A method for increasing the yield of yellow pigment in liquid fermentation of monascus yam powder

Abstract

The present invention provides a method for increasing the yield of yellow pigment in the liquid fermentation of yam powder with Monascus spp., and belongs to the technical field of microbial fermentation. The present invention provides the application of flavonoid compounds in increasing the yield of yellow pigment in Monascus fermentation, wherein the flavonoid compounds include luteolin and / or genistein. The present invention adds flavonoid compounds to the liquid fermentation medium of Monascus spp., and after the liquid fermentation of Monascus spp., the yield of yellow pigment is significantly increased. Therefore, the method provided by the present invention realizes the maximization of flavonoid resource utilization while improving the yellow pigment production efficiency, and has important application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for improving the yield of yellow pigment in liquid fermentation of monascus yam powder. Background Art

[0002] Monascus pigment is a high-quality natural food pigment with high safety, non-toxicity and no teratogenicity. It is widely used in China, Japan and other Southeast Asian countries. It is widely used as a substitute for nitrite in colorants, food additives and meat products in the food industry, and is used as a dye in cosmetics and textile industries. Monascus mainly produces six related pigments, which can be divided into three types according to color: yellow pigment (monascusin and monascus yellow pigment), orange pigment (monascus red pigment and pan red pigment) and red pigment (erythroplasmin and monascus red pigment). Among them, yellow pigment, as an important food pigment variety, accounts for 60% of the market demand, so the development and research of monascus yellow pigment has extremely broad market prospects and significant economic benefits. However, there is no report on the industrial production of monascus yellow pigment in my country at present, and it only stays in the strain breeding stage. Therefore, the strain breeding and culture medium optimization are important methods to improve the yield of monascus yellow pigment.

[0003] Flavonoids are a class of polyphenols with various structures, which mainly exist in plants in the form of glycosides. Flavonoids can be found in most edible fruits, grains and vegetables, but the types of flavonoids obtained from different dietary sources are different. Common flavonoids include quercetin, luteolin, genistein, baicalein, catechins and naringenin. Flavonoids have multiple functions such as antioxidant, antibacterial, anti-diabetic, liver protection, heart protection and retinal protection. At present, there are few reports on the use of flavonoids in microbial fermentation, and there are no reports on the influence of flavonoid content in liquid fermentation of Monascus. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an application of flavonoid compounds in increasing the yield of yellow pigment in Monascus fermentation.

[0005] The present invention also aims to provide a Monascus liquid fermentation method, which optimizes fermentation parameters and greatly improves the yellow pigment yield.

[0006] The invention provides application of flavonoid compounds in improving yellow pigment yield in Monascus fermentation. The flavonoid compounds include luteolin and / or genistein.

[0007] Preferably, in the fermentation system, the final concentration of the flavonoid compound is not less than 1.0 g / L.

[0008] Preferably, the final concentration of the flavonoids is 1.5 to 20.0 g / L.

[0009] Preferably, the mass ratio of luteolin to genistein is (1-3):1.

[0010] Preferably, the fermentation medium of the Monascus fermentation comprises the following components: 10.0-100.0 g / L iron stick yam powder, 1.0-5.0 g / L NaNO3, 0.1-1.0 g / L KH2PO4, 0.1-2.0 g / L K2HPO4, 0.1-2.0 g / LMgSO4·7H2O, and the pH value is 6.5-6.8.

[0011] The present invention provides a method for producing yellow pigment by liquid fermentation of Monascus spp., comprising the following steps:

[0012] Inoculating Monascus seed liquid into a fermentation medium containing flavonoid compounds, and fermenting and culturing under shaking conditions to obtain a fermentation liquid; the flavonoid compounds include luteolin and / or genistein;

[0013] The fermentation liquid and the alcohol solvent are mixed, the solid and the liquid are separated, and the obtained liquid phase is filtered through a membrane to obtain the fermentation product yellow pigment.

[0014] Preferably, the fermentation medium comprises the following ingredients: 10.0-100.0 g / L iron stick yam powder, 1.0-5.0 g / L NaNO3, 0.1-1.0 g / L KH2PO4, 0.1-2.0 g / L K2HPO4, 0.1-2.0 g / L MgSO4·7H2O, 1.0-20.0 g / L flavonoids, and the pH value is 6.5-6.8.

[0015] Preferably, the inoculation amount of the Monascus seed solution is 2% to 10%;

[0016] The fermentation temperature is 28-32° C., the rotation speed of the oscillation condition is 150-250 rpm, and the fermentation time is 6-14 days.

[0017] Preferably, the volume ratio of the fermentation broth to the alcohol solvent is 1:2;

[0018] The alcohol solvent includes chromatography grade methanol.

[0019] The present invention provides an application of flavonoid compounds in increasing the yield of yellow pigment in Monascus fermentation, wherein the flavonoid compounds include luteolin and / or genistein. The present invention experiments show that after adding luteolin and genistein alone or in combination, Monascus undergoes liquid fermentation to obtain a liquid fermentation product with significantly increased yellow pigment yield. The experiment shows that the yellow pigment content of the group adding 2.0g / L luteolin is 6.50U / mL, which is 26.08 times higher than that of the blank group; the yellow pigment content of the group adding 2.0g / L genistein is 0.51U / mL, which is 1.13 times higher than that of the blank group; the simultaneous addition of 1.0g / L luteolin and 1.0g / L genistein increases the yellow pigment content of Monascus by 3.42 times; the simultaneous addition of 1.5g / L luteolin and 0.5g / L genistein has the most significant effect on increasing the yellow pigment content of Monascus, increasing it by 16.8 times. It can be seen that adding luteolin and genistein during the liquid fermentation of Monascus can improve the ability of Monascus to produce yellow pigment, greatly improving the production efficiency of yellow pigment, which has important application value. At the same time, it can effectively utilize flavonoid resources and maximize resource utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 To compare the effects of adding luteolin and genistein on the yellow pigment production of Monascus purpurogenum 30141 in the medium of iron yam powder and inorganic salts;

[0021] Figure 2 This paper compares the effects of different doses of luteolin and genistein on the production of yellow pigment by Monascus purpurogenum 30141 in inorganic salt medium of iron yam powder. DETAILED DESCRIPTION

[0022] The invention provides application of flavonoid compounds in improving yellow pigment yield in Monascus fermentation. The flavonoid compounds include luteolin and / or genistein.

[0023] In the present invention, the structural formulas of luteolin and genistein are as follows:

[0024]

[0025] The genistein (≥97%) and luteolin (≥98%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0026] In the present invention, the Monascus is preferably Monascus purpurus.

[0027] In the present invention, in the fermentation system, the final concentration of the flavonoids is preferably not less than 1.0 g / L, preferably 1.5 to 20.0 g / L, and more preferably 2.0 g / L. As the concentration of flavonoids increases, the yield of yellow pigment also increases. The final concentration of the flavonoids. The mass ratio of luteolin and genistein is preferably (1 to 3): 1, and more preferably 3: 1. Different types of luteolin and genistein will also affect the yield of yellow pigment. Experiments show that at the same concentration of luteolin and genistein, the yield of yellow pigment is ranked from high to low as luteolin> luteolin+genistein>genistein. The reason for the difference in the two flavonoids in increasing the yield of yellow pigment may be the difference in the structure of luteolin and genistein, which are mainly in the binding site (positions 3 and 4) between the B ring and the C ring and the number of hydroxyl groups connected to the B ring. The B ring of luteolin is connected to the 4th position of the C ring and is bound to two hydroxyl groups on the B ring; while the B ring of genistein is connected to the 3rd position of the C ring and is only bound to one hydroxyl group on the B ring.

[0028] In the present invention, the fermentation medium of the Monascus fermentation preferably comprises the following components: 10.0-100.0 g / L iron stick yam powder, 1.0-5.0 g / L NaNO3, 0.1-1.0 g / L KH2PO4, 0.1-2.0 g / L K2HPO4, 0.1-2.0 g / L MgSO4·7H2O, and the pH value is 6.5-6.8, more preferably 20.0-80.0 g / L iron stick yam powder, 2.0-4.0 g / L NaNO3, 0.2-0.8 g / L KH2PO4, 0.5-1.5 g / L K2HPO4, 0.5-1.5 g / L MgSO4·7H2O, and most preferably 20.0 g / L iron stick yam powder, 2.0 g / L NaNO3, 0.5 g / L KH2PO4, 1.0 g / L K2HPO4, 1.0 g / L MgSO4·7H2O. The present invention has no particular limitation on the preparation method of the fermentation medium, and any preparation method known in the art can be used.

[0029] In the present invention, luteolin and genistein are used alone or in combination in fermentation, which can well increase the yield of flavonoids and effectively save flavonoid resources, thereby maximizing resource utilization.

[0030] The present invention provides a method for producing yellow pigment by liquid fermentation of Monascus spp., comprising the following steps:

[0031] Inoculating Monascus seed liquid into a fermentation medium containing flavonoid compounds, and fermenting and culturing under shaking conditions to obtain a fermentation liquid; the flavonoid compounds include luteolin and / or genistein;

[0032] The fermentation liquid and the alcohol solvent are mixed, the solid and the liquid are separated, and the obtained liquid phase is filtered through a membrane to obtain the fermentation product yellow pigment.

[0033] In the present invention, the Monascus is preferably purple Monascus, and the species and source are the same as above. The method for preparing the Monascus seed solution preferably includes the following steps: inoculating the Monascus into an MPPY medium for shaking culture, applying the obtained culture solution to a malt wort solid medium and culturing until spores are generated, eluting the spores with physiological saline to obtain a spore suspension; inoculating the spore suspension into a seed culture medium, culturing under shaking conditions, and obtaining a Monascus seed solution. The rotation speed of the shaking condition is preferably 180 to 200 rpm. The culture temperature is preferably 28 to 32°C, more preferably 30°C. The seed culture medium is preferably prepared by adding 20.0-40.0 g glucose, 1.0-3.0 g KH2PO4, 2.0-4.0 g NaNO3, 0.1-1.0 g KCl, and 0.1-1.0 g MgSO4·7H2O per liter of culture medium, and sterilized at 121°C for 20 min. More preferably, 30.0 g glucose, 2.0 g KH2PO4, 3.0 g NaNO3, 0.5 g KCl, and 0.5 g MgSO4·7H2O are added per liter of culture medium. The concentration of the spore suspension is preferably 1.0×10 5 -1.0×10 8 The seed liquid culture time is preferably 36 to 72 hours, more preferably 48 to 56 hours. The malt extract solid culture medium is preferably 100 mL of double distilled water and 2.0 g of agar, which is sterilized at 121° C. for 20 minutes and then poured into a plate, and the malt extract solid culture medium is obtained after cooling.

[0034] In the present invention, the fermentation medium preferably comprises the following ingredients: 10.0-100.0 g / L iron stick yam powder, 1.0-5.0 g / L NaNO3, 0.1-1.0 g / L KH2PO4, 0.1-2.0 g / L K2HPO4, 0.1-2.0 g / LMgSO4·7H2O, 1.0-20.0 g / L flavonoids, and a pH value of 6.5-6.8; more preferably: 20.0-80.0 g / L iron stick yam powder, 2.0 ~4.0g / LNaNO3, 0.2~0.8g / LKH2PO4, 0.5~1.5g / LK2HPO4, 0.5~1.5g / L MgSO4·7H2O, 1.5~10.0g / L flavonoids; more preferably 20.0g / L iron stick yam powder, 2.0g / LNaNO3, 0.5g / LKH2PO4, 1.0g / LK2HPO4, 1.0g / L MgSO4·7H2O, 2g / L flavonoids. The flavonoids are preferably 2g / L luteolin, 2g / L genistein or a composition formed by 1.5g / L luteolin and 0.5g / L genistein.

[0035] In the present invention, the inoculation amount of the Monascus seed liquid is preferably 2-10%, more preferably 4-7%, more preferably 5%. The temperature of the fermentation culture is 28-32°C, more preferably 30°C. The rotation speed of the oscillation condition is preferably 150-250rpm, more preferably 180rpm-200rpm; the fermentation culture time is 6-14d, more preferably 8-12h, and most preferably 10h.

[0036] In the present invention, the volume ratio of the fermentation broth and the alcohol solvent is preferably 1:2. The alcohol solvent includes chromatographic grade methanol. After the mixing, it is preferably treated in a water bath. The temperature of the water bath is preferably 58-62°C. The time of the water bath is preferably 50-70min, more preferably 60min. The solid-liquid separation is preferably centrifugation. The speed of the centrifugation is preferably 5000-7000rpm, more preferably 6000rpm. The time of the centrifugation is preferably 14-16min, more preferably 15min. The pore size of the filter membrane for membrane filtration is preferably 0.45μm. The yellow pigment content is preferably determined by ultraviolet spectrophotometry of the extracted yellow pigment, specifically detecting the absorbance value of the extract at 410nm, with methanol as a blank control. The yellow pigment content is the absorbance value multiplied by the dilution multiple of the extracted yellow pigment.

[0037] In the present invention, the yellow pigment content of the group with 2.0g / L luteolin was 6.50U / mL, which was 26.08 times higher than that of the blank group; the yellow pigment content of the group with 2.0g / L genistein was 0.51U / mL, which was 1.13 times higher than that of the blank group. The simultaneous addition of 1.0g / L luteolin and 1.0g / L genistein increased the yellow pigment content of Monascus by 3.42 times, which was 1.15U / mL; the simultaneous addition of 1.5g / L luteolin and 0.5g / L genistein had the most significant effect on increasing the yellow pigment content of Monascus, which was increased by 16.8 times. The results show that the addition of luteolin and genistein in the liquid fermentation of Monascus can improve the ability of Monascus to produce yellow pigment.

[0038] The following is a detailed description of a method for increasing the yellow pigment yield in liquid fermentation of Monascus yam powder provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] A method for producing yellow pigment by liquid fermentation of purple Monascus

[0041] 1) Preparation of seed culture medium: Add 30.0 g glucose, 2.0 g KH2PO4, 3.0 g NaNO3, 0.5 g KCl, and 0.5 g MgSO4·7H2O per liter of culture medium, dissolve in distilled water, dispense into 250 mL conical flasks, and sterilize at 121°C for 20 min.

[0042] 2) Preparation of seed solution: Take 1.0 mL of purple Monascus 30141 (purchased from ACCC China Agricultural Microbial Culture Collection Management Center) with a spore concentration of 1.0×10 6 The spore liquid of each / mL was inoculated into a 250mL conical flask containing 100mL of seed culture medium, and fermented and cultured in a constant temperature shaking incubator (180rpm, 30°C) for 48h to obtain the Monascus seed liquid.

[0043] 3) Preparation of fermentation medium: 20.0 g iron yam powder, 2.0 g NaNO3, 0.5 g KH2PO4, 1.0 g K2HPO4, 1.0 g MgSO4·7H2O, 2.0 g luteolin or 2.0 g genistein (at least four replicates per group) were added to each liter of medium, divided into 250 mL conical flasks, and sterilized at 121°C for 20 min. At the same time, a fermentation medium without flavonoids was prepared as a blank group.

[0044] 4) Inoculate 5% of the Monascus seed liquid into the fermentation medium, and culture in a shaking flask at 30° C. and 180 rpm for 12 days to obtain the Monascus fermentation liquid.

[0045] 5) Extraction method of Monascus yellow pigment: Take 2.0 mL of filtered fermentation broth and add 2 volumes of chromatographic grade methanol. Mix thoroughly and place in a 60°C water bath for 1 hour. After cooling, centrifuge at 6000 rpm for 15 minutes. Take the supernatant and filter with a 0.45 μm organic filter membrane to obtain the fermentation extract.

[0046] The yellow pigment of Monascus was determined by ultraviolet spectrophotometry. 100 μL of fermentation liquid was taken into a 96-well ELISA plate, methanol was used as a blank control, and the absorbance of each group of fermentation liquid was measured by a full-wavelength ELISA instrument. The detection wavelength of yellow pigment was 410 nm. The content of Monascus yellow pigment was the absorbance value multiplied by the dilution multiple of the fermentation extract.

[0047] The blank group (without flavonoids added) in the fermentation broth obtained in the above example had the lowest yield of red pigment yellow, which was 0.24 U / mL; the yellow pigment content of the group with 2.0 g / L luteolin was 6.50 U / mL, which was 26.08 times higher than that of the blank group; the yellow pigment content of the group with 2.0 g / L genistein was 0.51 U / mL, which was 1.13 times higher than that of the blank group (see Figure 1 ).

[0048] Example 2

[0049] A method for producing yellow pigment by liquid fermentation of purple Monascus

[0050] 1) Preparation of seed culture medium: Add 30.0 g glucose, 2.0 g KH2PO4, 3.0 g NaNO3, 0.5 g KCl, and 0.5 g MgSO4·7H2O per liter of culture medium, dissolve in double distilled water, dispense into 250 mL Erlenmeyer flasks, and sterilize at 121°C for 20 min.

[0051] 2) Preparation of seed solution: Take 1.0 mL of Monascus purpurogenum 30141 spores with a concentration of 1.0×10 6 The spore liquid of each / mL was inoculated into a triangular flask containing 100 mL of sterile seed culture medium, and cultured at 180 rpm and 30° C. for 48 h to obtain the Monascus seed liquid.

[0052] 3) Preparation of fermentation medium: 20.0 g iron stick yam powder, 2.0 g NaNO3, 0.5 g KH2PO4, 1.0 g K2HPO4, 1.0 g MgSO4·7H2O, 1.5 g or 2 g of a combination of luteolin and genistein (each concentration was repeated at least four times, including 0.5 g / L luteolin + 1.0 g / L genistein, 0.5 g / L luteolin + 1.5 g / L genistein, 1 g / L luteolin + 1 g / L genistein and 1.5 g / L luteolin + 0.5 g / L genistein) were added per liter of culture medium, divided into 250 mL Erlenmeyer flasks, and sterilized at 121°C for 20 min.

[0053] 4) 5% of the Monascus seed liquid was inoculated into the fermentation medium, and the culture was carried out in a shaking flask at 30° C. and 180 rpm for 12 days to obtain Monascus fermentation liquid. The yellow pigment was detected according to the method of Example 1.

[0054] In the method for increasing the yellow pigment content in liquid fermentation of Monascus involved in the present invention, the yield of Monascus yellow pigment in the blank group (without flavonoids added) in the fermentation broth obtained in the above embodiment is the lowest, which is 0.26U / mL; the Monascus yellow pigment content in the fermentation broth with different doses of luteolin and genistein is higher than that of the blank group. Among them, when 0.5g / L luteolin and 1.0g / L or 1.5g / L genistein are added at the same time, the yellow pigment content is 0.88U / mL and 1.09U / mL, respectively, which is 2.39 times and 3.19 times higher than that of the blank group; the simultaneous addition of 1.0g / L luteolin and 1.0g / L genistein increases the Monascus yellow pigment content by 3.42 times, which is 1.15U / mL; the simultaneous addition of 1.5g / L luteolin and 0.5g / L genistein has the most significant effect on increasing the Monascus yellow pigment content, which is increased by 16.8 times (see Figure 2 ). The results showed that the addition of luteolin and genistein in the liquid fermentation of Monascus could improve the yellow pigment production capacity of Monascus.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of flavonoid compounds in increasing the yield of yellow pigment in Monascus fermentation, wherein the flavonoid compounds are luteolin or a combination of luteolin and genistein; The Monascus is purple Monascus; the final concentration of the flavonoid compound is 1.5-2.0 g / L; the fermentation medium of the Monascus fermentation comprises the following components: 10.0-100.0 g / L iron stick yam powder, 1.0-5.0 g / L NaNO3, 0.1-1.0 g / L KH2PO4, 0.1-2.0 g / L K2HPO4, 0.1-2.0 g / L MgSO4·7H2O, and the pH value is 6.5-6.8; when the flavonoid compound is a combination of luteolin and genistein, the mass ratio of luteolin to genistein is (1-3):

1.

2. A method for producing yellow pigment by liquid fermentation of Monascus spp., characterized in that: The following steps are involved: The Monascus seed liquid is inoculated into a fermentation medium containing flavonoid compounds, and fermented and cultured under shaking conditions to obtain a fermentation liquid; the flavonoid compound is luteolin or a combination of luteolin and genistein; the Monascus is purple Monascus; the final concentration of the flavonoid compound is 1.5-2.0 g / L; the fermentation medium of the Monascus fermentation comprises the following components: 10.0-100.0 g / L iron stick yam powder, 1.0-5.0 g / L NaNO3, 0.1-1.0 g / L KH2PO4, 0.1-2.0 g / L K2HPO4, 0.1-2.0 g / L MgSO4·7H2O, and a pH value of 6.5-6.8; when the flavonoid compound is a combination of luteolin and genistein, the mass ratio of luteolin to genistein is (1-3):1; The fermentation liquid and the alcohol solvent are mixed, the solid and the liquid are separated, and the obtained liquid phase is filtered through a membrane to obtain the fermentation product yellow pigment.

3. The method according to claim 2, characterized in that: The inoculation amount of the Monascus seed liquid is 2% to 10%; The fermentation temperature is 28-32° C., the rotation speed of the oscillation condition is 150-250 rpm, and the fermentation time is 6-14 days.

4. The method according to claim 2 or 3, characterized in that: The volume ratio of the fermentation broth to the alcohol solvent is 1:2; The alcohol solvent includes chromatography grade methanol.

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