A method for processing radix sophorae tonkinensis by fermentation based on adjusting ammonia nitrogen content in fermentation environment

Abstract

The application relates to a fermentation processing method of Radix Sophorae Tonkinensis based on adjusting the ammonia nitrogen content in a fermentation environment. And Radix Sophorae Tonkinensis processed by the fermentation processing method. The fermentation process of Radix Sophorae Tonkinensis is finely controlled, and the main feature is that the ammonia nitrogen content in the environment during the fermentation process is controlled. Aspergillus oryzae TJTSW001 is used as a fermentation strain, and ammonia nitrogen is introduced by adding glutamic acid in the culture environment. Compared with the fermentation method without adding glutamic acid, the content of soybean isoflavone aglycone in the prepared Radix Sophorae Tonkinensis sample is significantly improved, and the antipyretic effect is also improved.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine processing technology, specifically relating to a fermentation processing method for light-colored fermented soybeans based on adjusting the ammonia nitrogen content in the fermentation environment. Background Technology

[0002] Soybeans, scientifically known as *Glycine max*, are also called yellow soybeans, green-kernel black soybeans, mud soybeans, and horse feed soybeans, belonging to the legume family of herbaceous plants. In ancient times, they were called "shu". They are high in protein and are one of China's most important food crops. my country's soybean production accounts for about one-tenth of the world's total, reaching approximately 17.3 million to 20 million tons per year, earning them the title of "King of Soybeans". Soybeans themselves have high nutritional value, and are praised as "King of Beans", "Meat of the Field", "Green Milk", and even hailed as the best food to save humanity and the planet. Fermented soy products are not only richer in nutrients but also more easily absorbed by the human body, greatly increasing the absorption rate of their protein and minerals to as high as 92% to 96%.

[0003] Soybean paste (Sojae Semen Praeparatum), also known as fragrant soybean (from *Shang Han Lun*), light soybean (from *Ben Cao Gang Mu*), soybean paste (from *Pu Ji Ben Shi Fang*), and soybean paste (from *Fan Wang Fang*), is made from the mature seeds of soybean (Glycine max (L.) Merr.), a plant in the genus *Glycine* of the legume family, through fermentation with mulberry leaves and artemisia annua. Since the Liang Dynasty, soybean paste has been widely used clinically. In ancient prescriptions, it was often used to treat typhoid fever, headache, chills and fever, malaria, malignant poisoning, and various symptoms of fullness and stuffiness.

[0004] Douchi is a product made from soybeans as the main raw material and Artemisia annua, mulberry leaves, etc. as auxiliary materials through fermentation and processing. The Ministry of Health stipulates that douchi is a variety that can be used both as food and medicine, with the effects of relieving exterior syndrome, dispelling vexation, and diffusing the stagnated heat, and is used for colds, aversion to cold and fever with headache, restlessness and chest distress, and vexation and insomnia. In Li Shizhen's Compendium of Materia Medica, there is a record that "douchi has curative effects such as appetizing and increasing food intake, promoting digestion and resolving food stagnation, inducing sweating and relieving exterior syndrome, dispelling vexation and relieving dyspnea, etc.". There are differences between medicinal and edible douchi. For medicinal douchi, 100 kg of soybeans need to be soaked and absorbed with the decoction of 7 kg - 10 kg of mulberry leaves and Artemisia annua each; while there is no such requirement for edible douchi. In terms of morphology, most douchi are complete fermented beans, but there are also those in the shape of soybean halves; classified by processing raw materials, they can be divided into two categories: yellow soybean douchi and black soybean douchi; according to taste, they can be divided into three categories: light douchi, salty douchi, and wine douchi. Light douchi, also known as home-made douchi, is made by natural fermentation of cooked soybeans or black beans. Salty douchi is made by first making koji from cooked soybeans, and then adding spices such as salt, white wine, chili, ginger, etc., and fermenting and sun-drying in a vat, and the salt content is generally higher than 8%. Soaking salty douchi in yellow rice wine for several days and then taking it out and drying it can make wine douchi; classified by fermenting microorganisms, douchi can be divided into four categories: Mucor-type douchi, Aspergillus-type douchi, Rhizopus-type douchi, and Bacteria-type douchi; classified by the moisture content of the finished product, they can be divided into three categories: dry douchi, wet douchi, and water douchi.

[0005] Usually, the fermentation of (light) douchi is the result of the joint action of multiple microorganisms. Except for the main microorganisms, the growth of other secondary microorganisms also occurs. During the koji-making and post-fermentation processes, basically, microorganisms such as molds, bacteria, and yeasts are involved. However, in different open environments, different microbial flora will be formed, which also determines the diversity of its enzyme systems and produces more diverse metabolites. Currently, the identified fermenting strains of light douchi include Aspergillus niger, Aspergillus oryzae, Mucor, Rhizopus, Lobster saucesubtilis, Bacillus subtilis, Lactic acid bacteria, and Micrococcus, etc. The research situation of douchi fermenting microorganisms at home and abroad is shown in the following table.

[0006]

[0007] Douchi has a long manufacturing history, and the processing methods mainly include steaming and fermentation. There are many types of douchi and the brewing process is complex, but it is mainly divided into three stages: pretreatment (i.e., raw material treatment), koji-making, and post-fermentation. Currently, the light douchi produced in China varies due to differences in the environment, climate, uses, and fermentation methods in different regions, and soybeans are greatly affected by factors such as strains, temperature, humidity, and acidity during the fermentation process.

[0008] The medicinal fermented soybeans currently used in clinical practice are processed according to the method outlined in Part I of the 2010 edition of the Pharmacopoeia of the People's Republic of China. The processing method is as follows: Take 70g-100g each of mulberry leaves and artemisia annua, add water and decoct, filter, mix the decoction with 1000g of clean soybeans, let them absorb all the decoction, steam thoroughly, take out, let cool slightly, then place in a container, cover with the decocted mulberry leaves and artemisia annua residue, let ferment until a yellow skin forms, take out, remove the residue, wash clean, place in a container and let ferment for another 15-20 days until fully fermented and fragrant, take out, steam slightly, and dry to obtain the medicinal fermented soybeans.

[0009] The processes for preparing fermented soybeans using various microorganisms are summarized below:

[0010] (1) Fermentation process of Mucor-type fermented soybeans

[0011] Mucor-type fermented black beans have the largest production volume among similar products nationwide and are a specialty of Sichuan Province, primarily represented by Yongchuan and Tongchuan fermented black beans. They are highly popular due to their rich, mellow aroma, abundant ester fragrance, and oily, melt-in-your-mouth texture. Mucor requires relatively low temperatures and a long fermentation time to grow, making it unsuitable for production in many parts of my country. The main production process for Mucor-type fermented black beans is as follows:

[0012] Black soybeans (yellow soybeans) → screening → washing → soaking → draining → steaming → cooling → inoculation → koji making → koji washing → adding auxiliary materials (mixing salt, etc.) → post-fermentation (6℃, 10-12 months) → packaging → sterilization → finished product.

[0013] The inoculated *Mucor racemosus* strain is generally *Mucor racemosus*, along with other molds with high cellulase activity and a small amount of bacteria. The fermentation time is generally 10–20 days, with an initial temperature of 2–6℃ and a final product temperature of 5–15℃. White mold spots are visible on the soybeans 3–5 days after entering the fermentation chamber; mycelial growth is uniform from 8 to 12 days, with a small amount of brown spores produced; and the *Mucor* matures from 16 to 20 days, and the mycelium turns from white to gray, at which point it can be removed from the fermentation chamber.

[0014] When washing the koji, break the fermented koji into granules and pour it into a basin. For every 50 kg of dry soybeans, add 9 kg of salt, 0.5 kg of white wine, and 0.5 kg to 2.5 kg of water. Mix well and rub by hand to remove as many spores and mycelium as possible. Otherwise, the product will not be of pure color, will have a strong bitter and musty taste, and will have a poor appearance after drying.

[0015] (2) Aspergillus-type fermented soybean fermentation process

[0016] Aspergillus-based fermented black beans originated earliest and are most widely distributed, with Yangjiang fermented black beans from Guangdong and Liuyang fermented black beans from Hunan being the most famous in China. Aspergillus oryzae-based fermented black beans primarily use Aspergillus oryzae AS3.951 and AS3.042. The pure Aspergillus oryzae fermentation method involves inoculating cultured Aspergillus oryzae spores for fermentation. During the production of Aspergillus-based fermented black beans, Aspergillus oryzae exhibits a strong ability to produce neutral and alkaline proteases, but low activity of acidic proteases. After the Aspergillus oryzae fermentation is complete and the koji (fermentation starter) is made, certain amounts of salt, fermented glutinous rice, and distilled spirits are added for further fermentation. The product is generally in the form of salted fermented black beans, mainly used for processing flavored fermented black beans and other condiments. The main production process of Aspergillus-based fermented soybeans is as follows: Select soybeans → Soak (40℃, 2h) → Steam (121℃, 30min) → Cool → Inoculate (Aspergillus oryzae, Aspergillus niger, etc.) → Make koji → Wash koji → Add auxiliary materials (mix with salt) → Post-fermentation → Packaging → Sterilization → Finished product.

[0017] (3) Rhizopus-type fermented soybean fermentation process

[0018] The representative product of Rhizopus-based fermented soybeans is tempeh, a fermented soybean food widely consumed as a staple food in Southeast Asia, including Indonesia, Malaysia, and Thailand. Its main fermentation microorganisms are Rhizopus oryze, Rhizopus oligosporus, and Mucor indicus. Because Southeast Asia's year-round temperature of 20℃~30℃ is ideal for Rhizopus growth, but Rhizopus lacks the enzymes to break down the soybean skin, it can only grow well on peeled soybeans. Therefore, a peeling process is included in tempeh production. Tempeh production mainly uses a salt-free solid-state fermentation method, resulting in a relatively short fermentation cycle. The traditional tempeh production process is: selecting soybeans → soaking → peeling → boiling → draining → cooling and covering with banana leaves or other leaves → fermentation (1-2 days) → maturation → finished product.

[0019] Because natural fermentation is difficult to control and easily leads to the growth of miscellaneous bacteria and spoilage, pure culture fermentation currently mainly uses Rhizopus oligosporus as the inoculum for the production of tamsui. Lactic acid is added or lactic acid bacteria are inoculated to acidify the pH value, thereby lowering the pH value and inhibiting the growth of miscellaneous bacteria. The modern tamsui production process is as follows: Select soybeans → Soak → Acidify the substrate (add 1% lactic acid) → Peel → Steam (121℃ high pressure steaming for 10 min) → Drain → Cool → Inoculate (Rhizopus oligosporus) → Mix well → Package → Constant temperature incubation (36±1)℃ for 30 h) → Maturation → Finished product.

[0020] (4) Bacterial fermentation process for fermented soybeans

[0021] Bacterial fermented black beans (douchi) are mainly homemade douchi made in Yunnan, Guizhou, and Shandong provinces. Except for Shandong-style fermented black beans, most families can make and consume them. Japanese research on bacterial fermented black beans is more extensive. Currently, the Bacillus subtilis strain most commonly used for fermentation is Bacillus subtilis Natto, the main active bacteria in Japanese bacterial fermented black beans ("natto"). At high temperatures, it multiplies on steamed soybeans, and its strong protease system produces douchi with a unique flavor. The production process for bacterial fermented black beans (natto) is as follows: Select soybeans → Soak in running water for 14 hours → Steam (130℃, 55 minutes) → Inoculate and ferment (40℃, 95% relative humidity) → Ferment and cook natto (5℃, 24 hours) → Mature → Natto.

[0022] In making bacterial fermented soybeans, boiled soybeans are drained, wrapped in burlap sacks while still hot, and covered to maintain temperature. In this high-temperature, high-humidity environment, the growth of most microorganisms is inhibited, but Bacillus subtilis can multiply rapidly. After 2 days of cultivation, when the soybeans are covered with a sticky substance that can be pulled into threads and have a distinctive odor, salt, white wine, and spices are added. Fermentation continues for 5-7 days to produce fermented soybeans. The production process of bacterial fermented soybeans in my country is as follows: Selected soybeans → Soaking → Boiling → Draining → Wrapping in burlap sacks while still hot → High-temperature koji making (2 days) → Adding salt, white wine, and spices → Fermentation (5-7 days) → Fermented soybeans.

[0023] (5) Multi-strain fermentation process for fermented black soybeans

[0024] Traditional natural koji-making methods produce fermented black beans with a rich flavor, but they are limited by climate conditions, resulting in a long fermentation period and low yield. Fermented black beans made with a single strain of microorganisms lack flavor and are prone to spoilage and foul odor. Therefore, the use of multi-strain koji-making produces fermented black beans with a rich aroma, high nutritional value, delicious taste, and a smooth, residue-free texture. The fermentation period can be shortened from over a year to about 2-3 months. In recent years, research has begun on multi-strain fermentation processes for fermented black beans.

[0025] In modern Chinese patent medicines, light-colored fermented soybeans are often combined with other drugs to treat diseases such as colds, dysentery, and carbuncles. The Ming Dynasty's *Compendium of Materia Medica* records: "Shen said: Shaanxi's fermented soybean juice is far superior to ordinary fermented soybeans. The method is to steam soybeans until yellow, adding four liters of salt and four ounces of pepper to every dou (a unit of volume). It takes three days in spring, two days in summer, and five days in winter. When half-cooked, add five ounces of fresh ginger, making it both clean and refined." Shizhen said: "Fermented soybeans can be made from all kinds of soybeans, but black soybeans are used in medicine. There are light-colored and salty fermented soybeans; for treating diseases, light-colored and salty fermented soybean juices are often used, depending on the method. The 'heart' of the fermented soybean is taken from the center when the soybeans are combined, not from peeling the skin." This statement can be found in *Waitai Miyao*.

[0026] Method for making light fermented soybeans: Use two to three dou (a unit of dry measure) of black soybeans, wash them clean in June, soak them in water overnight, drain them, steam them until cooked, take them out and spread them on a mat, and cover them with a bamboo pole when they are slightly warm. Check them every three days, when the yellow skin has covered them completely, but not too much. Take them out and dry them in the sun and winnow them clean. Mix them with water until they are just the right consistency, the juice should be enough to seep between your fingers. Place them in a jar, pack them tightly, cover them with mulberry leaves three inches thick, seal them with mud, and sun-dry them for seven days. Take them out and sun-dry them for an hour, then mix them with water and put them back into the jar. Repeat this process seven times, steam them again, spread them out to remove the heat, seal the jar, and the fermented soybeans are ready.

[0027] Method for making salted fermented soybeans: Soak one dou (approximately 150 ml) of soybeans in water for three days, then rinse, steam, and spread out on a mat. Once they turn yellow, remove them, winnow them clean, rinse again, and sun-dry. For every four jin (approximately 2 catties) of soybeans, add one jin (approximately 500 ml) of salt, half a jin (approximately 250 ml) of shredded ginger, pepper, orange peel, perilla, fennel, and almonds, mix well, and place in a jar. Cover with water up to one inch above the surface, seal with leaves, and sun-dry for one month. Method for making fermented soybean juice: From October to January, use three dou (approximately 150 ml) of good quality fermented soybeans. Boil in clear sesame oil until the smoke stops, then mix with one sheng (approximately 300 ml) of the soybeans, steam, spread out to cool, and sun-dry. Mix again and steam three times. Add one dou (approximately 150 ml) of white salt, pound and mix, then add three to four dou (approximately 150 ml) of broth and pour into a clean pot. Add pepper, ginger, scallions, and orange peel, and simmer until reduced by one-third. Store in a non-greasy container. The aroma and flavor are exceptionally delicious. This can be made from various other types of fermented soybeans, such as bran-fermented soybeans, melon-fermented soybeans, and soy sauce-fermented soybeans, but these are only for food and not for medicinal use.

[0028] There are clear records of its medicinal use in the Han Dynasty. Fermented soybeans have the effects of relieving exterior syndromes, eliminating irritability, and dispersing stagnant heat. Clinically, it is often taken internally to treat colds, chest tightness, and headaches caused by heat stagnation.

[0029] The main components of light fermented soybeans include protein, fat, vitamin B1, vitamin B2, polysaccharides, and soy isoflavones. Among them, soy isoflavones, saponins, protein, γ-aminobutyric acid, oligosaccharides, soy fibrinolytic enzyme, and some volatile compounds are the main active substances. Isoflavones, as a component in light fermented soybeans that plays a very important regulatory role in the human body, are divided into free aglycones and bound glycosides.

[0030] Chinese Patent CN 104073444B discloses a strain of *Aspergillus oryzae* TJTSW001 and its application in the fermentation of the traditional Chinese medicine fermented soybean (*Dan Dou Chi*). This *Aspergillus oryzae* strain was deposited on March 20, 2014, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.8945. This *Aspergillus oryzae* strain was isolated from fermented soybean and screened through cobalt-60 and ultraviolet mutagenesis. This strain can hydrolyze soybean isoflavones into daidzein and sugars. The strain of this invention can play an important role in the fermentation production of the traditional Chinese medicine fermented soybean, yielding fermented soybean with a high daidzein content, thereby increasing its efficacy and showing broad application prospects.

[0031] Soy isoflavones exist in soybeans primarily in two forms: free aglycones, including genistein (also known as daidzein or genistein) and daidzein; and bound glycosides. Glycosides mainly exist as malonyl genistein, malonyl daidzein, genistein, and daidzein. Bound glycosides are the dominant form in soybeans, with free aglycones accounting for a relatively small proportion, approximately 2%–3% of the total. It is important to note that the activity of aglycones is much higher than that of glycosides. Bound glycosides require hydrolysis to become aglycones to exert their effects. Therefore, hydrolyzing glycosides into aglycones will yield higher physiological activity. Thus, obtaining more active soy isoflavone aglycones has significant practical implications for improving the efficacy of fermented soybean (douchi). Summary of the Invention

[0032] The applicant conducted a more in-depth study on the fermentation process of Aspergillus oryzae TJTSW001 to produce light-colored fermented soybeans. The study found that by adjusting the content of ammonia nitrogen in the fermentation environment, the content of soybean isoflavone aglycones in the prepared light-colored fermented soybeans could be further increased, and its antipyretic effect could be effectively enhanced.

[0033] This application first discloses a fermentation method for light-colored fermented soybeans based on adjusting the ammonia nitrogen content in the fermentation environment.

[0034] The fermentation and processing method consists of the following steps:

[0035] Step 1) Select mature, plump, uniform, undamaged, and moldy fresh black beans, rinse them quickly with clean water to remove surface dirt and impurities, and steam them until cooked.

[0036] Step 2) Inoculate Aspergillus oryzae TJTSW001 into a slant test tube and incubate at 35℃ for 5 days. After growth, wash off the surface spores with sterile water and inoculate the culture medium into 20ml of purified water containing 6% soybean flour and glutamic acid. Incubate at 35℃ and 180rpm for 3 days. Use this as the liquid inoculum.

[0037] Step 3) Take mulberry leaves and artemisia annua, add water and decoct, decoct 3 times, and combine the filtrates;

[0038] Step 4) Soak the black beans obtained in Step 1) in the liquid until the liquid is completely absorbed. Then steam them until cooked, and after they have cooled to room temperature, mix the liquid starter into them. Cover them with four layers of gauze to retain moisture.

[0039] Step 5) After the black soybeans from Step 4) are covered with a yellow skin, take them out and place them in a sealed container. Ferment them at 50°C for 15-20 days.

[0040] Step 6) Take out the black soybeans from the sealed container in Step 5), steam for 30 minutes, and then dry them to obtain the product.

[0041] The characteristic feature is that, in step 2), the liquid culture contains Aspergillus oryzae TJTSW0013×10 6 -4×10 6 cfu / mL.

[0042] In step 2), the mass percentage of added glutamic acid is 1%-10%.

[0043] In step 2), the mass percentage of added glutamic acid is 2%.

[0044] In steps 3) and 4), the mass ratio of mulberry leaves, artemisia annua, and black beans is 1:1:10.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] This application provides a method for fermenting light-colored fermented soybeans based on adjusting the ammonia nitrogen content in the fermentation environment. Using *Aspergillus oryzae* TJTSW001 as the fermentation strain, ammonia nitrogen is introduced by adding glutamic acid to its culture environment. Compared with fermentation methods without the addition of glutamic acid, the prepared light-colored fermented soybean samples show a significant increase in soybean isoflavone aglycone content, and its antipyretic effect is also enhanced.

[0047] Finally, this application provides a light fermented soybean product, which is produced by the aforementioned light fermented soybean fermentation process. Detailed Implementation

[0048] The present invention will be further described in detail below through embodiments. These embodiments are illustrative of the invention, but do not limit the invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0049] Example 1: Preparation of Lightly Fermented Soybeans

[0050] Step 1) Select mature, plump, uniform, undamaged, and moldy fresh black beans, rinse them quickly with clean water to remove surface dirt and impurities, and steam them until cooked.

[0051] Step 2) Inoculate Aspergillus oryzae TJTSW001 into a slant test tube and incubate at 35℃ for 5 days. After growth, wash off the surface spores with sterile water and inoculate the culture medium into 20ml of purified water containing 6% soybean flour and 2% glutamic acid. Incubate at 35℃ and 180rpm for 3 days. Use this as the liquid inoculum. The inoculum contains 3 × 10⁻⁶ Aspergillus oryzae TJTSW001. 6 -4×10 6 cfu / mL.

[0052] Step 3) Take 100 grams each of mulberry leaves and artemisia annua, add water and decoct three times, then combine the filtrates;

[0053] Step 4) Soak 1000g of the black beans obtained in Step 1) in the liquid until the liquid is completely absorbed. Then steam them until cooked, and after they have cooled to room temperature, mix the liquid starter into them. Cover the mixture with four layers of gauze to retain moisture.

[0054] Step 5) After the black soybeans from Step 4) are covered with a yellow skin, take them out and place them in a sealed container. Ferment them at 50°C for 15-20 days.

[0055] Step 6) Take out the black soybeans from the sealed container in Step 5), steam for 30 minutes, and then dry them to obtain the product.

[0056] Example 2: Preparation of light fermented soybeans

[0057] By modifying the fermentation and processing parameters of the light-colored fermented soybeans in Example 1, light-colored fermented soybean samples were prepared. The process parameters are shown in the table below:

[0058]

[0059]

[0060] Example 3: Determination of soybean isoflavone aglycones in fermented soybean (conducted according to CN 115054623 B).

[0061] The pharmacological effects of black soybeans are enhanced after they are processed into light fermented soybeans, mainly due to the increase in soybean isoflavone aglycone components. By testing the content of soybean isoflavone aglycone components, it can be seen that the effects of light fermented soybeans prepared by different methods are different.

[0062] 1. Preparation of test solution (samples 1-4 of light fermented soybean prepared in Examples 1 and 2 were prepared separately with consistent operating steps and conditions)

[0063] 1.1 Place the light fermented soybean samples 1-4 prepared in Example 1 and Example 2 in an oven and dry them at 60°C for 4 hours. Take them out, crush them with a pulverizer, pass them through an 80-mesh sieve, and put them in a wide-mouth bottle and seal it tightly for later use.

[0064] 1.2 Take 0.5g of light fermented soybean powder (passed through an 80-mesh sieve), accurately weigh it, place it in a 50mL volumetric flask, accurately add 50mL of petroleum ether (60-90℃), reflux for 1.5h, filter, discard the filtrate, and collect the residue.

[0065] 1.3 Let the filter residue stand to evaporate the solvent, accurately add 50 mL of 70% ethanol, shake well, weigh, extract ultrasonically for 2 h, remove and cool, add 70% ethanol to make up the weight, shake well, filter, evaporate the filtrate to dryness, and collect the filter residue.

[0066] 1.4 Dissolve the filter residue from step 1.3 in 20 mL of water, load it onto 8 mL of the prepared AB-8 macroporous adsorption resin, elute with 60 mL of water, discard the water washing solution, then elute with 80 mL of 70% ethanol, collect the ethanol washing solution and evaporate to dryness.

[0067] 1.5 Dissolve the substance collected in step 1.4 in 70% ethanol and quantitatively transfer it to a 10 mL volumetric flask. Make up to the mark, shake well, accurately pipette 1 mL into a 50 mL volumetric flask, dilute with 70% ethanol and make up to the mark, shake well, and the test solution is obtained. Label the solution according to the analyte number.

[0068] 2. Draw the standard curve

[0069] A curve was plotted with the absorbance of genistein (the main component of soybean isoflavone aglycones) as the ordinate and the concentration as the abscissa. The standard curve equation of genistein was obtained by regression processing.

[0070] 3. Content testing

[0071] 3.1 Take 10 mL of each of the numbered solutions obtained in step 1.5 and measure the absorbance at a wavelength of 263 nm using a UV-Vis spectrophotometer, and record the corresponding absorbance.

[0072] 3.2 Calculate the corresponding concentration using the standard curve equation based on the absorbance measured in step 3.1.

[0073] Test Results: Table 1. Content of soybean isoflavone aglycones (mg / g)

[0074]

[0075]

[0076] As shown in Table 1, the light-colored fermented soybeans prepared using the processing method disclosed in this invention, with glutamic acid added as ammonia nitrogen in the Aspergillus oryzae TJTSW001 culture medium, showed a significant increase in the content of soybean isoflavone aglycones in Examples 1 and 2-4 compared to Sample 1 of Example 2, which did not have glutamic acid added.

[0077] Example 4: Evaluation of the antipyretic effect of light fermented soybean sample on LPS-induced fever model (implemented according to CN 116019853 B).

[0078] 4.1 Laboratory Animals

[0079] SPF grade male mice, weighing 20-25g. Housing environment: temperature 20-28℃, humidity 45-55%, with ample water and feed.

[0080] 4.2 Preparation of Lightly Fermented Soybean Liquid

[0081] Take appropriate amounts of samples 1-4 prepared in Examples 1 and 2, add 15 times the amount of ultrapure water, soak for 30 minutes, then heat and decoct. Start timing when boiling, keep boiling gently for 1 hour, pour off the supernatant, extract the residue twice in the same way, combine the two extracts, and obtain the required concentration of the medicinal solution (each 1 ml of medicinal solution contains about 0.2 g of raw medicinal material) by rotary evaporation, concentration and volume adjustment.

[0082] 4.3 Animal modeling, grouping, and drug administration

[0083] The experimental animals were acclimatized for 3 days. For the first 3 days of the experiment, the mice were subjected to temperature measurement twice a day, morning and evening. They were fasted for 12 hours before the experiment but allowed free water. On the day of the experiment, their body temperature was measured 3 times before the onset of fever, and the average value was used as the basal body temperature. Mice with a basal body temperature >38℃ and a temperature fluctuation >0.5℃ were eliminated. Eight qualified mice were injected intraperitoneally with 0.9% sodium chloride injection (10ml / kg) as the blank group. The remaining mice were injected intraperitoneally with an equal volume of LPS (1μg / 10g) solution prepared with 0.9% sodium chloride injection to establish the fever model.

[0084] Mice were randomly divided into 8 groups (n=8 per group) using a stratified grouping method: model group, acetaminophen group (positive control), sample group of Example 1, sample group 1 of Example 2, sample group 2 of Example 2, sample group 3 of Example 2, and sample group 4 of Example 2. Immediately after grouping, the mice were administered the drugs via gavage, along with the blank control group, for a total of 8 groups. The blank control group and the model group were simultaneously administered an equal volume of physiological saline. Body temperature was then measured every 1 hour for 26 hours. The mean body temperature, standard deviation, and temperature changes at each monitoring point were calculated for each group.

[0085] 4.4 Results

[0086] Table 2 shows the changes in body temperature of febrile mice in each group at different time points after drug administration intervention.

[0087] Table 1. Body temperature of mice in each group induced by LPS (°C, x±s, n=10)

[0088]

[0089]

[0090] Table 2. Changes in body temperature of mice in each group induced by LPS (°C, mean ΔT, n=10)

[0091] Experimental Groups 1 hour after modeling 3 hours after modeling 6 hours after modeling 9 hours after modeling 26 hours after modeling Blank group -0.08 0.08 -0.06 -0.03 -0.1 Model control group 0.76 0.77 0.93 0.96 1 Acetaminophen group 0.87 0.42 0.12 0.01 -0.01 Example 1 Sample Group 0.34 0.27 0.11 0.07 0.01 Example 2 Sample 1 group 0.71 0.38 0.1 -0.05 -0.05 Example 2: 2 groups of samples 0.5 0.28 0.26 0.18 0.1 Example 2: 3 groups of samples 0.37 0.31 0.15 0.4 0.1 Example 2: 4 sample groups 0.2 0.15 0.07 0.04 -0.01

[0092] As shown in Table 2, the body temperature of mice in the model group remained elevated 1 hour after modeling. The body temperature of the positive control group (acetaminophen group), and samples 1-4 prepared in Examples 1 and 2 remained relatively stable after drug intervention, especially samples 2-4 prepared in Examples 1 and 2. This indicates that all four drugs had a certain intervention effect on LPS-induced fever in mice. Furthermore, in terms of the intervention effect on LPS-induced fever in mice, the order was: Sample 4 of Example 2 > Sample 3 of Example 2 > Sample 1 of Example 1 > Sample 2 of Example 2 > Sample 1 of Example 2 > Positive control group. Specifically, at 1 hour and 3 hours after modeling, from the perspective of the mean increase in body temperature of the model animals, the order was: Sample 4 of Example 2 < Sample 3 of Example 2 < Sample 1 of Example 1 < Sample 2 of Example 2 < Sample 1 of Example 2 < Positive control group, demonstrating that Sample 1 and Samples 2-4 of Example 2 had a better antipyretic effect compared to the positive control group and Sample 1 of Example 2.

[0093] The above description of the invention and embodiments illustrates the basic principles, main features, and advantages of this patent application. Those skilled in the art should understand that this patent application is not limited to the above embodiments. The embodiments and descriptions in the specification are merely the optimal technical solutions of this patent application. Various changes and improvements can be made to this patent application without departing from the spirit and scope of this patent application. That is, the process of fermenting and processing light-colored soybeans by appropriately controlling the ammonia nitrogen content in the fermentation environment of Aspergillus oryzae TJTSW001 falls within the scope of this patent application. The scope of protection of this patent application is defined by the appended claims and their equivalents.

Claims

1. A method for fermenting and processing light-colored fermented soybeans based on adjusting the ammonia nitrogen content in the fermentation environment, the fermentation and processing method comprising the following steps: Step 1) Select mature, plump, uniform, undamaged, and moldy fresh black beans, rinse them quickly with clean water to remove surface dirt and impurities, and steam them until cooked. Step 2) Inoculate Aspergillus oryzae into a slant test tube and culture it at 35°C for 5 days. After it has grown well, wash off the surface spores with sterile water and inoculate it into a liquid culture medium. The liquid culture medium is purified water containing 6% soybean powder and glutamic acid, with a volume of 20 ml. Culture it at 35°C and 180 rpm for 3 days to use it as a liquid inoculum. Step 3) Take mulberry leaves and artemisia annua, add water and decoct, decoct 3 times, and combine the filtrates; Step 4) Soak the black beans obtained in Step 1) in the filtrate obtained in Step 3) until the liquid is completely absorbed, then steam until cooked. After cooling to room temperature, mix in the liquid starter. Cover it with four layers of gauze to retain moisture; Step 5) After the black soybeans from Step 4) are covered with a yellow skin, take them out and place them in a sealed container. Ferment them at 50°C for 15-20 days. Step 6) Remove the black soybeans from the sealed container in Step 5), steam for 30 minutes, and then dry them to obtain the product; The characteristic feature is that the Aspergillus oryzae mentioned in step 2) is TJTSW001; the liquid culture contains 3 × 10⁻⁶ Aspergillus oryzae TJTSW001. 6 -4×10 6 cfu / mL; in step 2), the mass fraction of glutamic acid in purified water is 1%-10%.

2. The fermentation and processing method of light-colored fermented soybeans according to claim 1, characterized in that, In step 2), the mass fraction of glutamic acid in the purified water is 2%.

3. The fermentation and processing method of light-colored fermented soybeans according to claim 1, characterized in that, In steps 3) and 4), the mass ratio of mulberry leaves, artemisia annua, and black beans is 1:1:

10.

4. A light-colored fermented black bean sauce, characterized in that, It is prepared by any one of the fermentation and processing methods described in claims 1-3.

5. The use of the light fermented soybean as described in claim 4 for preparing antipyretic drugs.

Images