Research On Permeabilized Intact Bacterial Cells Catalyzing Sugar Hydrolysis And Isomerisation

At present,a large proportion of people worldwide are lactose intolerant,and this proportion is even higher in China.In recent years,with the gradual improvement of living standards,people have a higher pursuit of a healthy life,and the demand for sugar-free dairy products has also been on the rise year by year.The vast majority of sugar-free dairy products on the market are derived from commercial purified?-galactosidase hydrolyzed lactose,which is quite expensive.Using permeabilized Streptococcus thermophilus cells to replace commercially purified?-galactosidase has many advantages,such as low cost,high resource utilization,and reusability.More importantly,compared to the widespread use of purified?-galactosidase,this is a more natural and safe method because many of them are derived from genetically modified microorganisms(GMO).In this research a strain of Streptococcus thermophilus isolated from yogurt was used as the material,using a combination of nisin and heat treatment to permeabilize cells as a whole-cell catalyst to hydrolyze lactose in to obtain lactose-free dairy products.At the same time,this method of permeabilization was applied to Corynebacterium glutamicum containing?-galactosidase,xylose isomerase,and arabinose isomerase and successfully constructed Lactococcus lactis containing?-galactosidase and arabinose isomerase,a syrup containing glucose,galactose,fructose,and tagatose(GGFT syrup)and a syrup containing glucose,galactose and tagatose(GGT syrup)were obtained respectively.Also,the use of Mother liquor(ML),a waste material produced in the cheese production process,was studied as a carbon source and energy source to cultivate strains in this subject.Meanwhile,in order to reduce the cost of using commercial nisin,a fermentation broth from nisin-producing strain was used to replace commercial nisin to permeabilize cells.The research content and test results of this paper mainly include the following five aspects:(1)Streptococcus thermophilus strain was permeabilized as a whole-cell catalyst to hydrolyze lactose.The combination of nisin and heat treatment had the best effect on permeabilizing Streptococcus thermophilus.It can permeabilize the cells of Streptococcus thermophilus at a temperature of 50?with 2.5?g/m L nisin and act as a whole-cell catalyst to hydrolyze lactose in milk at 50?.It can hydrolyze more than 95%lactose within 4 h.This method not only meets the requirements for safety in food processing,but is also suitable for scale-up production.Other methods,such as adding permeabilization reagents or bead milling methods,have the disadvantages that permeabilization reagents remain in the final product,or the efficiency is low,which is not conducive to industrial scale-up production.At the same time,starting from the following two aspects,the purpose of more efficient hydrolysis of lactose has been achieved.First,the Streptococcus thermophilus strains with high?-galactosidase activity were isolated from commercial starter cultures.Second,the use of bioreactors for large-scale cultivation and preparation of target strains of Streptococcus thermophilus obtained higher biomass in a short time.(2)Corynebacterium glutamicum was permeabilized as a whole-cell catalyst to hydrolyze and isomerize sugar to prepare a syrup containing glucose,fructose,galactose,and tagatose.Nisin and heat treatment are combined to permeabilize JS156 to hydrolyze and isomerize the sugar in delactosed whey permeate(DWP)to obtain GGFT syrup.The composition of GGFT is 26%of glucose,29%of galactose,25%of fructose and 20%of tagatose.At the same time,a medium using residual whey permeate(RWP)as a carbon source for Corynebacterium glutamicum to achieve high biomass was optimized,and its composition was:50%RWP,(NH₄)₂SO₄,Mn SO₄,Fe SO₄,and a mixture of trace elements.(3)Lactococcus lactis strain containing?-galactosidase and arabinose isomerase was constructed to hydrolyze and isomerize sugar in dairy products.The plasmid expression vector p LC9 containing the genes encoding?-galactosidase and arabinose isomerase was successfully constructed and introduced into Lactococcus lactis,and the strain numbered CS2014-7 was identified.Using permeabilized CS2014-7,the sugar in the milk(52 g/L lactose)was hydrolyzed,and a lactose-free milk was obtained after 60 h,which contained27.9 g/L of glucose,18.6 g/L of galactose and 9.1 g/L of tagatose.Cell permeabilization technology was also used on DWP,and a GGT syrup containing 50%of glucose,32%of galactose and 18%of tagatose was obtained after hydrolysis and isomerization.(4)S.thermophilus ST057-1,C.glutamicum JS156 and L.lactis CS2014-7 were cultivated using ML,a waste material from cheese production,as the carbon source of the culture medium.S.thermophilus ST057-1,C.glutamicum JS156 and L.lactis CS2014-7were successfully cultivated by using waste materials as the carbon and energy source of the culture medium.This greatly saves the high cost of purchasing commercial medium.In addition,compared with DWP,the ML component contains more lactose and amino acids,which can be applied to the cultivation of other strains and the direction of medium optimization.(5)Fermentation broth of strains L.lactis IO-1,L.lactis SL28 and L.lactis SL242 that can produce nisin was used as a permeabilizing reagent to permeabilize S.thermophilus CS1980.The S.thermophilus CS1980 cells permeabilized by the fermentation broth of the nisin-producing strain L.lactis SL28 completed 70%of the milk lactose hydrolysis within 2h.The fermentation broth of the nisin-producing strain can be used as a permeabilized cell reagent to replace commercial nisin to permeabilize cells,which greatly saves costs.At the same time,after optimization,a medium containing 20%ML as a carbon source and 0.5 g/L yeast extract(YE)was obtained.Three nisin-producing strains can grow well on this medium.In addition,the fermentation broth of the nisin-producing strain L.lactis SL28 was reused,and the effect of permeabilizing cells was maintained at a high level during the previous repeated use.