Application of lactobacillus delbrueckii subsp. Bulgaricus KSFY09 in preparation of medicine, food or health care product for regulating organism immunity

Abstract

The invention provides application of lactobacillus delbrueckii subsp. Bulgaricus KSFY09 in preparation of drugs, food or health care products for regulating organism immunity, and relates to the technical field of biological medicines. The lactobacillus delbrueckii subsp. Bulgaricus KSFY09 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.15716. The research finds that the lactobacillus delbrueckii subsp. Bulgaricus KSFY09 can regulate the immunity of the organism and has a wide application prospect in medicines, foods or health-care products for improving the yield of the organism.

Description

Technical Field

[0001] The invention provides an application of Lactobacillus delbrueckii subspecies bulgaricus KSFY09 in preparing medicines, foods or health products for regulating body immunity, belonging to the technical field of biomedicine. Background Art

[0002] The Kashgar region in the Xinjiang Uygur Autonomous Region of my country is an area where ethnic minorities live. The integration of multi-ethnic eating habits has made the local naturally fermented yogurt rich in nutritional value and good health benefits. The fermenting microorganisms in the yogurt also have the characteristics of strong resistance and good diversity, and are of great value for development and utilization.

[0003] At present, the lactic acid bacteria in fermented milk from dairy sources in my country mainly include other genera such as Lactobacillus, Bifidobacterium, Lactococcus, Enterococcus, Leuconostoc, Streptococcus and Pediococcus. Lactobacillus delbrueckii belongs to the genus Lactobacillus and is one of the representative species in the genus Lactobacillus.

[0004] At present, six subspecies of Lactobacillus delbrueckii have been found, and the 16S rRNA sequence similarity between the six subspecies is 99.21-99.54%. Among them, the Bulgarian subspecies is mainly isolated from milk sources and used in dairy fermentation, and is an edible species specified by the National Health Commission of my country. Lactobacillus delbrueckii subspecies Bulgarian subspecies is one of the most studied bacteria with probiotic properties. It is considered to be an excellent probiotic for regulating the human intestinal environment and producing essential metabolites such as lactic acid, acetic acid, propionic acid and butyric acid. It provides a unique flavor for dairy products, while giving dairy products special therapeutic effects, and also improving the functional characteristics and nutritional value of dairy products. Some fermented milk formulas of Lactobacillus delbrueckii can reduce intestinal epithelial tissue destruction by inhibiting goblet cell degeneration, intestinal permeability and inflammatory cell infiltration. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a use of Lactobacillus delbrueckii subspecies bulgaricus KSFY09 in the preparation of medicines, foods or health products for regulating body immunity.

[0006] Cytokines are the main signaling molecules released by inflammatory cells and are involved in multiple functions. The main pro-inflammatory cytokines include IL-1β, IL-6, and TNF-α. TNF-α can induce the formation of a tumor immunosuppressive microenvironment by activating inflammation-related signaling pathways and promoting tumor angiogenesis and tumor spread, which is positively correlated with the degree of inflammation. There is evidence from animal models that inflammatory mediators produced by inflammatory cells play an important role in promoting tumor development. As a cytokine that stimulates inflammatory responses, abnormal concentrations and overactivation of IL-6 in the body can promote tumor autocrine secretion and growth. Therefore, IL-6 is considered to be one of the important cytokines connecting inflammation and cancer.

[0007] The research results of the present invention show that LDSB-KSFY09 can significantly reduce the expression levels of inflammatory cytokines IL-1β, IL-6, TNF-α and messenger molecule iNOS in mouse serum and colon tissue, and can significantly improve the body's immunity.

[0008] The Lactobacillus delbrueckii subsp. bulgaricus KSFY09 (LDSB-KSFY09) of the present invention is currently deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC NO.15716.

[0009] The colorectal cancer mouse model constructed by the present invention using the chemical inducer AOM / DSS can reliably reproduce the stages of intestinal tumor occurrence and development, highly simulate the pathological conditions of human colorectal cancer, and this model can cause weight loss, inflammation and edema of organs in cancer-bearing mice. Organ weight and organ index are one of the basic indicators of biomedical research and an important basis for research. Inflammation and edema can lead to an increase in organ weight and organ index. The results of the present invention show that oral gavage intervention of LDSB-KSFY09 can effectively reduce the increase in spleen and liver index caused by AOM / DSS (P<0.05), and improve the inflammation and edema symptoms of the spleen and liver in colorectal cancer mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The effect of LDSB-KSFY09 on the body weight and organ indexes of mice;

[0011] Figure 2 The effect of LDSB-KSFY09 on mouse colorectal tissues;

[0012] Figure 3 The effect of LDSB-KSFY09 on rectal tissue pathology in mice;

[0013] Figure 4 The effect of LDSB-KSFY09 on the serum concentration of inflammation-related factors in mice;

[0014] Figure 5 The effect of LDSB-KSFY09 on the colon concentration of inflammation-related factors in mice;

[0015] Figure 6 This is the effect of LDSB-KSFY09 on the relative expression of apoptosis mRNA in mouse colon tissue cells. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the present invention clearer, the preferred embodiments of the present invention are further described in detail in conjunction with the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] 1 Materials and reagents

[0018] Experimental strain: Lactobacillus delbrueckiisubsp.bulgaricus KSFY09 (LDSB-KSFY09) was isolated from naturally fermented yogurt in herdsmen's homes in Kashgar, Xinjiang Uygur Autonomous Region by the Collaborative Innovation Center for Children's Nutrition and Healthy Development. It is now preserved in the China General Microbiological Culture Collection Center, with the collection number: CGMCC NO.15716.

[0019] Table 1 Experimental materials and reagents

[0020]

[0021] 2. Instruments and Equipment

[0022] Table 2 Instruments and equipment

[0023]

[0024] 3 Experimental methods

[0025] 3.1 Animal Experiment Design

[0026] A total of 40 SPF-grade 4-week-old male C57BL / 6 mice were purchased from Chongqing Labit Biotechnology Co., Ltd., with a 12h / 12h light-dark cycle, a temperature of 22℃~25℃, and a humidity of 50%±10%. They were fed with standard feed and had free access to water. After 1 week of adaptation, colorectal cancer modeling was performed. The mice were randomly divided into 5 groups: normal group, model group, aspirin group, LDSB-KSFY09 low concentration group (LDSB-KSFY09L), and LDSB-KSFY09 high concentration group (LDSB-KSFY09H). The experimental modeling cycle was 10 weeks. Except for the normal group, the other four groups were intraperitoneally injected with 10 mg / kg AOM on the first day of the experimental cycle, and then fed with 2.5% DSS aqueous solution in the 2nd, 5th, and 8th weeks, respectively. The intragastric administration lasted for 10 weeks. The normal group and the model group were given sterile saline daily, and the aspirin group was given 67 mg / kg aspirin solution daily. The intragastric concentration of the LDSB-KSFY09L group was 1×108 CFU / kg, and the intragastric concentration of the LDSB-KSFY09H group was 1×10 9CFU / kg. The mice were allowed to drink water freely at all times except for the DSS cycle. The changes in the state of the mice during the experiment were closely observed, and the weight of the experimental mice was recorded regularly and analyzed. After the end of the 10th week, all mice were fasted for 16-24h and dissected after weighing.

[0027] 3.2 Determination of organ indicators

[0028] The large intestine, spleen and liver of the mouse were removed by dissection, the tissues around the organs were removed, washed with physiological saline and then dried with filter paper, and weighed respectively. The mouse organ index was calculated according to formula (1):

[0029]

[0030] 3.3 Histopathological observation of mouse rectal tissue

[0031] Rectal tissue with a length not exceeding 0.5 cm was immersed in 4% paraformaldehyde fixative solution for 48 h, dehydrated with ethanol and embedded in paraffin, cut into slices of approximately 2-3 μm with a microtome, stained with H&E and fixed on a slide to prepare mouse rectal pathological tissue sections, and the morphological changes were observed under an upright microscope.

[0032] 3.4 Determination of levels of inflammatory factors in mouse serum and colon tissue

[0033] The whole blood of mice was collected and allowed to stand at room temperature for 30 minutes, centrifuged at 4°C and 4000 r / min for 15 minutes, and the upper serum was aspirated and stored at -80°C after appropriate aliquoting. Serum and colon tissue samples were prepared according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kit, and the levels of IL-6, IL-1β, NF-κB and iNOS in mouse serum and colon tissue were determined.

[0034] 3.5 Determination of expression levels of genes related to apoptosis pathway in mouse colon tissue

[0035] Weigh an appropriate amount of mouse colon tissue, add TRIzol for homogenization to extract total RNA from the tissue, then use a spectrophotometer to measure the concentration and purity of the RNA extract, and reverse transcribe the RNA extract into cDNA according to the instructions of the reverse transcription kit. After reverse transcription, prepare the cDNA reaction system in an eight-tube strip, namely 10μL of Master mix, 1μL of upstream and downstream primers, 1μL of template cDNA, and 7μL of sterile enzyme-free water. After the reaction system is prepared, place it on the StepOnePlus Real-Time PCR System for amplification detection. Amplification conditions: pre-denaturation at 95℃ for 5min, then denaturation at 95℃ for 10s, annealing at 60℃ for 30s, and the whole process is repeated 40 times. Eef2 is used as the internal reference gene and the expression is expressed as 2 -△△Ct The relative expression intensity of each gene was calculated by the primer sequence used in this experiment.

[0036] Table 3 Primer sequences

[0037]

[0038] 3.6 Data processing and statistical analysis

[0039] All data were expressed as mean ± standard deviation (SD) and analyzed using IBM SPSS 23.0 statistical software. One-way analysis of variance (ANOVA) and Duncan's multiple range test were used for comparison between groups. The same lowercase letters in ae indicate no significant difference between the two groups, and different lowercase letters indicate significant difference between the two groups (P < 0.05).

[0040] 4 Results and analysis

[0041] 4.1 Effects of LDSB-KSFY09 on body weight and organ indexes of mice

[0042] like Figure 1 As shown in A, during the period of DSS administration (weeks 2, 5, and 8), the body weight of mice in each modeling group showed a downward trend. After the cessation of DSS administration, the body weight of mice in each group showed a trend of gradual increase and recovery. After the experimental modeling period, the body weight of mice in the model group was significantly lower than that in the normal group, while the mice in the aspirin and LDSB-KSFY09 intervention groups showed varying degrees of recovery, and the difference was significant compared with the model group (P<0.05). Among them, the body weight of LDSB-KSFY09H mice was heavier than that of the LDSB-KSFY09L concentration group, and there was no significant difference with the aspirin group. The results show that AOM / DSS can cause weight loss in mice, and aspirin and LDSB-KSFY09 intervention can alleviate the trend of weight loss in mice to a certain extent. Organ index is one of the basic indicators of biomedical research and an important basis for research. Figure 1As shown in B and C, compared with the normal group, the spleen index and liver index of the model group mice increased significantly (P<0.05), which is speculated that the treatment of DSS caused organ enlargement and inflammation in the mice. Compared with the model group, the organ indexes of the aspirin group, LDSB-KSFY09L group and LDSB-KSFY09H group were significantly reduced (P<0.05), indicating that LDSB-KSFY09 can effectively alleviate the increase of spleen and liver indexes caused by AOM / DSS.

[0043] 4.2 Effects of LDSB-KSFY09 on colorectal-related indicators and intestinal tumor incidence in mice

[0044] When the colorectum of mice is inflamed, the length of the colorectum shrinks and swells to a certain extent. Figure 2 As shown in A and B, the ratio of colorectal weight to colorectal length of mice in the model group was significantly different from that in the normal group (P<0.05). The colorectal length of mice in the healthy normal group was the longest among the five groups. After AOM / DSS modeling, the colorectal length of mice was significantly affected. LDSB-KSFY09 intervention can alleviate the shortening of the colorectum of mice and restore the colorectal weight and length ratio. The effect of high concentration of LDSB-KSFY09 was significantly better than that of low concentration (P<0.05). At the same time, the number of intestinal tumors produced in large numbers in the model group ( Figure 2 C) indicates that AOM / DSS successfully induced colorectal cancer in experimental mice. Compared with the control group, the number of intestinal tumors in mice was significantly reduced after intervention with aspirin and LDSB-KSFY09 (P<0.05). The number of intestinal tumors in the aspirin group was similar to that in the high-concentration LDSB-KSFY09 group, and the effect of the high-concentration LDSB-KSFY09 group was significantly better than that of the low-concentration LDSB-KSFY09 group (P<0.05). This indicates that the intervention of LDSB-KSFY09 can effectively reduce the number of intestinal tumors in mice.

[0045] 4.3 Effect of LDSB-KSFY09 on rectal histopathology in mice

[0046] H&E staining analysis showed that ( Figure 3), the rectal mucosal epithelial cells of the normal group mice were intact, the crypts were normal, the glands were neatly arranged, and there was no ulcer; in the model group, the rectal mucosa was severely eroded, the crypts were highly damaged, the goblet cells were sharply reduced, there was no clear glandular structure, there were a large number of inflammatory cells infiltrated, and tumors occupying the entire intestinal cavity were seen. After intervention with aspirin and LDSB-KSFY09, the pathological state of the rectum of mice was significantly improved, the crypts were relatively intact, clear glandular structures were visible, and they were neatly arranged, the number of goblet cells increased, and the structural integrity of the intestinal mucosa was effectively maintained. Among them, the aspirin group had a low degree of inflammatory cell infiltration and less crypt structure damage; although the LDSB-KSFY09 group had slight inflammatory infiltration, the crypt structure was relatively intact. Compared with low concentrations of LDSB-KSFY09, high concentrations of LDSB-KSFY09 improved the pathological damage of rectal tissue caused by AOM / DSS to a greater extent.

[0047] Effects of LDSB-KSFY09 on serum inflammatory cytokine levels in mice

[0048] This study analyzed the effect of LDSB-KSFY09 on the concentrations of serum inflammatory cytokines (IL-1β, IL-6, TNF-α, and iNOS) in mice with inflammation-related CRC. Figure 4 , Figure 5 ), after AOM / DSS modeling, the levels of serum IL-1β, IL-6 and TNF-α in the model group mice were significantly increased, and aspirin and LDSB-KSFY09 could effectively reduce their expression (P<0.05). The serum of the model group mice contained high concentrations of iNOS, and the iNOS concentrations in the serum of the normal group, aspirin group and LDSB-KSFY09 group mice were lower than those in the model group, and there were significant differences (P<0.05). Among them, the effect of high concentration of LDSB-KSFY09 on reducing inflammatory-related factors was significantly better than that of low concentration of LDSB-KSFY09L (P<0.05), indicating that high concentration of LDSB-KSFY09 can play an anti-inflammatory role by reducing the concentration of serum inflammatory factors.

[0049] Effects of LDSB-KSFY09 on the levels of severe inflammatory cytokines in the colon of mice

[0050] This study also analyzed the changes in IL-1β, IL-6, TNF-α and iNOS concentrations in mouse colon tissue ( Figure 5), induced by AOM / DSS, the concentrations of inflammatory cytokines IL-1β, IL-6, TNF-α and iNOS in the colon of the model group mice were significantly increased compared with the normal group (P<0.05). After oral administration of LDSB-KSFY09, the concentrations of IL-1β, IL-6, TNF-α and iNOS in the colon tissue of each group of mice were observed to decrease to varying degrees, which was significantly different from that of the model group mice, and the effect of high concentration LDSB-KSFY09 in reducing inflammatory cytokines was significantly better than that of low concentration (P<0.05). This result is consistent with the trend of inflammatory cytokines in mouse serum measured in the previous period, further indicating that LDSB-KSFY09 can effectively reduce the level of inflammation in mice.

[0051] 4.6 Effect of LDSB-KSFY09 on mRNA expression levels of apoptosis-related factors in mouse colon tissue

[0052] In this study, real-time fluorescence quantitative PCR was used to analyze the mRNA expression levels of Bcl-2, Bcl-xL, Bcl-w, and Bax in mouse colon tissues. The results showed that ( Figure 6 ), compared with the normal group, the relative mRNA expression of Bcl-2, Bcl-xL and Bcl-w in the colon tissue of the model group mice increased significantly, and the mRNA expression level of Bax decreased significantly. After the intervention of aspirin and LDSB-KSFY09, the expression levels of Bcl-2, Bcl-xL and Bcl-w were significantly downregulated, and the mRNA expression level of Bax was also significantly upregulated (P<0.05). Compared with the low concentration of LDSB-KSFY09, the effect of high concentration gavage was more significant.

[0053] In this study, the effect and mechanism of LDSB-KSFY09 isolated from traditional fermented yogurt in Xinjiang Uygur Autonomous Region on the intervention of inflammation-related colorectal cancer were studied in five aspects, including the body weight and organ index of mice, intestinal index and number of intestinal tumors, rectal tissue pathological analysis, serum and colon tissue inflammation-related cytokine analysis, and colon tissue cell apoptosis signaling pathway-related gene expression analysis, using AOM / DSS-induced mouse inflammation-related colorectal cancer model. The experimental results showed that LDSB-KSFY09 significantly improved the symptoms of weight loss, increased organ index, shortened colon and increased intestinal index caused by cancer in mice, effectively reduced the incidence of intestinal tumors in cancer-bearing mice, and improved rectal tissue pathological damage (P<0.05). At the same time, the intervention of LDSB-KSFY09 also significantly reduced the expression levels of inflammatory cytokines IL-1β, IL-6, TNF-α and messenger molecule iNOS in mouse serum and colon tissue (P<0.05). In addition, LDSB-KSFY09 can also significantly downregulate the mRNA expression of anti-apoptotic factors Bcl-2, Bcl-xL and Bcl-w in colon tissue at the gene level, and upregulate the mRNA expression of pro-apoptotic factor Bax (P<0.05). This study first suggested the significant effect of LDSB-KSFY09 in intervening in the development of inflammation-related colorectal cancer in mice, revealing the anti-inflammatory effect and the ensuing anti-tumor effect on inflammation-related colorectal cancer, and can be used as a potential strain for the prevention and adjuvant treatment of inflammation-related colorectal cancer. It provides scientific evidence for the healthy functionality of LDSB-KSFY09 on the intestine, and provides more scientific basis for the prevention and adjuvant treatment of colorectal cancer from the dietary aspect, which can be further studied from the clinical aspect in the future.

[0054] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. Application of Lactobacillus delbrueckii subspecies bulgaricus KSFY09 in the preparation of medicines, foods or health products for regulating body immunity, characterized in that: The Lactobacillus plantarum KSFY16 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number being CGMCC No.15716.

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