| Lactobacills helveticus is a kind of rod-shaped,non-spore-producing gram-positive bacteria,which has been widely used in food,medicine and other fields.High temperature can cause protein denaturation,cell membrane structure damage and enzyme activity loss of lactic acid bacteria,which seriously affects the activity of bacteria.Therefore,the fluidity and permeability of cell membrane,osmotic regulation substances(trehalose,soluble protein)and antioxidant enzyme activities of L.helveticus were analyzed after high temperature stress.The compensatory effect of L.helveticus under high temperature stress at the molecular level was explored by transcriptome sequencing.In addition,the viable counts,gastrointestinal digestion and storage properties of the powder prepared by spraying drying of L.helveticus after high temperature stress were investigated.The main research results were as follows:(1)Effects of high temperature stress on metabolism compensation of L.helveticus NCUF 210.1: The growth curve,permeability and fluidity of cell membrane,soluble protein content,trehalose content and superoxide dismutase(SOD)activity of L.helveticus NCUF210.1 were measured under high temperature stress to explore the metabolism compensation of L.helveticus under high temperature stress.The results showed that compared with the control group(42?),the growth of L.helveticus was significantly inhibited under high temperature stress(47?)(p<0.05),the fluidity of the cell membrane was reduced,the membrane permeability was improved,soluble protein content increased by 140.13%(10 h)(p<0.05),the trehalose content increased by 17.23%(10 h)(p<0.05),and SOD activity increased by 159.28%(10 h)(p<0.05).These results indicated that high temperature would lead to changes in the composition of membrane lipids and membrane protein structure of L.helveticus,which would increase the permeability of the membrane and cause leakage of some small molecules such as potassium salt and phosphate and biological macromolecules such as proteins,which maintain the normal growth of the cells.In addition,trehalose content increased under high temperature stress,thus protecting cells against adverse environmental effect.Compared with the control group,the acid resistance of L.helveticus under high temperature stress increased by 9.43%(8 h),17.71%(10 h)and 37.82%(12 h),respectively.Bile salt tolerance increased by 6.26%(8 h),6.31%(10 h)and 6.25%(12 h),respectively.And the ability of heat resistance increased by 41.91%(8 h),43.06%(10 h)and 35.96%(12 h),respectively.This may be because the metabolic regulation mechanism enhanced the resistance of L.helveticus to different stress conditions such as acid,bile salt and high temperature by increasing the transcription expression of stress genes under stress conditions.Scanning electron microscope(SEM)showed that after high temperature stress,L.helveticus became longer,and there were obvious morphological changes,which may be due to the metabolic regulation mechanism in the cells of the bacteria under stress conditions may have changed to enhance the stress resistance of the strain.(2)Changes of transcriptome level of L.helveticus NCUF 210.1 after high temperature stress: Transphenome sequencing was performed on L.helveticus NCUF210.1 cultured at 42?(control group)and 47?(experimental group)for 10 h to explore the compensation mechanism of L.helveticus under high temperature stress.Transcriptome sequencing showed that the transcriptional levels of 432 genes in L.helveticus under high temperature stress(47?)were altered compared with the control group(42?).Among them,the transcription levels of 219 genes were up-regulated and the transcription levels of213 genes were down-regulated.The GO functional classification analysis showed that the differentially expressed genes were concentrated in cellular component such as cellular anatomical entity and protein-containing complex.KEGG pathway analysis showed that there were 6 differentially up-regulated genes involved in membrane transport,which indicated that high temperature stress may lead to changes in the cell membrane composition of L.helveticus,so that L.helveticus had better adaptability and protection to high temperature environment.KEGG pathway analysis also showed that high temperature stress promoted the transcription level of genes related to metabolic pathways such as amino acid metabolism,carbohydrate metabolism and nucleotide metabolism of L.helveticus,which provided energy for L.helveticus to resist high temperature stress,thus enhancing metabolic compensation effect of cells to resist cell damage caused by high temperature.The transcriptional levels of heat shock proteins(HSPs)-related genes(dna K?dna J?gro L and rec A)were also up-regulated,and combined with the results of protein interaction network analysis indicated that HSPs could enhance the heat tolerance of L.helveticus by regulating the metabolic activities in cells.(3)Analysis of the properties of the powder prepared by spray drying of of L.helveticus NCUF 210.1 after high temperature stress: L.helveticus powder was prepared by spray drying of of L.helveticus NCUF 210.1 after high temperature stress.Under different inlet temperatures,the particle size of L.helveticus powder was uniform in the range of 100?400 nm.When the air inlet temperature was 110?,the water content of L.helveticus powder obtained by spray drying was higher than that of air inlet temperature of120? and 130?.Compared to the control group,the viable counts of L.helveticus in powder was increased by 0.61 log cfu/g(air inlet temperature was 110?),0.62 log cfu/g(air inlet temperature was 120?)and 0.92 log cfu/g(air inlet temperature was 130?).These results indicated that high temperature stress could improve the resistance of L.helveticus NCUF 210.1 to stress environment in spray drying.When the air inlet temperature was120?,the water content was low and the viable counts of spray dried powder was high,so this group was selected for simulated gastrointestinal digestion and storage stability experiments.After 180 min of digestion in simulated gastric and intestinal fluid,the viable counts of spray dried powder in the control group and in the experimental group decreased by 4.06 log cfu/g and 3.85 log cfu/g,respectively.Under different storage conditions at 4?,25? and 37?,the viable counts of L.helveticus in powder was higher than that of the control group.When stored at 4? for 28 d,the the viable counts of L.helveticus powder decreased slowly,the stability was better,and the loss of viable bacteria was less,which was beneficial to the long-term storage of L.helveticus. |
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