CN111748585A - Method for increasing yield of liquid fermentation monascus pigment - Google Patents

Method for increasing yield of liquid fermentation monascus pigment Download PDF

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Publication number
CN111748585A
CN111748585A CN201910233653.7A CN201910233653A CN111748585A CN 111748585 A CN111748585 A CN 111748585A CN 201910233653 A CN201910233653 A CN 201910233653A CN 111748585 A CN111748585 A CN 111748585A
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fermentation
pigment
liquid
monascus
monascus pigment
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李利
陈莎
舒彬
王俊敏
吴光旭
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Yangtze University
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Yangtze University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources

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  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Wood Science & Technology (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Health & Medical Sciences (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
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  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses a method for improving the yield of liquid fermentation monascus pigment, which takes monascus as a production strain and obtains the monascus pigment through spore culture, seed culture, liquid fermentation and extraction of the monascus pigment, wherein macroporous resin is added into a fermentation medium. The invention can greatly improve the yield of liquid fermentation of the monascus pigment, the monascus pigment is concentrated in the precipitate, namely resin and hypha after the fermentation product is subjected to solid-liquid separation, the water phase of the fermentation liquor does not contain the pigment, the separation and extraction of the downstream fermentation product and the treatment of the fermentation waste liquid are facilitated, and the invention has good market prospect and market value.

Description

Method for increasing yield of liquid fermentation monascus pigment
Technical Field
The invention relates to the technical field of monascus pigment production, and particularly relates to a method for improving the yield of liquid fermentation monascus pigment.
Background
The monascus pigment is a secondary metabolite of monascus (also called monascus), and is a safe pigment proved by human generation eating. The monascus pigment has good dyeing property, thermal stability, light stability, metal ion stability and redox agent stability, and also has certain antibacterial and antioxidant activities. As a food additive, monascus pigment is widely used in meat products, condiments, wines, pickled vegetables and flour products.
Research shows that monascus pigment produced by monascus is mainly alcohol-soluble components and accounts for 70-80%, and the water-soluble components account for a small proportion. In the liquid fermentation process, most of the alcohol-soluble monascus pigment components are kept in the thallus cells because of low solubility in the water phase of the fermentation liquor. In the production practice, only mycelium is collected and pigment is extracted from the mycelium, the pigment content in the residual fermentation liquid water phase is low, the extraction is not facilitated, and the pigment is treated as waste liquid, so certain pollution and waste exist. In addition, from the perspective of microbial metabolism, monascus pigment is accumulated in cells, so that pigment synthesis is inhibited by negative feedback, and the yield of the pigment is influenced, which may be one of the main reasons for the low yield of the monascus pigment produced by liquid fermentation at present. Therefore, a new monascus pigment liquid state fermentation method is found to eliminate negative feedback inhibition of products in the fermentation process, and has important significance for improving the yield of monascus pigment.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for improving the yield of liquid fermentation monascus pigment, which is not only beneficial to extraction, but also can effectively remove negative feedback inhibition of a product on the fermentation process, thereby greatly improving the yield of the monascus pigment.
The invention is realized by the following technical scheme: a method for improving the yield of monascus pigment by liquid fermentation specifically comprises the following steps:
s1, spore culture: inoculating monascus to a spore culture medium for culture, and collecting spores to prepare a spore suspension;
s2, seed culture: inoculating the spore suspension to a seed culture medium for culture to obtain a seed solution;
s3, liquid fermentation: adding the pretreated macroporous resin into a fermentation culture medium according to a certain proportion, sterilizing at high temperature, and inoculating the seed liquid into a liquid fermentation culture medium for culture;
s4, monascus pigment extraction: after fermentation, adjusting the pH value of the fermentation medium to 6, centrifuging to obtain a fermentation liquid water phase and a precipitate, ultrasonically extracting the precipitate with absolute ethyl alcohol, and performing suction filtration to obtain the monascus pigment extracting solution.
Preferably, the macroporous resin is any one or more of S-8, NKA-9, HP20, AB-8, CAD-40, D101 and HPD-100A.
More preferably, the macroporous resin is one or two of D101 and HP 20.
Preferably, the mass volume ratio of the macroporous resin to the fermentation medium is 0.5-10%.
Preferably, the pretreatment method of the macroporous resin comprises the following steps: soaking in 95% ethanol, washing with deionized water, soaking in 5% HCl solution, washing with deionized water to neutral, soaking in 5% NaOH solution, washing with deionized water to neutral, and filtering.
Compared with the prior art, the invention has the beneficial effects that:
1. by adding the macroporous resin for adsorbing the monascus pigment into the fermentation culture medium and carrying out in-situ adsorption separation on the product monascus pigment, the negative feedback inhibition of the product on the fermentation process can be effectively relieved, and the fermentation yield of the monascus pigment is greatly improved.
2. The macroporous resin can adsorb a large amount of monascus pigment, particularly can adsorb the pigment in the water phase of the fermentation liquor, so that the monascus pigment is concentrated in the solid phase precipitate, downstream product extraction and fermentation waste liquid treatment are simpler and easier, and pollution and waste are reduced.
The specific implementation mode is as follows:
the present invention is described in further detail with reference to the following examples, which are specific and detailed, but are not to be construed as limiting the scope of the present invention, and all technical solutions obtained by equivalents or equivalent transformations shall be included in the scope of the present invention as claimed in the claims.
The reagents used in the following examples of the present invention are commercially available products unless otherwise specified, and the preparation methods thereof are conventional methods unless otherwise specified, and the red koji mold described in the examples is red koji mold M-7, which is one of the most widely studied red koji mold strains.
Example 1: adding the monascus pigment of macroporous resin D101 for liquid fermentation.
Inoculating red monascus to 50mL of spore culture medium, performing shake culture at 28 ℃ and 200rpm for 5d, taking out, and performing static culture at 28 ℃ for 5 d. Wherein the spore culture medium specifically comprises: NaNO33.0g/L，K2HPO41.0g/L，KCl 0.5g/L，MgSO4·7H2O 0.5g/L，FeSO4·7H20.5g/L of O, 30.0g/L of cane sugar, 5.0g/L of yeast extract and natural pH.
Picking out cultured monascus purpureus hyphae, transferring the hyphae into a sterilized conical flask with glass beads, dispersing the hyphae by using an inoculating needle, adding 20mL of sterile water, shaking for 10-20 min, filtering by using double-layer mirror wiping paper to obtain a filtrate, namely a spore suspension, adjusting the concentration of the spore suspension to 105one/mL. 0.5mL of spore suspension was inoculated into 50mL of seed medium, and shake-cultured at 30 ℃ and 200rpm for 36 hours to obtain a seed solution. Wherein, the components of the seed culture medium are as follows: glucose 50.0g/L, peptone 20.0g/L, MgSO4·7H2O 0.5g/L，NaCl 2.0g/L，KH2PO48.0g/L，CH3COONa 0.5g/L, pH is natural.
The method for pretreating the macroporous resin comprises the following steps: soaking the macroporous resin in 95% ethanol for 24h to fully swell the macroporous resin, washing the macroporous resin with deionized water after filtering until no white turbid liquid and no ethanol smell appear, adding 5% HCl (V/V) solution after filtering, soaking the macroporous resin for 24h, washing the macroporous resin with deionized water to be neutral, soaking the macroporous resin with 5% NaOH (V/V) solution after filtering for 24h, washing the macroporous resin with deionized water to be neutral, and filtering to obtain the well-treated macroporous resin. Adding the treated macroporous resin into a fermentation culture medium according to the mass volume ratio of 5%, and sterilizing at 121 ℃ for 20min, wherein the fermentation culture medium specifically comprises the following components: glucose 50.0g/L, ammonium nitrate 3.0g/L, MgSO4·7H2O 0.5g/L，KCl 0.5g/L，KH2PO48.0g/L，FeSO40.01g/L, threonine 7.0g/L, pH6.6; wherein threonine is sterilized separately and added 24h after inoculation. Then, according to 7% of the connectionThe seed solution is inoculated into 75mL of fermentation liquid medium, and is cultured for 5d under the conditions of constant temperature shaking at 30 ℃ and 200 rpm.
After fermentation, adjusting the pH value of the fermentation medium to 6 by using HCl and NaOH, and centrifuging for 10min at 12000r/min to obtain a fermentation liquid water phase and a precipitate. And (3) carrying out ultrasonic assisted extraction on the precipitate part by 75mL of absolute ethyl alcohol (the volume of the precipitate part is the same as that of the fermentation liquor) for 4 times, wherein each time is 1 hour, carrying out suction filtration after extraction, and combining to obtain the monascus pigment extracting solution. The content of monascus pigment is measured by spectrophotometry, and the absorbance at 520nm is used to calculate the color value (U/mL) ═ OD520× dilution factor.
The test results are: the color value of the pigment in the water phase of the fermentation liquid is 53.0U/mL, the color value of the pigment in the precipitate is 1606.4U/mL, and the total amount of the pigment is 1659.4U/mL. Compared with comparative example 1, the total pigment yield is improved by 89%.
Example 2: adding monascus pigment of macroporous resin D101 under the condition of feeding for liquid fermentation.
This embodiment is the same as embodiment 1 except that: and (3) monitoring the consumption condition of glucose in the fermentation process, supplementing carbon source glucose and nitrogen source ammonium nitrate when the glucose is consumed in the 3 rd fermentation period, so that the concentration of the carbon source glucose and the nitrogen source ammonium nitrate reaches the concentration of the initial fermentation period, and continuing to ferment until the 7 th fermentation period is finished.
The test results are: the color value of the pigment in the water phase of the fermentation liquid is 106.9U/mL, the color value of the pigment in the precipitate is 2343.0U/mL, and the total amount of the pigment is 2449.9U/mL. Compared with comparative example 2, the total pigment yield is improved by 107%.
Example 3: adding monascus pigment liquid of macroporous resin HP20 for liquid fermentation under the condition of feeding.
The macroporous resin D101 in example 2 was replaced with HP20, and the other experimental conditions were identical to those of example 2. The test results are: the color value of the pigment in the water phase of the fermentation liquor is 56.8U/mL, the color value of the pigment in the precipitate is 1470.0U/mL, and the total amount of the pigment is 1526.8U/mL. Compared with comparative example 2, the total pigment yield is improved by 29%.
Comparative example 1:
no macroporous resin was added and the other conditions were identical to those of example 1. The test results are: the color value of the pigment in the water phase of the fermentation liquor is 373.8U/mL, the color value of the pigment in the precipitate is 502.8U/mL, and the total amount of the pigment is 876.6U/mL.
Comparative example 2:
no macroporous resin was added, the others were identical to example 2. The test results are: the color value of the pigment in the water phase of the fermentation liquor is 1014.0U/mL, the color value of the pigment in the precipitate is 167.6U/mL, and the total amount of the pigment is 1181.6U/mL.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the technical scope of the present invention, so that any minor modifications, equivalent changes and modifications made to the above embodiments according to the technical spirit of the present invention still fall within the technical scope of the present invention.

Claims (5)

1. A method for improving the yield of monascus pigment in liquid fermentation is characterized by comprising the following steps:
s1, spore culture: inoculating monascus to a spore culture medium for culture, and collecting spores to prepare a spore suspension;
s2, seed culture: inoculating the spore suspension to a seed culture medium for culture to obtain a seed solution;
s3, liquid fermentation: adding the pretreated macroporous resin into a fermentation culture medium according to a certain proportion, sterilizing at high temperature, and inoculating the seed liquid into the fermentation culture medium for fermentation;
s4, monascus pigment extraction: after fermentation, adjusting the pH value of the fermentation medium, centrifuging to obtain a fermentation liquid water phase and a precipitate, performing ultrasonic assisted extraction on the precipitate by using absolute ethyl alcohol, and performing suction filtration to obtain a monascus pigment extracting solution.
2. The method for improving the yield of monascus pigment for liquid fermentation according to claim 1, wherein the macroporous resin is one or more of S-8, NKA-9, HP20, AB-8, CAD-40, D101 and HPD-100A.
3. The method for improving the yield of monascus pigment for liquid fermentation according to claim 2, wherein the macroporous resin is one or two of D101 and HP 20.
4. The method for increasing the yield of monascus pigment for liquid fermentation according to claim 1, wherein the mass-to-volume ratio of the macroporous resin to the fermentation medium is 0.5% -10%.
5. The method for improving the yield of monascus pigment for liquid fermentation according to claim 1 or 4, wherein the pretreatment method of the macroporous resin comprises the following steps: soaking in 95% ethanol, washing with deionized water, soaking in 5% HCl solution, washing with deionized water to neutral, soaking in 5% NaOH solution, washing with deionized water to neutral, and filtering.
CN201910233653.7A 2019-03-26 2019-03-26 Method for increasing yield of liquid fermentation monascus pigment Pending CN111748585A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110021397A1 (en) * 2008-11-11 2011-01-27 Colgate-Palmolive Company Composition With A Color Marker
CN102321713A (en) * 2011-09-14 2012-01-18 上海交通大学 Method for producing Thailandepsin A by continuous adsorption coupling fermentation
CN106222098A (en) * 2016-10-08 2016-12-14 福州大学 One strain monascus sp bacteria strain and application thereof
CN106590020A (en) * 2016-11-02 2017-04-26 华南理工大学 Method of separating water soluble monascus pigment by the use of macroreticular resin and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110021397A1 (en) * 2008-11-11 2011-01-27 Colgate-Palmolive Company Composition With A Color Marker
CN102321713A (en) * 2011-09-14 2012-01-18 上海交通大学 Method for producing Thailandepsin A by continuous adsorption coupling fermentation
CN106222098A (en) * 2016-10-08 2016-12-14 福州大学 One strain monascus sp bacteria strain and application thereof
CN106590020A (en) * 2016-11-02 2017-04-26 华南理工大学 Method of separating water soluble monascus pigment by the use of macroreticular resin and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LI LI等: "Acidic conditions induce the accumulation of orange Monascus pigments during liquid-state fermentation of Monascus ruber M7", 《BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING》 *
SUO CHEN等: "A facile macroporous resin-based method for separation of yellow and orange Monascus pigments", 《FOOD SCI BIOTECHNOL》 *
容艳筠等: "大孔吸附树脂吸附红曲红色素提取废液的研究", 《中国食品添加剂试验研究》 *
王卫等: "赤霉素发酵与大孔树脂吸附过程耦合工艺优化", 《中国生物工程杂志》 *

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Application publication date: 20201009