Application of fermentation product of fermentation pichia pastoris 15B1 in promoting proliferation of probiotic intestinal bacteria

Abstract

The invention relates to application of a fermentation product of fermented pichia pastoris 15B1 in proliferation of intestinal probiotics, and belongs to the technical field of biology. The fermentation product of the fermented pichia pastoris 15B1 disclosed by the invention has the effect of promoting the proliferation of Collinsella and Bifidobacterium in intestinal bacteria, and can be used as a new prebiotic additive.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to the application of fermentation products of Pichia fermentans strain 15B1 in the regulation of intestinal flora. BACKGROUND

[0002] Intestinal flora in the human body is an important part of the human body and plays a crucial role in ensuring human health. Bifidobacterium is a relatively important probiotic in the human body and has biological barrier, nutritional, anti-tumor, immune enhancement, gastrointestinal function improvement, anti-aging and other important physiological functions. Collinsella is also a probiotic with special value discovered in recent years. A research team found that intestinal bacteria "Collinsella" may be able to prevent COVID-19 infection and its severe illness. In addition, a German scientific team found that heart failure patients are often accompanied by a significant decrease in Collinsella in intestinal flora, and it is speculated that the decrease of these bacteria may lead to the occurrence of heart failure, because inflammation and increased intestinal permeability are related to the pathogenesis of heart failure. In summary, Collinsella mainly exists in the human intestine, is the dominant bacteria in the phylum Actinobacteria, is a genus of gas-producing bacteria, can promote the production of beta-steroid dehydrogenase, accelerate the synthesis of ursodeoxycholic acid in the body, improve the conversion efficiency of material and energy metabolism in the body, and enhance the antioxidant capacity and immune function of the body. However, the growth environment of the above-mentioned probiotics is harsh, and it is difficult to store and proliferate in vitro, and it is difficult to be ingested orally. The intake of prebiotics to promote the proliferation of these probiotics in the intestine is a better method, especially Collinsella, and there is no report on prebiotics for promoting the proliferation thereof. SUMMARY

[0003] In view of the problems existing in the prior art, the present application develops a technical solution for regulating the proportion of intestinal flora probiotics by using the product of Pichia fermentans culture on the basis of the original application "Fermented Pichia and its application" (Patent No. ZL202110032614.8). The Pichia fermentans strain 15B1 has a preservation number of CGMCC No. 19317. The fermentation product of the Pichia fermentans strain 15B1 promotes the proliferation of Collinsella and Bifidobacterium intestinal flora. The fermentation product of the Pichia fermentans strain 15B1 of the present application contains a component for regulating flora and has the potential to be developed into prebiotics. DETAILED DESCRIPTION

[0004] The application will be further described in connection with the following examples. The examples below do not limit the application.

[0005] Example 1: Fermentation and crude extract preparation Take the 15B1 bacteria liquid frozen at -80℃, under sterile conditions, spread on PDA flat plate, wait for the colony to grow, pick a single colony to a 500 mL shake flask containing 200 mL PDB medium, 200 rpm, 32℃ culture for 2 days, the bacteria liquid is loaded into a centrifuge tube, centrifuged at 4000 rpm for 10 min, the supernatant is filtered through a sterile filter membrane with a pore size of 0.22 μM, and stored at -20℃ for standby.

[0006] Example 2: Intestinal bacteria test According to the formula in Table 1, weigh and add reagents, and load the liquid medium into an anaerobic bottle, pass high-purity nitrogen for 1 min, immediately cover, and sterilize under high pressure for standby; prepare 1% cysteine mother liquor according to Table 2, pass high-purity nitrogen for 1 min, immediately cover, and sterilize under high pressure for standby; Prepare the 96-well enzyme-labeled plate for intestinal bacteria test on the super-clean workbench as follows: (1) place the enzyme-labeled plate in an anaerobic bag for more than 24 h; (2) take out the enzyme-labeled plate, add 150 μL YCFA liquid medium to each well, and then add 20 μL of liquid paraffin and vitamin E, silicon oil, respectively; (3) add the fermentation liquid or concentrated liquid obtained in Example 1 to the 1% cysteine solution at a ratio of 1 / 2, take 20 μL and add to the culture well, cover, and put back into the anaerobic bag, and store in the cold or at room temperature for more than 12 h, until the oxygen indicator turns red, and then use; Table 1. YCFA (1L) general liquid medium for intestinal bacteria Reagent Amount Casitone 10g Lab-lemco 10g yeast extract 2.5g NaHCO3 4g NaAc 2.71g Sodium propionate 0.86g Glucose 2g Maltose 2g Cellobiose 2g K2HPO4 0.45g KH2PO4 0.45g NaCl 0.9g MgSO4.7 H2O 0.09g CaCl2 0.09g Haemin 10 mg Resazurin 1 mg Isobutyrate 0.09g Isovalerate 0.10g Valerate 0.10g .

[0007] Table 2. 1% cysteine mother liquor (1L) Reagent Amount K2HPO4 0.45g KH2PO4 0.45g NaCl 0.9g MgSO4.7 H2O 0.09g CaCl2 0.09g Haemin 10 mg Resazurin stock (1000x) 1ml Isobutyrate 0.09g Isovalerate 0.10g Valerate 0.10g Cysteine 10g Take fresh intestinal bacteria samples from volunteers, resuspend 3 g of fecal sample in 30 mL of 1% cysteine mother liquor at a ratio of 10%, mix uniformly, and stand for 3 min; take out the culture plate with faded oxygen indicator color from the anaerobic bag, add 15 μL of bacteria liquid supernatant; paste a layer of optical film on the front of the enzyme-labeled plate, and place the plate in the anaerobic bag, and culture at 37℃ for 30 min; set the automatic detection program of the Thermovarioskan enzyme-labeled instrument and preheat to 37℃, then place the enzyme-labeled plate into the detection, and detect the OD value every 6 h; after 24 h, transfer 15 μL of culture to new medium, repeat for 2 days, and continuously culture for 72 h; The intestinal flora samples of the culture wells were collected after centrifugation, and the DNA was extracted by CTAB method. The deep sequencing region was V3-V4 region of 16s rRNA, and the primers were: 341F: 5'-CCTACGGGNGGCWGCAG-3' and 805R: 5'-GACTACHVGGGTATCTAATCC-3'. After removing the primer adapter sequence and removing each low-quality base (PhredQuality Score=20), the raw sequencing data was spliced. After removing non-specific amplification sequences and chimeras, the effective sequence data of each sample was obtained. Using the Mothur software package, 97% was used as the threshold to divide the operational taxonomic unit (OTU) of 16S sequence. Using RDP classifier2.2, the OTU representative sequence at 97% similarity level was subjected to taxonomic analysis, and the species classification information corresponding to each OTU was obtained, and the bacterial community composition of each sample was statistically analyzed at the level of kingdom, phylum, class, order, family and genus. The percentage of bacteria at the genus level was classified and screened, and the average value and standard error of the repeated samples in the same treatment group were calculated. The screening rule is: according to the average value of the intestinal flora content of the treatment group and the ratio of the content to the control, two indicators are used as screening requirements, and the top four bacteria with the highest intestinal flora content treated by 15B1 are screened (generally more than 5%, i.e. 0.05), and the bacteria with a ratio of 15B1 / CK greater than 2 are considered to be significantly up-regulated. The four bacteria in the intestinal flora after 15B1 treatment are shown in Tables 3, 4 and 5. As can be seen from the table, Collinsella1 is significantly up-regulated after 24h, which is 4.6 times that of the control, reaches 15.6 times at 48h, and reaches as much as 54.9 times at 72h. Although Bifidobacterium does not change significantly at 24h, it increases significantly at 48h, and is significantly up-regulated to 5.6 times at 72h, becoming the second dominant genus in the flora. Considering that Bifidobacterium grows slowly, this result is also in line with the actual situation of bacterial growth. Therefore, the fermentation product of Pichia fermentans strain 15B1 has a significant effect on the proliferation of Collinsella and Bifidobacterium in the intestinal flora; Table 3. Proportion of different groups of intestinal flora in number after 24h (calculated according to sequencing number) OTU ID ck mean 15B1 mean 15B / CK Collinsella 0.014±0.005 0.063±0.006 4.6 Streptococcus 0.489±0.014 0.417±0.020 0.9 Escherichia-Shigella 0.171±0.030 0.128±0.006 0.7 Bifidobacterium 0.068±0.008 ]]> ​ 0.045±0.005 ]]> ​ 0.7 .

[0008] Table 4. Proportion of different groups of intestinal flora in number after 48h (calculated according to sequencing number) OTU ID ck mean 15B1 mean 15B / CK Collinsella 0.008±0.001 0.121±0.019 15.6 Escherichia 0.082±0.014 0.158±0.006 1.9 Bifidobacterium 0.100±0.019 0.110±0.014 1.1 Streptococcus 0.751±0.042 0.550±0.030 0.7 .

[0009] Table 5. Proportion of different groups of intestinal bacteria in number (calculated from the number of sequences) after 72h OTU ID ck mean 15B1 mean 15B / CK Collinsella 0.002±0.001 0.120±0.040 54.9 Escherichia-Shigella 0.010±0.001 0.053±0.010 5.6 Bifidobacterium 0.034±0.009 0.158±0.021 4.6 Streptococcus 0.835±0.176 0.650±0.061 0.8

Claims

1. Pichia fermentans strain deposited under CGMCC No. 19317 Pichia fermentans Use of the fermentation product of strain 15B1 in promoting the proliferation of intestinal bacteria Collinsella and Bifidobacterium.