CN117099949A - Methods and applications for preparing S-adenosylmethionine-rich foods through Rhizopus oryzae fermentation - Google Patents

Abstract

The invention discloses an application of Rhizopus oryzae (CICC 40316) in preparing a food rich in S-adenosylmethionine by fermentation, which comprises the following steps: 1) Addition of vitamin B to fermentation substrate ₁ Vitamin B ₁₂ Mixing methionine and lysine uniformly; 2) Inoculating rhizopus oryzae strain into the mixture of the step 1), and fermenting and culturing to obtain the food rich in S-adenosylmethionine. The invention firstly utilizes the rhizopus oryzae which is a common strain for food fermentation to ferment to generate SAMe, and the invention adds VB into the fermentation raw material ₁ 、VB ₁₂ Methionine and lysine, the rhizopus oryzae fermented cereal is induced to produce high-content SAMe, the raw materials used meet the national food safety standards, no food safety risk is caused, the produced product can be directly eaten, and the later extraction is avoidedHigh cost, production hazard, environmental hazard and other risks brought by purification and the like.

Description

Methods and applications for preparing S-adenosylmethionine-rich foods through Rhizopus oryzae fermentation

Technical field

The invention belongs to the field of food processing. More specifically, the present invention relates to a method and application for preparing food rich in S-adenosylmethionine through Rhizopus oryzae fermentation.

Background technique

Methionine is an important methyl donor in organisms, but it must first be activated into S-adenosylmethionine (SAMe) to exert physiological effects. SAMe was first found in the liver and is widely involved in various biochemical pathways and metabolic reactions in organisms. It has the functions of transmethylation, transsulfation and transaminopropyl. It is closely related to the metabolism of proteins, nucleic acids, phospholipids, vitamins and other substances in organisms. It is also the precursor of bioactive substances such as glutathione and coenzymes, and participates in various biological processes such as human gene regulation, neurotransmission and detoxification.

SAMe has been used in Europe as a prescription drug to treat arthritis and later as an antidepressant. At present, SAMe has been widely used in the medical field. It is used for the treatment of chronic diseases such as liver disease, arthritis, and depression. It is also used as a health care drug for the prevention of cancer, cardiovascular disease, and anti-aging. It has good effects and few side effects. In 1999, the US FDA approved its use as a dietary supplement. In 2000, China began to import SAMe as a food nutritional supplement and is widely used in the food field. In recent years, as people pay more and more attention to health, the consumer market's demand for products rich in SAMe ingredients has also grown rapidly.

At present, the production technologies of SAMe mainly include chemical synthesis, in vitro enzyme catalytic synthesis and microbial fermentation. The prospects for industrialization of chemical synthesis methods are relatively limited, because there are many isomers in the synthesized products, which are difficult to separate and purify and the molecules are unstable; in vitro enzymatic synthesis methods have advantages in industrial production compared to chemical synthesis methods. Potential, but is limited by the difficulty and cost control of a large number of highly active SAMe synthases. In recent years, the main way to produce SAMe in industry is microbial fermentation. It mainly uses Saccharomyces cerevisiae, genetically engineered Pichia pastoris, recombinant Escherichia coli engineering bacteria, etc. for industrial production, and then purifies through separation and purification. The SAMe products produced are very expensive at 2000-3500 yuan/kg and are not suitable for the daily health care and health needs of ordinary people. Therefore, it is very necessary to produce common foods rich in SAMe to meet people's health needs for daily healthy diets.

Contents of the invention

It is an object of the present invention to solve at least the above-mentioned problems and to provide at least the advantages to be explained later.

An object of the present invention is to provide an application of Rhizopus oryzae in the fermentation preparation of S-adenosylmethionine-rich foods, which increases the S-adenosine in cereal foods by fermenting cereal foods with Rhizopus oryzae. Glycoside methionine content.

In order to achieve these objects and other advantages of the present invention, the use of Rhizopus oryzae CICC 40316 in the fermentation of S-adenosylmethionine-rich food is provided.

A method for preparing S-adenosylmethionine-rich food through Rhizopus oryzae CICC 40316 fermentation, including the following steps:

1) Add vitamin B ₁ , vitamin B ₁₂ , methionine and lysine to the fermentation matrix and mix evenly;

2) Inoculate Rhizopus oryzae species into the mixture in step 1), conduct fermentation and culture, and obtain food rich in S-adenosylmethionine.

Preferably, the amount of vitamin B ₁ added is 5-15 mg/kg, the amount of vitamin B ₁₂ added is 1-10 μg/kg, the amount of methionine added is 0.5-2.0 g/kg, and the amount of lysine added is 0.5-2.0 g/kg. The amount is 0.5-2.0g/kg.

Preferably, the inoculum amount of Rhizopus oryzae is 0.01-1.0%.

Preferably, the fermentation temperature is 26-36°C and the fermentation time is 24-72h.

Preferably, the preparation of the fermentation substrate includes the following steps:

Step 1: Soak the grains in water for 8-24 hours, drain the water, and then pulverize them. Pass the pulverized material through a 8-20 mesh sieve to obtain the undersize;

Step 2: Steam the sieved items in a steamer for 20-60 minutes until the grains are cooked to obtain steamed grains;

Step 3: Add cold boiled water or pure water to the steamed grains. The mass ratio of steamed grains to water is 1:0.6~1.2. Stir to mix the steamed grains and water evenly to obtain a fermentation substrate.

Preferably, the cereals include, but are not limited to, barley, indica rice, japonica rice, glutinous rice, brown rice, colored rice, yellow millet, wheat, barley, oats, rye, corn, sorghum, millet, millet, yellow rice, and buckwheat. One or several.

The present invention at least includes the following beneficial effects:

First, for the first time, the present invention uses the commonly used food fermentation strain Rhizopus oryzae to ferment to produce SAMe, instead of the currently commonly used strains that produce SAMe, such as Saccharomyces cerevisiae, Candida primogenogenes, recombinant Escherichia coli, recombinant Pichia pastoris, and metabolic engineering. Bacillus amyloliquefaciens, metabolically engineered Corynebacterium glutamicum, etc.

Second, the current recombinant Pichia pastoris that produces SAMe often uses methanol as an inducer, which has certain production safety risks and environmental hazards. In the later stage, complex technologies are required to remove the methanol, which increases production costs. SAMe produced by recombinant E. coli is inside the bacteria, and E. coli is a pathogenic bacteria. Therefore, complex post-processing technology is required to produce SAMe products with food safety properties. To produce SAMe using yeast fermentation, the fermentation liquid needs to undergo filtration, crushing, cation exchange adsorption, elution, recrystallization and vacuum drying technology to obtain SAMe products with food safety attributes; and the purification technology requires the use of concentrated sulfuric acid, concentrated sulfuric acid, and vacuum drying technology. Hydrochloric acid, perchloric acid, methanol, chloroform and other substances are very harmful to the environment and can easily cause production safety accidents. By adding VB ₁ , VB ₁₂ , methionine and lysine to fermented raw materials, the present invention induces Rhizopus oryzae to ferment grains to produce high-content SAMe. The raw materials used are in compliance with national food safety standards, and there is no food safety risk. The products can be eaten directly and avoid risks such as high costs, production hazards and environmental hazards caused by later extraction and purification.

Third, the present invention adopts solid fermentation technology. Compared with commonly used liquid fermentation technology, there is no need to carry out downstream technical treatments such as separation, concentration, and purification commonly used in liquid fermentation, and no fermentation wastewater is produced, thus simplifying the production process. It reduces production costs and is eco-friendly.

Fourth, the present invention uses grains for fermentation to obtain ordinary food rich in SAMe, and the food obtained can be eaten directly; while other fermentation methods commonly used at present require complex raw materials, and the fermented products rich in SAMe cannot be directly eaten. It requires complex purification technology before it can be eaten.

Other advantages, objects, and features of the present invention will be apparent in part from the description below, and in part will be understood by those skilled in the art through study and practice of the present invention.

Detailed ways

The present invention will be described in further detail below so that those skilled in the art can implement it according to the text of the description.

It should be understood that terms such as "having," "comprising," and "including" as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

It should be noted that the experimental methods described in the following embodiments, unless otherwise specified, are all conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

The invention provides an application of Rhizopus oryzae CICC 40316 in the fermentation of food rich in S-adenosylmethionine.

The Rhizopus oryzae used in the present invention was purchased from the China Industrial Microbial Culture Collection Center, and the preservation number is CICC 40316.

A method for preparing S-adenosylmethionine-rich food through fermentation of Rhizopus oryzae CICC 40316, including the following steps:

1) Add vitamin B ₁ , vitamin B ₁₂ , methionine and lysine to the fermentation matrix and mix evenly;

2) Inoculate Rhizopus oryzae species into the mixture in step 1), conduct fermentation and culture, and obtain food rich in S-adenosylmethionine.

In another technical solution, the added amount of vitamin _B1 is 5-15mg/kg, the added amount of vitamin B12 is 1-10μg/kg, the added amount of methionine is 0.5-2.0g/kg, and the added amount of lysine is 0.5-2.0g/kg. The amount of acid added is 0.5-2.0g/kg.

In another technical solution, the inoculum amount of Rhizopus oryzae is 0.01-1.0%.

In another technical solution, the fermentation temperature is 26-36°C and the fermentation time is 24-72h.

In another technical solution, the preparation of the fermentation substrate includes the following steps:

Step 1: Soak the grains in water for 8-24 hours, drain the water, and then pulverize them. Pass the pulverized material through a 8-20 mesh sieve to obtain the undersize;

Step 2: Steam the sieved material in a steamer for 20-60 minutes until the grains are cooked and there are no white cores to obtain steamed grains;

Step 3: Add cold boiled water or pure water to the steamed grains. The mass ratio of steamed grains to water is 1:0.6~1.2. Stir to mix the steamed grains and water evenly to obtain a fermentation substrate.

In another technical solution, the cereals include but are not limited to barley, indica rice, japonica rice, glutinous rice, brown rice, colored rice, yellow millet, wheat, barley, oats, rye, corn, sorghum, millet, millet, and yellow rice. , one or more types of buckwheat.

Example 1

Method for preparing food rich in S-adenosylmethionine through fermentation of Rhizopus oryzae CICC 40316

Select barley with plump grains without insect damage and mildew as raw material, rinse it with running water, and then add tap water to soak it according to the volume ratio of material to water of 1:3 (v/v). After soaking for 12 hours, drain the water from the barley and then crush it. The crushed material passes through an 8-mesh sieve, and the material below the sieve is taken for the next step. Place the sifted contents into the steamer and steam for 30 minutes until the barley grains are cooked and no white cores appear, thus obtaining steamed barley. Add cold boiled water to the steamed barley at a ratio of material to water mass ratio of 1:0.9, stir while adding water, so that the water and steamed barley are fully mixed evenly to obtain a mixture. Then based on the mass of the mixture, weigh appropriate amounts of VB ₁ , VB ₁₂ , methionine, and lysine respectively, add them into the mixture, and then stir evenly to obtain a fermentation substrate. In the fermentation matrix, the content of vitamin B ₁ (VB ₁ ) is 9mg/kg, the content of vitamin B ₁₂ (VB ₁₂ ) is 5μg/kg, the content of methionine is 1.2g/kg, and the content of L-lysine The content is 1.0g/kg. When the fermentation substrate is cooled to 35°C, add Rhizopus oryzae at an amount of 5g/kg based on the quality of the fermentation substrate, and stir to mix evenly. Then put the inoculated fermentation substrate into a fermentation box and keep it fermented in a constant temperature room at 30°C for 48 hours to obtain fermented barley food rich in SAMe.

Example 2

Method for preparing food rich in S-adenosylmethionine through fermentation of Rhizopus oryzae CICC 40316

Select rice with full grains and no insect infestation or mildew as raw material, rinse it with running water, and then add tap water to soak it according to the volume ratio of material to water of 1:3 (v/v). After soaking for 12 hours, drain the rice and then crush it. The crushed material passes through a 20-mesh sieve, and the material below the sieve is taken for the next step. Place the sieved contents into the steamer and steam for 30 minutes until the rice grains are cooked and no white core is left, and steamed rice is obtained. Add cold boiled water to the steamed rice according to the ratio of material to water mass ratio of 1:0.9, stir while adding water, so that the water and steamed rice are fully mixed evenly to obtain a mixture. Then based on the mass of the mixture, weigh appropriate amounts of VB ₁ , VB ₁₂ , methionine, and lysine respectively, add them into the mixture, and then stir evenly to obtain a fermentation substrate. In the fermentation matrix, the content of vitamin B ₁ (VB ₁ ) is 9mg/kg, the content of vitamin B ₁₂ (VB ₁₂ ) is 5μg/kg, the content of methionine is 1.2g/kg, and the content of L-lysine The content is 1.0g/kg. When the fermentation substrate is cooled to 35°C, add Rhizopus oryzae at an amount of 5g/kg based on the quality of the fermentation substrate, and stir to mix evenly. Then put the inoculated fermentation substrate into a fermentation box and keep it fermented in a constant temperature room at 30°C for 48 hours to obtain fermented rice food rich in SAMe.

Example 3

Method for preparing food rich in S-adenosylmethionine through fermentation of Rhizopus oryzae CICC 40316

Select brown rice with plump grains without insect damage and mildew as the raw material, rinse it with running water, and then add tap water to soak it according to the volume ratio of material to water of 1:3 (v/v). After soaking for 12 hours, drain the brown rice and then crush it. The crushed material passes through a 20-mesh sieve, and the material below the sieve is taken for the next step. Place the sieved contents into the steamer and steam for 30 minutes until the brown rice grains are cooked to obtain steamed brown rice. Add pure water to the steamed brown rice at a ratio of 1:0.9 to water, and stir while adding water to fully mix the water and steamed brown rice to obtain a mixture. Then based on the mass of the mixture, weigh appropriate amounts of VB ₁ , VB ₁₂ , methionine, and lysine respectively, add them into the mixture, and then stir evenly to obtain a fermentation substrate. In the fermentation matrix, the content of vitamin B ₁ (VB ₁ ) is 9mg/kg, the content of vitamin B ₁₂ (VB ₁₂ ) is 5μg/kg, the content of methionine is 1.2g/kg, and the content of L-lysine The content is 1.0g/kg. When the fermentation substrate is cooled to 35°C, add Rhizopus oryzae at an amount of 5g/kg based on the quality of the fermentation substrate, and stir to mix evenly. Then put the inoculated fermentation substrate into a fermentation box and keep it fermented in a constant temperature room at 30°C for 48 hours to obtain fermented brown rice food rich in SAMe.

Example 4

Method for preparing food rich in S-adenosylmethionine through fermentation of Rhizopus oryzae CICC 40316

Select sorghum with full grains, no insects, and no mildew as raw material, rinse it with running water, and then add tap water to soak it according to the volume ratio of material to water of 1:3 (v/v). After soaking for 12 hours, drain the sorghum and then pulverize it. The pulverized material passes through an 8-mesh sieve, and the undersized material is taken for the next step. Place the sieved material into the steamer and steam for 30 minutes until the sorghum grains are cooked to obtain steamed sorghum. Add pure water to the steamed sorghum at a ratio of 1:0.9 to water, stir while adding water, so that the water and steamed sorghum are fully mixed evenly to obtain a mixture. Then based on the mass of the mixture, weigh appropriate amounts of VB ₁ , VB ₁₂ , methionine, and lysine respectively, add them into the mixture, and then stir evenly to obtain a fermentation substrate. In the fermentation matrix, the content of vitamin B ₁ (VB ₁ ) is 9mg/kg, the content of vitamin B ₁₂ (VB ₁₂ ) is 5μg/kg, the content of methionine is 1.2g/kg, and the content of L-lysine The content is 1.0g/kg. When the fermentation substrate is cooled to 35°C, add Rhizopus oryzae at an amount of 5g/kg based on the quality of the fermentation substrate, and stir to mix evenly. Then put the inoculated fermentation substrate into a fermentation box and keep it fermented in a constant temperature room at 30°C for 48 hours to obtain fermented sorghum food rich in SAMe.

Comparative example 1

The preparation method of Example 1 was adopted, but the difference from Example 1 was that VB ₁₂ , methionine and lysine were not added in Comparative Example 1.

Comparative example 2

The preparation method of Example 1 was adopted, but the difference from Example 1 was that VB ₁ , methionine and lysine were not added in Comparative Example 2.

Comparative example 3

The preparation method of Example 1 was adopted, but the difference from Example 1 was that VB ₁ , VB ₁₂ and lysine were not added in Comparative Example 3.

Comparative example 4

The preparation method of Example 1 was adopted, but the difference from Example 1 was that VB ₁ , VB ₁₂ and methionine were not added in Comparative Example 4.

Comparative example 5

The preparation method of Example 1 was adopted, but the difference from Example 1 was that VB ₁ , VB ₁₂ , methionine and lysine were not added in Comparative Example 5.

Comparative example 6

The preparation method of Example 1 was adopted, and the difference from Example 1 was that in Comparative Example 6, Rhizopus oryzae species were not inoculated.

Comparative example 7

The preparation method of Example 1 was adopted, but the difference from Example 1 was that methionine and lysine were not added in Comparative Example 7.

Comparative example 8

The preparation method of Example 1 was adopted, and the difference from Example 1 was that VB ₁₂ and lysine were not added in Comparative Example 8.

Comparative example 9

The preparation method of Example 1 was adopted, but the difference from Example 1 was that VB ₁₂ was not added in Comparative Example 9.

Comparative example 10

The preparation method of Example 1 was adopted, and the difference from Example 1 was that VB ₁ and VB ₁₂ were not added in Comparative Example 10.

SAMe in fermented foods is detected by high performance liquid chromatography. The specific detection technology is: weigh about 2.5g of fermented food, homogenize it with a homogenizer, then add 4 times the volume of 0.6mol/L perchloric acid solution, conduct ultrasonic extraction for 20 minutes, centrifuge the supernatant and pass it through a 0.45μm filter membrane. , the filtrate is reserved. Using 50mmol/L NaH ₂ PO ₄ buffer, 10mmol/L sodium heptane sulfonate and 20% (V/V) methanol as the mobile phase, use a C18 (4.6mm×250mm, 5μm) column for chromatographic separation, with a flow rate of 1.0ml /min, UV detection wavelength 254nm.

The test results are shown in Table 1.

Table 1 SAMe content in fermented foods

It can be seen from the results in Table 1 that the results of Comparative Example 5 and Comparative Example 1 show that VB ₁ has a certain promoting effect on the production of SAMe by Rhizopus oryzae. The results of Comparative Example 5 and Comparative Example 2 show that the promoting effect of VB ₁₂ on the production of SAMe by Rhizopus oryzae is relatively weak. The results of Comparative Example 5 and Comparative Example 3 show that methionine has a certain promoting effect on the production of SAMe by Rhizopus oryzae. The results of Comparative Example 5 and Comparative Example 4 show that lysine has a certain promoting effect on the production of SAMe by Rhizopus oryzae. The results of Comparative Examples 5 and 6 show that Rhizopus oryzae has the effect of fermenting barley to produce SAMe. The results of Comparative Example 2, Comparative Example 5, and Comparative Example 7 show that VB ₁ and VB ₁₂ have a certain promoting effect on Rhizopus oryzae production of SAMe, and the promotion is stronger than using VB ₁₂ alone. The results of Comparative Example 1, Comparative Example 3, Comparative Example 5, and Comparative Example 8 show that VB ₁ , methionine has a certain promoting effect on Rhizopus oryzae production of SAMe, and VB ₁ , the mixed promoting effect of methionine Stronger than VB ₁ or methionine alone. The results of Comparative Examples 1, 3, 4, 5, and 9 show that VB ₁ , methionine, and lysine have a certain promoting effect on the production of SAMe by Rhizopus oryzae, and VB1, A The promoting effect of the mixed use of thionine and lysine is stronger than that of VB ₁ , methionine or lysine alone. The results of Comparative Example 3, Comparative Example 5, and Comparative Example 10 show that methionine and lysine have a certain promoting effect on Rhizopus oryzae production of SAMe, but the promoting effect of methionine and lysine when used together is weak. Compared with the promoting effect of methagonine alone. The results of Example 1 and Comparative Examples 1-10 show that the joint use of VB ₁ , VB ₁₂ , methionine, and lysine has a certain promoting effect on the production of SAMe by Rhizopus oryzae, and VB ₁ , VB ₁₂ , A The promoting effect of the combined use of sulfate and lysine is better than the promoting effect of using VB ₁ , VB ₁₂ , methionine and lysine alone. In summary, Rhizopus oryzae can ferment grains to produce SAMe, and the combined use of VB ₁ , VB ₁₂ , methionine, and lysine can induce Rhizopus oryzae to increase SAMe production.

The present invention includes but is not limited to one or more of barley, indica rice, japonica rice, glutinous rice, brown rice, colored rice, yellow millet, wheat, barley, oats, rye, corn, sorghum, millet, millet, yellow rice, and buckwheat. The fermentation substrate prepared by each method can increase the SAMe content in the fermentation product after inoculation with Rhizopus oryzae for fermentation.

Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and embodiments. They can be applied to various fields suitable for the present invention. For those familiar with the art, they can easily Additional modifications may be made, and therefore the invention is not limited to the specific details without departing from the general concept defined by the claims and equivalent scope.

Claims (7)

1. Application of Rhizopus oryzae CICC 40316 in the fermentation of foods rich in S-adenosylmethionine. 2. A method for preparing food rich in S-adenosylmethionine through Rhizopus oryzae fermentation, which is characterized by comprising the following steps: 1) Add vitamin B ₁ , vitamin B ₁₂ , methionine and lysine to the fermentation matrix and mix evenly; 2) Inoculate Rhizopus oryzae species into the mixture in step 1), conduct fermentation and culture, and obtain food rich in S-adenosylmethionine. 3. The method for preparing food rich in S-adenosylmethionine by Rhizopus oryzae fermentation according to claim 2, characterized in that the addition of vitamin _B1 is 5-15mg/kg, and the addition of vitamin _B12 The amount is 1-10 μg/kg, the amount of methionine is 0.5-2.0g/kg, and the amount of lysine is 0.5-2.0g/kg. 4. The method for preparing S-adenosylmethionine-rich food by Rhizopus oryzae fermentation according to claim 2, characterized in that the inoculum amount of Rhizopus oryzae is 0.01-1.0%. 5. The method for preparing S-adenosylmethionine-rich food by Rhizopus oryzae fermentation according to claim 2, characterized in that the fermentation temperature is 26-36°C and the fermentation time is 24-72h. 6. The method for preparing S-adenosylmethionine-rich food by Rhizopus oryzae fermentation according to claim 2, characterized in that the preparation of fermentation substrate includes the following steps: Step 1: Soak the grains in water for 8-24 hours, drain the water, and then pulverize them. Pass the pulverized material through a 8-20 mesh sieve to obtain the undersize; Step 2: Steam the sieved items in a steamer for 20-60 minutes until the grains are cooked to obtain steamed grains; Step 3: Add cold boiled water or pure water to the steamed grains. The mass ratio of steamed grains to water is 1:0.6~1.2. Stir to mix the steamed grains and water evenly to obtain a fermentation substrate. 7. The method for preparing food rich in S-adenosylmethionine by Rhizopus oryzae fermentation according to claim 6, characterized in that the cereals are barley, indica rice, japonica rice, glutinous rice, brown rice, colored rice, yellow rice, etc. One or more of millet, wheat, barley, oats, rye, corn, sorghum, millet, millet, yellow rice, and buckwheat.