Camellia oleifera meal extract rich in antimicrobial peptides and tea saponins, and preparation method and application thereof

Abstract

The invention discloses a camellia oleifera meal extract rich in antimicrobial peptides and tea saponins and a preparation method thereof. The method adopts a continuous preparation method, on the one hand, hydrolyzing camellia oleifera meal protein to obtain antimicrobial peptides, and on the other hand, extracting tea saponins from the camellia oleifera meal, and mixing the camellia oleifera meal antimicrobial peptides and tea saponins preparations in an appropriate ratio to obtain the camellia oleifera meal extract rich in antimicrobial peptides and tea saponins. The camellia oleifera meal extract is rich in antimicrobial peptides and tea saponins, integrates the antibacterial advantages of the camellia oleifera meal antimicrobial peptides and tea saponins, has a broad-spectrum antibacterial activity against foodborne spoilage bacteria, and can effectively inhibit foodborne spoilage bacteria. The camellia oleifera meal extract rich in antimicrobial peptides and tea saponins can be used to prepare products with the function of inhibiting foodborne spoilage bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of natural preservatives, and particularly relates to a tea seed meal extract rich in antimicrobial peptides and tea saponins, a preparation method thereof, and an application of the extract in preparing a product having the function of inhibiting foodborne spoilage bacteria. Background Art

[0002] Microbial contamination is the main cause of food spoilage, which not only causes huge economic losses, but also the toxins produced by foodborne spoilage bacteria pose a threat to human health, and is the main cause of food safety accidents. In order to prevent microbial contamination, preservatives are usually added during food processing. At present, my country's food industry mainly relies on chemical food preservatives, such as nitrites, sorbic acid, benzoic acid and its salts, to solve the problem of food spoilage. However, chemical food preservatives have potential hazards, especially excessive use of chemical preservatives has safety issues such as food poisoning and carcinogenicity. Therefore, it is of great significance to develop natural antibacterial active substances to replace chemical preservatives.

[0003] Camellia oleifera is a perennial woody oil crop of the Theaceae family. It is one of the main economic forest tree species in my country. It is known as the world's four major woody edible oil crops along with olive, oil palm and coconut. Camellia oleifera seeds can be extracted or pressed to obtain tea oil, which not only has excellent color, fragrance and taste, but also has the effect of preventing and treating cardiovascular diseases such as hypertension, coronary heart disease and atherosclerosis. Camellia oleifera meal is a byproduct left after the edible oil is extracted from camellia oleifera seeds. In recent years, with the increase in the demand and output of tea oil, the byproduct camellia oleifera meal has also increased year by year.

[0004] Tea seed meal contains tea saponin, protein, polysaccharide and other functional ingredients. Tea saponin, as a natural and excellent surfactant, not only has strong foaming and emulsifying effects, but also has physiological activities such as antibacterial, anti-inflammatory, analgesic and anti-cancer, among which the antibacterial effect is the most significant; at the same time, the protein in tea seed meal can be hydrolyzed to prepare active peptides with antibacterial activity, namely tea seed meal antibacterial peptides. Tea saponin and tea seed meal antibacterial peptides are both good natural antibacterial agents, but their inhibitory effects on bacteria, molds and yeasts are different, and their broad-spectrum antibacterial effects are not strong. Summary of the invention

[0005] One of the purposes of the present invention is to provide a camellia oleifera meal extract rich in antimicrobial peptides and tea saponins. The camellia oleifera meal extract is rich in antimicrobial peptides and tea saponins, integrates the antimicrobial advantages of camellia oleifera meal and tea saponin antimicrobial peptides, and has a broad-spectrum antimicrobial activity against foodborne spoilage bacteria.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned tea oil cake extract rich in antimicrobial peptides and tea saponins. The method adopts a continuous preparation method. On the one hand, the tea oil cake protein is hydrolyzed to obtain antimicrobial peptides, and on the other hand, tea saponins are extracted from the tea oil cake.

[0007] The last object of the present invention is to provide the use of the above-mentioned tea seed meal extract rich in antimicrobial peptides and tea saponins in the preparation of products having the effect of inhibiting foodborne spoilage bacteria.

[0008] The first object of the present invention can be achieved by the following technical scheme: A method for preparing a tea seed meal extract rich in antimicrobial peptides and tea saponins, comprising the following steps:

[0009] (1) Pretreatment of tea-seed meal: pretreatment of tea-seed meal by hot pressing, steam explosion and mixed enzymatic hydrolysis;

[0010] (1.1) Hot pressing pretreatment: The camellia dregs, a byproduct of camellia oil production, are used as raw materials. The camellia dregs are crushed and placed in a high pressure sterilization device for hot pressing at a temperature of 115-121°C for 15-20 minutes.

[0011] (1.2) Steam explosion pretreatment: The tea-seed meal powder that has been pretreated by hot pressing is placed in a steam explosion device and subjected to explosion treatment using saturated water vapor. The steam explosion pressure is adjusted to 1.2-1.4 MPa and the treatment time is 40-50 seconds. After the treatment time is reached, the material is instantly decompressed to complete the explosion and obtain tea-seed meal powder with a loose structure.

[0012] (1.3) Mixed enzymatic hydrolysis pretreatment: Place loosely structured camellia oleifera meal powder in a container, add water, add xylanase, hemicellulase and cellulase under the condition that the original pH value of the water-camellia oleifera meal system is 6.0-6.5, and then place it in a 40-50℃ water bath for hydrolysis for 120-150min to obtain an enzymatic hydrolyzate;

[0013] (2) After the pretreatment of the camellia oleifera meal is completed, the pH value of the enzymatic hydrolyzate is adjusted to reach the isoelectric point of the camellia oleifera meal protein to maximize the amount of protein precipitation. After completion, centrifugation is performed to collect the supernatant and protein precipitate, respectively. The supernatant is used to extract tea saponin, and the protein precipitate is used to prepare camellia oleifera meal antimicrobial peptides.

[0014] (3) Take the protein precipitate obtained in step (2), add water and stir evenly, adjust the pH value to 8.0-8.5, add a composite protease containing alkaline protease and trypsin, and then place it in a water bath at 40-50°C for hydrolysis for 150-210 minutes. After the composite protease hydrolysis is completed, centrifuge and collect the supernatant. After the supernatant is filtered through a membrane separation device, collect the permeate after the membrane separation is completed, and obtain the antimicrobial peptide preparation solution, which is dried under low temperature vacuum to obtain the antimicrobial peptide preparation;

[0015] (4) Take the supernatant obtained in step (2) and add it to a chromatography column filled with AB-8 macroporous resin. After the supernatant is completely adsorbed in the chromatography column, first elute the soluble sugar adsorbed on the macroporous resin with distilled water until the eluate is neutral and no sugar is detected by anthrone-sulfuric acid method. Then elute the polyphenols adsorbed on the macroporous resin with a low concentration of ethanol aqueous solution until no polyphenol is detected by Folin phenol method. Then elute the tea saponin adsorbed on the macroporous resin with a high concentration of ethanol aqueous solution. Collect all the tea saponin eluate and distill off ethanol to obtain a tea saponin water extract concentrate. The tea saponin water extract concentrate is subjected to low-temperature vacuum drying to obtain a tea saponin preparation.

[0016] (5) The antimicrobial peptide preparation obtained in step (3) and the tea saponin preparation obtained in step (4) are respectively compounded and mixed in a weight ratio of 30-70:20-60 of the active ingredient antimicrobial peptide to tea saponin, thereby obtaining a tea seed meal extract rich in antimicrobial peptides and tea saponins that inhibits foodborne spoilage bacteria.

[0017] The present invention adopts a continuous preparation method. On the one hand, tea oil meal protein is hydrolyzed to obtain antimicrobial peptides, and on the other hand, tea saponin is extracted from the tea oil meal. The tea oil meal antimicrobial peptides and tea saponin preparations are combined in an appropriate ratio to prepare a tea oil meal extract rich in antimicrobial peptides and tea saponins, which has a broad-spectrum antibacterial activity and can effectively inhibit foodborne spoilage bacteria. The tea oil meal extract can be used to develop green and natural food preservatives and provide a new way for the high-value utilization of tea oil meal.

[0018] In the preparation method of the above-mentioned tea seed meal extract rich in antimicrobial peptides and tea saponins:

[0019] Optionally, the amount of water added in step (1.3) is 8 to 12 times the total mass of the camellia oleifera meal powder.

[0020] According to the specific situation that the lignin content in the tea oil cake is higher than hemicellulose, and the hemicellulose content is higher than cellulose, optionally, the dosage of xylanase, hemicellulase and cellulase in step (1.3) is 15.0~20.0 U / g tea oil cake, 9.0~12.0 U / g tea oil cake and 5.0~8.0 U / g tea oil cake, respectively.

[0021] Optionally, in step (2), the pH value of the enzymatic hydrolyzate is adjusted to 4.6 using a 0.5-1.0 mol / L hydrochloric acid solution to achieve an isoelectric point pI of 4.6 for the camellia oleifera meal protein.

[0022] Optionally, the amount of water added in step (3) is 8 to 10 times the total mass of the protein precipitate.

[0023] Optionally, in step (3), the pH value is adjusted to 8.0-8.5 using 1.0-1.5 mol / L sodium hydroxide solution.

[0024] Optionally, the amount of the composite protease added in step (3) is 5500 to 6000 U / g protein precipitate with a total enzyme activity.

[0025] Optionally, the enzyme activity ratio of alkaline protease to trypsin in the composite protease in step (3) is 3:2.

[0026] Optionally, in step (3), the membrane filtration parameters in step (3) are: pressure 1.5-2 kg / cm 2 The permeability is 30-35 mL / min and the permeability molecular weight is 3000 Da.

[0027] Optionally, the antimicrobial peptide preparation obtained in step (3) is determined by trichloroacetic acid precipitation method, wherein the antimicrobial peptide content is more than 90%, and the antimicrobial peptide preparation is mainly short peptides with a molecular weight of less than 1000 Da, mainly small peptides composed of 3 to 8 amino acids, and the content of small peptides is more than 70%.

[0028] In step (4) of the present invention, the supernatant obtained in the above step (2) is used to extract tea saponin. Since the protein in the tea dregs is precipitated, the lignin, hemicellulose and cellulose are enzymatically hydrolyzed into soluble oligosaccharides and small molecular sugars, the main components of this supernatant are tea saponin, soluble sugars and polyphenols. Therefore, by removing the soluble sugars and polyphenols therefrom, the purpose of removing impurities can be achieved, and a preparation with tea saponin as the main component can be obtained.

[0029] Optionally, in step (4), the supernatant is added at a ratio of 25-30 mL volume to 1 g mass of AB-8 macroporous resin wet resin, and the loading flow rate is 3.0-4.0 mL / min.

[0030] In view of the fact that the polarity of the three main components in the supernatant sample is ranked as follows: soluble sugar has the highest polarity, polyphenol has the second highest polarity, and tea saponin has the lowest polarity, water, low concentration ethanol, and high concentration ethanol can be used for elution respectively. Therefore, distilled water was first used to elute the soluble sugar adsorbed on the macroporous resin until the eluent was neutral and no sugar was detected by anthrone-sulfuric acid method; then 20% (volume percentage) ethanol solution was used to elute the polyphenol adsorbed on the macroporous resin until no polyphenol was detected by Folin phenol method; then 75-85% (volume percentage) ethanol solution was used to elute the tea saponin adsorbed on the macroporous resin, the volume ratio of the eluent 75-85% ethanol solution to the supernatant sample was 1.5-2:1, the flow rate of the eluent was 2.0-3.0 mL / min, and all the tea saponin eluent was collected and ethanol was distilled out by rotary evaporator to obtain tea saponin water extract concentrate.

[0031] Optionally, the volume percentage of the low concentration ethanol aqueous solution in step (4) is 20%.

[0032] Optionally, the volume percentage of the high-concentration ethanol aqueous solution in step (4) is 75-85%, and the volume ratio of the high-concentration ethanol aqueous solution to the supernatant is 1.5-2:1.

[0033] Optionally, the flow rate of the eluent in step (4) is 2.0-3.0 mL / min.

[0034] Optionally, the tea saponin preparation in step (4) has a tea saponin content of more than 90% as determined by spectrophotometry.

[0035] Optionally, the temperature of low-temperature vacuum drying in step (3) to step (4) is 4 to 10°C.

[0036] Furthermore, in step (5), the antimicrobial peptide and tea saponin are compounded and mixed in a mass ratio of 2:1 to obtain a tea seed meal extract rich in antimicrobial peptides and tea saponin that effectively inhibits foodborne spoilage bacteria.

[0037] The present invention has the following beneficial effects:

[0038] (1) The present invention adopts a continuous preparation method to precipitate proteins from tea oil meal to prepare antimicrobial peptides, and uses the solution after the protein is precipitated to extract tea saponin, thereby not only obtaining antimicrobial peptides, but also extracting tea saponin from the tea oil meal. Compared with the traditional single comprehensive utilization technology of only using tea oil meal to prepare antimicrobial peptides or extract tea saponin, the present invention makes more comprehensive use of tea oil meal and has higher resource utilization efficiency;

[0039] (2) Both tea seed cake antimicrobial peptides and tea saponins are good natural antibacterial agents, but their inhibitory effects on different food spoilage bacteria such as bacteria, molds, and yeasts vary in strength. Antimicrobial peptides interact with the cell walls and cell membranes of food spoilage bacteria, increase the permeability of the cell membranes, and allow substances in the cells to flow out, thereby achieving the effect of killing food spoilage bacteria; the surface activity of tea saponins changes the permeability of the cell membranes of food spoilage bacteria, thereby destroying the intracellular structure and enzyme system. At the same time, the organic acid produced by the hydrolysis of tea saponins acidifies the intracellular fluid, destroys the normal function and metabolism of each organelle, and achieves the effect of killing food spoilage bacteria. The present invention combines tea seed cake antimicrobial peptides and tea saponins in an appropriate ratio, utilizes the antibacterial synergistic effect of antimicrobial peptides and tea saponins, and prepares a tea seed cake extract rich in antimicrobial peptides and tea saponins, which has a broad-spectrum antibacterial activity and can effectively inhibit food-borne spoilage bacteria. The tea seed cake extract can be used to develop green and natural food preservatives;

[0040] (3) Before preparing antimicrobial peptides and tea saponins, the present invention uses hot pressing, steam explosion and mixed enzyme pretreatment on the tea seed meal. The tea seed meal protein undergoes denaturation during the hot pressing process. After denaturation, the spatial structure is destroyed, so that the amino acids are fully exposed, which can improve the efficiency of enzymatic hydrolysis to prepare antimicrobial peptides; at the same time, hot pressing, steam explosion and mixed enzyme pretreatment also effectively destroy the spatial structure and molecular structure of lignin, hemicellulose and cellulose in the tea seed meal, and promote the release of protein and tea saponins encapsulated therein, thereby improving the preparation efficiency of antimicrobial peptides and tea saponins, and further improving the resource utilization efficiency of tea seed meal. DETAILED DESCRIPTION

[0041] The following examples are only used to illustrate the present invention and do not limit the protection scope of the present invention. A person skilled in the art can achieve the purpose of the present invention according to the above disclosed scope.

[0042] Example 1

[0043] A tea seed meal extract rich in antimicrobial peptides and tea saponins that effectively inhibits food-borne spoilage bacteria is prepared according to the following steps:

[0044] (1) Pretreatment of tea seed meal:

[0045] The camellia meal, a byproduct of camellia oil production, was used as raw material. The camellia meal was crushed and placed in a high pressure sterilizer for hot pressing at 115°C for 20 minutes.

[0046] The tea-seed meal powder obtained by the hot pressing treatment is placed in a steam explosion machine, and subjected to explosion treatment using saturated water vapor. The steam explosion pressure is adjusted to 1.2 MPa, and the processing time of the pressure is maintained for 50 seconds. After the processing time is reached, the material is instantly decompressed to complete the explosion, and the tea-seed meal powder with loose structure after steam explosion treatment is obtained;

[0047] Place the loosely structured camellia oil meal powder in a container, add 8 times its mass of water (pure water is preferred), add xylanase, hemicellulase and cellulase under the condition that the original pH value of the water-camellia oil meal system is 6.1, and according to the specific situation that the lignin content in the camellia oil meal is higher than hemicellulose, and the hemicellulose content is higher than cellulose, the dosage of the above three enzymes is 16.0U / g camellia oil meal, 10.0U / g camellia oil meal and 5.5U / g camellia oil meal respectively, and then place it in a 40℃ water bath for hydrolysis for 150min.

[0048] (2) After the pretreatment of camellia oleifera meal, the pH value was adjusted to 4.6 with 0.5 mol / L hydrochloric acid solution to reach the isoelectric point pI 4.6 of camellia oleifera meal protein and maximize the amount of protein precipitation. After completion, the meal was separated in a centrifuge to collect the supernatant and protein precipitate. The supernatant was used to extract tea saponin, and the protein precipitate was used to prepare camellia oleifera meal antimicrobial peptides.

[0049] (3) Take the protein precipitate obtained in step (2) above, add 8 times the mass of water (preferably purified water) and stir evenly, adjust the pH value to 8.1 with 1.0 mol / L sodium hydroxide solution, add a composite protease of alkaline protease and trypsin, wherein the ratio of alkaline protease to trypsin is 3:2, and the added amount is 5500 U / g protein precipitate with total enzyme activity, and then place it in a 40°C water bath for hydrolysis for 210 min.

[0050] After the composite protease hydrolysis is completed, the mixture is placed in a centrifuge for separation, and the supernatant is collected. The collected supernatant is filtered through a membrane separation device. The membrane filtration parameters are: pressure 1.5 kg / cm 2 , the permeability is 30mL / min, and the permeability molecular weight is 3000Da.

[0051] After membrane separation is completed, the permeate is collected to obtain the antimicrobial peptide preparation solution. At this time, the tea seed meal antimicrobial peptide preparation solution is vacuum dried at low temperature (4°C) to obtain the antimicrobial peptide preparation.

[0052] The obtained antimicrobial peptide preparation was determined by trichloroacetic acid precipitation method, and the antimicrobial peptide content was 93.63±1.47%. The antimicrobial peptide was mainly short peptides with a molecular weight of less than 1000 Da, mainly composed of 3-8 amino acids, with a content of 71.45±1.34%.

[0053] (4) Take the supernatant obtained in the above step (2) and add it to the chromatography column filled with AB-8 macroporous resin (wet resin). The supernatant is added in a ratio of 25 mL volume (mL) to AB-8 macroporous resin wet resin weight (g) of 1 g. The sample flow rate is 3 mL / min. After the sample solution is adsorbed in the chromatography column, the soluble sugar adsorbed on the macroporous resin is first eluted with distilled water until the eluent is neutral and no sugar is detected by anthrone-sulfuric acid method. Then, the polyphenols adsorbed on the macroporous resin are eluted with 20% (volume percentage, the same below) ethanol solution until no polyphenols are detected by Folin phenol method. Then, the tea saponin adsorbed on the macroporous resin is eluted with 75% (volume percentage, the same below). The volume ratio of the eluent 75% ethanol solution to the supernatant sample solution is 1.6:1. The flow rate of the eluent is 2.0 mL / min, collect all the tea saponin eluate and distill out the ethanol using a rotary evaporator to obtain the tea saponin water extract concentrate.

[0054] At this time, the tea saponin water extract concentrate is vacuum dried at low temperature (4°C) to obtain a tea saponin preparation, and the tea saponin content is determined by spectrophotometry to be 95.48±1.39%.

[0055] (5) The antimicrobial peptide obtained in step (3) and the tea saponin preparation obtained in step (4) are respectively compounded and mixed evenly in a mass ratio of the effective ingredient antimicrobial peptide to tea saponin of 2:1, thereby obtaining a tea seed meal extract rich in antimicrobial peptides and tea saponins that is effective in inhibiting foodborne spoilage bacteria.

[0056] Example 2

[0057] A tea seed meal extract rich in antimicrobial peptides and tea saponins that effectively inhibits food-borne spoilage bacteria is prepared according to the following steps:

[0058] (1) Pretreatment of tea seed meal:

[0059] The camellia meal, a byproduct of camellia oil production, was used as raw material. The camellia meal was crushed and placed in a high pressure sterilizer for hot pressing at 118°C for 17 minutes.

[0060] The oil-tea camellia meal powder obtained by the hot pressing treatment is placed in a steam explosion machine and subjected to explosion treatment using saturated water vapor. The steam explosion pressure is adjusted to 1.3 MPa and the processing time of maintaining the pressure is 45 seconds. After the processing time is reached, the material is instantly decompressed to complete the explosion, thereby obtaining the oil-tea camellia meal powder treated with steam explosion and having a loose structure.

[0061] Place the loosely structured camellia oil meal powder in a container, add water (preferably pure water) 10 times its mass, add xylanase, hemicellulase and cellulase under the condition that the original pH value of the water-camellia oil meal system is 6.3, and according to the specific situation that the lignin content in the camellia oil meal is higher than hemicellulose, and the hemicellulose content is higher than cellulose, the dosage of the above three enzymes is 18.0U / g camellia oil meal, 11.0U / g camellia oil meal and 7.0U / g camellia oil meal respectively, and then place it in a 45℃ water bath for hydrolysis for 140min.

[0062] (2) After the pretreatment of camellia oleifera meal, the pH value was adjusted to 4.6 with 0.7 mol / L hydrochloric acid solution to reach the isoelectric point pI 4.6 of camellia oleifera meal protein and maximize the amount of protein precipitation. After completion, the meal was separated in a centrifuge to collect the supernatant and protein precipitate. The supernatant was used to extract tea saponin, and the protein precipitate was used to prepare camellia oleifera meal antimicrobial peptides.

[0063] (3) Take the protein precipitate obtained in the above step (2), add 9 times the mass of purified water (preferably pure water) and stir evenly, adjust its pH value to 8.3 with 1.2 mol / L sodium hydroxide solution, add a composite protease of alkaline protease and trypsin, wherein the enzymatic activity ratio of alkaline protease to trypsin is 3:2, and the added amount is 5700 U / g precipitate, and then place it in a 45°C water bath for hydrolysis for 180 min.

[0064] After the composite protease hydrolysis is completed, the mixture is placed in a centrifuge for separation, and the supernatant is collected. The collected supernatant is filtered through a membrane separation device. The membrane filtration parameters are: pressure 1.7 kg / cm 2 The permeability is 32mL / min and the permeability molecular weight is 3000Da.

[0065] After membrane separation is completed, the permeate is collected to obtain the antimicrobial peptide preparation solution. At this time, the tea seed meal antimicrobial peptide preparation solution is vacuum dried at low temperature (6°C) to obtain the antimicrobial peptide preparation.

[0066] The obtained antimicrobial peptide preparation was determined by trichloroacetic acid precipitation method, and the antimicrobial peptide content was 94.73±1.52%. The antimicrobial peptide was mainly short peptides with a molecular weight of less than 1000 Da, mainly composed of 3-8 amino acids, with a content of 73.28±1.41%.

[0067] (4) Take the supernatant obtained in the above step (2) and add it to the chromatography column filled with AB-8 macroporous resin (wet resin). The supernatant is added in a ratio of 28 mL of supernatant volume (mL) to AB-8 macroporous resin wet resin weight (g) of 1 g. The sample flow rate is 3.5 mL / min. After the sample solution is adsorbed in the chromatography column, the soluble sugar adsorbed on the macroporous resin is first eluted with distilled water until the eluent is neutral and no sugar is detected by anthrone-sulfuric acid method. Then, the polyphenols adsorbed on the macroporous resin are eluted with 20% ethanol solution until no polyphenols are detected by Folin phenol method. Then, the tea saponin adsorbed on the macroporous resin is eluted with 80% ethanol solution. The volume ratio of the eluent 80% ethanol solution to the supernatant sample solution is 1.8:1. The flow rate of the eluent is 2.5 mL / min, collect all the tea saponin eluate and distill out the ethanol using a rotary evaporator to obtain the tea saponin water extract concentrate.

[0068] At this time, the tea saponin water extract concentrate is vacuum dried at low temperature (8°C) to obtain a tea saponin preparation, and the tea saponin content is determined by spectrophotometry to be 93.96±1.35%.

[0069] (5) The antimicrobial peptide obtained in step (3) and the tea saponin preparation obtained in step (4) are respectively compounded and mixed evenly in a mass ratio of the effective ingredient antimicrobial peptide to tea saponin of 2:1, thereby obtaining a tea seed meal extract rich in antimicrobial peptides and tea saponins that effectively inhibits foodborne spoilage bacteria.

[0070] Example 3

[0071] A tea seed meal extract rich in antimicrobial peptides and tea saponins that effectively inhibits food-borne spoilage bacteria is prepared according to the following steps:

[0072] (1) Pretreatment of tea seed meal:

[0073] The camellia meal, a byproduct of camellia oil production, was used as raw material. The camellia meal was crushed and placed in a high pressure sterilizer for hot pressing at 120°C for 15 minutes.

[0074] The tea-seed meal powder obtained by the hot pressing treatment is placed in a steam explosion machine and subjected to explosion treatment using saturated water vapor. The steam explosion pressure is adjusted to 1.4 MPa and the processing time of the pressure is maintained for 40 seconds. After the processing time is reached, the material is instantly decompressed to complete the explosion, thereby obtaining tea-seed meal powder treated with steam explosion and having a loose structure.

[0075] The loosely structured camellia oil meal powder is placed in a container, and 12 times its mass of purified water is added. Under the condition that the original pH value of the water-camellia oil meal system is 6.5, xylanase, hemicellulase and cellulase are added. According to the specific situation that the lignin content in the camellia oil meal is higher than hemicellulose, and the hemicellulose content is higher than cellulose, the dosage of the above three enzymes is 19.0U / g camellia oil meal, 12.0U / g camellia oil meal and 7.5U / g camellia oil meal respectively. Then, it is placed in a 50℃ water bath for hydrolysis for 130min.

[0076] (2) After the pretreatment of camellia oleifera meal, the pH value was adjusted to 4.6 with 0.9 mol / L hydrochloric acid solution to reach the isoelectric point pI 4.6 of camellia oleifera meal protein and maximize the amount of protein precipitation. After completion, the meal was separated in a centrifuge to collect the supernatant and protein precipitate. The supernatant was used to extract tea saponin, and the protein precipitate was used to prepare camellia oleifera meal antimicrobial peptides.

[0077] (3) Take the protein precipitate obtained in step (2) above, add 10 times the mass of purified water and stir evenly, adjust the pH value to 8.5 with 1.5 mol / L sodium hydroxide solution, add a composite protease of alkaline protease and trypsin, wherein the enzyme activity ratio of alkaline protease to trypsin is 3:2, and the added amount is 5900 U / g precipitate, and then place it in a 50°C water bath for hydrolysis for 160 min.

[0078] After the composite protease hydrolysis is completed, the mixture is placed in a centrifuge for separation, and the supernatant is collected. The collected supernatant is filtered through a membrane separation device. The membrane filtration parameters are: pressure 1.9 kg / cm 2 , the permeability is 35mL / min, and the permeability molecular weight is 3000Da.

[0079] After membrane separation is completed, the permeate is collected to obtain the antimicrobial peptide preparation solution. At this time, the tea seed meal antimicrobial peptide preparation solution is vacuum dried at low temperature (8°C) to obtain the antimicrobial peptide preparation.

[0080] The obtained antimicrobial peptide preparation was determined by trichloroacetic acid precipitation method, and the antimicrobial peptide content was 92.87±1.45%. The antimicrobial peptide was mainly short peptides with a molecular weight of less than 1000 Da, mainly composed of 3-8 amino acids, with a content of 72.59±1.38%.

[0081] (4) Take the supernatant obtained in step (2) above and add it to the chromatography column filled with AB-8 macroporous resin (wet resin). Add the supernatant according to the ratio of supernatant volume (mL) to AB-8 macroporous resin wet resin weight (g) of 30mL:1g. The sample loading flow rate is 4.0 mL / min, after the sample solution has completed adsorption in the chromatographic column, the soluble sugar adsorbed on the macroporous resin is first eluted with distilled water, and the eluent is washed with water until it is neutral and the anthrone-sulfuric acid method detects the absence of sugars; then the polyphenols adsorbed on the macroporous resin are eluted with 20% ethanol solution until the eluent is free of polyphenols by the Folin phenol method; then the tea saponin adsorbed on the macroporous resin is eluted with 85% ethanol solution, the volume ratio of the eluent 85% ethanol solution and the supernatant sample solution is 1.9:1, the flow rate of the eluent is 3.0mL / min, all the tea saponin eluent is collected and the ethanol is distilled out by rotary evaporator to obtain the tea saponin water extract concentrate.

[0082] At this time, the tea saponin water extract concentrate is vacuum dried at low temperature (6°C) to obtain a tea saponin preparation, and the tea saponin content is determined by spectrophotometry to be 95.14±1.48%.

[0083] (5) The antimicrobial peptide obtained in step (3) and the tea saponin preparation obtained in step (4) are respectively compounded and mixed evenly in a mass ratio of the effective ingredient antimicrobial peptide to tea saponin of 2:1, thereby obtaining a tea seed meal extract rich in antimicrobial peptides and tea saponins that is effective in inhibiting foodborne spoilage bacteria.

[0084] Example 4

[0085] In order to investigate the antibacterial effects of tea seed meal antimicrobial peptides, tea saponins and their different ratios of compounding, food-borne spoilage bacteria such as Escherichia coli, Salmonella, Bacillus subtilis, Staphylococcus aureus, Aspergillus niger, Aspergillus oryzae, Penicillium citrinum, Saccharomyces cerevisiae, Rhodotorula glutinosus, Candida albicans and Saccharomyces cerevisiae were used as test bacteria. The antimicrobial peptide preparation obtained in step (3) of the above-mentioned experimental example 1, the tea saponin preparation obtained in step (4), the tea seed meal extract obtained in step (5) (the present invention) and compound extracts in other ratios were respectively taken to conduct antibacterial effect comparison experiments. The antibacterial activity evaluation results are shown in Table 1.

[0086] Table 1 MIC (minimum inhibitory concentration, μg / mL) of the antimicrobial peptide preparations, tea saponin preparations and tea seed meal extracts prepared in Example 1

[0087]

[0088] As can be seen from Table 1, the antimicrobial peptide preparation (antimicrobial peptide content>90%), the tea saponin preparation (tea saponin content>90%) and the tea oil seed meal extract (antimicrobial peptide mass percentage 30-70%, tea saponin mass percentage 20-60%) all have obvious antibacterial effects on the test bacteria, molds and yeasts and other food-borne spoilage bacteria. The MIC (minimum inhibitory concentration) of the antimicrobial peptide preparation is 50-350 μg / mL, the MIC (minimum inhibitory concentration) of the tea saponin preparation is 12.5-325 μg / mL, and the MIC (minimum inhibitory concentration) of the tea oil seed meal extract of the present invention with an antimicrobial peptide: tea saponin mass ratio of 2:1 is 12.5-75 μg / mL.

[0089] It can also be seen from Table 1 that the MIC of tea saponin preparations against bacteria such as Escherichia coli, Salmonella, Bacillus subtilis, Staphylococcus aureus, and yeasts such as Saccharomyces cerevisiae, Rhodotorula glutinosus, Candida albicans, and Saccharomyces cerevisiae are all lower than those of antimicrobial peptide preparations, indicating that tea saponin preparations have stronger inhibitory effects on the above four bacteria and four yeasts than antimicrobial peptide preparations; while the MIC of antimicrobial peptide preparations against molds such as Aspergillus niger, Aspergillus oryzae, and Penicillium citrinum are all lower than those of tea saponin preparations, indicating that antimicrobial peptide preparations have stronger inhibitory effects on the above three molds than tea saponin preparations. It can be seen that tea saponin and tea oil cake antimicrobial peptide preparations are both good natural antibacterial agents, but their inhibitory effects on bacteria, molds, and yeasts vary in strength.

[0090] What is particularly important is that the results in Table 1 show that after the antimicrobial peptide and the tea saponin preparation are compounded, the inhibitory effect on the test bacteria is significantly stronger than the weaker inhibitory effect when the two are used alone, and is stronger than or equal to the better inhibitory effect when the two are used alone; at the same time, the antibacterial effect after compounding is related to the ratio of antimicrobial peptide and tea saponin. The tea seed meal extract obtained by compounding the antimicrobial peptide and tea saponin at a mass ratio of 2:1 adopted in the present invention has the best antibacterial effect. It can be seen that the tea seed meal extract containing the two main components of antimicrobial peptide and tea saponin has a better antibacterial effect. When the two main components of antimicrobial peptide and tea saponin exist at the same time, a significant synergistic antibacterial effect is exhibited, and its antibacterial spectrum is larger, with a broader spectrum and more efficient antibacterial effect.

[0091] Example 5

[0092] In order to investigate the beneficial effects of the tea seed meal pretreatment technology of the present invention, the antimicrobial peptide and tea saponin preparations of Example 1 were taken, and under the same conditions as Example 1 of the present invention, control preparation experimental groups without hot pressing pretreatment, steam explosion pretreatment, mixed enzymatic hydrolysis pretreatment, and none of the above three pretreatments were set up, respectively. The yield of the preparation and the content of the effective ingredients (antimicrobial peptides, tea saponins) in the preparation were measured, respectively. The yield of the preparation was calculated as the weight percentage of the effective ingredients (antimicrobial peptides, tea saponins) in the tea seed meal dry basis, and the content of the effective ingredients (antimicrobial peptides, tea saponins) in the preparation was calculated as the weight percentage of the effective ingredients (antimicrobial peptides, tea saponins) in the preparation dry basis. The average value was taken after three repeated experimental measurements.

[0093] Table 2 Effects of tea seed meal pretreatment on the yield and effective ingredient content of the preparation

[0094]

[0095] The results in Table 2 show that the yield of effective ingredients of the antimicrobial peptide and tea saponin preparations prepared by the method of the present invention and the content of effective ingredients in the preparations are significantly higher than those of the control experimental group without pretreatment. The yield of antimicrobial peptides of the antimicrobial peptide preparation of the present invention (based on the dry basis of camellia oleifera cake) is 3.57±0.06%, and the content of antimicrobial peptides in the preparation reaches 93.63±1.47%; the yield of tea saponin of the tea saponin preparation of the present invention (based on the dry basis of camellia oleifera cake) is 12.60±0.25%, and the content of tea saponin in the preparation reaches 95.48±1.39%; while the yield of antimicrobial peptides of the control experimental group without hot pressing pretreatment, steam explosion pretreatment and mixed enzymatic pretreatment did not exceed 2.5%, the yield of tea saponin did not exceed 8%, the content of antimicrobial peptides in the preparation did not reach 80%, and the content of tea saponin did not reach 75%. In particular, the yield of effective ingredients and the content of effective ingredients in the preparation of the control group without any of the three pretreatments were the lowest.

[0096] It can be seen that the hot pressing, steam explosion and mixed enzyme pretreatment of the camellia oleifera meal in the present invention significantly improve the yield of the effective ingredients of the antimicrobial peptide and tea saponin preparations and the content of the effective ingredients in the preparations, and effectively improve the utilization rate of the camellia oleifera meal and the quality of the preparations.

[0097] The above description is only a non-limiting embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements without departing from the principle of the present invention, and these improvements are all considered to be within the protection scope of the present invention.

Claims

1. A method for preparing a tea seed meal extract rich in antimicrobial peptides and tea saponins, characterized in that The following steps are involved: (1) Pretreatment of tea-seed meal: pretreatment of tea-seed meal by hot pressing, steam explosion and mixed enzymatic hydrolysis; (1.1) Hot pressing pretreatment: The camellia dregs, a byproduct of camellia oil production, are used as raw materials. The camellia dregs are crushed and placed in a high pressure sterilization device for hot pressing at a temperature of 115-121°C for 15-20 minutes. (1.2) Steam explosion pretreatment: The tea-seed meal powder that has been pretreated by hot pressing is placed in a steam explosion device and subjected to explosion treatment using saturated water vapor. The steam explosion pressure is adjusted to 1.2-1.4 MPa and the treatment time is 40-50 seconds. After the treatment time is reached, the material is instantly decompressed to complete the explosion and obtain tea-seed meal powder with a loose structure. (1.3) Mixed enzymatic hydrolysis pretreatment: Place loosely structured camellia oleifera meal powder in a container, add water, add xylanase, hemicellulase and cellulase under the condition that the original pH value of the water-camellia oleifera meal system is 6.0-6.5, and then place it in a 40-50℃ water bath for hydrolysis for 120-150min to obtain an enzymatic hydrolyzate; (2) After the pretreatment of the camellia oleifera meal is completed, the pH value of the enzymatic hydrolyzate is adjusted to reach the isoelectric point of the camellia oleifera meal protein to maximize the amount of protein precipitation. After completion, centrifugation is performed to collect the supernatant and protein precipitate, respectively. The supernatant is used to extract tea saponin, and the protein precipitate is used to prepare camellia oleifera meal antimicrobial peptides. (3) Take the protein precipitate obtained in step (2), add water and stir evenly, adjust the pH value to 8.0-8.5, add a composite protease containing alkaline protease and trypsin, and then place it in a water bath at 40-50°C for hydrolysis for 150-210 minutes. After the composite protease hydrolysis is completed, centrifuge and collect the supernatant. After the supernatant is filtered through a membrane separation device, collect the permeate after the membrane separation is completed, and obtain the antimicrobial peptide preparation solution, which is dried under low temperature vacuum to obtain the antimicrobial peptide preparation; (4) Take the supernatant obtained in step (2) and add it to a chromatography column filled with AB-8 macroporous resin. After the supernatant is completely adsorbed in the chromatography column, first elute the soluble sugar adsorbed on the macroporous resin with distilled water until the eluate is neutral and no sugar is detected by anthrone-sulfuric acid method. Then elute the polyphenols adsorbed on the macroporous resin with a low concentration of ethanol aqueous solution until no polyphenol is detected by Folin phenol method. Then elute the tea saponin adsorbed on the macroporous resin with a high concentration of ethanol aqueous solution. Collect all the tea saponin eluate and distill off ethanol to obtain a tea saponin water extract concentrate. The tea saponin water extract concentrate is subjected to low-temperature vacuum drying to obtain a tea saponin preparation. (5) respectively taking the antimicrobial peptide preparation obtained in step (3) and the tea saponin preparation obtained in step (4), compounding and mixing them in a weight ratio of 2:1 between the active ingredient antimicrobial peptide and the tea saponin, thereby obtaining a tea seed meal extract rich in antimicrobial peptides and tea saponins that inhibits foodborne spoilage bacteria; In step (1.3), the amount of water added is 8 to 12 times the total mass of the camellia oleifera meal powder, and the amounts of xylanase, hemicellulase and cellulase used are 15.0 to 20.0 U / g camellia oleifera meal, 9.0 to 12.0 U / g camellia oleifera meal and 5.0 to 8.0 U / g camellia oleifera meal, respectively; In step (2), the pH value of the enzymatic hydrolyzate is adjusted to 4.6 with a 0.5-1.0 mol / L hydrochloric acid solution to achieve an isoelectric point pI of 4.6 for the camellia meal protein; In step (3), the amount of water added is 8 to 10 times the total mass of the protein precipitate, the pH value is adjusted to 8.0 to 8.5 with a 1.0 to 1.5 mol / L sodium hydroxide solution, the amount of the composite protease added is 5500 to 6000 U / g protein precipitate with a total enzyme activity, and the enzyme activity ratio of alkaline protease to trypsin in the composite protease is 3:2; The membrane filtration parameters in step (3) are: pressure 1.5-2 kg / cm 2 , the penetration rate is 30-35 mL / min, and the penetration molecular weight is 3000 Da; The antimicrobial peptide preparation obtained in step (3) is determined by trichloroacetic acid precipitation method, wherein the antimicrobial peptide content is more than 90%, and the antimicrobial peptide preparation is mainly short peptides with a molecular weight of less than 1000 Da, mainly small peptides composed of 3 to 8 amino acids, and the content of small peptides is more than 70% as determined by gel chromatography; In step (4), the supernatant is added in a ratio of 25-30 mL volume to 1 g mass of AB-8 macroporous resin wet resin, and the loading flow rate is 3.0-4.0 mL / min; the volume percentage of the low-concentration ethanol aqueous solution is 20%, the volume percentage of the high-concentration ethanol aqueous solution is 75-85%, the volume ratio of the high-concentration ethanol aqueous solution to the supernatant is 1.5-2:1, the flow rate of the eluent is 2.0-3.0 mL / min, and the tea saponin preparation, as determined by spectrophotometry, has a tea saponin content of more than 90%.

2. The tea seed cake extract rich in antimicrobial peptides and tea saponins prepared by the method according to claim 1.

3. Use of the tea seed meal extract rich in antimicrobial peptides and tea saponins as claimed in claim 2 in the preparation of a product having the effect of inhibiting foodborne spoilage bacteria.

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