A strain of tropical Candida and its use, as well as an astragalus polysaccharide fermentation product and its preparation method

Abstract

This application belongs to the field of biology and discloses a strain of Candida tropicalis, which is classified and named candida tropicalis The deposit number is GDMCC NO: 65030; the deposit date is August 20, 2024, and it is deposited at the Guangdong Provincial Center for Microbiological Culture Collection, with the deposit address being: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. When fermented with this tropical yeast, Astragalus polysaccharide can enhance the immune-enhancing effect of Astragalus polysaccharide, stimulate animals to produce higher antibody titers after immunization, and reduce adverse immune reactions. The present invention also provides an Astragalus polysaccharide fermentation product, a preparation method, and applications.

Description

Technical Field

[0001] The present invention relates to the biological field, in particular to Candida tropicalis and its application, 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, which is a patent application for an inactivated poultry vaccine immunopotentiator, its preparation method and application, and publication number CN116531498A, which is a patent application for an immunopotentiator, its preparation method and application, disclose 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 immune enhancer. It is generally used together with other polysaccharides or functional components 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 present invention aims to provide a strain of Candida tropicalis, which can make Astragalus polysaccharide exhibit a more excellent immune enhancement effect after fermentation of Astragalus polysaccharide by the Candida tropicalis, 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:

[0008] A strain of Candida tropicalis, classified as C. andida tropicalis , 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.

[0009] At the same time, the present invention also discloses the use of the tropical candida fermenting astragalus polysaccharide.

[0010] In addition, the present invention also discloses a method for preparing an astragalus polysaccharide fermentation product, which comprises anaerobically fermenting the astragalus polysaccharide with the above-mentioned Candida tropicalis to obtain the astragalus polysaccharide fermentation product.

[0011] In the above-mentioned preparation method, the method is specifically as follows:

[0012] Step 1: Activate Candida tropicalis to obtain a viable cell count of 1~10*10 8 CFU / mL of bacterial solution;

[0013] 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.

[0014] In the above preparation method, the culture medium is composed 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.

[0015] 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%.

[0016] In the above preparation method, the weight ratio of the bacterial liquid to the culture medium is 1-5:100.

[0017] In the above preparation method, the activation method in step 1 is: inoculating Candida tropicalis into malt wort liquid culture medium, and culturing on a shaker at 28-37° C. for 6-24 hours.

[0018] At the same time, the present invention also discloses an astragalus polysaccharide fermentation product, which is prepared by adopting the above preparation method.

[0019] Finally, the present invention also discloses the use of the astragalus polysaccharide fermentation product described above in preparing an immunopotentiator.

[0020] 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.

[0021] In the above-mentioned use, the animal is chicken, duck or pig.

[0022] This application has at least the following beneficial effects:

[0023] 1. The present invention uses tropical yeast 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.

[0024] 2. The fermentation product of the astragalus polysaccharide of the present invention can also increase the growth rate of animals and reduce the feed-to-egg ratio. DETAILED DESCRIPTION

[0025] The present invention will be described clearly and completely below in conjunction with the examples of the present invention. In the description of the present invention, it should be noted that, where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0026] The strains used in the present invention are derived from:

[0027] Candida tropicalis (GDMCC 65030): Classification and designation candida tropicalis , 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;

[0028] 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;

[0029] Bacillus subtilis (GDMCC 65032): Classification and designation 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.

[0030] Example

[0031] The bacterial liquid is inoculated into a culture medium containing sterilized astragalus polysaccharide. The fermentation temperature is controlled at 30-35°C in the absence of oxygen for 6 days, and the fermentation is sterilized after completion to obtain an astragalus polysaccharide fermentation product. 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 an inorganic salt, wherein the inorganic salt is 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 entire astragalus root, and its composition is: solid content 35wt%, including approximately 30wt% astragalus polysaccharide and approximately 0.07wt% astragaloside IV.

[0032] 2 kg of bacterial solution was added to every 100 kg of culture medium. The activation method of the above-mentioned strains was as follows: each strain that grew well after activation was selected and inoculated into the malt wort liquid culture medium. Among them, the culture temperature of Enterococcus faecium was 28-30°C and the culture conditions were 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 solutions was 8*10 8 CFU / mL or so.

[0033] Finally, astragalus polysaccharide fermentation products 1 to 3 are obtained, wherein:

[0034] The fermentation strain of Astragalus polysaccharide fermentation product 1 was Candida tropicalis (GDMCC 65030);

[0035] The fermentation strain of Astragalus polysaccharide fermentation product 2 was Enterococcus faecium (GDMCC 65031);

[0036] The fermentation strain of astragalus polysaccharide fermentation product 3 is Bacillus subtilis (GDMCC 65032).

[0037] Immune enhancement experiments

[0038] 1. Chicken immune enhancement experiment

[0039] 1.1 Application in production at Shandong Hanting broiler breeder farm.

[0040] A total of 5,000 48-week-old Lifeng broiler breeder chickens were selected and divided into five groups. The control group did not use fermented astragalus polysaccharide powder, while 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 and experimental groups, and the serum was separated and the antibody titer was determined.

[0041] The experimental groups are:

[0042] Immunity enhancement group 1: chicken diets supplemented with Astragalus polysaccharide fermentation product 1;

[0043] Immunity enhancement group 2: chicken diets supplemented with Astragalus polysaccharide fermentation product 2;

[0044] Immunity enhancement group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3;

[0045] Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet;

[0046] Blank group: fed only with diet.

[0047] The experimental results are shown in Table 1 below;

[0048] Table 1 Antibody levels before and after immunization Unit: log2

[0049] 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

[0050] 1.2 Application in production at Shandong Hanting broiler breeder farm.

[0051] A total of 5,000 48-week-old Lifeng broiler breeder chickens were selected and divided into five groups. The control group did not use fermented astragalus polysaccharide powder, while 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 and experimental groups, and the serum was separated and the antibody titer was determined.

[0052] The experimental groups are:

[0053] Immunity enhancement group 1: chicken diets supplemented with Astragalus polysaccharide fermentation product 1;

[0054] Immunity enhancement group 2: chicken diets supplemented with Astragalus polysaccharide fermentation product 2;

[0055] Immunity enhancement group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3;

[0056] Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet;

[0057] Blank group: fed only with diet.

[0058] The experimental results are shown in Table 2 below;

[0059] Table 2 Antibody levels before and after immunization Unit: log2

[0060] 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

[0061] 1.3 Application in production at Shandong Hanting broiler breeder farm.

[0062] A total of 5,000 48-week-old Lifeng broiler breeder chickens were selected and divided into five groups. The control group did not use fermented astragalus polysaccharide powder, while the experimental group added 0.1wt% fermented astragalus polysaccharide powder to the feed for five days before and after immunization with the vaccine (H9 avian influenza). The antibody titer was monitored 7 days after immunization. 50 blood samples were randomly collected from each of the control and experimental groups, and the serum was separated and the antibody titer was determined.

[0063] The experimental groups are:

[0064] Immunity enhancement group 1: chicken diets supplemented with Astragalus polysaccharide fermentation product 1;

[0065] Immunity enhancement group 2: chicken diets supplemented with Astragalus polysaccharide fermentation product 2;

[0066] Immunity enhancement group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3;

[0067] Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet;

[0068] Blank group: fed only with diet.

[0069] The experimental results are shown in Table 3 below;

[0070] Table 3 Antibody levels before and after immunization Unit: log2

[0071] 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

[0072] 1.4 Application in production at Linquan Layer Chicken Farm in Yutian County, Hebei Province.

[0073] A total of 8,000 35-week-old Hy-Line Brown chickens 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 their 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 and experimental groups, and the serum was separated and the antibody titer was determined.

[0074] The experimental groups are:

[0075] Immunity enhancement group 1: chicken diets supplemented with Astragalus polysaccharide fermentation product 1;

[0076] Immunity enhancement group 2: chicken diets supplemented with Astragalus polysaccharide fermentation product 2;

[0077] Immunity enhancement group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3;

[0078] Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet;

[0079] Blank group: fed only with diet.

[0080] The experimental results are shown in Table 4 below;

[0081] Table 4 Antibody levels before and after immunization Unit: log2

[0082] 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

[0083] 1.5 Application in production at Linquan Layer Chicken Farm in Yutian County, Hebei Province.

[0084] A total of 8,000 35-week-old Hy-Line Brown laying hens were divided into five groups. The control group received no fermented Astragalus polysaccharide powder, while the experimental group received 0.1 wt% fermented Astragalus polysaccharide powder in their feed for five days before and after vaccination (ND Newcastle disease). Antibody titers were monitored seven days after immunization. Sixty blood samples were randomly collected from each of the control and experimental groups, and serum was separated and antibody titers were measured. Average egg production rates were also observed from days 7 to 9 after immunization.

[0085] The experimental groups are:

[0086] Immunity enhancement group 1: chicken diets supplemented with Astragalus polysaccharide fermentation product 1;

[0087] Immunity enhancement group 2: chicken diets supplemented with Astragalus polysaccharide fermentation product 2;

[0088] Immunity enhancement group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3;

[0089] Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet;

[0090] Blank group: fed only with diet.

[0091] The experimental results are shown in Table 5 below;

[0092] Table 5 Antibody levels before and after immunization Titer unit: log2

[0093] 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

[0094] 1.6. Application in production at a yellow-feathered broiler breeder farm in Guangxi.

[0095] A total of 5,000 43-week-old yellow-feathered broiler breeders 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, and feed-to-egg ratio) was observed.

[0096] The experimental groups are:

[0097] Immunity enhancement group 1: chicken diets supplemented with Astragalus polysaccharide fermentation product 1;

[0098] Immunity enhancement group 2: chicken diets supplemented with Astragalus polysaccharide fermentation product 2;

[0099] Immunity enhancement group 3: chicken diets supplemented with Astragalus polysaccharide fermentation product 3;

[0100] Immunity enhancement group 4: Astragalus polysaccharide extract was added to the chicken diet;

[0101] Blank group: fed only with diet.

[0102] The experimental results are shown in Table 6 below;

[0103] Table 6 Production performance table

[0104] 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

[0105] Result analysis:

[0106] 1. Based on the experimental results of the immune effects in Tables 1 to 6 above, the astragalus polysaccharide fermentation product 1 obtained by fermenting the Candida tropicalis screened by the present invention can significantly increase the immune titer after immunization, and the improvement is more than 50% compared with other fermentation bacteria;

[0107] 2. Based on 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.

[0108] 3. Based on the egg production and production efficiency results in Tables 5 and 6, the use of the astragalus polysaccharide fermentation product 1 of the present invention can significantly reduce the feed-to-egg ratio and significantly increase the egg production rate and egg weight compared to other strain fermentation extracts;

[0109] Combining the data of 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.

[0110] Therefore, from the perspective of the dual purpose of improving immune effect and production efficiency, the strains screened by the present invention have great advantages.

[0111] The applicant declares that while the above-described embodiments illustrate the process of the present invention, the present invention is not limited to the above-described process steps, nor does it imply that the present invention must rely on the above-described process steps for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for the raw materials used, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. A strain of Candida tropicalis, characterized in that Classification named candida tropicalis , 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 astragalus polysaccharide fermented by Candida tropicalis 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 Candida tropicalis 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 Candida tropicalis to obtain a viable cell 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 as follows: inoculating Candida tropicalis into a malt wort liquid culture medium, and culturing on a shaker at 28-37° C. for 6-24 hours.