| Stachyose is a non-reducing functional oligosaccharide from natural plant of Lamiaceae Stachys L.,which has many functions such as proliferating intestinal probiotics,inhibiting the growth of spoilage bacteria,promoting intestinal motility,and so on.Also,stachyose shows good characteristics,including natural,green,low usage,and no side effect.In recent years,more and more attention has been payed to stachyose that is used as ingredients of functional food,and it has great values of application and development.The purity of stachyose directly extracted from natural raw materials is very low and other no-functional sugars present in stachyose product reduce its functional properties,which limit its application in industry.Therefore,it will be of great significance for the stachyose application to develop a new technology of improving the purity of stachyose.This study aimed at building a new technology of improving the purity of stachyose,to provide the theoretical and practical guide for large-scale production of high-purity stachyose.In this study,stachyose solution was obtained through extracting Stachys sieboldii Miq in high temperature(102-105 ~0C),and the optimal rehydration time was considered to be 3 h,according to the results of total sugar and protein content in extract.The optimal fermentation strains were screened to purify stachyose based on the high retention rate of stachyose and the high degradation rate of sucrose.The strains include Aspergillus niger,Aspergillus oryzae,lactic acid bacteria,Lactobacillus helveticus and.Lactobacillus rhamnosus.The results showed that compared with lactic acid bacteria,Aspergillus had a stronger ability to hydrolyze sucrose,but lower ability to consume fructose,which led to the fructose accumulation during the Aspergillus fermentation.And the lactic acid bacteria(Streptococcus thermophilus and Lactobacillus bulgaricus)and Lactobacillus rhamnosus could rapidly consume fructose,but they had low sucrose utilization.Therefore,these two kinds of strains could be considered to form complementary advantages in the purification of stachyose.Meanwhile,Aspergillus niger and lactic acid bacteria had higher potential in the purification of stachyose among five strains.After 36 h fermentation for Aspergillus niger,the retention rate of stachyose and degradation rate of sucrose were 91.23%and 65.59%,respectively.After 36 h fermentation for lactic acid bacteria,the retention rate of stachyose and degradation rate of sucrose were 94.33%and 28.39%.Furthermore,it was not feasible that the combination of?-fructosyltransferase with microbial fermentation was to purify stachyose.Based on metabolic properties of each strain for fructose,glucose,sucrose,raffinose and stachyose,the appropriate strain combinations were selected for mixed bacteria fermentation,and the mixed bacteria fermentation processes were analyzed and optimized.The results showed that low inoculation amount was more beneficial to the stachyose purification than the high inoculation amount.The optimal combination of microorganisms was 0.01%Aspergillus niger and 0.01%lactic acid bacteria.The result of purification of stachyose by mixed bacteria fermentation was better than that of single bacteria fermentation.The addition of EDTA could keep the balance between the hydrolysis activity of extracellular enzymes secreted by microorganisms and the rate of monosaccharides consumption which resulted from microorganisms,then reducing the rate of stachyose hydrolyzed.The effect of purification of stachyose through synchronous fermentation was better than that of lag fermentation,and the retention rate of synchronous fermentation was higher 10.12%than that of lag fermentation after 36 h.Controlling pH of fermentation broth between 5.5 and 7.0would be beneficial to delay the stachyose hydrolysis and have not impact on the remove of sucrose.Compared with static fermentation,shaker fermentation could provide enough O2 for microorganisms and lower the activity of glucoamylase,which would contribute to purify the stachyose.The result of purification of stachyose through fermentation with slag of Stachys sieboldii Miq was better than that of traditional fermentation with only extract.In the system of fermentation broth with slag of Stachys sieboldii Miq,the stachyose could dissolve out from raw materials,and other nutrients such as Vitamins and minerals also penetrated out to provide enough nutrition and harmonious environment for growth of microorganism.Finally,activated carbon,microfiltration membrane and ion exchange resin were studied and used to remove the pigment,proteins(animo acids)and ionic compounds in fermentation broth,respectively.The results showed that the composition of proteins in stachyose solution obtained through extracting Stachys sieboldii Miq in high temperature was mainly the amino acid and peptides with small molecular weight,and the percentage of charged amino acid was higher than that of uncharged amino acid in all free amino acids.The content of glutamate increased significantly after fermentation.The condition of activated carbon decolorization included 1%addition amount,55 ~0C and 90 min.Strong-acid cation exchange resin DOO1and weak-alkali anion exchange resin D301 were screened from five types of ion exchange resins,such as 732,IR120 and D001 cation exchange resins,and 717 and D301 anion exchange resins to be applied to separate these impurities from sugar fermentation solution.When temperature of extract and rate of adsorption elution were 35 ~0C and 3.77 BV/H,respectively,to deal with the extract,the remove rate of proteins,salt compounds and pigments were up to 93.70%,97.81%,99.50%,respectively.Contents of Fe and Cu were very low in the freeze-dried sample of extracts from Stachys sieboldii Miq.The content of Na was high,but most could be removed by the exchange resins and its remove rate was up to 98.57%.The freeze-dried sample of extract from Stachys sieboldii Miq was rich in phenolic acid compounds,but these phenolic acid compounds could be separated completely through the fermentation of microorganisms,decolorization of activated carbon and the combined use of cation exchange resin DOO1 and anion exchange resin D301.The purity of stachyose was up to 78.13%of total solid after 36 h co-fermentation of0.01%Aspergillus niger and 0.01%lactic acid bacteria,with centrifugation,filtration,decolorization of activated carbon,the filtration of 100 KDa microfiltration membrane and the use of cation exchange resin DOO1 combined with anion exchange resin D301. |
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