CN111518860A - Preparation method of cowberry fruit extract - Google Patents
Preparation method of cowberry fruit extract Download PDFInfo
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- CN111518860A CN111518860A CN202010387274.6A CN202010387274A CN111518860A CN 111518860 A CN111518860 A CN 111518860A CN 202010387274 A CN202010387274 A CN 202010387274A CN 111518860 A CN111518860 A CN 111518860A
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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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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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
- 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
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
A preparation method of a cowberry extract takes cowberry biological enzyme fermentation liquor as a raw material to prepare the cowberry extract, and the cowberry biological enzyme fermentation liquor comprises the following processing procedures: the method comprises the following steps: crushing cowberry berries and/or processing residues, and fermenting and filtering by using mixed bacteria to obtain a fermentation extract a, wherein the mixed bacteria are strains capable of producing cellulase, hemicellulase and pectinase; step two: and (3) carrying out secondary fermentation and filtration on the fermentation extracting solution a to obtain a fermentation extracting solution b, and carrying out fine extraction on the fermentation extracting solution b to obtain the cowberry fruit extract, wherein the secondary fermentation process adopts a process of inoculating the apple venturia inaequalis spore suspension into the fermentation extracting solution a for fermentation. The anthocyanin substances in the extract obtained by the invention have high yield and good purification effect, and meanwhile, the waste is reduced, so that the extract is green and environment-friendly.
Description
Technical Field
The invention belongs to the technical field of plant extraction and refining, and particularly relates to a preparation method of a cowberry fruit extract.
Background
Vaccinium, a plant of the genus Vaccinium of the family Ericaceae, comprising: more than 400 varieties of vaccinium myrtillus, vaccinium americanum, vaccinium sibiricum and the like are preferred, and vaccinium myrtillus produced in Sweden, Finland, Norway and the like is the best. The average weight of the cowberry fruit is 0.5-2.5g, the fruit is beautiful in color, the fruit is fine and smooth, the seeds are extremely small, and the edible rate reaches 100%. The cowberry contains rich bioactive constitution, has extremely strong medicinal value and nutrition and health care functions besides being eaten, and is listed as one of five kinds of human health foods by the international food and agriculture organization. The cowberry contains amino acid, vitamin, anthocyanin, flavone, organic selenium, fruit acid and other special nutrient components which are incomparable with any plant, and has more excellent antioxidant activity than common plants including blueberries. At present, people have higher and higher attention on the cowberry, and related industries are gradually increased. The development market of the blueberry deep processing is wide, and the deep processing will become the key development direction of the blueberry development in future.
Anthocyanins, or anthocyanins and anthocyanidins, are abundant in Vaccinium myrtillus and have a structure in which anthocyanins are bound to saccharides via glycosidic bonds. Due to its unique functionality, it is used to scavenge free radicals in vivo, resist tumors, cancers, inflammation, inhibit lipid peroxidation and platelet aggregation, prevent diabetes, reduce weight, protect vision, etc. Anthocyanin is a natural pigment, is safe and nontoxic, has a plurality of health care functions for human body, and is applied to industries of food, health care products, cosmetics, medicines and the like.
The existing preparation process of anthocyanin basically adopts organic solvent extraction or acidified alcohol aqueous solution extraction, and obtains a product by utilizing a spray drying technology after purification and concentration through macroporous resin, for example, Chinese patent application No. 201310461582.9 discloses a method for extracting anthocyanin from vaccinium myrtillus, wherein vaccinium myrtillus is taken as a raw material, ethanol with 9 times of the raw material amount is heated, refluxed and extracted for 2-3 times, reduced pressure concentration is carried out, D101 resin is adsorbed, desorbed, concentrated and dried to obtain a crude extract, and the crude extract is extracted by acetone to obtain vaccinium myrtillus extract with total anthocyanin content of about 70 percent; the Chinese patent application No. 201410253728.5 discloses a vaccinium myrtillus extract and a preparation method thereof, the method takes vaccinium myrtillus as a raw material, acidified ethanol with the volume fraction of 60 percent which is 3 times of that of fruit pulp is added, heating reflux extraction is carried out for 2 times at 50 ℃, self-made macroporous resin with polystyrene as a main framework is used for adsorption after concentration, then acid water is used for washing, acid ethanol, methanol or acetone are used for desorption, spray drying is carried out to obtain the vaccinium myrtillus extract with the total anthocyanin content of 55-95 percent, and a spectrogram accords with the specification of European pharmacopoeia, but the vaccinium myrtillus extract obtained by the method has high anthocyanin purity, the extraction by adopting the heating reflux method can easily cause the anthocyanin as the main active component to be heated and decomposed, the total yield of the anthocyanin is influenced, secondly, hydrochloric acid is used for acidifying a solvent in the extraction, water washing and desorption processes, and chloride ions are easily brought into products, leading the chloride ions of the product to exceed the standard; the Chinese patent application No. 201710522268.5 discloses a preparation method of a vaccinium myrtillus extract, which uses vaccinium myrtillus as a raw material, 95% ethanol with the volume amount of 3 times (4.5 times) of the weight of fruits is stirred and extracted at normal temperature, sulfuric acid is required to be added for extraction when the first extraction is carried out, saturated sodium citrate solution is used for regulating the pH of an extracting solution to 3.0-3.5 after the extraction is finished, centrifugal filtration and concentration are carried out, water is added into concentrated solution for dilution to obtain feed liquid, the feed liquid is adsorbed by HPD100 resin, citric acid aqueous solution with the pH of 3.0-3.5 is eluted, 80% -90% ethanol is desorbed, and the desorbed feed liquid is concentrated and dried to obtain the vaccinium myrtillus extract.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a preparation method of a cowberry fruit extract, which has the advantages of high anthocyanin substance yield and good purification effect in the obtained extract, reduces the generation of waste materials, and is green and environment-friendly.
The technical concept of the invention is as follows:
a preparation method of a cowberry extract takes cowberry biological enzyme fermentation liquor as a raw material to prepare the cowberry extract, and the cowberry biological enzyme fermentation liquor comprises the following processing procedures:
the method comprises the following steps: crushing cowberry berries and/or processing residues, and fermenting and filtering by using mixed bacteria to obtain a fermentation extract a, wherein the mixed bacteria are strains capable of producing cellulase, hemicellulase and pectinase;
step two: and (3) carrying out secondary fermentation and filtration on the fermentation extracting solution a to obtain a fermentation extracting solution b, and carrying out fine extraction on the fermentation extracting solution b to obtain the cowberry fruit extract, wherein the secondary fermentation process adopts a process of inoculating the apple venturia inaequalis spore suspension into the fermentation extracting solution a for fermentation.
In the design of the scheme, the mixed strain can be purchased from various commercial sources, for example, the mixed strain can be purchased from China center for Industrial culture Collection of microorganisms (CICC), and the Venturia inaequalis is locally collected and is obtained by single spore isolation test tube expansion culture.
In the design of the scheme, the beneficial microorganism fermentation is assisted by composite biological enzyme and enzymolysis, the fermentation environment is acidic due to continuously dissolved organic acid in the fermentation process, and the blueberry procyanidin cannot be lost under the normal temperature condition; the raw materials are further decomposed by cellulase, hemicellulase and pectinase generated by fermentation, so that the effective components in the cowberry are fully released, and the content of the proanthocyanidins in the fermentation extracting solution a can reach 70-85mg/ml by determination. The invention adopts the cerrena malus to carry out secondary fermentation on the fermentation extract a, and aims to purify the anthocyanin extract, because the cerrena malus not only has acid resistance and higher activity at normal temperature, but also can generate high-activity pectinase and cutinase, the pectinase can further decompose the fermentation liquor, and the cutinase plays a role in catalyzing the esterification reaction of phenolic hydroxyl on the proanthocyanidin and organic acid to generate a stable esterification product which is dissolved in the fermentation liquor, thereby reducing the adverse effect of low anthocyanin yield caused by easy decomposition of the proanthocyanidin, and the generated esterification product can be decomposed and extracted to obtain the anthocyanin extract in a weakly alkaline environment.
Preferably, the Venturia inaequalis spore suspension is the Venturia inaequalis spore suspension prepared by carrying out Venturia inaequalis induction spore production on filter residue left after preparing the fermentation extracting solution a as an induction spore production culture medium.
In the design of the scheme, the filter residue is used as the induced spore production culture medium of the venturia inaequalis, so that the extraction waste can be fully utilized,
the invention saves resources, and the inventor tests and verifies that the filter residue can be used as the induced sporulation culture medium of the venturia inaequalis to the conventional culture medium, and the reason is that the extracted waste contains ammonia source and carbon source required by the venturia inaequalis, and the waste has the mineral substances produced by organic acid, organic selenium and thalli with equivalent content, and has the effect of being beneficial to the sporulation of the venturia inaequalis.
As preferred in the present invention, the alternaria mali spore suspension: the fermentation extract a is 1: 15 to 25.
Preferably, the concentration of the Venturia inaequalis spore suspension is 0.8-1.2 per ml.
As a preferred aspect of the present invention, the component for inducing sporulation culture comprises: filter residue and barley grains, wherein the filter residue: the ratio of barley grains is 1-2: 2.
Preferably, the step one mixed bacterium comprises: two or more of yeast, Lactobacillus plantarum, Trichoderma viride, Streptococcus thermophilus, and Bacillus bifidus.
As a further preferred aspect of the present invention, the mixed bacteria in the first step include: yeast, lactobacillus plantarum, trichoderma viride.
As a further preferred aspect of the present invention, the yeast: lactobacillus plantarum: the ratio of the trichoderma viride to the trichoderma viride is 3:2: 1.
The invention has the following beneficial effects:
the blueberry extract is prepared by fermenting a blueberry mixture by adopting the complex enzyme, further purifying and fermenting the obtained fermentation liquor, protecting procyanidin under a mild enzymolysis condition, catalyzing an esterification process of procyanidin in a secondary fermentation process, and converting anthocyanin in the fermentation liquor into stable anthocyanin esterified substance, so that the anthocyanin substance in the finally obtained extract is high in yield and good in purification effect, and the obtained anthocyanin esterified substance can be hydrolyzed and reduced into anthocyanin and anthocyanin under a weak alkali environment.
According to the method, the filter residue obtained after primary fermentation of the cowberry fruit mixture is used as the induced spore production culture medium of the venturia inaequaling to the conventionally used induced spore production culture medium, and meanwhile, the generation of waste materials is reduced, so that the method is green and environment-friendly.
Detailed Description
Example 1
A method for preparing an extract of fructus Citri Tangerinae,
(1) preparing materials: the method is characterized in that the European cowberry jelly is used as a raw material, and the mass volume ratio of material liquid (W: V) is 1: 2, adding hot water at the temperature of 50-60 ℃ for unfreezing, feeding and crushing, and then carrying out sterilization treatment to obtain the cowberry fruit mixed solution.
(2) Activation of mixed strains: respectively activating yeasts such as Saccharomyces cerevisiae (YPD culture medium), Lactobacillus plantarum (LB culture medium) and Trichoderma viride (LB culture medium), culturing to middle and late logarithmic growth period, and preparing into mixed microbial inoculum (yeast (1.5 × 10)8cfu/ml), Lactobacillus plantarum (1.0 x 10)8cfu/ml), Trichoderma viride (0.5 x 10)8cfu/ml））。
(3) Biological enzymolysis: adding the mixed microbial inoculum prepared in the step (2) into the cowberry mixed liquor obtained in the step (1) according to the ratio of 15:1, uniformly stirring, sealing and fermenting at 20 ℃ for 5d, stirring once every 5h from the beginning of fermentation to the third day, stirring once every 8h every fourth day, and stirring once every 12h every fifth day to obtain fermentation liquor and filter residue;
(4) preparation of apple Venturia: collecting apple scab leaves, separating and purifying by PDA, storing in 5 deg.C refrigerator, and activating on PDA culture medium before inducing sporulation.
(5) Preparation of a spore production culture medium: taking the filter residue obtained in the step (3), washing with water for 3 times, drying until the water content is about 30-35%, and then preparing a culture medium for inducing apple venturia inaequalis to produce spores: 30g of treated filter residue and 40g of barley grains soaked in water for 1 hour are placed in a triangular flask, 20ml of 6% honey diluent and 20ml of 1% peptone solution are added, and then sterilization treatment is carried out.
(6) Inoculating the activated Venturia nigra into the three culture bottles prepared in the step (5), culturing in the dark at 25 ℃ for 10d, transferring into a PDA culture dish, inducing to produce spores by a black light lamp (365 nm) for 5d, and preparing conidia into a spore suspension with the concentration of 106One per ml.
(7) Adding the spore suspension obtained in the step (6) into the fermentation liquor obtained in the step (3), wherein the ratio of the fermentation liquor to the spore suspension is: spore suspension =20:1, the ratio of spores,
mixing, fermenting at 25 deg.C and pH of 5.0 for 70 hr, stirring once every 5 hr, and filtering to obtain secondary fermentation liquid.
(8) Sterilizing the obtained secondary fermentation liquor, adding 70% ethanol, extracting for 2 times at normal temperature for 2 hours, performing centrifugal filtration after extraction to obtain an extracting solution, adding sodium bicarbonate to adjust the pH value to be about 7.5-8.0, reacting for 2 hours, performing reduced pressure concentration, adding excipient maltodextrin, and performing spray drying to obtain the cowberry fruit extract.
Example 2
The difference between the embodiment and the embodiment 1 is that the filter residue is washed with water for 3 times and then dried until the water content is about 30-35%, and then the culture medium for inducing the spore production by the venturia inaequalis is prepared: 20g of treated filter residue and 50g of barley grains soaked in water for 1 hour are placed in a triangular flask, 20ml of 6% honey diluent and 20ml of 1% peptone solution are added, and then sterilization treatment is carried out.
Example 3
The difference between the embodiment and the embodiment 1 is that the filter residue is washed with water for 3 times and then dried until the water content is about 30-35%, and then the culture medium for inducing the spore production by the venturia inaequalis is prepared: 40g of treated filter residue and 30g of barley grains soaked in water for 1 hour are placed in a triangular flask, 20ml of 6% honey diluent and 20ml of 1% peptone solution are added, and then sterilization treatment is carried out.
Example 4
The difference between the embodiment and the embodiment 1 is that the filter residue is washed with water for 3 times and then dried until the water content is about 30-35%, and then the culture medium for inducing the spore production by the venturia inaequalis is prepared: 50g of treated filter residue and 20g of barley kernels soaked in water for 1 hour are placed in a triangular flask, 20ml of 6% honey diluent and 20ml of 1% peptone solution are added, and then sterilization treatment is carried out.
Example 5
The difference between the embodiment and the embodiment 1 is that the filter residue is washed with water for 3 times and then dried until the water content is about 30-35%, and then the culture medium for inducing the spore production by the venturia inaequalis is prepared: 70g of the treated filter residue is placed in a triangular flask, 20ml of 6% honey diluent and 20ml of 1% peptone solution are added, and then sterilization treatment is carried out.
Example 6
This example is different from example 1 in that the spore production medium is 70g of barley grains soaked in water for 1 hour, placed in a triangular flask, added with 20ml of 6% honey diluent and 20ml of 1% peptone solution, and then sterilized.
Example 7
This example is different from example 1 in that the time for the secondary fermentation in step (7) was 50 hours.
Example 8
This example is different from example 1 in that the time for the secondary fermentation in step (7) was 60 hours.
Example 9
This example is different from example 1 in that the time for the secondary fermentation in step (7) was 80 hours.
Example 10
This example is different from example 1 in that the time for the secondary fermentation in step (7) was 90 hours.
The results of anthocyanin and anthocyanin measurements for examples 1 to 10 were as follows:
the above data show that: examples 1-6 compare and find that using the residue as culture medium in example 1 at the proper ratio is equivalent to using conventional barley culture medium, whereas if the residue: if the proportion of the barley is more than 3/4, the spore production effect of the alternaria alternata can be influenced; the comparison of examples 7-10 shows that the fermentation time has obvious influence on the yield of anthocyanin and anthocyanin, and the fermentation yield is good within 60-80 h, preferably 70 h.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (8)
1. The preparation method of the cowberry extract is characterized in that the cowberry extract is prepared by taking cowberry biological enzyme fermentation liquor as a raw material, and the cowberry biological enzyme fermentation liquor comprises the following processing procedures:
the method comprises the following steps: crushing cowberry berries and/or processing residues, and fermenting and filtering by using mixed bacteria to obtain a fermentation extract a, wherein the mixed bacteria are strains capable of producing cellulase, hemicellulase and pectinase;
step two: and (3) carrying out secondary fermentation and filtration on the fermentation extracting solution a to obtain a fermentation extracting solution b, and carrying out fine extraction on the fermentation extracting solution b to obtain the cowberry fruit extract, wherein the secondary fermentation process adopts a process of inoculating the apple venturia inaequalis spore suspension into the fermentation extracting solution a for fermentation.
2. The method for preparing a cowberry fruit extract as claimed in claim 1, wherein the venturia inaequalis spore suspension is prepared by using the filter residue left after the fermentation extracting solution a is prepared as a culture medium for inducing the production of Venturia inaequalis to induce the production of Venturia inaequalis spore.
3. The method of claim 2, wherein the malus pumilus spore suspension comprises: the fermentation extract a is 1: 15 to 25.
4. The method of claim 3, wherein the concentration of the Alternaria inaequalis spore suspension is 0.8-1.2/ml.
5. The method for preparing the cowberry fruit extract as claimed in claim 2, wherein the component for inducing the spore-forming nutrient comprises: filter residue and barley grains, wherein the filter residue: the ratio of barley grains is 1-2: 2.
6. The method as claimed in claim 1, wherein the step one of mixing the bacteria comprises: two or more of yeast, Lactobacillus plantarum, Trichoderma viride, Streptococcus thermophilus, and Bacillus bifidus.
7. The method as claimed in claim 6, wherein the step one of mixing the bacteria comprises: yeast, lactobacillus plantarum, trichoderma viride.
8. The method of claim 7, wherein the yeast: lactobacillus plantarum: the ratio of the trichoderma viride to the trichoderma viride is 3:2: 1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113786371A (en) * | 2021-07-05 | 2021-12-14 | 中国科学院烟台海岸带研究所 | Composition for preparing blueberry seaweed mask, mask and preparation method |
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