A lactic acid bacterium producing aminopeptidase and its application in fermentative debittering of oligopeptide feed

Abstract

The present invention discloses a lactic acid bacterium producing aminopeptidase and its application in the fermentation and debittering of oligopeptide feed. The lactic acid bacterium uses Enterococcus faecium as an expression host and pQE-30 as an expression vector, and recombinantly expresses an aminopeptidase gene with a nucleotide sequence as shown in SEQ ID NO.1. The present invention applies the engineered lactic acid bacterium producing aminopeptidase to feed fermentation, making the feed rich in oligopeptides and debittering the oligopeptides, which not only improves the nutritional value of the feed, but also improves the overall palatability of the feed, and further increases the probiotics and prebiotics in the feed, having extremely high application prospects.

Description

Technical Field

[0001] The present invention relates to the technical fields of genetic engineering and microecological fermentation technology, and in particular to a lactic acid bacterium producing aminopeptidase and its application in oligopeptide feed fermentation for debittering. Background Art

[0002] Soybean meal is a by-product after soybean oil is extracted from soybeans, and is often used as the main raw material in the preparation of animal feed, accounting for more than 60% of the protein sources in the feed industry. Due to its high protein content and good nutritional characteristics, it is known as the king of vegetable protein feeds. Among the proteins contained in soybean meal, the proportion of water-soluble proteins is relatively high, but these water-soluble protein molecules have large molecular weights and complex structures. If soybean meal is directly used to feed animals, the proportion of its protein absorbed and utilized is not high, which will cause waste of soybean meal resources and indirectly lead to an increase in feeding costs. In addition, there are certain amounts of anti-nutritional factors in soybean meal, which hinder the digestion and absorption of feed protein by animals.

[0003] Research shows that animals have better absorption effects on oligopeptides and small molecule peptides than on macromolecular proteins. The existing technology is to use enzymatic hydrolysis and microbial fermentation methods to process soybean meal to prepare high-efficiency animal feed. For example, using enzymes in combination with Bacillus coagulans to produce high-efficiency protein feed, using the method of mixed fermentation of microbial live dry bacteria to prepare soybean meal feed, using compound probiotics to ferment soybean meal feed to improve the conversion rate of soybean isoflavones, and using the method of enzymatic hydrolysis to prepare oligopeptide soybean meal for aquatic feed. By enzymatic hydrolysis and fermentation treatment of soybean meal, firstly, macromolecular proteins can be converted into oligopeptides and small molecule peptides, and secondly, anti-nutritional factors in soybean meal can be eliminated. Therefore, the absorption efficiency of animals for feed nutrients can be greatly improved. However, when macromolecular proteins are hydrolyzed, a certain amount of bitter peptides will be produced, thus affecting the palatability of animals.

[0004] In the existing technology, aminopeptidase is used to treat protein hydrolysates to achieve the effect of debittering. For example, aminopeptidase is used to treat the intermediate product of soybean meal fermentation for debittering. Due to the high price of aminopeptidase, the cost of debittered protein feed is increased.

[0005] Existing aminopeptidases generally have problems such as low yield, low enzyme activity, and high price. For the heterologous expression research of aminopeptidase, most of them focus on microorganisms such as Escherichia coli and Bacillus subtilis, which have the advantages of clear genetic background, fast reproduction speed, and short fermentation cycle. However, Escherichia coli can only express intracellularly, and heterologous expression is prone to form inclusion bodies, and there are endotoxins in Escherichia coli, which limits its application in the food industry; although Bacillus subtilis has strong secretion ability, due to its own ability to produce a large amount of extracellular proteases that can recognize and degrade foreign proteins, only a few heterologous proteins can be produced on an industrial scale. Currently, food-grade excretory aminopeptidase gene engineering strains are rare. Summary of the Invention

[0006] To solve the problem of bitter peptides existing in fermented feed, the object of the present invention is to provide a lactic acid bacterium producing aminopeptidase and its application in debittering oligopeptide feed fermentation

[0007] To achieve the above object, a lactic acid bacterium producing aminopeptidase of the present invention uses Enterococcus faecium ( Enterococcus faecium ) as an expression host and pQE-30 as an expression vector, and recombines the aminopeptidase gene with the nucleotide sequence shown in SEQ ID NO.1.

[0008] Furthermore, the construction method of the lactic acid bacterium includes:

[0009] 1) Obtaining of template DNA fragment: According to the aminopeptidase gene sequence (SEQ ID NO.1, GenBank:UIN45439.1) derived from Bacillus licheniformis ;

[0010] 2) Designing primers according to the aminopeptidase gene sequence (SEQ ID NO.1), upstream primer PF: 5’-CGC GGATCC ATGTTTTATGCCTTTAAAGATTTCG-3’; downstream primer PR: 5’-CGG AAGCTT CTATAAATGATCCGAAAACCGCTCG-3’;

[0011] 3) Amplifying the target gene fragment by PCR reaction;

[0012] 4) Double-digesting the amplified target gene fragment and plasmid pQE-30 with Bam HⅠ and Hin dⅢ, purifying the enzyme digestion products and then ligating them with T4 ligase. The recombinant plasmid is transformed into Escherichia coli DH5α by electroporation, and the plasmid is extracted for preliminary identification of the recombinant by double digestion. After sequencing, the recombinant vector pQE30-AP is obtained;

[0013] 5) Preparing Enterococcus faecium competent cells;

[0014] 6) Mixing the recombinant vector pQE30-AP and Enterococcus faecium competent cells in a ratio of 1:100, coating them on an MRS screening plate containing ampicillin after electroporation, picking transformants for colony PCR rapid verification after culturing, and the successfully amplified ones are recombinant strains, namely lactic acid bacteria producing aminopeptidase.

[0015] Further, the step of amplifying the target gene fragment by PCR reaction in step 3) is as follows: PCR reaction system: 10 μL of 5×PrimeSTAR buffer, 2 μL of dNTP mix (10 mM), 1 μL of upstream primer PF, 1 μL of downstream primer PR, 1 μL of plasmid template, 0.5 μL of PrimeSTAR HS DNA polymerase (2.5 U / μl), and make up the volume to 50 μL with water; PCR reaction program: pre-denaturation at 96 °C for 4 min, denaturation at 96 °C for 20 sec, annealing at 55 °C for 15 sec, extension at 72 °C for 1 min, final extension at 72 °C for 5 min, for 32 cycles.

[0016] Further, in step 5), the preparation steps of Enterococcus faecalis competent cells include:

[0017] 1) Pick up Enterococcus faecalis strain and inoculate it into 50 mL of MRS broth, culture it at 37 °C with shaking at 200 r / min for 24 h to prepare a seed solution;

[0018] 2) Transfer the seed solution to 100 mL of MRS broth at an inoculation amount of 5%, culture it at 37 °C with shaking at 200 r / min until the OD 550 reaches 0.5 - 0.6;

[0019] 3) Take 10 mL of the bacterial solution and place it in a sterile centrifuge tube, centrifuge at 4 °C and 6000 r / min for 5 min, and discard the supernatant;

[0020] 4) Suspend and wash the bacterial cells with 10 mL of pre-cooled sterile water, place them on ice for 30 min, centrifuge at 4 °C and 6000 r / min for 5 min, and discard the supernatant;

[0021] 5) Suspend and wash the bacterial cells with 1 mL of pre-cooled 10% sterile glycerol, centrifuge at 4 °C and 6000 r / min for 5 min, and discard the supernatant;

[0022] 6) Suspend the bacterial cells with 1 mL of pre-cooled 10% sterile glycerol to obtain Enterococcus faecalis competent cells.

[0023] The present invention provides an application of the above-mentioned lactic acid bacteria in the process of fermenting and debittering oligopeptide feed.

[0024] Further, the process of fermenting and debittering oligopeptide feed includes the following steps:

[0025] 1) Preparation of lactic acid bacteria seed solution: Inoculate the activated Enterococcus faecalis slant strain into MRS broth, and culture it in a shaker at 37 °C and 150 - 180 r / min for 28 - 30 h;

[0026] 2) High-density fermentation of lactic acid bacteria to produce aminopeptidase

[0027] 3) Substrate fermentation: The substrate consists of soybean meal powder, wheat bran, sucrose, lactic acid bacteria fermentation broth, and acid protease;

[0028] 4) After the substrate is mixed evenly, deionized water is added until the moisture content of the total fermentation material ranges from 35% to 45%. After mixing evenly, fermentation and debittering treatment are carried out. The conditions are: ferment at 37 °C for 4 - 6 d to obtain the debittered oligopeptide fermented feed.

[0029] Furthermore, the fermentation medium formula in step 2) is: yeast extract 3%, peptone 1%, lactose 4%, dipotassium hydrogen phosphate 0.2%, sodium acetate 0.5%, ammonium citrate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.005%, pH 6.2; The lactic acid bacteria seed liquid is inoculated into the fermentation medium at an inoculation amount of 5%, and the liquid filling amount of the fermenter is 70%. The culture conditions are: 37 °C, stirring speed 120 - 150 r / min, ventilation volume 0.5 - 0.6 m 3 / h, culture for 18 - 20 h to obtain the lactic acid bacteria fermentation broth rich in aminopeptidase.

[0030] Furthermore, in step 2), the aminopeptidase activity in the lactic acid bacteria fermentation broth is not less than 1200 U / mL, and the density of Enterococcus faecium is not less than 2.5×10 9 CFU / mL.

[0031] Furthermore, in step 3), the proportion of oligopeptides with a molecular weight below 3000 Da in the debittered oligopeptide fermented feed accounts for not less than 30% of the total protein content of the feed.

[0032] Furthermore, in step 4), the debittered oligopeptide fermented feed needs to be dried. The drying temperature range is 50 - 60 °C, and the moisture content of the feed is not higher than 12%.

[0033] The technical solution of the present invention has the following advantages and effects:

[0034] (1) A lactic acid bacteria producing aminopeptidase provided by the present invention is an engineered bacterium with Enterococcus faecium ( Enterococcus faecium ) as the expression host, which can express aminopeptidase in an excretory manner, and has the characteristics of simultaneous fermentation and enzymatic debittering. It can improve the two-step process of prior fermentation followed by enzymatic debittering in the traditional process to a one-step method of simultaneous fermentation and debittering, shortening the production process and saving costs;

[0035] (2)The defatted oligopeptide feed prepared by the production process of the present invention has a high oligopeptide content, accounting for more than 30% of the total protein content of the feed, which is beneficial to the rapid absorption of the fed animals. Moreover, it is basically not bitter after fermentation and defatting. With the fermentation of lactic acid bacteria, the overall palatability of the feed is improved. At the same time, probiotics and prebiotics are added to the feed, enhancing the nutritional efficacy and quality of the feed. Detailed implementation manners

[0036] The technical solutions of the present invention will be described clearly and completely below. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention. It should be understood that the specific implementation manners described herein are only for explaining and illustrating the present invention and are not used to limit the present invention.

[0037] Example 1:

[0038] This example relates to a lactic acid bacterium producing aminopeptidase, which uses Enterococcus faecium ( Enterococcus faecium ) as an expression host and pQE-30 as an expression vector, and recombines the aminopeptidase gene with the nucleotide sequence shown in SEQ ID NO.1.

[0039] This example relates to a lactic acid bacterium producing aminopeptidase, and the construction method involves the following steps:

[0040] 1. Obtaining of the template DNA fragment: According to the aminopeptidase gene sequence (SEQ ID NO.1, GenBank:UIN45439.1) derived from Bacillus licheniformis , Guangzhou Da'an Gene Co., Ltd. was entrusted to perform artificial synthesis by chemical synthesis method.

[0041] 2. Designing primers according to the aminopeptidase gene sequence (SEQ ID NO.1):

[0042] Upstream primer PF: 5’-CGC GGATCC ATGTTTTATGCCTTTAAAGATTTCG-3’ (the underlined sequence represents Bam the HⅠ restriction enzyme site);

[0043] Downstream primer PR: 5’-CGG AAGCTT CTATAAATGATCCGAAAACCGCTCG-3’ (the underlined sequence represents Hin the dⅢ restriction enzyme site).

[0044] 3. Amplify the target gene fragment by PCR reaction; PCR reaction system: 10 μL of 5×PrimeSTAR buffer, 2 μL of dNTPmix (10 mM), 1 μL of upstream primer PF, 1 μL of downstream primer PR, 1 μL of plasmid template, 0.5 μL of PrimeSTAR HS DNA polymerase (2.5 U / μL), make up the volume to 50 μL with water; PCR reaction program: pre-denaturation at 96°C for 4 min, denaturation at 96°C for 20 sec, annealing at 55°C for 15 sec, extension at 72°C for 1 min, final extension at 72°C for 5 min, 32 cycles.

[0045] 4. Digest the amplified target gene fragment and plasmid pQE-30 with Bam HⅠ and Hin dⅢ by double digestion. After purification of the digestion products, ligate them with T4 ligase. The recombinant plasmid is transformed into Escherichia coli DH5α by electroporation. Extract the plasmid and perform double digestion for preliminary identification of the recombinant. Send the recombinant plasmid to Shanghai Sangon Biotech Co., Ltd. for sequencing. If the sequence is correct, it is the recombinant vector pQE30-AP.

[0046] 5. Preparation of Enterococcus faecium competent cells, and the specific steps include:

[0047] (1) Pick an Enterococcus faecium strain and inoculate it into 50 mL of MRS broth. Incubate it at 37°C with shaking at 200 r / min for 24 h to prepare a seed culture.

[0048] (2) Transfer the seed culture to 100 mL of MRS broth at an inoculation amount of 5%. Incubate it at 37°C with shaking at 200 r / min until the OD 550 reaches 0.5 - 0.6.

[0049] (3) Take 10 mL of the bacterial solution and place it in a sterile centrifuge tube. Centrifuge it at 4°C at 6000 r / min for 5 min, and discard the supernatant.

[0050] (4) Suspend and wash the above-mentioned bacterial cells with 10 mL of pre-cooled sterile water, place them on ice for 30 min, centrifuge at 4°C at 6000 r / min for 5 min, and discard the supernatant.

[0051] (5) Suspend and wash the above-mentioned bacterial cells with 1 mL of pre-cooled 10% sterile glycerol, centrifuge at 4°C at 6000 r / min for 5 min, and discard the supernatant.

[0052] (6) Suspend the above-mentioned bacterial cells with 1 mL of pre-cooled 10% sterile glycerol to obtain Enterococcus faecium competent cells.

[0053] 6. Preparation of recombinant engineering strain: Mix the recombinant plasmid pQE30-AP and Enterococcus faecium competent cells at a ratio of 1:100, coat the mixture on an MRS screening plate containing ampicillin after electrotransformation, pick the transformants for rapid verification by colony PCR after culture, and the successfully amplified ones are recombinant strains, namely lactic acid bacteria producing aminopeptidase.

[0054] Example 2:

[0055] This example relates to a production process for fermentative debittering of oligopeptide feed, including the following steps:

[0056] 1. Preparation of lactic acid bacteria seed liquid: Inoculate the activated Enterococcus faecium recombinant strain slant culture into MRS broth, place it in a shaker at 37 °C and 150 - 180 r / min for oscillating culture for 28 - 30 h to obtain lactic acid bacteria seed liquid.

[0057] 2. High-density fermentation of lactic acid bacteria producing aminopeptidase in liquid state: The fermentation medium formula is: yeast extract 3%, peptone 1%, lactose 4%, dipotassium hydrogen phosphate 0.2%, sodium acetate 0.5%, ammonium citrate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.005%, pH 6.2; Inoculate the lactic acid bacteria seed liquid into the fermentation medium at an inoculation amount of 5%, the liquid filling amount of the fermenter is 70%, and the culture conditions are: 37 °C, stirring speed 120 - 150 r / min, ventilation volume 0.5 - 0.6 m 3 / h, culture for 18 - 20 h to obtain lactic acid bacteria fermentation broth rich in aminopeptidase; The aminopeptidase activity in the lactic acid bacteria fermentation broth is not less than 1200 U / mL, and the density of Enterococcus faecium is not less than 2.5×10 9 CFU / mL.

[0058] Determination of aminopeptidase activity in lactic acid bacteria fermentation broth: The LNA method is used to determine the enzyme activity of the enzyme solution in the fermentation broth. a. Prepare a series of standard solutions of p-nitroaniline, measure the absorbance at a wavelength of 405 nm, draw a standard curve, and obtain the regression equation; b. Centrifuge the lactic acid bacteria fermentation broth at 5000 r / min and 4 °C for 10 min, collect the supernatant, which is the enzyme solution to be measured; c. In a stoppered test tube, add Tris-HC1 buffer solution, L-leucine p-nitroaniline and the enzyme solution to be measured in sequence, react in a 37 °C water bath for 10 min, terminate the reaction with 30% acetic acid, measure the absorbance at a wavelength of 405 nm, and calculate the enzyme activity according to the regression equation of the standard curve. Definition of enzyme activity: Under the condition of 37 °C, the amount of enzyme required to decompose the substrate to produce 1 nmoL p-nitroaniline in 1 min is defined as one enzyme activity unit;

[0059] Determination of Enterococcus faecium density in lactic acid bacteria fermentation broth: Refer to the method in the national food safety standard GB 4789.35-2016.

[0060] 3. Substrate Fermentation: The substrate consists of soybean meal powder (passed through a 40-mesh sieve), wheat bran, sucrose, lactic acid bacteria fermentation broth, and acid protease, with the following weight parts for each component: 60 parts of soybean meal powder, 3 parts of wheat bran, 2 parts of sucrose, 10 parts of lactic acid bacteria fermentation broth, and 0.5 part of acid protease. After mixing the above substrates evenly, deionized water is added until the moisture content of the total fermentation material is 45%. After mixing evenly, solid-state fermentation is carried out at 37 °C for 4 days to obtain a defatted oligopeptide fermentation feed; among them, the content of oligopeptides with a molecular weight below 3000 Da accounts for no less than 30% of the total protein content of the feed, and the feed does not show bitterness (bitterness score 0.44).

[0061] Determination of Oligopeptide Content in Fermentation Feed: Take 100 g of the fermentation feed, add 300 g of deionized water, shake well to dissolve, centrifuge at 4000 r / min for 10 min, and transfer the supernatant to a clean container; suspend the feed precipitate with another 300 g of deionized water, shake well, centrifuge at 4000 r / min for 10 min, and combine the supernatants; repeat the operation for the feed precipitate once more and combine the supernatants from the three times; subject all the supernatants to ultrafiltration treatment with a membrane having a molecular weight cut-off of 3000 Da. The obtained oligopeptide retentate is used to determine the amount of oligopeptides by the Kjeldahl method, and at the same time, the total protein content in the fermentation feed is determined by the Kjeldahl method. The ratio of the two is the oligopeptide content in the fermentation feed.

[0062] Determination of Bitterness Value of Fermentation Feed: A panel of 9 experienced personnel uses the sensory analysis method for determination. The fermentation feed and drinking water are mixed evenly at a solid-liquid ratio of 1:2, filtered to obtain the filtrate. After rinsing their mouths with distilled water, the appraisers take an appropriate amount of the filtrate to be evaluated and hold it in their mouths, then spit it out after 10 s. Using a series of quinine sulfate concentration solutions as the standard reference substances, if the bitterness of the sample solution to be measured is similar to that of a certain concentration standard solution, the bitterness intensity of the sample solution to be measured can be considered as the bitterness intensity of that standard solution. Finally, the average value of the 9 appraisers is taken as the bitterness value of the fermentation feed.

[0063] The bitterness standards of the series of quinine sulfate concentration solutions as standard reference substances are as shown in Table 1 below:

[0064] Concentration of quinine sulfate solution (mol / L) <![CDATA[8×10 -4 > <![CDATA[4×10 -4 > <![CDATA[2×10 -4 > <![CDATA[1×10 -4 > <![CDATA[5×10 -5 > <![CDATA[2.5×10 -5 > <![CDATA[1.25×10 -5 > Bitterness description Very bitter Bitter Rather bitter Moderately bitter Slightly bitter Slightly bitter None Bitterness score 6 5 4 3 2 1 0

[0065] 4. Drying: Place the above defatted oligopeptide fermentation feed in a dryer at 50 - 60 °C for drying treatment until the moisture content is not higher than 12% to obtain the dried defatted oligopeptide fermentation feed.

[0066] 5. Packaging: Cool down and pulverize the above dried feed, and pack it into bags to obtain the finished product of defatted oligopeptide fermentation feed.

[0067] Example 3:

[0068] This embodiment relates to a production process for fermenting and debittering oligopeptide feed, comprising the following steps:

[0069] 1. Preparation of lactic acid bacteria seed liquid: Inoculate the slant culture of the recombinant Enterococcus faecium strain after activation into MRS broth, and place it in a shaker at 37 °C and 150 r / min for oscillating culture for 30 h to obtain lactic acid bacteria seed liquid.

[0070] 2. High-density fermentation of lactic acid bacteria for producing aminopeptidase: The fermentation medium formula is as follows: yeast extract 3%, peptone 1%, lactose 4%, dipotassium hydrogen phosphate 0.2%, sodium acetate 0.5%, ammonium citrate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.005%, pH 6.2; inoculate the lactic acid bacteria seed liquid into the fermentation medium at an inoculation amount of 5%, with the liquid volume in the fermenter being 70%, and the culture conditions being: 37 °C, stirring speed 120 r / min, aeration volume 0.6 m 3 / h, culture for 18 h to obtain lactic acid bacteria fermentation broth; among them, the enzyme activity of aminopeptidase in the fermentation broth is 1295 U / mL, and the density of Enterococcus faecium is 2.7×10 9 CFU / mL.

[0071] Determination of the enzyme activity of aminopeptidase in the lactic acid bacteria fermentation broth: The same as in Example 2.

[0072] Determination of the density of Enterococcus faecium in the lactic acid bacteria fermentation broth: The same as in Example 2.

[0073] 3. Substrate fermentation: The substrate consists of soybean meal powder (screened through 40 meshes), wheat bran, sucrose, lactic acid bacteria fermentation broth, and acidic protease, and the weight parts of each component are as follows: 50 parts of soybean meal powder, 2.5 parts of wheat bran, 1.5 parts of sucrose, 5 parts of lactic acid bacteria fermentation broth, and 0.25 part of acidic protease. After the substrate is mixed evenly, add deionized water until the moisture content of the total fermentation material is 35%, mix evenly, and perform solid-state fermentation at 37 °C for 6 d to obtain debittered oligopeptide fermented feed; among them, the content of oligopeptides with a molecular weight below 3000 Da accounts for 30.5% of the total protein content of the feed, and the feed does not show bitterness (bitterness score 0.33).

[0074] Determination of the oligopeptide content in the fermented feed: The same as in Example 2.

[0075] Determination of the bitterness value of the fermented feed: The same as in Example 2.

[0076] 4. Drying: Place the above-mentioned debittered oligopeptide fermented feed in a dryer at 50 °C for drying treatment until the moisture content is lower than 12% to obtain dried debittered oligopeptide fermented feed;

[0077] 5. Packaging: Cool and pulverize the above-mentioned dried feed, and pack and bag it to obtain the finished product of debittered oligopeptide fermented feed.

[0078] The above are only the preferred embodiments of the present invention, and do not impose any formal restrictions on the present invention. For any ordinary person skilled in the art, any local changes, combinations, modifications, process transformations, etc. made by using the content of the specification and embodiments of the present invention, directly or indirectly applied to other related technical fields, shall be included within the protection scope of the present invention.

Claims

1. A lactic acid bacterium producing aminopeptidase, characterized in that, The lactic acid bacterium uses Enterococcus faecium as the expression host and pQE-30 as the expression vector, and recombines the aminopeptidase gene with the nucleotide sequence shown in SEQ ID NO.

1.

2. A method for constructing the lactic acid bacterium producing aminopeptidase according to claim 1, characterized in that, It includes: 1) Obtaining the template DNA fragment: According to the aminopeptidase gene sequence SEQ ID NO.1 derived from Bacillus licheniformis; 2) Design primers according to the aminopeptidase gene sequence. Forward primer PF: 5’-CGC GGATCC ATGTTTTATGCCTTTAAAGATTTCG-3’; Reverse primer PR: 5’-CGGAAGCTTCTATAAATGATCCGAAAACCGCTCG-3’; 3) Amplifying the target gene fragment by PCR reaction; 4) Double-digesting the amplified target gene fragment and plasmid pQE-30 with BamHⅠ and HindⅢ. After purification of the digestion products, they are ligated with T4 ligase. The recombinant plasmid is transformed into Escherichia coli DH5α by electroporation. The plasmid is extracted and preliminarily identified by double digestion for the recombinant, and after sequencing, the recombinant vector pQE30-AP is obtained; 5) Preparing competent Enterococcus faecium cells; 6) Mixing the recombinant vector pQE30-AP and competent Enterococcus faecium cells at a ratio of 1:100, spreading them on an MRS screening plate containing ampicillin after electroporation, picking transformants for rapid verification by colony PCR after cultivation, and the ones with successful amplification are recombinant strains, namely lactic acid bacteria producing aminopeptidase.

3. A method for constructing the lactic acid bacterium producing aminopeptidase according to claim 2, characterized in that, The steps for amplifying the target gene fragment by PCR reaction in step 3) are as follows: PCR reaction system: 10 μL of 5×PrimeSTAR buffer, 2 μL of 10 mM dNTPmix, 1 μL of upstream primer PF, 1 μL of downstream primer PR, 1 μL of plasmid template, 0.5 μL of 2.5 U / μL PrimeSTAR HS DNA polymerase, and make up the volume to 50 μL with water; PCR reaction program: pre-denaturation at 96°C for 4 min, denaturation at 96°C for 20 sec, annealing at 55°C for 15 sec, extension at 72°C for 1 min, extension at 72°C for 5 min, 32 cycles.

4. A method for constructing the lactic acid bacterium producing aminopeptidase according to claim 2, characterized in that, In step 5), the steps for preparing competent Enterococcus faecium cells include: 1) Picking Enterococcus faecium strains and inoculating them into 50 mL of MRS broth, culturing them at 37°C with shaking at 200 r / min for 24 h to prepare a seed solution; 2) Transfer the seed solution to 100 mL of MRS broth at an inoculation amount of 5%, and culture it with shaking at 37 °C and 200 r / min until the OD 550 reaches 0.5 - 0.6; 3) Taking 10 mL of the bacterial solution and placing it in a sterile centrifuge tube, centrifuging at 4°C and 6000 r / min for 5 min, and discarding the supernatant; 4) Suspending and washing the bacterial cells with 10 mL of pre-cooled sterile water, placing them on ice for 30 min, centrifuging at 4°C and 6000 r / min for 5 min, and discarding the supernatant; 5) Suspending and washing the bacterial cells with 1 mL of pre-cooled 10% sterile glycerol, centrifuging at 4°C and 6000 r / min for 5 min, and discarding the supernatant; 6) Suspending the bacterial cells with 1 mL of pre-cooled 10% sterile glycerol to obtain competent Enterococcus faecium cells.

5. The application of the lactic acid bacterium according to claim 1 or the construction method according to any one of claims 2 - 4 in the process of oligopeptide feed fermentation and debittering.

6. The application according to claim 5, characterized in that, The steps of the oligopeptide feed fermentation and debittering process include: 1) Preparation of lactic acid bacteria seed solution: Inoculating the activated Enterococcus faecium slant strain into MRS broth, and culturing it in a shaker at 37°C and 150 - 180 r / min for 28 - 30 h; 2) High-density fermentation of lactic acid bacteria for producing aminopeptidase in liquid state; 3) Substrate fermentation: The substrate consists of soybean meal powder, wheat bran, sucrose, lactic acid bacteria fermentation broth, and acidic protease; 4) After the base materials are evenly mixed, deionized water is added until the moisture content of the total fermented feed ranges from 35% to 45%. After mixing evenly, fermentation and debittering treatment are carried out under the conditions of: fermenting at 37°C for 4 - 6 days to obtain the debittered oligopeptide fermented feed.

7. The application according to claim 6, characterized in that, In step 2), the fermentation medium formula is as follows: yeast extract 3%, peptone 1%, lactose 4%, dipotassium hydrogen phosphate 0.2%, sodium acetate 0.5%, ammonium citrate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.005%, pH 6.2; the lactic acid bacteria seed liquid is inoculated into the fermentation medium at an inoculation amount of 5%, the liquid filling amount in the fermentation tank is 70%, and the culture conditions are: 37 °C, stirring speed 120-150 r / min, ventilation volume 0.5-0.6 m 3 / h, culture for 18-20 h to obtain a lactic acid bacteria fermentation broth rich in aminopeptidase.

8. The application according to claim 6, characterized in that, In step 2), the aminopeptidase activity in the lactic acid bacteria fermentation broth is not less than 1200 U / mL, and the density of Enterococcus faecium is not less than 2.5×10 9 CFU / mL.

9. The application according to claim 6, characterized in that, In the step 3) described above, the proportion of oligopeptides with a molecular weight below 3000 Da in the debittered oligopeptide fermented feed accounts for no less than 30% of the total protein content of the feed.

10. The application according to claim 6, characterized in that, In the step 4) described above, the debittered oligopeptide fermented feed needs to be dried. The drying temperature range is 50 - 60°C, and the moisture content of the feed is not higher than 12%.