Candida tropicalis, application of candida tropicalis, astragalus polysaccharide fermentation product and preparation method of astragalus polysaccharide fermentation product

Abstract

The invention belongs to the field of biology, and discloses a strain of candida tropicalis, which is classified and named as Candida tropicalis, and has a preservation number of GDMCC NO: 65030. The preservation date is August 20, 2024, and the strain is preserved in the Guangdong Microbial Culture Collection Center at the fifth floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou City. After the candida tropicalis is used for fermenting the astragalus polysaccharide, the astragalus polysaccharide can show a more excellent immunological enhancement effect, animals can be stimulated to generate higher antibody titer after immunization, and immune adverse reaction is relieved. Meanwhile, the invention further provides an astragalus polysaccharide fermentation product, a preparation method and application.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to tropical candida and its use, as well as an astragalus polysaccharide fermentation product and a preparation method. Background Art

[0002] Astragalus polysaccharide is widely used as an immunopotentiator in the agricultural field; for example, publication number CN118903404A, the subject of which is a patent application for an inactivated vaccine immunopotentiator for poultry, its preparation method and application, and publication number CN116531498A, the subject of which is a patent application for an immunopotentiator, its preparation method and application, discloses that the use of astragalus polysaccharide as an immunopotentiator can enable veterinary vaccines to produce high levels of humoral, cellular and mucosal immune responses.

[0003] In the prior art, it is relatively rare to use Astragalus polysaccharide as a single immunopotentiator. It is generally used together with other polysaccharides or functional ingredients to improve the immune effect of the vaccine.

[0004] The technical problem solved in this case is: how to further improve the immune-enhancing effect of Astragalus polysaccharides. Summary of the invention

[0005] The purpose of the present invention is to provide a tropical Candida yeast, which can make the astragalus polysaccharide show a more excellent immune enhancement effect after being used for fermentation of astragalus polysaccharide, can stimulate animals to produce higher antibody titers after immunization, and reduce adverse immune reactions.

[0006] At the same time, the present invention also provides an astragalus polysaccharide fermentation product, a preparation method and an application.

[0007] To achieve the above objectives, this application discloses: A strain of Candida tropicalis, taxonomically named C andida tropocalis , the deposit number is GDMCC NO: 65030; the deposit date is August 20, 2024, and it is deposited in Guangdong Provincial Microbiological Culture Collection Center, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] Meanwhile, the invention also discloses the use of fermenting astragalus polysaccharide with the tropical candida as mentioned above.

[0009] In addition, the present invention also discloses a method for preparing an astragalus polysaccharide fermentation product, which uses the tropical candida to anaerobically ferment the astragalus polysaccharide to obtain the astragalus polysaccharide fermentation product.

[0010] In the above-mentioned preparation method, the method is specifically: Step 1: Activate Candida tropicalis to obtain a viable count of 1~10*10 8CFU / mL of bacterial solution; Step 2: inoculate the bacterial liquid into a culture medium containing sterilized astragalus polysaccharide, control the fermentation temperature at 30-35°C in an oxygen-free state, and ferment for 5-10 days to obtain an astragalus polysaccharide fermentation product.

[0011] In the above preparation method, the culture medium consists of 100 parts by weight of astragalus polysaccharide extract, 2 to 4 parts by weight of glucose, 1 to 2 parts by weight of molasses, 3 to 5 parts by weight of peptone, and 0.1 to 0.3 parts by weight of inorganic salts.

[0012] Astragalus polysaccharide extract is produced by our company, and its composition is: solid content is about 35wt%, of which astragalus polysaccharide is about 30wt% and astragaloside IV is about 0.07wt%.

[0013] In the above preparation method, the weight ratio of the bacterial liquid to the culture medium is 1-5:100.

[0014] In the above preparation method, the activation method in step 1 is: inoculating tropical Candida into malt wort liquid culture medium, and culturing on a shaker at 28-37° C. for 6-24 hours.

[0015] Meanwhile, the invention also discloses an astragalus polysaccharide fermentation product, which is prepared by adopting the above-mentioned preparation method.

[0016] Finally, the present invention also discloses the use of the astragalus polysaccharide fermentation product as described above in preparing an immunopotentiator.

[0017] In the above-mentioned use, the immunopotentiator is added to animal feed in the form of a feed additive before or after the animal is vaccinated.

[0018] In the above-mentioned use, the animal is chicken, duck or pig.

[0019] This application has at least the following beneficial effects: 1. The present invention uses tropical Candida to ferment astragalus polysaccharide, which can significantly improve the immune enhancement effect of the fermentation product, increase the antibody titer after immunization, and reduce adverse immune reactions.

[0020] 2. The fermentation product of astragalus polysaccharide of the present invention can also increase the growth rate of animals and reduce the feed-egg ratio. DETAILED DESCRIPTION

[0021] The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that, if no specific conditions are specified in the embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0022] The strains used in the present invention are derived from: Candida tropicalis (GDMCC 65030): Classification and naming candida tropocalis , the deposit number is GDMCC NO: 65030; the deposit date is August 20, 2024, and it is deposited in Guangdong Provincial Microbiological Culture Collection Center, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Enterococcus faecium (GDMCC 65031): Classification and naming Enterococcus faecium , the deposit number is GDMCCNO: 65031; the deposit date is August 20, 2024, and it is deposited in Guangdong Provincial Microbiological Culture Collection Center, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Bacillus subtilis (GDMCC 65032): Classification and naming Bacillus subtilis , the deposit number is GDMCCNO: 65032; the deposit date is August 20, 2024, and it is deposited in Guangdong Provincial Microbiological Culture Collection Center, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0023] Example The bacterial liquid is inoculated into a culture medium containing sterilized astragalus polysaccharide, and the fermentation temperature is controlled at 30-35°C under the condition of oxygen isolation, and the fermentation is carried out for 6 days. After the fermentation is completed, the culture medium is sterilized to obtain an astragalus polysaccharide fermentation product, wherein the culture medium is composed of 100 parts by weight of astragalus polysaccharide extract, 3 parts by weight of glucose, 2 parts by weight of molasses, 5 parts by weight of peptone, and 0.2 parts by weight of inorganic salt, wherein the inorganic salt is composed of sodium chloride and dipotassium hydrogen phosphate in a weight ratio of 1:1. The astragalus polysaccharide extract is produced by our company and is an extract extracted from the whole components of astragalus, and its composition is: solid content 35wt%, of which astragalus polysaccharide is about 30wt%, and astragaloside IV is about 0.07wt%.

[0024] 2 kg of bacterial liquid was added to every 100 kg of culture medium; the activation method of the above strains was as follows: each strain that grew well after activation was selected and inoculated into the malt juice liquid culture medium, wherein the culture temperature of Enterococcus faecium was 28-30°C and the culture condition was about 8 hours; the culture temperature of Candida tropicalis and Bacillus subtilis was 35-37°C and the culture time was about 12 hours; after measurement, the number of viable bacteria in the three bacterial liquids was 8*10 8 CFU / mL or so.

[0025] Finally, astragalus polysaccharide fermentation products 1 to 3 are obtained, wherein: The fermentation strain of astragalus polysaccharide fermentation product 1 was Candida tropicalis (GDMCC 65030); The fermentation strain of Astragalus polysaccharide fermentation product 2 was Enterococcus faecium (GDMCC 65031); The fermentation strain of astragalus polysaccharide fermentation product 3 is Bacillus subtilis (GDMCC 65032).

[0026] Immunity enhancement experiment 1. Chicken immune enhancement experiment 1.1 Application in production at Shandong Hanting broiler breeder farm.

[0027] A total of 5,000 Lifeng broiler breeders aged 48 weeks were selected and divided into five groups. The control group did not use fermented astragalus polysaccharide powder, and the experimental group added 0.1wt% fermented astragalus polysaccharide powder to the feed for five days before and after immunization with vaccine (ND Newcastle disease). The antibody titer was monitored 7 days after immunization. 50 blood samples were randomly collected from each of the control group and the experimental group, and the serum was separated and the antibody titer was determined.

[0028] The experimental groups are: Immunoenhancing group 1: chicken diets supplemented with astragalus polysaccharide fermentation product 1; Immunoenhanced group 2: Astragalus polysaccharide fermentation product 2 was added to the chicken diet; Immunoenhanced group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3; Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet; Blank group: only fed with diet.

[0029] The experimental results are shown in Table 1 below; Table 1 Antibody levels before and after immunization Unit: log2 Immune Enhancement Group 1 Immune Enhancement Group 2 Immune Enhancement Group 3 Immune Enhancement Group 4 Blank group Before immunization 10.3 10.7 11.2 10.9 10.8 7d 11.4 11.2 11.8 11.6 11.1 Titer change value 1.1 0.5 0.6 0.7 0.3

[0030] 1.2 Application in production at Shandong Hanting broiler breeder farm.

[0031] A total of 5,000 Lifeng broiler breeders aged 48 weeks were selected and divided into five groups. The control group did not use fermented astragalus polysaccharide powder, and the experimental group added 0.1wt% fermented astragalus polysaccharide powder to the feed for five days before and after immunization with the vaccine (H5 avian influenza). The antibody titer was monitored 7 days after immunization. 50 blood samples were randomly collected from each of the control group and the experimental group, and the serum was separated and the antibody titer was determined.

[0032] The experimental groups are: Immunoenhancing group 1: chicken diets supplemented with astragalus polysaccharide fermentation product 1; Immunoenhanced group 2: Astragalus polysaccharide fermentation product 2 was added to the chicken diet; Immunoenhanced group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3; Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet; Blank group: only fed with diet.

[0033] The experimental results are shown in Table 2 below; Table 2 Antibody levels before and after immunization Unit: log2 Immune Enhancement Group 1 Immune Enhancement Group 2 Immune Enhancement Group 3 Immune Enhancement Group 4 Blank group Before immunization 9.1 9.7 9.6 9.3 9.6 7d 10.1 10.2 10.3 9.9 9.8 Titer change value 1 0.5 0.7 0.6 0.2

[0034] 1.3 Application in production at Shandong Hanting broiler breeder farm.

[0035] A total of 5,000 Lifeng broiler breeders aged 48 weeks were selected and divided into five groups. The control group did not use fermented astragalus polysaccharide powder, and the experimental group added 0.1wt% fermented astragalus polysaccharide powder in the feed for five days before and after immunization with vaccine (H9 avian influenza). The antibody titer was monitored 7 days after immunization. 50 blood samples were randomly collected from the control group and the experimental group, and the serum was separated and the antibody titer was determined.

[0036] The experimental groups are: Immunoenhancing group 1: chicken diets supplemented with astragalus polysaccharide fermentation product 1; Immunoenhanced group 2: Astragalus polysaccharide fermentation product 2 was added to the chicken diet; Immunoenhanced group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3; Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet; Blank group: only fed with diet.

[0037] The experimental results are shown in Table 3 below; Table 3 Antibody levels before and after immunization Unit: log2 Immune Enhancement Group 1 Immune Enhancement Group 2 Immune Enhancement Group 3 Immune Enhancement Group 4 Blank group Before immunization 11.2 11.6 11.9 11.5 11.4 7d 11.9 12.1 12.4 12 11.6 Titer change value 0.7 0.5 0.5 0.5 0.2

[0038] 1.4 Application in production at Linquan laying hen farm in Yutian County, Hebei Province.

[0039] A total of 8,000 Hy-Line brown chickens aged 35 weeks were selected and divided into five groups. The control group did not use fermented astragalus polysaccharide powder, while the experimental group added 0.1% fermented astragalus polysaccharide powder in the feed for five days before and after immunization with the vaccine (H7 avian influenza). The antibody titer was monitored 7 days after immunization. 60 blood samples were randomly collected from each of the control group and the experimental group, and the serum was separated and the antibody titer was determined.

[0040] The experimental groups are: Immunoenhancing group 1: chicken diets supplemented with astragalus polysaccharide fermentation product 1; Immunoenhanced group 2: Astragalus polysaccharide fermentation product 2 was added to the chicken diet; Immunoenhanced group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3; Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet; Blank group: only fed with diet.

[0041] The experimental results are shown in Table 4 below; Table 4 Antibody levels before and after immunization Unit: log2 Immune Enhancement Group 1 Immune Enhancement Group 2 Immune Enhancement Group 3 Immune Enhancement Group 4 Blank group Before immunization 9 9.2 9.4 9.1 9.4 7d 10.1 9.8 10.1 9.9 9.7 Titer change value 1.1 0.6 0.7 0.8 0.3

[0042] 1.5 Application in production at Linquan laying hen farm in Yutian County, Hebei Province.

[0043] 8,000 35-week-old Hy-Line brown laying hens were selected and divided into 5 groups. The control group did not use fermented astragalus polysaccharide powder, and the experimental group added 0.1wt% fermented astragalus polysaccharide powder to the feed for five days before and after immunization with vaccine (ND Newcastle disease). The antibody titer was monitored 7 days after immunization. 60 blood samples were randomly collected from the control group and the experimental group, and the serum was separated to determine the antibody titer. The average egg production rate was observed on the 7th to 9th day after immunization.

[0044] The experimental groups are: Immunoenhancing group 1: chicken diets supplemented with astragalus polysaccharide fermentation product 1; Immunoenhanced group 2: Astragalus polysaccharide fermentation product 2 was added to the chicken diet; Immunoenhanced group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3; Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet; Blank group: only fed with diet.

[0045] The experimental results are shown in Table 5 below; Table 5 Antibody levels before and after immunization Titer unit: log2 Immune Enhancement Group 1 Immune Enhancement Group 2 Immune Enhancement Group 3 Immune Enhancement Group 4 Blank group Before immunization 8 9 9 9.1 9 7d 10 10 10 10 9.3 Titer change value 2 1 1 0.9 0.3 Egg production rate% 94.8 94.2 94.3 94.2 93.9

[0046] 1.6. Application in production at a yellow-feathered broiler breeder farm in Guangxi.

[0047] A total of 5000 yellow-feathered broiler breeders aged 43 weeks were selected and divided into 5 groups. The control group did not use fermented astragalus polysaccharide powder, while the experimental group added 0.1% fermented astragalus polysaccharide powder to the feed for 12 weeks. There were 100 chickens in each control group and observation group, and their production performance (egg production rate, egg weight, feed-egg ratio) was observed.

[0048] The experimental groups are: Immunoenhancing group 1: chicken diets supplemented with astragalus polysaccharide fermentation product 1; Immunoenhanced group 2: Astragalus polysaccharide fermentation product 2 was added to the chicken diet; Immunoenhanced group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3; Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet; Blank group: only fed with diet.

[0049] The experimental results are shown in Table 6 below; Table 6 Production performance table Immune Enhancement Group 1 Immune Enhancement Group 2 Immune Enhancement Group 3 Immune Enhancement Group 4 Blank group Average egg production rate% 91.5 90.5 90.6 89.3 88.5 Average egg weight g 59.8 58.6 58.9 58.8 57.9 Feed to Egg Ratio 1.92 2.02 2.08 2.10 2.25

[0050] Result analysis: 1. According to the experimental results of the immune effects in Tables 1 to 6 above, the astragalus polysaccharide fermentation product 1 obtained by fermenting the tropical Candida obtained by the present invention can significantly improve the immune titer after immunization, and the improvement rate is more than 50% compared with other fermentation bacteria; 2. Combined with the data of immune enhancement groups 2 to 5, the immune enhancement effect obtained by fermenting the extract with different strains is not certain. According to the data in Table 1 and Table 4, the use of other strains to ferment Astragalus polysaccharides cannot make the effect better than that of the immune enhancement group using only the extract.

[0051] 3. Based on the results of egg production and production efficiency in Table 5 and Table 6, the use of the astragalus polysaccharide fermentation product 1 of the present invention can significantly reduce the feed-egg ratio and significantly increase the egg production rate and egg weight compared to other strains of fermentation extracts; Combining the data from immune enhancement groups 2 to 4, we can draw a general conclusion: in terms of production performance, the product obtained by fermenting the extract with conventional strains will be better than the extract itself.

[0052] Therefore, from the perspective of the dual purpose of improving the immune effect and production efficiency, the strains screened by the present invention have great advantages.

[0053] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, and it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A strain of Candida tropicalis, characterized in that Classification Name C andida tropocalis , the deposit number is GDMCC NO: 65030; the deposit date is August 20, 2024, and it is deposited in Guangdong Provincial Microbiological Culture Collection Center, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the tropical candida fermented astragalus polysaccharide as claimed in claim 1.

3. A method for preparing an astragalus polysaccharide fermentation product, characterized in that: The astragalus polysaccharide is anaerobic fermented by the tropical candida as claimed in claim 1 to obtain an astragalus polysaccharide fermentation product.

4. The preparation method according to claim 3, characterized in that: The method is specifically as follows: Step 1: Activate tropical Candida to obtain a viable count of 1~10*10 8 CFU / mL of bacterial solution; Step 2: inoculate the bacterial liquid into a culture medium containing sterilized astragalus polysaccharide, control the fermentation temperature at 30-35°C in an oxygen-free state, and ferment for 5-10 days to obtain an astragalus polysaccharide fermentation product.

5. The preparation method according to claim 4, characterized in that: The culture medium consists of 100 parts by weight of astragalus polysaccharide extract, 2-4 parts by weight of glucose, 1-2 parts by weight of molasses, 3-5 parts by weight of peptone, and 0.1-0.3 parts by weight of inorganic salt.

6. The preparation method according to claim 4, characterized in that: The weight ratio of the bacterial liquid to the culture medium is 1-5:

100.

7. The preparation method according to claim 4, characterized in that: The activation method in step 1 is: inoculating tropical Candida into malt wort liquid culture medium, and culturing on a shaker at 28-37° C. for 6-24 hours.

8. An astragalus polysaccharide fermentation product, characterized in that: The preparation is obtained by the preparation method according to any one of claims 3 to 7.

9. Use of the astragalus polysaccharide fermentation product as claimed in claim 8 in preparing an immunopotentiator.

10. The use according to claim 9, characterized in that The immunopotentiator is used to be added to animal feed in the form of a feed additive before or after the animal is vaccinated, and the animal is a chicken, a duck or a pig.