Construction Of A Multilayer Structure Vector To Enhance Lactobacillus Acidophilus JYLA-191 Stress Resistance Studies

With the continuous expansion of the probiotics market,various probiotic foods have entered people’s lives.Due to their beneficial effects such as anti-cancer,anti-obesity,and anti-diabetes,probiotics have gained widespread attention and recognition.However,as with all things,probiotics have both positive and negative aspects.Despite their numerous health benefits,their activity is easily lost during processing,storage,and digestion,making it difficult to ensure that enough live bacteria reach the colon after ingestion to exhibit their probiotic properties.To protect probiotics as much as possible and maintain their activity,encapsulating probiotics in a multi-layer structure carrier is considered a viable method.This paper proposes the use of coaxial dripping and double emulsion methods to construct multi-layer structure carriers for the encapsulation of Lactobacillus JYLA-191,and two types of microcapsules were prepared and evaluated for their morphology and strain stability.The main results of preparing core-shell structure dropping pills based on coaxial dropping method are as follows:1.In order to obtain the best technological conditions for preparing double-layer dropping pills of Lactobacillus acidophilus JYLA-191,the effects of sodium alginate concentration,pectin concentration and extruding ratio on the roundness and the survival rate of Lactobacillus acidophilus JYLA-191 after gastrointestinal digestion were studied.According to their comprehensive scores,the single factor and response surface optimization experiments were carried out,and the optimum conditions were obtained as follows:the concentration of pectin1.25%,the concentration of sodium alginate 1.3%,the extruding ratio of inner and outer layers1:4,after digestion,the survival rate of Lactobacillus acidophilus JYLA-191 was 92.37±0.18%,the roundness of dropping pills was 89.63±0.21%,and the comprehensive score was 91.27%.2.To verify the superior protection effect of double-layer microcapsules on Lactobacillus JYLA-191,compared with single-layer microcapsules,double-layer microcapsules were prepared using the optimal process conditions.Morphological analysis indicated that both wet single-layer and double-layer microcapsules were spherical and uniform in size,with the double-layer microcapsules exhibiting a clear core-shell structure,with Lactobacillus JYLA-191 evenly distributed in the inner oil-gelatin core.After processing,the activity of Lactobacillus JYLA-191 in the single-layer and double-layer microcapsules decreased by 2.11lg（CFU/g）and 1.02 lg（CFU/g）,respectively.In vitro digestion studies showed that both types of microcapsules could smoothly pass through the upper gastrointestinal tract and achieve colon-targeted release,with single-layer and double-layer microcapsules achieving live cell counts of 6.38 lg（CFU/g）and 8.43 lg（CFU/g）,respectively,after continuous digestion.In high-concentration bile salt,heat treatment,and different temperature storage conditions,compared with single-layer microcapsules,the Lactobacillus JYLA-191 in the double-layer microcapsules exhibited better stability,and cell stability was higher at 4°C.The main conclusions of the preparation of multilayer microcapsules based on double emulsion method are as follows:1.Single-factor experiments were conducted to optimize the encapsulation material for microcapsules,and the optimal process conditions were determined to be a pectin concentration of 1.5%,a sodium alginate concentration of 2%,and a soybean oligosaccharide concentration of 3%.Under the optimal conditions,the protective effect of double-emulsion microcapsules and the addition of soybean oligosaccharides on Lactobacillus JYLA-191 were investigated,and four types of microcapsules were prepared:single-layer and double-emulsion microcapsules without soybean oligosaccharides（S₁ and S₃）,and single-layer and double-emulsion microcapsules with soybean oligosaccharides（S₂ and S₄）.Scanning electron microscopy showed that S₁had a higher concentration of Lactobacillus JYLA-191 on its surface,while the addition of soybean oligosaccharides reduced the number of probiotics on the surface.The surface of the double-emulsion microcapsules（S₃ and S₄）did not have any probiotics,but had more protrusions.Thermogravimetric analysis showed that the thermal stability of the double-emulsion microcapsules（S₃ and S₄）was poor.Freezing and drying,continuous digestion in the gastrointestinal tract,heat treatment,and long-term storage at different temperatures showed that the double-emulsion microcapsules had better resistance to adverse environments compared to the single-layer microcapsules,and the addition of soybean oligosaccharides provided better protection for the probiotics.Compared to storage at 25°C,the cells were more stable when stored at 4°C.2.Applied research was conducted on the four Lactobacillus JYLA-191 microcapsules mentioned above,and they all showed good cholesterol-lowering ability.Compared to free bacteria,the cholesterol degradation ability of the dissolved microcapsules was improved to a certain extent.The addition of free bacteria,S₁,S₂,S₃,and S4 to tea water caused a significant decrease in p H after being cultured at 37°C for 6 days.During storage,the microcapsules provided better protection for Lactobacillus JYLA-191,and the double-emulsion microcapsules with soybean oligosaccharides added exhibited the best protective effect.After being stored for6 days at 37°C,the live cell count of S4 decreased by only 2.51 lg（CFU/g）.