Preparation method of walnut protein powder

Abstract

The invention relates to a preparation method of walnut protein powder, and relates to the technical field of walnut processing. The preparation method comprises the following steps: S1, soaking walnut kernels in warm water, then soaking the walnut kernels in a pretreatment solution for ultrasonic treatment, and then sequentially carrying out liquid nitrogen quick freezing and vacuum microwave treatment to obtain peeled walnut kernels; and S2, squeezing and crushing the peeled walnut kernels to obtain walnut cake meal, performing ultrasonic treatment, putting the walnut cake meal into a compound enzyme solution, performing microwave treatment, then adding polyethylene glycol and magnesium sulfate, performing extraction to obtain supernate and precipitate, adjusting the pH of the supernate, performing standing, and sequentially performing centrifugation, filtration, concentration, sterilization and spray drying to obtain the walnut protein powder. The seed coat is removed without destroying the protein structure as much as possible, the treatment time is short, the method is mild, green and safe, and the natural structure of the protein is maintained as much as possible; and the enzyme consumption is reduced, so that the cost benefit of the whole process is low. The method is suitable for large-scale factory application.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and particularly relates to a preparation method of walnut protein powder. Background Art

[0002] Walnut is one of the four major nuts in the world and has high nutritional value. The kernel of walnut contains 60% - 70% of oil, and the content of unsaturated fatty acids in the oil can reach more than 90%. The protein content in the kernel of walnut is about 15%, and the proportion of arginine in walnut protein is as high as 12% - 13%, which can be used as a good source of nutritional supplement for essential amino acids of infants.

[0003] At present, walnut oil is mostly extracted from walnut kernels by pressing method, leaching method, supercritical CO2 extraction method, etc., and then walnut protein in walnut meal is extracted by alkali solution acid precipitation method, aqueous enzymatic method, etc., or walnut peptides are prepared. These processes generally do not carry out dehulling treatment or partial dehulling treatment. There are many polyphenol substances in walnut seed coat, such as tannins, etc., which will reduce the quality and taste of walnut oil, and will also interact with proteins to form insoluble substances, affecting the extraction of walnut protein and the color of the obtained products, which puts higher requirements on the subsequent treatment of products. The methods for dehulling walnut kernels mainly include alkali soaking method, microwave method, baking method, etc. The dehulling effect of microwave method and baking method is not very ideal; although the alkali soaking method has good dehulling effect, it will cause different degrees of browning of walnut kernels and reduce the quality of walnut oil and walnut protein. Summary of the Invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a preparation method of walnut protein powder. The present invention removes the seed coat without damaging the protein structure as much as possible, has a short treatment time, is mild, green and safe, and can maintain the natural structure of the protein as much as possible; reduces the amount of enzyme used, making the cost - benefit of the whole process low. It is suitable for large - scale industrial application.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The purpose of the present invention is to provide a preparation method of walnut protein powder, comprising the following steps:

[0007] S1. Soak walnut kernels in warm water, then put the walnut kernels into a pretreatment solution and immerse them in ultrasonic waves, and then carry out liquid nitrogen quick - freezing and vacuum microwave treatment in sequence to obtain dehulled walnut kernels;

[0008] S2. Press and crush the dehulled walnut kernels to obtain walnut cake meal, put it into a complex enzyme solution and carry out microwave treatment after ultrasonic treatment, then add polyethylene glycol and magnesium sulfate for extraction to obtain supernatant and precipitate. Adjust the pH of the supernatant, let it stand, and then carry out centrifugation, filtration, concentration, sterilization in sequence, and spray - dry to obtain walnut protein powder.

[0009] The beneficial effects of the present invention are as follows: The present invention removes the seed coat while minimizing damage to the protein structure, with a short treatment time, being mild, green, and safe, and maintaining the natural structure of the protein as much as possible; reducing the amount of enzyme used, resulting in a low cost-benefit for the entire process. It is suitable for large-scale industrial applications.

[0010] Further, the soaking time in step S1 is 10 - 15 min, and the temperature of the warm water is 38 - 45 °C.

[0011] Further, the pretreatment liquid comprises the following raw materials in parts by mass: 5 - 6 parts of choline chloride, 10 - 12 parts of lactic acid, 0.1 - 2 parts of polyglycerol fatty acid ester, and 0.1 - 0.8 parts of sodium percarbonate.

[0012] The beneficial effects of adopting the above further scheme are as follows: Choline chloride is used to decompose the cortical structure. However, the adhesion between the walnut seed coat and the pulp is relatively strong. Therefore, lactic acid is added to degrade pectin, reducing the cortical adhesion, being environmentally friendly and having strong solubility for polyphenols. Sodium percarbonate, as an oxidant, gently releases H2O2 to degrade pigments and tannins, preventing the oxidation of the oil in walnuts to form oil oxides that combine with proteins.

[0013] Further, the pH of the pretreatment liquid is 8 - 9.

[0014] Further, in step (1), the frequency of the ultrasonic wave is 38 - 42 kHz, and the time is 4 - 6 min.

[0015] The beneficial effects of adopting the above further scheme are as follows: It assists the penetration of the pretreatment liquid and shortens the reaction time.

[0016] Further, in step (1), the temperature of the liquid nitrogen quick-freezing is -50 °C to -60 °C, and the time is 5 - 10 min.

[0017] The beneficial effects of adopting the above further scheme are as follows: Using liquid nitrogen to quickly freeze walnuts, taking advantage of the thermal shrinkage difference between cellulose and lipids, making the cortex brittle and generating microcracks. The residual water between the cortices forms ice, increasing in volume, and further expanding the gap between the cortex and the walnut pulp.

[0018] Further, in step (1), the frequency of the vacuum microwave treatment is 500 - 800 W, and the time is 5 - 10 seconds.

[0019] The beneficial effects of adopting the above further scheme are as follows: The brittle cortex expands and vaporizes through vacuum microwave treatment to achieve peeling.

[0020] Further, in step (2), the frequency of the vacuum microwave treatment is 500 - 800 W, and the time is 5 - 10 seconds.

[0021] The beneficial effects of adopting the above further scheme are as follows: The microwave treatment enhances enzyme penetration.

[0022] Furthermore, the dosage ratio of the walnut cake meal, the complex enzyme solution, and the polyethylene glycol is 400-500 g: 1000-1200 mL: 500-600 g; the mass ratio of the polyethylene glycol to the magnesium sulfate is 500-600: 25-30.

[0023] The beneficial effect of adopting the above further scheme is that polyethylene glycol and magnesium sulfate are used for aqueous two-phase extraction, the protein is enriched in the PEG phase, the seed coat precipitates in the lower layer, and the seed coat residue is separated by centrifugation.

[0024] Furthermore, the complex enzyme solution comprises the following raw materials in parts by mass: 30-40 parts of lactic acid, 25-35 parts of malic acid, 3-5 parts of laccase, 1-3 parts of xylanase, and 0.5-1 part of lactic acid.

[0025] The beneficial effect of adopting the above further scheme is that the degradation rate of the seed coat using the complex enzyme solution reaches 98%, and the bound protein is released synchronously.

[0026] Furthermore, the pH of the complex enzyme solution is 4-5. Detailed implementation mode

[0027] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For the reagents or instruments not indicating the manufacturer, they are all conventional products that can be purchased through regular channels.

[0028] Example 1: Preparation of walnut protein powder I

[0029] S1. 500 parts by mass of walnut kernels are soaked in warm water at 40 °C for 10 minutes, and then the walnut kernels are taken out and immersed in 800 ml of a pretreatment solution (the mass ratio of choline chloride, lactic acid, polyglycerol fatty acid ester, sodium percarbonate, and water is 5: 10: 0.5: 0.5: 770) at a temperature of 38 °C and a pH of 8, and treated with ultrasonic waves at 40 kHz for 5 minutes, and then left standing for 17 minutes. The walnut kernels are quickly taken out and frozen at -60 °C with liquid nitrogen for 5 min, and then treated with vacuum microwave at 650 W for 8 seconds to obtain peeled walnut kernels;

[0030] S2. After mechanically pressing and peeling the walnuts, they are crushed and sieved through a 80-mesh sieve to obtain walnut cake meal. After ultrasonic treatment at 40 kHz and 200 W for 10 minutes, it is put into 1200 parts by mass of a composite enzyme solution (35 parts by mass of lactic acid, 30 parts by mass of malic acid, 4 parts by mass of laccase, 2 parts by mass of xylanase, 0.7 parts by mass of pectinase, and the rest is water), and treated with 800 W microwave for 5 s. Then, 500 parts by mass of polyethylene glycol and 25 parts by mass of magnesium sulfate are added for extraction to obtain a supernatant and a precipitate. The pH of the supernatant is adjusted to 4.5 and then allowed to stand, and then centrifuged at 8000 g for 15 min. Subsequently, it is successively filtered by intercepting with a 30 kDa ultrafiltration membrane and concentrated with a 5 kDa nanofiltration membrane until the solid content > 25%. Then, it is sterilized, pre-frozen at -50 °C and then lyophilized to obtain walnut protein powder.

[0031] Example 2: Preparation of walnut protein powder II

[0032] S1. 500 parts by mass of walnuts are soaked in warm water at 40 °C for 10 minutes, and then the walnuts are taken out and immersed in 800 ml of a pretreatment solution (the mass ratio of choline chloride, lactic acid, polyglycerol fatty acid ester, sodium percarbonate, and water is 6:12:0.1:0.8:760) at 35 °C and pH 8.5, combined with ultrasonic treatment at 38 kHz for 6 minutes, and then allowed to stand for 15 minutes. It is quickly taken out and frozen at -55 °C with liquid nitrogen for 8 min, and then treated with 500 W vacuum microwave for 10 s to obtain peeled walnuts;

[0033] S2. After mechanically pressing and peeling the walnuts, they are crushed and sieved through a 80-mesh sieve to obtain walnut cake meal. After ultrasonic treatment at 40 kHz and 200 W for 10 minutes, it is put into 1200 parts by mass of a composite enzyme solution (30 parts by mass of lactic acid, 25 parts by mass of malic acid, 3 parts by mass of laccase, 1 part by mass of xylanase, 0.5 parts by mass of pectinase, and the rest is water), and treated with 800 W microwave for 5 s. Then, 500 parts by mass of polyethylene glycol and 25 parts by mass of magnesium sulfate are added for extraction to obtain a supernatant and a precipitate. The pH of the supernatant is adjusted to 4.5 and then allowed to stand, and then centrifuged at 8000 g for 15 min. Subsequently, it is successively filtered by intercepting with a 30 kDa ultrafiltration membrane and concentrated with a 5 kDa nanofiltration membrane until the solid content > 25%. Then, it is sterilized, pre-frozen at -50 °C and then lyophilized to obtain walnut protein powder.

[0034] Example 3: Preparation of walnut protein powder III

[0035] S1. Soak 500 parts by mass of walnut kernels in warm water at 38 °C for 12 minutes, then take out the walnut kernels and immerse them in 800 ml of a pretreatment solution (mass ratio of choline chloride, lactic acid, polyglycerol fatty acid ester, sodium percarbonate, and water is 5.5:11:2:0.1:770) at a temperature of 40 °C and a pH of 9. Combine with ultrasonic treatment at 42 kHz for 4 minutes, then let it stand for 20 minutes. Quickly take out and use liquid nitrogen to freeze at -50 °C for 10 min, and then perform 800W vacuum microwave treatment for 5 seconds to obtain peeled walnut kernels;

[0036] S2. After mechanically pressing the peeled walnut kernels, crush them and pass through an 80-mesh sieve to obtain walnut cake meal. After ultrasonic treatment at 40 kHz and 200W for 10 minutes, put it into 1200 parts by mass of a complex enzyme solution (40 parts by mass of lactic acid, 35 parts by mass of malic acid, 5 parts by mass of laccase, 3 parts by mass of xylanase, 1 part by mass of pectinase, and the rest is water) and perform 800W microwave treatment for 5s. Then add 500 parts by mass of polyethylene glycol and 25 parts by mass of magnesium sulfate for extraction to obtain supernatant and precipitate. Adjust the pH of the supernatant to 4.5 and let it stand, then centrifuge at 8000g for 15 min. Subsequently, perform ultrafiltration and retention filtration using a 30 kDa ultrafiltration membrane and nanofiltration concentration using a 5 kDa nanofiltration membrane until the solid content > 25%. Then sterilize, pre-freeze at -50 °C, and perform sublimation drying to obtain walnut protein powder.

[0037] Comparative Example 1: Preparation of walnut protein powder

[0038] This comparative example is the same as Example 1, except that the treatment process in step S1 is different. Specifically, it does not undergo soaking in the pretreatment solution. The rest of the steps and raw materials are the same as in Example 1. The specific steps of S1 are as follows:

[0039] S1. Soak 500 parts by mass of walnut kernels in warm water at 40 °C for 10 minutes, then combine with ultrasonic treatment at 40 kHz for 5 minutes, and then let it stand for 17 minutes. Quickly take out and use liquid nitrogen to freeze at -60 °C for 5 min, and then perform 650W vacuum microwave treatment for 8 seconds to obtain peeled walnut kernels.

[0040] Comparative Example 2: Preparation of walnut protein powder

[0041] This comparative example is the same as Example 1, except that the treatment process in step S1 is different. Specifically, it does not undergo ultrasonic treatment. The rest of the steps and raw materials are the same as in Example 1. The specific steps of S1 are as follows:

[0042] S1. Soak 500 parts by mass of walnut kernels in warm water at 40 °C for 10 minutes, then take out the walnut kernels and immerse them in 800 ml of a pretreatment solution (the mass ratio of choline chloride, lactic acid, polyglycerol fatty acid ester, sodium percarbonate, and water is 5:10:0.5:0.5:770) at a temperature of 38 °C and a pH of 8, and let it stand for 17 minutes. Quickly take out and use liquid nitrogen to freeze at -60 °C for 5 min, and then perform vacuum microwave treatment at 650 W for 8 seconds to obtain peeled walnut kernels.

[0043] Comparative Example 3: Preparation of walnut protein powder

[0044] This comparative example is the same as Example 1, and the only difference lies in the treatment process in step S1, specifically that the liquid nitrogen quick-freezing treatment is not carried out. The remaining steps and raw materials are the same as those in Example 1. The specific steps of S1 are as follows:

[0045] S1. Soak 500 parts by mass of walnut kernels in warm water at 40 °C for 10 minutes, then take out the walnut kernels and immerse them in 800 ml of a pretreatment solution (the mass ratio of choline chloride, lactic acid, polyglycerol fatty acid ester, sodium percarbonate, and water is 5:10:0.5:0.5:770) at a temperature of 38 °C and a pH of 8, combine with ultrasonic treatment at 40 kHz for 5 minutes, and then let it stand for 17 minutes. Quickly take out and perform vacuum microwave treatment at 650 W for 8 seconds to obtain peeled walnut kernels.

[0046] Comparative Example 4: Preparation of walnut protein powder

[0047] This comparative example is the same as Example 1, and the only difference lies in the treatment process in step S1, specifically that the vacuum microwave treatment is not carried out. The remaining steps and raw materials are the same as those in Example 1. The specific steps of S1 are as follows:

[0048] S1. Soak 500 parts by mass of walnut kernels in warm water at 40 °C for 10 minutes, then take out the walnut kernels and immerse them in 800 ml of a pretreatment solution (the mass ratio of choline chloride, lactic acid, polyglycerol fatty acid ester, sodium percarbonate, and water is 5:10:0.5:0.5:770) at a temperature of 38 °C and a pH of 8, combine with ultrasonic treatment at 40 kHz for 5 minutes, and then let it stand for 17 minutes. Quickly take out and use liquid nitrogen to freeze at -60 °C for 5 min to obtain peeled walnut kernels.

[0049] Comparative Example 5: Preparation of walnut protein powder

[0050] This comparative example is the same as Example 1, and the only difference lies in the treatment process in step S2, specifically that the microwave treatment is not carried out. The remaining steps and raw materials are the same as those in Example 1. The specific steps of S2 are as follows:

[0051] S2. After mechanically pressing and peeling walnuts, they are pulverized and passed through an 80-mesh sieve to obtain walnut cake meal. After ultrasonic treatment at 40 kHz and 200 W for 10 min, it is placed in 1200 parts by mass of a composite enzyme solution (35 parts by mass of lactic acid, 30 parts by mass of malic acid, 4 parts by mass of laccase, 2 parts by mass of xylanase, 0.7 parts by mass of pectinase, and the rest is water) and allowed to stand. Subsequently, 500 parts by mass of polyethylene glycol and 25 parts by mass of magnesium sulfate are added for extraction to obtain a supernatant and a precipitate. The pH of the supernatant is adjusted to 4.5 and then allowed to stand for 5 min, followed by centrifugation at 8000 g for 15 min. Subsequently, it is successively filtered by retention using a 30 kDa ultrafiltration membrane and concentrated using a 5 kDa nanofiltration membrane until the solid content > 25%. Subsequently, it is sterilized, pre-frozen at -50 °C, and then lyophilized to obtain walnut protein powder.

[0052] Comparative Example 6: Preparation of walnut protein powder

[0053] This comparative example is the same as Example 1, except that the treatment process in step S2 is different. Specifically, the composite enzyme solution is replaced with an alkali solution, and the remaining steps and raw materials are the same as those in Example 1. The specific step S2 is as follows:

[0054] S2. After mechanically pressing and peeling walnuts, they are pulverized and passed through an 80-mesh sieve to obtain walnut cake meal. After ultrasonic treatment at 40 kHz and 200 W for 10 minutes, it is placed in 1200 parts by mass of an alkali solution and treated with 800 W microwave for 5 s. Subsequently, 500 parts by mass of polyethylene glycol and 25 parts by mass of magnesium sulfate are added for extraction to obtain a supernatant and a precipitate. The pH of the supernatant is adjusted to 4.5 and then allowed to stand for 5 min, and then centrifuged at 8000 g for 15 min. Subsequently, it is successively filtered by retention using a 30 kDa ultrafiltration membrane and concentrated using a 5 kDa nanofiltration membrane until the solid content > 25%. Subsequently, it is sterilized, pre-frozen at -50 °C, and then lyophilized to obtain walnut protein powder.

[0055] Comparative Example 7: Preparation of walnut protein powder:

[0056] This comparative example is the same as Example 1, except that the raw materials of the pretreatment solution in step S1 are different. Specifically, choline chloride and lactic acid are not added, and the remaining steps and raw materials are the same as those in Example 1.

[0057] Comparative Example 8: Preparation of walnut protein powder:

[0058] This comparative example is the same as Example 1, except that the raw materials of the composite enzyme solution in step S2 are different. Specifically, laccase, xylanase, and pectinase are not added, and the remaining steps and raw materials are the same as those in Example 1.

[0059] Test Example:

[0060] (1) Weigh the final protein powder quality of Example 1 and Comparative Examples 1-9 and calculate the extraction rate. Subsequently, test the protein powders prepared in Example 1 and Comparative Examples 1-9. The determination of the solubility of the protein powder is carried out according to the following method: Weigh 1 g of the spray-dried protein powder, add 50 mL of water, stir at room temperature of 25 °C for 1 h, take a part of the supernatant and centrifuge it at 10000×g for 15 min, and then refer to the first method, the Kjeldahl method, in GB / T 5009.5-2016 "National Food Safety Standard - Determination of Protein in Foods" for testing. The solubility calculation formula of the protein is: (protein concentration in the supernatant / protein concentration in the whole solution) × 100%, and parallel test 3 groups, take the average value, and the results are shown in Table 1:

[0061] Table 1

[0062]

[0063]

[0064] (2) Quality inspection

[0065] According to the national quality standards GB 5009.5-2016 "National Food Safety Standard - Determination of Protein in Foods", GB 5009.6-2016 "National Food Safety Standard - Determination of Fat in Foods", and GB 5009.4-2016 "National Food Safety Standard - Determination of Ash in Foods", test the walnut protein powders prepared in Example 1 and Comparative Examples 1-10 respectively, and the obtained data are shown in Table 2:

[0066] Table 2

[0067]

[0068]

[0069] It can be seen from Tables 1-2 that:

[0070] (1) From the data in Table 1, it can be seen that compared with Example 1, in Comparative Example 1, the skin removal treatment was carried out without adding the pretreatment solution, and the skin removal rate was relatively low, which not only affected the taste of the protein powder product, but also the protein in the seed coat combined with the oil oxide, resulting in a decrease in the final protein extraction rate. Compared with Example 1, in Comparative Example 2, ultrasonic treatment was not carried out during the pretreatment solution immersion. Since the immersion time in the pretreatment solution was short, it was not conducive to the penetration of the pretreatment into the seed coat, resulting in poor final reaction results. In Comparative Example 3, liquid nitrogen quick-freezing was not carried out, and in Comparative Example 4, vacuum microwave treatment was not carried out after liquid nitrogen quick-freezing, and the skin removal rate was also relatively low.

[0071] (2) Compared with Example 1, microwave treatment was not carried out in Step S2 in Comparative Example 5. Compared with Example 1, the complex enzyme was replaced with water in Comparative Example 6 to synchronously release the bound protein. In Comparative Example 7, choline chloride and lactic acid were not added to the pretreatment solution. In Comparative Example 8, no enzyme was added to the complex enzyme solution for treatment. It can be seen that the quality test results of the protein powder in each comparative example are worse than those in Example 1, and the extraction rate and protein solubility are also poor. It can be seen that the present invention removes the seed coat without damaging the protein structure as much as possible, has a short treatment time, is mild, green and safe, and has low cost. Moreover, the protein extraction rate is high and the quality is good, which is suitable for large-scale industrial application.

[0072] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A preparation method of walnut protein powder, characterized in that, The preparation method comprises the following steps: S1. Soak walnut kernels in warm water, then immerse the walnut kernels in a pretreatment solution and perform ultrasonic treatment. Subsequently, perform liquid nitrogen quick-freezing and vacuum microwave treatment in sequence to obtain peeled walnut kernels; S2. Press and crush the peeled walnut kernels to obtain walnut cake meal. After ultrasonic treatment, place it in a composite enzyme solution and perform microwave treatment. Then, add polyethylene glycol and magnesium sulfate for extraction to obtain a supernatant and a precipitate. Adjust the pH of the supernatant, let it stand, and then perform centrifugation, filtration, concentration, and sterilization in sequence. Spray drying yields walnut protein powder.

2. The preparation method of a walnut protein powder according to claim 1, wherein, The pretreatment solution comprises the following raw materials in parts by mass: 5 - 6 parts of choline chloride, 10 - 12 parts of lactic acid, 0.1 - 2 parts of polyglycerol fatty acid ester, and 0.1 - 0.8 part of sodium percarbonate.

3. The preparation method of a walnut protein powder according to claim 2, characterized in that, The pH of the pretreatment solution is 8 - 9.

4. The preparation method of a walnut protein powder according to claim 3, characterized in that, In step (1), the frequency of the ultrasonic treatment is 38 - 42 kHz, and the time is 4 - 6 min.

5. The preparation method of a walnut protein powder according to claim 1, characterized in that, In step (1), the temperature of the liquid nitrogen quick-freezing is -50°C to -60°C, and the time is 5 - 10 min.

6. The preparation method of a walnut protein powder according to claim 1, characterized in that, In step (1), the frequency of the vacuum microwave treatment is 500 - 800 W, and the time is 5 - 10 seconds.

7. A method for preparing walnut protein powder according to claim 1, characterized in that, In step (2), the frequency of the vacuum microwave treatment is 500 - 800 W, and the time is 5 - 10 seconds.

8. A method for preparing walnut protein powder according to claim 1, characterized in that, The dosage ratio of the walnut cake meal, the composite enzyme solution, and the polyethylene glycol is 400 - 500 g: 1000 - 1200 mL: 500 - 600 g; the mass ratio of the polyethylene glycol to the magnesium sulfate is 500 - 600: 25 - 30.

9. The preparation method of a walnut protein powder according to claim 5, characterized in that, The composite enzyme solution comprises the following raw materials in parts by mass: 30 - 40 parts of lactic acid, 25 - 35 parts of malic acid, 3 - 5 parts of laccase, 1 - 3 parts of xylanase, and 0.5 - 1 part of pectinase.

10. The preparation method of a walnut protein powder according to claim 1, characterized in that, The pH of the composite enzyme solution is 4 - 5.

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