CN112335810B - Edible antioxidant and preparation method and application thereof - Google Patents
Edible antioxidant and preparation method and application thereof Download PDFInfo
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- CN112335810B CN112335810B CN202011127338.5A CN202011127338A CN112335810B CN 112335810 B CN112335810 B CN 112335810B CN 202011127338 A CN202011127338 A CN 202011127338A CN 112335810 B CN112335810 B CN 112335810B
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- propionibacterium
- fermentation
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- enzymolysis
- slurry
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to an edible antioxidant and a preparation method and application thereof. The preparation method of the edible antioxidant takes grain powder with natural sources as a fermentation raw material, the fermentation raw material contains various elements and compounds required by growth and metabolism of propionibacterium, additional supplement of a nutrient medium is not needed, and the preparation method has the advantages of wide sources and low cost.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to an edible antioxidant and a preparation method and application thereof.
Background
With the improvement of the living standard of people and the continuous attention of safety and health, consumers gradually abandon foods added with chemical additives, more and more favor fresh natural foods, and more food brands advocate concepts such as 'natural', 'organic', 'artificial ingredient free' and 'no chemical/pesticide residue'. Products containing the cleaning labels, such as natural extraction and microbial fermentation, gradually become a great trend in the food industry, and have better development prospects.
The oxidative rancidity of oil-containing food is one of the important reasons for food deterioration, the food oxidative rancidity not only can greatly reduce the nutrition of the food, but also can generate small molecular aldehydes, ketones, acids and the like with unpleasant odor, namely rancid odor, so that the flavor and the appearance of the food are deteriorated, even the commodity value is lost, and more importantly, the food oxidative rancidity can cause greater harm to the health of a human body, such as increasing the occurrence probability of various inflammations, aging, anaphylactic reactions, atherosclerosis, cancers and the like.
In order to improve the oxidation resistance of food, antioxidants are often added in the processes of food processing, storage and fresh keeping, and currently, synthetic antioxidants such as Butyl Hydroxy Anisole (BHA), dibutyl hydroxy toluene (BHT), propyl Gallate (PG) and tert-butyl hydroquinone (TBHQ) are used more frequently, but the safety of the antioxidants is questioned and the antioxidants have many disadvantages: BHA, BHT, PG and TBHQ have poor thermal stability and are extremely volatile and ineffective in hot oil with the temperature of above 80 ℃; synthetic antioxidants (commonly used BHA and BHT) have large toxic and side effects and have adverse effects on human liver, spleen, lung and the like; low oxidation resistance and poor bacteriostatic effect; the application range has many limitations, and western countries such as the European Union and Japan have limited import of products such as foods processed by using synthetic antioxidants in the food import-related inspection. Therefore, natural edible antioxidants having high safety, high antioxidant ability, and no side effects have been attracting attention and expected.
Disclosure of Invention
The invention aims to provide an edible antioxidant, a preparation method and application thereof, and aims to solve the technical problem of poor safety of the existing artificially synthesized antioxidant.
In order to achieve the above object, according to one aspect of the present invention, there is provided a method for preparing an edible antioxidant, comprising the steps of:
mixing the grain powder with water to obtain slurry;
carrying out protease enzymolysis treatment, amylase liquefaction treatment and saccharification enzyme enzymolysis treatment on the slurry to obtain an enzymolysis solution;
inoculating propionibacterium into the enzymolysis liquid for fermentation culture to obtain fermentation liquid;
and removing propionate in the fermentation liquor to obtain the edible antioxidant.
In another aspect of the present invention, an edible antioxidant is provided, which is prepared by the preparation method of the edible antioxidant of the present invention.
In a final aspect of the invention, there is provided the use of the edible antioxidant of the invention in food antioxidation.
The preparation method of the edible antioxidant provided by the invention has the following advantages:
firstly, the method takes grain powder with natural sources as a fermentation raw material, the fermentation raw material contains various elements and compounds required by growth and metabolism of propionibacterium, additional supplement of a nutrient medium is not needed, and the method has the advantages of wide sources and low price, and can greatly reduce the production cost; secondly, the method can decompose carbohydrate and protein in the fermentation raw materials by performing protease enzymolysis treatment, amylase liquefaction treatment and saccharification enzyme enzymolysis treatment on the fermentation raw materials to enable the carbohydrate and protein to be used as a carbon source and a nitrogen source required by the fermentation culture of the propionibacterium, so that the additional supplement of the carbon source and the nitrogen source is not needed in the fermentation culture process, and the production link is simplified; thirdly, the fermentation raw materials and the treatment mode of the method are designed according to the specific nutritional requirements of the propionibacterium, so that the effect of directionally regulating and controlling the yield of the target product can be realized; finally, the method adopts natural, green and environment-friendly biological enzyme to treat the fermentation raw materials, does not need strict anaerobic operation in the fermentation process, does not need nitrogen gas to be introduced for protection, does not need stirring in the whole fermentation process, has the advantages of simplicity, high efficiency, safety, energy conservation, environmental protection and strong controllability, can fully utilize the fermentation raw materials, shortens the production time, saves the production process, and is favorable for realizing industrial mass production. In addition, the method can obtain the edible antioxidant and propionate byproducts, can be used as raw materials in various fields such as food additives, medical supplies and the like, is favorable for fully utilizing resources, and reduces the risks of waste and environmental pollution.
The edible antioxidant provided by the invention is obtained by fermenting grains from natural sources through propionibacterium, belongs to a biological natural antioxidant, has the special faint scent of a fermentation product, has good inoxidizability, does not have organic solvent residues, and has higher safety and stability compared with the traditional artificially synthesized antioxidant. The edible antioxidant has the main components of vitamin B12, short-chain small-molecule active peptide substances and a small amount of organic acid salt, and the components play an antioxidant effect through mutual synergistic action so as to resist the oxidation of various free radical ions on food. Has good application prospect.
The edible antioxidant provided by the invention is used for food antioxidation, can obviously reduce the oxidation rancidity speed of oil and fat components in food, ensures the food safety, can replace the traditional chemical antioxidant, and is used for prolonging the shelf life of the food. Meanwhile, the edible antioxidant provided by the invention is a biological natural antioxidant obtained by a natural fermentation method, and has the advantages of high safety, good stability, wide and easily available sources and the like.
Drawings
FIG. 1 is a graph showing the results of measuring the antioxidant effect of the edible antioxidant obtained in example 1 of the present invention on the conditioned meat;
FIG. 2 is a graph showing the results of measuring the antioxidant effect of the edible antioxidant obtained in example 1 of the present invention on a roast meat cake.
Detailed Description
In order to make the objects, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described, and the embodiments described below are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention. The examples do not indicate specific conditions, and the conventional conditions or conditions suggested by the manufacturer are followed; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the description of the present invention, the term "and/or" describing an association relationship of associated objects means that there may be three relationships, for example, a and/or B, may mean: a is present alone, A and B are present simultaneously, and B is present alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
In the description of the present invention, "at least one" means one or more, "a plurality" means two or more. "at least one of the following" or similar expressions refer to any combination of these items, including any combination of the singular or plural items. For example, "at least one (a), b, or c", or "at least one (a), b, and c", may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, and c may be single or plural, respectively.
It should be understood that the weight of the related components mentioned in the embodiments of the present invention may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, it is within the scope of the disclosure that the content of the related components is scaled up or down according to the embodiments of the present invention. Specifically, the weight described in the embodiments of the present invention may be a unit of mass known in the chemical field such as μ g, mg, g, kg, etc.
In addition, unless the context clearly uses otherwise, an expression of a word in the singular is to be understood as including the plural of the word. The terms "comprises" or "comprising" are intended to specify the presence of stated features, quantities, steps, operations, elements, portions, or combinations thereof, but are not intended to preclude the presence or possible addition of one or more other features, quantities, steps, operations, elements, portions, or combinations thereof.
The embodiment of the invention provides a preparation method of an edible antioxidant, which comprises the following steps:
s1, mixing grain powder and water to obtain slurry;
s2, carrying out protease enzymolysis treatment, amylase liquefaction treatment and saccharification enzyme enzymolysis treatment on the slurry to obtain an enzymolysis solution;
s3, inoculating propionibacterium into the enzymolysis liquid for culture fermentation to obtain fermentation liquid;
and S4, removing propionate in the fermentation liquor to obtain the edible antioxidant.
The preparation method of the edible antioxidant provided by the embodiment of the invention has the following advantages:
firstly, the method takes grain powder with natural sources as a fermentation raw material, the fermentation raw material contains various elements and compounds required by growth and metabolism of propionibacterium, additional supplement of a nutrient medium is not needed, and the method has the advantages of wide sources and low price, and can greatly reduce the production cost; secondly, the method can decompose carbohydrate and protein in the fermentation raw materials by performing protease enzymolysis treatment, amylase liquefaction treatment and saccharification enzyme enzymolysis treatment on the fermentation raw materials, so that the carbohydrate and protein can be used as a carbon source and a nitrogen source required by fermentation culture of propionibacterium, and therefore, additional supplement of the carbon source and the nitrogen source is not needed in the fermentation culture process, and the production link is simplified; thirdly, the fermentation raw materials and the treatment mode of the method are designed according to the specific nutritional requirements of the propionibacterium, and the effect of directionally regulating and controlling the yield of the target product can be realized; finally, the method adopts natural, green and environment-friendly biological enzyme to treat the fermentation raw materials, does not need strict anaerobic operation in the fermentation process, does not need nitrogen gas to be introduced for protection, does not need stirring in the whole fermentation process, has the advantages of simplicity, high efficiency, safety, energy conservation, environmental protection and strong controllability, can fully utilize the fermentation raw materials, shortens the production time, saves the production process, and is beneficial to realizing industrial mass production.
In S1, the grain powder is used as a fermentation raw material, so that various elements and compounds can be provided for the growth and metabolism of the propionibacterium, and a nutrient medium does not need to be supplemented additionally. In some embodiments, the cereal flour is selected from flours made from at least one of wheat, rice, corn, tapioca. In some embodiments, when wheat flour (i.e., flour) is used as the cereal flour, no additional nutrients are required because the flour contains sufficient nutrients such as protein for the growth and fermentation of propionibacteria; when the powder made of at least one of rice, corn and cassava is used as the grain powder, enough nutrition can be provided for the growth and fermentation of propionibacterium by supplementing nutrients containing protein, and the supplemented amount accounts for 0.2-2% of the mass of the grain powder. The nutrient containing protein can specifically select yeast extract, soybean protein meal and/or soybean cake powder, wherein the yeast extract has rich nutrient components and relatively small addition amount, and optionally, the addition amount of the yeast extract accounts for 0.2-0.5 percent of the mass of the grain powder; the nutritional ingredients of the soybean protein meal and the soybean cake powder are slightly less than those of the yeast extract, so the addition amount of the soybean protein meal and the soybean cake powder is relatively more, and optionally, the addition mass of the soybean protein meal and/or the soybean cake powder accounts for 0.5-2% of the mass of the grain powder.
The cereal powder is mixed with water to be processed into slurry, so that the nutrient substances in the cereal powder can be subjected to enzymolysis and subsequent fermentation and separation. In some embodiments, the mass concentration of the cereal flour in the slurry is between 2% and 20%, preferably between 3% and 17%, most preferably between 6% and 12%. In some embodiments, the slurry may be formulated by controlling the ratio of the cereal flour to water mass to be (1-10): 50, preferably (3-17): 100, most preferably (6-12.7): 100. By optimizing the mass ratio of the grain powder to the water, the slurry with proper solid content can be obtained, the utilization rate of the grain powder and the fermentation efficiency of the propionibacterium can be improved, and the yield and the quality of the microbial fermentation agent are improved. In addition, it is preferable that 80% of the formulated slurry may pass through a 60-mesh sieve to increase the dispersion degree and fineness of the grain powder in the slurry, thereby improving the treatment effect and efficiency of the protease enzymolysis treatment, the amylase liquefaction treatment, and the saccharification enzyme enzymolysis treatment in S2.
In S2, by performing protease enzymolysis treatment, amylase liquefaction treatment and saccharification enzyme enzymolysis treatment on the slurry, macromolecules such as protein, starch, glucose and the like contained in the grain powder can be degraded to obtain nutritional ingredients such as a carbon source, a nitrogen source and the like required by growth and fermentation of the propionibacterium, so that the propionibacterium does not need to be additionally supplemented with the carbon source and the nitrogen source in the fermentation culture process. In the present example, the order of the steps of enzymatic treatment of the slurry is critical and will have a significant impact on the fermentation product and the composition of the resulting edible antioxidant. The pH of the starch-containing slurry is constantly changing throughout the enzymatic process, and thus the acidity of the slurry is adjusted prior to the protease, amylase, and carbohydrase enzymatic processes. According to the embodiment of the invention, the order of the protease enzymolysis treatment, the amylase liquefaction treatment and the saccharification enzyme enzymolysis treatment is adopted, so that the addition of an acidity regulator can be reduced, the cost is reduced, the reaction operation is facilitated, the saccharification enzyme enzymolysis treatment is carried out after the amylase liquefaction treatment, and the reaction can be promoted to be carried out fully and quickly, so that the edible antioxidant is obtained.
In some embodiments, the method of protease enzymatic treatment comprises: adjusting the pH value of the slurry to 8.0-11.0, adding protease, and performing enzymolysis at 40-55 deg.C until the pH value is 6.0-7.0 and the pH value does not decrease within half an hour; preferably, the pH of the slurry is adjusted to 9.0 to 11.0, and then protease is added, followed by an enzymatic reaction at 50 ℃. In some embodiments, the particular protease may be selected based on the pH of the slurry. When the pH value of the slurry is 10.0-12.0, selecting alkaline protease for enzymolysis; when the pH value of the slurry is 4.0-6.0, selecting acid protease; when the pH value of the slurry is 6.0-9.0, neutral protease, a compound of the neutral protease and bromelain or a compound of the neutral protease and papain are selected for enzymolysis.
In some embodiments, a method of amylase liquefaction treatment comprises: adjusting the pH value of the slurry subjected to protease enzymolysis treatment to 5.0-7.5, adding amylase, and performing hydrolysis reaction at 60-110 ℃ for 10-30 min; preferably, the pH of the slurry after the protease enzymolysis treatment is adjusted to 6.0, and then amylase is added to perform hydrolysis reaction at 90 ℃ for 20min. In some embodiments, the amylase used may be a high temperature amylase and/or an alpha-amylase; the test can be carried out by using dilute iodine solution in the process of liquefaction treatment, and when the liquid is no longer blue, the hydrolysis reaction is completed.
In some embodiments, the method of saccharification enzymatic hydrolysis treatment comprises: adjusting the pH of the slurry after the amylase liquefaction treatment to 3.5-6.0, adding 10U-200U of glucoamylase into each gram of starch according to the mass of the starch in the grain powder, then carrying out the enzymolysis reaction for 12-50 h at 30-65 ℃, preferably adjusting the pH of the slurry after the amylase liquefaction treatment to 4.5, adding 10U-200U of glucoamylase into each gram of starch according to the mass of the starch in the grain powder, and then carrying out the enzymolysis reaction for 20-24 h at 50 ℃. In some embodiments, the reducing sugar and total sugar are measured during the enzymatic hydrolysis of the saccharification enzyme, and the enzymatic hydrolysis is completed when the amount of reducing sugar reaches 80% of the total sugar.
And in the step S3, the propionibacterium is inoculated into the enzymolysis liquid obtained in the step S2, and fermentation culture is carried out to obtain fermentation liquid. The process does not need strict anaerobic treatment, so nitrogen does not need to be introduced for protection, stirring treatment is not needed, and the production process and cost are greatly saved. It will be appreciated that in order to ensure growth and fermentation for propionibacteria, the enzymatic hydrolysate should be in a sterile environment. In some embodiments, the enzymolysis liquid obtained in S2 can be subjected to inactivation and sterilization treatment, and the treatment mode not only kills microorganisms in the enzymolysis liquid and provides a good growth environment for propionibacterium, but also causes inactivation of various enzymes added in S2 under a high temperature condition, thereby avoiding influence of the enzymes on growth of propionibacterium and facilitating obtaining of fermentation liquid with high purity and good performance. In some embodiments, the method for inactivation and sterilization comprises autoclaving the enzymatic hydrolysate at 115-121 deg.C, and inactivating the enzyme for 20-30 min.
In some embodiments, the Propionibacterium is selected from at least one of Propionibacterium (Propionibacterium acidipronici), propionibacterium freudenreichii subsp.
In some embodiments, the method further comprises the step of activating the propionibacterium. This is because the culture conditions are often different from the preservation conditions of the strains, and it is necessary to revive the strains by an activation treatment to gradually adapt to a new culture environment. The activation treatment method adopted by the embodiment of the invention is specially designed for fermenting the grain powder provided by the embodiment of the invention by propionibacterium, and the specific method is as follows: inoculating propionibacterium into a seed culture medium, and culturing at 30-34 ℃ for 40-50 h, wherein the preferable culture time is 48h. Wherein the seed culture medium comprises 0.5-1.0% of glycerol, 1-2% of yeast extract, 0.5-1% of peptone, 0.3-0.5% of dipotassium hydrogen phosphate and 0.25-0.3% of monopotassium phosphate, and the balance of water, and the pH of the seed culture medium is 6.0-7.0, preferably the pH is 7.0, based on 100% of the mass of the seed culture medium.
In some embodiments, the method further comprises the step of performing scale-up culture on the propionibacterium. The propionibacterium can be stably grown by carrying out amplification culture on the propionibacterium, the cell concentration of the propionibacterium is expanded, the lag phase of the propionibacterium in the fermentation culture process is shortened, and the fermentation culture efficiency is improved. In some embodiments, the scale-up culture can be divided into a first-stage scale-up culture and a second-stage scale-up culture to further improve the efficiency of the subsequent fermentation culture. Wherein, the specific method of the first-stage amplification culture is as follows: the propionibacterium is inoculated into a triangular flask containing a seed culture medium and cultured for 24 hours at the temperature of 30-34 ℃. Wherein the seed culture medium comprises 0.5-1.0% of glycerol, 1-2% of yeast extract, 0.5-1% of peptone, 0.3-0.5% of dipotassium hydrogen phosphate, 0.25-0.3% of monopotassium phosphate and the balance of water, and the weight of the seed culture medium is 100%, and the pH of the seed culture medium is 7.0. The specific method of the secondary amplification culture is as follows: inoculating the propionibacterium after the first-stage amplification culture into a seeding tank containing a seed culture medium, and culturing for 48h at the temperature of 30-34 ℃.
The conditions of the fermentation culture have a significant impact on the composition of the food antioxidant produced. The components of the fermentation liquor can have significant differences under different fermentation conditions. In some embodiments, when propionibacterium is inoculated into the enzymolysis liquid for fermentation culture, the method for fermentation culture is as follows: the culturing is carried out at 30 ℃ to 32 ℃ at a pH of 6.0 to 7.0, preferably pH =7.0. And detecting reducing sugar and propionate in the culture process, and ending fermentation when the mass of the reducing sugar is less than or equal to 0.1% and the content of the propionate is not increased any more. The edible antioxidant obtained under the fermentation culture condition has better antioxidant performance than the edible antioxidants obtained under other conditions. In some embodiments, ca (OH) can be added by flowing 2 The pH of the fermentation broth is controlled in a (sterile) manner to 6.0-7.0.
And S4, removing propionate in the fermentation liquor obtained in the S3, wherein the remainder is the edible antioxidant. Among them, it can be understood that, because the fermentation liquid obtained in S3 still contains a large amount of viable propionibacterium, the propionibacterium should be inactivated to obtain a safer edible antioxidant. In some embodiments, the method of inactivating the propionibacterium is by heating the fermentation broth to 60 ℃ for inactivation.
Furthermore, thalli and insoluble substances in the inactivated fermentation liquor can be removed, so that the purity of the edible antioxidant is improved. The removing method comprises the following steps: and (3) filtering or centrifugally separating the inactivated fermentation liquor by a filter press to obtain supernatant, namely the fermentation liquor from which the thalli and insoluble substances are removed.
In some embodiments, the method of removing propionate from a fermentation broth is: and crystallizing the fermentation liquor after vacuum evaporation and concentration to obtain propionate, and then centrifuging at the rotating speed of 2000rpm-2500rpm for 5min-8min to separate the propionate from the edible antioxidant.
The edible antioxidant obtained in this case is in liquid form, and it is understood that the liquid edible antioxidant can be made into other forms, including but not limited to solid form, by various methods according to the actual application requirements.
Correspondingly, the embodiment of the invention also provides an edible antioxidant which is prepared by the preparation method of the edible antioxidant.
The edible antioxidant provided by the embodiment of the invention is obtained by fermenting grains of natural sources through propionibacterium, belongs to a biological natural antioxidant, has the special faint scent of a fermentation product, has good inoxidizability, does not have organic solvent residues, and has higher safety and stability compared with the traditional artificially synthesized antioxidant. The edible antioxidant has the advantages that the main components of the edible antioxidant are vitamin B12, small molecular active peptide, antioxidant peptide, amino acid and a small amount of organic acid salts such as calcium lactate and calcium propionate, the components have an antioxidant effect through mutual synergistic effect, can resist the oxidation of various free radical ions on food, and has good application prospect.
Correspondingly, the embodiment of the invention also provides the application of the edible antioxidant in food antioxidation.
The edible antioxidant provided by the embodiment of the invention is used for food antioxidation, can obviously reduce the oxidation rancidity speed of oil components in food, ensures the food safety, can replace the traditional chemical antioxidant, and is used for prolonging the shelf life of the food. Meanwhile, the edible antioxidant provided by the embodiment of the invention is a biological natural antioxidant obtained by a natural fermentation method, and has the advantages of high safety, good stability, wide and easily-obtained raw material sources and the like.
In order to make the above implementation details and operations of the present invention clearly understood by those skilled in the art and to make the progress of the edible antioxidant and its preparation method and application obvious in the examples of the present invention, the above technical solutions are illustrated by the following examples.
Example 1
A preparation method of an edible antioxidant comprises the following steps:
(11) Mixing flour and water according to the mass ratio of 12.7;
(12) Adjusting the pH value of the slurry to 11.0, adding alkaline protease, controlling the temperature at 50 ℃, and stopping the reaction when the pH value is reduced to below 7.0 and is not reduced within half an hour; adjusting the pH value of the slurry to 6.0, adding high-temperature amylase, rapidly heating the slurry to 90 ℃, liquefying for 20min, and testing the slurry with dilute iodine solution until the slurry does not show blue; adjusting the pH value of the slurry to 4.5, adding 200U/g of glucoamylase, reacting for 15h, and finishing the reaction when reducing sugar reaches 80% of the total sugar amount; adding the obtained slurry into a 2 ton fermentation tank, and autoclaving at 121 deg.C for 30min;
(13) Storing Propionibacterium acidipropionici in glycerin tube, and storing in a refrigerator at-80 deg.C for use; 5mL of the seed culture was inoculated into a shake flask containing 200mL of seed medium and cultured at 30 ℃ for 48 hours. Seed culture medium: 1.0% of glycerol, 2% of yeast extract, 1% of peptone, 0.5% of dipotassium hydrogen phosphate, 0.3% of monopotassium phosphate and the balance of water; the pH value is 7.0;
(14) Inoculating the activated strain into a 2L first-stage triangular flask containing a seed culture medium, and standing and culturing at 30 ℃ for 24h. During the culture period, nitrogen does not need to be introduced, and stirring is not needed; then, the cells were inoculated into a 200L seed tank containing a seed medium and subjected to static culture at 30 ℃ for 48 hours. During the culture period, nitrogen is not required to be introduced, and stirring is not required;
(15) Inoculating the cultured propionibacterium strain obtained in the step (14) into a fermentation tank, and culturing and fermenting at 30 ℃; automatic feeding 20% Ca (OH) 2 Controlling the pH value of the fermentation liquor to be 7.0 by using the sterile suspension; when the amount of reducing sugar in the fermentation tank is reduced to be below 0.1 percent and the content of calcium propionate is not increased any more, the fermentation is finished; heating the obtained fermentation liquor to 60 ℃ to inactivate the zymocyte, and centrifugally separating thalli and insoluble substances to obtain supernatant;
(16) Evaporating the supernatant under reduced pressure, concentrating, crystallizing to obtain propionate, centrifuging at 2000rpm for 8min, and separating propionate byproduct from supernatant which is edible antioxidant.
The detection result of the ingredients of the obtained edible antioxidant shows that the main active ingredients of the edible antioxidant are vitamin B12, small molecular active peptide, antioxidant peptide, various amino acids (mainly comprising 6 human essential amino acids: tyrosine, alanine, methionine, aspartic acid, threonine and lysine) and a small amount of organic acid salt (mainly comprising calcium lactate and calcium propionate). The edible antioxidant is liquid, and the mass of the main active ingredients of the edible antioxidant accounts for about 10% of the total mass by detection.
Example 2
A preparation method of an edible antioxidant comprises the following steps:
(21) Mixing a mixture of rice flour, corn flour and cassava flour with water according to a mass ratio of 6;
(22) Adjusting the pH value of the slurry to 9.0, adding neutral protease, controlling the temperature at 50 ℃, and stopping the reaction when the pH value is reduced to below 6.0 and is not reduced within half an hour; adjusting the pH value of the slurry to 6.0, adding high-temperature amylase, rapidly heating the slurry to 90 ℃, liquefying for 20min, and testing the slurry with dilute iodine solution until the slurry does not show blue; adjusting the pH value of the slurry to 4.5, adding 100U/g of glucoamylase, reacting for 24h, and finishing the reaction when the reducing sugar reaches 80% of the total sugar content; adding the obtained slurry into a 2 ton fermentation tank, and autoclaving at 121 deg.C for 30min;
(23) The Propionibacterium freudenreichii subsp. Shermanii is stored in glycerin tube and stored in refrigerator at-80 deg.C for use; 5mL of the seed culture was inoculated into a shake flask containing 200mL of seed medium and cultured at 34 ℃ for 48 hours. Seed culture medium: 0.5% of glycerol, 1% of yeast extract, 0.5% of peptone, 0.3% of dipotassium hydrogen phosphate, 0.25% of potassium dihydrogen phosphate and the balance of water; the pH value is 7.0;
(24) Inoculating the activated strain into a 2L first-stage triangular flask containing a seed culture medium, and standing and culturing at 34 deg.C for 24h. During the culture period, nitrogen does not need to be introduced, and stirring is not needed; then, the cells were inoculated into a 200L seed tank containing a seed medium and subjected to static culture at 34 ℃ for 48 hours. During the culture period, nitrogen does not need to be introduced, and stirring is not needed;
(25) Inoculating the cultured propionibacterium strain obtained in the step (24) into a fermentation tank, and culturing and fermenting at 32 ℃; automatic feeding 30% Ca (OH) 2 Controlling the pH value of the fermentation liquor to be 7.0 by using the sterile suspension; when the amount of reducing sugar in the fermentation tank is reduced to be below 0.1 percent and the content of calcium propionate is not increased any more, the fermentation is finished; heating the obtained fermentation liquor to 60 ℃ to inactivate the zymocyte, and centrifugally separating thalli and insoluble substances to obtain supernatant;
(26) Evaporating the supernatant under reduced pressure, concentrating, crystallizing to obtain propionate, centrifuging at 2500rpm for 5min, and separating propionate byproduct from supernatant which is edible antioxidant.
The detection result of the components of the edible antioxidant shows that the main components of the edible antioxidant are vitamin B12, small molecular active peptide substances, antioxidant peptides, various amino acids (mainly comprising 6 human essential amino acids: tyrosine, alanine, methionine, aspartic acid, threonine and lysine) and a small amount of organic acid salts (mainly comprising calcium lactate and calcium propionate). The edible antioxidant is in liquid state, and the mass of the main active ingredients of the edible antioxidant accounts for about 10% of the total mass through detection.
Application Experimental example 1
The edible antioxidant obtained in example 1, sodium D-erythorbate and BHT were added to the ingredients for conditioning meat for treatment, respectively, and the antioxidant effect of the three antioxidants on the conditioned meat was compared, and the results are shown in fig. 1. The steps of treating the conditioned meat are as follows: raw and auxiliary materials → pretreatment (removing fascia tissues and the like) → weighing (the proportion of the fat lining of the pig is 20%) → mincing → adding ingredients → chopping → forming → packaging → low-temperature storage at 8 ℃. Wherein, taking the quality of the pork as a reference, the quality of the ingredients accounts for the following mass percent of the pork: 0.6% of granulated sugar, 0.5% of monosodium glutamate, 2% of cooking wine, 2% of ginger, 6% of green onion, 2% of salt, 8% of starch, 20% of crushed ice and an antioxidant. The first group is blank, i.e. no antioxidant is added; in the second group, the antioxidant in the ingredients was the edible antioxidant obtained in example 1, and the addition amount was 12g/kg (i.e. the addition amount of the active ingredient was 1.2 g/kg); in the third group, the antioxidant in the ingredients is D-sodium erythorbate, and the addition amount is 0.8g/kg; in the fourth group, the antioxidant in the formulation was BHT, added at 0.2g/kg.
As can be seen from FIG. 1, the edible antioxidant obtained in example 1 of the present invention has a good antioxidant effect after being treated on the conditioned meat, and the TBA value of the conditioned meat added with the edible antioxidant obtained in example 1 of the present invention is slightly different from the TBA value of the conditioned meat added with BHT and sodium D-erythorbate, but not significantly different from the TBA value of the conditioned meat stored under refrigeration for 15 days. Therefore, the edible antioxidant obtained in the embodiment 1 of the invention can greatly reduce the oxidative deterioration of oil components in the meat product and improve the edible safety of the meat product.
Application Experimental example 2
The edible antioxidant obtained in example 2, sodium D-erythorbate and BHT were added to the ingredients of the roast cake for treatment, respectively, and the antioxidant effect of the three antioxidants on the roast cake was compared, and the results are shown in fig. 2. The steps of processing the roast cake are as follows: the pig fat and lean meat (the proportion of the pig fat is 20%) → cutting, chopping → adding ingredients → chopping → extrusion molding (cake shape) → baking (190 ℃,15-20min, the central temperature of the meat cake reaches 75 ℃) → cooling → packaging → 17 ℃ storage. Wherein, taking the quality of pork as a reference, the quality of the ingredients accounts for the following mass percent of the pig: 6% of starch, 2% of table salt, 0.2% of white pepper powder, 0.26% of mixed composite phosphate, 0.3% of green onion, 13% of crushed ice and an antioxidant, wherein four groups are set in the experimental example, and the difference lies in selection of the antioxidant in the formula of the added ingredients. The first group is blank, i.e. no antioxidant is added; in the second group, the antioxidant in the ingredient is the edible antioxidant obtained in example 2, and the addition amount is 12g/kg (namely the addition amount of the active ingredient is 1.2 g/kg); in the third group, the antioxidant in the ingredients is D-sodium erythorbate, and the addition amount is 0.8g/kg; in the fourth group, the antioxidant in the formulation was BHT, added at 0.2g/kg.
As can be seen from FIG. 2, the edible antioxidant obtained in example 2 of the present invention has a good antioxidant effect after the treatment of the roast cake. The BBQ cake added with the edible antioxidant obtained in the example 2 of the invention has no significant difference in TBA value from the BBQ cake added with BHT and sodium D-erythorbate when stored for 21 days at 17 ℃. The edible antioxidant obtained in the embodiment 2 of the invention can greatly reduce the oxidative deterioration of oil components in the baked meat cake, the antioxidant effect of the edible antioxidant can completely replace the traditional chemical antioxidant BHT, and the edible safety of the baked meat cake can be improved.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (3)
1. The preparation method of the edible antioxidant is characterized by comprising the following steps:
mixing the grain powder with water to obtain slurry; wherein the mass ratio of the grain powder to the water is (6-12.7) 100, the grain powder is selected from at least one of flour, rice flour, corn flour and tapioca flour, and when the grain powder is selected from at least one of rice flour, corn flour and tapioca flour, yeast extract accounting for 0.2% of the mass of the grain powder is also added;
sequentially carrying out protease enzymolysis treatment, amylase liquefaction treatment and saccharification enzymolysis treatment on the slurry to obtain an enzymolysis solution;
sterilizing the enzymolysis solution under high pressure at 115-121 ℃, and inactivating the enzyme for 20-30 min; inoculating propionibacterium into the enzymolysis liquid for fermentation culture to obtain fermentation liquid; the Propionibacterium is selected self-producing propionibacterium (A), (B), (C)Propionibacterium acidipropionici) Or Propionibacterium freudenreichii subspecies sjohnsonia (Propionibacterium freudenreichii subsp.shermanii) The fermentation culture process comprises a first-stage amplification culture and a second-stage amplification culture, wherein the first-stage amplification culture process comprises the following steps: inoculating the propionibacterium into a triangular flask containing a seed culture medium, and culturing for 24 hours at the temperature of 30-34 ℃, wherein the seed culture medium comprises 0.5-1.0% of glycerol, 1-2% of yeast extract, 0.5-1% of peptone, 0.3-0.5% of dipotassium hydrogen phosphate and 0.25-0.3% of monopotassium phosphate, and the balance of water, the seed culture medium has the pH of 7.0, and the secondary amplification culture process comprises the following steps: inoculating the propionibacterium after the first-stage amplification culture into a seeding tank containing a seed culture medium, and culturing for 48 hours at the temperature of 30-34 ℃;
heating the fermentation liquor to 60 ℃ to inactivate the propionibacterium, carrying out reduced pressure evaporation and concentration on the fermentation liquor, crystallizing propionate, and then centrifuging at the rotating speed of 2000rpm-2500rpm for 5min-8min to remove the propionate in the fermentation liquor, so as to obtain the edible antioxidant;
the protease enzymolysis treatment method comprises the following steps: adjusting the pH value of the slurry to 9.0-11.0, adding protease, carrying out enzymolysis reaction at 50 ℃ until the pH value is 6.0-7.0, and the pH value does not drop within half an hour, wherein the protease is selected from alkaline protease or neutral protease;
the method for liquefying amylase comprises the following steps: adjusting the pH value of the slurry subjected to protease enzymolysis to 6.0, adding amylase, and performing hydrolysis reaction at 90 ℃ for 20min, wherein the amylase is high-temperature amylase;
the method for the enzymatic hydrolysis treatment of the saccharifying enzyme comprises the following steps: adjusting the pH value of the slurry after the amylase liquefaction treatment to 4.5, adding 10U-200U of glucoamylase into each gram of starch according to the mass of the starch in the grain powder, and then carrying out enzymolysis reaction at 50 ℃ for 20-24 h.
2. An edible antioxidant prepared by the method of claim 1.
3. Use of the edible antioxidant according to claim 2 in food products for antioxidant purposes.
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