CN114891841A - Method for increasing yield of yellow pigment in liquid fermentation of monascus and yam powder - Google Patents
Method for increasing yield of yellow pigment in liquid fermentation of monascus and yam powder Download PDFInfo
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- genistein
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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Abstract
The invention provides a method for improving the yield of yellow pigment in liquid fermentation of monascus and yam powder, and belongs to the technical field of microbial fermentation. The invention provides application of flavonoids compounds in improving the yield of yellow pigment in monascus fermentation, wherein the flavonoids compounds comprise luteolin and/or genistein. According to the method, the flavonoid compound is added into the liquid fermentation culture medium of the monascus so that the yield of the yellow pigment is obviously improved after the monascus is subjected to liquid fermentation. Therefore, the method provided by the invention improves the production efficiency of the yellow pigment, realizes the maximization of flavone resource utilization and has important application value.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for improving the yield of yellow pigment in liquid fermentation of monascus and yam powder.
Background
The monascus pigment is a high-quality natural edible pigment, has high safety, no toxicity and no distortion, and is widely applied to China, Japan and other southeast Asia countries. Are widely used in the food industry as colorants, food additives and substitutes for nitrite in meat products, and as dyes in the cosmetic and textile industries. Monascus mainly produces six related pigments, which can be divided into three types according to color: yellow pigments (monascin and monascin), orange pigments (monascorubin and panrubin), and red pigments (rubropunctatin and monascorubin). Wherein, the yellow pigment as an important edible pigment variety accounts for 60 percent of the market demand, so the development and research of the monascus yellow pigment has extremely wide market prospect and great economic benefit. However, at present, no report of industrial production of monascus yellow pigment exists in China, and the method only stays in a strain breeding stage, so strain breeding and culture medium optimization are important methods for improving the yield of the monascus yellow pigment.
The flavonoids are polyphenols with various structures, and are mainly present in plants in the form of glucoside. Flavonoid shadows are found in most edible fruits, grains and vegetables, but the types of flavonoids obtained from different dietary sources vary. The common flavonoids include quercetin, luteolin, genistein, baicalein, catechin, naringenin, etc. The flavonoids have multiple functions of resisting oxidation, resisting bacteria, resisting diabetes, protecting liver, protecting heart, protecting retina and the like. At present, few reports about flavonoid compounds used in microbial fermentation exist, and no report about influence on the content of yellow pigment in monascus liquid fermentation exists.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of a flavonoid compound in increasing the yield of yellow pigment in monascus fermentation.
The invention also aims to provide a monascus liquid state fermentation method, which optimizes fermentation parameters and greatly improves the yield of the yellow pigment.
The invention provides application of flavonoids compounds in improving the yield of yellow pigment in monascus fermentation, wherein the flavonoids compounds comprise luteolin and/or genistein.
Preferably, in a fermentation system, the final concentration of the flavonoid compound is not less than 1.0 g/L.
Preferably, the final concentration of the flavonoid compound is 1.5-20.0 g/L.
Preferably, the mass ratio of the luteolin to the genistein is (1-3) to 1.
Preferably, the fermentation medium for fermenting the monascus comprises the following components: 10.0-100.0 g/L Tiegun Yam powder, 1.0-5.0 g/L NaNO 3 、0.1～1.0g/LKH 2 PO 4 、0.1～2.0g/LK 2 HPO 4 、0.1～2.0g/LMgSO 4 ·7H 2 O, the pH value is 6.5-6.8.
The invention provides a method for producing yellow pigment by monascus liquid fermentation, which comprises the following steps:
inoculating monascus seed liquid into a fermentation culture medium containing flavonoid compounds, and performing fermentation culture under the oscillation condition to obtain a fermentation liquid; the flavonoid compound comprises luteolin and/or genistein;
and mixing the fermentation liquor and the alcohol solvent, carrying out solid-liquid separation, and filtering the obtained liquid phase by a membrane to obtain a fermentation product yellow pigment.
Preferably, the fermentation medium comprises the following components: 10.0-100.0 g/L Tiegun yam powder, 1.0-5.0 g/L NaNO 3 、0.1～1.0g/L KH 2 PO 4 、0.1～2.0g/L K 2 HPO 4 、0.1～2.0g/L MgSO 4 ·7H 2 O, 1.0-20.0 g/L flavonoid compound, and the pH value is 6.5-6.8.
Preferably, the inoculation amount of the monascus seed solution is 2-10%;
the temperature of the fermentation culture is 28-32 ℃, and the rotating speed of the oscillation condition is 150-250 rpm; the fermentation culture time is 6-14 days.
Preferably, the volume ratio of the fermentation liquor to the alcohol solvent is 1: 2;
the alcohol solvent comprises chromatographic grade methanol.
The invention provides application of flavonoids compounds in improving the yield of yellow pigment in monascus fermentation, wherein the flavonoids compounds comprise luteolin and/or genistein. Experiments prove that after the luteolin and the genistein are added singly or in combination, the liquid fermentation product with obviously improved yield of the yellow pigment is obtained after the monascus is subjected to liquid fermentation. Experiments prove that the content of the luteolin yellow pigment added in the amount of 2.0g/L is 6.50U/mL, which is improved by 26.08 times compared with the blank group; the content of the genistein group yellow pigment added by 2.0g/L is 0.51U/mL, which is improved by 1.13 times compared with the blank group; meanwhile, the content of the monascus yellow pigment is increased by 3.42 times by adding 1.0g/L of luteolin and 1.0g/L of genistein; meanwhile, the increase effect of 1.5g/L luteolin and 0.5g/L genistein on the content of monascus yellow pigment is most obvious, and is increased by 16.8 times. Therefore, the addition of luteolin and genistein in the liquid fermentation of monascus can improve the yellow pigment producing capability of monascus, greatly improve the production efficiency of yellow pigment, have important application value, and effectively utilize flavone resources to maximize resource utilization.
Drawings
FIG. 1 is a graph showing the comparison of production of uranidin in inorganic salt medium of Tiegun yam powder by monascus purpureus 30141 with addition of luteolin and genistein alone;
FIG. 2 is a graph comparing the production of yellow pigment by various combinations of luteolin and genistein on monascus purpureus 30141 in mineral salts medium of Tiegun yam flour.
Detailed Description
The invention provides application of flavonoids compounds in improving the yield of yellow pigment in monascus fermentation, wherein the flavonoids compounds comprise luteolin and/or genistein.
In the invention, the structural formulas of the luteolin and the genistein are as follows:
the genistein (not less than 97%) and luteolin (not less than 98%) are available from Shanghai Aladdin Biotechnology GmbH.
In the present invention, the Monascus is preferably Monascus purpureus (Monascus purpureus).
In the invention, in a fermentation system, the final concentration of the flavonoid compound is preferably not less than 1.0g/L, preferably 1.5-20.0 g/L, and more preferably 2.0 g/L. With the increase of the concentration of the flavonoid compounds, the yield of the yellow pigment is also increased. The final concentration of the flavonoid. The mass ratio of luteolin to genistein is preferably (1-3) to 1, and more preferably 3 to 1. The different kinds of luteolin and genistein also influence the yield of yellow pigment, and experiments show that the yield of yellow pigment is ranked from high to low as luteolin + genistein. The analysis reason for the difference of the two flavonoids in improving the yield of the yellow pigment is probably the difference of the structures of luteolin and genistein, and the two flavonoids are mainly at the binding sites (3, 4 sites) of B ring and C ring and the number of the connected hydroxyl on the B ring. The B ring of luteolin is connected with the 4-site of the C ring and is combined with two hydroxyl groups; while the B ring of genistein is linked to the 3-position of the C ring and only one hydroxyl group is attached to the B ring.
In the present invention, the fermentation medium for the fermentation of monascus preferably comprises the following components: 10.0-100.0 g/L Tiegun Yam powder, 1.0-5.0 g/L NaNO 3 、0.1～1.0g/LKH 2 PO 4 、0.1～2.0g/LK 2 HPO 4 、0.1～2.0g/LMgSO 4 ·7H 2 O, pH 6.5-6.8, preferably 20.0-80.0 g/L Tiegun yam powder and 2.0-4.0 g/L NaNO 3 、0.2～0.8g/L KH 2 PO 4 、0.5～1.5g/L K 2 HPO 4 、0.5～1.5g/L MgSO 4 ·7H 2 O, most preferably 20.0g/L Tiegun Yam powder and 2.0g/L NaNO 3 、0.5g/L KH 2 PO 4 、1.0g/L K 2 HPO 4 、1.0g/L MgSO 4 ·7H 2 And O. The method for preparing the fermentation medium is not particularly limited, and the method for preparing the fermentation medium can be a method well known in the art.
In the invention, the luteolin and the genistein are used singly or in combination in fermentation, so that the yield of the yellow pigment can be well improved, the flavone resource can be effectively saved, and the maximization of resource utilization is achieved.
The invention provides a method for producing yellow pigment by monascus liquid fermentation, which comprises the following steps:
inoculating monascus seed liquid into a fermentation culture medium containing flavonoid compounds, and performing fermentation culture under the oscillation condition to obtain a fermentation liquid; the flavonoid compound comprises luteolin and/or genistein;
and mixing the fermentation liquor and the alcohol solvent, carrying out solid-liquid separation, and filtering the obtained liquid phase by a membrane to obtain a fermentation product yellow pigment.
In the present invention, the monascus is preferably monascus purpureus, and the species and source are the same. The preparation method of the monascus seed liquid preferably comprises the following steps: inoculating monascus to an MPPY culture medium for shake culture, coating the obtained culture solution in a malt wort solid culture medium for culture until spores are generated, and eluting the spores by using normal saline to obtain a spore suspension; inoculating the spore suspension into a seed culture medium, and culturing under a shaking condition to obtain monascus seed liquid. The rotation speed of the oscillation condition is preferably 180-200 rpm. The temperature of the culture is preferably 28-32 ℃, and more preferably 30 ℃. The seed culture medium is preferably prepared by adding 20.0-40.0 g of glucose and 1.0-3.0 g of KH to each liter of culture medium 2 PO 4 ，2.0～4.0g NaNO 3 ，0.1～1.0g KCl，0.1～1.0g MgSO 4 ·7H 2 O, sterilizing at 121 deg.C for 20min, preferably adding 30.0g glucose and 2.0g KH per liter culture medium 2 PO 4 ，3.0g NaNO 3 ，0.5g KCl，0.5g MgSO 4 ·7H 2 And O. The concentration of the spore suspension is preferably 1.0X 10 5 -1.0×10 8 one/mL. The time for culturing the seed liquid is preferably 36-72 h, and more preferably 48-56 h. The malt wort solid culture medium is preferably selected from 100mL of redistilled water and 2.0g of agar, sterilized at 121 ℃ for 20min, poured into a flat plate, and cooled to obtain the malt wort solid culture medium.
In the present invention, the fermentation medium preferably comprises the following components: 10.0-100.0 g/L Tiegun Yam powder, 1.0-5.0 g/L NaNO 3 、0.1～1.0g/LKH 2 PO 4 、0.1～2.0g/LK 2 HPO 4 、0.1～2.0g/LMgSO 4 ·7H 2 O, 1.0-20.0 g/L flavonoid compound, and the pH value is 6.5-6.8; more preferably: 20.0-80.0 g/L Tiegun Yam powder, 2.0-4.0 g/L NaNO 3 、0.2～0.8g/LKH 2 PO 4 、0.5～1.5g/LK 2 HPO 4 、0.5～1.5g/LMgSO 4 ·7H 2 O, 1.5-10.0 g/L of flavonoid compound; more preferably 20.0g/L Tiegun Yam powder and 2.0g/L NaNO 3 、0.5g/LKH 2 PO 4 、1.0g/LK 2 HPO 4 、1.0g/LMgSO 4 ·7H 2 O, 2g/L flavonoid compound. The flavonoid compound is preferably 2g/L luteolin and 2g/L genistein or a composition formed by 1.5g/L luteolin and 0.5g/L genistein.
In the invention, the inoculation amount of the monascus seed solution is preferably 2-10%, more preferably 4-7%, and more preferably 5%. The temperature of the fermentation culture is 28-32 ℃, and more preferably 30 ℃. The rotating speed of the oscillation condition is preferably 150-250 rpm, more preferably 180-200 rpm; the fermentation culture time is 6-14 d, more preferably 8-12 h, and most preferably 10 h.
In the present invention, the volume ratio of the fermentation broth to the alcohol solvent is preferably 1: 2. The alcohol solvent comprises chromatographic grade methanol. After the mixing, a water bath treatment is preferably performed. The temperature of the water bath is preferably 58-62 ℃. The time of the water bath is preferably 50-70 min, and more preferably 60 min. The solid-liquid separation is preferably centrifugation. The rotation speed of the centrifugation is preferably 5000-7000 rpm, and more preferably 6000 rpm. The time for centrifugation is preferably 14-16 min, and more preferably 15 min. The pore size of the membrane for membrane filtration is preferably 0.45. mu.m. The content of yellow pigment is preferably determined by ultraviolet spectrophotometry, specifically detecting absorbance value of the extractive solution at 410nm, and using methanol as blank control. The yellow pigment content is the absorbance value multiplied by the dilution factor of the extracted yellow pigment.
In the invention, the content of luteolin yellow pigment added by 2.0g/L is 6.50U/mL, which is improved by 26.08 times compared with blank group; the content of genistein yellow pigment of 2.0g/L is 0.51U/mL, which is increased by 1.13 times compared with blank group. Meanwhile, the content of the monascus yellow pigment is increased by 3.42 times to be 1.15U/mL by adding 1.0g/L of luteolin and 1.0g/L of genistein; meanwhile, the increase effect of 1.5g/L luteolin and 0.5g/L genistein on the content of monascus yellow pigment is most obvious, and is increased by 16.8 times. The result shows that the addition of luteolin and genistein in the liquid fermentation of monascus can improve the yellow pigment producing capability of monascus.
The following examples are provided to illustrate the method for increasing the yield of yellow pigment in liquid fermentation of monascus yam powder according to the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
Method for producing yellow pigment by liquid fermentation of monascus purpureus
1) Preparing a seed culture medium: adding 30.0g glucose and 2.0g KH per liter culture medium 2 PO 4 ，3.0g NaNO 3 ，0.5g KCl，0.5g MgSO 4 ·7H 2 Dissolving O in distilled water, subpackaging in 250mL conical flask, and sterilizing at 121 deg.C for 20 min.
2) Preparing a seed solution: 1.0mL of Monascus purpureus 30141 (purchased from ACCC China agricultural microorganism culture Collection management center) with spore concentration of 1.0 × 10 6 Inoculating the spore solution per mL into a 250mL conical flask containing 100mL seed culture medium, and fermenting and culturing in a constant temperature shaking (180rpm, 30 ℃) incubator for 48h to obtain the monascus seed solution.
3) Preparing a fermentation medium: adding 20.0g Tiegun yam powder and 2.0g NaNO into each liter of culture medium 3 ，0.5gKH 2 PO 4 ，1.0g K 2 HPO 4 ，1.0g MgSO 4 ·7H 2 O, adding 2.0g luteolin or 2.0g genistein (at least four in each group), subpackaging in 250mL conical bottles, and sterilizing at 121 deg.C for 20 min. Meanwhile, a fermentation medium without flavonoid compounds is prepared to be used as a blank group.
4) Inoculating 5% monascus seed solution into a fermentation medium, and performing shake-flask culture for 12d at 30 ℃ and 180rpm to obtain monascus fermentation liquor.
5) The extraction method of the monascus yellow pigment comprises the following steps: adding 2 times volume of chromatographic grade methanol into 2.0mL of filtered fermentation liquid, mixing well, placing in 60 deg.C water bath for 1h, cooling, centrifuging at 6000rpm for 15min, collecting supernatant, and filtering with 0.45 μm organic filter membrane to obtain fermentation extract.
The monascus yellow pigment is measured by an ultraviolet spectrophotometry. And (3) absorbing 100 mu L of fermentation liquor into a 96-hole enzyme label plate, taking methanol as a blank reference, and measuring the absorbance of each group of fermentation liquor by using a full-wavelength enzyme label instrument, wherein the detection wavelength of the yellow pigment is 410 nm. The content of monascus yellow pigment is the absorbance value multiplied by the dilution multiple of the fermentation extract.
The yield of the monascus yellow pigment in the blank group (without flavone) in the fermentation liquid obtained in the embodiment is the lowest, and is 0.24U/mL; the content of luteolin yellow pigment added in the solution is 2.0g/L, which is 6.50U/mL and is improved by 26.08 times compared with the blank solution; the content of genistein yellow pigment of 2.0g/L is 0.51U/mL, which is increased by 1.13 times compared with blank group (see figure 1).
Example 2
Method for producing yellow pigment by liquid fermentation of monascus purpureus
1) Preparing a seed culture medium: adding 30.0g glucose, 2.0gKH per liter culture medium 2 PO 4 ，3.0g NaNO 3 ，0.5g KCl，0.5g MgSO 4 ·7H 2 Dissolving O in redistilled water, packaging in 250mL triangular flask, and sterilizing at 121 deg.C for 20 min.
2) Preparing a seed solution: 1.0mL of purple monascus 30141 with spore concentration of 1.0 × 10 6 Inoculating each/mL spore solution into a triangular flask containing 100mL sterile seed culture medium, and performing shake culture at 180rpm and 30 ℃ for 48h to obtain monascus seed solution.
3) Preparing a fermentation medium: adding 20.0g Tiegun yam powder and 2.0g NaNO into one liter of culture medium 3 ，0.5g KH 2 PO 4 ，1.0g K 2 HPO 4 ，1.0g MgSO 4 ·7H 2 O, 1.5g or 2g of a combination of luteolin and genistein (each concentration being repeated at least four times, wherein each concentration comprises 0.5g/L of luteolin +1.0g/L of genistein, 0.5g/L of luteolin +1.5g/L of genistein, 1g/L of luteolin +1g/L of genistein and 1.5g/L of luteolin0.5g/L genistein), subpackaging into 250mL triangular bottles, and sterilizing at 121 ℃ for 20 min.
4) Inoculating 5% monascus seed solution into a fermentation medium, and performing shake-flask culture for 12d at 30 ℃ and 180rpm to obtain monascus fermentation liquor. Yellow pigment was detected according to the method of example 1.
The yield of monascus yellow pigment in the blank group (not added with flavone) in the fermentation liquid obtained in the embodiment in the method for improving the content of yellow pigment in monascus liquid fermentation related by the invention is the lowest, and is 0.26U/mL; the content of monascus yellow pigment in the combined fermentation broth added with different dosages of luteolin and genistein is higher than that of the blank group. Wherein, 0.5g/L luteolin and 1.0g/L or 1.5g/L genistein are simultaneously added, the content of the yellow pigment is respectively 0.88U/mL and 1.09U/mL, which are respectively improved by 2.39 times and 3.19 times compared with the blank group; meanwhile, the content of the monascus yellow pigment is increased by 3.42 times to be 1.15U/mL by adding 1.0g/L of luteolin and 1.0g/L of genistein; the addition of 1.5g/L luteolin and 0.5g/L genistein has the most obvious effect of improving the content of monascus yellow pigment, and the content is improved by 16.8 times (see figure 2). The results show that the addition of luteolin and genistein in the liquid fermentation of monascus can improve the yellow pigment producing ability of monascus.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. Application of flavonoids compounds in improving the yield of yellow pigment in monascus fermentation, wherein the flavonoids compounds comprise luteolin and/or genistein.
2. The use according to claim 1, wherein the final concentration of flavonoids in the fermentation system is not less than 1.0 g/L.
3. The use according to claim 2, wherein the final concentration of the flavonoids is 1.5-20.0 g/L.
4. The use according to claim 3, wherein the mass ratio of luteolin to genistein is (1-3): 1.
5. The use according to any one of claims 1 to 4, wherein the fermentation medium for the fermentation of the monascus comprises the following components: 10.0-100.0 g/L Tiegun yam powder, 1.0-5.0 g/L NaNO 3 、0.1～1.0g/L KH 2 PO 4 、0.1～2.0g/L K 2 HPO 4 、0.1～2.0g/L MgSO 4 ·7H 2 O, the pH value is 6.5-6.8.
6. A method for producing yellow pigment by monascus liquid fermentation is characterized by comprising the following steps:
inoculating monascus seed liquid into a fermentation culture medium containing flavonoid compounds, and performing fermentation culture under the oscillation condition to obtain a fermentation liquid; the flavonoid compound comprises luteolin and/or genistein;
and mixing the fermentation liquor and the alcohol solvent, carrying out solid-liquid separation, and filtering the obtained liquid phase by a membrane to obtain a fermentation product yellow pigment.
7. The method of claim 6, wherein the fermentation medium comprises the following components: 10.0-100.0 g/L Tiegun yam powder, 1.0-5.0 g/L NaNO 3 、0.1～1.0g/L KH 2 PO 4 、0.1～2.0g/L K 2 HPO 4 、0.1～2.0g/L MgSO 4 ·7H 2 O, 1.0-20.0 g/L flavonoid compound, and the pH value is 6.5-6.8.
8. The method according to claim 6, wherein the inoculation amount of the monascus seed solution is 2-10%;
the temperature of the fermentation culture is 28-32 ℃, and the rotating speed of the oscillation condition is 150-250 rpm; the fermentation culture time is 6-14 days.
9. The method according to any one of claims 6 to 8, wherein the volume ratio of the fermentation broth to the alcohol solvent is 1: 2;
the alcohol solvent comprises chromatographic grade methanol.
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