Cell wall-broken saccharomyces boulardii feed additive, and preparation method and application thereof

Abstract

The present application belongs to the field of microbial engineering, and discloses a broken wall red phaffia yeast feed additive, a preparation method and application thereof. The preparation method of the broken wall red phaffia yeast feed additive comprises the following steps: strain culture: inoculating red phaffia yeast into YM liquid culture medium to obtain seed liquid through culture; fermentation expansion culture: transferring the seed liquid into new YM liquid culture medium to carry out fermentation culture, and centrifuging to obtain bacterial body precipitate; wall breaking treatment: adding hydrochloric acid into the bacterial body precipitate, heating, cooling, centrifuging to obtain precipitate; freeze-drying to obtain the broken wall red phaffia yeast feed additive. The broken wall red phaffia yeast feed additive, the preparation method and application thereof can significantly improve the growth rate and survival rate of seedlings, improve the antioxidant stress resistance and immunity of seedlings, and enhance the stress resistance of seedlings.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial engineering, and particularly relates to a broken-wall red Phaffia rhodozyma feed additive and a preparation method and application thereof. BACKGROUND

[0002] Tachysurus fulvidraco, also known as yellow croaker, is an important freshwater aquaculture fish, which is favored by breeders and consumers due to its delicious meat and no intermuscular spines. However, with the development of intensive aquaculture, breeders continuously increase the stocking density of fry to further improve economic efficiency, and widely use high-energy feed to further save costs. Long-term poor feeding environment makes Tachysurus fulvidraco prone to various diseases during the breeding process, especially during the fry period, when the fish body has not fully developed and the ability to cope with environmental stress is low. Therefore, developing a feed additive for Tachysurus fulvidraco fry period to improve the growth rate and stress resistance during the fry period can effectively improve the breeding efficiency.

[0003] In fish, the most common method to improve immunity is still the oral administration of chemical preparations, which has seriously affected the safety of the breeding environment and the quality of the products. Probiotics are widely used in human and veterinary medicine fields, and they can effectively promote growth and improve immunity without adverse effects on host animals. For example, Bacillus subtilis and photosynthetic bacteria have been widely used in the field of aquaculture, but the types of probiotics suitable for fry cultivation are still few. Therefore, developing a new probiotic additive for Tachysurus fulvidraco fry period to improve fry quality and survival rate is an urgent problem in the Tachysurus fulvidraco fry industry. SUMMARY

[0004] The present application aims to provide a preparation method of a broken-wall red Phaffia rhodozyma feed additive and its application, to solve the problems of slow growth and low immunity of Tachysurus fulvidraco fry, and to improve the survival rate and breeding efficiency of fry.

[0005] In order to achieve the above-mentioned application purposes, the present application provides a preparation method of a broken-wall red Phaffia rhodozyma feed additive, comprising the following steps:

[0006] S1, strain culture: inoculate red Phaffia rhodozyma into YM liquid medium, and culture at 180 rpm and 20°C for 48 hours to obtain seed liquid;

[0007] S2, fermentation expansion: transfer the seed liquid into new YM liquid medium at a volume ratio of 1:10, and ferment and culture at 180 rpm and 20°C for 96 hours, then centrifuge at 8000 rpm for 10 minutes, and wash twice with 1xPBS, and collect the bacterial precipitate;

[0008] S3, wall breaking treatment: 3 mol / L hydrochloric acid with a volume ratio of 2.5:1 is added to the bacteria obtained in step S2, heated for 4 minutes and then cooled to room temperature, centrifuged at 8000 rpm for 10 minutes, and the precipitate is collected;

[0009] S4, freeze-drying: the wall-broken bacteria precipitate is treated by freeze-drying to obtain the wall-broken red Phaffia yeast feed additive.

[0010] Further, the application also provides the wall-broken red Phaffia yeast feed additive prepared by the preparation method.

[0011] Further, the application also provides the application of the wall-broken red Phaffia yeast feed additive in the preparation of a product for promoting the growth of yellow catfish fry and / or improving the immunity of the yellow catfish fry.

[0012] Further, the application also provides a yellow catfish fry feed containing the wall-broken red Phaffia yeast feed additive.

[0013] Further, the application also provides a preparation method of a yellow catfish fry feed, and the steps are as follows:

[0014] The wall-broken red Phaffia yeast feed additive is dissolved in 95% ethanol, uniformly sprayed on the opening bait of the yellow catfish, and then dried in the shade to obtain the yellow catfish feed containing the wall-broken red Phaffia yeast.

[0015] Further, the addition amount of the wall-broken red Phaffia yeast feed additive is 0.1-10 g / 100 g of feed. After the feed is dried in the shade, the yellow catfish is fed, and this addition method can make the wall-broken red Phaffia yeast uniformly distributed in the bait, which is convenient for the fry to feed.

[0016] Further, the application also provides a preparation method of a yellow catfish fry feed, and the steps are as follows:

[0017] The wall-broken red Phaffia yeast feed additive is added to the brine shrimp hatching liquid with a salinity of 20‰, and the addition amount is 0.1-10 mg / L of salinity hatching brine shrimp. After hatching for 18 hours, they are incubated together for 12 hours to obtain the red Phaffia yeast biological feed taking brine shrimp as the biological carrier.

[0018] Further, the addition amount of the wall-broken red Phaffia yeast feed additive is 0.1-10 mg / L.

[0019] By taking the brine shrimp as the carrier, the uptake rate of the fry to the wall-broken red Phaffia yeast can be improved, and the effect is further enhanced.

[0020] The wall-broken red Phaffia yeast feed additive, the preparation method and the application thereof have the advantages that:

[0021] (1) The red Phaffia yeast as a feed additive for yellow catfish fry can significantly improve the growth rate of yellow catfish fry and improve the survival rate of fry to a certain extent.

[0022] (2) The red Phaffia yeast as a feed additive for yellow catfish fry can assist fry in coping with oxidative stress, improve the antioxidant stress resistance and immunity of fry, and enhance the stress resistance of fry.

[0023] (3) The red Phaffia yeast as a feed additive for yellow catfish fry can significantly improve the intestinal flora environment of fry. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The growth difference chart of the feed containing red Phaffia yeast and the conventional feeding group in the embodiment of the present application;

[0025] Figure 2 The yellow catfish fry feeding treatment scheme diagram in the embodiment of the present application;

[0026] Figure 3 The liver and intestinal tissue section chart of the feed containing red Phaffia yeast and the conventional feeding group in the embodiment of the present application;

[0027] Figure 4 The difference chart of the immune enzyme activity of the feed containing red Phaffia yeast and the conventional feeding group in the embodiment of the present application, wherein A is the lysozyme LZM activity result, and B is the alkaline phosphatase AKP activity result;

[0028] Figure 5 The difference chart of the digestive enzyme activity of the feed containing red Phaffia yeast and the conventional feeding group in the embodiment of the present application, wherein A is the lipase LPS activity result, and B is the amylase AMS activity result;

[0029] Figure 6 The difference chart of the antioxidant enzyme activity of the feed containing red Phaffia yeast and the conventional feeding group in the embodiment of the present application, wherein A is the SOD enzyme activity result, and B is the dismutase GSH activity result;

[0030] Figure 7 The difference chart of the intestinal flora composition of the feed containing red Phaffia yeast and the conventional feeding group in the embodiment of the present application, wherein A is the principal component analysis result of intestinal microbial detection of each group, and B is the alpha diversity analysis result of intestinal microbial detection of each group. DETAILED DESCRIPTION

[0031] The technical solutions of the present application are further described below through the drawings and examples.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0033] The red Phaffia yeast (P. rhodozyma) strain of the present application is provided by Xiamen Junhecheng Biotechnology Co., Ltd. Phaffia rhodozyma

[0034] Example 1 Preparation of broken-wall red Phaffia yeast feed additive

[0035] 1. Strain culture: inoculate the red Phaffia yeast (P. rhodozyma) into 10 mL of YM liquid medium, and culture at 180 rpm and 20°C for 48 h to obtain a seed liquid; Phaffia rhodozyma

[0036] 2. Fermentation expansion: inoculate the seed liquid into 1 L of new YM liquid medium at a volume ratio of 1:10, and ferment and culture at 180 rpm and 20°C for 96 h. Centrifuge at 8000 rpm for 10 min, and wash twice with 1xPBS. Collect the precipitate, which is the cell body of the red Phaffia yeast;

[0037] 3. Wall-breaking treatment: add 400 mL of 3 mol / L hydrochloric acid to the cell body at a volume ratio of 2.5:1 of the culture medium, heat for 4 min, cool to room temperature, centrifuge at 8000 rpm for 10 min, and collect the precipitate;

[0038] 4. Freeze-drying: freeze-dry the wall-broken cell body precipitate to obtain the broken-wall red Phaffia yeast feed additive;

[0039] Example 2 Application of the feed additive in the cultivation of yellow catfish fry

[0040] 1. Feed addition method:

[0041] (1) Dissolve the broken-wall red Phaffia yeast in 95% ethanol, and uniformly spray it on the yellow catfish opening feed at an addition amount of 1 g / 100 g of feed. After drying in the shade, it is ready for use.

[0042] (2) Add the broken-wall red Phaffia yeast to the brine shrimp hatching liquid with a salinity of 20‰ at an addition amount of 1 mg / L of salinity hatching brine shrimp. After hatching for 18 hours, incubate together for 12 hours to obtain brine shrimp containing broken-wall red Phaffia yeast.

[0043] 2. Growth traits:

[0044] Select mature individuals of yellow catfish, inject them with two needles to induce spawning, and obtain yellow catfish fertilized eggs by half-dry fertilization. After hatching in flowing water at 26°C for about 50 h, obtain the hatched yellow catfish fry. Divide the hatched fry into 6 groups, with 50 fry in each group. Through three stages of feeding treatment, the treatment scheme is as follows​​Figure 2 and Table 1, each feeding once a day, and the feeding period is 30 days.

[0045] Table 1

[0046]

[0047] After the feeding period, the survival rate and growth traits of each group were counted. In the early stage, 50 yellow catfish raised for 30 days after feeding were all dead in groups 1 and 2 due to the lack of feed. In groups 3 and 4, the survival rate was 12% due to the lack of high-quality nutrition intake during the opening process. In group 3 (early rotifer opening), the average body length was 1.5 cm, and the average body weight was 0.05 g. In group 4 (adding egg yolk group), the average body length was 1.5 g, and the average body weight was 0.05 g. In group 5, there was a short period of brine shrimp transition, and then the survival rate was 48% after the transition to regular feed. The body length was 2.0-2.5 cm, and the average body weight was 0.2 g. In group 6, the survival rate was 52% after adding broken red fava yeast, the body length was 2.3-2.5 cm, and the average body weight was 0.21 g. The growth status of each group is shown in Table 1. Figure 1

[0048] 3. Liver and intestinal sections:

[0049] The liver and gonadal tissues of each group of large-size yellow catfish fry were dissected and stored in 4% paraformaldehyde solution. After 16 h of fixation, the tissues were dehydrated in 70%, 90%, 95%, and 100% ethanol, transparentized with xylene, and immersed in paraffin. The tissues were then embedded in paraffin and cut into 5-micron sections using a paraffin sectioning machine. After baking the paraffin sections at 42°C, they were stained with hematoxylin and eosin to produce tissue sections.

[0050] Histological results showed that after the brine shrimp transition, the yellow catfish fry showed an increase in liver tissue volume due to more mature individual development, and the liver tissue development was more complete. The addition of broken red fava yeast significantly increased the length of the intestinal villi and the thickness of the intestinal wall, as shown in Table 1. Figure 3

[0051] 4. Liver immune enzyme activity detection:

[0052] The livers of each group of large-size yellow catfish fry were dissected and stored in a -80°C freezer. When detecting liver immune enzyme activity, the livers were taken out, 0.85x PBS was added at a ratio of 1:9, and homogenized to prepare enzyme activity detection samples. The specific operation refers to the instruction manual (lysozyme (LZM) activity detection kit (Nanjing Jiancheng Nanjing Jiancheng Biological Engineering Institute, A050-1-1) alkaline phosphatase (AKP) detection kit (Nanjing Jiancheng Nanjing Jiancheng Biological Engineering Institute, A059-1-1)).

[0053] ​​The results of the comparison of immune enzyme activities in different groups of liver showed that the lysozyme activity in the liver of yellow catfish fry was significantly increased after brining with Artemia worms. Figure 4 (A) This may be because the yellow catfish fry are more mature, with more complete liver development and more complete liver function. However, alkaline phosphatase activity tests showed that yellow catfish without artemia transition exhibited stronger alkaline phosphatase activity. This may be because the group without artemia transition showed slower development, incomplete liver development, and a greater number of stem cells, leading to higher alkaline phosphatase activity. Figure 4 (B) While the liver is fully developed after Artemia transition, its overall alkaline phosphatase activity is low. However, the addition of Pharbitis rubescens can increase liver alkaline phosphatase activity to some extent. Figure 4 (A)

[0054] 5. Detection of intestinal digestive enzyme activity:

[0055] Intestinal tissue was harvested from large-sized yellow catfish fry in each group and stored at -80℃. When the intestinal digestive enzyme activity was to be tested, the tissue was removed, mixed with 0.85×PBS at a 1:9 ratio, and homogenized to prepare enzyme activity detection samples. Specific procedures were performed according to the instructions (Lipase (LPS) Activity Assay Kit (Nanjing Jiancheng Biotechnology Institute, A054-2-1) α-Amylase (AMS) Assay Kit (Nanjing Jiancheng Biotechnology Institute, C016-1-1)).

[0056] like Figure 5 As shown in Figure A, the intestinal lipase activity of the Artemia transition group of yellow catfish fry was higher, and the addition of broken-cell wall Pharbitis pyrenoidosa further increased the intestinal lipase activity. This may be because Pharbitis pyrenoidosa can improve the intestinal health of yellow catfish fry and increase the abundance of intestinal flora. The results of amylase activity detection showed that the overall intestinal amylase activity of yellow catfish fry was low. Figure 5 (B) This may be one of the reasons for the low survival rate of yellow catfish fry in the first feeding stage.

[0057] 6. Detection of intestinal antioxidant enzyme activity:

[0058] Intestinal tissue was harvested from large-sized yellow catfish fry in each group and stored at -80℃. When the intestinal antioxidant enzyme activity was to be detected, the tissue was removed, mixed with 0.85×PBS at a 1:9 ratio, and homogenized to prepare enzyme activity detection samples. Specific procedures were performed according to the instructions (SOD activity detection kit (Nanjing Jiancheng Biotechnology Institute, A001-3-1) and GSH detection kit (Nanjing Jiancheng Biotechnology Institute, A005-1-1)).

[0059] like Figure 6As shown, although the Artemia transitional post-Pelteobagrus yuan-gonggensi fingerlings have a faster development speed, long-term feeding of feed during the later feeding process can cause nutritional imbalance and cause oxidative stress, and the SOD activity cannot respond to oxidative stress due to the lack of trace elements, while the dismutase (GSH) increases sharply in oxidative stress. The addition of broken cell red Phaffia yeast can neutralize the oxygen free radicals in the body, thereby reducing the stress of the fingerlings to produce oxidative stress, and further improving the stress resistance of the fingerlings. Figure 6

[0060] 7. Intestinal flora diversity analysis:

[0061] Intestinal tissues of each group of Pelteobagrus yanguanggensi large-sized fingerlings were dissected and stored in a-80°C refrigerator. When the intestinal microorganisms were detected, whole genome DNA was extracted, the intestinal flora 16S sequence was amplified using universal primers 343F and 798R as primers, and sequencing was performed by using an Illumina NovaSeq 6000 sequencing platform.

[0062] The comparison results of the intestinal microbial abundance show that the intestinal flora abundance of the rotifer opening group (CG) and the rotifer + egg yolk group (EG) is very different. Although the intestinal microbial abundance of individual individuals is poor, there are also individuals with high abundance. The Artemia transitional post-Pelteobagrus yuan-gonggensi fingerlings have a faster growth and development speed, and the loss of intestinal flora diversity during the subsequent long-term feeding (AG) process will be more obvious, and the addition of broken cell red Phaffia yeast (FG) can meet the components required for the development of intestinal flora, and the intestinal flora abundance is significantly improved and more uniform. Figure 7

[0063] Therefore, the present application adopts the above-mentioned broken cell red Phaffia yeast feed additive, its preparation method and application, through strain culture, fermentation expansion, hydrochloric acid wall breaking and freeze-drying process, to obtain broken cell red Phaffia yeast rich in active ingredients. The additive can significantly improve the intestinal health of Pelteobagrus yanguanggensi fingerlings, improve the activity of lipase, the length of intestinal villi and the thickness of intestinal wall, reduce oxidative stress and protect intestinal flora diversity. The feed additive can be added by directly spraying the opening bait or incubating with Artemia, which can effectively improve the survival rate, growth rate and stress resistance of the fingerlings, solve the problems of intestinal damage and low survival rate in intensive culture, and provide a safe and economical solution for efficient Pelteobagrus yanguanggensi culture.

[0064] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.​​

Claims

1. A method for promoting the growth of and improving the immunity of yellow catfish fry by using a cell wall broken Phaffia rhodozyma feed additive, characterized in that, Be applied to the preparation of open period feed; The application method is as follows: ① 1-3 DPH, feeding soybean milk + rotifer; ② 3-10 DPH, feeding red Phaffia yeast biological feed taking Artemia as a biological carrier; ③ 10-30 DPH, feeding yellow catfish feed containing broken red Phaffia yeast; Each feeding is carried out once in the morning and evening, and after the feeding is completed, the survival rate of yellow catfish is 52%; The preparation method of the broken red Phaffia yeast feed additive comprises the following steps: S1, strain culture: inoculate the red Phaffia yeast into YM liquid medium, and culture at 180 rpm and 20°C for 48 hours to obtain a seed liquid; S2, fermentation expansion: transfer the seed liquid into new YM liquid medium, and ferment and culture at 180 rpm and 20°C for 96 hours, then centrifuge at 8000 rpm for 10 minutes and wash twice with 1*PBS, collect the bacterial precipitate, and the inoculation volume ratio of the seed liquid is 1:10; S3, wall breaking treatment: add 3 mol / L hydrochloric acid to the bacterial body obtained in step S2, heat for 4 minutes, then cool to room temperature, centrifuge at 8000 rpm for 10 minutes, collect the precipitate, and the volume ratio of the bacterial body to hydrochloric acid is 2.5:1; S4, freeze-drying: freeze-dry the broken bacterial body precipitate to obtain the broken red Phaffia yeast feed additive; The preparation method of the red Phaffia yeast biological feed taking Artemia as a biological carrier is as follows: add the broken red Phaffia yeast feed additive to Artemia hatching liquid with a salinity of 20‰, incubate for 18 hours, then co-incubate for 12 hours to obtain the red Phaffia yeast biological feed taking Artemia as a biological carrier, and the addition amount of the broken red Phaffia yeast feed additive is 1-10 mg / L; The preparation method of the yellow catfish feed containing broken red Phaffia yeast is as follows: dissolve the broken red Phaffia yeast feed additive in 95% ethanol, uniformly spray it on the open bait of yellow catfish, and obtain the yellow catfish feed containing broken red Phaffia yeast after shade drying, and the addition amount of the broken red Phaffia yeast feed additive is 1-10 g / 100 g feed.

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