Application of papain in removing toxicity of patulin
By using the method of mixing papain with apple juice, the problem of high cost and low efficiency of penicillin removal in apple juice in the prior art is solved, and an efficient, safe and environmentally friendly detoxification effect is achieved, and the quality of apple juice is maintained.
Patent Information
- Application Number
- CN202411855373.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art has problems such as high cost, expensive equipment, and may lead to secondary contamination and destruction of food nutrition in the process of removing penicillin from apple juice.
Papain is used as a detoxicant and mixed with apple juice to remove penicillin through the degradation of enzymes. This method is simple to operate, low cost, safe and environmentally friendly, and has no negative impact on the quality of apple juice.
The efficient penicillin detoxin rate has been achieved, reaching 89.4%, while maintaining the quality of apple juice, and is suitable for a wide range of penicillin removal applications in apple juice.
Smart Images
Figure CN119605943A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bioenzyme detoxification, in particular to the application of papain in detoxification of patulin. Background Art
[0002] Patulin, also known as Patulin (PAT), is a water-soluble fungal toxin with a molecular formula of C 7 H 6 O 4 , mainly produced by the metabolism of Penicillium expansum. PAT is easily soluble in water, has stable chemical properties, and has significant toxicity. It may cause serious short-term and long-term damage to humans, including immunotoxicity, genotoxicity, neurotoxicity, and mutagenicity. Acute symptoms of PAT poisoning include convulsions, congestion, gastrointestinal dilatation, intestinal bleeding, etc. Chronic toxicity manifests itself in neurological, immunotoxicity, genotoxicity, immunosuppression, teratogenicity, and mutagenicity. Toxicological experiments have shown that the median lethal dose of PAT to rats is LD50. 50 The range is about 15-25 mg / kg. Due to its potential carcinogenicity to humans, the International Agency for Research on Cancer (IARC) has classified it as a Class III carcinogen. PAT is widely found in rotten fruits (mainly apples, but also pears, grapes, tomatoes, hawthorns, etc.) and their processed products (juice, jam, puree, etc.), among which apples and their products are the most seriously contaminated.
[0003] my country is the world's largest apple producer and the world's most important apple juice producer, processor and exporter. The amount of concentrated apple juice exported has long been ranked first in the world. Due to the solubility of PAT in water and its stability in acidic media and high temperatures, PAT can enter apple products through processing. Drinking apple juice contaminated with PAT will be harmful to human health. At present, PAT contamination in apple juice has become an important food safety issue. The presence of PAT has been detected in apple juice in many countries and regions, and it has become one of the important factors in the development of the apple juice processing industry. Regulatory agencies in China (GB 2761-2017), the United States (CPG Sec.510.150) and the European Union (Commission Regulation [EU] 2023 / 915) set the maximum allowable content of PAT in apple products at 50μg / kg.
[0004] Currently, PAT detoxification methods are mainly divided into three categories: physical method, chemical method and biological method.
[0005] Physical methods include clarification filtration, heat treatment, ultraviolet radiation and pulsed light. Patras's group used ultraviolet (UV-C) to detoxify and found that 400mJ / cm 2The UV-C dose successfully reduced the PAT concentration by 69.47%. However, physical methods usually require expensive equipment and maintenance costs, and some methods also have disadvantages such as secondary pollution, destruction of nutritional and functional components in food, and general detoxification effects.
[0006] The chemical method mainly achieves the purpose of detoxification by adding chemical reagents such as sulfur dioxide, potassium permanganate, ozone, and ascorbic acid. Wei et al. found that adding ascorbic acid and ferrous iron to apple juice containing 0.08 or 0.4 μg / mL PAT resulted in a 75-94% PAT degradation rate. The chemically treated apple juice has the disadvantages of serious loss of phenolic compounds and organic acids with flavor and taste and unclear safety properties.
[0007] The biological detoxification method is based on biotechnology to detoxify PAT in food by degradation or adsorption. It mainly includes microbial and enzymatic detoxification. Liu et al. found that pig liver esterase, horseradish peroxidase, Aspergillus niger lipase A, and Mucor lipase M have the ability to degrade PAT in apple juice, with degradation rates of 97.8%, 53.2%, 76.3% and 68.2%, respectively. In comparison, the biological detoxification method has the advantages of being more gentle, efficient, safe and environmentally friendly, and has a broader application prospect in the field of food detoxification.
[0008] Papain is an active cysteine protease that can be extracted from the stems, leaves and roots of papaya. Papain is a white or light yellow powder in solid state, easily soluble in glycerol and water, and poorly soluble in organic solvents such as chloroform and ethanol. Papain is widely used in the food industry because of its high enzyme activity, good thermal stability, natural safety and economic benefits. At present, there is no report on the use of papain for PAT detoxification. Summary of the invention
[0009] The purpose of the present invention is to provide an application of papain in removing patulin toxicity to solve the problems existing in the above-mentioned prior art. The present invention provides a new method and idea for removing patulin from apple juice, which has the advantages of high detoxification rate, simple operation, low cost, safety, environmental protection and wide application range.
[0010] To achieve the above object, the present invention provides the following solutions:
[0011] One of the technical solutions of the present invention is the use of papain in removing the toxicity of patulin.
[0012] The second technical solution of the present invention is a method for removing patulin from fruit products by using papain, which comprises mixing the fruit products with papain and reacting them.
[0013] Based on the above technical solution, the present invention has the following technical effects:
[0014] The papain used in the present invention is one of the most widely used enzymes in the food industry, and has the advantages of being natural, safe, low-priced, and environmentally friendly. The embodiments of the present invention show that the detoxification rate of patulin using papain can reach 89.4%, and the quality of the apple juice after detoxification does not change significantly, indicating that the enzyme will not have a negative impact on the quality of apple juice. The method of the present invention has high detoxification efficiency, low cost, and is green and safe, and can be widely used for the removal of patulin in apple juice. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0016] Figure 1 This is a comparison chart of the detoxification ability of papain in Example 1 of the present invention at different detoxification times.
[0017] Figure 2 This is a graph showing the effect of papain concentration on detoxification ability in Example 2 of the present invention.
[0018] Figure 3 This is a graph showing the effect of temperature on the detoxification ability of papain in Example 3 of the present invention.
[0019] Figure 4 This is a graph showing the effect of PAT concentration in apple juice on the detoxification ability of papain in Example 4 of the present invention. DETAILED DESCRIPTION
[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0021] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.
[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0025] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.
[0026] The embodiment of the present invention provides the use of papain in removing the toxicity of patulin.
[0027] The embodiment of the present invention also provides a method for removing patulin from a fruit product by using papain, which comprises mixing the fruit product with papain and reacting the mixture.
[0028] In some specific embodiments, the amount of papain used is 400-720 U / mL based on the volume of the fruit product.
[0029] In some specific embodiments, the reaction conditions of the light-proof reaction include: a reaction temperature of 30 to 70° C. and a reaction time of 36 to 48 hours.
[0030] In some specific embodiments, the fruit preparation is a fruit preparation containing patulin.
[0031] In some specific embodiments, the fruit preparations include juices, jams and purees.
[0032] In some specific embodiments, the fruit includes apples, pears, grapes, tomatoes and hawthorns.
[0033] Example 1
[0034] Detoxification ability of papain at different detoxification times:
[0035] Step 1: mix apple juice (pH 3.2) and patulin standard solution to obtain a working solution with a final concentration of 5 μg / mL, and store at 4°C.
[0036] Step 2, dissolve papain (800 U / mg) in Tris-HCl buffer (1 mol / L, pH 7.0) to prepare 5 mg / mL enzyme solution, and store it at 4°C.
[0037] Step 3, take 2 mL of working solution and mix it with 200 μL of enzyme solution, place it on a constant temperature shaking shaker at 37°C, 200 rpm, and react in the dark for 12, 24, 36, and 48 h.
[0038] Step 4, extract patulin in apple juice three times with equal volumes of ethyl acetate, vortex shake for 3 minutes, further centrifuge (4000 rpm, 5 minutes), collect the supernatant and blow dry under nitrogen, dissolve it in acidic water (pH 3.8), filter it with a 0.22 μm filter, and store it at -20°C.
[0039] Step 5, high performance liquid chromatography (HPLC) was used to analyze the detoxification efficiency. The chromatographic column was Welch-C18 (250x4.6mm, 5μm); mobile phase A (water), mobile phase B (acetonitrile); elution conditions were 5% B (0min~13min), 100% B (13min~15min), 5% B (15min~20min); flow rate was 0.8mL / min; column temperature was 40℃; injection volume was 20μL; detection wavelength was 276nm. Calculate the detoxification rate:
[0040] Detoxification rate = (PAT concentration before detoxification - PAT concentration after detoxification) / PAT concentration before detoxification × 100%.
[0041] The results are as follows Figure 1 As shown, the detoxification rate increases with the increase of detoxification time. When the time is as long as 36 hours, the detoxification rate of patulin in apple juice can reach more than 80%.
[0042] Example 2
[0043] Effect of papain concentration on detoxification ability:
[0044] Step 1: mix apple juice (pH 3.2) and patulin standard solution to obtain a working solution with a final concentration of 5 μg / mL, and store at 4°C.
[0045] Step 2, dissolve papain (800 U / mg) in Tris-HCl buffer (1 mol / L, pH 7.0) to prepare 1, 3, 5, 7, and 9 mg / mL enzyme solutions, and store them at 4°C.
[0046] Step 3, take 2 mL of working solution and mix it with 200 μL of enzyme solution, place it on a constant temperature shaking shaker at 37°C, 200 rpm, and react for 36 hours in the dark.
[0047] Step 4 and step 5 are the same as in Example 1.
[0048] The results are as follows Figure 2 As shown, the detoxification rate increases with the increase of enzyme solution concentration, and the detoxification rate reaches more than 70% when the enzyme concentration is greater than 5 mg / mL.
[0049] Example 3
[0050] Effect of temperature on the detoxification ability of papain:
[0051] Step 1: mix apple juice (pH 3.2) and patulin standard solution to obtain a working solution with a final concentration of 5 μg / mL, and store at 4°C.
[0052] Step 2, dissolve papain (800 U / mg) in Tris-HCl buffer (1 mol / L, pH 7.0) to prepare 5 mg / mL enzyme solution, and store it at 4°C.
[0053] Step 3, take 2 mL of working solution and mix it with 200 μL of enzyme solution, place it at different temperatures (30°C, 40°C, 50°C, 60°C, 70°C) in the dark for 36 hours.
[0054] Step 4 and step 5 are the same as in Example 1.
[0055] The results are as follows Figure 3 As shown, the detoxification rate increases with the increase of temperature. The detoxification efficiency is best when the detoxification temperature is 70°C, reaching 88.8%.
[0056] Example 4
[0057] Effect of PAT concentration in apple juice on the detoxification ability of papain:
[0058] Step 1, mix apple juice (pH 3.2) with patulin standard solution to obtain working solutions with final concentrations of 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL, and store at 4°C.
[0059] Step 2, dissolve papain (800 U / mg) in Tris-HCl buffer (1 mol / L, pH 7.0) to prepare 5 mg / mL enzyme solution, and store it at 4°C.
[0060] Step 3, take 2 mL of working solution and mix with 200 μL of enzyme solution, place on a constant temperature shaking table at 37°C, 200 rpm, and react for 36 hours in the dark.
[0061] Step 4 and step 5 are the same as in Example 1.
[0062] The results are as follows Figure 4 As shown, the detoxification rate of apple juice containing 5 different concentrations of PAT can reach more than 70%, indicating that papain has a certain ability to remove PAT in apple juice.
[0063] Example 5
[0064] Evaluation of the quality of apple juice after papain detoxification:
[0065] Step 1: mix apple juice (pH 3.2) and patulin standard solution to obtain a working solution with a final concentration of 5 μg / m, and store it at 4°C.
[0066] Step 2, dissolve papain (800 U / mg) in Tris-HCl buffer (1 mol / L, pH 7.0) to prepare 5 mg / mL enzyme solution, and store it at 4°C.
[0067] Step 3, take 2 mL of working solution and mix with 200 μL of enzyme solution, place on a constant temperature shaking table at 37°C, 200 rpm, and react for 36 hours in the dark.
[0068] Step 4, comparing the quality parameters (soluble solids, transmittance, color value, pH and total acid) of apple juice before and after removal of PAT.
[0069] Table 1 Quality parameters of apple juice before and after detoxification with papain
[0070]
[0071] It can be seen from the data in Table 1 that the soluble solids content, transmittance, color value, and pH of apple juice do not change significantly when the PAT detoxification method provided by the present invention is adopted, and the pH and titratable acid change slightly. The pH value of apple juice is generally 3.2-4.5, and the change is within a reasonable range. It shows that the use of papain in the detoxification process has no negative impact on the quality of apple juice. Obviously, the above embodiments of the present invention are only examples made to clearly illustrate the present invention, and are not limitations on the embodiments of the present invention. For ordinary technical users in the field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the claims of the present invention.
Claims
1. Application of papain in detoxification of patulin.
2. A method for removing patulin from fruit products using papain, characterized in that: Mix the fruit product with papain and allow to react in a dark place.
3. The method according to claim 2, characterized in that Calculated by the volume of the fruit product, the dosage of the papain is 400-720 U / mL.
4. The method according to claim 2, characterized in that: The reaction conditions of the light-proof reaction include: a reaction temperature of 30 to 70° C. and a reaction time of 36 to 48 hours.
5. The method according to claim 2, characterized in that: The fruit product is a fruit product containing patulin.
6. The method according to claim 5, characterized in that The fruit products include juice, jam and puree.
7. The method according to claim 5, characterized in that The fruits include apples, pears, grapes, tomatoes and hawthorns.