CN109593795B - Ferulic acid and preparation method thereof - Google Patents
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- CN109593795B CN109593795B CN201910065788.7A CN201910065788A CN109593795B CN 109593795 B CN109593795 B CN 109593795B CN 201910065788 A CN201910065788 A CN 201910065788A CN 109593795 B CN109593795 B CN 109593795B
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Abstract
The invention belongs to the field of bioengineering, and particularly relates to ferulic acid and a preparation method thereof, wherein the ferulic acid is prepared by fermenting vinegar residue and/or bean dregs by taking a Neurospora sitophila strain No. 17 as a strain, the Neurospora sitophila strain No. 17 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CCMCC No.1836, and the preservation date is 2006, 10 and 12 days. The raw materials used for producing the ferulic acid are the leftovers of vinegar residue and/or bean dregs for brewing the table vinegar and producing the bean products, so that the production cost of the ferulic acid can be reduced, the additional value of the raw materials is increased, the pollution caused by improper treatment of the raw materials is reduced, and a new way is provided for the development and utilization of the resources; the used strain Neurospora sitophila strain has safe sources and short growth period, and the purified ferulic acid has high purity; the ferulic acid produced by the preparation method is an extracellular product and is beneficial to later-stage bioseparation.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to ferulic acid and a preparation method thereof.
Background
Ferulic Acid (Ferulic Acid), which is one of the derivatives of cinnamic Acid (also known as cinnamic Acid, 3-phenyl-2-propenoic Acid), has the chemical name of 4-hydroxy-3-methoxycinnamic Acid, and is a phenolic Acid widely found in plants, originally found in seeds and leaves of plants. Ferulic acid cross-links with polysaccharides in the cell wall form part of the fibres in the cell wall, in the form of esters, with a very low content of free ferulic acid. Ferulic acid has the effects of resisting platelet aggregation, inhibiting the release of platelet 5-hydroxytryptamine, inhibiting the generation of platelet thromboxane a2(txa2), enhancing the activity of prostaglandin, relieving pain, relieving vasospasm and the like, and is a basic raw material for producing medicaments for treating cardiovascular and cerebrovascular diseases, leukopenia and the like. The natural ferulic acid extracted from plants has good pharmacological action and biological activity and high safety, so the ferulic acid has wide application in the aspects of medicines, health products, cosmetic raw materials, food additives and the like.
Neurospora (Neurospora) belongs to the kingdom Fungi (Fungi), the phylum Ascomycota (Ascomycota), the class Scleromycetes (Pyrenomycetes), the order Sphaeriales. The FDA organization considers neurospora parva to be a few of the safe organisms that can be used, a class of organisms that is important to medicine, agriculture, and the environment, as well as a very important model organism in genetic and biochemical research. The Neurospora sitophila has strong capability of decomposing plant cellulose and hemicellulose, and has good development prospect in producing ferulic acid by utilizing the Neurospora sitophila.
Disclosure of Invention
The invention aims to provide ferulic acid and Neurospora sitophila (A)Neurospora sitophila) The method for preparing natural ferulic acid by biotransformation of dregs takes vinegar dregs and/or bean dregs as raw materials, has low production cost, can reduce the pollution to the environment caused by improper treatment of the vinegar dregs and/or the bean dregs by utilizing the vinegar dregs and/or the bean dregs, and has simple preparation method.
In order to achieve the purpose, the invention adopts the following technical scheme:
the ferulic acid is prepared by fermenting vinegar residue and/or bean dregs by taking a Neurospora parvum (Neurospora sitophila) strain No. 17 as a strain, wherein the Neurospora parvum strain No. 17 is preserved in the China general microbiological culture Collection center (CCMCC) No.1836 with the preservation date of 2006, 10 months and 12 days.
Preferably, the method for preparing ferulic acid comprises the following steps: taking the neurospora parvum strain No. 17 as a strain, and degrading hemicellulose in the vinegar residue and/or the bean dregs to produce ferulic acid by solid fermentation culture.
Preferably, the fermentation medium used for the solid fermentation culture contains 80-100% of dry vinegar residue and 0-20% of dry bean dregs, the ratio of material to water is 1:3, the initial pH of the culture medium is 5.5-6.5, the culture medium is sterilized at 121 ℃ for 20-30 min and then cooled, and 10 percent of dry matter is inoculated according to per gram6～107Inoculating the quantity of the good Neurospora sitophila spores, culturing for 72-84 h at 28-30 ℃, and obtaining supernatant through leaching and centrifugation. Wherein the percentage content of the dry vinegar residue and the dry bean dregs refers to the mass percentage of the dry vinegar residue or the dry bean dregs in the total amount of the dry vinegar residue and the dry bean dregs (dry matter).
Preferably, the fermentation medium used for the solid fermentation culture contains 80-90% of dry vinegar residue and 10-20% of dry bean dregs in a material-water ratio of 1: 3.
Preferably, the solid fermentation culture is preceded by a slant culture.
Preferably, the slant culture medium used for slant culture contains boiled potato juice 20%, glucose 2%, and agar 2%, has natural pH, is sterilized at 121 deg.C for 30min, is cooled and inoculated, and is cultured at 28-30 deg.C for 3-5 days. Wherein, in the percentages referred to, the numbers preceding the percentage (%) refer to the grams of solute contained in a 100mL solution.
The invention has the following beneficial effects:
(1) the present ferulic acid obtaining approach mainly comprises three methods of extraction, chemical synthesis or biological synthesis from plant raw materials with high ferulic acid content, while the raw materials used for producing the ferulic acid are vinegar residue and/or bean dregs which are leftovers of brewing vinegar and producing bean products, so that the production cost of the ferulic acid can be reduced, the additional value of the raw materials can be increased, the pollution caused by improper treatment of the raw materials can be reduced, and a new approach is provided for the development and utilization of the resources;
(2) the used strain, namely the Neurospora sitophila strain, has safe sources, the culture time is 72-84 h, the growth period is short, and the purity of the purified ferulic acid is high;
(3) the ferulic acid produced by the preparation method is an extracellular product, does not need cell disruption, can obtain a ferulic acid crude product after removing solid matters through centrifugation, can obtain high-purity ferulic acid through impurity removal, and is convenient to separate;
(4) the yield of the ferulic acid produced by the preparation method is high, when the ferulic acid is produced by singly culturing and degrading the hemicellulose in the vinegar residue through solid fermentation, the yield of the ferulic acid in the solid fermentation dry matter can reach more than 226.4 mug/g, and when the ferulic acid is produced by culturing and degrading the hemicellulose in the vinegar residue and the bean dregs through common solid fermentation, the yield of the ferulic acid in the solid fermentation dry matter can reach more than 340.2 mug/g;
(5) the method provided by the invention has the advantages of simple related production equipment, low capital investment and contribution to industrial production.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is a HPLC elution profile of ferulic acid standard.
FIG. 2 is an HPLC elution profile of the fermentation product extract of example 1.
Detailed Description
For ease of understanding, the present invention is further illustrated below with reference to the following examples.
The culture No. 17 good Neurospora sitophila used by the invention has been provided for preservation by the applicant to China general microbiological culture Collection center (CGMCC) in 2006, 10 and 19, and the preservation number is CGMCC No. 1836.
Example 1
(1) Slant culture: the slant culture medium contains boiling potato juice 20%, glucose 2% and agar 2%, has natural pH, is sterilized at 121 deg.C for 30min, is cooled and inoculated, and is cultured at 28-30 deg.C for 3 days.
(2) Solid fermentation culture: the culture medium comprises 100% of dry vinegar residue, the water addition amount is 3 times of the dry vinegar residue, and the initial p of the culture mediumH5.5, sterilizing at 121 ℃ for 30min, cooling, inoculating 10 per gram of dry culture medium6Culturing the neurospora europaea spores at 28-30 ℃ for 72h, adding 10 times of distilled water into the culture after the culture is finished, extracting, centrifuging at 4 ℃ and 10000rpm for 10min to obtain supernatant, wherein the content of ferulic acid in the supernatant is 11.32 mu g/mL, and the yield of the ferulic acid in solid fermentation dry substances (the total amount of dry vinegar residues and dry bean residues) is 226.4 mu g/g.
The fermentation product extract obtained in example 1 was analyzed by high performance liquid chromatography.
Preparing a ferulic acid standard curve: accurately weighing 10mg ferulic acid standard, diluting to 100mL with methanol to obtain 100, 80, 60, 40 and 20 mug/mL ferulic acid standard solutions respectively.
The type of chromatographic column used is: hypersil BDSC 185 μm E2616643, size: 4.6X 250mm, mobile phase: 0.2% glacial acetic acid-100% methanol (28: 72), detection wavelength: 314nm, temperature: 30 ℃, flow rate: 1mL/min, sample size: and (5) carrying out sample injection for 3 times on the standard substance with each concentration. The ferulic acid content is taken as the ordinate, and the peak area is taken as the abscissa to make a standard curve. The analysis and determination of ferulic acid in the sample adopt the chromatographic conditions of the same standard substance.
FIG. 1 is a HPLC elution profile of ferulic acid standard, and FIG. 2 is a HPLC elution profile of fermentation product extract of example 1. The HPLC retention times of the ferulic acid standard in FIG. 1 and the fermentation product extract of the invention in FIG. 2 are both 3.353min, indicating that the fermentation product extract of the invention contains ferulic acid. And substituting the measured HPLC elution peak area of the sample into a standard curve to obtain the content of the ferulic acid.
Example 2
(1) Slant culture: the slant culture medium contains boiling potato juice 20%, glucose 2% and agar 2%, has natural pH, is sterilized at 121 deg.C for 30min, is cooled and inoculated, and is cultured at 28-30 deg.C for 5 days.
(2) Solid fermentation culture: the culture medium comprises 80% of dry vinegar residue and 20% of dry bean dregs, the water addition amount is 3 times of the total mass of the dry vinegar residue and the dry bean dregs, the initial pH of the culture medium is 6.5, the culture medium is cooled after being sterilized at 121 ℃ for 20min, and each gram of the dry culture mediumAccess 107Culturing the neurospora europaea spores at 28-30 ℃ for 78h, adding 10 times of distilled water into the culture after the culture is finished, extracting, centrifuging at 4 ℃ and 10000rpm for 10min to obtain supernatant, wherein the content of ferulic acid in the supernatant is 17.01 mu g/mL, and the yield of the ferulic acid in solid fermentation dry substances (the total amount of dry vinegar residues and dry bean residues) is 340.2 mu g/g.
Example 3
(1) Slant culture was performed as in example 1.
(2) Solid fermentation culture: the culture medium comprises 90% of dry vinegar residue and 10% of dry bean dregs, the water amount is 3 times of the total weight of the dry vinegar residue and the dry bean dregs, the initial pH of the culture medium is 6, the culture medium is sterilized at 121 ℃ for 25min and then cooled, and 10 g of the dry culture medium is inoculated into each gram of the culture medium7Culturing the neurospora europaea spores at 28-30 ℃ for 84h, adding distilled water with the mass being 10 times of that of the culture after the culture is finished, leaching, and centrifuging at 4 ℃ and 10000rpm for 10min to obtain supernatant, wherein the content of ferulic acid in the supernatant is 18.22 mu g/mL, and the yield of the ferulic acid in solid fermentation dry substances (the total amount of dry vinegar residue and dry bean residue) is 364.4 mu g/g.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (3)
1. A method for preparing ferulic acid is characterized in that: the ferulic acid takes a Neurospora sitophila (Neurospora sitophila) strain No. 17 as a fermentation strain, the Neurospora sitophila strain No. 17 is preserved in the China general microbiological culture Collection center, the preservation number is CGMCC No.1836, and the preservation date is 2006, 10 and 12 days;
the preparation method of the ferulic acid comprises the following steps: taking a neurospora parvum strain No. 17 as a fermentation strain, and performing solid fermentation culture to degrade hemicellulose in vinegar residue and bean dregs to produce ferulic acid;
the fermentation medium used for solid fermentation culture contains 80-90% of dry vinegar residue and 10-20% of dry bean dregs, the material-water ratio is 1:3, the initial pH of the culture medium is 5.5-6.5, the culture medium is sterilized at 121 ℃ for 20-30 min and then cooled, and 10 percent of dry matter is inoculated into the culture medium per gram6～107Inoculating the quantity of the good Neurospora sitophila spores, culturing for 72-84 h at 28-30 ℃, and obtaining supernatant through leaching and centrifugation;
the percentage content of the dry vinegar residue and the dry bean dregs refers to the mass percentage of the dry vinegar residue or the dry bean dregs in the total amount of the dry vinegar residue and the dry bean dregs.
2. The method of claim 1, wherein: before the solid fermentation culture, slant culture is carried out.
3. The method of claim 2, wherein: the slant culture medium used for slant culture contains 20% of boiled potato juice, 2% of glucose and 2% of agar, has natural pH, is sterilized for 30min at 121 ℃, is cooled and inoculated, and is cultured for 3-5 days at 28-30 ℃; wherein the numbers preceding the percentage numbers refer to the grams of solute contained in a 100mL solution.
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| WO2018217588A1 (en) * | 2017-05-21 | 2018-11-29 | Innovanutra Inc. | Methods and compositions of incorporating a bioactive agent and use thereof |
| CN109022501A (en) * | 2018-07-13 | 2018-12-18 | 江南大学 | A method of ferulic acid is obtained using waste |
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- 2019-01-24 CN CN201910065788.7A patent/CN109593795B/en active Active
Patent Citations (7)
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| CN101091510A (en) * | 2007-05-30 | 2007-12-26 | 齐齐哈尔大学 | Feedstuff of zeaxanthin powder fermented by Neurospora sitophila, and preparation method |
| CN101863758A (en) * | 2010-03-23 | 2010-10-20 | 南京泽朗农业发展有限公司 | Technology for preparing ferulic acid and phytic acid from rice bran |
| CN105543293A (en) * | 2016-03-10 | 2016-05-04 | 南京林业大学 | Method for preparing ferulic acid |
| WO2018217588A1 (en) * | 2017-05-21 | 2018-11-29 | Innovanutra Inc. | Methods and compositions of incorporating a bioactive agent and use thereof |
| CN109022501A (en) * | 2018-07-13 | 2018-12-18 | 江南大学 | A method of ferulic acid is obtained using waste |
Non-Patent Citations (3)
| Title |
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| Stefan Coghe等.Ferulic Acid Release and 4-Vinylguaiacol Formation during Brewing and Fermentation: Indications for Feruloyl Esterase Activity in Saccharomyces cerevisiae.《J. Agric. Food Chem.》.ACS,2004,第52卷(第3期),第602-608页. * |
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