CN114456988A - Functional flora for producing kaempferol and preparation and application thereof - Google Patents
Functional flora for producing kaempferol and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a functional flora for producing kaempferol, and preparation and application thereof, and belongs to the technical field of producing kaempferol by microbial fermentation. The method takes potato and glucose as substrates, utilizes bacillus to change cell membrane permeability and rhizobium mediation effect, and produces kaempferol by co-fermentation with fusarium. The production method is simple, low in production cost, environment-friendly and capable of producing various other flavonoid compounds at high yield. The kaempferol produced by the method has the advantage that the yield of kaempferol of fusarium is obviously improved. The invention solves the problems of serious pollution and high cost in the production process of kaempferol, can provide a more natural and more sustainable substitute for consumers, promotes the industrial development of the biological fermentation technology, and can realize the large-scale production of kaempferol.
Description
Technical Field
The invention relates to a functional flora for producing kaempferol, and preparation and application thereof, and belongs to the technical field of producing kaempferol by microbial fermentation.
Background
The flavonoid compounds are the key points of research and development in the fields of medicine and nutritional products at home and abroad at present. The related components of the compound are widely applied, and comprise product formulas of skin, inflammation, immunity and the like. As can be seen from market data published externally by InsightSLICE, by 2031, the market share of flavonoids worldwide is expected to achieve 5.5% composite annual growth rate, and the market size will reach $ 14.5 billion.
Kaempferol is a polyalcohol hydroxy flavonoid compound widely existing in various plants, and has various pharmacological activities of resisting cancer, resisting oxidation, lowering blood pressure, reducing blood lipid, resisting inflammation, etc. The terminal products are mainly divided into medicine grade, food grade and other market segments, wherein the medicine grade accounts for the highest percentage. According to data released by global market research companies, 98% of kaempferol market demand in the united states comes from the pharmaceutical industry, and nutritional supplements, functional food and beverage products, topical cosmetic creams and the like are becoming new development directions. Kaempferol has great application potential, for example in the nutritional supplement industry, and is mainly used in immune support and inflammation formula products. In summary, kaempferol represents a worldwide consumer market of $ 57 billion from a global market perspective, with enormous development potential.
Currently, the main global production process for kaempferol is dominated by chemical synthesis. It is known that most chemically synthesized kaempferols start with hydroxybenzoic acid as a synthesis precursor. Hydroxybenzoic acid, in turn, is primarily derived from the chemical reaction of phenol and carbon dioxide, both chemicals being derived from petroleum and other fossil fuels. As consumer demand for natural health products increases, it becomes important how to produce the feedstock in a more natural and environmentally friendly process. At the end of 2021, danish biotechnology company Biosyntia and the french white biotechnology company Lantana Bio stated that a new technology platform was created to develop a more sustainable flavonoid production process through fermentation technology. Biosyntia is responsible for raw material production and commercialization, the first commercial raw material being kaempferol. In the near future of commercialization of this material, kaempferol from fermentation technology was also introduced by Conagen, USA, at the beginning of 2022. Although the whole process avoids the use of fossil fuel derivatives, the fermentation process is still complex, natural raw materials cannot be directly utilized, specific sugar needs to be extracted from plants, and then a series of fermentation processes are carried out, so that the application cost is high. But not negligible is that biofermentation is more sustainable than the use of fossil products and products of plant extract origin. Therefore, by developing and applying the proprietary biological fermentation process, a more natural and sustainable substitute can be provided for consumers, the industrial development of the biological fermentation technology is promoted, and the method has a wide application prospect.
Disclosure of Invention
The invention mainly aims to provide a functional flora for producing kaempferol. The flora takes potato and glucose as substrates, utilizes bacillus to change cell membrane permeability and rhizobium mediation, builds a co-fermentation process, and can realize that the yield of kaempferol produced by fusarium is obviously increased.
The object of the present invention is achieved as follows.
A functional flora for producing kaempferol comprises Bacillus velezensis (Bacillus velezensis), Rhizobium huanensis (Rhizobium mesonium) and Fusarium solani (Fusarium solani).
Further, the functional flora comprises: bacillus velezensis DSM 28326, Rhizobium huanensis (Rhizobium mesonium) LMG 24135 and Fusarium (Fusarium solani) ATCC 36031.
Furthermore, the functional flora is characterized in that two kinds of bacteria and fusarium are cultured independently, when the three kinds of bacteria reach logarithmic growth phase, the two kinds of bacteria are inoculated into fermentation liquor of the fusarium according to the inoculation amount of 5-10%, and finally, a co-fermentation system is constructed through culturing.
The second purpose of the invention is to provide the preparation method of the functional flora, which comprises the steps of independently culturing the Bacillus belgii, the Rhizobium huazhou and the fusarium, and inoculating the Bacillus belgii and the Rhizobium huazhou into a fermentation system containing the fusarium according to the inoculation amount of 5-10% when the Bacillus belgii, the Rhizobium huazhou and the fusarium all reach the logarithmic growth phase.
Furthermore, the dry weight of fusarium in the fermentation system reaches 2-3g/100mL, and the fusarium is used for inoculating bacillus beiLeisi and rhizobium huazhouensis.
According to the preparation method, seed culture media of the bacillus belgii, the rhizobium huazhou and the fusarium are all potato glucose culture media added with 0.1-0.2g/L chloramphenicol. So as to control the composition of metabolites of the strain and the colony morphology of fusarium when fermenting. With the addition of chloramphenicol, fusarium fermentation broth is not dark purple and yellow, and is dark purple without the addition of chloramphenicol, the bacterial liquid concentration is low, and the hyphae are few.
The preparation method comprises the steps of respectively inoculating bacillus belgii and rhizobium huazhou into a culture medium to culture so as to obtain a seed solution, wherein the culture conditions are as follows: the temperature is 25-30 ℃, the rotation speed is 160-180rpm, the initial pH value is 6.5-7.5, and the culture time is 1-2 days;
the previous-stage individual culture conditions of the fusarium are that the temperature is 25-30 ℃, the rotating speed is 140-160rpm, the initial pH value is 6.5-7.0, and the culture time is 2-4 days.
According to the preparation method, before co-fermentation, seed solutions of Bacillus belgii and Rhizobium huazhou need to be centrifuged to remove supernatant, a sterile culture medium is added, after heavy suspension, centrifugation is carried out again, washing is repeated for 3-6 times, after washing is finished, collected thalli are diluted into bacterial suspension by the sterile culture medium, and then inoculation is carried out.
As a further improvement, before the co-fermentation, the seed liquid of the Bacillus belgii and the Rhizobium huazhou needs to be centrifuged for 8-10 minutes at 3000-8-1×109CFU·mL-1Suspending the bacteria and then inoculating.
The third purpose of the invention is to provide the application method of the functional flora for producing kaempferol, wherein the functional flora is fermented to produce kaempferol, and the preferred co-fermentation culture conditions are as follows: the temperature is 25-30 ℃, the rotation speed is 160-180rpm, the initial pH value is 6.5-8.0, and the culture time is 2-4 days. The fermentation culture medium is potato glucose culture medium added with 0.1-0.2g/L chloramphenicol.
In the application method, the strain is strictly shielded from light in the processes of independent culture and mixed fermentation.
As a further improvement, the final fermentation liquor is filtered by gauze for separating thalli. 100mL of the filtrate was taken, and ethyl acetate was added in an amount equivalent to the amount of the filtrate to extract the filtrate 3 to 4 times. Performing rotary evaporation on the extract liquor for 15 minutes at 60 ℃ by using a rotary evaporator until no liquid drops are seen at the bottom of the flask, adding 3mL of chromatographic grade methanol for redissolving, and filtering by using a 0.22 mu m filter membrane to obtain 2mL of chromatographic bottles to be detected; washing the thallus with gauze for several times to retain fungus thallus, baking in 55 deg.C oven for 4-8 hr, grinding, sieving with 400 mesh sieve, extracting flavonoid glycoside with Soxhlet extraction method, and detecting kaempferol content with liquid chromatography. The detection conditions were as follows: the automatic sample injector is set to be 10 mu L of sample injection volume, and the flow rate is 1.0 mL/min; the column temperature of the chromatographic column is 25 ℃; the detector sets the detection wavelength to 360 nm; the extraction and elution program is set to be 0-20 min, and the mobile phase B: 5% -70%; 20-23 min, mobile phase B: 70% -5%; and (5) stopping for 25 min. Mobile phase A: 0.05mol/L sodium acetate (pH5.5), mobile phase B: 90% acetonitrile.
The invention aims to solve the problems of serious pollution and high cost in the kaempferol production process, provides a flora capable of obviously improving the kaempferol production by fusarium and an application method thereof, has simple culture medium components, low cost and simple and convenient operation process, can ensure the stability and safety of raw materials, can provide a more natural and more sustainable substitute for consumers, promotes the industrial development of a biological fermentation technology, and has wide application prospect.
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FIG. 1 is a high performance liquid chromatography chromatogram detection profile of a flavonoid compound;
FIG. 2 is a graph of the effect of co-fermentation on kaempferol yield;
FIG. 3 shows the effect of chloramphenicol on Fusarium fermentation broth.
The left figure is not added and the right figure is added.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments, which are only illustrative and are not intended to limit the scope of the present invention.
Example 1: preliminary screening of functional microorganisms
In order to solve the problems of serious pollution and high cost in the kaempferol production process, a proper strain needs to be selected firstly. According to the existing research basis, the kaempferol-producing fusarium is determined as the main fermentation strain. The co-fermentation strain mainly considers the capability of hydrolyzing cell walls, changing cell membrane permeability and resisting pressure (adapting to severe fermentation conditions), can antagonize chloramphenicol, can generate a large amount of biological membranes, can be symbiotic with fusarium, and has vigorous growth and metabolism capability. The main focus has been on various bacilli and rhizobia. Furthermore, antagonism among strains is reduced, the synergistic effect of the strains is exerted, and bacillus and rhizobia are mainly selected to be fermented together. The co-fermentation effect of different combinations is as follows:
in the following combinations, bacillus and rhizobia are inoculated and mixed according to 5% of equal proportion, and cultured and fermented under the same conditions.
Mixing group A: the fusarium, the bacillus belgii and the rhizobium huazhou are subjected to a co-fermentation test, and the measurement result of the fermentation liquid shows that the yield of kaempferol is increased by 189.23-195.46%;
mixing group B: the fusarium, the bacillus belgii and the austenitic rhizobium are subjected to a co-fermentation test, and the measurement result of the fermentation liquid shows that the yield of kaempferol is increased by 131.16-145.28%;
mixing group C: fusarium, bacillus pumilus and rhizobium huazhou carry out co-fermentation test, and the measurement result of the fermentation liquid shows that the yield of kaempferol is increased by 22.53-32.41%;
mixing group D: fusarium, bacillus pumilus and austenitorhizobium are subjected to a co-fermentation test, and the measurement result of the fermentation liquid shows that the yield of kaempferol is increased by 30.60-33.16%;
mixing group E: fusarium, bacillus subtilis and rhizobium huazhou perform co-fermentation tests, and the determination result of fermentation liquid shows that the yield of kaempferol is increased by 113.34-115.11%;
mixing group F: fusarium, bacillus subtilis and austenitzobium are subjected to co-fermentation test, and the determination result of the fermentation liquid shows that the yield of kaempferol is increased by 126.23-137.69%;
the above yield increases are compared to the blank of fusarium fermentation alone, 10 replicates for each set of experiments, taking the minimum and maximum values.
And selecting the mixed group A as a co-fermentation strain for producing the kaempferol according to the result, and optimizing the fermentation process again to finally obtain the controllable fermentation process capable of obviously improving the yield of the kaempferol.
Example 2: composition and source of functional microorganisms
The microorganisms involved in the invention are all purchased from various strain depositories, and are Fusarium (Fusarium solani) ATCC 36031, Bacillus belgii (Bacillus velezensis) DSM 28326, Bacillus pumilus (Bacillus pumilus) ATCC 7061, Bacillus subtilis ATCC6051, Rhizobium austeniticum (Rhizobium alamini) LMG 24466 and Rhizobium huazhongo (Rhizobium mesotrionum) LMG 24135.
Strains purchased from the American Type Culture Collection (ATCC) at 5 months in 2018 included: fusarium (Fusarium solani) ATCC 36031, Bacillus pumilus (Bacillus pumilus) ATCC 7061 and Bacillus subtilis (Bacillus subtilis) ATCC 6051.
Strains purchased from the german collection of microorganisms and cells (DSM) in 2017 at 10 months include: bacillus velezensis DSM 28326.
Purchased from the National Type Culture Collection (NTCC) in 2017 at 10 months and including: rhizobium austeniticum (Rhizobium alamii) LMG 24466, Rhizobium huanensis (Rhizobium mesotrionum) LMG 24135.
Example 3: method for culturing microorganism strains
Inoculating Bacillus belgii and Rhizobium huazhou into a culture medium to culture so as to obtain a seed solution, wherein the culture conditions are as follows: the temperature was 26 ℃, the rotation speed was 160rpm, the initial pH was 7.0, and the cells were incubated in the dark to log phase. In the whole fermentation process of the fusarium, the early-stage individual culture condition is that the temperature is 26 ℃, the rotating speed is 160rpm, the initial pH value is 6.5, and the light-proof culture time is 3 days; the culture mediums used separately by the Bacillus belgii, Rhizobium huazhoi and Fusarium were potato glucose culture mediums added with 0.12g/L chloramphenicol.
Example 4: seed liquid preparation of two kinds of functional bacteria
Centrifuging the two cultured functional bacteria of example 3 at 4000rpm for 10 min by a high-speed centrifuge, removing the supernatant, adding an equal amount of PDB culture medium, re-suspending, re-centrifuging, washing for 6 times, and after washing, further re-suspending and diluting the collected bacteria to 1 × 10 with PDB culture medium9CFU·mL-1The left and right bacterial suspensions were inoculated into a fermentation tank of Fusarium (the dry weight of Fusarium in the fermentation system was 2g per 100mL for inoculation of Bacillus belgii and Rhizobium huazhouense) at an inoculum size of 5%. The co-fermentation culture conditions are as follows: the temperature was 26 ℃, the rotation speed was 160rpm, the initial pH was 7.0, and the incubation time was 3 days. The light-resistant treatment is carried out in the whole fermentation process. The fermentation medium is a potato glucose medium added with 0.12g/L chloramphenicol.
Example 5: effect of Co-fermentation Process on Kaempferol yield
According to a high performance liquid chromatography detection map of fusarium flavonoids, fusarium itself can produce a plurality of flavonoids while producing kaempferol, as shown in fig. 1, (in fig. 1, single fermentation refers to single fermentation of fusarium). After the self-fermentation is finished, filtering the final fermentation liquor by using gauze, and carrying out thallus separation. 100mL of the filtrate was taken, and ethyl acetate was added in an amount equivalent to the amount of the filtrate to extract the filtrate 3 to 4 times. Performing rotary evaporation on the extract liquor for 15 minutes at 60 ℃ by using a rotary evaporator until no liquid drops are seen at the bottom of the flask, adding 3mL of chromatographic grade methanol for re-dissolution, and filtering by using a 0.22 mu m organic filter membrane to obtain 2mL of chromatographic bottles to be detected; washing the thallus with gauze for several times to retain fungus thallus, baking in 55 deg.C oven for 4-8 hr, grinding, sieving with 400 mesh sieve, extracting flavonoid glycoside with Soxhlet extraction method, and detecting kaempferol content with liquid chromatography. Repeated experiments show that the yield of kaempferol produced by co-fermentation of the three strains is significantly higher than that of a control group (i.e. fusarium which is not co-cultured with bacillus and rhizobia) in the case of excluding the possibility of producing kaempferol by functional bacteria, as shown in fig. 2. The content of kaempferol obtained by extracting the bacterial liquid can reach 644.49 mug/L, the content of kaempferol in the thalli can reach 25.78 mug/g, and the yield of kaempferol is improved by at least 200 percent on the whole. Further detecting the capability of producing kaempferol by mixing single bacteria, the yield difference of kaempferol is not significant compared with that of a control group in both the combination of fusarium and rhizobium huazhou and the combination of fusarium and bacillus beiLevensis (the addition amount of the rhizobium huazhou or the bacillus beivensis is equal to the total addition amount of the bacillus beivensis and the rhizobium huazhou). Therefore, the fermentation process for producing kaempferol by fusarium can be obviously improved.
Claims (10)
1. A functional flora for producing kaempferol is characterized by comprising Bacillus subtilis, Rhizobium mesonium and Fusarium solani.
2. The functional flora according to claim 1, comprising: bacillus belgii (Bacillus velezensis) DSM 28326, Rhizobium huanensis (Rhizobium mesonium) LMG 24135 and Fusarium (Fusarium solani) ATCC 36031.
3. The functional flora according to claim 1, wherein the two bacteria and the fusarium are cultured separately, and when the three bacteria reach logarithmic growth phase, the two bacteria are inoculated into the fusarium fermentation broth according to the inoculum concentration of 5-10%, and finally the co-fermentation system is constructed by culturing.
4. The method for preparing functional flora according to any one of claims 1 to 3, wherein Bacillus belgii, Rhizobium huazhou and Fusarium are cultured separately, and when the three reach logarithmic growth phase, the Bacillus belgii and Rhizobium huazhou are inoculated into the fermentation system containing Fusarium according to an inoculation amount of 5-10%.
5. The process according to claim 4, wherein the fermentation system comprises Fusarium weighing up to 2-3g/100mL in dry weight for inoculation with Bacillus belgii and Rhizobium huazhou.
6. The method according to claim 4, wherein the seed culture medium of Bacillus belgii, Rhizobium huanii and Fusarium is a potato dextrose medium supplemented with 0.1-0.2g/L chloramphenicol.
7. The method according to claim 4, wherein the seed solution is obtained by inoculating Bacillus bleekii and Rhizobium huazhoi to a culture medium and culturing under the following conditions: the temperature is 25-30 ℃, the rotation speed is 160-180rpm, the initial pH value is 6.5-7.5, and the culture time is 1-2 days;
the previous-stage individual culture conditions of the fusarium are that the temperature is 25-30 ℃, the rotating speed is 140-160rpm, the initial pH value is 6.5-7.0, and the culture time is 2-4 days.
8. The method according to claim 4, wherein the seed solution of Bacillus belgii and Rhizobium huazhou is centrifuged to remove the supernatant before the co-fermentation, a sterile medium is added, the suspension is resuspended and centrifuged again, the washing is repeated 3 to 6 times, and after the washing is completed, the collected cells are diluted with the sterile medium to form a cell suspension, and then the inoculation is performed.
9. The method for the use of kaempferol-producing functional flora according to any of claims 1-3, wherein kaempferol is produced by fermenting the functional flora, preferably under co-fermentation conditions: the temperature is 25-30 ℃, the rotation speed is 160-180rpm, the initial pH value is 6.5-8.0, the culture time is 2-4 days, and the fermentation culture medium is a potato glucose culture medium added with 0.1-0.2g/L chloramphenicol.
10. The use of the method according to claim 9, wherein the strain is strictly lightproof both during the culture alone and during the whole fermentation.
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| CN114480219A (en) * | 2022-03-15 | 2022-05-13 | 中南大学 | Microbial functional flora for improving terpene lactone content of ginkgo in ginkgo leaves and preparation and application thereof |
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| CN110637082A (en) * | 2017-01-04 | 2019-12-31 | 诺维信生物农业公司 | Bacillus isolate and uses thereof |
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| CN114480219A (en) * | 2022-03-15 | 2022-05-13 | 中南大学 | Microbial functional flora for improving terpene lactone content of ginkgo in ginkgo leaves and preparation and application thereof |
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