated were able to transform sucrose into glucan,and 6 strains showed citrate-metabolizing ability.The 15 strains were identified as L.mesemteroides according to their morphological,physiological and biochemical features and 16s rRNA sequence.Among these strains used in this study, 4 strains,named as L.mesemteroides DM1-2,PC13,L4 and 6055 were chosen for further study in terms of their high dextransucrase activity,glucan mass,and the synthesis rate of glucan.(2) Nutrients and fermentation conditions affected the growth of L. mesemteroides cells.Controlled-pH and aeration by shaking culture greatly increased the propagation and biomass of L.mesemteroides cell when sucrose was used as carbon source,and soybean peptone and yeast extract as nitrogen source.The effect of Mg²⁺ and Mn²⁺ on the cell propagation was not remarkable,but MnSO₄,NaCl and FeSO₄ notably promoted the level of dextransucrase by L.mesemteroides.Tween80 and Ca²⁺ significantly improved the stability of dextransucrase activity.K₂HPO₄,due to efficiently buff the pH of media,was the essential ingredient of fermentation media in producing dextransucrase as if maltose played an essential role as acceptor in the process of generating oligosaccharides.So,the medium MS was confirmed to be the optimal fermentation medium for producing oligosacchaddes by L.mesemteroides. The optimized conditions for generating enzyme were 28℃plus shaking at the speed of 100r/min.(3) SDS-PAGE,together with double staining and PAS staining proved the existence of specific reaction bands which indicated dextransucrase.It was seen that strain 6055 generated three kinds of dextransucrases whose molecular weights were 151 kDa,142kDa and 117kDa,respectively.Among them,the former two ones were found both in supematants and cell wall,while the later was only found in the fermentation liquor.Strain DM 1-2 synthesized two kinds of dextransucrases,and their molecular weights were 183kDa and 142kDa,respectively.The former existed both in cell wall and out of cell,but the later only was located on cell surface.Strain PC 13 produced three kinds of dextransucrases,and their molecular weights were 148kDa, 138kDa and 115kDa,respectively.The former two ones were ectoenzyme while the later was linked to cell.Resembled to strain 6055,strain L4 produced two types of dextransucrases coexisting in supernatants and cell wall with molecular weights of 145 kDa and 136kDa,respectively,but the dextransucrase of 115kDa was only found in free-cells supernatants.(4) The activities of dextransucrases from strains 6055,DM1-2,PC13 and L4 in fermentation liquor containing complete cells were 5.204 U/mL,1.638 U/mL,3.244 U/mL and 3.250 U/mL,respectively;while Those in the supematants were 4.710U/mL,1.352U/mL,1.118U/mL and 1.006U/mL,respectively.The proportion held by ectoenzymes was 90.5%,82.5%,34.5%and 31%respectively.The enzyme dextransucrase in cell-free supernatants was purified by saturated NH₄SO₄,centrifugal separation,dialysis-PEG concentration and lyophilization.The activities of dextransucrases yielded from strains 6055,DM1-2,PC13 and L4,calculated in cell dry weight,were 10.7 U/mg,2.7 U/mg,2.8 U/mg and 2.5U/mg,respectively.The activities of dextransucrases,in terms of contents of protein,were 24.4 U/mg for strain 6055,6.5 U/mg for strain DM1-2,4.2 U/mg for strain PC13 and 4.0U/mg for strain L4.The recoveries pertinent to the strains were 32%,32%,44%and 43%, respectively.After stored at -18℃for 2 weeks,the activities of dextransucrases decreased by 31.6%for strain 6055,48.2%for strain DM1-2,41.2%for strain PC13 and 37.2%for strain L4.Ultra-filtration at low temperature(Molecular Weight Cut-Off,100kDa,-4℃) was forwarded to concentrate dextransucrase from the supernatants.The activities of concentrated dextransucrase from 500 mL fermenting liquor were 75.8 U/mL for strain 6055,18.7 U/mL for strain DM1-2,15.8 U/mL for strain PC13 and 13.9 U/mL for strain L4.The recoveries of the enzyme for strains 6055,DM1-2,PC13 and L4 were 76%,74%,69%and 73%,respectively. Conservation for 2 weeks at -18℃decreased the activities of the concentrated dextransucrase by 6.6%for strain 6055,11.5%for strain DM1-2,9.1%for strain PC13 and 6.4%for strain L4.(5) When the ratio of sucrose to maltose was 2 to 1,the monitoring of the substrates and end-products by L.mesemteroides indicated that sucrose and maltose were consumed rapidly in log phase and there was great deal of oligosaccharides to be generated.At the terminal stage of fermentation,all the strains metabolized sucrose except for a little of maltose residue.After fermentation stopped,the biomass of the strains 6055,DM1-2,PC13 and L4 averaged about 1.7g/L,and the yields of corresponding oligosaccharides were 69.8g/L,38.2g/L,48.4g/L and 54.7g/L, respectively.Theoretically,the oligosaccharides harvested were 75%for strain 6055, 41%for strain DM1-2,51%for strain PC13 and 58%for strain L4.(6) When the ratio of sucrose to maltose(S/M) was evaluated to 7:1,strain PC13 was found to generate three kinds of oligosaecharides whilst other 3 strains to generate two kinds of oligosaccharides.In the media in which S/M was 2,strain 6055 produced 4 kinds of oligosaccharides,but other strains produced two kinds of oligosaccharides.Increasing the ratio of sucrose to maltose was conducive to generating the oligosaccharides with relatively high degree of polymerization.In the media in which S/M was 7,the oligosaccharides yields from strain 6055,DM1-2, PC13 and L4 were 45.4g/L,35.5g/L,57.3g/L and 48.8g/L,respectively.Compared to the medium whose ratio of sucrose to maltose was 7:1,other 3 strains got the relatively higher oligosaccharides yields in the medium whose ratio of sucrose to maltose was 2:1 except for strain PC13.(7) A purification scheme including decoloration by active carbon and diatomite, ion-exchange demineralization,mannitol removal by crystallization,and yeast fermentation was conduced to collecting oligosacchadde formed by L.mesemteroides strains.One of advantages of this scheme was that Saccharomyces cerevisiae fermented selectively sugars except for the oligosaccharides generated by L. mesemteroides.It was found that the fructose,maltose and panose in media formed by strains 6055 and DM1-2 were scavenged completely after yeast inoculation for 10h to 12h.Fructose and maltose in media produced by strains PC13 and L4 were completely consumed by yeast cell after 10h incubation.The tri-maltose sugars formed by the two strains were eaten completely after 14h inoculation of yeast cells. The percentage of panose produced by strains 6055,DM1-2,PC13 and L4 in the total oligosaccharides yields was 7.6%,22%,32%and 30%,respectively.Throughout the whole fermentation,no significant change in the oligosaccharides whose DP was greater than 3(DP＞3) occurred.It indicated that the oligosaccharides whose DP was greater than 3(DP＞3) had good resistance to various metabolizing enzymes,and so they were the reliable source of functional oligosaccharides.Finally,the gluco-oligosaccharides' yields by strains 6055,DM1-2,PC13 and L4,especially after the selective removal of sugars from fermenting liquors by S.cerevisiae,were124g/L, 58g/L,66g/L and 83g/L,respectively.Data from nuclear magnetic resonance illustrated that the oligosaccharide product were linked byα-1,6 linkages in the backbone with several branching structures linked byα-1,4 linkages,and theα-1,4 linkages locad at the reducing end of oligosaccharides.