Application of phospholipid hydroglutathione peroxidase in feed additive

Abstract

The invention provides application of phospholipid hydroglutathione peroxidase in a feed additive, and belongs to the technical field of feed. The method comprises the following steps: constructing recombinant pichia pastoris for expressing deacetylated phospholipid hydroglutathione peroxidase, adding the expressed deacetylated phospholipid hydroglutathione peroxidase into crushed feed raw materials, stirring and uniformly mixing, decomposing trypsin inhibiting factors in the feed, and then preparing the feed. The deacetylated phospholipid hydroglutathione peroxidase is not added into a control group, and the trypsin activity is only 59.13% after the feed reacts with the trypsin; after the deacetylated phospholipid hydroglutathione peroxidase group feed is added to act with the trypsin, the trypsin activity is 95.39%. The feed conversion ratio of the Chang-bai piglets fed with the feed prepared by adding the deacetylated phospholipid hydroglutathione peroxidase is reduced compared with the feed conversion ratio of the Chang-bai piglets fed with the feed without adding the deacetylated phospholipid hydroglutathione peroxidase, the weight gain of the piglets is increased by 33.06%, and the breeding benefits are effectively increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of feeds, and particularly relates to the application of phospholipid hydroperoxide glutathione peroxidase in feed additives. Background Art

[0002] Soybean meal is a by-product obtained after soybean is pressed to produce soybean oil. The crude protein content in soybean meal is as high as 30 - 50%, which is the main nitrogen source for making animal feeds, occupies a dominant position in animal feeds, and is not easily replaced. While supplying animals with nutrients such as amino acids and vitamins, soybean meal also contains some anti-nutritional factors. Among them, trypsin inhibitor is the main anti-nutritional factor in soybean meal. It can inhibit the activity of trypsin in the animal digestive tract, affect the digestion of proteins, cause nutritional waste, and seriously reduce the breeding efficiency. The specific types of trypsin inhibitors in soybean meal are not yet clear, and the methods for eliminating trypsin inhibitors in soybean meal feeds are still in the research stage.

[0003] Phospholipid hydroperoxide glutathione peroxidase (PHGPx / GPx4) is a key antioxidant enzyme, belonging to the glutathione peroxidase (GPx) family, catalyzing the reduction of phospholipid hydroperoxides, and playing a core role in cellular antioxidant defense and lipid peroxidation regulation. It mainly reduces phospholipid hydroperoxides, such as oxidized phospholipids and cholesterol ester peroxides, and can also degrade small molecule hydroperoxides such as H 2 O 2。 Phospholipid hydroperoxide glutathione peroxidase has a certain effect on eliminating trypsin inhibitors in soybean meal feeds. The post-translational modifications of phospholipid hydroperoxide glutathione peroxidase include acetylation, phosphorylation and other modifications to regulate its activity, among which acetylation seriously reduces its reducing ability. Therefore, by modifying the acetylation sites of phospholipid hydroperoxide glutathione peroxidase and preparing phospholipid hydroperoxide glutathione peroxidase by genetic engineering bacteria for feed raw material treatment, the purpose of eliminating trypsin inhibitors in soybean meal feeds can be achieved. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of phospholipid hydroperoxide glutathione peroxidase in feed additives, belonging to the technical field of feeds.

[0005] To achieve the above purpose, the present invention provides the following technical solutions: First, the present invention provides the application of phospholipid hydroperoxide glutathione peroxidase in feed additives, and the phospholipid hydroperoxide glutathione peroxidase is deacetylated phospholipid hydroperoxide glutathione peroxidase expressed by recombinant Pichia pastoris.

[0006] Further, the amino acid sequence of the deacetylated phospholipid hydroperoxide glutathione peroxidase is SEQ ID NO.2.

[0007] Further, the nucleotide sequence of the expression regulatory gene of the phospholipid hydroperoxide glutathione peroxidase is SEQ ID NO. 3.

[0008] Further, the deacetylated phospholipid hydroperoxide glutathione peroxidase can be used to reduce the inhibitory effect of trypsin inhibitor in feed on the activity of animal trypsin.

[0009] Further, the preparation method of the feed is as follows: (1) The recombinant Pichia pastoris expressing deacetylated phospholipid hydroperoxide glutathione peroxidase is inoculated into YPD culture medium at a ratio of 1% by volume, and cultured in a shaker at 30 °C and 250 r / min for 24 h as the primary seed solution; (2) According to the fermentation volume, the primary seed solution is inoculated into YPD culture medium at a ratio of 1% by volume to prepare the secondary seed solution and multiple-stage seed solutions; (3) The multiple-stage seed solution is inoculated into YPD culture medium containing 50 mg / L sodium selenite at a ratio of 1% by volume, and fermented for 120 h; (4) The fermented bacterial liquid is broken by a high-pressure homogenizer, and the mass ratio of the feed raw material to the high-pressure homogenized broken bacterial liquid is 10:1. It is stirred and mixed evenly by a mixer, and acts at 37 °C for 12 hours to decompose the trypsin inhibitor in the feed, and then granulated by a granulator to form the feed.

[0010] Second, the present invention provides a gene for expressing deacetylated phospholipid hydroperoxide glutathione peroxidase, and the nucleotide sequence of the expression regulatory gene of the deacetylated phospholipid hydroperoxide glutathione peroxidase is SEQ ID NO. 3.

[0011] The beneficial effect of the present invention is that by constructing a recombinant Pichia pastoris expressing deacetylated phospholipid hydroperoxide glutathione peroxidase, the expressed deacetylated phospholipid hydroperoxide glutathione peroxidase has significantly enhanced the effect of degrading trypsin inhibitor in soybean meal compared with the natural phospholipid hydroperoxide glutathione peroxidase. In the control group without adding deacetylated phospholipid hydroperoxide glutathione peroxidase, the trypsin activity was only 59.13% after the feed reacted with trypsin; the trypsin activity was 95.39% after the feed prepared by adding deacetylated phospholipid hydroperoxide glutathione peroxidase reacted with trypsin. The feed conversion ratio of the feed prepared by adding deacetylated phospholipid hydroperoxide glutathione peroxidase for feeding Landrace piglets was reduced compared with the non-added group, and the weight gain of the piglets increased by 33.06%. The application of deacetylated phospholipid hydroperoxide glutathione peroxidase in feed raw material treatment can effectively reduce the trypsin inhibitor existing in grains such as soybean meal in feed raw materials, thereby improving the digestion and absorption rate of animals for proteins in feed, reducing the feed conversion ratio, and improving the breeding efficiency. Description of the Drawings

[0012] Figure 1 : Effect of Deacetylated Phosphatidylhydrogen Glutathione Peroxidase on Trypsin Activity in Soybean Meal.

[0013] Figure 2 : Effect of Deacetylated Phosphatidylhydrogen Glutathione Peroxidase on Trypsin Activity in Feed. Detailed Implementation Manner Example 1: Design of Deacetylated Phosphatidylhydrogen Glutathione Peroxidase The GenBank accession number of the amino acid sequence of porcine phosphatidylhydrogen glutathione peroxidase is: CAA53596.2, and the amino acid sequence is SEQ ID NO.1. The 46th U is selenocysteine, and the 48th K and 90th K are potential acetylation sites. To eliminate the acetylation sites, the 48th K and 90th K were mutated to H, and the 48th K and 90th K were designed to obtain the amino acid sequence of deacetylated phosphatidylhydrogen glutathione peroxidase as SEQ ID NO.2.

[0014] Example 2: Construction of Recombinant Pichia pastoris Expressing Phosphatidylhydrogen Glutathione Peroxidase 1.1 Gene Design for Expressing Deacetylated Phosphatidylhydrogen Glutathione Peroxidase According to the amino acid sequence of deacetylated phosphatidylhydrogen glutathione peroxidase SEQ ID NO.2 designed in Example 1, the 46th U is selenocysteine, and its coding sequence is TGA. In eukaryotic cells, TGA is transcribed into mRNA as UGA, which is a stop codon during protein translation and cannot synthesize selenocysteine U, and protein translation will terminate here. The GenBank accession number of the selenoprotein P gene sequence is: NM_005410.4, and its 1217 - 1974bp is the selenocysteine UGA translation regulatory region sequence, which regulates the translation of UGA into selenocysteine during protein translation. Tandemly connecting the 1217 - 1974bp selenocysteine UGA translation regulatory region sequence of NM_005410.4 after the deacetylated phosphatidylhydrogen glutathione peroxidase coding gene can regulate the translation of UGA into selenocysteine during the protein translation process of phosphatidylhydrogen glutathione peroxidase, thereby obtaining active deacetylated phosphatidylhydrogen glutathione peroxidase. The nucleotide sequence of the expression regulatory gene of deacetylated phosphatidylhydrogen glutathione peroxidase was designed as SEQ ID NO.3, and the nucleotide sequence of the expression regulatory gene of natural phosphatidylhydrogen glutathione peroxidase was designed as SEQ ID NO.4, with Eco RI and Not I restriction enzyme sites added at the upstream and downstream, and commissioned Sangon Biotech (Shanghai) Co., Ltd. to synthesize and clone them into the Pichia pastoris expression vector pGAPZA to obtain pGAPZ - GPxMut and pGAPZ - GPx plasmids.

[0015] 1.2 Construction of recombinant Pichia pastoris strains expressing phospholipid hydroperoxide glutathione peroxidase The pGAPZ-GPxMut and pGAPZ-GPx plasmids were respectively electrotransformed into competent cells of Pichia pastoris X33. 100 μL of electrotransformed cells were taken and spread on a bleomycin YPD screening plate with a concentration of 100 μg / mL, and incubated at 25 - 30 °C for 3 - 5 days. Single colonies on the bleomycin YPD plate were picked and inoculated into shake flasks with a 50 mL capacity and an air-permeable membrane hole on the lid. 5 mL of YPD medium was added to each flask, and the cultures were incubated at 30 °C and 250 rpm for 48 hours as seed solutions, named X33-GPxMut and X33-GPx respectively.

[0016] 1.3 Expression of phospholipid hydroperoxide glutathione peroxidase (1)1 mL of seed solution of each of the yeast strains X33, X33-GPxMut and X33-GPx was taken and inoculated into 100 mL of YPD culture medium (containing sodium selenite at a concentration of 50 mg / L) at a ratio of 1% by volume, and incubated at 30 °C and 250 r / min on a shaker for 24 h. Each strain was inoculated into 3 culture flasks for replication.

[0017] (2)The shaker temperature was adjusted to 25 °C, and fermentation culture was carried out on a shaker at 250 r / min for 120 h.

[0018] (3)The fermented bacterial liquid was harvested and disrupted by a high-pressure homogenizer.

[0019] (4)1 g of pulverized soybean meal dry powder was weighed, 1 mL of the fermented bacterial liquid disrupted by high-pressure homogenization was added, and the mixture was incubated at 37 °C for 12 h. After equilibration at 25 °C for 30 minutes, 1 mL of a 1% trypsin solution was added, and the mixture was mixed by a vortex oscillator and incubated at 25 °C for 30 minutes.

[0020] (5)The relative activity of trypsin in each group was detected according to the method for determining the titer of trypsin in the second part of the Chinese Pharmacopoeia (2020 edition).

[0021] From Figure 1 It can be seen that the trypsin activity in the group where the lysate of the fermented bacterial liquid of the wild-type yeast strain X33 reacted with soybean meal was only 61.93%; the trypsin activity in the group where the fermented bacterial liquid of the X33-GPx strain expressing phospholipid hydroperoxide glutathione peroxidase reacted with soybean meal was 82.71%, and the trypsin activity in the group where the fermented bacterial liquid of the X33-GPxMut strain expressing deacetylated phospholipid hydroperoxide glutathione peroxidase reacted with soybean meal was 96.74%. The deacetylated phospholipid hydroperoxide glutathione peroxidase expressed by Pichia pastoris significantly reduced the inhibitory effect of trypsin inhibitor in soybean meal on trypsin activity.

[0022] Example 3: Fermentation Expression of Phosphatidylhydrogen Glutathione Peroxidase (1) Inoculate 0.1 ml of X33-GPxMut seed liquid into 10 ml of YPD culture medium at a ratio of 1% by volume, and culture it in a shaker at 30 °C and 250 r / min for 24 h as the primary seed liquid.

[0023] (2) Transfer 10 ml of the primary seed liquid to 1000 ml of YPD culture medium at a ratio of 1% by volume, and culture it in a shaker at 30 °C and 250 r / min for 24 h as the secondary seed liquid.

[0024] (3) Calibrate the pH electrode and dissolved oxygen electrode of the fermenter, and calibrate the flow rate of the peristaltic pump.

[0025] (4) Prepare 70 L of YPD medium (containing 50 mg / L of sodium selenite), add it to a 100 L fermenter, and sterilize the medium, fermenter and pipeline at 121 °C for 30 min.

[0026] (5) When the culture medium in the fermenter cools down to 30 °C, add 700 ml of the secondary seed liquid to the fermenter, start the fermenter culture, and set the fermenter parameters as follows: stirring speed 800 r / min, tank pressure 9 psi, temperature 30 °C, set the DO value (dissolved oxygen) above 30%, and ferment for 24 h.

[0027] (6) Adjust the fermentation temperature to 25 °C and ferment for 120 h.

[0028] (7) Harvest the fermented bacterial liquid, and break it with a high-pressure homogenizer to obtain the crude enzyme liquid of phosphatidylhydrogen glutathione peroxidase.

[0029] (8) Weigh 1 g of crushed soybean meal dry powder, add 1 ml of the crude enzyme liquid of phosphatidylhydrogen glutathione peroxidase, and react at 37 °C for 12 h. After equilibrating at 25 °C for 30 min, add 1 ml of 1% trypsin solution, mix well with a vortex oscillator, and react at 25 °C for 30 min. At the same time, set a control group by weighing 1 g of crushed soybean meal dry powder and adding 1 ml of YPD medium.

[0030] (9) Detect the trypsin activity according to the method for determining the titer of trypsin in the second part of the Chinese Pharmacopoeia (2020 edition). The relative activity of trypsin measured after adding trypsin to the crude enzyme liquid of phosphatidylhydrogen glutathione peroxidase reacting with soybean meal is 96.92%, while the relative activity of trypsin measured after adding trypsin to soybean meal without treatment with phosphatidylhydrogen glutathione peroxidase is 61.18%.

[0031] Example 4: Preparation of Low Trypsin Inhibitor Feed by Adding Deacetylated Phosphatidylhydrogen Glutathione Peroxidase 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. Stir and mix the feed raw materials with a mixer, crush them with a crusher, and granulate them. 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. Stir and mix the feed raw materials with a mixer, crush them with a crusher, add crude deacetylated phospholipid hydroperoxidase glutathione peroxidase solution in a ratio of 10:1 by weight of the feed raw materials to the weight of the crude enzyme solution, stir and mix with a mixer, react at 37°C for 12 hours, dry, and granulate.

[0032] Weigh 5 g of each of the two feeds and grind them into powder. Weigh 1 g of each of the ground feed powders, add 1 ml of deionized water, add 1 ml of 1% trypsin solution, mix well with a vortex oscillator, and react at 25°C for 30 minutes. Detect the relative activity of trypsin in each group according to the method for determining the titer of trypsin in the second part of the Chinese Pharmacopoeia (2020 edition). Repeat the measurement of the two feeds 3 times.

[0033] From Figure 2 It can be seen that in the control group without adding deacetylated phospholipid hydroperoxidase glutathione peroxidase, the trypsin activity was only 59.13% after the feed reacted with trypsin; the trypsin activity in the feed additive group with added deacetylated phospholipid hydroperoxidase glutathione peroxidase was 95.39% after the feed reacted with trypsin. Adding deacetylated phospholipid hydroperoxidase glutathione peroxidase to the feed raw materials for treatment significantly reduced the inhibitory effect of trypsin inhibitor in the feed on trypsin activity.

[0034] Example 5: Effect of feed added with deacetylated phospholipid hydroperoxidase glutathione peroxidase on the growth performance of piglets Select 200 28-day-old weaned Landrace piglets with a body weight of 8 ± 0.2 kg, randomly divide them into 2 groups, 100 pigs per group, and raise them in isolation; feed the two feeds in Example 4 to the two groups respectively, feed them once at 8:00 and 16:00 every day, and allow free access to food and water. The test period is 30 days. Observe the feeding and mental state of the test pigs every day during the period, and count the feed-to-gain ratio and weight gain of each group of pigs (both are geometric means) at the end of the experiment.

[0035] Table 1 Effect of feed added with deacetylated phospholipid hydroperoxidase glutathione peroxidase on the growth performance of piglets

[0036] As can be seen from Table 1, the feed conversion ratio of the Landrace piglets fed with the feed prepared by adding deacetylated phospholipid hydroperoxide glutathione peroxidase is lower than that of the group without addition, and the weight gain of the piglets has increased by 33.06%, effectively increasing the breeding efficiency.

Claims

1. An application of phospholipid hydrolyzate glutathione peroxidase in feed additives, characterized in that: The phospholipid hydrogen glutathione peroxidase is a deacetylated phospholipid hydrogen glutathione peroxidase expressed by recombinant Pichia pastoris, and the amino acid sequence of the deacetylated phospholipid hydrogen glutathione peroxidase is SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the expression regulatory gene of deacetylated phospholipid hydrogen glutathione peroxidase is SEQ ID NO.

3.

3. The use according to claim 1, wherein the deacetylated phospholipid hydrolyzed 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 deacetylated phospholipid hydrogen 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-level seed solution is inoculated with YPD culture solution at a volume ratio of 1% to prepare the second-level seed solution and multi-level seed solution; (3) The multi-grade seed liquid was inoculated with YPD culture medium containing 50 mg / L sodium selenite at a volume ratio of 1%, and fermented for 120 h; (4) The fermented bacterial liquid is crushed by a high-pressure homogenizer. The mass ratio of the feed raw material: the high-pressure homogenized and crushed bacterial liquid is 10:

1. The mixture is stirred and mixed by a mixer. The mixture is incubated at 37°C for 12 hours to decompose the trypsin inhibitor in the feed. The feed is then granulated by a granulator to form the feed.

5. A gene for expressing deacetylated phospholipid hydrolyzate glutathione peroxidase, characterized in that: The nucleotide sequence of the expression regulatory gene of deacetylated phospholipid hydrogen glutathione peroxidase is SEQ ID NO.3.

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