Pecan astringency removal method

Abstract

The invention discloses a pecan astringency removal method, which is characterized in that tannase and pecan kernels react at about 40 DEG C, so that the contents of main astringency component substances such as condensed tannin, flavone and phenolic compounds in pecan endopleura can be effectively reduced, and the original rich nutrition and unique flavor of pecan are maintained. The invention also discloses a degradation catalysis area of tannase in a deastringency process. The method provided by the invention can solve the problems existing in the existing hickory nut deastringency processing technology, retains better quality and taste of hickory nut compared with the traditional water boiling hickory nut deastringency, still retains partial substances such as saccharides, soluble protein, fatty acid and the like with nutritional value while deastringency, reduces water consumption, reduces wastewater pollution, and is suitable for industrial production. And the green and healthy processing purpose is achieved.

Description

Technical Field

[0001] The invention relates to the field of pecan processing, and in particular to a method for removing astringency from pecans. Technical Background

[0002] Carya cathayensis, belonging to the genus CaryaNutt of the family Juglandaceae, is an economic tree species unique to China. Its seed kernel is rich in nutrients, deeply loved by consumers, and has high economic value. However, the seed coat that is tightly bonded to the pecan embryo is rich in a variety of astringent substances, such as phenolic compounds, flavonoids, tannins, etc. Therefore, pecans must be de-astringent before they can be eaten.

[0003] High-temperature boiling and high-pressure steaming are currently the most commonly used methods for removing the astringency of pecan kernels. However, during the processing, some high-nutrient substances are dissolved in boiling water and lost, and high-concentration organic wastewater is generated, causing great environmental pollution and significantly increasing the cost of processing companies. How to improve the technology for removing the astringency of pecan kernels is an urgent problem to be solved. Summary of the invention

[0004] The purpose of the present invention is to design a new pecan deastringency process by using purchased tannase (which meets the requirements of food additives) in view of the deficiencies in the prior art, remove the condensed tannins, flavonoids and phenolic compounds in the pecan seed coat, solve the problems existing in the existing pecan deastringency processing technology, and retain better pecan quality and taste compared with traditional water-boiled pecan deastringency. While deastringency, the present invention still retains some sugars, soluble proteins, fatty acids and the like with nutritional value, reduces water consumption, reduces wastewater pollution, and achieves the purpose of green and healthy processing.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for removing astringency from walnuts comprises the following steps:

[0007] Step (1), dissolving tannase dry powder in water, preparing a tannase solution with a mass fraction of 1% to 5% at 40-45° C., and dividing the solution into two portions;

[0008] Step (2), placing pecan kernels in the first tannase solution, reacting at 40-45° C. to remove condensed tannins, flavonoids and phenolic compounds in the pecan kernels; wherein the mass volume ratio of the pecan kernels to the tannase solution is 3 g: 5-10 mL;

[0009] Step (3), after the reaction is completed, taking out the pecan kernels and placing them in a second portion of tannase solution, reacting at 40-45° C. to further remove condensed tannins, flavonoids and phenolic compounds in the pecan kernels; wherein the mass volume ratio of the pecan kernels to the tannase solution is 3 g: 5-10 mL;

[0010] Step (4): After the reaction is completed, take out the pecan kernels and wash them.

[0011] Preferably, the mass fraction of the tannase solution is 1%.

[0012] Preferably, the reaction time of step (2) is 20 min, the reaction temperature is 40° C., and the mass volume ratio of pecan kernel to tannase solution is 3 g:5 mL.

[0013] Preferably, the reaction time of step (3) is 20 min, the reaction temperature is 40° C., and the mass volume ratio of pecan kernels to tannase solution is 3 g:5 mL.

[0014] Preferably, the entire catalytic region of the tannase is composed of PHE110, SER111, SER301, PHE304, PHE306, ARG316, GLU319, SER367, ASP368, THR387 and HIS501.

[0015] Compared with the prior art, the beneficial effects of the present invention are at least:

[0016] The present invention provides a method for effectively degrading most of the astringent substances in the pecan seed coat to condense tannins, flavonoids and phenolic compounds, while still retaining a large amount of nutrients in the pecan kernel, such as protein, lipids and sugars. Compared with traditional water-boiled pecan kernels, the pecan kernels treated by the method of the present invention have lower astringency, better quality, better taste, less nutrient loss, and significantly reduced processing costs. At the same time, the present invention also discusses the catalytic degradation area of ​​tannase, and discloses for the first time its catalytic area when de-astringing, degrading and condensing tannins, flavonoids and phenolic compounds, filling the gap in research in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of different pH on the catalytic effect of tannase.

[0018] Figure 2 The effect of different temperatures on the catalytic effect of tannase.

[0019] Figure 3 The effect of different concentrations on the catalytic effect of tannase.

[0020] Figure 4The effect of tannase on the content of astringent substances in pecan kernels, where A is flavonoids, B is phenolic compounds, and C is condensed tannin.

[0021] Figure 5 The effect of tannase on the nutrient content in pecan kernels, where A: sugar content; B: soluble protein content; C: oil content; D: related fatty acid content.

[0022] Figure 6 is the frequency of contacting amino acids in molecular docking. DETAILED DESCRIPTION

[0023] The following embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

[0024] In the following examples, pecan kernels were used as the reaction object of the tannase-based de-astringency preparation. Pecans were sourced from farmers in Lin'an. The tannase, also known as tannase, is a tannic acid acyl hydrolase that hydrolyzes tannins. It is a highly efficient biological preparation prepared by deep liquid fermentation and refined extraction. It was purchased from Beijing Winova Biotechnology Co., Ltd. (http: / / www.winovazyme.com / index.php / Index / pshow / catid / 2 / xid / 0 / id / 30).

[0025] Example 1: Screening of optimal degradation conditions for tannase

[0026] Accurately prepare 100mM citric acid and 100mM sodium citrate solution, accurately weigh the volume ratio of citric acid solution and sodium citrate solution of 18.6:1.4, and mix them to form a citric acid-sodium citrate buffer solution with a pH of 3; accurately weigh the volume ratio of citric acid solution and sodium citrate solution of 13.1:6.9, and mix them to form a citric acid-sodium citrate buffer solution with a pH of 4; accurately weigh the volume ratio of citric acid solution and sodium citrate solution of 8.2:11.8, and mix them to form a citric acid-sodium citrate buffer solution with a pH of 5; accurately weigh the volume ratio of citric acid solution and sodium citrate solution of 3.8:12.2, and mix them to form a citric acid-sodium citrate buffer solution with a pH of 6; add 4% NaOH solution to the citric acid-sodium citrate buffer solution with a pH of 6 to accurately adjust the citric acid-sodium citrate buffer solution to a pH of 7-10, and obtain a citric acid-sodium citrate buffer solution with a pH value of 3-10. 200ppm tannase solution was prepared with different pH citric acid-sodium citrate buffers, and the enzyme activity was tested with propyl gallate as substrate. 10μL of 100mM propyl gallate, 50μL of 200ppm tannase solution, 40μL of corresponding pH citric acid-sodium citrate buffer, total volume 100μL, fully mixed and reacted at 40℃ for 10min, 100μL of methanol solution was added to terminate the reaction, and the amount of gallic acid produced was detected by gas chromatography-mass spectrometry.

[0027] The results of gallic acid (GA) content detected by liquid chromatography-mass spectrometry are as follows: Figure 1 As shown. The concentration of gallic acid generated in different pH values ​​shows that in the acidic environment of pH 2, the catalytic activity of tannase is inhibited, and the content of gallic acid generated is 0.1957μg / mL. The catalytic activity of tannase is the strongest in pH 3-4, and reaches a peak in the environment of pH 4, with the content of gallic acid generated being 0.6002μg / mL and 1.542μg / mL, respectively. In the environment where the pH exceeds 4, the activity of tannase begins to decline. In the environment with a pH value of 5-7, the catalytic activity of tannase has been inhibited, and the content of gallic acid generated is 0.2533μg / mL, 0.1728μg / mL and 0.2645μg / mL, respectively. In the environment with a pH value of 8-10, the catalytic activity of tannase has been severely inhibited, and the content of gallic acid generated is 0.0506μg / mL, 0.4115μg / mL and 0.0299μg / mL, respectively.

[0028] A 200 ppm tannase solution was prepared with ultrapure water, and propyl gallate (PG) was used as a substrate to test the enzyme activity. Temperature treatment groups were set at 20°C, 30°C, 40°C, 50°C and 60°C. First, 50 μL of 200 ppm tannase solution was added and preheated with 40 μL of ultrapure water at the corresponding temperature for 20 min. Then, 10 μL of 100 mM propyl gallate was added and mixed. The total reaction volume was 100 μL. After 10 min of reaction, 100 μL of methanol solution was added to terminate the reaction. The amount of gallic acid (GA) produced was detected by gas chromatography-mass spectrometry.

[0029] The concentration of gallic acid generated by tannase-catalyzed propyl gallate at different temperatures detected by liquid chromatography-mass spectrometry is as follows: Figure 2 As shown, the catalytic activity of tannase is low at a temperature of 20℃-30℃, and the production contents of gallic acid are 0.0139μg / mL and 0.0193μg / mL, respectively. The catalytic activity of tannase increases after 30℃. The suitable reaction temperature for tannase catalysis is 40℃-50℃, and the most suitable reaction temperature is 40℃, and the production contents of gallic acid are 3.0596μg / mL and 0.8556μg / mL, respectively. After the reaction temperature exceeds 40℃, the catalytic activity of tannase gradually decreases. At 60℃, the catalytic activity of tannase has been inhibited, and the production content of gallic acid is 0.4152μg / mL.

[0030] 1%, 3% and 5% tannase solutions were prepared with water, and the enzyme activity was tested with condensed tannin as substrate. The results were as follows: Figure 3 As shown, when the mass fraction of the tannase solution is 1%, 3% and 5%, the condensed tannins decrease significantly, and there is no difference in the condensed tannin content of the material ratios of 1%, 3% and 5%. For optimal cost control, 1% tannase solution can be used for de-astringency.

[0031] Therefore, the present invention uses 1%-5% tannase in a pH value of 4.2-5.6 and a temperature of 40-45°C to remove the astringency of walnuts, and the more preferred conditions are 1% tannase, pH value of 4.2 and reaction temperature of 40°C.

[0032] Example 2: Effect of tannase on astringent substances

[0033] Set up the tannase treatment group:

[0034] Prepare a 1% tannase solution with a pH of 4.2, take 50 mL and soak 30 g of pecan kernels, keep the reaction temperature at 40°C, and keep stirring to ensure sufficient reaction. After 30 minutes of reaction, remove the pecan kernels and immerse them in a new 1% tannase solution. After 30 minutes of reaction, remove the pecan kernels to complete the de-astringency.

[0035] To set up the boiling treatment group:

[0036] Soak 30g of pecan kernels in 50mL of pure water, keep the temperature at 40℃, and keep stirring to ensure sufficient reaction. Remove the pecan kernels after 1 hour.

[0037] Set up a blank control group:

[0038] The pecan kernels are not processed in any way.

[0039] The pecan kernels of the tannin treatment group, the boiled group and the blank control group were ground into powder, thoroughly defatted and deoiled, and then freeze-dried. The NaNO2-AlCl3-NaOH method was used to determine the difference in the content of total flavonoids in the pecan kernels with different treatments in an enzyme-labeled instrument. The Folin-sodium carbonate method was used to determine the difference in the content of total phenolic substances in the pecan kernels with different treatments in an enzyme-labeled instrument. The DMACA method was used to determine the difference in the content of condensed tannin substances in the pecan kernels with different treatments in an enzyme-labeled instrument.

[0040] The results are as follows Figure 4 shown; see Figure 4 A in the table, the highest average content of total flavonoids in untreated pecan kernels was 2.6659 mg / g, which was significantly higher than the 2.2235 mg / g of the tannase treated group and the 2.2322 mg / g of the water boiled group. There was no significant difference in the total flavonoids content between the tannase treated and water boiled pecan kernels, and both groups were significantly lower than those of the untreated pecan kernels.

[0041] See Figure 4 In B, the total phenolic content in the untreated pecan kernels was the highest at 47.9150 mg / g, significantly higher than the 32.0600 mg / g in the tannin treatment group and the 21.5125 mg / g in the water-boiled group. In the tannin treatment and water-boiled treatment, the total phenolic content decreased to a certain extent, and the total phenolic content in the water-boiled pecan kernels was significantly lower than that in the tannin treatment group. The total phenolic content of the tannin-treated pecan kernels was between the untreated pecan kernels and the water-boiled pecan kernels.

[0042] See Figure 4 After calculation, the content of condensed tannins in the untreated pecan kernels was 98.4858 mg / g, which was significantly higher than the 55.1491 mg / g of the tannin treatment group and the 24.1958 mg / g of the water-boiled group. In the tannin treatment and water-boiled treatments, the content of condensed tannins in the water-boiled pecan kernels was significantly lower than that in the tannin treatment group, and the content of condensed tannins in the tannin treatment pecan kernels was between the untreated pecan kernels and the water-boiled pecan kernels.

[0043] Example 3: Effect of Tannase on Nutrients

[0044] Set up a control group:

[0045] Soak 30g of pecan kernels in 50mL of pure water, keep the temperature at 40℃, and keep stirring to ensure sufficient reaction. Remove the pecan kernels after 30 minutes.

[0046] Set up the tannase treatment group:

[0047] Place 30g of pecan kernels in 50mL of 1% tannase solution with a pH of 4.2. Keep the reaction temperature at 40°C and stir continuously to ensure sufficient reaction. After 20 minutes of reaction, remove the pecan kernels and place them in another pot of 1% tannase solution. After 20 minutes, place them in 500mL of clean water, stir them thoroughly and then remove them.

[0048] To set up the boiling treatment group:

[0049] Place 30g of pecan kernels in 90mL of water, boil for 30min, remove from the water, and place in 500mL of clean water and stir thoroughly.

[0050] The pecan kernels in the control group, tannase treatment group and boiled water treatment group were ground into powder, thoroughly defatted and deoiled, and then freeze-dried. 0.02 g of the kernels were weighed and all organic matter was extracted with 80% acetone.

[0051] Figure 5 It represents the changes in nutrient content in the control group, tannase treatment group and boiling treatment group. The protein in the tannase treatment group is almost the same as that in the control group, and it also retains more fat than the boiling treatment group. The composition ratio of fatty acids in the control group, tannase treatment group and boiling treatment group remains almost unchanged.

[0052] In summary, the contents of the main astringent substances, condensed tannins, flavonoids, and phenolic compounds in pecan kernels treated with tannase have decreased to a certain extent, which are significantly lower than those in pecan kernels that have not undergone any de-astringency treatment. After sensory evaluation, the astringency sensation has decreased significantly, reaching a level that can be accepted by the human oral cavity. Although the main astringent substances in boiled pecans have decreased the most, in the process of de-astringency during steaming, a large amount of nutritious substances are also lost, and a large amount of organic wastewater is caused to pollute the environment, and the taste is poor. Using tannase to remove astringency from pecan kernels can not only remove a large amount of astringency, but also retain a large amount of nutritious protein, oil and sugar, with a high reuse rate, saving water and reducing costs, and at the same time, the taste of pecan kernels is better.

[0053] Example 4: Catalytic region of tannase

[0054] In order to further study how tannase binds to phenolic acids, flavonoids and tannins, the accurate molecular models found by 30 different substances of pecans treated with tannase were docked using AutoDockvina, and the docking results were visualized using PyMol software to observe the small molecule docking pockets and obtain the interaction sites. The results were statistically analyzed, as shown in Table 1 and Figure 6 shown.

[0055] Table 1 Molecular docking sites

[0056]

[0057]

[0058] The top ten small molecule contact amino acid sites with the highest docking scores are:

[0059] Platycaryanin A: TYR103, GLY108, SER111, SER114, ASP115, GLU192, GYS194, PHE304, AGR316, TYR450, CYS502 and TYR512;

[0060] Casuarin (Strictinin): TYR103, GLY108, GLU192, CYS194, SER195, PHE304, ASN308, VAL314, ARG316, ALA318 and GLU319;

[0061] 1,2,6-Tri-O-galloyl-β-D-glucose: PRO247, LYS251, LYS261, SER301, LEU302, GLY303, LEU366, ASN383, SER386, PRO385 and SER506;

[0062] Corilagin: GLY107, GLY108, SER111, ALA135, PHE136, ASP137, ASN138, ASN308, ALA318, and GLU319;

[0063] Isocorilagin: GLY107, GLY108, SER111, ALA135, PHE136, ASP137, ASN138, ASN308, ARG316, ALA318, and GLU319;

[0064] Catechingallate: GLY108, PHE110, SER111, LEU112, ALA135, PHE304 and ARG316;

[0065] Quercetin-3-O-galactoside: ARG316, GLU319, SER111, SER113, SER114, PHE110, GLY108 and GLY107;

[0066] 1,4,6-Tri-O-galloyl-β-D-glucose: PHE306, ASP367, ASP368, LEU302, ASN383, SER386, PRO456, and SER506;

[0067] Quercetin-3-O-β-D-galactopyranoside: GLY108, SER111, SER139, CYS194, SER195, PHE304, PHE306, SER307, ASN308, SER312, GLU319, HIS501, and CYS502;

[0068] 1,6-Di-O-galloyl-2-O-Cinnamoyl-β-D-glucose: LYS251, VAL254, THR300, SER301, LEU302, GLY303, SER307, GLU365, LEU366, SER367, THR387, ALA500 and SER506.

[0069] The statistical results showed that SER111 appeared 15 times, GLU319 appeared 13 times, PHE306 appeared 13 times, SER367 appeared 11 times, GLY108 appeared 11 times, ARG316 appeared 10 times, PHE304 appeared 9 times, PHE110 appeared 8 times, SER301 appeared 6 times, and THR387 appeared 6 times.

[0070] It can be seen from the results of this example that the entire catalytic region of the tannase is composed of PHE110, SER111, SER301, PHE304, PHE306, ARG316, GLU319, SER367, ASP368, THR387 and HIS501.

[0071] In addition to the above preferred embodiments, the present invention has other implementation modes. Those skilled in the art may make various changes and modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope defined in the claims of the present invention.

Claims

1. A method for removing astringency from walnuts, characterized in that: The method comprises the following steps: Step (1), dissolving tannase dry powder in water, preparing a tannase solution with a mass fraction of 1% to 5% at 40-45° C., and dividing the solution into two portions; Step (2), placing pecan kernels in the first tannase solution, reacting at 40-45° C. to remove condensed tannins, flavonoids and phenolic compounds in the pecan kernels; wherein the mass volume ratio of the pecan kernels to the tannase solution is 3 g: 5-10 mL; Step (3), after the reaction is completed, taking out the pecan kernels and placing them in a second portion of tannase solution, reacting at 40-45° C. to further remove condensed tannins, flavonoids and phenolic compounds in the pecan kernels; wherein the mass volume ratio of the pecan kernels to the tannase solution is 3 g: 5-10 mL; Step (4): After the reaction is completed, take out the pecan kernels and wash them.

2. The method for removing astringency from walnuts according to claim 1, characterized in that: The mass fraction of the tannase solution is 1%.

3. The method for removing astringency from walnuts according to claim 1, characterized in that: The reaction time in step (2) is 20 min.

4. The method for removing astringency from walnuts according to claim 1, characterized in that: The reaction temperature in step (2) is 40°C.

5. The method for removing astringency from walnuts according to claim 1, characterized in that: In step (2), the mass volume ratio of walnut kernel to tannase solution is 3g:5mL.

6. The method for removing astringency from walnuts according to claim 1, characterized in that: The reaction time in step (3) is 20 min.

7. The method for removing astringency from pecan nuts according to claim 1, characterized in that: The reaction temperature in step (3) is 40°C.

8. The method for removing astringency from walnuts according to claim 1, characterized in that: In step (3), the mass volume ratio of walnut kernel to tannase solution is 3g:5mL.

9. The method for removing astringency from walnuts according to any one of claims 1 to 8, characterized in that: In the process of removing astringency from pecan, the catalytic region of the tannase is composed of PHE110, SER111, SER301, PHE304, PHE306, ARG316, GLU319, SER367, ASP368, THR387 and HIS501.

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