Method for producing monacolin K by bidirectional fermentation of medicinal and edible traditional Chinese medicine honeysuckle flower-monascus purpureus

Abstract

The invention belongs to the technical field of microorganism and traditional Chinese medicine fermentation, and particularly relates to a method for producing monacolin K through bidirectional fermentation of medicinal and edible traditional Chinese medicine honeysuckle flower-monascus purpureus. Honeysuckle is used as an exogenous additive to be subjected to bidirectional co-fermentation with monascus purpureus, and fermentation conditions such as an additive pretreatment method, addition concentration and culture time are optimized, so that the growth of thalli is promoted, and meanwhile, the expression of monacolin K synthesis related genes is regulated and controlled; and the yield of the monacolin K in the monascus purpureus fermentation liquor is obviously improved to 536.10 mg / L. The contents of monascus pigment, total flavonoids and total polyphenols in the obtained fermentation liquor are obviously increased, and the obtained fermentation liquor has relatively strong antioxidant activity. Two-way fermentation of the honeysuckle and the monascus purpureus is utilized, the monascus purpureus can be used for producing the monacolin K, a plurality of effective active ingredients are contained in fermentation liquor, and the monacolin K has wide application prospects in the field of fermentation of medicinal and edible products.

Description

Technical Field

[0001] The present invention relates to the technical fields of microorganisms and traditional Chinese medicine fermentation, and particularly relates to a method for bidirectional fermentation of honeysuckle, a traditional Chinese medicine with both medicinal and edible properties, and Monascus purpureus to produce monacolin K. Background Art

[0002] Monascus is a filamentous fungus that can be used as both medicine and food, and can produce a variety of metabolites with application value. Secondary metabolites such as Monascus pigments, γ-aminobutyric acid, and monacolin K produced by Monascus during the secondary metabolic process have been the focus of research in recent years. Monacolin K is a polyketide isolated from the fermentation broth of Monascus ruber, and has been proven to have a good effect on inhibiting cholesterol synthesis. Through research, it has been found that monacolin K can control the level of cholesterol synthesis by inhibiting the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase related to cholesterol synthesis.

[0003] According to the industry standard of our country, "QB / T2847-2007 Functional Monascus rice (powder)", the ability of Monascus to ferment and synthesize monacolin K is the key to determining the quality of functional Monascus. However, the current production capacity of monacolin K by existing Monascus strains is relatively low, resulting in a corresponding increase in the industrial production cost, severely restricting the development of the functional Monascus industry, and also becoming a major obstacle to the application of Monascus in the fields of food and health products. Therefore, it is of great significance to explore effective methods to increase the yield of monacolin K to solve the current situation of low production in traditional fermentation. In recent years, researchers have done a lot of in-depth exploration on improving the quality of functional Monascus from two aspects: exogenous and endogenous. One is to promote the synthesis of monacolin K by Monascus by optimizing the fermentation conditions, such as controlling environmental factors or improving the composition and ratio of the culture medium; the other is to select high-yield strains by mutagenesis breeding methods or genetic engineering techniques, and fundamentally and directionally transform Monascus at the gene level in order to achieve the efficient production of monacolin K.

[0004] The co-fermentation of traditional Chinese medicine resources and microorganisms is of great significance, which can not only make full use of resources, but also expand their application potential. The Qu preparation with a long history of clinical application is one of the traditional Chinese medicines that embody the characteristics of fermentation. The co-fermentation of traditional Chinese medicine and microorganisms can, on the one hand, provide substrates for the growth and metabolism of microorganisms, and on the other hand, the microorganisms themselves or their metabolic enzymes also contribute to the transformation of active ingredients in traditional Chinese medicine. Active ingredients in traditional Chinese medicine can effectively promote the growth and metabolism of Monascus, but how to achieve their synergistic application is still a key problem to be solved. The present invention has deeply studied the application of honeysuckle, a traditional Chinese medicine, as an exogenous additive in the liquid fermentation medium of Monascus, significantly improving the production efficiency of monacolin K, and at the same time significantly increasing the growth rate, biomass of the bacteria and the synthesis of secondary metabolites, providing technical support for the development of high-value-added fermented products with both medicinal and edible properties. Summary of the Invention

[0005] The present invention relates to the technical fields of microorganisms and traditional Chinese medicine fermentation. Specifically, it is a method for producing monacolin K by the bidirectional fermentation of honeysuckle and Monascus purpureus, which not only increases the content of monacolin K, but also increases the contents of active ingredients such as monascus pigments, total polyphenols, and total flavonoids, and can be used to develop high-value-added fermented food and medicine homologous products.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for producing monacolin K by the bidirectional fermentation of the food and medicine homologous traditional Chinese medicine honeysuckle - Monascus purpureus, which comprises the following steps:

[0008] Weigh a certain amount of honeysuckle, add it to the Monascus purpureus fermentation medium after pretreatment, observe the morphological changes of the mycelium, measure the content of monacolin K in the fermentation broth after co-fermentation, and analyze the transcriptional situation of the genes related to monacolin K synthesis; detect the growth indexes of Monascus purpureus, and measure the contents of active ingredients such as monascus pigments, total polyphenols, and total flavonoids, and evaluate the in vitro antioxidant activity of the fermentation broth;

[0009] The pretreatment of the honeysuckle can be prepared by powder grinding or juice decocting. Juice decocting: Weigh 5 g of honeysuckle, wash it with clean water, add 500 mL of distilled water and soak for 1 h, heat and keep it boiling for 30 min, filter out the juice; repeat the above operation, combine the juices decocted twice, and add distilled water to make up the volume to 1000 mL, sterilize at 121 °C for 15 min for standby; Powder grinding: Weigh a certain amount of honeysuckle, wash it with clean water and air-dry it naturally, use a pulverizer to grind it into powder and pass through an 80-mesh sieve, sterilize at 121 °C for 15 min for standby.

[0010] After the pretreatment of the honeysuckle, add it to the Monascus purpureus fermentation medium, and add 2 - 20% of the juice or 0 - 1 g of honeysuckle powder at different fermentation times; the fermentation times are 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, measure the content of monacolin K in the fermentation broth, and analyze the transcriptional level of the genes related to monacolin K synthesis in Monascus purpureus;

[0011] Specifically, the strain is: wild-type Monascus purpureus strain M1 (CGMCC 3.0568); inoculate the strain into the seed medium for fermentation, the shaking speed of the shaker is 200 rpm, and culture at 30 °C for 48 h until the seed liquid shows a light pink state, inoculate it into the fermentation medium at an inoculation amount of 10%, culture at 30 °C, 150 rpm for 2 d, and then set the temperature to 25 °C and culture for 15 d;

[0012] The seed culture medium is as follows: 70 g / L of glycerol, 30 g / L of glucose, 15 g / L of soybean powder, 10 g / L of peptone, KH 2 PO 4 2, 2 g / L of NaNO 3 2, 1 g / L of MgSO 4 ·7H 2 O, sterilized at 115 °C under high pressure for 20 min;

[0013] The liquid fermentation medium is as follows: 90 g / L of glycerol, 10 g / L of peptone, 20 g / L of rice flour, KH 2 PO 4 2.5 g / L, 5 g / L of NaNO 3 5, 2 g / L of ZnSO 4 ·7H 2 O, 1 g / L of MgSO 4 ·7H 2 O, sterilized at 121 °C under high pressure for 15 min;

[0014] More specifically, in 50 mL of the liquid fermentation medium, 1 mL, 2 mL, 4 mL, 6 mL, 8 mL, and 10 mL (volume fractions of 2%, 4%, 8%, 12%, 16%, and 20%, respectively) of honeysuckle juice, and 0 g, 0.2 g, 0.4 g, 0.6 g, 0.8 g, and 1.0 g of honeysuckle powder were added respectively, and the indexes were measured according to the method. After determining the optimal form and addition amount of honeysuckle addition based on the experimental results, the corresponding honeysuckle was added at different fermentation times (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h), and the content of monacolin K in the fermentation broth was measured;

[0015] Preferably, adding 0.8 g of honeysuckle powder at 48 h of fermentation showed a significant promoting effect; on the 15th day of fermentation, the yield of monacolin K reached 536.10 mg / L, which was nearly 1.54 times higher than that of the non-added group;

[0016] For the transcriptional level of the monacolin K synthesis gene, the fermentation broth of Monascus purpureus at different culture days was placed in a 2 mL centrifuge tube, washed and centrifuged with sterile water until the supernatant was no longer red, and the residual water in the centrifuge tube was aspirated. The RNA of Monascus purpureus was extracted and then reverse transcribed into cDNA for fluorescence quantitative analysis.

[0017] The biomass of the thallus was measured by the dry weight method. 3 mL of the fermentation broth was aspirated with a pipette, filtered with a sterilized filter paper, washed 3 times with distilled water, and then dried until a constant weight was reached to obtain the dry weight of the mycelium;

[0018] During the two-way fermentation of honeysuckle and Monascus purpureus, the mycelial growth indexes were detected, including mycelial biomass and mycelial morphology;

[0019] For the mycelial morphology, the fermentation broth on the 8th day of fermentation was collected to obtain mycelial cells, which were then processed. Take 2 mL of the fermentation broth and centrifuge it at 12,000 rpm for 5 min, and discard the waste liquid. Resuspend and fix the mycelia with 2.5% glutaraldehyde solution and keep it for 12 h. Then rinse the cells with 0.1 M phosphate buffer solution (PBS, pH 7.2), and repeat once. Then dehydrate the cells in turn. The dehydration operation uses ethanol solutions with different concentration gradients, namely 30%, 50%, 70%, 80%, 90%, and 100%. After each addition, let it stand for about 10 min and then centrifuge at 12,000 rpm for 5 min to discard the supernatant, and repeat the same concentration twice. Then resuspend the mycelia with a 1:1 volume ratio of isoamyl acetate and ethanol, and then add it to the isoamyl acetate solution to replace ethanol in the cells. Finally, add the solvent hexamethyldisilazane so that its amount covers the sample, use absorbent cotton to plug the top of the centrifuge tube to improve water absorption, and place it in a 60 °C oven to dry until the sample becomes powdery for observation.

[0020] In a specific embodiment, the in vitro antioxidant activity indexes of the fermentation broth of honeysuckle-Monascus purpureus were detected, including monascus pigments, total polyphenol content, total flavonoid content, DPPH free radical scavenging activity, superoxide anion free radical scavenging activity, ABTS free radical scavenging activity, and total antioxidant capacity;

[0021] The monascus pigments are mainly monascus yellow, monascus orange, and monascus red pigments, which are detected using a UV spectrophotometer. The measurement wavelengths are: 410 nm, 448 nm, and 505 nm respectively. Take 1 mL of the fermentation broth of Monascus purpureus, dilute it 9 times with 70% ethanol solution, put it in a 60 °C constant temperature water bath for extraction for 1 h, let it cool, centrifuge at 4,000 rpm for 15 min, and keep it in the dark and static for detection;

[0022] For the detection of the total polyphenol content, pipette 100 μL of the sample into a centrifuge tube, add 50 μL of Folin-Ciocalteu reagent, mix well and react at room temperature for 5 min. Then, add 150 μL of 20% Na 2 CO 3 solution, pipette and mix well, let it react statically for 20 min, and measure the absorbance value (765 nm) of the reaction solution by an enzyme-linked immunosorbent assay (ELISA) reader. Using the same method, a standard curve is prepared with gallic acid as the standard and a standard curve is prepared with rutin as the standard, and the total polyphenol content in the fermentation broth of different groups is calculated accordingly;

[0023] For the detection of the total flavonoid content, the total flavonoid content in the fermentation broth of different groups is measured using the aluminum nitrate colorimetric method. According to the sample solution: 5% NaNO2 Solution: 10% Al(NO 3 ) 3 The solution was prepared for the reaction system in a ratio of 1:1.5:1.5. Each time a solution was added, after mixing, it was allowed to stand in the dark for 5 min for the reaction. Finally, 2 mL of 4% NaOH solution was added and allowed to stand for 15 min for the reaction. The absorbance value (510 nm) of the reaction solution was measured by an enzyme-linked immunosorbent assay (ELISA) reader. Using the same method, a standard curve was prepared with rutin as the standard, and the total flavonoid content in the fermentation broth of different groups was calculated therefrom;

[0024] Detection of DPPH radical scavenging activity: A blank group, a control group, a measurement group, and a positive control group were set up, with at least 3 parallels in each group. A 0.1 μmol / L DPPH solution was prepared with absolute ethanol. 200 μL of solutions with different concentrations were taken respectively, 200 μL of DPPH solution was added, and after mixing evenly, the supernatant was taken to measure the absorbance value at 517 nm after standing at room temperature for 30 min;

[0025] Detection of superoxide anion radical scavenging activity: 200 μL of sample solution, 900 μL of Tris-HCl buffer solution, 200 μL of 8 mmol / L HCl solution, and 100 μL of 3 mmol / L pyrogallol solution were added, and the reaction was accompanied by shaking for 5 min. The absorbance of the mixture was measured at 320 nm.

[0026] Advantages of the present invention

[0027] The present invention provides a method for producing Monacolin K by the bi-directional fermentation of honeysuckle and Monascus purpureus. By optimizing the pretreatment of additives, addition concentration and fermentation conditions, the content of Monacolin K and other active ingredients in the fermentation broth is significantly increased, realizing the innovative development of fermented medicinal and edible homologous products. Using the medicinal and edible homologous Chinese medicine honeysuckle as an exogenous additive, after being prepared by powder grinding or juice decocting, it is added to the liquid fermentation medium of Monascus purpureus. Through systematic optimization, the highest yield of Monacolin K in the fermentation broth reaches 536.10 mg / L, which is greatly improved compared with the traditional fermentation process. At the same time, experiments found that the addition of honeysuckle effectively promotes the growth rate of Monascus purpureus cells, and the mycelial biomass is increased by 85% compared with the non-added group. In terms of fermentation products, the addition of honeysuckle significantly increases the content of active ingredients such as Monascus pigments, total flavonoids and total polyphenols in the fermentation broth. Among them, the contents of total flavonoids and total polyphenols are increased by 3.93 times and 2.01 times respectively, and the antioxidant activity of the fermentation broth is significantly improved. The present invention successfully develops a high-value-added fermented medicinal and edible homologous product by combining the nutritional and active ingredients of honeysuckle with the efficient metabolic ability of Monascus purpureus. This method has strong popularization potential. It is not only applicable to the large-scale production of Monacolin K, but also can be widely used in the development of functional foods and health products, with broad market prospects and important social and economic value. Brief Description of the Drawings

[0028] The following further elaborates on the specific embodiments of the present invention with reference to the drawings.

[0029] Figure 1 Evaluating the effects of honeysuckle after different pretreatments on the growth of Monascus purpureus and the yield of Monacolin K. (a) Changes in the biomass of Monascus purpureus after adding honeysuckle juice; (b) Changes in the yield of Monacolin K after adding honeysuckle juice; (c) Changes in the biomass of Monascus purpureus after adding honeysuckle powder; (d) Changes in the yield of Monacolin K after adding honeysuckle powder.

[0030] Figure 2 Comparative analysis of the expression levels of genes mokA - mokI related to the synthesis of Monacolin K in Monascus purpureus (the experimental group is the group added with honeysuckle powder, and the control group is the non-added group).

[0031] Figure 3 Effect of the addition of honeysuckle on the growth of Monascus purpureus. (a) Biomass; (b) pH of the fermentation broth; (c) Residual sugar concentration; (d) Protein concentration. (The experimental group is the group added with honeysuckle, the control group is the group without adding honeysuckle, and the blank group is the non-inoculated group)

[0032] Figure 4Comparison of the mycelial morphology of Monascus purpureus after the addition of honeysuckle powder under electron microscopy. (a) Control group (1500×); (b) Addition group (1500×); (c) Control group (5000×); (d) Addition group (5000×).

[0033] Figure 5 Effect of the addition amount of honeysuckle on the pigment synthesis of Monascus purpureus. (a) Yellow pigment; (b) Orange pigment; (c) Red pigment.

[0034] Figure 6 Changes in the in vitro antioxidant activity of the co-fermentation products of honeysuckle-Monascus purpureus. (a) Total polyphenols; (b) Total flavonoids; (c) Total antioxidant capacity; (d) ABTS radical scavenging activity; (e) DPPH radical scavenging activity; (f) Superoxide anion radical scavenging activity. Detailed implementation manners

[0035] Example 1 Preparation of culture medium and seed liquid

[0036] The wild-type Monascus purpureus strain M1 (CGMCC 3.0568) is preserved in this laboratory; Liquid seed medium: glycerol 70 g / L, glucose 30 g / L, soybean powder 15 g / L, peptone 10 g / L, KH 2 PO 4 2, NaNO 3 2 g / L, MgSO 4 ·7H 2 O 1 g / L, autoclaved at 115 °C for 20 min; Liquid fermentation medium: glycerol 90 g / L, peptone 10 g / L, rice flour 20 g / L, KH 2 PO 4 2.5 g / L, NaNO 3 5 g / L, ZnSO 4 ·7H 2 O 2 g / L, MgSO 4 ·7H 2 O 1 g / L, autoclaved at 121 °C for 15 min; The culture medium used in the control group is the same as above. The experimental group and the blank group add the traditional Chinese medicine honeysuckle according to the experimental requirements on the basis of the above liquid fermentation medium. Monascus purpureus M1 is cultured on PDA medium until the 2nd generation and then inoculated. The inoculated seed liquid is fermented, and the shaking speed of the shaker is 200 rpm, and the temperature is 30 °C for 48 h until the seed liquid shows a light pink state, and then inoculated into the fermentation broth at an inoculation amount of 10%, and cultured at 30 °C and 150 rpm for 2 d, and then the temperature is set at 25 °C for 15 d.

[0037] Example 2 Optimization of the two-way fermentation of the traditional Chinese medicine honeysuckle-Monascus purpureus

[0038] Optimize the fermentation conditions of Monascus purpureus by considering factors such as the state, concentration, and addition time of honeysuckle. Pretreat the honeysuckle by powder grinding or juice decoction. Juice decoction: Weigh 5 g of honeysuckle, wash it with water, add 500 mL of distilled water, soak for 1 h, heat to boiling and maintain for 30 min, then filter out the juice; repeat the above operation, combine the juices from the two decoctions, add distilled water to make up to 1000 mL, and sterilize at 121 °C for 15 min for standby. Powder grinding: Weigh a certain amount of honeysuckle, wash it with water, air-dry it naturally, grind it into powder with a grinder and pass through an 80-mesh sieve, and sterilize at 121 °C for 15 min for standby. In the liquid fermentation medium, add honeysuckle juice with volumes of 1 mL, 2 mL, 4 mL, 6 mL, 8 mL, 10 mL (volume fractions are 0%, 2%, 4%, 8%, 12%, 16%, 20%) respectively, and honeysuckle powder with masses of 0 g, 0.2 g, 0.4 g, 0.6 g, 0.8 g, 1.0 g respectively, and measure the indicators according to the method. According to the experimental results, after determining the optimal form and addition amount of honeysuckle addition, add the corresponding honeysuckle at different fermentation times (0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h) respectively, and measure the content of monacolin K in the fermentation broth to determine the optimal addition time.

[0039] Example 3 Determination of Monacolin K Content

[0040] Evaluate the effects of honeysuckle pretreated in different ways on the growth of Monascus purpureus and the production of monacolin K. Take 5 mL of the fermentation broth of Monascus purpureus in the addition group and the non-addition group respectively, dilute it 4 times with 75% chromatographic grade methanol, ultrasonically disrupt the cells in an ultrasonic cleaner for 30 min, and let it stand overnight in the dark. From Figure 1 It is found that the promoting effect of honeysuckle juice is not completely positively correlated with the addition amount, and even shows an inhibitory effect with the increase of the addition amount; on the contrary, honeysuckle powder not only promotes the growth of the bacteria, but also greatly increases the production of monacolin K. Under the condition of powder addition, the biomass of the bacteria shows an upward trend with the increase of the addition amount, and the promoting effect is the best at the highest level of 1.0 g, and the biomass reaches up to 39.42 mg / L, which is 1.29 times higher than that of the control group. Adding 0.8 g of honeysuckle powder has the strongest promoting effect on the synthesis of monacolin K. At this addition amount, the synthesis of monacolin K at each time point shows the most significant promoting effect, and the yield reaches 536.10 mg / L on the 15th day of fermentation, which is nearly 1.54 times higher than that of the control group. Finally, choose to add 0.8 g of powder at 48 h of fermentation as the optimal condition for honeysuckle addition fermentation.

[0041] Example 4 Transcription Analysis of Monacolin K Synthesis Gene

[0042] To further clarify the effect of honeysuckle on the synthesis of monacolin K, the expression levels of 9 key genes mokA - mokI on its synthetic gene cluster were analyzed, and the results are as follows. Figure 2 As shown, the addition of honeysuckle basically promoted the expression of 9 key genes related to monacolin K synthesis, but there were differences in the transcriptional levels due to different genes. Among them, the transcriptional levels of mokA, mokC, and mokE genes changed significantly in the early stage of fermentation. The change degree of mokE gene was the largest on the 8th day of fermentation, and the transcriptional level increased by 13.4% compared with the control group. mokA and mokC also increased by 3.54% and 0.94% respectively. The function of mokH was comprehensively explored, and it was found that the overexpression of this gene increased the yield of monacolin K by 82%. Among several genes, the expression of mokC changed little, but there was a slight down - regulation of the expression of mokG in the honeysuckle group. It is worth noting that the coding product of mokI plays an efflux role, and its transcriptional level increased in the late stage of fermentation under the influence of honeysuckle, realizing the extracellular transport of monacolin K and reducing the feedback inhibition of the product.

[0043] Example 5 Determination of Bacterial Biomass and Fermentation Broth Components

[0044] Evaluate the effect of adding honeysuckle on the biomass of Monascus purpureus bacteria, as well as the changes in protein content, residual sugar content, and pH in the fermentation broth ( Figure 3 ). After adding honeysuckle to Monascus purpureus, the material utilization rate was greatly improved, and the growth rate of the bacteria accelerated. Even at the end of fermentation, the biomass increased by 85% compared with the control group. Take 1 mL of Monascus purpureus fermentation broth, add 9 mL of 1×PBS buffer respectively, ultrasonically extract for 30 min, centrifuge at 10000 rpm for 10 min at room temperature, take the supernatant for use, and measure the residual sugar content and protein concentration of the fermentation broth in different groups at different fermentation stages. As Figure 4 can be seen, although the residual sugar content in the fermentation broth of the experimental group was relatively high, in the early growth stage, its sugar consumption increased by 38% compared with the control group. The protein concentration in the experimental group reached 3.93 mg / mL on the 12th day of fermentation, which was 59.1% higher than that of the control group and 51.1% higher than that of the blank group. In addition, substances such as chlorogenic acid and caffeic acid rich in honeysuckle created a fermentation environment with a lower pH for Monascus purpureus, and the acidic environment was proven to effectively promote the growth of Monascus purpureus bacteria and the synthesis of secondary metabolites. The results show that the addition of honeysuckle greatly promoted the growth of Monascus purpureus bacteria.

[0045] Example 6 Observation of Mycelium Morphology

[0046] The morphological changes of Monascus purpureus mycelium after adding honeysuckle were observed by electron microscopy. The fermentation broth on the 8th day of fermentation was selected to collect the mycelial cells for treatment. Take 2 mL of the fermentation broth and centrifuge it at 12,000 rpm for 5 min, and discard the waste liquid. Resuspend and fix the mycelium with 2.5% glutaraldehyde solution for 12 h. Then rinse the cells with 0.1 M phosphate buffer (PBS, pH 7.2) and repeat once. Then dehydrate the cells in sequence. The dehydration operation uses ethanol solutions with different concentration gradients, namely 30%, 50%, 70%, 80%, 90%, and 100%. After each addition, let it stand for about 10 min and then centrifuge at 12,000 rpm for 5 min to discard the supernatant, and repeat the same concentration twice. Then resuspend the mycelium with a 1:1 volume ratio of isoamyl acetate and ethanol, and then add it to the isoamyl acetate solution to perform ethanol replacement on the cells. Finally, add the solvent hexamethyldisilazane so that its amount covers the sample, use absorbent cotton to plug the top of the centrifuge tube to improve water absorption, and place it in an oven at 60 °C to dry until the sample becomes powdery for observation. From Figure 4 It can be seen that when observing the overall state of the mycelium at low magnification, although the addition of honeysuckle increased the mycelial density, the tightness between the mycelia was lower than that of the control group, and the independence of the mycelia was relatively high, and the growth and metabolism would not be affected by mutual entanglement. At the same time, the increase in the mycelial gap can increase the permeability of the cell membrane, which is helpful for the secretion of synthetic products, further promoting the synthesis of substances such as monacolin K, and more secreted substances accumulate on the mycelial surface. When observing the morphology of the mycelium at high magnification, compared with the control group, the spore heads and the surfaces of the mycelia in the experimental group were relatively smooth, and the phenomena of depression and folds were not obvious, and the individual differences of the mycelia were not significant. The morphology of fungal mycelia has a certain impact on their biological properties and will change under different fermentation conditions.

[0047] Detection of in vitro antioxidant activity indexes of fermentation products in Example 7

[0048] The active ingredients in the fermentation broth of Monascus purpureus, such as monascus pigments, total flavonoids, and total polyphenols, have strong antioxidant activities. Among the three pigments in monascus pigments (monascus yellow, monascus orange, and monascus red pigment), the peak value was reached when the addition amount was 0.4 g. The color value of the red pigment, which had the largest increment change, increased to 109.8 U / mL at most, which was 52.5% higher than that of the control group ( Figure 5 ). As Figure 6As shown, the increments of total flavonoids and total polyphenols were relatively obvious in the early stage of fermentation, and the contents increased by 3.93 and 2.01 times respectively compared with the control group. The ability of the co-fermentation product of Monascus purpureus Went - Lonicera japonica Thunb. to scavenge DPPH free radicals was improved, but except on the 5th day of fermentation, its activity remained at a level lower than that of the Lonicera japonica Thunb. blank group. On the contrary, after fermentation, the ability of the co-fermentation product to scavenge ABTS free radicals was significantly improved, with a maximum increase of 1.61 times. Through the detection of the total antioxidant capacity index, it can be clearly seen that the co-fermentation of Lonicera japonica Thunb. and Monascus purpureus Went promoted each other in two directions and reached the peak on the 15th day. The above results indicate that the co-fermentation of Lonicera japonica Thunb. and Monascus purpureus Went improved the antioxidant activity of the product.

Claims

1. A method for producing monacolin K by bidirectional fermentation of honeysuckle and purple Monascus, which is characterized by: A certain amount of honeysuckle was weighed, pretreated and added to the fermentation medium of purple Monascus, the morphological changes of mycelium were observed, the content of monacolin K in the fermentation broth after co-fermentation was determined, and the transcription of genes related to monacolin K synthesis was analyzed; the growth index of purple Monascus, the color value of red pigment and the metabolite index were detected, and the in vitro antioxidant activity of the fermentation broth was evaluated.

2. The method according to claim 1, characterized in that Honeysuckle can be prepared by powder grinding or juice decoction; the powder grinding is to grind the honeysuckle into powder and pass it through an 80-mesh sieve, sterilize it at 121°C for 15 minutes for later use. The juice decoction is to add distilled water to decoct the honeysuckle, combine the decoctions of the two times, add distilled water to make the volume 1000 mL, sterilize it at 121°C for 15 minutes for later use.

3. The method according to claim 1, characterized in that The strain was inoculated into the seed culture medium for fermentation. The shaking speed was 200 rpm and the temperature was 30°C for 48 h until the seed liquid showed a light pink state. The inoculation amount was inoculated into the fermentation medium at 10%, and the culture was carried out at 30°C and 150 rpm for 2 days. The temperature was set to 25°C for 15 days. The strains are: wild-type purple Monascus strain M1 (CGMCC 3.0568); The seed culture medium is: 70 g / L glycerol, 30 g / L glucose, 15 g / L soybean meal, 10 g / L peptone, 2 g / L KH2PO4, 2 g / L NaNO3, 1 g / L MgSO4·7H2O, sterilized at 115°C for 20 min; The liquid fermentation medium comprises: 90 g / L glycerol, 10 g / L peptone, 20 g / L rice flour, 2.5 g / L KH2PO4, 5 g / L NaNO3, 2 g / L ZnSO4·7H2O, 1 g / L MgSO4·7H2O, and is sterilized at 121°C under high pressure for 15 min.

4. The method according to claim 1, wherein 2-20% of honeysuckle juice or 2-20% of honeysuckle powder is added to the fermentation medium at different fermentation times; the fermentation time is 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h, and the content of monacolin K in the fermentation broth is determined, and the highest yield reaches 536.10 mg / L.

5. The method according to claim 1, wherein the mycelium biomass is detected by the dry weight method, the fermentation liquid is absorbed, filtered with sterilized filter paper, washed with distilled water and then dried to obtain the dry matter weight of the mycelium; after the addition of honeysuckle, the growth rate of the purple Monascus thalli is accelerated, and the fermentation enters the final stage, and the biomass increases by 85% compared with the group without addition.

6. The method according to claim 1, wherein after adding honeysuckle, the spore heads and the surface of the thalli of purple Monascus are relatively smooth, the depression and wrinkle phenomena are not obvious, the size of the thalli is not much different, and there is obvious accumulation of secretions on the surface.

7. According to the method as claimed in claim 1, after adding honeysuckle, the contents of total flavonoids and total polyphenols in the fermentation broth increased by 3.93 and 2.01 times respectively compared with the group without addition; the scavenging ability of the fermentation broth on ABTS free radicals was significantly improved, with the highest increase of 1.61 times.

8. The method according to claims 1-8, wherein the Chinese medicine honeysuckle is used as an exogenous additive in the purple Monascus fermentation process, which not only effectively promotes the significant increase in the content of monacolin K in the fermentation broth, but also significantly improves the contents of active ingredients such as monascus pigment, total polyphenols, and total flavonoids. The Chinese medicine honeysuckle-purple Monascus bidirectional fermentation method can be used to develop high value-added fermented food and medicine products.

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