Application of glutathione peroxidase in feed preparation
By using recombinant Pichia glutathione peroxidase expressed by recombinant Pichia cerevisiae in feed, the problem of inhibition of trypsin activity is solved, and the nutritional digestibility and breeding benefits of the feed are improved.
Patent Information
- Application Number
- CN202510215362.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-26
- Publication Date
- 2025-04-22
AI Technical Summary
The trypsin inhibitors present in soybean meal can inhibit the activity of trypsin in the animal's digestive tract, affect protein digestion, lead to nutrient waste, and reduce breeding benefits.
The recombinant Pichia glutathione peroxidase expressed in vitro was prepared and added to the feed raw material. The trypsin inhibitor in the feed was decomposed by mixing the high-pressure homogenized rupture solution and the feed raw material.
It significantly reduces the inhibitory effect of trypsin inhibitors in feed on trypsin activity, improves the digestion and absorption rate of nutrients in feed, reduces the feed-to-meat ratio, advances the chicken slaughter time, and improves the breeding efficiency and benefits.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of feed, and in particular relates to application of glutathione peroxidase in feed preparation. Background Art
[0002] Soybean meal is a byproduct obtained after soybeans are squeezed to make soybean oil. The crude protein content in soybean meal is as high as 30-50%, and it is the main nitrogen source for making animal feed. Soybean meal is slightly cheaper than other feed nitrogen sources, and its supply is stable, easy to store, process and transport. It occupies a dominant position in high-protein animal feed and is not easily replaced.
[0003] Soybean meal not only provides amino acids, vitamins and other nutrients to animals, but also contains some nutritional inhibitors. Among them, trypsin inhibitor is the main nutritional inhibitor in soybean meal, which can inhibit the activity of trypsin in the animal digestive tract, affect protein digestion, cause nutritional waste, and seriously reduce breeding benefits. The specific types of trypsin inhibitors in soybean meal are still unclear, and the method of eliminating trypsin inhibitors in soybean meal feed is still in the research stage.
[0004] Glutathione peroxidase (GSH-Px) is an important peroxide decomposing enzyme in the body. The active center of GSH-Px is selenocysteine, and its activity can reflect the selenium level of the body. Selenium is a component of the GSH-Px enzyme system. It can catalyze GSH to GSSG, reducing toxic peroxides to non-toxic hydroxyl compounds, thereby protecting the structure and function of the cell membrane from interference and damage by peroxides. GSH-Px mainly includes four types: cytoplasmic GSH-Px, plasma GSH-Px, phospholipid hydroperoxide GSH-Px and gastrointestinal-specific GSH-Px. Among them, gastrointestinal-specific GSH-Px is a tetramer composed of four subunits with a molecular weight of 22kDa. It only exists in the gastrointestinal tract of rodents and its function is to protect animals from damage caused by the ingestion of lipid peroxides. Gastrointestinal-specific glutathione peroxidase has a certain effect on eliminating trypsin inhibitors in soybean meal feed. Therefore, the in vitro preparation of glutathione peroxidase for feed raw material processing can achieve the purpose of eliminating trypsin inhibitors in soybean meal feed. Summary of the invention
[0005] The invention aims to provide an application of glutathione peroxidase in feed preparation, belonging to the technical field of feed.
[0006] To achieve the above object, the present invention provides the following technical solutions:
[0007] Firstly, the present invention provides an application of glutathione peroxidase in feed preparation, wherein the glutathione peroxidase is glutathione peroxidase expressed by recombinant Pichia pastoris, and the amino acid sequence of the glutathione peroxidase is SEQ ID NO.1.
[0008] Furthermore, the nucleotide sequence of the glutathione peroxidase expression regulatory gene is SEQ ID NO.2.
[0009] Furthermore, the glutathione peroxidase can be used to reduce the inhibitory effect of trypsin inhibitor in feed on animal trypsin activity.
[0010] Furthermore, the preparation method of the feed is as follows:
[0011] (1) Recombinant Pichia pastoris expressing glutathione peroxidase was inoculated with YPD culture medium at a volume ratio of 1%, and cultured at 30°C and 250 rpm for 24 h as the primary seed liquid;
[0012] (2) According to the fermentation volume, the first-stage seed solution was inoculated with YPD culture solution at a volume ratio of 1% to prepare the second-stage seed solution and multi-stage seed solution;
[0013] (3) The multi-grade seed solution was inoculated with YPD culture medium containing 50 mg / L sodium selenite at a volume ratio of 1%, and the fermentation was carried out for 120 h;
[0014] (4) The fermented bacterial liquid is crushed by a high-pressure homogenizer, and the mass ratio of the feed raw material to the high-pressure homogenized and crushed bacterial liquid is 10:1. The mixture is stirred and mixed by a mixer, and the mixture is incubated at 37°C for 12 hours to decompose the trypsin inhibitor in the feed. The mixture is then granulated by a granulator to form the feed.
[0015] Secondly, the present invention provides a gene for expressing glutathione peroxidase, and the nucleotide sequence of the expression regulatory gene of glutathione peroxidase is SEQ ID NO.2.
[0016] The beneficial effect of the present invention is that by constructing a recombinant Pichia yeast expressing glutathione peroxidase, the expressed glutathione peroxidase is added to the crushed feed raw materials and stirred and mixed by a mixer, and the feed is made into feed at 37°C for 12 hours to decompose the trypsin inhibitor in the feed. After the feed without glutathione peroxidase is reacted with trypsin, the trypsin activity is only 60.40%; after the feed prepared by adding glutathione peroxidase is reacted with trypsin, the trypsin activity is 95.09%. Adding glutathione peroxidase to the feed raw materials for treatment significantly reduces the inhibitory effect of the trypsin inhibitor in the feed on the trypsin activity. The feed-to-meat ratio of white-feathered broilers fed with feed prepared by adding glutathione peroxidase is lower than that of the group without adding glutathione peroxidase, and the chickens in the group with adding glutathione peroxidase are marketed two days earlier than those in the group without adding glutathione peroxidase, which reduces feed consumption, improves breeding efficiency, and effectively increases breeding benefits. The application of glutathione peroxidase in feed can, on the one hand, improve the digestion and absorption rate of nutrients in feed by animals, reduce the feed-to-meat ratio, and improve breeding efficiency; on the other hand, it can reduce the amount of protein raw materials such as soybean meal in feed, effectively reduce the amount of soybean meal in feed, and reduce feed costs. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 : Effect of soybean meal supplemented with glutathione peroxidase on trypsin activity.
[0018] Figure 2 : Effect of feed supplemented with glutathione peroxidase on trypsin activity. DETAILED DESCRIPTION
[0019] Example 1: Construction of recombinant Pichia pastoris expressing glutathione peroxidase
[0020] 1.1 Gene design for expression of glutathione peroxidase
[0021] The GenBank accession number of the amino acid sequence of glutathione peroxidase is: NP_109602.2, the amino acid sequence is SEQ ID NO.1, the 40th amino acid is selenocysteine U, and the coding sequence is TGA. In eukaryotic cells, TGA is transcribed into mRNA as UGA, which is a stop codon during protein translation, and selenocysteine U cannot be synthesized, and protein translation will terminate here. The GenBank accession number of the selenoprotein P gene sequence is: NM_005410.4, and its 1217-1974 bp is the selenocysteine UGA translation regulatory region sequence, which regulates the translation of UGA into selenocysteine during protein translation. The 1217-1974 bp selenocysteine UGA translation regulatory region sequence of the NM_005410.4 sequence is connected in series after the glutathione peroxidase coding gene, which can regulate the translation of UGA into selenocysteine during the translation of glutathione peroxidase protein, thereby obtaining active glutathione peroxidase. The nucleotide sequence of the expression regulatory gene of glutathione peroxidase was designed to be SEQ ID NO.2, EcoRI and NotI restriction endonuclease sites were added upstream and downstream, respectively, and it was commissioned to Sangon Biotechnology (Shanghai) Co., Ltd. for synthesis and cloned into the pUC57 plasmid to obtain the pUC57-GSH-Px plasmid.
[0022] 1.2 Construction of recombinant expression vector
[0023] (1) Double digest the pUC57-GSH-Px plasmid with restriction endonucleases EcoRI and NotI to recover the GSH-Px gene fragment;
[0024] (2) using restriction endonucleases EcoRI and NotI to double-digest the expression vector pGAPZA and recover the gene fragment;
[0025] (3) The GSH-Px gene fragment and the pGAPZA nucleic acid fragment were connected with T4 DNA ligase and transformed into E. coli DH5α competent cells. The plasmid was extracted and identified by double restriction enzyme digestion with EcoRI and NotI to obtain the recombinant expression plasmid pGAPZA-GSH-Px.
[0026] 1.3 Construction of recombinant Pichia pastoris strain expressing glutathione peroxidase
[0027] After the recombinant plasmid glutathione peroxidase was identified as correct by enzyme digestion and sequencing, it was electrotransformed into Pichia pastoris X33 competent cells. 100 μL of electrotransformed cells were spread on 100 μg / mL bleomycin YPD screening plates and cultured at 25-30°C for 3-5 days. Single colonies on the bleomycin YPD plates were picked and inoculated into 50 mL shaking tubes with air permeable membrane holes on the lids. 5 mL of YPD medium was added to each tube and cultured at 30°C, 250 rpm for 48 hours as seed solution, named X33-GSH-Px.
[0028] 1.4 Expression of glutathione peroxidase
[0029] (1) Take 1 ml of seed liquid of yeast strains X33 and X33-GSH-Px respectively, inoculate 100 ml of YPD culture medium (containing 50 mg / L sodium selenite) at a volume ratio of 1%, and culture at 30°C, 250 r / min shaking for 24 h. Each strain is inoculated in three culture bottles.
[0030] (2) The shaking incubator temperature was adjusted to 25°C and the fermentation was carried out at 250 rpm for 120 h.
[0031] (3) The fermented bacterial liquid is harvested and crushed by a high-pressure homogenizer.
[0032] (4) Weigh 1 g of crushed soybean meal powder, add 1 ml of high-pressure homogenized fermentation liquid, and incubate at 37°C for 12 hours. After equilibration at 25°C for 30 minutes, add 1 ml of 1% trypsin solution, mix well with a vortex oscillator, and incubate at 25°C for 30 minutes.
[0033] (5) The relative activity of trypsin in each group was detected according to the trypsin potency determination method in Part II of the 2020 edition of the Pharmacopoeia of the People's Republic of China.
[0034] from Figure 1 It can be seen that the trypsin activity of the wild-type yeast strain X33 fermentation broth lysate and soybean meal group was only 61.63%, while the trypsin activity of the X33-GSH-Px strain fermentation broth expressing glutathione peroxidase and soybean meal group was 96.21%. The glutathione peroxidase expressed by Pichia pastoris significantly reduced the inhibitory effect of the trypsin inhibitor in soybean meal on trypsin activity.
[0035] Example 2: Fermentation expression of glutathione peroxidase
[0036] (1) Take 0.1 ml of seed solution and inoculate 10 ml of YPD culture medium at a volume ratio of 1%, and culture at 30°C and 250 r / min in a shaking incubator for 24 h as the primary seed solution.
[0037] (2) 10 ml of the first-level seed solution was transferred to 1000 ml of YPD culture medium at a volume ratio of 1%, and cultured in a shaking incubator at 30°C and 250 r / min for 24 h as the second-level seed solution.
[0038] (3) Calibrate the pH electrode and dissolved oxygen electrode of the fermenter, and perform flow calibration of the peristaltic pump.
[0039] (4) Prepare 70 L of YPD medium (containing 50 mg / L sodium selenite), add it to a 100 L fermenter, and sterilize the medium, fermenter, and pipes at 121°C for 30 min.
[0040] (5) When the culture solution in the fermentation tank is cooled to 30°C, 700 ml of the secondary seed solution is added to the fermentation tank and the fermentation tank culture is started. The fermentation tank parameters are set to a stirring speed of 800 r / min, a tank pressure of 9 psi, a temperature of 30°C, and a DO value (dissolved oxygen) set above 30%, and the fermentation culture is carried out for 24 hours.
[0041] (6) The fermentation temperature was adjusted to 25°C and the fermentation was carried out for 120 h.
[0042] (7) The fermented bacterial liquid is harvested and crushed by a high-pressure homogenizer to obtain a crude glutathione peroxidase enzyme liquid.
[0043] (8) Weigh 1 g of crushed soybean meal powder, add 1 ml of glutathione peroxidase crude enzyme solution, and incubate at 37°C for 12 hours. After equilibration at 25°C for 30 minutes, add 1 ml of 1% trypsin solution, mix on a vortex oscillator, and incubate at 25°C for 30 minutes. At the same time, weigh 1 g of crushed soybean meal powder and add 1 ml of YPD medium to the control group.
[0044] (9) The activity of trypsin was detected according to the trypsin titer determination method in Part II of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The relative activity of trypsin measured by adding trypsin to the crude glutathione peroxidase solution after reacting with soybean meal was 96.87%, while the relative activity of trypsin measured by adding trypsin to soybean meal that was not treated with glutathione peroxidase was 60.73%.
[0045] Example 3: Preparation of low trypsin inhibitor feed by adding glutathione peroxidase
[0046] Feed 1: Mix 60% corn, 10% wheat bran, 15% soybean meal, 10% fish meal, 1.5% bone meal, 3% yeast powder, and 0.5% salt. Mix the mixed feed materials in a mixer, crush them in a grinder, and granulate them.
[0047] Feed 2: Mix 60% corn, 10% wheat bran, 15% soybean meal, 10% fish meal, 1.5% bone meal, 3% yeast powder and 0.5% salt in a mixer, grind with a grinder, add glutathione peroxidase crude enzyme solution in a ratio of 10:1 between the weight of the feed raw materials and the weight of the crude glutathione peroxidase solution, mix with a mixer, let stand at 37°C for 12 hours, dry and granulate.
[0048] Weigh 5g of each feed and grind it. Weigh 1g of each of the two grinded feed powders, add 1ml of deionized water, add 1ml of 1% trypsin solution, mix on a vortex oscillator, and act at 25°C for 30 minutes. The relative activity of trypsin in each group was detected according to the trypsin titer determination method in Part II of the 2020 edition of the Pharmacopoeia of the People's Republic of China.
[0049] from Figure 2 It can be seen that after the feed without glutathione peroxidase reacted with trypsin, the trypsin activity was only 60.40%; after the feed prepared with glutathione peroxidase reacted with trypsin, the trypsin activity was 95.09%. Adding glutathione peroxidase to the feed raw materials to treat it significantly reduced the inhibitory effect of the trypsin inhibitor in the feed on the trypsin activity.
[0050] Example 4: Effect of glutathione peroxidase-added feed on growth performance of broiler chickens
[0051] 400 one-day-old healthy white-feathered broiler chickens were randomly divided into 4 groups, 100 in each group, and raised in isolation. They were fed once at 8:00 and 16:00 each day, with free access to food and water. The experimental period was 40 days. During the rearing period, the immunization and feeding management were carried out according to the conventional immunization procedures for white-feathered broiler chickens. At the end of the experiment, the feed-to-meat ratio of the experimental chickens and the number of days to reach the market weight were calculated (both were geometric means).
[0052] Table 1 Effect of glutathione peroxidase-added feed on growth performance of broiler chickens
[0053] Grouping Glutathione Peroxide Feed to meat ratio Days to reach market weight Feed 1 No Addition 1.86:1 40 Feed 2 Add to 1.84:1 38
[0054] As can be seen from Table 1, the feed-to-meat ratio of white-feathered broilers fed with feed prepared with glutathione peroxidase was lower than that of the group without addition, and the chickens in the group with glutathione peroxidase were marketed two days earlier than those in the group without addition, which reduced feed consumption, improved breeding efficiency, and effectively increased breeding benefits.
Claims
1. An application of glutathione peroxidase in feed preparation, characterized in that: The glutathione peroxidase is a glutathione peroxidase expressed by recombinant Pichia pastoris, and the amino acid sequence of the glutathione peroxidase is SEQ ID NO.
1.
2. The use according to claim 1, characterized in that: The nucleotide sequence of the glutathione peroxidase expression regulatory gene is SEQ ID NO.
2.
3. The use according to claim 1, wherein the glutathione peroxidase can be used to reduce the inhibitory effect of trypsin inhibitor in feed on animal trypsin activity.
4. The use according to claim 1, characterized in that: The preparation method of the feed is as follows: (1) Recombinant Pichia pastoris expressing glutathione peroxidase was inoculated with YPD culture medium at a volume ratio of 1%, and cultured at 30°C and 250 rpm for 24 h as the primary seed liquid; (2) According to the fermentation volume, the first-stage seed solution was inoculated with YPD culture solution at a volume ratio of 1% to prepare the second-stage seed solution and multi-stage seed solution; (3) The multi-grade seed solution was inoculated with YPD culture medium containing 50 mg / L sodium selenite at a volume ratio of 1%, and the fermentation was carried out for 120 h; (4) The fermented bacterial liquid is crushed by a high-pressure homogenizer, and the mass ratio of the feed raw material to the high-pressure homogenized bacterial liquid is 10:
1. The mixture is stirred and mixed by a mixer, and the mixture is incubated at 37°C for 12 hours to decompose the trypsin inhibitor in the feed. The mixture is then granulated by a granulator to form the feed.
5. A gene for expressing glutathione peroxidase, characterized in that: The nucleotide sequence of the glutathione peroxidase expression regulatory gene is SEQ ID NO.2.