Method for increasing content of lovastatin in functional red yeast rice

Abstract

A method for increasing the content of lovastatin in functional red yeast rice, comprising the steps of pretreatment, inoculation and fermentation. During pretreatment, rice and an auxiliary solution accounting for 5-15% of the mass of the rice are mixed and then sterilized to serve as a rice culture medium for later use, wherein the auxiliary solution contains corn steep liquor, monopotassium phosphate, and magnesium sulfate heptahydrate, and the pH value of the auxiliary solution is 4.5-5.5; and monascus is activated and then inoculated to the rice culture medium, and fermentation is carried out, thereby obtaining a red yeast rice product. According to the method, an added auxiliary solution and rice are used to prepare a unique rice culture medium, and specific variable-temperature fermentation and turning processes are adopted in combination, so that the content of lovastatin in a functional red yeast rice product can be effectively increased, and lovastatin in the product is mainly present in an acid structure.

Description

A method for increasing the content of lovastatin in functional red yeast rice Technical Field

[0001] The invention relates to the technical field of red yeast rice fermentation, and in particular to a method for increasing the content of lovastatin in functional red yeast rice. Background Art

[0002] Red yeast rice, also known as red yeast rice, is a traditional fermented product in my country. Commonly used as a food colorant, it also possesses various pharmacological properties. Based on its primary use, it is categorized as functional red yeast rice, pigment red yeast rice, and brewed red yeast rice. Functional red yeast rice primarily refers to red yeast rice containing a high content (>4 mg / g) of lovastatin (Lovastatin or Monacolin K, MK), which can be developed into pharmaceuticals and various health foods. Lovastatin in red yeast rice can exist in two structural conformations: acidic and lactonic. The acidic form of lovastatin has a spatial structure similar to that of HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl coenzyme A reductase), inhibiting cholesterol synthesis through competitive binding. However, the lactonic form of lovastatin requires hydrolysis by hydroxyesterases in the body to the acidic form in order to exert its inhibitory effect. In addition, lovastatin also has other physiological functions, such as accelerating the decomposition and inhibition of lipid synthesis in human liver cancer cells; inhibiting the growth of human gliomas and reducing tumor deterioration and metastasis of lung cancer cells.

[0003] Currently, the production of functional red yeast rice primarily relies on solid-state fermentation based on rice. Limited by the specificity of Monascus growth and metabolism, the production of functional red yeast rice still relies primarily on traditional bottled fermentation. The lovastatin content in this product is relatively low, ranging from 4mg / g to 30mg / g. Large-scale solid-state fermentation technology has yet to achieve industrial production. Therefore, how to increase the lovastatin content in the product, maintain product stability, and facilitate large-scale processing and production is a challenge in research and development.

[0004] Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a method for increasing the content of lovastatin in functional red yeast rice, so as to increase the content of lovastatin in the product, maintain product stability and facilitate the large-scale processing and production of functional red yeast rice.

[0006] The technical problem solved by the present invention is achieved by adopting the following technical solutions:

[0007] A method for increasing the lovastatin content in functional red yeast rice comprises pretreatment, inoculation and fermentation steps. In the pretreatment step, rice is mixed with an auxiliary solution accounting for 5-15% of the rice mass and then sterilized to prepare a rice culture medium for standby use; the auxiliary solution comprises corn steep liquor, potassium dihydrogen phosphate and magnesium sulfate heptahydrate, and the pH value of the auxiliary solution is 4.5-5.5; Monascus is activated and inoculated into the rice culture medium, and then fermented to obtain a red yeast rice product.

[0008] Furthermore, the method for increasing the lovastatin content in functional red yeast rice comprises the following steps:

[0009] Pretreatment: mixing rice with an auxiliary solution accounting for 5 to 15% of the weight of the rice and sterilizing the mixture to prepare a rice culture medium; the auxiliary solution comprises corn steep liquor, potassium dihydrogen phosphate and magnesium sulfate heptahydrate, and the pH value of the auxiliary solution is 4.5 to 5.5;

[0010] Inoculation: activating Monascus to obtain Monascus seed liquid, and inoculating the Monascus seed liquid into the rice culture medium; preferably, the Monascus seed liquid is inoculated at 10-15% v / w of the mass of the rice.

[0011] Fermentation: After inoculation, ferment in the dark at 28-32° C. and 70-85% humidity for 2-3 days, then ferment in the dark at 22-26° C. and 70-85% humidity for 22-26 days, supplementing glacial acetic acid solution during the fermentation process; and drying after fermentation to obtain the fermented red yeast rice product.

[0012] Furthermore, the mass proportion of corn steep liquor in the auxiliary solution is 55-65%, the concentration of potassium dihydrogen phosphate is 1.4-1.6 g / L, and the concentration of magnesium sulfate heptahydrate is 2.8-3.2 g / L.

[0013] Furthermore, the Monascus is one of Monascus ruber with a strain number of CICC 41649 or Monascus purpureus with a strain number of CGMCC 3.0568.

[0014] Furthermore, in the inoculation step, after the Monascus is activated on the PDA plate, the bacterial block is placed in a seed culture medium, and then cultured in the dark at 30-32° C. and 150-250 rpm for 40-50 hours to obtain the Monascus seed liquid.

[0015] Furthermore, the rice is indica rice.

[0016] Furthermore, in the fermentation step, variable temperature culture is used during the fermentation process. Before the variable temperature culture, the koji is turned over every 10 to 14 hours, and after the variable temperature culture, the koji is turned over every 22 to 26 hours. During each turning over, glacial acetic acid solution is added, wherein the glacial acetic acid content in the glacial acetic acid solution is 0.1%, and the amount of glacial acetic acid solution added each time is 0.8 to 1.2% v / w of the weight of the rice. This ensures that the humidity and pH value of the culture medium are stably maintained at a level suitable for the growth of Monascus.

[0017] Furthermore, the temperature change time during the temperature-variable fermentation is based on the time when the surface of the rice culture medium turns red.

[0018] Furthermore, the material after fermentation is dried at a low temperature of 40-50° C., so that the moisture content in the red yeast rice product is less than 10%.

[0019] Preferably, the red yeast rice product contains 4.25 mg / g of lovastatin, of which the content of acid lovastatin is 4.10 mg / g and the content of lactone lovastatin is 0.15 mg / g.

[0020] Beneficial effects: The method for increasing the content of lovastatin in functional red yeast rice of the present invention has a simple process operation. It creatively adopts an added auxiliary solution and rice to prepare a unique rice culture medium, combines it with a specific temperature-variable fermentation and koji turning process, thereby forming a synthesis environment suitable for the synthesis of lovastatin, effectively promoting the synthesis of lovastatin, and effectively increasing the content of lovastatin in the functional red yeast rice product. In addition, the lovastatin in the product mainly exists in an acid structure, further improving the product quality of the functional red yeast rice. The product meets the quality requirements of the "Light Industry Industry Standard of the People's Republic of China" QB / T 2847-2007 (currently valid). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG1 is a liquid chromatogram of a standard substance of acid lovastatin;

[0022] FIG2 is a liquid chromatogram of 10 mg / L lactone lovastatin;

[0023] FIG3 is a liquid chromatogram of lovastatin in red yeast rice obtained in Example 1;

[0024] FIG4 is a liquid chromatogram of lovastatin in red yeast rice obtained in Example 2;

[0025] FIG5 is a liquid chromatogram of lovastatin in red yeast rice obtained in Reference Example 1;

[0026] FIG6 is a liquid chromatogram of lovastatin in red yeast rice obtained in Comparative Example 2. DETAILED DESCRIPTION

[0027] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific embodiments.

[0028] Example 1

[0029] A method for increasing the content of lovastatin in functional red yeast rice comprises the following steps:

[0030] Preprocessing:

[0031] Wash indica rice three times, drain the water, weigh 200 g and place it in a 1 L Erlenmeyer flask for later use; add an auxiliary solution to the Erlenmeyer flask containing the rice at a ratio of 10% v / wt of the rice mass and stir evenly; the auxiliary solution has a corn steep liquor mass concentration of 60%, a potassium dihydrogen phosphate concentration of 1.5 g / L, and a magnesium sulfate heptahydrate concentration of 3.0 g / L; and the pH value of the auxiliary solution is 5.0.

[0032] Seal the conical flask containing rice culture medium with 8 layers of gauze and sterilize it at 121℃ for 30 minutes. After sterilization, stir the culture medium while it is hot and let it cool for later use.

[0033] Vaccination:

[0034] The Monascus ruber strain used was CICC 41649, which was obtained from the China Industrial Microbiology Culture Collection Center. After the strain was activated, it was transferred to a 90 mm PDA plate and cultured for 5 days at 30 ° C. After the Monascus had grown all over the culture plate, 1 cm 2 Place the large and small fungus blocks in a 250 mL Erlenmeyer flask containing 100 mL of seed culture medium and incubate in a constant temperature shaking incubator at 30°C and 200 rpm in the dark for 48 hours to obtain Monascus seed solution. The seed culture medium contains 40 g / L glucose, 20 g / L peptone, and 10 g / L soluble starch.

[0035] The Monascus seed liquid is inoculated into the rice culture medium at 12% v / w of the mass of rice and stirred evenly.

[0036] Fermentation:

[0037] The inoculated rice culture was placed in a mold incubator at 30°C and approximately 80% humidity in the dark for fermentation for 2-3 days. The culture was shaken and turned every 12 hours. After the rice surface turned substantially red, the temperature was lowered to 25°C and fermented for another 24 days. The culture was shaken and turned every 48 hours. Each time the culture was turned, 2 mL of 0.1% glacial acetic acid solution was added.

[0038] After the fermentation, the culture was dried in an oven at 40-50°C to obtain a red yeast rice product. The red yeast rice product was ground through a 40-60 mesh sieve and then quality tested. The results are shown in FIG3 . The lovastatin content in the red yeast rice product was 2.39 mg / g, of which the acid lovastatin content was 2.27 mg / g and the lactone lovastatin content was 0.12 mg / g.

[0039] The results of multiple parallel tests and amplification tests were relatively stable.

[0040] Example 2

[0041] A method for increasing the content of lovastatin in functional red yeast rice comprises the following steps:

[0042] Preprocessing:

[0043] Wash indica rice three times, drain the water, weigh 200 g and place it in a 1 L Erlenmeyer flask for later use; add an auxiliary solution to the Erlenmeyer flask containing the rice at a ratio of 10% v / wt of the rice mass and stir evenly; the auxiliary solution has a corn steep liquor mass concentration of 60%, a potassium dihydrogen phosphate concentration of 1.5 g / L, and a magnesium sulfate heptahydrate concentration of 3.0 g / L; and the pH value of the auxiliary solution is 5.0.

[0044] Seal the conical flask containing rice culture medium with 8 layers of gauze and sterilize it at 121℃ for 30 minutes. After sterilization, stir the culture medium while it is hot and let it cool for later use.

[0045] Vaccination:

[0046] The Monascus purpureus strain numbered CGMCC 3.0568 was used and was obtained from the China General Microbiological Culture Collection Center. After the strain was activated, it was transferred to a 90 mm PDA plate and cultured for 5 days at 30 ° C. After the Monascus had grown all over the culture dish, 1 cm 2 Place the large and small fungus blocks in a 250 mL Erlenmeyer flask containing 100 mL of seed culture medium and incubate in a constant temperature shaking incubator at 30°C and 200 rpm in the dark for 48 hours to obtain Monascus seed solution. The seed culture medium contains 40 g / L glucose, 20 g / L peptone, and 10 g / L soluble starch.

[0047] The Monascus seed liquid is inoculated into the rice culture medium at 15% v / w of the mass of rice and stirred evenly.

[0048] Fermentation:

[0049] The inoculated rice culture was placed in a mold incubator at 30°C and approximately 80% humidity in the dark for fermentation for 2-3 days. The culture was shaken and turned every 12 hours. After the rice surface turned substantially red, the temperature was lowered to 25°C and fermented for another 24 days. The culture was shaken and turned every 48 hours. Each time the culture was turned, 2 mL of 0.1% glacial acetic acid solution was added.

[0050] After the fermentation, the culture was dried in an oven at 45°C to obtain a red yeast rice product. The red yeast rice product was ground through a 40-60 mesh sieve and then quality tested. The results are shown in FIG4 . The lovastatin content in the red yeast rice product was 4.25 mg / g, of which the acid lovastatin content was 4.10 mg / g and the lactone lovastatin content was 0.15 mg / g.

[0051] The results of multiple parallel tests and amplification tests were relatively stable.

[0052] Comparative Example 1

[0053] Compared with Example 1, in this reference example, no auxiliary solution was added to the rice culture medium, but water was added, and the specific steps included:

[0054] Preprocessing:

[0055] Wash the indica rice three times, drain the water, weigh 200 g and put it into a 1 L Erlenmeyer flask for later use; add water to the Erlenmeyer flask containing the rice at a ratio of 10% v / wt of the rice mass and stir evenly.

[0056] Seal the conical flask containing rice culture medium with 8 layers of gauze and sterilize it at 121℃ for 30 minutes. After sterilization, stir the culture medium while it is hot and let it cool for later use.

[0057] Vaccination:

[0058] The Monascus ruber strain used was CICC 41649, which was obtained from the China Industrial Microbiology Culture Collection Center. After the strain was activated, it was transferred to a 90 mm PDA plate and cultured for 5 days at 30 ° C. After the Monascus had grown all over the culture plate, 1 cm 2 Place the large and small fungus blocks in a 250 mL Erlenmeyer flask containing 100 mL of seed culture medium and incubate in a constant temperature shaking incubator at 30°C and 200 rpm in the dark for 48 hours to obtain Monascus seed solution. The seed culture medium contains 40 g / L glucose, 20 g / L peptone, and 10 g / L soluble starch.

[0059] Inoculate the Monascus seed liquid into the rice culture medium at 12% v / w of the rice mass and stir evenly.

[0060] Fermentation:

[0061] The inoculated rice culture was placed in a mold incubator at 30°C and approximately 80% humidity in the dark for fermentation for 2-3 days. The culture was shaken and turned every 12 hours. After the rice surface turned substantially red, the temperature was lowered to 25°C and fermented for another 24 days. The culture was shaken and turned every 48 hours. Each time the culture was turned, 2 mL of 0.1% glacial acetic acid solution was added.

[0062] After fermentation, the culture was dried in a 45°C oven to obtain the red yeast rice product. The red yeast rice product was then ground through a 40-60 mesh sieve and quality tested. The results, as shown in Figure 5, showed a lovastatin content of 0.74 mg / g, including 0.70 mg / g of acid lovastatin and 0.04 mg / g of lactone lovastatin. This indicates that the addition of the auxiliary solution has a crucial impact on the lovastatin yield in the product.

[0063] Comparative Example 2

[0064] Compared with Example 2, in this reference example, no auxiliary solution was added to the rice culture medium, but water was added, and the specific steps included:

[0065] Preprocessing:

[0066] Wash the indica rice three times, drain the water, weigh 200 g and put it into a 1 L Erlenmeyer flask for later use; add water to the Erlenmeyer flask containing the rice at a ratio of 10% v / wt of the rice mass and stir evenly.

[0067] Seal the conical flask containing rice culture medium with 8 layers of gauze and sterilize it at 121℃ for 30 minutes. After sterilization, stir the culture medium while it is hot and let it cool for later use.

[0068] Vaccination:

[0069] The Monascus purpureus strain numbered CGMCC 3.0568 was used and was obtained from the China General Microbiological Culture Collection Center. After the strain was activated, it was transferred to a 90 mm PDA plate and cultured for 5 days at 30 ° C. After the Monascus had grown all over the culture dish, 1 cm 2 Place the large and small fungus blocks in a 250 mL Erlenmeyer flask containing 100 mL of seed culture medium and incubate in a constant temperature shaking incubator at 30°C and 200 rpm in the dark for 48 hours to obtain Monascus seed solution. The seed culture medium contains 40 g / L glucose, 20 g / L peptone, and 10 g / L soluble starch.

[0070] The Monascus seed liquid is inoculated into the rice culture medium at 15% v / w of the mass of rice and stirred evenly.

[0071] Fermentation:

[0072] The inoculated rice culture was placed in a mold incubator at 30°C and approximately 80% humidity in the dark for fermentation for 2-3 days. The culture was shaken and turned every 12 hours. After the rice surface turned substantially red, the temperature was lowered to 25°C and fermented for another 24 days. The culture was shaken and turned every 48 hours. Each time the culture was turned, 2 mL of 0.1% glacial acetic acid solution was added.

[0073] After fermentation, the culture was dried in a 45°C oven to obtain the red yeast rice product. The red yeast rice product was then ground through a 40-60 mesh sieve and quality tested. The results, as shown in Figure 6, showed a lovastatin content of 1.39 mg / g, including 1.31 mg / g of acid lovastatin and 0.08 mg / g of lactone lovastatin. This indicates that the addition of the auxiliary solution has a crucial impact on the lovastatin yield in the product.

[0074] Comparative Example 3

[0075] In this embodiment, rice was mixed with an auxiliary solution accounting for 20% of the mass of rice and then sterilized to prepare a rice culture medium. The rest was the same as in Example 1. As a result, the fermentation failed and no red yeast rice product could be obtained.

[0076] Comparative Example 4

[0077] In this embodiment, the amount of glacial acetic acid solution added each time was 2.0% v / w of the mass of rice, and the rest was the same as in Example 1. As a result, the fermentation failed and no red yeast rice product could be obtained.

[0078] In each embodiment and control example, the detection of lovastatin in the functional red yeast rice product and the lovastatin standard product Lovastatin (L0790000, Sigma) was carried out with reference to the determination method for the content of moracolin (Monacolin K) in functional red yeast rice in Appendix A of the "Light Industry Standard of the People's Republic of China" QB / T2847-2007. The liquid chromatograms of the acid lovastatin standard and 10 mg / L lactone lovastatin are shown in Figures 1 and 2, respectively.

[0079] The process and product indicators of Examples 1-2 and Comparative Examples 1-2 are shown in Table 1.

[0080] Table 1

[0081] In the red yeast rice product prepared by the present invention, the lovastatin content is 2.39 mg / g and 4.25 mg / g, respectively, and the acid lovastatin content is relatively high. When no auxiliary solution is added and water is used instead, the lovastatin content is only 0.74 mg / g and 1.39 mg / g, which is much lower than the lovastatin content in the product of the present invention. It is explained that in the technical solution of the present invention, by adding the auxiliary solution, it is unexpected that during the fermentation process using red Monascus ruber (Monascus ruber) with a strain number of CICC 41649 or Monascus purpureus (Monascus purpureus) with a strain number of CGMCC 3.0568 as the strain, lovastatin synthesis is greatly promoted, thereby increasing lovastatin production.

[0082] In contrast, in Comparative Examples 3 and 4, improper addition of the auxiliary solution or improper addition of the glacial acetic acid solution during the fermentation process resulted in fermentation failure, indicating that the process of the present invention is the result of proper coordination of each step.

[0083] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for increasing the content of lovastatin in functional red yeast rice, characterized in that: The method comprises pretreatment, inoculation and fermentation steps. In the pretreatment process, rice is mixed with an auxiliary solution accounting for 5-15% of the weight of the rice and then sterilized to prepare a rice culture medium for standby use; the auxiliary solution comprises corn steep liquor, potassium dihydrogen phosphate and magnesium sulfate heptahydrate, and the pH value of the auxiliary solution is 4.5-5.5; Monascus is activated and inoculated into the rice culture medium, and then fermented to obtain a red yeast rice product.

2. The method for increasing the content of lovastatin in functional red yeast rice according to claim 1, characterized in that: The steps include: Pretreatment: mixing rice with an auxiliary solution accounting for 5-15% of the weight of the rice and sterilizing the mixture to prepare a rice culture medium; the auxiliary solution comprises corn steep liquor, potassium dihydrogen phosphate and magnesium sulfate heptahydrate, and the pH value of the auxiliary solution is 4.5-5.5; Inoculation: activating Monascus to obtain Monascus seed liquid, and inoculating the Monascus seed liquid into the rice culture medium; Fermentation: After inoculation, ferment in the dark at 28-32° C. and 70-85% humidity for 2-3 days, then ferment in the dark at 22-26° C. and 70-85% humidity for 22-26 days, supplement with glacial acetic acid solution during the fermentation process; dry after fermentation to obtain the fermented red yeast rice product.

3. The method for increasing the content of lovastatin in functional red yeast rice according to claim 1, characterized in that: The mass proportion of corn steep liquor in the auxiliary solution is 55-65%, the concentration of potassium dihydrogen phosphate is 1.4-1.6 g / L, and the concentration of magnesium sulfate heptahydrate is 2.8-3.2 g / L.

4. The method for increasing the content of lovastatin in functional red yeast rice according to claim 1, characterized in that: The Monascus is a red Monascus (Monascus ruber) with a strain number of CICC 41649, or a purple Monascus (Monascus purpureus) with a strain number of CGMCC 3.0568.

5. The method for increasing the content of lovastatin in functional red yeast rice according to claim 1, characterized in that: In the inoculation step, after the Monascus is activated on the PDA plate, the bacterial block is placed in a seed culture medium, and then cultured in the dark at 30-32° C. and 150-250 rpm for 40-50 hours to obtain the Monascus seed liquid.

6. The method for increasing the content of lovastatin in functional red yeast rice according to claim 1, characterized in that: The rice is indica rice.

7. The method for increasing the content of lovastatin in functional red yeast rice according to claim 1, characterized in that, in the fermentation step, variable temperature culture is adopted during the fermentation process, and before the variable temperature culture, the yeast is turned over every 10 to 14 hours, and after the variable temperature culture, the yeast is turned over every 22 to 26 hours; glacial acetic acid solution is added each time the yeast is turned over, the content of glacial acetic acid in the glacial acetic acid solution is 0.1%, and the amount of glacial acetic acid solution added each time is 0.8 to 1.2% v / w of the mass of rice.

8. The method for increasing the content of lovastatin in functional red yeast rice according to claim 7, characterized in that: The temperature change time during the temperature-changing fermentation is based on the time when the surface of the rice culture medium turns red.

9. The method for increasing the content of lovastatin in functional red yeast rice according to claim 2, characterized in that: The material after fermentation is dried at low temperature at 40-50°C to make the moisture content of the red yeast rice product less than 10%.

10. The method for increasing the content of lovastatin in functional red yeast rice according to claim 2, characterized in that: The red yeast rice product has a lovastatin content of 4.25 mg / g, of which the acid lovastatin content is 4.10 mg / g and the lactone lovastatin content is 0.15 mg / g.

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