CN115029370A

CN115029370A - Feed additive for animal feeding and resistance replacement and preparation method thereof

Info

Publication number: CN115029370A
Application number: CN202210592867.5A
Authority: CN
Inventors: 杨春玉; 李亚茹; 李春芳; 刘远翔
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2022-09-09
Anticipated expiration: 2042-05-27
Also published as: CN115029370B

Abstract

The invention relates to the technical field of biological medicines, in particular to a feed additive for animal feeding attraction and resistance replacement and a preparation method thereof. The bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 is utilized, and high yield is obtained through pichia pastoris multi-copy overexpression and fermentation. The method can directly utilize the supernatant of the enzyme-containing fermentation liquor to catalyze a substrate S-allyl-L-cysteine sulfoxide S-allyl-L-cysteine to synthesize a high-concentration sterilization substance dialkyl thiosulfinate and a compound allyl mercaptan with an attractant effect, and can also spray-dry the fermentation liquor to form a soluble enzyme powder solid product. The method realizes high conversion rate synthesis of antibacterial substances dialkyl sulfo-sulfinate and attractant compound allyl mercaptan by a biocatalytic in-situ catalysis mode, is safe, simple and convenient, is easy to realize industrialization, solves the defect of instability of products, and has good application prospect in animal breeding, especially in the aspect of antibiotic substitution.

Description

Feed additive for animal feeding and resistance replacement and preparation method thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a feed additive for animal feeding attraction and resistance replacement and a preparation method thereof.

Background

For a long time, the feed antibiotics have seriously contributed to the breeding production benefit, and the feed antibiotics can inhibit the growth and reproduction of microorganisms to some extent, regulate immunity and improve the tissue structure of the digestive tract so as to promote the digestion and absorption of nutrient substances and the growth of animals. However, the risks of drug resistance and the like are substantially existed, and before the application, the 194 th civilization issued by rural agricultural departments definitely stipulates that the feed circulated and used 12-month-31-day-later 2020 does not contain growth-promoting drug feed additives and the resistance to hammer landing is forbidden. The farm needs to establish a corresponding alternative antibiotic scheme for maintaining the production benefit, and the alternative antibiotic scheme is selected from the advantages of functional action on the target antibiotic and low cost of the target antibiotic. Many types of substitute antibiotic products are currently presented, such as fermented feed, enzyme preparations, antibacterial peptides, acidifiers, microecologics, Chinese herbal medicines, plant extracts and the like, but low-cost substitute antibiotic products with broad-spectrum bacteriostasis, high efficiency and high biological safety are lacked.

Many inventions and researches indicate that the dialkyl sulphosulfinate is an ideal feed substitute anti-bacterial additive which is green, has no residue, has broad-spectrum bacteriostasis, sterilization, animal disease generation rate reduction, animal growth promotion and immunity improvement. And can attract animals to generate appetite through smell, thereby increasing the feed intake and improving the feed conversion rate. At present, the plant extracts from garlic, onion and the like are mostly relied on, for example, patent CN103037856A utilizes the extraction of dialkyl thiosulfinate from garlic to combat parasites in poultry, monogastric animals and ruminants. However, the method has certain disadvantages by processing from plants: the preparation process is complicated, the extraction yield is low, the preparation cost is high, the stability of the diallyl thiosulfinate is poor, and the defects greatly limit the application of the diallyl thiosulfinate in the field of animal feeding.

Since dialkyl thiosulfinate in plants such as garlic and onion is produced by catalyzing substrate S-allyl-L-cysteine sulfoxide by alkyl cysteine sulfoxide, researchers consider obtaining plant-derived alkyl cysteine sulfoxide biologically in order to obtain diallyl thiosulfinate with high cost performance. However, the results show that the inclusion body of the recombinant protein is a serious problem in the heterologous expression in E.coli, and the enzyme activity is lower than that of the enzyme extracted from the plant by a chemical method. There are also examples of constructing alkyl cysteine sulfoxylates of plant origin into Saccharomyces cerevisiae and Pichia pastoris, but the final enzyme activity is lower than that of the native enzyme. For example, in patent CN105838729A, wild alkyl cysteine sulfoxidase of garlic bulbs is expressed in a bacillus subtilis expression system and pichia pastoris by using a random mutation technology, and finally, the specific enzyme activity is increased by 50% compared with that of the wild type, but the specific enzyme activities in bacillus subtilis and pichia pastoris are only 105.34U/mg and 173.72U/mg at the highest respectively.

Therefore, how to prepare a low-cost alternative antibiotic product with high stability, simple preparation process, broad-spectrum bacteriostasis, high efficiency and high biological safety is urgent.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a feed additive for animal feeding and resistance replacement and a preparation method thereof. A microbial fermentation method is utilized to obtain a high-yield bacteria source S-alkyl-L-cysteine sulfoxide lyase LCC1, and then a high-concentration bactericidal substance diallyl thiosulfinate and a feeding attractant compound allyl mercaptan with rich garlic flavor are synthesized in a biocatalysis manner, so that the aim of preparing the high-efficiency feeding attractant feed additive with high stability and low cost is fulfilled.

The technical scheme of the invention is as follows:

a feed additive for animal feeding and resisting is prepared from bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1, and substrate S-allyl-L-cysteine and S-allyl-L-cysteine sulfoxide through catalytic reaction.

Wherein the protein sequence of the S-alkyl-L-cysteine sulfoxide lyase LCC1 is as follows:

MKDLVYLNYAATSYKKFPATIEALTAYLAENQFMNYGRNAPLLREGLPLLETRQLLADFFQAPSAAQITFTNNATTSLNLALAGILQPGDHVITTMLEHHAVARPLHLLEKERGISVTYVACQKTGLLDVEDIQRAWRTNTKALVMTHASNVLGTILPIEECFQWAQQKGLLTVLDAAQTAGFLPIKMTQMAIDVLAFTGHKSLYGLAGIGGLAFSERGAEAVKPLMAGGTGSHSNSFDQPSFLPDKFEAGTLNSLGILSLNSSIKELNKIGLAAIQKHERTLMQNFLNGLSGLPVTILGTKDVAQTVPVVSITLWNQEETVVAQQLAEQYGIMTRAGLHCAPLAHETAGTLATGTLRFSFGWQTTPEEITWTIHALQELLI。

the molecular formulas of the substrates S-allyl-L-cysteine and S-allyl-L-cysteine sulfoxide are respectively C ₆ H ₁₁ NO ₂ S、C ₆ H ₁₁ NO ₃ S。

As a preferable technical scheme, the plant growth regulator further comprises an activator PLP, and the ratio of the S-alkyl-L-cysteine sulfoxide lyase LCC1 to the activator PLP is 1000 (1-5).

A preparation method of a feed additive for animal feeding and resistance replacement comprises the following steps:

bacterial strain construction, fermentation and enzyme production

The S-alkyl-L-cysteine sulfoxide lyase LCC1 is obtained by expressing an expression vector in a host cell; further, the host cell may be escherichia coli or pichia pastoris; further, the host cell may be Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae; further, the expression vector may be pHT43, pHT01, pHT08, pHT9, pHT10, pYES-DEST52, pPIC9, pPIC9K, pHIL-S1, pPICZ α A, or the like.

Further, the specific preparation process comprises the following steps:

(1) selecting a biologically safe strain Pichia pastoris GS115 and an integration vector pPIC9K to realize the integration expression of S-alkyl-L-cysteine sulfoxide lyase LCC 1;

(2) carrying out codon optimization synthesis on a protein sequence, and then carrying out integration expression construction; obtaining an integrated expression strain with antibiotic tolerance capacity reaching 1500 mu g/ml by high resistance screening of geneticin, and obtaining 5400U/mg of high specific enzyme activity by optimizing different fermentation culture media and different fermentation processes;

(3) after fermentation is finished, spray drying the fermentation liquor to obtain enzyme powder: adding fluidizing agent (w/w) into the fermentation liquor: 5-20% of dextrin, 15-25% of corn starch and stirring for 20-30 min at 100 rpm/min;

(4) spray drying: the temperature of the air inlet is 150-180 ℃, the temperature of the air outlet is 70-80 ℃, and the rotating speed of the peristaltic pump is 2000 ml/h.

In order to improve the protein expression quantity, the protein sequence is subjected to codon optimization synthesis and then integrated expression construction is carried out; obtaining an integrated expression strain with antibiotic tolerance capacity reaching 1500 mug/ml by high resistance screening of the geneticin, which shows that the screened strain is an integrated strain with multi-copy insertion and the protein expression quantity is obviously improved; the high specific enzyme activity of 5400U/mg is obtained by optimizing different fermentation culture media and different fermentation processes.

Preparation of substrate

1. Method for preparing S-allyl-L-cysteine

And (4) in the step (5): 1-4: 1, adding sodium hydroxide particles with the final concentration of 0.8-4.5 mol/L into the ethanol water solution, stirring and dissolving, adding L-cysteine solid with the final concentration of 0.4-2.5 mol/L, adding bromopropylene with the same molar concentration as cysteine after the L-cysteine solid is completely dissolved, and reacting for 5-12 hours while stirring. Adjusting the pH value of the solution to 4-6 with acetic acid, filtering, and drying in an oven to obtain the S-allyl-L-cysteine.

2. Method for preparing S-allyl-L-cysteine sulfoxide

Taking S-allyl-L-cysteine prepared in the step 1 and hydrogen peroxide according to the mass volume ratio of (1-3) g: (6-2) ml, and reacting for 5 minutes. Adding precooled ethanol, and drying to obtain the S-allyl-L-cysteine sulfoxide.

Preparation of allyl mercaptan by biocatalysis

Under the condition of room temperature, the reaction between S-alkyl-L-cysteine sulfoxide lyase LCC1 and a substrate S-allyl-L-cysteine is as follows (1-10): (10-1) and reacting for 15-30 minutes. The PLP is added into a reaction system according to the ratio of the enzyme to the PLP of 1000 (1-5) to improve the enzyme activity, and the PLP is used as a coenzyme of S-alkyl-L-cysteine sulfoxide lyase LCC1, and the existence of the PLP can obviously improve the activity of S-alkyl-L-cysteine sulfoxide lyase LCC 1. Allyl mercaptan having a strong garlic flavor generated by the catalytic reaction attracts animals to produce appetite through odor, thereby increasing food intake and increasing feed conversion ratio.

Preparation of diallyl thiosulfinate by biocatalysis

The method comprises the following steps of (1-10) carrying out S-alkyl-L-cysteine sulfoxide lyase LCC1 and a substrate S-allyl-L-cysteine sulfoxide: (10-1) for 20-60 minutes, and adding an activator PLP into the reaction system to obtain the diallyl thiosulfinate.

Fifthly, preparation of products

And (3) mixing the spray-dried dry powder, S-allyl-L-cysteine sulfoxide and an activator PLP according to the ratio of (1-10): (10-1): (10-1): (0.001-0.005) in proportion. The product can be used for producing feed premix and pellet feed, and the addition amount is 100-300 g/ton feed. When the water-saving drinking water is used, the water-saving drinking water can be added into animal drinking water, and the daily addition amount of the water-saving drinking water is 100-300 mg/l. The feed additive is used for treating animal enteritis diarrhea, chicken coccidiosis, chicken leucocytozoonosis, fish, shrimp and crab feeding in aquaculture, fish, shrimp and crab disease control and the like caused by pathogenic bacteria, and the dosage is 1-5 g/l, and is 1-3 ml for each dosage.

And (2) mixing the fermentation liquor, S-allyl-L-cysteine sulfoxide, an activator PLP and dextrin according to the weight ratio of (1-50): (10-1): (10-1): (0.001-0.005): (0.1-0.5) to form an animal drinking water additive product. When the drinking water is used, the drinking water is added into animal drinking water, and the daily addition amount of the drinking water is 100-300 mg/l.

The invention has the beneficial effects that:

(1) compared with the prior art, the invention realizes the high activity of the novel S-alkyl-L-cysteine sulfoxide lyase LCC1 through pichia pastoris multi-copy integrated expression and fermentation optimization, and the specific enzyme activity is as high as 5400U/mg, which is obviously higher than 105.34U/mg and 173.72U/mg of the specific enzyme activities in bacillus subtilis and pichia pastoris in the patent CN 105838729A. The use of bacterial source S-alkyl-L-cysteine sulfoxide lyase LCC1 and the high yield expression obtained are the first in China.

(2) Calculated by the content of the substrate S-allyl-L-cysteine sulfoxide, the MIC values of the product on animal intestinal pathogenic bacteria escherichia coli, clostridium perfringens and staphylococcus aureus respectively reach 11.5 mu g/ml, 2.85 mu g/ml and 5.7 mu g/ml, and the product has outstanding inhibition effects on gram positive bacteria and gram negative bacteria.

(3) Animal experiments prove that the product has obvious effect on preventing and treating intestinal pathogenic bacteria infection, obviously reduces the chicken morbidity, and has high curative rate of enteritis and diarrhea. The product is a natural edible compound, is biosynthesized, has high biosecurity, and can be applied to the addition of various feed products.

(4) The method adopts the mode that S-alkyl-L-cysteine sulfoxide lyase LCC1 and a substrate react with each other to generate the diallyl thiosulfinate, simply and effectively avoids the defect of unstable antibacterial activity of the diallyl thiosulfinate, and belongs to the initiative in the field.

(5) The technical combination of adopting allyl mercaptan with strong garlic flavor to cooperate with diallyl thiosulfinate to increase the feed intake of animals is pioneered in the field.

(6) The high-efficiency and low-cost enzyme is obtained by adopting the conventional fermentation operation, and the blank that the isozyme of the enzyme plant has poor heterologous expression and is difficult to industrially apply is filled.

Drawings

FIG. 1 is a transformant verification diagram;

FIG. 2 shows the enzyme activity assay of sample samples at different times during fermentation;

FIG. 3 shows the MIC values of the product for each test strain.

Detailed Description

In order to make the technical means, technical features, objects and technical effects of the present invention easily understandable, the present invention is further described below with reference to the specific drawings.

Example 1:

construction of integrated expression strain of Pichia pastoris GS115, wherein the protein sequence of S-alkyl-L-cysteine sulfoxide lyase LCC1 is as follows:

(1) carrying out codon optimization synthesis on an LCC1 protein sequence of the S-alkyl-L-cysteine sulfoxide lyase according to P.pastoris GS115, designing a primer, and carrying out industrial synthesis.

(2) pPIC9K expression vector plasmid extraction, and Xho I/EcoR I double digestion, running

Agarose gel electrophoresis, and use

The vector DNA after double enzyme digestion is recovered by a Quick Gel Extraction Kit (all-type gold) Gel recovery Kit.

Digestion reaction system (100. mu.l): transformant plasmid DNA 80. mu.l; xho I5. mu.l; EcoR I5. mu.l; 10 × Buffer 10 μ l.

The enzyme digestion reaction conditions are as follows: 60min at 37 ℃.

(3) Using Lcc1-Xho I F/Lcc1-EcoR I R (each 20. mu.M) as primers, and S-alkyl-L-cysteine sulfoxide lyase LCC1 gene sequence as template, pcr amplified S-alkyl-L-cysteine sulfoxide lyase LCC1 gene.

PCR reaction (50. mu.l): 0.5. mu.l of template DNA; lcc1-Xho I F1. mu.l; lcc1-Eco R I R1 mu l; 5. mu.l of 2.5mM dNTPs; 5 × TransStartFastpfu Buffer 10 μ l; TransStart Fastpfu DNApolymerase 1. mu.l; ddH ₂ O make up to 50. mu.l.

PCR conditions were as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 53 ℃ for 30s, 72 ℃ for 90 s; 72 deg.C

5min。

And (3) running TAE glue on the product after PCR, and recovering the target gene by using a glue recovery kit.

(4) The recovered S-alkyl-L-cysteine sulfoxide lyase LCC1 gene fragment and the pPIC9K plasmid fragment were ligated.

Ligation reaction (10. mu.l): lcc15.5 μ l; pPIC9K 1.5. mu.l; 1. mu.l of T4 DNA ligase; 5 × buffer 2 μ l.

The connection reaction conditions are as follows: at 25 ℃ for 40 min.

(1) Coli DH5 α.

(2) Transformants were picked up in LB medium (50. mu.g.ml) ^-1 Kanamycin), 12h at 37 ℃ with shaking, and plasmid DNA was extracted.

(3) Xho I/EcoR I double digestion transformant plasmid pPIC9K-Lcc1, 0.5 × TBE agarose gel electrophoresis detection of digestion results.

(4) The correct transformants were stored in 15% glycerol and kept at-20 ℃ until use, while gene fragment full length sequencing was performed by the batch worker.

(5) Carrying out Sac I single enzyme digestion linearization on recombinant plasmid containing S-alkyl-L-cysteine sulfoxide lyase LCC1 gene sequence

And (4) glue, and recovering by using a glue recovery kit.

(6) Creating competent cell of P.pastoris GS115, electrically transducing the linearized recombinant expression vector into competent cell of Pichia pastoris, recovering, and coating YPD plate (200 ug/ml) ^-1 Geneticin).

(7) Transformants were picked up on YPD medium (200. mu.g.ml) ^-1 Geneticin) was cultured with shaking at 30 ℃ and the genome was extracted.

(8) The correct transformants No. 1 and No. 2 were confirmed by PCR and electrophoresis as shown in FIG. 1, the correct transformants were stored in 15% glycerol and kept at-20 ℃ until use.

(9) The correct transformant cultures were resistant at different concentrations (600, 800, 1000, 1200, 1500,

2000μg·ml ^-1 Geneticin), and storing the transformant which is tolerant to the high-resistance condition for later use.

Example 2:

fermenting to obtain enzyme with high enzyme activity

First-order seed culture: picking single colony on YPD plate to 3ml YPD culture medium, culturing at 30 deg.C and 250rpm for 12 hr;

secondary seed culture: transferring the first-stage seed culture solution into BMGY second-stage seed culture solution with a liquid containing volume of 500ml and a liquid containing volume of 100ml according to the inoculation amount of 3%, culturing at 30 ℃ and 220rpm until OD is reached ₆₀₀ To the range of 10-12;

5L fermentation tank fermentation: inoculating the secondary seed solution into a 5L fermentation tank with 10% inoculation amount, wherein the fermentation medium is BSM medium. The temperature is controlled at 28 ℃, the pH value is controlled at 6.0, and the dissolved oxygen is above 20 percent. After the glycerol in BSM is exhausted and the dissolved oxygen rises, 50% (W/V) glycerol containing 12ml/L (V/V) PTM1 is added in a flowing way until OD is reached ₆₀₀ Stopping adding glycerol when the time reaches 125, starving and culturing for 0.5-2h, adding 100% methanol containing 12ml/L (V/V) PTM1 at the rate of 8 g/(L.h) for induction, sampling every 12h during the induction to determine the enzyme activity, and as can be seen from figure 2, obtaining the highest specific enzyme activity 5400.13U/mg after 96h of induction.

Definition of enzyme activity: the S-alkyl-L-cysteine sulfoxide lyase LCC1 can generate 2 molecules of pyruvic acid when catalyzing 1 molecule of S-allyl-L-cysteine sulfoxide to react, so that the enzyme activity can be calculated by measuring the concentration of the pyruvic acid. The enzyme activity of the invention is therefore defined as: S-alkyl-L-cysteine sulfoxide lyase LCC1 catalyzes the reaction of S-allyl-L-cysteine sulfoxide at 37 ℃ and the production of L μ g pyruvate per minute is defined as 1 activity unit (U).

Example 3:

experiment of pathogenic bacteria inhibition

Three chicken-origin pathogenic bacteria, namely escherichia coli, clostridium perfringens and staphylococcus aureus, are selected as test strains. Bacteriostatic experiments were performed according to CLSI standards. The method is characterized in that a broth turbidimetric dilution method is applied in a 96-well plate, the principle is that a product with a certain concentration and a liquid culture medium containing test bacteria are subjected to a series of multiple dilutions, whether the test bacteria can be seen by naked eyes grow or not is observed after the culture, and the lowest concentration of the product capable of inhibiting the visible growth of the test bacteria by naked eyes is defined as the lowest (or Minimum) Inhibitory Concentration (MIC) of the product. 96 wellsThe plate had 12 wells per row, and the test inoculum was diluted to a concentration of 5X 10 per well according to the CLSI standard ⁵ cfu/ml. And then adding bacterial liquid into 1-11 holes, wherein 190 mu l of bacterial liquid is added into the first hole, 100 mu l of bacterial liquid is added into each of the 2-11 holes, no liquid medicine is added into the 11 th hole as a positive control, and sterile MH culture medium is added into the 12 th hole as a blank control.

Taking enzyme dry powder, S-allyl-L-cysteine sulfoxide and PLP according to the ratio of (1-10): (10-1): (10-1): (0.001-0.05), adding a certain amount of water to completely dissolve, taking 10 mu l of the product, adding the product into the first hole, fully sucking, uniformly mixing, taking 100 mu l of the product, adding the product into the next hole, sucking, uniformly mixing, fully diluting according to a 2-time dilution method, taking 100 mu l of the product after the product is uniformly sucked and blown by the 10 th hole, removing the product, and not processing the 11 th hole. The drug concentration in the first well was controlled to 0.023g/ml and the drug concentration in the subsequent wells was successively halved, calculated according to the substrate S-allyl-L-cysteine sulfoxide. And after the sample is added, putting the 96-well plate into a constant-temperature incubator at 37 ℃ for culturing for 16-20 h. The positive control should be observed for bacterial growth, the negative control (blank MH broth) should be free of bacterial growth, then the test sample is observed, the bacterial growth is visually observed as turbid, the growth is visually not observed as clear, and the corresponding lowest sample concentration in the clear well is judged as the MIC value.

It was calculated that the MIC values, which are the lowest substrate concentrations capable of inhibiting these three bacteria, were 11.5. mu.g/ml, 2.85. mu.g/ml and 5.7. mu.g/ml, respectively, as shown in FIG. 3, based on the amount of the substrate, S-allyl-L-cysteine sulfoxide. Therefore, the product has excellent inhibition effect on gram-positive bacteria and gram-negative bacteria, but has better inhibition effect on gram-positive bacteria such as aureostan and clostridium perfringens.

Example 4:

product experiment for treating enteritis of meat-eating chickens

Test animals: the broiler breeder chicks with the chicken enteritis are bred from 0 day old to 7 days old, and the number of the bred breeder chicks is 500. Test drugs: a substitute antibiotic product (a product of the patent), a market product 1, a market product 2, and ampicillin and apramycin.

And (3) test grouping: 1. the groups 3 and 4 are experimental groups. Group 2 was drug control group and antibiotics were used. Each replicate group had 20 chickens, each replicate 5. 1 group is prepared according to the concentration ratio of the antibiotics product to water of 1g/L, drinking water is concentrated, and the medicines are continuously used for 7 days; the 2 groups of antibiotics are prepared according to the concentration ratio of 7g/L of water, and the ratio of the two antibiotics is 1: 1, drinking water in a centralized way for 7 days continuously; 3 groups of market products 1 are prepared with water at the concentration ratio of 6g/L, and drinking water is concentrated for 7 days; 4 groups of market products 2 are prepared according to the concentration ratio of 0.4g/L of water, and drinking water is concentrated for 7 days; group 5 was blank without any treatment. All subjects were dosed with 100g of feed and 200kg of water for 7 consecutive days.

The measuring index and the method are as follows: (1) the stool state was observed. (2) Growth indexes are as follows: body weight changes were recorded daily. And (4) counting the feed consumption and calculating the feed weight ratio before and after the administration.

Average daily feed intake/average daily gain

As a result: as can be seen from Table 1 and the feces conditions, the average daily gain of the 1 group of patent products is the best after the test, and the material-to-weight ratio is the lowest; the fecal condition is optimal in the experimental group, the overall curative effect is best, and the overall curative effect is equivalent to that of the antibiotic group. The two selected market anti-corrosion products have poor effect, the material weight ratio is lowest, and the situation of rare events is serious.

TABLE 1 summary of test results

In summary, the embodiments of the present invention are merely exemplary and should not be construed as limiting the scope of the invention. All equivalent changes and modifications made according to the content of the claims of the present invention should fall within the technical scope of the present invention.

Claims

1. A feed additive for animal food calling and resistance replacement is characterized in that: comprises bacteria source S-alkyl-L-cysteine sulfoxide lyase LCC1 and substrates S-allyl-L-cysteine and S-allyl-L-cysteine sulfoxide; the combination ratio of the S-alkyl-L-cysteine sulfoxide lyase LCC1, the S-allyl-L-cysteine and the S-allyl-L-cysteine sulfoxide is (1-10): (10-1): (10-1), wherein the protein sequence of the S-alkyl-L-cysteine sulfoxide lyase LCC1 is as follows:

MKDLVYLNYAATSYKKFPATIEALTAYLAENQFMNYGRNAPLLREGLPLLETRQLLADFFQAPSAAQITFTNNATTSLNLALAGILQPGDHVITTMLEHHAVARPLHLLEKERGISVTYVACQKTGLLDVEDIQRAWRTNTKALVMTHASNVLGTILPIEECFQWAQQKGLLTVLDAAQTAGFLPIKMTQMAIDVLAFTGHKSLYGLAGIGGLAFSERGAEAVKPLMAGGTGSHSNSFDQPSFLPDKFEAGTLNSLGILSLNSSIKELNKIGLAAIQKHERTLMQNFLNGLSGLPVTILGTKDVAQTVPVVSITLWNQEETVVAQQLAEQYGIMTRAGLHCAPLAHETAGTLATGTLRFSFGWQTTPEEITWTIHALQELLI，

2. The feed additive for animal feeding attraction and resistance replacement according to claim 1, wherein: the composition also comprises an activator pyridoxal phosphate (PLP), and the ratio of S-alkyl-L-cysteine sulfoxide lyase LCC1 to the PLP is 1000 (1-5).

3. The feed additive for animal feeding attraction and resistance replacement according to claim 1, wherein: the S-alkyl-L-cysteine sulfoxide lyase LCC1 is obtained by expressing an expression vector in a host cell; the host cell is one of bacillus subtilis, pichia pastoris and saccharomyces cerevisiae; the expression vector is one of pHT43, pHT01, pHT08, pHT9, pHT10, pYES-DEST52, pPIC9, pPIC9K, pHIL-S1 or pPICZ alpha A.

4. The feed additive for animal feeding attraction and resistance replacement according to claim 3, wherein: the specific preparation process of the S-alkyl-L-cysteine sulfoxide lyase LCC1 is as follows:

5. The feed additive for animal feeding and resistance replacement according to claim 1, wherein: the preparation method of the S-allyl-L-cysteine comprises the following steps:

(1) the medium is 5: 1-4: 1, adding sodium hydroxide particles with the final concentration of 0.8-4.5 mol/l into the ethanol water solution, and stirring and dissolving;

(2) adding L-cysteine solid with the final concentration of 0.4-2.5 mol/L;

(3) after the materials are completely dissolved, adding bromopropylene with the same molar concentration as the cysteine, and reacting for 5-12 hours while stirring;

(4) adjusting the pH value of the solution to 4-6 by using acetic acid;

(5) filtering and drying in a drying oven.

6. The feed additive for animal feeding attraction and resistance replacement according to claim 1, wherein: the preparation method of the S-allyl-L-cysteine sulfoxide comprises the following steps:

(1) taking the S-allyl-L-cysteine prepared in claim 4

(2) And 30% hydrogen peroxide according to the mass volume ratio of (1-3) g: (6-2) ml, and reacting for 5 minutes;

(3) adding precooled ethanol, filtering and drying.

7. The use of the feed additive for animal feeding and fighting according to any of claims 1 to 6 in any of the following aspects:

(1) is used as an additive component of a premix feed,

(2) is used for adding drinking water for feeding animals,

(3) is used for feeding fishes, shrimps and crabs in aquaculture.

8. Use of the feed additive for animal feeding and resistance replacement according to any one of claims 1 to 6 in the following fields:

(1) the prevention and treatment of livestock and poultry animal diseases caused by fungi and bacterial pathogenic bacteria, such as enteritis, diarrhea and the like,

(2) preventing and treating parasitic diseases of livestock and fowl such as chicken coccidiosis and chicken leucocytozoonosis,

(3) preventing and treating pathogenic bacteria and parasite of aquatic animals such as fish, shrimp, crab, etc.