| Probiotics have a variety of biological functions and are widely used in food,medicine,health care and other fields.At present,domestic research on the biological activity of probiotics is mainly limited to living bacteria,and research on inactivation of probiotics has rarely been reported.It has been found that probiotics or dead cells have many similar physiological functions,and inactivated probiotics have great advantages in safety,storage,and maintenance of microenvironmental flora balance.In this experiment,a commercially available strain of Bacillus subtilis was firstly cultured and identified,and then SPF Kunming mice were selected as test animals,setting high(1.0×10~8 CFU/mL),medium(1.0×10~7CFU/mL),and low(1.0×10~6 CFU/mL)dose inactivated bacteria group(IVC)and live bacteria control group(VC)(1.0×10~7 CFU/mL)and blank control group(BC)were administered to test mice to study on the effect of heat-inactivated Bacillus subtilis on immune index in mice,the main test results are as follows:1.Cultivation and identification of test strains.The purchased test strains were proliferated and cultured to prepare a bacterial growth curve.The colony and bacterial morphological characteristics were observed,and the bacterial 16S rDNA sequence was determined and the bacteria were identified.The results showed that the optimal culture time of the test strain was 10-14 h,and its morphological observation was consistent with the morphological characteristics of Bacillus subtilis;The sequence similarity between the 16S rDNA amplification sequence of the test strain and the standard strain Bacillus(ID:KY283146.1)was 100%,and the test strain was determined to be Bacillus subtilis.2.Heat-inactivated Bacillus subtilis safety evaluation.The levels of serum ALT,BUN and CREA in each test group were measured by biochemical analyzer.Small intestine tissue sections were made and the integrity of the small intestinal mucosa was observed.The results showed that there was no significant difference in serum biochemical parameters between the experimental group and the control group;In the low power field,the small intestine wall of each group was clear,the epithelial mucosa was clear,there was no inflammatory cell infiltration,the small intestine villus was intact,and the lamina propria cells were closely arranged,which indicated that the heat inactivated Bacillus subtilis had higher safety.3.The effect of heat-inactivated Bacillus subtilis on various immune indexes in mice.40mice were randomly divided into 10 groups.According to the dose of 0.3 mL/piece/day,The experimental group and the living bacteria group were infused with high,middle and low doses of Bacillus subtilis and medium dose of live bacteria,respectively,and the blank control group were given LB broth.The immune indicators were tested after 7 days and 14 days of gavage:(1)The number of IgA cells and expression intensity of positive intestinal mucosa.Immunohistochemistry was used to detect the number and expression intensity of positive IgA cells in the small intestinal mucosa of each group.The results showed that the H-score scores of the IVC groups were higher than those of the BC control group after 7 days and 14 days of gavage,and were highest in the IVC medium dose group.This indicates that heat-inactivated Bacillus subtilis can increase the spleen index of mice,and the effect is most significant in the middle dose..(2)Determination of spleen index.The body weight of each group of mice was weighed.The mice were sacrificed after routine eyeball collection.The spleen was aseptically removed from the clean bench,the surface adipose tissue was removed,and the spleen weight of each group was recorded.The results showed that the spleen index of IVC middle dose group was significantly higher than that of other groups after 7 days of gavage(P<0.05).The spleen index of IVC group was higher than that of BC control group after 14 days of gavage,but the difference was not significant(P>0.05).This indicates that heat-inactivated Bacillus subtilis can promote the growth of spleen in mice,and the effect is most significant in the middle dose.(3)Determination of whole blood lymphocytes and neutrophils.The number of lymphocytes and neutrophils in the whole blood samples of each test group was measured using a blood cell analyzer.The results showed that after 7 and 14 days of intragastric administration,the number of whole blood lymphocytes and neutrophils in the middle and high dose groups of IVC were higher than those in the VC group and the BC group,and the IVC high dose group was significantly higher than the BC group(P<0.05).This indicates that heat-inactivated Bacillus subtilis can increase the number of blood lymphocytes and neutrophils in mice,and the high dose effect is most significant.(4)Determination of serum cytokine levels such as IL-2,IL-6,and IFN-γ.The assay was carried out by enzyme-linked immunosorbent assay(ELISA).The results showed that the levels of serum cytokines in IVC group were higher than those in BC group after 7 and 14days of intragastric administration,and the high dose group of IVC except IFN-γwas significantly higher than that of BC group(P<0.05).This indicates that heat-inactivated Bacillus subtilis can significantly increase the levels of serum cytokines such as IL-2,IL-6and IFN-γin mice,and the effect is significant at high doses.(5)Relative expression level of IL-2 mRNA.Refer to the RNA extraction kit procedure to extract total RNA,and reverse-transcribe total RNA into cDNA,then amplify the sample cDNA,record the number of cycles(Ct value)when the fluorescence signal reaches the threshold in the system,and useβ-Actin as the internal reference.The relative expression level of IL-2 was calibrated.The results showed that after 7 days and 14 days of intragastric administration,the relative expression of IL-2 mRNA in the middle and high dose groups of IVC was up-regulated in the BC group,which was consistent with the serum IL-2 content. |
