Albumin pichia pastoris feed additive as well as preparation method and application thereof

Abstract

The invention provides an albumin pichia pastoris feed additive as well as a preparation method and application thereof, and relates to the field of biological medicines. The pichia pastoris strain for expressing the recombinant human albumin is subjected to high-density fermentation culture and then is subjected to microwave-hot air combined drying to obtain the albumin pichia pastoris feed additive, and the feed additive is prepared from pichia pastoris produced by producing a thallus albumin product, so that the waste of resources is reduced, and the environmental pollution is effectively prevented; the method can be used in pig raising production, and provides a new feed research direction for animal breeding.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an albumin Pichia pastoris feed additive and a preparation method and application thereof. Background Art

[0002] Microbial pharmaceutical residues are solid wastes from the production process of fermentation pharmaceutical production enterprises. They are the residues after microorganisms ferment on a culture medium composed of glucose, soybeans, peanut cakes, starch, etc., and then acidify and filter to extract the active ingredients of the drug. They are mainly antibiotic residues, and also include vitamins, amino acids and other drug residues. These residues contain a large amount of water, bacteria, nutrients and bacterial metabolites, some of which are toxic substances and will cause serious environmental pollution.

[0003] Most of the mushroom residues are disposed of by landfill or incineration. Landfilling not only causes the most direct pollution to the soil, but also causes varying degrees of pollution to groundwater, and even leads to enrichment and drug resistance in organisms. If direct incineration is used, the unconsumed nutrients in the mushroom residues cannot be reused, resulting in a huge waste of resources.

[0004] The increasing demand for bacterial protein products has led to the emergence of various biopharmaceuticals. The albumin Pichia pastoris used to produce bacterial albumin products will also produce a large amount of residue after processing. However, the development and utilization of these Pichia pastoris residues are currently rare, resulting in a large amount of waste. Therefore, the development and utilization of the Pichia pastoris residues produced by the production of bacterial albumin products for feed research is beneficial to preventing environmental pollution and improving the economic benefits of enterprises. Summary of the invention

[0005] The purpose of the present invention is to provide a Pichia albumin feed additive and a preparation method and application thereof in view of the deficiencies of the prior art. The Pichia albumin feed additive can be used in pig production.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: The present invention provides a method for preparing a Pichia albumin feed additive, comprising the following steps: The Pichia pastoris strain expressing recombinant human albumin is cultured by high-density fermentation, solid-liquid separation is performed, the bacteria are collected, and a microwave-hot air combined drying method is used for drying to reduce the moisture content to less than 10%, thereby obtaining the albumin Pichia pastoris feed additive.

[0007] Furthermore, the steps of drying in the microwave-hot air combined drying method include: Firstly, microwave drying is used to dry the bacteria to a moisture content of about 50-60%, and then hot air drying is used to dry the bacteria to a moisture content of less than 10%, thereby obtaining the albumin Pichia yeast feed additive.

[0008] Preferably, the microwave density during the microwave drying process is 0.9-1.1 W / g.

[0009] More preferably, the microwave density during the microwave drying process is 0.9 W / g.

[0010] Preferably, the hot air drying temperature is 60-70°C.

[0011] More preferably, the hot air drying temperature is 70°C.

[0012] On the other hand, an albumin Pichia pastoris feed additive is prepared by the above method.

[0013] On the other hand, the above-mentioned albumin Pichia yeast feed additive is used in pig production.

[0014] Furthermore, the amount of the albumin Pichia feed additive added to the diet of nursery pigs is 0.01-6%; the amount of the albumin Pichia feed additive added to the diet of fattening pigs is 0.01-9%.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The albumin Pichia pastoris feed additive provided by the present invention is prepared from Pichia pastoris produced by producing bacterial albumin products, which reduces the waste of resources and effectively prevents environmental pollution. The albumin Pichia pastoris feed additive can be used in pig production, providing a new feed research direction for animal breeding.

[0016] 2. The albumin Pichia pastoris feed additive provided by the present invention can be used in pig production to replace part of the soybean meal protein in the diet without affecting the growth performance of pigs or the digestibility of nutrients.

[0017] 3. The present invention adopts a microwave-hot air combined drying method to dry the bacteria. Compared with the simple hot air or microwave drying method, the drying conditions are scientifically and reasonably set by using microwaves first and then hot air, which can greatly retain the nutrients in the product and reduce nutrient loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings described herein are used to provide further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute improper limitations on the present application.

[0019] Figure 1It is a structural diagram of the expression vector pPIC9-HSA of the present invention. DETAILED DESCRIPTION

[0020] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the invention of the present invention based on the disclosed content, and they should also fall within the scope of the present invention.

[0021] In a specific embodiment of the present invention, the medium formula used is as follows: YPD medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and the rest is purified water.

[0022] YPG medium: peptone 20g / L, yeast extract powder 10g / L, glycerol 20g / L, and the rest is purified water.

[0023] PTM1 trace element solution: copper sulfate pentahydrate 6.0g / L, sodium iodide 0.08g / L, manganese sulfate monohydrate 3.0g / L, sodium molybdate dihydrate 0.2g / L, boric acid 0.02g / L, cobalt chloride 0.5g / L, zinc chloride 20.0g / L, ferrous sulfate heptahydrate 65.0g / L, biotin 0.2g / L. Add the above raw materials while stirring, take 5mL of concentrated sulfuric acid and slowly add it to the solution, continue stirring until it is completely dissolved, make up to 1L with purified water, store at 4℃, and filter and sterilize with a 0.22μm filter before use.

[0024] BSM culture medium: 85% phosphoric acid 26.7 mL / L, calcium sulfate 0.93 g / L, potassium sulfate 18.2 g / L, magnesium sulfate 14.9 g / L, potassium hydroxide 4.13 g / L, glycerol 40.0 g / L, defoaming agent 0.15 mL / L, and the rest is purified water.

[0025] Glycerol feed: 60% glycerol, defoamer 0.15mL / L, the balance is purified water, after sterilization, add PTM1 trace element solution at 12mL / L.

[0026] Methanol feed: Methanol with 0.15 mL / L defoamer added.

[0027] The present invention is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels.

[0028] Example 1 Construction of recombinant human albumin Pichia pastoris strain S1. The human albumin (HSA) coding gene sequence (as shown in SEQ ID NO: 1) was optimized for Pichia pastoris codon preference and artificially synthesized to obtain a DNA sequence encoding recombinant human albumin (as shown in SEQ ID NO: 2), which was inserted into the multiple cloning site of the vector pPIC9 (purchased from Hunan Fenghui Biotechnology Co., Ltd., catalog number JM008) to form an expression vector pPIC9-HSA. The structure is shown in the figure Figure 1 shown.

[0029] SEQ ID NO: 1 is: SEQ ID NO:2 is: S2. The expression vector was transferred into competent E. coli cells, spread on LB plates, cultured upside down at 37°C, and cultured on a shaker overnight. Based on the alkaline lysis method for lysing cells, the HSA expression vector was extracted in large quantities using silicon matrix membrane adsorption technology, and linearized using restriction endonucleases in a 37°C water bath.

[0030] S3. Use YPD liquid medium to culture GS115 (purchased from Thermo Fisher Scientific, catalog number: C18100) at 30°C overnight, centrifuge the cell culture at 4°C, resuspend the bacterial pellet with ice-cold sterile water and centrifuge, resuspend the bacterial pellet with ice-cold 1M sorbitol solution and centrifuge, and finally resuspend the bacteria with 1M sorbitol to obtain competent GS115. Mix the linearized HSA expression vector and competent GS115, add them to the electroporation cup, and perform electroporation. After electroporation, spread the bacteria on the MD plate, invert and culture at 30°C, screen the positive clones and culture, extract the yeast genome, amplify the HSA gene fragment by PCR technology, and entrust Sangon Biotech (Shanghai) Co., Ltd. to sequence the PCR product. The results show that the pPIC9-HSA vector has been successfully recombined into the Pichia pastoris strain.

[0031] Example 2 Recombinant human albumin Pichia pastoris fermentation culture S1. Pick a single colony of GS115 (pPIC9-HSA) and inoculate it into 50 mL YPG medium, and culture it at 28°C and 220 rpm for about 27 hours. Take 5 mL of the above culture and inoculate it into 500 mL YPG liquid medium, and culture it at 28°C and 220 rpm until the bacterial liquid OD reaches 600 ≥12, used as seed liquid for tank loading.

[0032] S2. Use 50L triple fermentation system for fermentation tank culture Prepare 20L BSM culture medium and put it into the fermenter. Adjust the pH to 5.0 with ammonia water, sterilize at 121℃ for 30min, and correct the dissolved oxygen to 0%. Cool after sterilization, set the temperature to 28℃, the ventilation volume to 20L / min, and the tank pressure to 0.05Mpa. Add 87mL of sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 5.0 again. Discard about 500mL of the culture medium in the fermenter through the sampling port, so that the volume in the tank before inoculation is about 19.5L. Pump the shake flask seeds into the fermenter, correct the dissolved oxygen to 100%, set the speed to 300rpm, and start the batch fermentation stage. During this period, the dissolved oxygen is maintained above 30% by adjusting the speed (300rpm~1000rpm) and ventilation.

[0033] When the dissolved oxygen suddenly increases and the rotation speed suddenly drops, the glycerol fed fermentation stage is entered. The glycerol feed addition method is constant dissolved oxygen (the flow rate maintains dissolved oxygen>30%), and the parameter requirements remain unchanged. After 10 hours of glycerol feed addition, the mixed fed fermentation stage is entered, that is, glycerol feed and methanol feed are mixed and added at a ratio of 1:10 (w / w), and the total flow is added for 2 hours. The flow rate is adjusted to control the dissolved oxygen above 30%. At this time, the culture temperature is reduced to 24°C, the pH is raised to 6.0, and other parameters remain unchanged.

[0034] After mixed feeding, the methanol feeding fermentation stage begins. Methanol feeding is performed, and various fermentation parameters are monitored in real time. The methanol flow rate is adjusted according to the dissolved oxygen content (if the DO value rises by more than 10% within 1 minute after stopping methanol feeding, it means that the carbon source is limited, otherwise it means that the methanol is excessive). If the carbon source is limited, the methanol feeding rate is accelerated, and if the methanol is excessive, the methanol feeding rate is slowed down. Fermentation to OD 600 When the wet weight stops increasing, the material is collected and the tank is opened. The fermentation product is centrifuged at 8000rpm for 20min, the cells are collected and stored at -20℃.

[0035] Example 3 Drying and inactivation of Pichia albumin The cells collected in Example 2 were first dried to a moisture content of 40-60% (i.e., the moisture conversion point) by hot air drying, microwave drying, microwave-hot air combined drying, and hot air-microwave combined drying, and then further dried to a moisture content of less than 10% to obtain albumin Pichia inactivated bacterial residue; the crude protein and true protein contents of each group of albumin Pichia inactivated bacterial residue were detected with reference to GB / T6432-2018 and DB13 / T1098-2009 methods, and the specific experimental groups and test results are shown in Table 1 below. The moisture content was determined by direct drying method, expressed as dry basis moisture content, and the calculation formula is as follows:

[0036] Where: M t : Dry basis moisture content after drying to time t, %; m t : mass when dried to time t, g; m: mass when dried to constant weight, g.

[0037] Table 1. Protein content of inactivated Pichia pastoris residue

[0038] Table 1

[0039] The results in Table 1 show that compared with the methods of hot air drying, microwave drying, and hot air-microwave combined drying, the albumin Pichia pastoris inactivated bacterial residue prepared by microwave-hot air combined drying in this application has better effect, and the true protein content is stable at more than 40%; among them, group 23 has the best drying effect, with a true protein content of about 45% and less nutritional loss. It can also be seen that in the microwave-hot air combined drying process, the temperature and microwave density are either too high or too low, and the change of the moisture conversion point will significantly reduce the true protein content in the bacterial residue, which means that in this application, microwave drying (microwave density 0.9~1.1W / g) is first used to dry the bacteria to about 50~60% moisture, and then hot air drying (60~70℃) is used to dry to less than 10% moisture, which helps to reduce the loss of true protein during the drying process and retain the nutrients to the greatest extent.

[0040] The inactivated bacterial residue of albumin Pichia pastoris used in subsequent experiments in this application was prepared using the scheme of Group 23, that is, the bacteria were first dried to about 50% moisture by microwave drying (microwave density 0.9 W / g), and then dried to less than 10% moisture by hot air drying (70°C).

[0041] Test Example 1 Brief nutrient analysis of inactivated Pichia albuminus residue and test of feed hygiene standards The composition and safety of the inactivated bacteria residue of Pichia albuminum were tested. The nutrients of the inactivated bacteria residue of Pichia albuminum were analyzed with reference to GB / T6435-2014, GB / T6438-2007, GB / T6432-2018, GB / T6433-2006 and GB / T6434-2006 methods. The results are shown in Table 2. The inactivated bacteria residue of Pichia albuminum was tested for feed hygiene standards with reference to GB13078-2017 method. The results are shown in Table 3. The test showed that the content of heavy metals, harmful bacteria and pesticide residues that are harmful to animals in the inactivated bacteria residues are within the qualified range of national standards.

[0042] Table 2. Nutrient analysis of inactivated Pichia albuminosa residue

[0043] Table 3. Hygienic safety of inactivated Pichia albumin residue feed

[0044] Test Example 2 Acute toxicity test 1. Test method: Acute toxicity test was conducted according to GB / T23179-2008 and GB 15193.3-2014. The specific steps are as follows: Healthy mice of similar size and weight were selected and raised in cages according to gender, with a total of 6 groups, 30 mice in each group, half male and half female. Pre-feed for 5 days, keep fasting for 16 hours before gavage, gavage mice with gavage needles, and each group was gavaged with albumin Pichia inactivated bacterial residue according to 0g / kg, 2g / kg, 4g / kg, 6g / kg, 8g / kg, and 10g / kg body weight, respectively. The gavage volume was 20mL / kg, and the solvent was 1% sodium carboxymethyl cellulose. During the experiment, the mental state and death of the mice, as well as their eating and drinking conditions were observed. After gavage, the mice were killed by cervical dislocation 24 hours later, and the internal organs and immune organs were observed after dissection. The organ coefficient is the ratio of organ weight to animal weight.

[0045] 2. Test results 2.1 General Observations The experiment found that when the mice were gavaged with 10.0g / kg body weight (maximum dose) of albumin-killed Pichia yeast residue, their mental state was good and no deaths occurred.

[0046] 2.2 Feed intake Table 4 shows the effect of inactivated Pichia albumin residue on the food intake of mice. The results show that the food intake of mice did not change significantly after oral gavage and the mice drank water normally.

[0047] Table 4. Food intake of mice

[0048] 2.3 Organ weight and organ coefficient Tables 5 and 6 show the effects of albumin Pichia pastoris inactivated bacteria residue on organ weight and organ coefficient of mice, respectively. The results show that there was no difference in organ weight and organ coefficient among the groups 24 hours after oral gavage, and the highest dose of albumin Pichia pastoris inactivated bacteria residue had no significant toxic effect on the animal body, which means that the test sample albumin Pichia pastoris inactivated bacteria residue is a practical non-toxic substance and can be directly fed to animals without causing obvious damage.

[0049] Table 5. Mouse organ weights

[0050] Table 6. Mouse organ coefficients

[0051] Test Example 3 Quantitative analysis of active substances and functional amino acids in the inactivated residue of Pichia pastoris The active substances and functional amino acids in the inactivated residue of Pichia albuminum were quantitatively analyzed, and the results are shown in Table 7. The results showed that the content of mannose, the main active substance in the inactivated residue of Pichia albuminum, was 10.44%, and the contents of 9 functional amino acids were detected, among which lysine was the first limiting amino acid for pigs, and the lysine content in the inactivated residue of Pichia albuminum was 3.36%, which was better than plant proteins such as soybean meal, but contained less sulfur amino acids.

[0052] Table 7. Content of active substances and functional amino acids in albuminous Pichia yeast residue

[0053] Test Example 4 Effects of inactivated Pichia albumin residue on growth performance of nursery pigs 48 three-way crossbred pigs with an average initial weight of 10.23 kg were selected and randomly divided into 4 treatment groups, each treatment was divided into 4 pens, and each pen had 3 pigs. The nursery pig feeding experiment (i.e., P10 group, P20 group, and P30 group) was carried out by replacing 10%, 20%, and 30% of the soybean meal in the diet with albumin Pichia inactivated bacteria residue, and the diet was used as the control group (i.e., P0 group). The pigs were fed for 21 days, and the growth status of the nursery pigs was recorded. The formula of the experimental diet for nursery pigs is shown in Table 8, and the growth performance results are shown in Table 9. The results showed that there was no significant change in the weekly weight of the nursery pigs in each experimental group compared with the control group. The albumin Pichia inactivated bacteria residue in the nursery pig diet can replace 30% of the soybean meal protein, that is, the addition amount can reach 6%.

[0054] Table 8. Nursery pig diet formula

[0055] Table 9. Growth performance of nursery pigs

[0056] Test Example 5 Effects of inactivated Pichia albumin residue on growth performance of finishing pigs Thirty-six three-way hybrid pigs with an average initial weight of 46.8 kg were selected and randomly divided into three treatment groups, each treatment was divided into four pens, and each pen had three pigs. The fattening pig feeding experiment was carried out by replacing 50% and 100% of the soybean meal in the diet with the inactivated bacterial residue of Pichia pastoris (i.e., P50 group and P100 group), and the diet was used as the control group (i.e., P0 group). The growth of growing and fattening pigs was observed for 21 days. The formula of the fattening pig experimental diet is shown in Table 10, the growth performance results are shown in Table 11, and the nutrient digestibility results are shown in Table 12. The results show that replacing 50% of the soybean meal protein in the fattening pig feed with the inactivated bacterial residue of Pichia pastoris will not affect the growth performance of the pigs, nor the digestibility of nutrients; replacing 100% of the soybean meal in the diet with the inactivated bacterial residue of Pichia pastoris will significantly reduce the digestibility of nutrients. That is, albumin-containing inactivated Pichia pastoris residue in the diet of fattening pigs can replace 50% of the soybean meal protein, that is, the added amount can reach 9%.

[0057] Table 10. Experimental diet formula for finishing pigs

[0058] Table 11. Growth performance of finishing pigs

[0059] Table 12 Nutrient digestibility of fattening pigs

[0060] In summary, the feed hygiene index of the inactivated bacterial residue of albumin Pichia pastoris provided by the present invention meets the national standards, is safe and non-toxic, and can be used as a feed additive in pig production. The present invention provides a new feed research direction for animal breeding, which is beneficial to preventing environmental pollution and improving the economic benefits of pharmaceutical companies.

[0061] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a Pichia albumin feed additive, characterized in that: The steps include: The Pichia pastoris strain expressing recombinant human albumin is cultured by high-density fermentation, solid-liquid separation is performed, the bacteria are collected, and a microwave-hot air combined drying method is used for drying to reduce the moisture content to less than 10%, thereby obtaining the albumin Pichia pastoris feed additive.

2. The method for preparing the albumin Pichia pastoris feed additive according to claim 1, characterized in that: The steps of drying in the microwave-hot air combined drying method include: Firstly, microwave drying is used to dry the bacterial body to a moisture content of 50-60%, and then hot air drying is used to dry the bacterial body to a moisture content of less than 10%, thereby obtaining the albumin Pichia yeast feed additive.

3. The method for preparing the albumin Pichia pastoris feed additive according to claim 2, characterized in that: The microwave density during the microwave drying process is 0.9-1.1 W / g.

4. The method for preparing the albumin Pichia pastoris feed additive according to claim 3, characterized in that: The microwave density during the microwave drying process is 0.9 W / g.

5. The method for preparing the albumin Pichia pastoris feed additive according to claim 2, characterized in that: The temperature of the hot air drying is 60-70°C.

6. The method for preparing the albumin Pichia pastoris feed additive according to claim 5, characterized in that: The hot air drying temperature is 70°C.

7. An albumin Pichia pastoris feed additive, characterized in that: The invention is prepared by the method for preparing the albumin Pichia pastoris feed additive according to any one of claims 1 to 6.

8. Use of the albumin Pichia yeast feed additive according to claim 7 in pig production.

9. The use of the albumin Pichia yeast feed additive in pig production according to claim 8, characterized in that: The albumin Pichia yeast feed additive is added in an amount of 0.01-6% in the daily diet of nursery pigs.

10. The use of the albumin Pichia yeast feed additive in pig production according to claim 8, characterized in that: The albumin Pichia yeast feed additive is added in an amount of 0.01-9% in the daily diet of fattening pigs.

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