阅读说明：本技术 一种在制备饲料级胰酶中的灭活病毒的方法 (Method for inactivating virus in preparation of feed-grade pancreatin ) 是由 蔡青和 陈远庆 朱琳娜 于 2019-11-07 设计创作，主要内容包括：本发明提供一种从胰渣中提取饲料级胰酶过程中的灭活病毒的方法,它将原料胰渣经过粉碎、溶解、板框压滤后,上清液加入有机溶剂和活性剂进行病毒灭活,再经超滤、制粒、包衣和干燥,分装得制品。通过有机溶剂和活性剂破坏病毒的脂质包膜,同时保持胰酶的结构和功能,使制品纯度和活性提高,不仅病毒灭活操作简单,处理时间短,经济实用,而且该发明具有污染小、设备简单、生产周期短、无需添加激活剂、性质稳定便于保存、安全性好等特点。(The invention provides a method for inactivating virus in the process of extracting feed-grade pancreatin from pancreatin slag, which comprises the steps of crushing, dissolving and plate-and-frame filter pressing the raw material pancreatin slag, adding an organic solvent and an active agent into supernate to inactivate the virus, and then carrying out ultrafiltration, granulation, coating, drying and subpackaging to obtain a product. The method destroys the lipid envelope of the virus by the organic solvent and the active agent, and simultaneously keeps the structure and the function of the pancreatin, so that the purity and the activity of the product are improved, the virus inactivation operation is simple, the treatment time is short, and the method is economical and practical, and the method has the characteristics of small pollution, simple equipment, short production period, no need of adding the active agent, stable property, convenient storage, good safety and the like.)

1. A method for inactivating viruses in the process of extracting feed-grade pancreatin from pancreatin residues is characterized by comprising the following steps:

A. raw material treatment: the raw material is frozen pancreas residue obtained after extracting angiotensin from crude enzyme powder, the raw material is crushed by a flat plate crusher or a flaker, dissolved by distilled water and stirred by a stirrer for 15-60 minutes, or ground and dissolved by a pulping machine to obtain a dissolved solution;

B. centrifuging: centrifuging the solution obtained in the step A by using a centrifugal machine or filtering and removing slag by using a plate-and-frame filter press to obtain supernatant filtrate;

C. and (3) precipitation and concentration: adding organic solvent ethanol and an active agent into the supernatant filtrate obtained in the step B, uniformly stirring, precipitating for 0.5-2.5 hours at the temperature of 5-20 ℃, and collecting the precipitate by ultrafiltration;

D. granulating, coating and drying: and C, mixing the precipitate obtained in the step C with a carrier for feed, granulating by using a granulating device, coating the product by using a coating device, and finally drying by using a drying device to obtain the pancreatin.

2. The process of claim 1 for the extraction of feed-grade pancreatin from pancreatin pomace, wherein: the raw material is frozen pancreas residue obtained after extracting angiotensin from crude enzyme powder, and organic solvent ethanol is adopted for extraction in the raw material obtaining process.

3. The process of claim 1 for the extraction of feed-grade pancreatin from pancreatin pomace, wherein: the dissolving temperature of the step A is controlled to be 5-20 ℃, and the ratio of distilled water to raw materials is controlled to be 10-1: 1.

4. The process of claim 1 for the extraction of feed-grade pancreatin from pancreatin pomace, wherein: and the rotating speed of the centrifuge in the step B is controlled to be 1000-3000 r/min.

5. The process of claim 1 for the extraction of feed-grade pancreatin from pancreatin pomace, wherein: and C, the concentration of the organic solvent ethanol in the step C is 60-100%.

6. The process of claim 1 for the extraction of feed-grade pancreatin from pancreatin pomace, wherein: the active agent in the step C is a lipophilic nonionic surfactant (fatty acid mono/diglyceride, glyceryl monostearate, glycerin fatty acid ester and the like), the concentration of the active agent is 2g/L-10g/L, and the active agent is a wide non-specific feed additive for preventing and treating animal viruses.

7. The process of claim 1 for the extraction of feed-grade pancreatin from pancreatin pomace, wherein: and D, any one of corn starch, silicon dioxide, gelatin and rice hull powder is used as the carrier for the feed in the step D.

8. The process of claim 1 for the extraction of feed-grade pancreatin from pancreatin pomace, wherein: and D, concentrating by adopting ultrafiltration, wherein the drying device is a blast drying device, a vacuum drying device or a fluidized bed drying device.

Technical Field

The invention relates to pancreatin and a method for inactivating viruses in the process of producing the pancreatin, belongs to the technical field of biology, and particularly relates to a method for inactivating viruses in the process of preparing feed-grade pancreatin by using an organic solvent and an active agent.

Background

The digestive tract of young animals such as suckling piglets, chicks, ducklings, dairy cows and the like is underdeveloped, an endogenous digestive enzyme system is not established, and the young animals are very easy to have nutritional diarrhea and diarrhea caused by other reasons due to feeding stress such as early weaning, and antibiotics are usually used for feeding or treating in actual production, so that the antibiotic residues and the excessive use are caused. By adding suitable enzyme preparation, the occurrence of nutritional diarrhea in young animals can be prevented, thereby reducing the use of antibiotics.

Pancreatin, a protein with special functions. Pancreatin in the pharmaceutical industry is mainly used for dyspepsia, inappetence and digestive disorder caused by liver and pancreas diseases, and is also suitable for congenital pancreatic insufficiency, and pancreatic insufficiency caused by abdominal operation and traumatic pancreatic resection. Or pancreatic insufficiency caused by non-surgical excision and chronic pancreatitis caused by alcoholism. Also, pancreatin has a wide range of applications in the feed industry, not only providing a protein source to animals, but also because it contains a large number of biologically active components, such as proteases, amylases and lipases and active peptides.

At present, the traditional preparation process of industrial pancreatin mostly takes pig or ox pancreas as a raw material and comprises a plurality of procedures of raw material treatment (flaking, grinding into thick liquid), activation, extraction, separation (filtration, precipitation, squeezing), degreasing, granulation, drying, crushing and the like. The raw material for extracting angiotensin for medicine is derived from crude enzyme powder (derived from pig pancreas), and the crude enzyme powder contains angiotensin, trypsin, pancreatic amylase and pancreatic lipase. The process for extracting the angiotensin mainly adopts water washing and specific molecular filter membrane filtration, and the enzyme in the angiotensin is slightly damaged. The pancreatic residue left after the crude enzyme powder extracts the angiotensin for medicine is used as a raw material for preparing the pancreatin, has certain economic value and feasibility, fully and comprehensively utilizes pancreatic residue resources, and has great significance in the aspects of environmental protection and resource saving.

In order to ensure the safety of the product, in addition to controlling the pig feeding environment and the quality of the raw material pancreatin dregs, how to effectively remove or inactivate viruses in the raw material in the production process of the pancreatin is a key problem for obtaining a safe and reliable feed pancreatin product.

With the gradual increase of products prepared by animal-derived tissues and the lack of sufficient attention paid to the quality control of raw materials of animal-derived products, the risk of virus infection of livestock and poultry by the animal-derived products and the potential problem of iatrogenic infection become increasingly prominent. To ensure the safety of the product, it is necessary to add effective virus inactivation/removal measures in the production process. At present, the relatively mature methods for removing and inactivating viruses at home and abroad mainly comprise a chemical method and a physical method.

1. Chemical process

Chemical inactivation is the modification of some structures of microorganisms and active substances by chemicals or enzymes, thereby losing vitality, infectivity, toxicity or activity. The chemical inactivation effect is true, the method is simple and most commonly used, but the chemical inactivation effect is usually influenced by factors such as the type, dosage, action temperature, pH, time and the like of the inactivator, and the type, nitrogen content and organic matter existence of microorganisms are related, so that the optimal inactivation condition needs to be screened.

1.1 organic solvent/surfactant (S/D) Process

The S/D method starts to be developed and applied as early as the middle of the 80' S in the 20 th century, and is still a core virus inactivation method in blood products at present. The basic principle of the method is as follows: the mixture of organic solvent and nonionic surfactant can break down the lipid membrane of the lipid-enveloped virus, thereby causing the lipid to be stripped off the virus surface, and the virus loses the ability to adhere to and infect cells. Roberts PL (2008) examined the effectiveness of the S/D method for viral inactivation of highly pure clotting factors. The results show that after the blood coagulation factor preparation is treated for 30min at 22 ℃ by using 0.3% of TN-BP and 1% of Triton-X100 as S/D reagents, simulated lipid envelope viruses such as vaccinia virus, herpes simplex virus, Sindbis virus and the like in the blood coagulation factor preparation can be effectively inactivated, and the robustness and the effectiveness of the S/D method for inactivating the lipid envelope viruses are verified. The S/D method is mainly used for inactivating lipid envelope viruses of blood products such as blood coagulation factor products, protease inhibitors, human immunoglobulins and the like.

1.2 Low pH incubation

The low pH incubation prevented the polymerization of human immunoglobulins during their preparation for injection in the early 80's of the 20 th century, which was subsequently found to have an inactivating effect on most of the lipo-enveloped viruses, which were then used for viral inactivation of blood products. The inactivation mechanism is that certain virus components are deteriorated when the temperature is kept for more than 20 hours under the conditions of pH4 and the temperature of 30-37 ℃, so that the replication of the virus is influenced, and finally the infectivity of the virus is lost. Roberts et al (2012) examined the incubation method and showed that the continuous treatment of intravenous immunoglobulin at pH5 and 30 ℃ for 14d was effective in inactivating both lipid-enveloped and partially non-enveloped viruses. The method can be used as the terminal treatment of the production process and is combined with an S/D method and an ion exchange chromatography method to be jointly used for virus inactivation of the immunoglobulin for injection, thereby ensuring the safety of the immunoglobulin product. This method requires that the activity of the protein product be stable at low pH and is therefore generally used only for the inactivation of immunoglobulin lipid envelope viruses.

1.3 treatment of octanoic acid

As early as the 90 s of the 20 th century, Lundblad et al (1991) reported a mechanism for inactivating 4 lipid envelope proteins by sodium caprylate and uncovered the use of caprylic acid in virus inactivation, which is lipophilic in a non-ionic form and can enter into the lipid envelope of viruses to destroy the integrity of lipid bilayers and related proteins and lose infectivity of lipid envelope viruses to inactivate lipid envelope viruses under low pH conditions, Dichtelm ü ler et al (2002) used 3 batches of immunoglobulin preparations for injection as research subjects and examined the effectiveness of caprylic acid method for virus inactivation of immunoglobulin solutions, which showed that 7.45G/kg of caprylic acid solution can effectively inactivate human immunodeficiency viruses, bovine viral diarrhea viruses, Sindbis viruses, pseudorabies viruses in a very short time (1 min.) As caprylic acid method needs to act under low pH conditions (usually pH < 6), it is required that protein products can maintain their stability under low pH conditions, and is therefore currently used mainly for virus inactivation of immunoglobulin M and immunoglobulin G.

1.4 photochemical method

Methylene Blue (MB)/visible light treatment, psoralen/UVA treatment, riboflavin/UV treatment, etc. are currently commonly used photochemical methods (tan mazu et al, 2012), mainly for virus inactivation of whole plasma and platelet products. The principle of virus inactivation by MB/visible light method (mourning, etc., 2003) is that MB is a photosensitive phenothiazine dye with positive charge, can pass through the envelope of virus to be combined with nucleic acid, and can produce photochemical reaction under the irradiation of light with certain intensity to produce hydroxyl radical and singlet oxygen to prevent the replication of nucleic acid so as to achieve the purpose of virus inactivation. The method has good inactivation effect on lipid-enveloped viruses and has undesirable inactivation effect on non-enveloped viruses. In addition, for unstable protein products, the loss of functional activity may be caused, and there are many reports in the literature (chenyugen et al, 2007) that the influence of the virus inactivation method on plasma proteins changes.

2. Physical method

2.1 pasteurization

The pasteurization method is characterized in that a protein solution is continuously heated for at least 10 hours at the temperature of 60 ℃ to denature virus proteins, so that the replication of virus genetic materials is inhibited, and finally viruses are not infected, Chandra et al (2002) carry out detailed review on the pasteurization method through relevant literature search, and the result shows that the pasteurization method only needs to control few process parameters, the sterilization process is easy to monitor, and the pasteurization method has inactivation effects on both lipid-enveloped viruses and non-enveloped viruses, and has a wide sterilization range.

2.2 Dry Heat treatment

The dry heat method was used for the inactivation of hepatitis virus in coagulation factor preparations first in the 80 th of the 20 th century, and was approved for the inactivation of HIV in 1984. The principle is that the preparation is heated after being freeze-dried, so that the molecules and the structures required by virus replication are changed, and the replication process is inhibited. The dry heating method usually uses the processing conditions of 60 ℃, 96h, 80 ℃, 72h and 100 ℃, 30 min. Seop et al (2008) found that, by performing dry heat virus inactivation on the freeze-dried blood coagulation factor, under the treatment conditions of 100 ℃ and 30min, the virus inactivation method has a good inactivation effect on other viruses except for non-ideal inactivation on porcine parvovirus, and the inactivation of the blood coagulation factor by the method is only about 5%, and no change in physical or biological properties of a protein product is found, so that the dry heat method is proved to be a key step for final virus inactivation, and the virus safety of the blood coagulation factor product can be ensured. Due to the limitation of virus inactivation capacity, this method is commonly used as a helper virus inactivation method for blood coagulation factor preparations.

3. Other methods

The preparation process of protein products usually comprises a precipitation step and also has the function of virus removal. During cold ethanol precipitation, various viruses may be separated into waste fractions, and partial virus removal may be performed while protein purification is performed. Deep filtration is an important protein purification means, and the filter material can adsorb part of viruses so as to remove the viruses. The chromatographic technology is a protein purification method which is rapidly developed at present, a protein product is adsorbed on a chromatographic column in the purification process, viruses exist in a mobile phase, and the viruses are removed while the protein is purified. Roberts et al (2012) found that, after the immunoglobulin product for intravenous injection was subjected to ion exchange chromatography, the titer of herpes simplex virus type 1 and bovine papilloma virus decreased by > 5log, which was effective in removing viruses, and had a certain effect of removing other mock viruses, but did not achieve effective removal.

The cold ethanol precipitation, deep filtration, chromatography and the like are all production processes of protein products, and have the virus removal effect.

Tijsen et Al (US 2014/0017223Al) disclose a method for preparing pancreatin (PEP) comprising reacting propriocene acetate (BPL) with a preparation containing one or more pancreatin for a time sufficient to reduce viral infectivity in the preparation.

Ramsch et Al (US 2011/0268844Al) disclose treatment of pancreatin with high pressure and/or screen filtration followed by treatment at 15 ℃ for 5 minutes with 4000, 5000 or 6000 known high pressures. According to Ramsch, this applies to all viral forms, such as DNA and RNA viruses, enveloped and non-enveloped viruses as well as bacteria and fungi, and comprises at least 50% biological activity. He further discloses the unpredictability of success in inactivating certain viruses using high pressure treatment. Due to the different compressibility of the sample, different process conditions must be chosen depending on whether the sample is a liquid or a solid.

Kurfurst et Al (US 2009/0233344Al) discloses a method for reducing viral and microbial contamination of a sample by treating the sample with 0.5 wt% or less residual moisture by heat treating pancreatin at a temperature of 84 ℃ (preferably 80 ℃ and below) for 48 hours or 30 hours, wherein the activity of the resulting pancreatin is at least 50% biological activity. The disclosed pancreatin virus infectivity is reduced by more than llog₁₀Log of (a)₁₀Reduction factor (reduction factor).

Mann (US 2009/6749851) discloses a method for sterilizing digestive enzyme preparations to reduce the levels of active biological contaminants such as viruses, bacteria, yeasts, molds and fungi. Treatment of a composition comprising a digestive enzyme comprises stabilizing the composition by (a) reducing the temperature, (b) reducing the solvent, or (c) adding a stabilizing agent to the composition, followed by irradiation of the composition.

Lewis (US 1971/3956483) discloses a process for membrane enzyme preparation processing and bacterial reduction while maintaining amylolytic, proteolytic and lipolytic activity. The method comprises heating the membrane enzyme preparation to a sufficiently high temperature between 49-82 ℃. However, Lewis does not provide a means to inactivate or reduce the number of viruses.

In summary, the S/D method, the low pH incubation method, the octanoic acid method, the photochemical method, the pasteurization method and the dry heat method in the physical method are the core virus inactivation methods in the blood product preparation process. The pasteurization method and the dry heat method both belong to a method for inactivating viruses by heating, namely, virus protein is denatured through a certain temperature so as to destroy the virus structure to achieve the purpose of inactivating the viruses, and meanwhile, a certain temperature can denature protein products and reduce the biological activity, so when the two methods are used for inactivating the viruses, special treatment (coating, adding protective agents and the like) is usually needed to be carried out on the protein products, and meanwhile, the protective agents protect the proteins and simultaneously protect the viruses, the virus inactivation rate is reduced, and the difficulty of virus inactivation is increased. The photochemical method is a method which utilizes strong affinity action of certain photosensitizer rancour virus surface and virus nucleic acid structure, is easy to activate under the illumination of proper wavelength, and destroys the virus structure contacted with the photosensitizer by photochemical action, but the method has large loss of protein activity.

Thus, it is a particular challenge to inactivate or reduce viruses from a biological extract matrix in which the active substance is a mixture of enzymes, without destroying or altering the enzymatic activity or proportion of the protein in the process.

The organic solvent/active agent (S/D method) is to destroy the virus lipid envelope by the compatibility of the organic solvent and the active agent, so that the virus lipid envelope is not infected, thereby achieving the purpose of inactivating the virus. The method has good protein compatibility, can effectively inactivate virus, and has little influence on the activity of biological products.

The active agent used in the invention is a lipophilic nonionic surfactant, is an excellent emulsifier, is a safe and efficient broad-spectrum antibacterial agent, is not limited by pH, still has a good antibacterial effect under a neutral or slightly alkaline condition, has an inhibiting effect on replication and transmission of enveloped viruses, and can be used as a feed additive to be added into feed.

At present, the virus inactivation process for extracting and producing feed-grade pancreatin in a large scale from animal-derived biochemical extracted products, particularly pancreatin residues, is not reported at home and abroad, and the invention provides a method for reducing or inactivating the virus pollution of the feed-grade pancreatin without influencing the activity of the enzyme contained in the pancreatin products and changing the composition of the enzyme contained in the pancreatin products. Furthermore, the process does not produce any toxic compounds or residues in the final product.

Disclosure of Invention

Aiming at the defects of long operation time, easy inactivation of bioactive substances, incomplete virus inactivation, low extraction amount of the active substances and the like in the existing biological product for virus removal and inactivation, the invention provides the method which is simple, quick, economic and wide in application range, can obtain the highest-yield active pancreatin in the process of extracting feed-grade pancreatin from pancreatin residues and simultaneously inactivate the viruses existing in the pancreatin residues, so as to ensure that the pancreatin products can be applied to the production of feed additives more safely and effectively.

The technical scheme adopted by the invention for solving the technical problems is as follows: a method for inactivating viruses in preparation of feed-grade pancreatin, which is characterized by comprising the following steps:

A. raw material treatment: the raw material is frozen pancreas residue obtained after extracting angiotensin from crude enzyme powder, the raw material is crushed by a flat plate crusher or a flaker, dissolved by distilled water and stirred by a stirrer for 15-60 minutes, or ground and dissolved by a pulping machine to obtain a dissolved solution;

B. centrifuging: centrifuging the solution obtained in the step A by using a centrifugal machine or filtering and removing slag by using a plate-and-frame filter press to obtain supernatant filtrate;

C. and (3) precipitation and concentration: adding organic solvent ethanol and an active agent into the supernatant filtrate obtained in the step B, uniformly stirring, precipitating for 0.5-2.5 hours at the temperature of 5-20 ℃, and collecting the precipitate by ultrafiltration;

D. granulating, coating and drying: and C, mixing the precipitate obtained in the step C with a carrier for feed, granulating by using a granulating device, coating the product by using a coating device, and finally drying by using a drying device to obtain the pancreatin.

Preferably, the raw material is frozen pancreas residue obtained after angiotensin is extracted from crude enzyme powder, and organic solvent ethanol is adopted for extraction in the raw material obtaining process.

Preferably, the dissolving temperature of the step A is controlled to be 5-20 ℃, and the ratio of distilled water to raw materials is controlled to be 10-1: 1. .

Preferably, the rotating speed of the centrifuge in the step B is controlled at 1000-3000 r/min.

Preferably, the concentration of the organic solvent ethanol in the step C is 60-100%; the active agent in the step C is a lipophilic nonionic surfactant (fatty acid mono/diglyceride, glyceryl monostearate, glycerin fatty acid ester and the like), the concentration of the active agent is 2g/L-10g/L, the active agent has a good control effect on viruses with envelope, and the active agent is a wide non-specific feed additive for controlling animal viruses, does not cause stress on animals to influence the growth of the animals, and can promote the growth of the animals.

Preferably, the carrier for feed in step D is any one of corn starch, silica, gelatin and rice hull powder.

Preferably, step D is performed by ultrafiltration, and the drying device is a forced air drying device, a vacuum drying device or a fluidized bed drying device.

Compared with the prior art: the preparation process for extracting the feed-grade pancreatin from the pancreatin residue can destroy lipid envelopes of viruses by using an organic solvent and an active agent in the process of preparing pancreatin products to lose infectivity, thereby completely inactivating the residual viruses in the pancreatin residue raw materials, and simultaneously keeping the structure and the function of protein.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein, and it will be apparent to those of ordinary skill in the art that the present invention may be practiced without departing from the spirit and scope of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.