Method for inactivating lectin in mixed bean cells through cooperation of high hydrostatic pressure and mild acid treatment

Abstract

The invention discloses a method for inactivating lectin in mixed bean cells through cooperation of high hydrostatic pressure and mild acid treatment. Soaking the mixed beans in an acidic buffer solution to obtain the mixed beans subjected to mild acid treatment; or preparing mixed beans into mixed bean cell powder, and soaking the mixed bean cell powder in an acidic buffer solution to obtain the mixed bean cell powder subjected to mild acid treatment; or soaking mixed beans in an acidic buffer solution, and then preparing mixed bean cell powder to obtain mixed bean cell powder subjected to mild acid treatment; placing the miscellaneous beans subjected to mild acid treatment or miscellaneous bean cell powder subjected to mild acid treatment in a salt solution, and performing high hydrostatic pressure treatment and drying to obtain the intracellular lectin inactivated miscellaneous bean whole food or miscellaneous bean cell powder subjected to high hydrostatic pressure and mild acid treatment. The lectin activity of the intracellular lectin inactivated mixed bean whole food prepared by the method can be less than 430HU / mg, and a new efficient, environment-friendly and safe way for inactivating the intracellular lectin of the mixed beans is provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and particularly relates to a method for inactivating intracellular lectin in miscellaneous beans by high hydrostatic pressure combined with mild acid treatment. Background Art

[0002] Bean lectins have a negative impact on the digestion and absorption of nutrients through multiple mechanisms such as binding to intestinal glycoproteins, inhibiting the activity of digestive enzymes, and promoting erythrocyte agglutination. Intake of high concentrations of active lectins may damage the integrity of the intestinal mucosa, reduce the absorption efficiency of nutrients, and trigger an immune response. Such immune responses can lead to food allergies, causing the body to produce immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies, and further causing clinical symptoms such as nausea, vomiting, diarrhea, abdominal pain, and anemia caused by erythrocyte agglutination. Leguminous crops (such as kidney beans, broad beans, peas, chickpeas, and lentils) have relatively high lectin contents, and the lectin content in kidney beans can account for 1% of its components. Therefore, it is crucial to perform appropriate processing to reduce the sensitization risk of bean lectins and ensure the edible safety of bean foods.

[0003] Miscellaneous bean whole foods are rich in high-quality protein, high fiber, and low fat. The rigid structure of their cell walls acts as a physical barrier, which can effectively reduce the damage and loss of nutrients in miscellaneous bean whole foods during the processing, and retain their flavor and functionality to the greatest extent. However, traditional processing techniques have not made good use of this characteristic of the cell wall. During the processing, miscellaneous beans are usually ground into powder, seriously damaging the integrity of the cell walls of miscellaneous beans. Steaming is a commonly used method to reduce the sensitization of miscellaneous bean lectins. Research shows that boiling beans for 30 minutes can eliminate the immunoglobulin E (IgE) reactivity of some allergens (such as Len c 1 in lentils). However, some bean lectins have partial resistance to heat denaturation and need to extend the heating time to ensure their inactivation, while long-term steaming may lead to cell rupture and reduction of nutrient components.

[0004] High hydrostatic pressure treatment is an emerging non-thermal processing technology that changes the conformational structure of lectin allergens by disrupting non-covalent protein interactions (hydrogen bonds, ionic bonds, and hydrophobic bonds), reducing their sensitization potential. When the high hydrostatic pressure is less than 200 MPa, the primary and secondary structures of the lectin allergen proteins remain basically intact; while when the high hydrostatic pressure is between 200 MPa and 600 MPa, the tertiary structures and intermolecular interactions of these allergens change, resulting in the unfolding of the molecular structure, exposing hydrophobic amino acids and intramolecular sulfhydryl groups, and significantly reducing the IgE binding ability. However, due to the strong and thick cell wall structure of miscellaneous bean whole foods, high hydrostatic pressure needs to be combined with other processing technologies, such as cell disruption, to ensure lectin inactivation.

[0005] Acid treatment has also been proven to reduce lectin allergenicity to a certain extent. After purified black kidney bean lectin is incubated in a low pH (1.0 - 3.5) solution, its IgE binding ability decreases.

[0006] Currently, existing studies on legume lectins mainly use completely crushed legumes as raw materials, and there are very few studies on the inactivation of intracellular lectins in whole legume foods. Therefore, it is very meaningful to provide an environmentally friendly and efficient method for inactivating lectins that does not require high temperature or chemical additives and retains the nutritional components and flavors of whole legume foods. Summary of the Invention

[0007] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a method for inactivating intracellular lectins in legumes by combining high hydrostatic pressure with mild acid treatment. This preparation method is environmentally friendly and efficient, does not require high temperature or chemical additives, and uses the synergistic effect of high hydrostatic pressure combined with mild acid treatment. While retaining the nutritional components and flavors of whole legume foods, the intracellular lectin activity in the prepared whole legume foods can be <430 HU / mg.

[0008] The specific technical solutions for the present invention to achieve the above purposes are as follows:

[0009] A method for inactivating intracellular lectins in legumes by combining high hydrostatic pressure with mild acid treatment, comprising the following steps:

[0010] (1) Mild acid treatment

[0011] (1.1) Soak the legumes in an acidic buffer solution with a pH of 1.0 - 3.0 to obtain mildly acid-treated legumes;

[0012] Or (1.2) Prepare legume cell powder from the legumes, and then soak it in an acidic buffer solution with a pH of 1.0 - 3.0 to obtain mildly acid-treated legume cell powder;

[0013] Or (1.3) Soak the legumes in an acidic buffer solution with a pH of 1.0 - 3.0, and then prepare legume cell powder to obtain mildly acid-treated legume cell powder;

[0014] (2) High hydrostatic pressure treatment

[0015] Place the mildly acid-treated legumes or mildly acid-treated legume cell powder obtained in step (1) in a salt solution to prepare a suspension of whole legume components, and perform high hydrostatic pressure treatment at 300 MPa - 600 MPa for 5 min - 30 min, and then dry to obtain whole legume foods or legume cell powder with inactivated intracellular lectins by combining high hydrostatic pressure with mild acid treatment.

[0016] Preferably, the legumes in step (1) are one or more of large white kidney beans, kidney beans, adzuki beans, black beans, mung beans, peas, chickpeas, and broad beans.

[0017] Preferably, the acidic buffer solution in step (1) is a hydrochloric acid - potassium chloride solution or a hydrochloric acid - sodium chloride solution;

[0018] Preferably, the mass - to - volume ratio of the miscellaneous beans or miscellaneous bean cell powder to the acidic buffer solution in step (1) is 1 g: 4 - 12 mL;

[0019] Preferably, the concentration of the acidic buffer solution in step (1) is 0.01 mol / L - 0.5 mol / L, and the pH is 1.0 - 2.0;

[0020] More preferably, the concentration of the acidic buffer solution in step (1) is 0.01 mol / L, and the pH is 1.0;

[0021] Preferably, the soaking time in step (1) is 8 - 24 h; the soaking temperature is 4°C - 10°C.

[0022] Preferably, the salt solution in step (2) is a sodium chloride solution or a sodium dihydrogen phosphate - disodium hydrogen phosphate buffer solution; the mass - to - volume ratio of the mildly acid - treated miscellaneous beans or mildly acid - treated miscellaneous bean cell powder to the salt solution is 1 g: 5 mL - 15 mL.

[0023] Preferably, the concentration of the salt solution in step (2) is 0.01 mol / L - 0.5 mol / L, and the pH is 6.0 - 8.0.

[0024] More preferably, the concentration of the salt solution in step (2) is 0.01 mol / L, and the pH is 7.0.

[0025] Preferably, the method for preparing the miscellaneous bean cell powder in step (1) includes the following steps:

[0026] In step (1.2), the miscellaneous beans are soaked in a salt solution and then peeled, or in step (1.3), the mildly acid - treated miscellaneous beans are peeled and heated in a salt solution at 60°C - 80°C, crushed into a miscellaneous bean paste, and passed through a double - layer sieve to obtain the miscellaneous bean cell powder.

[0027] Preferably, the suspension of the complete components of the miscellaneous beans in step (2) is put into a double - layer polyethylene sealed bag, vacuum - sealed, and then subjected to high - hydrostatic - pressure treatment.

[0028] Preferably, the pressure of the high - hydrostatic - pressure treatment in step (2) is 300 MPa - 500 MPa, and the time is 10 min - 30 min.

[0029] Preferably, the drying in step (2) is to dry until the moisture content ≤ 9%;

[0030] The whole pulse food or pulse cell powder with intracellular lectin inactivated by high hydrostatic pressure combined with mild acid treatment prepared by the above method.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) In the process of inactivating intracellular lectin in pulses by high hydrostatic pressure combined with mild acid treatment of the present invention, intact pulse seeds are first soaked in an acidic buffer solution to obtain mildly acid-treated pulses, or pulse cell powder is prepared from pulses and then soaked in an acidic buffer solution to obtain mildly acid-treated pulse cell powder, or pulses are soaked in an acidic buffer solution and then pulse cell powder is prepared to obtain mildly acid-treated pulse cell powder; then the mildly acid-treated pulses or mildly acid-treated pulse cell powder are mixed with a phosphate buffer solution to prepare a suspension of the whole pulse components, and through high hydrostatic pressure treatment, the tertiary structure and molecular interactions of intracellular lectin in pulses are changed, exposing hydrophobic amino acids and sulfhydryl groups, reducing its immunoglobulin (IgE) binding ability, thereby more effectively reducing its allergenicity, and finally obtaining a whole pulse food with intracellular lectin inactivated, and its intracellular lectin activity can be < 430 HU / mg.

[0033] (2) The process of inactivating intracellular lectin in pulses by high hydrostatic pressure combined with mild acid treatment of the present invention is efficient, environmentally friendly and safe, without high temperature or chemical additives, can retain the nutritional components and flavor of pulses, and has convenient operation, large processing capacity and stable effect, improving the effective utilization rate and processing adaptability of pulses. Description of the Drawings

[0034] Figure 1 It is a bar chart of the lectin activity of the whole pulse food / pulse cells with intracellular lectin inactivated in Examples 1 to 5 and the samples in Comparative Examples 1 to 5.

[0035] Figure 2 It is a bar chart of the content of secondary structures (α-helix, β-sheet and random coil) of the whole pulse food / pulse cells with intracellular lectin inactivated in Examples 1 to 5 and the samples in Comparative Examples 1 to 5. Detailed Embodiments

[0036] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0037] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention.

[0038] In one aspect of the present invention, a method for inactivating intracellular lectin in miscellaneous beans by high hydrostatic pressure combined with mild acid treatment is provided, including the following steps:

[0039] (1) Soak the miscellaneous beans in 0.01 mol / L hydrochloric acid-potassium chloride buffer solution overnight, with a pH of 1.0 - 3.0, to obtain mildly acid-treated miscellaneous beans;

[0040] Or prepare the miscellaneous beans into miscellaneous bean cell powder, and then soak it in 0.01 mol / L hydrochloric acid-potassium chloride buffer solution overnight, with a pH of 1.0 - 3.0, to obtain mildly acid-treated miscellaneous bean cell powder;

[0041] Or soak the miscellaneous beans in 0.01 mol / L hydrochloric acid-potassium chloride buffer solution overnight, with a pH of 1.0 - 3.0, and then prepare it into miscellaneous bean cell powder to obtain mildly acid-treated miscellaneous bean cell powder;

[0042] The miscellaneous beans are minor legumes other than soybeans, and can be one or more of large white kidney beans, kidney beans, adzuki beans, black beans, mung beans, peas, chickpeas, and broad beans.

[0043] The material-liquid ratio of the miscellaneous beans or miscellaneous bean cell powder to the hydrochloric acid-potassium chloride buffer solution is 1 g: 4 - 12 mL; the soaking time is 8 - 24 h, and the soaking temperature is 4°C - 10°C; preferably, the material-liquid ratio of the miscellaneous beans to the hydrochloric acid-potassium chloride buffer solution is 1 g: 9 mL, the soaking time is 24 h, and the soaking temperature is 4°C.

[0044] (2) Place the mildly acid-treated miscellaneous beans or mildly acid-treated miscellaneous bean cell powder obtained in step (1) into 0.01 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.0 to prepare a suspension of the complete components of the miscellaneous beans;

[0045] In this step, the material-liquid ratio of the mildly acid-treated miscellaneous beans or mildly acid-treated miscellaneous bean cell powder to the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution is 1 g: 5 mL - 15 mL; preferably, the material-liquid ratio of the miscellaneous beans to the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution is 1 g: 10 mL.

[0046] (3) Put the suspension of the complete components of the miscellaneous beans obtained in step (2) into a double-layer polyethylene sealed bag, vacuum seal it, apply a high hydrostatic pressure of 300 MPa - 600 MPa, with a treatment time of 5 min - 30 min, and fully dry it until the moisture content ≤ 9%, to obtain the miscellaneous bean whole food or miscellaneous bean cell powder with inactivated intracellular lectin by high hydrostatic pressure combined with mild acid treatment.

[0047] The high hydrostatic pressure in this step is 300 MPa to 500 MPa, and the treatment time is 10 min to 30 min; preferably, the high hydrostatic pressure treatment time is 15 min.

[0048] The activity of lectin depends on its three-dimensional structure, especially the active site that binds to glycosyl. High hydrostatic pressure treatment destroys the non-covalent bonds (such as hydrogen bonds and hydrophobic interactions) within the lectin molecule through high pressure, causing conformational changes in the lectin, thereby weakening or destroying its binding ability to carbohydrates. When the high hydrostatic pressure ≥ 300 MPa, the tertiary structure and intermolecular interactions of the lectin change, resulting in the unfolding of the molecular structure, exposing hydrophobic amino acids and sulfhydryl groups, significantly reducing the binding ability of the whole legume food to immunoglobulin (IgE), thereby potentially reducing its allergenicity. The length of the high hydrostatic pressure treatment time has little effect on the reduction of lectin activity. To save time costs, a moderate 15 min was selected as the high hydrostatic pressure treatment time in step (3).

[0049] The performance test methods for the intracellular lectin-inactivated whole legume foods or legume cell powders in the following examples and comparative examples are as follows:

[0050] 1. Determination of lectin activity in the intracellular lectin-inactivated whole legume foods or legume cell powders by high hydrostatic pressure combined with mild acid treatment

[0051] In the embodiments of the present invention, the determination of lectin activity was slightly modified based on the method proposed by He et al. (He S, Shi J, Walid E, et al. Reverse micellar extraction of lectin from black turtle bean (Phaseolus vulgaris): Optimisation of extraction conditions by response surface methodology[J]. Food Chemistry, 2015, 166:93 - 100.). The specific implementation steps are as follows: First, weigh 2 mg of the ground sample and place it in a 5 mL centrifuge tube. Then, pipette 1 mL of 0.01 mol / L phosphate buffer solution (pH 7.2) and mix it with the sample to prepare a 2.0 mg / mL sample solution. Then, add 25 μL of 0.01 mol / L phosphate buffer solution (pH 7.2) to each well of a 96 - well microtiter plate, and then add 25 μL of a series of two - fold dilutions of the sample solution. Finally, add 25 μL of 2% rabbit red blood cell suspension. After mixing, incubate at 25 °C for 2 hours. The 0.01 mol / L phosphate buffer solution (pH 7.2) without the sample solution is used as a negative control. The lectin activity is expressed in terms of the activity units per milligram of protein (HA, HU / mg) as follows:

[0052]

[0053] Where n (n≥1) is the highest well number showing hemagglutination, c is the concentration of the sample solution, and v is the volume of the sample solution added to each well.

[0054] 2. Characterization of the thermal stability of lectin in intracellular lectin - inactivated whole pulse foods or pulse cell powders by high hydrostatic pressure combined with mild acid treatment

[0055] The thermal stability of lectin in intracellular lectin - inactivated whole pulse foods or pulse cell powders was characterized by the degree of enthalpy change measured using a differential scanning calorimeter equipped with an internal cooler. The specific implementation steps are as follows: Accurately weigh 2.0 mg of the sample and place it in a coated aluminum sample pan. Then, add 10 μL of 0.01 mol / L phosphate buffer solution (pH 7.2). At the same time, use a sealed empty aluminum pan as a reference. The scanning temperature range is 25 °C to 120 °C, and the scanning rate is 5 °C / min. The denaturation temperature (T d, the peak temperature) and the enthalpy of denaturation (ΔH) were extracted from the thermogram by Universal Analysis 2000 software (version 4.1D, TA Instruments-Waters LLC) after baseline correction. All experiments were performed in triplicate.

[0056] 3. Characterization of the secondary structure of lectin in intracellular lectin-inactivated miscellaneous bean whole foods or miscellaneous bean cell powders by high hydrostatic pressure combined with mild acid treatment

[0057] The secondary structure of lectin in intracellular lectin-inactivated miscellaneous bean whole foods or miscellaneous bean cell powders was characterized by the contents of α-helix, β-sheet and random coil determined using a far-ultraviolet (190 nm - 250 nm) circular dichroism spectrometer (CD). In the examples of the present invention, the determination of the secondary structure of lectin was carried out with slight modifications based on the method proposed by Sun et al. (Sun X, He S, Ye Y, et al. Combined effects of pH and thermal treatments on IgE-binding capacity and conformational structures of lectin from black kidney bean (Phaseolus vulgaris L.) [J]. Food Chemistry, 2020, 329: 127183). The specific implementation steps were as follows: First, 0.2 mg of the sample was weighed and placed in a 5 mL centrifuge tube, and 1 mL of 0.01 mol / L phosphate buffer solution (pH 7.2) was pipetted and mixed with the sample to prepare a sample solution with a concentration of 0.2 mg / mL, and 0.01 mol / L sodium chloride was added to ensure the transparency of the buffer solution in the far-ultraviolet region. The measurement was carried out at 25 °C, and a quartz cuvette with a path length of 1.0 mm was used to hold the sample. The acquisition conditions for the CD spectrum were: scanning speed 50 nm / min, bandwidth 1.0 nm, data interval 0.5 nm. The baseline was corrected using the spectrum of the phosphate buffer solution under the same conditions (scanning speed 50 nm / min, bandwidth 1.0 nm, path length 1.0 mm, test temperature 25 °C). Each scan was repeated 5 times to eliminate signal noise, and the results of each sample were averaged. The OMNIC software and CDNN tool were used to estimate the contents of α-helix, β-sheet and random coil in the sample. The CONTIN algorithm and SP175 reference data set were used in the analysis.

[0058] Example 1

[0059] This example provides a method for inactivating intracellular lectin in large white kidney beans by high hydrostatic pressure combined with mild acid treatment, which includes the following steps:

[0060] (1) Soak the white kidney beans in a 0.01 mol / L hydrochloric acid - potassium chloride buffer solution at a material - to - liquid ratio of 1:9 g / mL for 24 h. The soaking temperature is 4°C and the pH is 3.0 to obtain mildly acid - treated white kidney beans.

[0061] (2) Add the mildly acid - treated white kidney beans obtained in step (1) to a 0.01 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution at a material - to - liquid ratio of 1:10 g / mL, with a pH of 7.0, to prepare a white kidney bean homogenate.

[0062] (3) Put the white kidney bean homogenate obtained in step (2) into a double - layer polyethylene sealed bag, vacuum - seal it, and then apply a high hydrostatic pressure of 300 MPa for 15 min. Dry it thoroughly until the moisture content ≤ 9% to obtain the white kidney beans with inactivated intracellular lectin by high hydrostatic pressure combined with mild acid treatment in this example.

[0063] Example 2

[0064] This example provides a method for inactivating intracellular lectin in white kidney beans by high hydrostatic pressure combined with mild acid treatment, which includes the following steps:

[0065] (1) Soak the white kidney beans in a 0.01 mol / L hydrochloric acid - potassium chloride buffer solution at a material - to - liquid ratio of 1:9 g / mL for 24 h. The soaking temperature is 4°C and the pH is 1.0 to obtain mildly acid - treated white kidney beans.

[0066] (2) Add the mildly acid - treated white kidney beans obtained in step (1) to a 0.01 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution at a material - to - liquid ratio of 1:10 g / mL, with a pH of 7.0, to prepare a white kidney bean homogenate.

[0067] (3) Put the white kidney bean homogenate obtained in step (2) into a double - layer polyethylene sealed bag, vacuum - seal it, and then apply a high hydrostatic pressure of 300 MPa for 15 min. Dry it thoroughly until the moisture content ≤ 9% to obtain the white kidney beans with inactivated intracellular lectin by high hydrostatic pressure combined with mild acid treatment in this example.

[0068] Example 3

[0069] This example provides a method for inactivating intracellular lectin in white kidney beans by high hydrostatic pressure combined with mild acid treatment, which includes the following steps:

[0070] (1) Soak the white kidney beans in a 0.01 mol / L hydrochloric acid - potassium chloride buffer solution at a material - to - liquid ratio of 1:9 g / mL for 24 h. The soaking temperature is 4°C and the pH is 1.0 to obtain mildly acid - treated white kidney beans.

[0071] (2) Add the mildly acid-treated white kidney beans obtained in step (1) to a 0.01 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution at a solid-liquid ratio of 1:10 g / mL, with a pH of 7.0, to prepare a white kidney bean homogenate.

[0072] (3) Put the white kidney bean homogenate obtained in step (2) into a double-layer polyethylene sealed bag, vacuum seal it, and then apply a high hydrostatic pressure of 500 MPa for 15 min. After thoroughly drying until the moisture content ≤ 9%, the white kidney beans with inactivated intracellular lectin treated by high hydrostatic pressure in combination with mild acid in this example are obtained.

[0073] Example 4

[0074] This example provides a method for inactivating intracellular lectin in white kidney beans by combining high hydrostatic pressure with mild acid treatment, which includes the following steps:

[0075] (1) Immerse white kidney beans in a 0.01 mol / L hydrochloric acid - potassium chloride buffer solution at a solid-liquid ratio of 1:9 g / mL for 24 h at an immersion temperature of 4°C and a pH of 1.0 to obtain mildly acid-treated white kidney beans.

[0076] (2) Add the mildly acid-treated white kidney beans obtained in step (1) to a 0.01 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution at a solid-liquid ratio of 1:10 g / mL, with a pH of 7.0, to prepare a white kidney bean homogenate.

[0077] (3) Put the white kidney bean homogenate obtained in step (2) into a double-layer polyethylene sealed bag, vacuum seal it, and then apply a high hydrostatic pressure of 500 MPa for 10 min. After thoroughly drying until the moisture content ≤ 9%, the white kidney beans with inactivated intracellular lectin treated by high hydrostatic pressure in combination with mild acid in this example are obtained.

[0078] Example 5

[0079] This example provides a method for inactivating intracellular lectin in white kidney bean cells by combining high hydrostatic pressure with mild acid treatment, which includes the following steps:

[0080] (1) Immerse white kidney beans in a 0.01 mol / L hydrochloric acid - potassium chloride buffer solution at a solid-liquid ratio of 1:9 g / mL for 24 h, peel them, at an immersion temperature of 4°C and a pH of 1.0, to obtain the cotyledons of mildly acid-treated white kidney beans.

[0081] (2) Place the cotyledons of white kidney beans obtained in step (1) at a solid-liquid ratio of 1:4 g / mL in distilled water at 60°C and heat for 1 h, then crush them through a 2.2 mm sieve hole to make white kidney bean puree.

[0082] (3) Rinse the white kidney bean puree obtained in step (2) 5 times through a 100-mesh sieve under running water to obtain white kidney bean coarse pulp;

[0083] (4) Pass the white kidney bean coarse pulp obtained in step (3) through a 300-mesh sieve 5 times, collect the residue on the sieve to obtain white kidney bean cell powder;

[0084] (5) Add the white kidney bean cell powder obtained in step (4) to a 0.01 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution according to a solid-liquid ratio of 1:10 g / mL, with a pH of 7.0, to prepare a white kidney bean homogenate;

[0085] (6) Put the white kidney bean homogenate obtained in step (5) into a double-layer polyethylene sealed bag, vacuum seal it, and then apply a high hydrostatic pressure of 500 MPa for 15 min, and fully dry it until the moisture content ≤ 9%, thus obtaining the white kidney bean cell powder with intracellular lectin inactivated by high hydrostatic pressure combined with mild acid treatment in this example.

[0086] Comparative Example 1

[0087] This comparative example provides white kidney beans without high hydrostatic pressure treatment compared to Example 3, and its preparation method includes the following steps:

[0088] (1) Immerse white kidney beans in a 0.01 mol / L hydrochloric acid - potassium chloride buffer solution according to a solid-liquid ratio of 1:9 g / mL, soak for 24 h, with a soaking temperature of 4°C and a pH of 1.0, to obtain mildly acid-treated white kidney beans;

[0089] (2) Add the mildly acid-treated white kidney beans obtained in step (1) to a 0.01 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution according to a solid-liquid ratio of 1:10 g / mL, with a pH of 7.0, to prepare a white kidney bean homogenate;

[0090] (3) Fully dry the white kidney bean homogenate obtained in step (2) until the moisture content ≤ 9%, thus obtaining the white kidney beans with intracellular lectin inactivated in this comparative example.

[0091] Comparative Example 2

[0092] This comparative example provides white kidney beans without mild acid treatment compared to Example 3, and its preparation method includes the following steps:

[0093] (1) Immerse white kidney beans in a 0.01 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution according to a solid-liquid ratio of 1:9 g / mL, soak for 24 h, with a soaking temperature of 4°C and a pH of 7.0;

[0094] (2) Add the white kidney beans obtained in step (1) to a 0.01 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution at a solid - to - liquid ratio of 1:10 g / mL, with a pH of 7.0, to prepare a white kidney bean homogenate.

[0095] (3) Put the white kidney bean homogenate obtained in step (2) into a double - layer polyethylene sealed bag, vacuum - seal it, and then apply a high hydrostatic pressure of 500 MPa for 15 min. Dry it thoroughly until the moisture content ≤ 9%, thus obtaining the white kidney beans with inactivated intracellular lectin in this comparative example.

[0096] Comparative Example 3

[0097] This comparative example provides a sample using white kidney bean powder with damaged cell structure and subjected to boiling treatment. Its preparation method includes the following steps:

[0098] (1) Soak the white kidney beans at a solid - to - liquid ratio of 1:9 g / mL in distilled water for 24 h at an immersion temperature of 4°C.

[0099] (2) Crush the white kidney beans obtained in step (1) through a 1.3 - mm sieve hole to make white kidney bean powder.

[0100] (3) Place the white kidney bean powder obtained in step (2) at a solid - to - liquid ratio of 1:4 g / mL in distilled water at 100°C and heat for 1 h. Dry it thoroughly until the moisture content ≤ 9%, thus obtaining the white kidney bean powder with inactivated intracellular lectin in this comparative example.

[0101] Comparative Example 4

[0102] This comparative example provides white kidney bean cells without mild acid treatment compared to Example 5. Its preparation method includes the following steps:

[0103] (1) Soak the white kidney beans at a solid - to - liquid ratio of 1:9 g / mL in a 0.01 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution for 24 h, peel them, at an immersion temperature of 4°C and a pH of 7.0.

[0104] (2) Place the cotyledons of the white kidney beans obtained in step (1) at a solid - to - liquid ratio of 1:4 g / mL in distilled water at 60°C and heat for 1 h, then crush them through a 2.2 - mm sieve hole to make white kidney bean puree.

[0105] (3) Rinse the white kidney bean puree obtained in step (2) 5 times through a 100 - mesh sieve under running water to obtain white kidney bean thick pulp.

[0106] (4) Pass the white kidney bean thick pulp obtained in step (3) through a 300 - mesh sieve 5 times, collect the residue on the sieve, and obtain white kidney bean cell powder.

[0107] (5) Add the white kidney bean cell powder obtained in step (4) to a 0.01 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution at a material - liquid ratio of 1:10 g / mL, with a pH of 7.0, to prepare a white kidney bean homogenate;

[0108] (6) Put the white kidney bean homogenate obtained in step (5) into a double - layer polyethylene sealed bag, vacuum - seal it, and then apply a high hydrostatic pressure of 500 MPa for 15 min. Dry it thoroughly until the moisture content ≤ 9%, and thus obtain the white kidney bean cell powder with inactivated intracellular lectin of this comparative example.

[0109] Comparative Example 5

[0110] This comparative example provides a method for inactivating intracellular lectin in white kidney beans by combining high hydrostatic pressure and mild acid treatment, which includes the following steps:

[0111] (1) Immerse white kidney beans in a 0.01 mol / L hydrochloric acid - potassium chloride buffer solution at a material - liquid ratio of 1:9 g / mL for 24 h, with an immersion temperature of 4°C and a pH of 5.0, to obtain white kidney beans treated with mild acid;

[0112] (2) Add the white kidney beans treated with mild acid obtained in step (1) to a 0.01 mol / L disodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution at a material - liquid ratio of 1:10 g / mL, with a pH of 7.0, to prepare a white kidney bean homogenate;

[0113] (3) Put the white kidney bean homogenate obtained in step (2) into a double - layer polyethylene sealed bag, vacuum - seal it, and then apply a high hydrostatic pressure of 100 MPa for 15 min. Dry it thoroughly until the moisture content ≤ 9%, and thus obtain white kidney beans with inactivated intracellular lectin by combining high hydrostatic pressure and mild acid treatment of this comparative example.

[0114] Product testing and characterization:

[0115] (1) Characterization of lectin activity and lectin content in the whole food of miscellaneous beans or miscellaneous bean cell powder with inactivated intracellular lectin

[0116] Table 1 and Figure 1Analysis of the activity of lectins in the miscellaneous bean whole foods / miscellaneous bean cells prepared in Examples 1-5 and the miscellaneous bean whole foods / miscellaneous bean powders / miscellaneous bean cells prepared in Comparative Examples 1-5. Previous studies have shown that heating at 100 °C for 30 min can completely inactivate the allergenicity of miscellaneous bean lectins, indicating that the lectin activity value of the sample in Comparative Example 3 has reached a safe edible level. The lectin activities of the samples in Examples 2-5 are all lower than that in Comparative Example 3. Among them, Example 3 has the lowest lectin activity in the miscellaneous bean whole food samples, indicating that mild acid treatment (pH 1.0) combined with high hydrostatic pressure treatment (500 MPa) can more effectively reduce the lectin activity of miscellaneous bean whole foods than heat treatment (100 °C), possibly by disrupting the interaction between lectins and carbohydrates and changing the lectin structure. Comparing Comparative Example 4, Example 3 and Example 5, it shows that the 60 °C water bath in the cell extraction process of miscellaneous bean whole foods also reduces the allergenicity of lectins to a certain extent. Comparing Example 2 and Example 3, it shows that as the high hydrostatic pressure increases, the lectin activity value decreases significantly, possibly because the high-pressure treatment (500 MPa) disrupts the three-dimensional structure of the lectin, and this natural structure is partially related to the blood coagulation activity of the lectin molecule. Comparing Example 1 and Example 2, it shows that as the pH of the acidic buffer solution for soaking decreases, the lectin activity value decreases, possibly because the low pH (pH 1.0) treatment will cause the exposure of hydrophobic residues, thus effectively masking or destroying the lectin epitope, thereby reducing the recognition ability of antibodies. Comparing Example 3 and Example 4, it shows that the action time of high hydrostatic pressure has little effect on the lectin activity value.

[0117] As can be seen from Table 1, only acid treatment (pH 1.0) in Comparative Example 1 can inactivate 295.2 HU / mg of lectin, salt soaking in step (1) of Comparative Example 2 can inactivate 75.6 HU / mg of lectin, and high hydrostatic pressure treatment in steps (2) and (3) can inactivate 269 HU / mg of lectin, that is, only high hydrostatic pressure treatment can inactivate 269 HU / mg of lectin, while acid treatment (pH 1.0) + high hydrostatic pressure treatment in Example 3 can inactivate 606.7 HU / mg of lectin, which is significantly higher than the sum of the inactivation of lectin by only acid treatment (pH 1.0) and only high hydrostatic pressure treatment (564.2 HU / mg). Moreover, based on the marginal effect, the value of inactivating lectin after a simple combination of the two will be lower than 564.2 HU / mg, indicating that acid treatment (pH 1.0) + high hydrostatic pressure treatment in Example 3 for inactivating lectin can have a synergistic effect. The possible reason is that acid treatment changes the cell wall permeability, and at the same time, under the condition of pH 1.0, the carboxyl groups of lectins are protonated, resulting in the exposure of the internal hydrophobic regions, which not only changes the tertiary structure of the lectins but also increases their surface hydrophobicity, making the antigen epitopes originally buried inside more easily destroyed and degraded by high hydrostatic pressure, and thus more effectively inactivating the lectins.

[0118] Table 1

[0119]

[0120] (2) Characterization of the thermal stability of lectins in intracellular lectin-inactivated whole pulses or pulse cell powders

[0121] Table 2 shows the table of thermodynamic properties of lectins determined by differential scanning calorimetry in the whole pulses / pulse cells prepared in Examples 1 to 5 and the whole pulses / pulse powders / pulse cells prepared in Comparative Examples 1 to 5. Thermal stability determines the sensitization potential of lectins. The denaturation peak temperature (T d ) can be used to monitor the thermal stability of lectins in samples, while the enthalpy change (ΔH) is related to the proportion of undenatured proteins or the degree of ordered protein structure. Compared with the samples of Comparative Example 3, the samples of Examples 2 to 5 showed lower denaturation peak temperatures (T d ) and lower enthalpy changes (ΔH). Among them, Example 3 had the lowest denaturation peak temperature (T d ) and enthalpy change (ΔH) in the samples of whole pulses, indicating that mild acid treatment (pH 1.0) combined with high hydrostatic pressure treatment (500 MPa) can promote the change of the secondary structure of lectins and the exposure of hydrophobic groups more effectively than heat treatment (100 °C), reducing their thermal stability. The possible reason is that the thermal stability of lectins decreases after treatment at pH 1.0, and the structure is more likely to unfold under high hydrostatic pressure treatment, resulting in the exposure of more cleavage sites, accelerating the digestion and degradation of antigenic epitopes, and thus more effectively inactivating lectins. Comparing Example 2 with Example 3 and Example 1, it shows that as the high hydrostatic pressure increases and the pH of the acidic buffer solution for soaking decreases, the T d and ΔH of lectins in whole pulses decrease. Previous studies have pointed out that the decrease in ΔH is related to the denaturation of lectins, reflecting the unfolding of the lectin structure in whole pulses caused by the combination of high hydrostatic pressure and mild acid treatment.

[0122] Table 2

[0123]

[0124]

[0125] (3) Characterization of the secondary structure of lectins in intracellular lectin-inactivated whole pulses or pulse cell powders

[0126] Figure 2The α-helix and β-sheet contents of lectins in the whole pulse foods / pulse cells prepared in Examples 1-5 and the whole pulse foods / pulse powders / pulse cells prepared in Comparative Examples 1-5. The results show that compared with the samples of Comparative Example 3, the samples of Examples 2-5 exhibit lower α-helix content and higher β-sheet content, indicating that mild acid treatment (pH 1.0) in combination with high hydrostatic pressure treatment (500 MPa) can increase the looseness of the lectin structure more than heat treatment (100 °C), thereby interfering with the spatial arrangement of its antigenic epitopes and reducing the possibility of allergic reactions. The possible reason is that comparing Example 2 with Examples 3 and 1 shows that as the high hydrostatic pressure increases and the pH of the acidic buffer solution for soaking decreases, the α-helix content of the lectin in the whole pulse foods decreases and the β-sheet content increases, indicating that high hydrostatic pressure combined with mild acid treatment may change the lectin conformation, and by increasing β-sheet formation and reducing the α-helix ordered region, the lectin epitopes are more easily destroyed and degraded, thus reducing allergic reactions.

[0127] Based on the above, it can be shown that compared with the existing preparation methods, this preparation method is efficient and environmentally friendly, without the need for high temperature or chemical additives. By using the synergistic effect of high hydrostatic pressure combined with mild acid treatment, while retaining the nutritional components and flavors of the whole pulse foods, the intracellular lectin activity in the prepared whole pulse foods can be <430 HU / mg, and it is convenient to operate, has a large processing capacity, stable effects, and improves the effective utilization rate and processing adaptability of the whole pulse foods.

[0128] The above examples are only used to specifically illustrate the technical solutions of the present invention and should not be construed as limiting the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the claims.

Claims

1. A method for inactivating intracellular lectins of miscellaneous beans by high hydrostatic pressure combined with mild acid treatment, characterized in that: The steps include: (1) Mild acid treatment (1.1) Soaking the beans in an acidic buffer solution with a pH of 1.0 to 3.0 to obtain mildly acid-treated beans; or (1.2) preparing miscellaneous beans into miscellaneous bean cell powder, and then soaking the miscellaneous beans in an acidic buffer solution with a pH of 1.0 to 3.0 to obtain a mildly acid-treated miscellaneous bean cell powder; or (1.3) soaking the beans in an acidic buffer solution with a pH of 1.0 to 3.0, and then preparing the beans into cell powder to obtain mildly acid-treated beans cell powder; (2) High hydrostatic pressure treatment The mildly acid-treated miscellaneous beans or mildly acid-treated miscellaneous bean cell powder obtained in step (1) is placed in a salt solution to prepare a miscellaneous bean complete component suspension, and is subjected to a high hydrostatic pressure treatment of 300 MPa to 600 MPa for 5 min to 30 min, and then dried to obtain a miscellaneous bean whole food or miscellaneous bean cell powder in which intracellular lectins are inactivated by high hydrostatic pressure and mild acid treatment.

2. The method for inactivating intracellular lectins of miscellaneous beans by high hydrostatic pressure and mild acid treatment according to claim 1, characterized in that: The miscellaneous beans in step (1) are one or more of large white kidney beans, kidney beans, adzuki beans, black beans, mung beans, peas, chickpeas, and broad beans.

3. The method for inactivating intracellular lectins of miscellaneous beans by high hydrostatic pressure and mild acid treatment according to claim 1, characterized in that: The acidic buffer solution in step (1) is a hydrochloric acid-potassium chloride solution or a hydrochloric acid-sodium chloride solution; the mass volume ratio of the miscellaneous beans or miscellaneous bean cell powder to the acidic buffer solution is 1g:4-12mL.

4. The method for inactivating intracellular lectins of miscellaneous beans by high hydrostatic pressure and mild acid treatment according to claim 1, characterized in that: The concentration of the acidic buffer solution in step (1) is 0.01 mol / L to 0.5 mol / L, and the pH is 1.0 to 2.0; The soaking time in step (1) is 8 to 24 hours; the soaking temperature is 4° C. to 10° C.

5. The method for inactivating intracellular lectins of miscellaneous beans by high hydrostatic pressure combined with mild acid treatment according to claim 1, characterized in that: The saline solution in step (2) is a sodium chloride solution or a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution; the mass volume ratio of the mildly acid-treated miscellaneous beans or mildly acid-treated miscellaneous bean cell powder to the saline solution is 1 g: 5 mL to 15 mL; The concentration of the salt solution in step (2) is 0.01 mol / L to 0.5 mol / L, and the pH is 6.0 to 8.

0.

6. The method of inactivating intracellular lectins of miscellaneous beans by high hydrostatic pressure and mild acid treatment according to claim 1, characterized in that: The method for preparing miscellaneous bean cell powder in step (1) comprises the following steps: In step (1.2), the miscellaneous beans are soaked in a salt solution and then peeled, or in step (1.3), the miscellaneous beans treated with mild acid are peeled, heated in a salt solution at 60° C. to 80° C., crushed to form miscellaneous bean paste, and passed through a double-layer sieve to obtain the miscellaneous bean cell powder.

7. The method for inactivating intracellular lectins of miscellaneous beans by high hydrostatic pressure combined with mild acid treatment according to claim 1, characterized in that: The mixed bean complete component suspension in step (2) is placed in a double-layer polyethylene sealed bag, vacuum-sealed, and then subjected to high hydrostatic pressure treatment.

8. The method of inactivating intracellular lectins of miscellaneous beans by high hydrostatic pressure combined with mild acid treatment according to claim 1, characterized in that: The high hydrostatic pressure treatment in step (2) is performed at a pressure of 300 MPa to 500 MPa and for a time of 10 min to 30 min.

9. The method for inactivating intracellular lectins of miscellaneous beans by high hydrostatic pressure combined with mild acid treatment according to claim 1, characterized in that: The drying in step (2) is to dry the mixture to a moisture content of ≤ 9%.

10. Whole bean food or bean cell powder prepared by the method according to any one of claims 1 to 9, wherein the intracellular lectin is inactivated by high hydrostatic pressure and mild acid treatment.

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