Wheat sauce with anti-oxidation and anti-inflammatory effects as well as preparation method and application of wheat sauce

Abstract

The invention provides a wheat sauce with anti-oxidation and anti-inflammatory effects as well as a preparation method and application of the wheat sauce. According to the preparation technology of the wheat sauce, the quality of the produced wheat sauce is stable, large-scale production is facilitated, the equipment is simple, and the cost is low; the produced wheat sauce is reddish brown or reddish brown, moderate in thickness, rich in wheat fragrance, and mellow in ester fragrance and sauce fragrance. The amino acid nitrogen content of the wheat sauce is obviously higher than the lowest limit value specified by the state and is higher than that of most commercially available wheat sauces; the salt content is lower than that of most commercially available wheat sauce. The wheat sauce disclosed by the invention also has the effects of resisting oxidation, resisting inflammation, diminishing swelling and reducing oxidative stress injury, is a functional seasoning sauce, and can also be used for preparing an anti-oxidation and anti-inflammatory preparation. The nutritional and healthy functions of the wheat sauce are deeply excavated, healthy development, transformation and upgrading of traditional fermented seasonings are promoted, the economic benefits of the product are improved, and scientific and technological support is provided for protecting and inheriting national traditional food.

Description

Technical Field

[0001] The invention relates to the technical field of food engineering, and in particular to a wheat sauce with anti-oxidation and anti-inflammatory effects, and a preparation method and application thereof. Background Art

[0002] As people's health awareness continues to increase, condiments will inevitably develop in the direction of functionalization. Functional condiments can not only bring rich taste and flavor to food, but also provide various nutritional and health benefits to the human body. At present, the conventional method of developing functional condiments is to add micronutrients to condiments to enhance nutritional functions, but this method has high R&D costs and low market acceptance. However, the functional exploration and development of traditional fermented condiments is less, and they have great market potential.

[0003] Although modern processing technology can unify processing conditions and stabilize product quality, the products are usually not as mellow, unique, and rich in taste as traditional methods. Many traditional processed foods have many advantages: (1) They carry the wisdom and regional culture of generations, are a vivid embodiment of traditional culture, and are of great significance to cultural inheritance; (2) They rely more on natural ingredients and simple physical methods, use less chemical additives, reduce food safety risks, and retain the nutritional components of ingredients; (3) They usually go through complex processes and involve complex and rich microorganisms. The finished products contain a variety of enzymes and nutrients and have certain health benefits; (4) The equipment required is simple and the investment is small, which reduces production costs and makes the product price more affordable; (5) Most of them are labor-intensive and can absorb a large number of workers, drive employment, and promote local economic development.

[0004] At present, there are literatures that study the functional effects of soy sauce and vinegar in traditional fermented condiments, but there is a lack of research on the functionality of other solid sauces and semi-solid sauces. The functional effects of soy sauce and vinegar are mainly studied in terms of antioxidant function, and less attention is paid to anti-inflammatory ability. Solid sauce and semi-solid sauce convert and release the functional active ingredients in the raw materials through fermentation, and their functional effects need to be explored urgently.

[0005] Wheat sauce is made from wheat grains through the steps of steaming, drying, grinding, and mixing and fermenting raw and auxiliary materials. The product is not only delicious, but also nutritious, convenient to eat, economical, and can replace soy sauce, and is deeply loved by residents in the production area. The production method of wheat sauce is mainly family workshops, and the processing methods of each family are not uniform. Even the quality of wheat sauce products made by the same family is unstable. Therefore, large-scale production has not been achieved and it is not well-known outside the production area.

[0006] In summary, the current production methods of wheat paste are not uniform, the product quality is unstable, and the potential nutritional and health functions of wheat paste have not been explored. Based on this, the present invention is specially proposed. Summary of the invention

[0007] In view of this, the present invention provides a wheat paste with antioxidant and anti-inflammatory effects and a preparation method and application thereof, so as to solve or at least partially solve the defects existing in the prior art.

[0008] In a first aspect, the present invention provides a method for preparing a wheat paste having antioxidant and anti-inflammatory effects, comprising the following steps:

[0009] Add water to the wheat until it covers the wheat and heat and cook;

[0010] The steamed wheat is naturally fermented in a sealed environment at a temperature of 25 to 35°C and a relative humidity of 70% to 90% for 3 to 7 days;

[0011] The naturally fermented wheat is dried in the sun, cleaned, dried again, crushed, and sieved to obtain the sauce noodles;

[0012] After crushing the fresh pepper at room temperature, adding salt and marinating for 2-6 hours, removing excess water to obtain the marinated pepper;

[0013] The noodle sauce, Sichuan peppercorns and pickled chili peppers are mixed and fermented in a sealed environment at a temperature of 25-35°C for more than 20-35 days. After the fermentation is completed, the wheat sauce with antioxidant and anti-inflammatory effects is obtained.

[0014] Preferably, the cooked wheat is spread out in a sealed environment to a wheat pile with a thickness of 2 to 6 cm, and naturally fermented for 3 to 7 days at a temperature of 25 to 35° C. and a relative humidity of 70% to 90%;

[0015] During the fermentation period, when the center temperature of the wheat pile is higher than 30°C, it is turned over twice a day; during the fermentation period, when the center temperature of the wheat pile is ≤30°C, it is turned over once a day.

[0016] Preferably, the mass ratio of the sauce noodles, salt, pepper and fresh pepper is 1:(0.14-0.17):(0.004-0.02):(1.4-1.7).

[0017] Preferably, water is added to the wheat until the wheat is covered, and then the wheat is heated and cooked at 90-100° C. for 20-50 minutes.

[0018] Preferably, the naturally fermented wheat is air-dried, washed, air-dried again, crushed, and passed through a 40-90 mesh sieve to obtain the sauce noodles.

[0019] Preferably, the sauce noodles, peppercorns and pickled peppers are mixed and fermented in a sealed environment at a temperature of 25-35° C. for more than 20-35 days. When the color of the fermented product changes from bright red to reddish brown or tan, the fermentation is completed, and a wheat sauce with antioxidant and anti-inflammatory effects is obtained.

[0020] In a second aspect, the present invention further provides a wheat paste with antioxidant and anti-inflammatory effects, which is prepared by the preparation method.

[0021] In a third aspect, the present invention also provides a use of the wheat paste having antioxidant and anti-inflammatory effects in the preparation of antioxidant and anti-inflammatory preparations.

[0022] Preferably, the application, the method for preparing the antioxidant and anti-inflammatory preparation comprises the following steps:

[0023] The wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, and the first filtrate is concentrated by rotary evaporation and freeze-dried to obtain an ethanol extract, which is an antioxidant and anti-inflammatory preparation;

[0024] Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate and a filter residue, water is added to the filter residue, extracted in a homogenizer, filtered to obtain a second filtrate, and the second filtrate is rotary evaporated and freeze-dried to obtain a water extract, which is an antioxidant and anti-inflammatory preparation;

[0025] Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, the first filtrate is concentrated by rotary evaporation to obtain an ethanol extract, the ethanol extract is dissolved in water, petroleum ether is added for extraction multiple times, the petroleum ether phases are combined, vacuum rotary evaporation and freeze-dried to obtain a petroleum ether extract, which is an antioxidant and anti-inflammatory preparation;

[0026] Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, the first filtrate is concentrated by rotary evaporation to obtain an ethanol extract, the ethanol extract is dissolved in water, petroleum ether is added to extract multiple times, the aqueous phases are combined, the aqueous phases are extracted multiple times with ethyl acetate, the ethyl acetate phases are combined, and vacuum rotary evaporation and freeze-drying are performed to obtain an ethyl acetate extract, which is an antioxidant and anti-inflammatory preparation;

[0027] Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, the first filtrate is concentrated by rotary evaporation to obtain an ethanol extract, the ethanol extract is dissolved in water, petroleum ether is added and extracted multiple times, the aqueous phases are combined, the aqueous phases are extracted multiple times with ethyl acetate, the extracted aqueous phases are combined and extracted multiple times with n-butanol, the n-butanol phases are combined, and vacuum rotary evaporation and freeze-drying are performed to obtain n-butanol extract, which is an antioxidant and anti-inflammatory preparation.

[0028] The wheat sauce with antioxidant and anti-inflammatory effects and its preparation method and application have the following advantages over the prior art:

[0029] Beneficial effects:

[0030] 1. The method for preparing the wheat sauce with antioxidant and anti-inflammatory effects of the present invention standardizes and unifies the preparation process of wheat sauce, stabilizes the quality of wheat sauce products, and is conducive to large-scale production; the wheat sauce with antioxidant and anti-inflammatory effects prepared by the present invention is reddish brown or reddish brown, and has moderate thickness, rich wheat aroma, mellow ester aroma, pleasant salty and fresh taste, and a long aftertaste. The amino acid nitrogen content is higher than the minimum limit of amino acid nitrogen stipulated by the state for fermented sauces, and higher than most commercially available wheat sauces; the salt content is lower than most commercially available wheat sauces; the content of aflatoxin B1 in the wheat sauce meets the limit requirements for brewing sauces in GB 2761-2017;

[0031] 2. The wheat sauce prepared by the present invention has the effects of anti-oxidation, anti-inflammatory and detumescence, and reducing oxidative stress damage. It is a functional seasoning sauce and can also be used to prepare antioxidant and anti-inflammatory preparations. The present invention deeply explores the nutritional and health functions of wheat sauce, deepens the understanding of wheat sauce, promotes the healthy development and transformation and upgrading of traditional fermented condiments, improves the economic benefits of products, and provides scientific and technological support for the protection and inheritance of national geographical indication products.

[0032] 3. The present invention uses different polar reagents to extract new malt sauce (storage time one year) and old malt sauce (storage time two years) to obtain different extracts of malt sauce, and detects OH free radicals, ABTS + The antioxidant activity of wheat paste and its extracts was evaluated by free radical, DPPH free radical and iron ion reducing ability. The extracts of new and old wheat pastes all had antioxidant capacity, and the antioxidant capacity of ethyl acetate extract and petroleum ether extract was more prominent among the extracts.

[0033] 4. The present invention clarifies the anti-inflammatory activity of wheat paste by detecting the changes in NO content after the new and old wheat paste extracts acted on the lipopolysaccharide-induced RAW264.7 cell inflammation model. The new paste extracts are generally more anti-inflammatory than the old paste extracts. The anti-inflammatory effects of the new paste extracts are as follows from large to small: ethyl acetate extract > n-butanol extract > water extract > ethanol extract > petroleum ether extract. The ethyl acetate extract of the new paste has the strongest inhibitory effect on NO. When the concentration is 100μg / mL, the NO content decreases by 76%; when the concentration is 300μg / mL, the NO content decreases by 98.2%;

[0034] 5. The present invention established a xylene-induced mouse ear swelling inflammation model, and applied the extracts of the new sauce to treat the mice by applying. It was found that each extract could significantly reduce the swelling rate of the mouse ears and reduce the NO content in the mouse serum. Among them, the best effect was the petroleum ether extract of wheat sauce, with the highest swelling inhibition rate of 48% and the lowest NO content of 3.33μmol / L;

[0035] 6. The present invention studies the anti-inflammatory and detumescent effects of the new sauce ethyl acetate extract on mice by intragastric administration. The extract has significant anti-inflammatory and detumescent effects. The low-dose group can reduce the ear swelling rate to 55%, and the high-dose group can reduce the swelling rate to 38%. Both the low-dose and high-dose groups can reduce the levels of inflammatory factors NO and PEG2, inflammatory-related enzymes INOS and COX-2, and oxidative stress factor MDA to a certain extent, indicating that the wheat sauce extract has the effects of anti-inflammatory and detumescent, and reducing oxidative stress damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0037] Figure 1 The amino acid nitrogen content of different wheat sauce products;

[0038] Figure 2 The results of salt content determination for different wheat sauce products;

[0039] Figure 3 Aflatoxin B1 content in wheat paste;

[0040] Figure 4 Flow chart of extraction of different extracts of wheat paste;

[0041] Figure 5 The scavenging effect of the extracts of new sauce and old sauce on OH free radicals;

[0042] Figure 6 The scavenging effect of the extracts of new sauce and old sauce on DPPH free radicals;

[0043] Figure 7 ABTS for each extract of new sauce and old sauce + Free radical scavenging effect;

[0044] Figure 8 The NO content of RAW264.7 cells stimulated by LPS after different extracts of new and old wheat paste acted on them;

[0045] Fig. 9 The inhibitory effects of different extracts of Xinjiang on mouse ear swelling by application (A) and the changes in NO content in serum (B);

[0046] Fig.10 The inhibitory effect of the ethyl acetate extract of Xinjiang on ear swelling in mice by oral administration;

[0047] Fig.11 This study is the effect of new sauce ethyl acetate extract on the inflammatory factors NO (A), PEG2 (B), INOS (C), COX-2 (D), and oxidative stress factors SOD (E) and MDA (F) in the serum of mice with ear swelling model by oral gavage. DETAILED DESCRIPTION

[0048] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of the present application, the term "including" means "including but not limited to". Various embodiments of the present invention may be in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated in this article, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0050] The present invention provides a method for preparing a wheat paste having antioxidant and anti-inflammatory effects, comprising the following steps:

[0051] S1. Add water to the wheat until it covers the wheat and heat and cook;

[0052] S2, fermenting the steamed wheat in a sealed environment at a temperature of 25 to 35° C. and a relative humidity of 70% to 90% for 3 to 7 days;

[0053] S3, drying the naturally fermented wheat, washing it, drying it again, crushing it, and sieving it to obtain noodles;

[0054] S4, crushing the fresh peppers at room temperature, adding salt and marinating for 2 to 6 hours, and then removing excess water to obtain marinated peppers;

[0055] S5. Mix the sauce noodles, peppercorns and pickled chili peppers, and ferment them in a sealed environment at a temperature of 25-35° C. for more than 20-35 days. After the fermentation is completed, the wheat sauce with antioxidant and anti-inflammatory effects is obtained.

[0056] In some embodiments, the cooked wheat is spread out in a sealed environment to a wheat pile with a thickness of 2 to 6 cm, and naturally fermented for 3 to 7 days at a temperature of 25 to 35° C. and a relative humidity of 70% to 90%;

[0057] During the fermentation period, when the center temperature of the wheat pile is higher than 30°C, it is turned over twice a day; during the fermentation period, when the center temperature of the wheat pile is ≤30°C, it is turned over once a day.

[0058] In some embodiments, the mass ratio of the sauce noodles, salt, peppercorns, and fresh chili peppers is 1:(0.14-0.17):(0.004-0.02):(1.4-1.7).

[0059] In some embodiments, water is added to the wheat until the wheat is covered, and then the wheat is heated and cooked at 90-100° C. for 20-50 min.

[0060] In some embodiments, the naturally fermented wheat is air-dried, washed, air-dried again, crushed, and passed through a 40-90 mesh sieve to obtain the sauce noodles.

[0061] In some embodiments, the sauce noodles, peppercorns and pickled peppers are mixed and fermented in a sealed environment at a temperature of 25-35°C for more than 20-35 days. When the color of the fermented product changes from bright red to reddish brown or brown, the fermentation is completed, and a wheat sauce with antioxidant and anti-inflammatory effects is obtained.

[0062] In some embodiments, a method for preparing a wheat paste having antioxidant and anti-inflammatory effects is provided, comprising the following steps:

[0063] S1. Wheat impurity removal and cleaning: Select wheat with full grains, wash with clean water, and remove floating matter and dust on the upper layer;

[0064] S2. Steaming: Add water until the surface of the wheat is covered and cook at 90-100°C. Continue steaming for 20-50 minutes after boiling until the internal cross section of the wheat is transparent and has no white color. Be careful not to cook until the wheat blooms.

[0065] S3, natural fermentation: put the cooked wheat into the room to cool, and spread it flat to a pile thickness of 2 to 6 cm. The temperature is maintained at 25 to 35 degrees Celsius, and the relative humidity is 70% to 90%. Ferment in a closed room for 3 to 7 days. During the fermentation period, the wheat pile is turned over and stirred twice a day when the center temperature is higher than 30 degrees Celsius, and once a day for the rest. When the surface of the wheat is covered with yellow-green mold powder, spores fly when it is turned over and stirred, and it has a koji aroma, the fermentation process can be ended;

[0066] S4, drying: placing the naturally fermented wheat in the sun to dry to remove some of the moisture, making it easier to wash;

[0067] S5. Cleaning: Wash until there is no visible mold and spores and impurities introduced from the environment;

[0068] S6. Drying: Dry the wheat until the surface is dry, there is a crisp sound when touched, and the color is slightly white;

[0069] S7, grinding: grind the dried wheat, pass it through a 40-90 mesh sieve, and seal the obtained noodles in a cool and dry place for later use;

[0070] S8. Pepper processing: select fresh peppers, remove impurities, wash and chop or crush at room temperature, add salt and marinate for 2 to 6 hours, then remove excess water and set aside;

[0071] S9, mixed fermentation: put the sauce noodles obtained in step S7 and the pickled peppers obtained in step S8 into a jar or other container and mix them; seal and place them at an ambient temperature of 25-35° C. during fermentation for more than 20-35 days, and fermentation is completed when the color of the wheat sauce changes from bright red to reddish brown or brownish brown, and the shape changes from dry and dispersed to viscous, and there is a distinct sauce flavor; wherein the mass ratio of the sauce noodles, salt, pepper, and fresh pepper is 1:(0.14-0.17):(0.004-0.02):(1.4-1.7).

[0072] Based on the same inventive concept, the present invention also provides a wheat sauce with antioxidant and anti-inflammatory effects, which is prepared by the above-mentioned preparation method.

[0073] Based on the same inventive concept, the present invention also provides an application of the above-mentioned wheat paste with antioxidant and anti-inflammatory effects in the preparation of antioxidant and anti-inflammatory preparations.

[0074] In some embodiments, the method for preparing the antioxidant and anti-inflammatory preparation comprises the following steps:

[0075] The wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, and the first filtrate is concentrated by rotary evaporation and freeze-dried to obtain an ethanol extract, which is an antioxidant and anti-inflammatory preparation;

[0076] Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate and a filter residue, water is added to the filter residue, extracted in a homogenizer, filtered to obtain a second filtrate, and the second filtrate is rotary evaporated and freeze-dried to obtain a water extract, which is an antioxidant and anti-inflammatory preparation;

[0077] Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, the first filtrate is concentrated by rotary evaporation to obtain an ethanol extract, the ethanol extract is dissolved in water, petroleum ether is added for extraction multiple times, the petroleum ether phases are combined, vacuum rotary evaporation and freeze-dried to obtain a petroleum ether extract, which is an antioxidant and anti-inflammatory preparation;

[0078] Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, the first filtrate is concentrated by rotary evaporation to obtain an ethanol extract, the ethanol extract is dissolved in water, petroleum ether is added to extract multiple times, the aqueous phases are combined, the aqueous phases are extracted multiple times with ethyl acetate, the ethyl acetate phases are combined, and vacuum rotary evaporation and freeze-drying are performed to obtain an ethyl acetate extract, which is an antioxidant and anti-inflammatory preparation;

[0079] Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, the first filtrate is concentrated by rotary evaporation to obtain an ethanol extract, the ethanol extract is dissolved in water, petroleum ether is added and extracted multiple times, the aqueous phases are combined, the aqueous phases are extracted multiple times with ethyl acetate, the extracted aqueous phases are combined and extracted multiple times with n-butanol, the n-butanol phases are combined, and vacuum rotary evaporation and freeze-drying are performed to obtain n-butanol extract, which is an antioxidant and anti-inflammatory preparation.

[0080] In some embodiments, 100 g of wheat paste (the dry weight of wheat paste is about 45 g), 900 mL of anhydrous ethanol is added at a solid-liquid ratio of 1:20 (i.e., the dry weight of wheat paste: anhydrous ethanol = 45 g: 900 mL = 1 g: 20 mL), and the mixture is extracted with a high-speed homogenizer at 10000 rpm / min for 3 min, filtered to obtain a first filtrate, and concentrated and freeze-dried by rotary evaporation to obtain an ethanol extract; 800 mL of ultrapure water is added to the filter residue obtained by filtration after alcohol extraction, and extracted with a high-speed homogenizer at 10000 rpm / min for 3 min, and a second filtrate is obtained by suction filtration, and the water extract is obtained by rotary evaporation and freeze-drying; A portion of the ethanol extract is hot-dissolved with a certain volume of ultrapure water (just dissolve), extracted three times with three volumes of petroleum ether (double the volume each time), the petroleum ether phases are combined, and the petroleum ether extract is obtained by rotary evaporation in vacuum and freeze-drying; the remaining aqueous phase is extracted three times with three volumes of ethyl acetate (double the volume each time), the ethyl acetate phases are combined, and the ethyl acetate is removed by rotary evaporation in vacuum and freeze-dried to obtain an ethyl acetate extract; the remaining aqueous phase is extracted three times with three volumes of n-butanol (double the volume each time), the n-butanol phases are combined, the n-butanol extraction layer is vacuum rotary evaporated and freeze-dried to remove the n-butanol, and the n-butanol extract is obtained.

[0081] The following further illustrates the antioxidant and anti-inflammatory wheat paste of the present application and its preparation method and application with specific examples. This section further illustrates the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0082] Example 1

[0083] The present invention provides a method for preparing a wheat paste having antioxidant and anti-inflammatory effects, comprising the following steps:

[0084] S1. Add water to the wheat until it covers the wheat, and heat and cook at 98°C for 30 minutes;

[0085] S2, spreading the cooked wheat in a sealed environment to a wheat pile with a thickness of 6 cm, and naturally fermenting it for 7 days at a temperature of 30° C. and a relative humidity of 70%;

[0086] During the fermentation period, when the temperature at the center of the wheat pile is higher than 30°C, the wheat pile is turned over twice a day; during the fermentation period, when the temperature at the center of the wheat pile is ≤30°C, the wheat pile is turned over once a day;

[0087] S3, drying the naturally fermented wheat, washing it, drying it again, crushing it, and passing it through a 40-mesh sieve to obtain a sauce noodle;

[0088] S4, crushing the fresh peppers at room temperature (23° C.), adding salt and marinating for 6 hours, and then removing excess water to obtain marinated peppers;

[0089] S5. Mix the sauce noodles, Chinese prickly ash and pickled peppers, and ferment them in a sealed environment at a temperature of 28° C. for more than 28 days. When the color of the wheat sauce changes from bright red to reddish brown or tan, a wheat sauce (denoted as XJ-1) with antioxidant and anti-inflammatory effects is obtained; wherein the mass ratio of the sauce noodles, salt, Chinese prickly ash and fresh peppers is 1:0.15:0.01:1.6.

[0090] Example 2

[0091] The present invention provides a method for preparing a wheat paste having antioxidant and anti-inflammatory effects, comprising the following steps:

[0092] S1. Add water to the wheat until it covers the wheat, and heat and cook at 100°C for 20 minutes;

[0093] S2, spreading the cooked wheat in a sealed environment to a wheat pile with a thickness of 5 cm, and naturally fermenting it for 3 days at a temperature of 35° C. and a relative humidity of 90%;

[0094] During the fermentation period, when the temperature at the center of the wheat pile is higher than 30°C, the wheat pile is turned over twice a day; during the fermentation period, when the temperature at the center of the wheat pile is ≤30°C, the wheat pile is turned over once a day;

[0095] S3, drying the naturally fermented wheat, washing it, drying it again, crushing it, and passing it through an 80-mesh sieve to obtain a sauce noodle;

[0096] S4, crushing the fresh peppers at room temperature (25° C.), adding salt and marinating for 4 hours, and then removing excess water to obtain marinated peppers;

[0097] S5. Mix the sauce noodles, Chinese prickly ash and pickled peppers, and ferment them in a sealed environment at a temperature of 32 ° C for more than 32 days. When the color of the wheat sauce changes from bright red to reddish brown or tan, a wheat sauce (denoted as XJ-2) with antioxidant and anti-inflammatory effects is obtained; wherein the mass ratio of the sauce noodles, salt, Chinese prickly ash and fresh peppers is 1:0.16:0.02:1.4.

[0098] Example 3

[0099] The present invention provides a method for preparing a wheat paste having antioxidant and anti-inflammatory effects, comprising the following steps:

[0100] S1. Add water to the wheat until it covers the wheat, and heat and cook at 90°C for 50 minutes;

[0101] S2, spreading the cooked wheat in a sealed environment to a wheat pile with a thickness of 2 cm, and naturally fermenting it for 6 days at a temperature of 25° C. and a relative humidity of 75%;

[0102] During the fermentation period, when the temperature at the center of the wheat pile is higher than 30°C, the wheat pile is turned over twice a day; during the fermentation period, when the temperature at the center of the wheat pile is ≤30°C, the wheat pile is turned over once a day;

[0103] S3, drying the naturally fermented wheat, washing it, drying it again, crushing it, and passing it through a 90-mesh sieve to obtain a sauce noodle;

[0104] S4, crushing the fresh peppers at room temperature (20° C.), adding salt and marinating for 2 hours, and then removing excess water to obtain marinated peppers;

[0105] S5. Mix the sauce noodles, Chinese prickly ash and pickled peppers, and ferment them in a sealed environment at a temperature of 25 ° C for more than 35 days. When the color of the wheat sauce changes from bright red to reddish brown or tan, a wheat sauce (denoted as XJ-3) with antioxidant and anti-inflammatory effects is obtained; wherein the mass ratio of the sauce noodles, salt, Chinese prickly ash and fresh pepper is 1:0.17:0.015:1.7.

[0106] Example 4

[0107] The present invention provides a method for preparing a wheat paste having antioxidant and anti-inflammatory effects, comprising the following steps:

[0108] S1. Add water to the wheat until it covers the wheat, and heat and cook at 100°C for 40 minutes;

[0109] S2, spreading the cooked wheat in a sealed environment to a wheat pile with a thickness of 4 cm, and naturally fermenting it for 5 days at a temperature of 32° C. and a relative humidity of 80%;

[0110] During the fermentation period, when the temperature at the center of the wheat pile is higher than 30°C, the wheat pile is turned over twice a day; during the fermentation period, when the temperature at the center of the wheat pile is ≤30°C, the wheat pile is turned over once a day;

[0111] S3, drying the naturally fermented wheat, washing it, drying it again, crushing it, and passing it through a 70-mesh sieve to obtain a sauce noodle;

[0112] S4, crushing the fresh peppers at room temperature (25° C.), adding salt and marinating for 5 hours, and then removing excess water to obtain marinated peppers;

[0113] S5. Mix the sauce noodles, Chinese prickly ash and pickled peppers, and ferment them in a sealed environment at a temperature of 35° C. for more than 20 days. When the color of the wheat sauce changes from bright red to reddish brown or tan, a wheat sauce (denoted as XJ-4) with antioxidant and anti-inflammatory effects is obtained; wherein the mass ratio of the sauce noodles, salt, Chinese prickly ash and fresh peppers is 1:0.14:0.004:1.5.

[0114] Example 5

[0115] The present application provides an application of a wheat paste having antioxidant and anti-inflammatory effects in the preparation of an antioxidant and anti-inflammatory preparation, and the application specifically comprises the following steps:

[0116] Preparation of ethanol extract: 100 g of XJ-1 wheat paste (wheat paste dry weight 45 g) prepared in Example 1 was stored for 1 or 2 years (storage for 1 year is new sauce, storage for 2 years is old sauce), 900 mL of anhydrous ethanol was added at a solid-liquid ratio of 1:20, and extracted for 3 minutes using a high-speed homogenizer at 10000 rpm / min, and the filtrate was filtered and concentrated by rotary evaporation to obtain an ethanol extract (referred to as VY-E), which is an antioxidant and anti-inflammatory preparation;

[0117] Or, the preparation of the water extract, according to the above method, collect the filter residue after ethanol extraction (that is, take 100g of XJ-1 wheat paste prepared in Example 1 (wheat paste dry weight is 45g), add 900mL of anhydrous ethanol at a solid-liquid ratio of 1:20, use a high-speed homogenizer at 10000rpm / min to extract for 3min, filter to obtain a filtrate, and filter to obtain a filter residue), add 800mL of ultrapure water to the filter residue, use a high-speed homogenizer at 10000rpm / min to extract for 3min, filter to obtain a filtrate, and rotary evaporate to obtain a water extract (recorded as VY-L), which is an antioxidant and anti-inflammatory preparation;

[0118] Or, the preparation of petroleum ether extract, a part of ethanol extract (specifically 1 / 4 of the mass of ethanol extract) is hot-dissolved (dissolved) with a certain volume of ultrapure water, extracted three times with three volumes of petroleum ether (double the volume each time), the petroleum ether layers are combined, and vacuum rotary evaporated to obtain the petroleum ether extract (VY-L), which is an antioxidant and anti-inflammatory preparation;

[0119] Or, the preparation of the ethyl acetate extract, after extraction with petroleum ether, the remaining aqueous phase (i.e., a portion of the ethanol extract is hot-dissolved with a certain volume of ultrapure water (just dissolve), extracted 3 times with three volumes of petroleum ether, and the aqueous phases are combined) is then extracted 3 times with three volumes of ethyl acetate (double the volume each time), the ethyl acetate phases are combined, and the ethyl acetate extraction layer is vacuum-rotated to remove the ethyl acetate to obtain the ethyl acetate extract (VY-EA), which is an antioxidant and anti-inflammatory preparation;

[0120] Alternatively, the preparation of the n-butanol extract is carried out by extracting with ethyl acetate, and then extracting the remaining aqueous phase (i.e., after extraction with petroleum ether, the remaining aqueous phase is extracted three times with three volumes of ethyl acetate, and the aqueous phases are combined) with three volumes of n-butanol for three times (double the volume each time), combining the n-butanol phases, and vacuum rotary evaporating the n-butanol extraction layer to remove the n-butanol to obtain the n-butanol extract (VY-N), which is an antioxidant and anti-inflammatory preparation.

[0121] Specifically, the preparation process of ethanol extract, water extract, petroleum ether extract, ethyl acetate extract, and n-butanol extract is as follows Figure 4 .

[0122] Performance Characterization

[0123] 1. Characterization of sensory organs, amino acid nitrogen content, salt content, and aflatoxin B1 content of wheat sauces XJ-1, XJ-2, XJ-3, and XJ-4

[0124] Sensory evaluation was performed on the wheat sauces XJ-1, XJ-2, XJ-3 and XJ-4 with antioxidant and anti-inflammatory effects prepared in Examples 1 to 4; the amino acid nitrogen content and salt content of the wheat sauces XJ-1, XJ-2, XJ-3 and XJ-4 with antioxidant and anti-inflammatory effects prepared in Examples 1 to 4 and commercially available wheat sauces were tested. Figures 1-2 As shown; the amino acid nitrogen content of the finished malt sauce was determined by formaldehyde potentiometric titration, and the operating method referred to GB5009.235-2016 "National Food Safety Standard Determination of Amino Acid Nitrogen in Food"; the salt content of malt sauce was determined by precipitation method, and the operating steps referred to GB / T5009.40-2003 "Analysis Method of Sauce Hygiene Standard".

[0125] Figures 1-2 Among them, BJ-1 is Dafang sun-dried sauce, produced in Dafang County, Bijie City, Guizhou Province; BK-1 and BK-2 are spicy wheat sauce and spicy wheat sauce respectively, produced in Baokang County, Xiangyang City, Hubei Province; JZ-1 and JZ-2 are farmhouse pure wheat sauce (original flavor) and farmhouse pure wheat sauce (slightly spicy), produced in Siche Village, Chengji Town, Jianli City, Hubei Province.

[0126] Sensory evaluation results of wheat sauce: the wheat sauce prepared by the present invention is reddish brown or reddish brown, and has moderate thickness, rich wheat aroma, mellow ester aroma, pleasant salty and fresh taste and long aftertaste. The spiciness of the wheat sauce can be accepted by most people, avoiding the problem of limited sales of products due to spicy discomfort caused by differences in regions and eating habits.

[0127] Amino acid nitrogen refers to the content of nitrogen in the form of amino acids. It is a standard for measuring product quality and flavor, and is also a key indicator for evaluating production processes and product quality. The higher the amino acid nitrogen content, the better the product quality. Figure 1 is the amino acid nitrogen content of different wheat sauce finished products. The amino acid nitrogen contents of XJ-1, XJ-2, XJ-3 and XJ-4 are 0.51g / 100g, 0.55g / 100g, 0.48g / 100g and 0.48g / 100g, respectively, indicating that the amino acid nitrogen content of the wheat sauce prepared by the present invention is stable. All samples are higher than the minimum amino acid nitrogen content (0.3g / 100g) of fermented sauce products specified in GB 2718-2014. The amino acid nitrogen content of the wheat sauce samples is higher than that of most commercially available wheat sauces.

[0128] Salt can provide flavor to food and inhibit the growth of harmful bacteria during the fermentation process, ensuring that the product will not spoil within a certain period of time. Excessive salt content will inhibit microbial enzyme activity, causing the fermentation cycle to become longer, masking the sweetness of the food and being detrimental to human health. Figure 2The salt content of the wheat paste prepared by the present invention is in the range of 4.45-6.64 g / 100 g, with an average value of 5.65 g / 100 g. The average salt content of commercially available wheat paste is 8.42 g / 100 g, and the wheat paste is more in line with the development trend of low-salt food.

[0129] The aflatoxin B1 content of the wheat sauces XJ-1, XJ-2, XJ-3, and XJ-4 with antioxidant and anti-inflammatory effects prepared in Examples 1 to 4 after being stored for one year and XJ-1 and XJ-4 after being stored for two years was tested. The results are as follows Figure 3 As shown; the aflatoxin B1 content was determined by referring to the enzyme-linked immunosorbent screening method in "GB 5009.22-2016 National Food Safety Standard Determination of Aflatoxins Group B and Group G in Foods" and was determined according to the instructions of the ELISA detection kit.

[0130] Figure 3 The content of aflatoxin B1 (AFB1) in wheat paste at different storage times. The content of AFB1 in wheat paste is between 3.43 and 4.79 μg / kg, which meets the limit requirements for the content of AFB1 in brewing sauce in "GB 2761-2017 National Food Safety Standard Limits of Mycotoxins in Food" (≤5 μg / kg). The highest content is XJ-1 stored for one year, and the lowest content is XJ-4 stored for two years. The AFB1 content of wheat paste stored for two years is lower than that of wheat paste stored for one year. It is speculated that the reason may be that during the storage process, AFB1 is degraded due to microorganisms or metabolism in the sauce.

[0131] 2. Extraction yield of different extracts from wheat paste

[0132] According to the method in Example 5, the new sauce was extracted to obtain water extract, ethanol extract, petroleum ether extract, ethyl acetate extract, and n-butanol extract respectively; each extract was freeze-dried to obtain freeze-dried powder of each part, and the yield was calculated according to the dry weight of the malt sauce (taking the ethanol extract as an example, the yield is the percentage of the ratio of the mass of the ethanol extract after freeze-drying to the mass of the dry weight of the malt sauce used for extraction); the results are shown in Table 1.

[0133] Table 1- Extraction yield of different extracts from wheat paste

[0134] Solvents Yield (%) Distilled water 30.17 Ethanol 40.78 Petroleum ether 4.11 Ethyl acetate 0.69 n-Butanol 4.41

[0135] 3. Characterization of the antioxidant activity of new and old sauce extracts in vitro

[0136] 3.1 Preparation of Vc solution and sample solution

[0137] Preparation of Vc solution: Accurately weigh 0.1 g of L-ascorbic acid solid, add distilled water to make up to 50 mL, prepare a 2 mg / mL standard solution, and set aside.

[0138] According to the method in Example 5, the new sauce / old sauce was extracted to obtain water extract, ethanol extract, petroleum ether extract, ethyl acetate extract, and n-butanol extract. The water extract was dissolved in ultrapure water to a concentration of 2 mg / mL, 3.125 mg / mL, 4 mg / mL, 6 mg / mL, 6.25 mg / mL, 8 mg / mL, 10 mg / mL, 12.5 mg / mL, 25 mg / mL, 50 mg / mL, 100 mg / mL, and 200 mg / mL, and the remaining extracts (i.e., ethanol extract, petroleum ether extract, ethyl acetate extract, and n-butanol extract) were dissolved in anhydrous ethanol to a concentration of 0.062 5mg / mL, 0.125mg / mL, 0.25mg / mL, 0.39mg / mL, 0.50mg / mL, 0.78mg / mL, 1mg / mL, 1.56mg / mL, 2mg / mL, 3.125mg / mL, 4mg / mL, 6mg / mL, 6.25mg / mL, 8mg / mL, 10mg / mL, 12.5mg / mL, 25mg / mL, 50mg / mL, and 100mg / mL to obtain sample solutions.

[0139] 3.2DPPH free radical scavenging test

[0140] Accurately weigh 0.0040g of DPPH solid powder and prepare a 1mmol / L DPPH mother solution in a 10mL brown volumetric flask using anhydrous ethanol as solvent. Accurately pipette 3mL of the mother solution, add 12mL of anhydrous ethanol, and dilute to a 200μmol / L DPPH solution for later use.

[0141] Pipette 50μL of sample solutions of different concentrations and 200μL DDPPH solution into a 96-well microplate, shake on a microplate reader for 10s to mix, react at room temperature in the dark for 30min, measure the absorbance A1 at a wavelength of 517nm, and use anhydrous ethanol as a reference. At the same time, measure the absorbance A0 after mixing 50μL 60% ethanol with 200μL DDPPH solution, and the absorbance A2 after mixing 50μL sample solution with 200μL anhydrous ethanol, with ascorbic acid solution as a positive control, and repeat the measurement three times.

[0142] DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0]×100.

[0143] 3.3 Hydroxyl free radical scavenging test

[0144] Take 50 μL of sample solutions with different concentration gradients into a 96-well plate, add 50 μL of salicylic acid anhydrous ethanol solution (6 mmol / L) and 50 μL of FeSO4 solution (6 mmol / L) respectively, mix well, add 50 μL of 7.5% H2O2, react in a 37°C water bath for 30 min, and measure the absorbance B1 at 536 nm. According to the same operating steps, measure the absorbance B2 when distilled water is used instead of H2O2, and the absorbance B0 when 60% ethanol solution is used instead of the sample solution. Use ascorbic acid solution as the positive control and repeat the measurement three times.

[0145] OH radical scavenging rate (%) = [1-(B1-B2) / B0]×100.

[0146] 3.4ABTS + Free radical scavenging assay

[0147] Prepare 2.45mmol / L potassium persulfate solution and 7mmol / L ABT + Solution, take equal amounts of potassium persulfate solution and ABTS + The solution was mixed and reacted for 12 to 16 hours in the dark as ABTS + Mother solution. Dilute the ABTS after the reaction in anhydrous ethanol and keep it away from light. + The mother solution was adjusted to an absorbance of 0.7 ± 0.002 as ABTS + The working solution is ready for use. Take 30 μL of different concentration gradient sample solutions and 200 μL ABTS + The working solution was shaken on a microplate reader for 10 seconds to mix, and the reaction was carried out in the dark for 10 minutes. The absorbance value C1 at 734 nm was measured. The same operation steps were followed to measure the absorbance value C1 instead of ABTS with distilled water. + The absorbance C2 of the working solution and the absorbance C0 of the sample solution replaced by 60% ethanol solution were measured three times with ascorbic acid solution as the positive control.

[0148] ABTS + Free radical scavenging rate (%) = [1-(C1-C2) / C0]×100.

[0149] 3.5 Determination of total antioxidant capacity (FRAP, ferric reducing capacity)

[0150] Acetic acid-sodium acetate buffer (pH 3.6): Take 1.02g of sodium acetate, add 4mL of glacial acetic acid, and then dilute to 50mL with water. FRAP reagent: Mix 300mmol / L sodium acetate buffer solution (pH 3.6), 10mmol / L TPTZ hydrochloric acid solution (hydrochloric acid concentration 40mmol / L), and 20mmol / LFeCl3 solution in a volume ratio of 10:1:1 to prepare the FRAP working solution, and store it at 37℃ for 1h.

[0151] Using a 96-well plate, 200 μL of FRAP reagent was mixed evenly with 50 μL of sample solutions of different concentrations. After incubation at room temperature for 30 minutes, the absorbance was measured at 593 nm. At the same time, ascorbic acid (Vc) was taken as a reference substance, and solutions of 1000, 500, 250, 125 and 62.5 μmol / L were prepared as reference solutions. A standard curve was drawn with Vc concentration as the horizontal axis and absorbance as the vertical axis. The final result was expressed as μmol / LVc equivalent antioxidant capacity (VcE).

[0152] Figure 5 The scavenging effect of each extract of fresh sauce and old sauce on hydroxyl free radicals according to the method in Example 5. Each extract has the effect of scavenging hydroxyl free radicals, and the effect gradually increases with the increase of sample concentration. 50 The value represents the concentration of sample required to reduce the initial absorbance value by 50%, IC 50 The smaller the value, the stronger its ability to scavenge free radicals, that is, the stronger its antioxidant activity.

[0153] Table 2 shows the IC values ​​of the scavenging ability of the extracts of new sauce and old sauce on hydroxyl free radicals. 50 IC value and Vc equivalent of the new sauce's hydroxyl radical scavenging ability 50 The order of size is: petroleum ether extract < water extract < ethyl acetate extract < n-butanol extract < ethanol extract, and the old sauce is slightly different. The ethyl acetate extract and petroleum ether extract of the old sauce are stronger in scavenging hydroxyl free radicals than the ethyl acetate extract and petroleum ether extract of the new sauce, but the other three extracts of the new sauce are stronger in scavenging hydroxyl free radicals than the old sauce. Ascorbic acid is used as a positive control, and its IC 50 The value is 1.05 mg / mL. According to Table 2, the IC values ​​of each extract for hydroxyl radicals can be obtained. 50 The results are converted to equivalents with ascorbic acid.

[0154] Table 2-IC of the scavenging ability of each extract of new sauce and old sauce on hydroxyl free radicals 50 Value and Vc equivalent

[0155]

[0156] Figure 6 The scavenging effect of each extract of fresh sauce and old sauce on DPPH free radicals according to the method in Example 5. The ability of each extract to reduce DPPH free radicals gradually increases with the increase of sample concentration. 50 The value represents the concentration of sample required to reduce the initial absorbance value by 50%, IC 50 The smaller the value, the stronger its ability to scavenge free radicals, that is, the stronger its antioxidant activity.

[0157] Table 3 shows the IC values ​​of the scavenging ability of the extracts of new sauce and old sauce on DPPH free radicals 50 IC values ​​and Vc equivalents of DPPH free radical scavenging ability of each extract of new sauce 50 The order of size is: ethyl acetate extract < n-butanol extract < petroleum ether extract < ethanol extract < water extract. IC values ​​of DPPH free radical scavenging ability of each old sauce extract 50 The order of size is: ethyl acetate extract < n-butanol extract < water extract < petroleum ether extract < ethanol extract. The water extract and petroleum ether extract of old sauce are stronger than those of new sauce in scavenging DPPH free radicals, but the new sauce of the other three extracts is stronger in scavenging DPPH free radicals than the old sauce. Ascorbic acid is used as a positive control, and its IC 50 The value is 0.11 mg / mL. According to Table 3, the IC values ​​of each extract for DPPH free radical can be obtained. 50 The results are converted to equivalents with ascorbic acid.

[0158] Table 3 - IC values ​​of DPPH free radical scavenging ability of each extract of new sauce and old sauce 50 Value and Vc equivalent

[0159]

[0160] Figure 7 According to the method in Example 5, the extracts of new sauce and old sauce were subjected to ABTS + Free radical scavenging effect. Each extract reduces ABTS + The ability of free radicals gradually increases with the increase of sample concentration. 50 The value represents the concentration of sample required to reduce the initial absorbance value by 50%, IC 50 The smaller the value, the stronger its ability to scavenge free radicals, that is, the stronger its antioxidant activity.

[0161] Table 4 shows the effects of each extract of new sauce and old sauce on ABTS + Free radical scavenging ability of IC 50 Value and Vc equivalent. ABTS in new sauce and old sauce + Free radical scavenging ability of IC 50 The order of size is: ethyl acetate extract < n-butanol extract < petroleum ether extract < water extract < ethanol extract. Among them, the water extract and ethanol extract of the old sauce are in the ABTS + The free radical scavenging ability of the extract was stronger than that of the water extract and ethanol extract of Xinjiang, but the difference between the other three was not significant. + IC of free radicals 50 The value is 0.09 mg / mL. According to Table 4, the ABTS +The IC50 of free radicals was converted to equivalent amount with ascorbic acid.

[0162] Table 4 - Effects of extracts from new sauce and old sauce on ABTS + Free radical scavenging ability of IC 50 Value and Vc equivalent

[0163]

[0164] According to the method in Example 5, the concentration of each extract of the new and old sauce was diluted to 10 mg / mL, and its FRAP value (iron ion reducing ability) was obtained according to the Vc equivalent. The larger the FRAP value, the stronger its antioxidant property. The Vc equivalent corresponding to the iron ion reducing ability of each extract of the new and old sauce is shown in Table 5, expressed as the antioxidant ability of each extract of the new and old sauce equivalent to millimoles of ascorbic acid per gram. The water extract of the old sauce is stronger than the water extract of the new sauce in terms of iron ion reducing ability, but the petroleum ether extract and ethyl acetate extract of the new sauce are stronger than the old sauce in terms of iron ion reducing ability.

[0165] Table 5 - Vc equivalent of iron ion reducing capacity of each extract of new and old sauce

[0166]

[0167] The above in vitro antioxidant capacity test showed that the extracts of new sauce and old sauce all have antioxidant capacity, and the extracts of different polar reagents have antioxidant capacity. The antioxidant capacity of new sauce, old sauce and each extract is different. In most evaluation methods, the ethyl acetate extract has the strongest antioxidant capacity; some data also show that the petroleum ether extract has the best antioxidant effect. The hydroxyl free radical scavenging ability of the ethyl acetate extract of the old sauce corresponds to a Vc equivalent of 214.72 mg Vc / g, and ABTS + The Vc equivalent corresponding to the free radical scavenging ability is 529.41 mg Vc / g, the Vc equivalent corresponding to the DPPH free radical scavenging ability is 100.00 mgVc / g, and the Vc equivalent for the iron ion reducing ability is 1.965 mmol / LVc / g.

[0168] 4. Characterization of anti-inflammatory activity of wheat paste extracts

[0169] 4.1 Effect of wheat paste extract on lipopolysaccharide-induced inflammation in RAW264.7 cells

[0170] Griess method was used to determine the NO content in RAW264.7 cells. Principle of determination: NO is easily oxidized to NO in vivo or in aqueous solution. 2-Under acidic conditions, NO can react with diazonium salt sulfonamide to form a diazo compound, and the diazo compound can react with naphthyl vinyl diamine to form a pink special substance with the highest absorption peak at 540nm. Within a certain concentration range, the concentration of this substance has a linear relationship with the NO concentration, and the absorbance value can reflect the concentration of the pink substance, so the NO concentration can be inferred from the absorbance value.

[0171] Effects of fresh and old soybean paste extracts on NO secretion in RAW264.7 cells

[0172] Take RAW267.4 cells and adjust the cell concentration to 1×10 6 / mL, and divided into a normal group (cells + culture medium), a positive group, and a sample-treated group according to the experimental design. 100 μL of cell suspension was added to the normal group, the positive group, and the sample-treated group, and placed in an incubator (37°C, 5% CO2) for 24 hours. The original culture medium was removed, and only 100 μL of fresh phenol red-free basal culture medium solution was added to the normal group, and 100 μL of phenol red-free basal culture medium with a final LPS concentration of 1 μg / mL was added to the positive group. The sample-treated group was added with different new sauce / old sauce extracts containing the same mass concentration (the new sauce / old sauce extracts were obtained by the method in Example 5) and a phenol red-free basal culture medium solution with a final LPS concentration of 1 μg / mL, 100 μL per well, 5 replicate wells were set, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. Then 80 μL of the supernatant was aspirated, and the NO content was detected by the Griess method. The test results are as follows Figure 8 shown.

[0173] Figure 8 The NO content of different extracts of new sauce / old sauce after acting on LPS-stimulated RAW264.7 cells. Figure 8As shown in the figure, compared with the NO content of RAW264.7 cells stimulated by LPS, the extracts of new and old sauces all had an inhibitory effect on the secretion of NO by RAW264.7 macrophages, and the inhibitory effect increased with the increase of concentration. At the same concentration, the inhibitory effect of each extract of new sauce on NO was better than that of each extract of old sauce. Among the extracts of old sauce, the ethyl acetate extract had the strongest inhibitory effect on NO. When the concentration was 100μg / mL, the NO content decreased by 46%, and when the concentration was 300μg / mL, the NO content decreased by 97%, which was almost able to completely inhibit the increase of NO content induced by LPS. The order of the inhibitory effect of each extract of old sauce on NO from large to small is: ethyl acetate extract > n-butanol extract > ethanol extract > petroleum ether extract > water extract. The ethyl acetate extract of new sauce has a better inhibitory effect on NO. When the concentration of ethyl acetate extract is 100μg / mL, the NO content is 9.71μmol / L, which decreases by 76%; when the concentration is 300μg / mL, the NO content decreases by 98.2%; the inhibitory effect of n-butanol extract on NO is second. When the concentration is 500μg / mL, the NO content is 18.47μmol / L, which decreases by 45%. The anti-inflammatory effects of the extracts of new sauce are as follows: ethyl acetate extract> n-butanol extract> water extract> ethanol extract> petroleum ether extract. Therefore, the ethyl acetate extract of new sauce was used for subsequent animal gavage experiments.

[0174] 4.2 Inhibitory test of the new sauce extract on xylene-induced ear swelling and inflammation model in mice by smearing

[0175] Trial Groups

[0176] Eighteen 4-week-old SPF KM mice, weighing 20±2g, were randomly divided into 6 groups: model group, wheat paste water extract group, wheat paste ethanol extract group, wheat paste petroleum ether extract group, wheat paste ethyl acetate extract group, and wheat paste n-butanol extract group, with 3 mice in each group. The dosage was 10 mg / ear.

[0177] Modeling and drug administration

[0178] After three days of adaptive feeding, on the fourth day, 20 μL of xylene was applied to both sides of the right auricle of each group of mice using a pipette tip to induce inflammation, and the left ear was used as a control. Then, except for the model group, the corresponding wheat paste extract (10 mg / ear) was applied to both ears of each group.

[0179] 30 minutes after inflammation, blood was collected from the orbits of mice in all groups and the mice were killed by cervical dislocation. Both ears were cut off along the base line of the auricle, and ear pieces were punched at the same position with a 6 mm diameter puncher and weighed immediately.

[0180] Index determination

[0181] The NO content in the serum of each group of mice was detected by Griess method, and the effects of each extract of wheat paste on the ear swelling of mice induced by xylene were observed. The swelling degree and swelling inhibition rate of each group were calculated as follows:

[0182] Swelling degree = mass of inflamed Pleurotus eryngii - mass of non-inflamed Pleurotus eryngii

[0183] Swelling rate = (weight of inflamed Pleurotus eryngii - weight of non-inflamed Pleurotus eryngii) / weight of non-inflamed Pleurotus eryngii × 100%

[0184] Swelling inhibition rate = (average swelling rate of blank group - average swelling rate of drug-treated group) / average swelling rate of blank group × 100%

[0185] Fig. 9 The inhibitory effect of different extracts of Xinjiang (the extracts of Xinjiang were obtained according to the method in Example 5) on mouse ear swelling by smearing (A) and the change of NO content in serum (B); wherein, Fig. 9 In the data, Model refers to the model group, VY-E refers to the ethanol extract group of wheat paste, VY-W refers to the water extract group of wheat paste, VY-L refers to the petroleum ether extract group of wheat paste, VY-EA refers to the ethyl acetate extract group of wheat paste, and VY-N refers to the n-butanol extract group of wheat paste.

[0186] like Fig. 9 As shown in (A), under the same dose (10 mg / ear) treatment, the swelling rate of each extract of wheat paste was significantly lower than that of the model group (P<0.05). Among them, the swelling inhibition rate of the VY-L group was the highest, reaching 48%, followed by the VY-EA group, with a swelling inhibition rate of 43%. Fig. 9 (B) The changes in NO content in the serum of each group of mice were detected. After the treatment with wheat paste extract, the NO content of each group was significantly reduced compared with the model group (P<0.05). Similar to the swelling inhibition rate, the NO content of the VY-L group was the lowest, at 3.33μmol / L, and the NO content of the VY-EA group was reduced to 3.74μmol / L. The study showed that VY-L was more likely to play an anti-inflammatory role when applied.

[0187] 4.3 Experiment on the inhibition of xylene-induced ear swelling in mice by oral administration of the ethyl acetate extract of Xinjiang (the ethyl acetate extract of Xinjiang was obtained according to the method in Example 5)

[0188] Experimental Grouping

[0189] Fifty 4-week-old SPF-grade male KM mice, weighing 20±2g, were randomly divided into 5 groups: blank group (oral administration of 0.2mol / L NaHCO3 solution), model group (oral administration of 0.2mol / L NaHCO3 solution), positive control group (aspirin 200mg / kg / dbw), low-dose group (ethyl acetate extract of malt sauce 300mg / kg / dbw), and high-dose group (ethyl acetate extract of malt sauce 600mg / kg / dbw), with 10 mice in each group.

[0190] Modeling and drug administration

[0191] After three days of adaptive feeding, the drug volume was 0.1 mL per 10 g of body weight, calculated according to the weight of the mice. At the same time, the model group and the blank group were given the same volume of 0.2 mol / L NaHCO3 solution, and the positive group was given 0.2 g / kgbw aspirin solution. The mice were gavaged for 7 consecutive days, once a day. During the drug administration period, they ate and drank normally. The weight of each mouse was recorded before gavage, and the diet and water intake of each cage of mice was recorded every day.

[0192] 1 hour after the last administration, except for the blank group, 20 μL of xylene was applied to both sides of the right auricle of the mice through a gun tip to induce inflammation, and the left ear was used as a control. 30 minutes after inflammation, blood was collected from the eye sockets of all groups of mice and the mice were killed by cervical dislocation. The two ears were cut along the auricle baseline, and the ear pieces were punched at the same position with a 6 mm diameter puncher, and weighed immediately. Then the tissue supernatant was extracted from the ears. The mouse liver was dissected and extracted, rinsed in physiological saline to remove excess blood stains, and weighed after absorbing excess water with filter paper, and stored in a -80℃ refrigerator.

[0193] Index determination

[0194] The NO content in the serum of each group of mice was detected by Griess method. The changes in the ALT, AST, SOD, MDA, PEG2, INOS, and COX-2 content in the serum of each group were detected by ELISA method. The organ index of the liver was calculated. The effect of ethyl acetate extract of malt sauce on the ear swelling of mice caused by xylene was observed. The swelling degree and swelling inhibition rate of each group were calculated. The formula for calculating the organ index is: organ index = organ weight (g) / mouse weight (g) × 100%.

[0195] Fig.10The inhibitory effect of the new sauce ethyl acetate extract on mouse ear swelling by gavage. After being treated with xylene, the swelling rate of the model group (Model) was significantly higher than that of the blank group (Control), reaching 87%, showing a very significant difference (P<0.001), indicating that the mouse ear swelling model was successfully established. The low-dose (L-VY-EA) and high-dose (H-VY-EA) groups of the new sauce ethyl acetate extract can significantly reduce the ear swelling rate (P<0.001), and show a dose-dependent manner, among which the low-dose group can reduce the swelling rate to 55%, and the high-dose group can reduce the swelling rate to 38%. The positive drug aspirin group (Aspirin) can reduce the mouse ear swelling rate to 3%. The experiment shows that the ethyl acetate extract of the new sauce has a good anti-inflammatory and detumescent effect.

[0196] Fig.11 The effect of ethyl acetate extract of malt sauce on inflammatory factors NO (A), PEG2 (B), INOS (C), COX-2 (D), oxidative stress factors SOD (E), and MDA (F) in the serum of mice with ear swelling model by oral gavage. After xylene treatment, as shown in Figure (A), the NO concentration in the model group increased to 4.34 μmol / L, which was significantly different from the NO concentration of 1.08 μmol / L in the blank group (P<0.001), indicating that the modeling was successful. The low (L-VY-EA) and high-dose (H-VY-EA) groups of ethyl acetate extract of malt sauce can significantly reduce the NO concentration (P<0.05) in a dose-dependent manner, and the high-dose group can reduce the NO concentration to 2.72 μmol / L. The NO concentration in the positive group (Aspirin) was 2.08 μmol / L.

[0197] As shown in Figure (B), the PEG2 concentration in the model group was significantly increased from 554ng / L to 685ng / L compared with the blank group (P<0.05). The positive drug aspirin can reduce the PEG2 concentration to 580ng / L (P<0.05), and the ethyl acetate extract of wheat paste can also reduce the PEG2 concentration stimulated by xylene, but there is no significant difference between the low-dose group (L-VY-EA) and the model group, and the high-dose group (H-VY-EA) can significantly reduce it to 607ng / L (P<0.05).

[0198] As shown in Figure (C), compared with the blank group, the INOS concentration in the model group was significantly increased from 23.97U / mL to 79.40U / mL (P<0.001). The positive group (Aspirin) and the low-dose (L-VY-EA) and high-dose (H-VY-EA) groups of ethyl acetate extract of malt sauce significantly reduced the INOS concentration (P<0.001). The average INOS concentration of the positive group was 27.86U / mL, the average INOS concentration of the low-dose group of ethyl acetate extract of malt sauce was 48.11U / mL, and the average INOS concentration of the high-dose group of ethyl acetate extract of malt sauce was 39.59U / mL, showing a dose-dependent relationship.

[0199] As shown in Figure (D), the COX-2 concentration in the model group was significantly increased from 56.70 ng / L to 69.59 ng / L (P<0.05) compared with the blank group. The positive drug aspirin can reduce the INOS concentration to 55.88 ng / L (P<0.05), and the ethyl acetate extract of wheat paste can also reduce the INOS concentration, but there is no significant difference between the low-dose group (L-VY-EA) and the model group, and the high-dose group (H-VY-EA) can significantly reduce it to 58.38 ng / L (P<0.05).

[0200] As shown in Figure (E), the average SOD concentration in the serum of mice in the blank group was 42.73U / mL, and the average SOD concentration in the serum of mice in the model group was 32.87U / mL, which was significantly lower (P<0.001). The low (L-VY-EA) and high-dose groups (H-VY-EA) of ethyl acetate extract of wheat paste can increase the SOD concentration in a dose-dependent manner, among which the high-dose group can significantly increase it to 40.43U / mL (P<0.05). The positive group (Aspirin) significantly increased the SOD concentration to 41.93U / mL (P<0.001).

[0201] As shown in Figure (F), the MDA concentration in the model group was significantly increased from 6.26 nmol / mL to 13.01 nmol / mL compared with the blank group (P<0.001). The positive group (Aspirin) and the high-dose group of ethyl acetate extract of malt sauce (H-VY-EA) were able to significantly reduce the MDA concentration (P<0.001), among which the average MDA concentration in the positive group was 7.31 nmol / mL, and the average MDA concentration in the high-concentration group of ethyl acetate extract of malt sauce was 9.60 nmol / mL. The average MDA concentration in the low-concentration group of ethyl acetate extract of malt sauce was 11.91 nmol / mL.

[0202] The above cell tests and animal tests all show that the extracts of wheat paste have certain anti-inflammatory and detumescent effects. The anti-inflammatory effects of some extracts are close to those of positive drugs, indicating that wheat paste has a good anti-inflammatory effect and is a functional seasoning. It can also be used to prepare anti-inflammatory preparations.

[0203] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing wheat paste with antioxidant and anti-inflammatory effects, characterized in that: The following steps are involved: Add water to the wheat until it covers the wheat and heat and cook; The steamed wheat is naturally fermented in a sealed environment at a temperature of 25 to 35°C and a relative humidity of 70% to 90% for 3 to 7 days; The naturally fermented wheat is dried in the sun, cleaned, dried again, crushed, and sieved to obtain the sauce noodles; After crushing the fresh pepper at room temperature, adding salt and marinating for 2-6 hours, removing excess water to obtain the marinated pepper; The noodle sauce, Sichuan peppercorns and pickled chili peppers are mixed and fermented in a sealed environment at a temperature of 25-35°C for more than 20-35 days. After the fermentation is completed, the wheat sauce with antioxidant and anti-inflammatory effects is obtained.

2. The method for preparing the wheat paste with antioxidant and anti-inflammatory effects according to claim 1, characterized in that: The steamed wheat is spread out in a sealed environment to a wheat pile with a thickness of 2 to 6 cm, and naturally fermented for 3 to 7 days at a temperature of 25 to 35° C. and a relative humidity of 70% to 90%; During the fermentation period, when the center temperature of the wheat pile is higher than 30°C, it is turned over twice a day; during the fermentation period, when the center temperature of the wheat pile is ≤30°C, it is turned over once a day.

3. The method for preparing the wheat paste with antioxidant and anti-inflammatory effects according to claim 1, characterized in that: The mass ratio of the sauce noodles, salt, pepper and fresh pepper is 1:(0.14-0.17):(0.004-0.02):(1.4-1.7).

4. The method for preparing the wheat paste with antioxidant and anti-inflammatory effects according to claim 1, characterized in that: Add water to the wheat until it covers the wheat, then heat and cook at 90-100°C for 20-50 minutes.

5. The method for preparing wheat paste with antioxidant and anti-inflammatory effects according to claim 1, characterized in that: The naturally fermented wheat is air-dried, washed, air-dried again, crushed, and passed through a 40-90 mesh sieve to obtain the sauce noodles.

6. The method for preparing the wheat paste with antioxidant and anti-inflammatory effects according to claim 1, characterized in that: The noodles, peppercorns and pickled chilies are mixed and fermented in a sealed environment at a temperature of 25-35°C for more than 20-35 days. When the color of the fermented product changes from bright red to reddish brown or brown, the fermentation is completed and a wheat sauce with antioxidant and anti-inflammatory effects is obtained.

7. A wheat sauce with antioxidant and anti-inflammatory effects, characterized in that: The preparation is obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the wheat paste with antioxidant and anti-inflammatory effects as claimed in claim 7 in the preparation of antioxidant and anti-inflammatory preparations.

9. The use according to claim 8, wherein the method for preparing the antioxidant and anti-inflammatory preparation comprises the following steps: The wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, and the first filtrate is concentrated by rotary evaporation and freeze-dried to obtain an ethanol extract, which is an antioxidant and anti-inflammatory preparation; Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate and a filter residue, water is added to the filter residue, extracted in a homogenizer, filtered to obtain a second filtrate, and the second filtrate is rotary evaporated and freeze-dried to obtain a water extract, which is an antioxidant and anti-inflammatory preparation; Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, the first filtrate is concentrated by rotary evaporation to obtain an ethanol extract, the ethanol extract is dissolved in water, petroleum ether is added for extraction multiple times, the petroleum ether phases are combined, vacuum rotary evaporation and freeze-dried to obtain a petroleum ether extract, which is an antioxidant and anti-inflammatory preparation; Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, the first filtrate is concentrated by rotary evaporation to obtain an ethanol extract, the ethanol extract is dissolved in water, petroleum ether is added to extract multiple times, the aqueous phases are combined, the aqueous phases are extracted multiple times with ethyl acetate, the ethyl acetate phases are combined, and vacuum rotary evaporation and freeze-drying are performed to obtain an ethyl acetate extract, which is an antioxidant and anti-inflammatory preparation; Alternatively, the wheat paste is added to anhydrous ethanol, extracted in a homogenizer, filtered to obtain a first filtrate, the first filtrate is concentrated by rotary evaporation to obtain an ethanol extract, the ethanol extract is dissolved in water, petroleum ether is added and extracted multiple times, the aqueous phases are combined, the aqueous phases are extracted multiple times with ethyl acetate, the extracted aqueous phases are combined and extracted multiple times with n-butanol, the n-butanol phases are combined, and vacuum rotary evaporation and freeze-drying are performed to obtain n-butanol extract, which is an antioxidant and anti-inflammatory preparation.

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