Molecular Modification Of Streptococcus Thermophilus β-galactosidase And Its Application In Yogurt

Yogurt is favored by consumers for its probiotic functions such as antioxidation,lowering cholesterol and promoting immunity.As an important food biocatalyst,β-galactosidase is often used in yogurt making.In addition to hydrolyze lactose,β-galactosidase has the transgalactosylation activity that can convert galactose in milk to galactooligosaccharide(GOS)to improve the yogurt quality.S.thermophilus is the main strain in yogurt making and it hydrolyze lactose into glucose and galactose by β-galactosidase,however,glucose is utilized in the glycolysis pathway,and galactose is pumped into the extracellular circumstance.Therefore,there are a large amount of residual galactose and unused lactose in yogurt which is not conducive to yogurt quality and human health.Therefore,enhancing the activity of βgalactosidase from S.thermophilus is of guiding significance for improving the yogurt quality.In this study,the effects of β-galactosidase hydrolytic and transglycosylation activity of S.thermophilus on yogurt quality were analyzed.The β-galactosidase BgaQ from S.thermophilus SDMCC050237 with the highest hydrolysis activity was genetically modified to improve its transglycosylation activity for low sugar and GOS-enriched yogurt making.The protective effects of GOS on yogurt starters and β-galactosidase in simulated gastrointestinal conditions were evaluated.In addition,the effect of common sugar alcohol sweeteners in yogurt on the thermal stability of β-galactosidase was also analysed in order to make a better transglycosylation.Finally,the transglycosylation of xylitol by β-galactosidase was discussed in order to reduce the content of galactose of yogurt and endow yogurt with more probiotic function.The innovative results are as follows:1.Diversity of S.thermophilus β-galactosidase and its effect on yogurt qualityIn order to explore the effects of S.thermophilus β-galactosidase activity on yogurt quality,the β-galactosidase activity of 28 strains of S.thermophilus in our laboratory was analyzed.It was found that the β-galactosidase hydrolytic activity was significantly different,but the transglycosylation activity was generally low.Three S.thermophilus strains SDMCC050237,SDMCC050219 and SDMCC050217 with high,middle and low β-galactosidase hydrolytic activity were fermented in the synthetic medium,the results showed that the S.thermophilus SDMCC050237 with highest β-galactosidase hydrolytic activity could hydrolyze lactose quickly to promote the growth of the bacteria and produce lactic acid faster to make the pH of the medium decrease rapidly.Results indicated that S.thermophilus with high hydrolytic activity of β-galactosidase had higher activity.The above three S.thermophilus strains were cofermented with Lb.bulgaricus ATCC 11842 to produce yogurt,respectively,and their effects on the quality of yogurt were analyzed.The results showed that the yogurt fermented with highβ-galactosidase hydrolytic activity of S.thermophilus had shorter curd time and richer flavor substances.This research provided a simple and convenient method to screen good yogurt starters by screening S.thermophilus with high β-galactosidase hydrolyzing activity based on the hydrolytic activity.However,the hydrolytic activity of β-galactosidase had no significant effect on the contents of lactose and galactose in yogurt.2.Site-directed mutation of β-galactosidase from S.thermophilus for GOS-enriched yogurt makingIn order to reduce the amount of galactose or lactose in yogurt for improving the yogurt quality,β-galactosidase BgaQ from S.thermophilus SDMCC050237 with high hydrolytic activity was modified to improve its transglycosylation activity,which could convert more galactose to GOS for the production of low sugar and high GOS yogurt.Firstly,BgaQ was analyzed by bioinformatics.Two amino acid sites,Y801 and P802,which were involved in the transgalactosylation reaction were identified and mutated into H801 and G802 by overlapping PCR,respectively.As a result,the mutant enzyme BgaQ-8012 was obtained.Enzymatic property analysis showed that there was no significant difference between BgaQ-8012 and BgaQ in hydrolytic activity.The optimal temperature and pH were 50℃ and was 8.5 respectively.However,the transgalactosylation activity of the mutated enzyme increased.Under 5%lactose concentration,the GOS yield of BgaQ-8012 was 30.2%higher than that of BgaQ,and the optimum temperature for GOS production changed from 50℃ to 42℃,which was consistent with the yogurt fermentation conditions.The purified β-galactosidase BgaQ and BgaQ-8012 were added to fermented milk,respectively,and then co-fermented with S.thermophilus SDMCC050237 and Lb.subsp.bulgaricus ATCC 11842.It was found that the contents of lactose and total sugar were reduced by 38.5%and 16.5%,respectively,and the GOS content was 3.7 g/L in yogurt fermented with starter alone.When BgaQ and BgaQ-8012 were added,lactose content decreased by 49.3%and 54.4%,total sugar content decreased by 26.6%and 40.1%,and GOS content reached 5.8 g/L and 8.3 g/L,respectively.The content of lactose and total sugar were lowest and the content of GOS were significantly highest in yogurt with BgaQ-8012 addition.Therefore,the addition ofβ-galactosidase with high transgalactosylation activity in the process of yogurt production could reduce the contents of lactose and total sugar in yogurt and produce more GOS,this study provided an effective method for the low-sugar and GOS-enriched yogurt making.3.Protective effect of GOS on yogurt starters and β-galactosidaseIn order to evaluate the function of GOS-enriched yogurt,the effects of GOS concentration in yogurt on the activity of yogurt starters and β-galactosidase in simulated gastrointestinal conditions were evaluated.According to the GOS production of yogurt with BgaQ and BgaQ8012,three kinds of yogurt G0,G5 and G10 with final GOS concentrations of 0 g/L,5 g/L and 10 g/L respectively were incubated with artificial gastrointestinal juice for 210 min,the viability of the starter strains and the β-galactosidase activity were determined.After incubation with artificial gastrointestinal juice,the viable numbers of S.thermophilus SDMCC050237 in G0,G5 and G10 decreased from 8.7 to 6.4,7.6 and 7.8 log CFU/g respectively,the number of viable bacteria of Lb.bulgaricus ATCC 11842 in G0,G5 and G10 decreased from 8.2 to 6.8,7.1 and 7.3 log CFU/g,respectively.The β-galactosidase activity in G0,G5 and G10 decreased to 8.9%,17.5%and 26.1%,respectively.The results suggested that GOS in yogurt was beneficial to the survival activity of starters and β-galactosidase in the simulated gastrointestinal conditions,so as to perform the probiotic function.Three yogurt(G0,G5 and G10)digested by the artificial gastroenteric fluid was cocultured with E.coli ATCC 25922 in the simulated large intestine for 48 h,the viable number of E.coli ATCC 25922 changed from 5 log CFU/mL to 8.47,3.87 and 3.33 log CFU/mL respectively.The result analysis showed that GOS could promote the growth of the starters,resulting in lactic acid accumulation and pH reduction,which inhibited the growth of E.coli.This study provided theoretical support for the probiotic effects of GOS-enriched yogurt.4.Effects of sugar alcohol on S.thermophilus β-galactosidaseImproving the thermal stability of β-galactosidase was beneficial for the formation of GOS in yogurt.The effect of three common sugar alcohol sweeteners in yogurt on the thermal stability of β-galactosidase BgaQ and BgaQ-8012 were analyzed.The results showed erythritol and xylitol could increase the activity retention of β-galactosidase under heat treatment,while sorbitol reduced the thermal stability.It was found that erythritol and xylitol could slow down the increase of the surface hydrophobicity of β-galactosidase,while sorbitol was opposite.The results of circular dichroism and fluorescence spectrum illustrated that erythritol and xylitol could stabilize the secondary and tertiary structures of β-galactosidase,thus improving the thermal stability of the enzyme under heat treatment.While sorbitol accelerated the secondary and tertiary structure changes of β-galactosidase during heat treatment.Therefore,the erythritol and xylitol could protect the β-galactosidase in yogurt,for making better to convert galactose to GOS in yogurt making.5.The transglycosylation of xylitol by β-galactosidase from S.thermophilusThe transglycosylation of β-galactosidase could act on different substrates.In this study,it was found that β-galactosidase BgaQ-8012 from S.thermophilus could transfer galactose molecules to xylitol for the production of the synthesized galactosylated xylitol.The molecular weight of the synthesized galactosylated xylitol was 315 by mass spectrometry analysis,and the galactosylated xylitol was presumed to be 1-O-β-D-galactopyranosyl-xylitol and 3-O-β-Dgalactopyranosyl-xylitol by NMR.The production conditions of galactosylated xylitol were optimized.The results showed that the optimum production conditions were as follows:pH 8.0,temperature 60℃,the dosage of BgaQ-8012 2 U/mL,lactose/xylitol concentration ratio 1:3.The glycosylated xylitol had antioxidant function in vitro,which could clear DPPH by 14.7%at maximum,and it had an inhibitory effect on Escherichia coli ATCC 25922.In this study,the galactosylated xylitol with antioxidant and antibacterial functions was generated by modified β-galactosidase BgaQ-8012,which enriched the application of S.thermophilus βgalactosidase in the synthesis of new functional compounds.