CN114164245A - Fermentation method for improving antioxidant stability of phycocyanin - Google Patents
Fermentation method for improving antioxidant stability of phycocyanin Download PDFInfo
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- CN114164245A CN114164245A CN202111678159.5A CN202111678159A CN114164245A CN 114164245 A CN114164245 A CN 114164245A CN 202111678159 A CN202111678159 A CN 202111678159A CN 114164245 A CN114164245 A CN 114164245A
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- phycocyanin
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- spirulina
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- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/41—Porphyrin- or corrin-ring-containing peptides
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P17/18—Antioxidants, e.g. antiradicals
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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Abstract
The invention belongs to the technical field of fermentation, and discloses a fermentation method for improving the antioxidant stability of phycocyanin. In particular discloses application of bacillus in improving the antioxidant stability of phycocyanin. The invention discloses the application of bacillus in improving the antioxidant stability of phycocyanin for the first time, and the bacillus and phycocyanin extracting solution are mixed and fermented to obtain the phycocyanin; not only can effectively improve the oxidation resistance of the phycocyanin, but also can effectively improve the oxidation resistance stability of the phycocyanin.
Description
Technical Field
The invention belongs to the technical field of fermentation, and particularly relates to a fermentation method for improving the antioxidant stability of phycocyanin.
Background
Spirulina (Spirulina) belongs to Oscilatoriaceae (Oscilatoriaceae) Spirulina of Cyanophyta (Cyanophyta) and is a prokaryote, mainly including Spirulina platensis (Spirulina platensis), Spirulina maxima (Spirulina maxima), Spirulina fusiformis (Spirulina fusiformis) and Spirulina pacifica (Spirulina pacifica), etc., of which Spirulina platensis is most commonly used in production and nutrition research. The spirulina has been proved to have no negative influence on the normal physiological function of animals in acute toxicology experiments and conventional drug experiments; the fertility and the fetal development of the pregnant rats are not affected by feeding the pregnant rats, and the pregnant rats have no reproductive toxicity. In addition, the spirulina contains various nutrient components including protein, saccharides, carotenoid, essential amino acid, vitamins and minerals, and has the effects of resisting oxidation, reducing blood fat, diminishing inflammation, resisting virus and cancer, regulating and enhancing immune system, and the like. Due to its safety and efficacy, it is widely cultivated and used as a dietary supplement around the world.
Among many nutritional ingredients of spirulina, the protein content is up to 60%, and the spirulina contains a very rare pigment protein Phycocyanin (Phycocyanin), also called Phycocyanin, which is a complex formed by protein and porphyrin pigment, is soluble in water, presents bright blue color, and has characteristic absorption near 620 nm. Because the amino acid composition in the phycocyanin is rich and complete, and the phycocyanin has activities of resisting oxidation, regulating immunity and the like, the phycocyanin is often used as a natural pigment and a functional raw material in the field of cosmetics. However, phycocyanin has a large molecular weight and a complex structure, the presence of a surfactant in a cosmetic formulation may destroy the high-grade structure of the protein, and the shelf life of cosmetics is usually long, so that the instability of phycocyanin during storage easily causes the stability of product quality and the efficacy to be difficult to guarantee.
Fermentation refers to the process by which a person prepares the microbial cells themselves, or direct or secondary metabolites, by virtue of the life activities of the microorganisms under aerobic or anaerobic conditions. The natural product can change the biological activity of the natural product through a fermentation process, namely, the metabolic process of the strain is fully utilized, such as the enrichment of active molecules is improved, the toxicity of the natural product is reduced, and the like. However, most of the existing fermentation of natural products has the characteristics of no design, blindness and the like, and the mechanism of changing the activity of the natural products by fermentation cannot be clarified, so that the improvement of the biological activity is limited.
Disclosure of Invention
The first aspect of the invention aims to provide application of bacillus in improving the stability of phycocyanin in oxidation resistance.
The second aspect of the present invention is to provide a method for preparing a phycocyanin fermentation broth.
The third aspect of the present invention is to provide a phycocyanobilin fermentation broth obtained by the production method of the second aspect of the present invention.
The fourth aspect of the present invention is to provide the application of the phycocyanin fermentation liquid of the third aspect of the present invention in the preparation of products.
The object of the fifth aspect of the invention is to provide a product.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, the application of bacillus in improving the stability of phycocyanin against oxidation is provided.
Preferably, the bacillus is at least one of bacillus amyloliquefaciens, bacillus pumilus, bacillus licheniformis and bacillus subtilis.
The second aspect of the present invention provides a preparation method of a phycocyanin fermentation liquid, comprising the following steps: mixing the culture medium and the bacterial liquid, and fermenting to obtain phycocyanin fermentation liquid; the culture medium contains phycocyanin extracting solution.
Preferably, the medium further comprises a carbon source.
Further preferably, the carbon source is a saccharide.
Even more preferably, the carbon source is glucose.
Preferably, the preparation method of the phycocyanin extracting solution comprises the following steps: mixing Spirulina with water, and crushing to obtain phycocyanin extractive solution.
Preferably, the mass ratio of the spirulina to the water is (3-10): 50.
further preferably, the mass ratio of the spirulina to the water is (4-8): 50.
more preferably, the mass ratio of the spirulina to the water is (4-6): 50.
preferably, the preparation method of the phycocyanin extracting solution further comprises the following steps: and (4) centrifuging and sterilizing.
Further preferably, the centrifugation condition is 10000-15000 rpm centrifugation for 7-15 min.
Further preferably, the centrifugation is performed at 10000-12000 rpm for 10-13 min.
Further preferably, the sterilization is performed by filtration sterilization with a 0.1-0.45 μm filter membrane.
More preferably, the sterilization is performed by filtration sterilization with a 0.1-0.22 μm filter membrane.
Preferably, the final concentration of phycocyanin in the culture medium is 1 wt% to 10 wt%.
Further preferably, the final concentration of phycocyanin in the culture medium is 1 wt% to 5 wt%.
Still more preferably, the final concentration of phycocyanin in the culture medium is 1 wt% to 3 wt%.
Preferably, the final concentration of the carbon source in the culture medium is 1 wt% to 8 wt%.
Further preferably, the final concentration of the carbon source in the culture medium is 1 wt% to 5 wt%.
Still more preferably, the final concentration of the carbon source in the medium is 1 wt% to 3 wt%.
Preferably, the inoculation amount of the bacterial liquid is 1 v/v% -8 v/v% of the culture medium.
More preferably, the inoculation amount of the bacterial liquid is 2 v/v% -8 v/v% of the culture medium.
More preferably, the inoculation amount of the bacterial liquid is 2 v/v% -5 v/v% of the culture medium.
Preferably, the number of viable bacteria in the bacterial liquid is (1-9) multiplied by 106CFU/mL。
More preferably, the number of viable bacteria in the bacterial liquid is (1-7) multiplied by 106CFU/mL。
More preferably, the number of viable bacteria in the bacterial liquid is (1-5) x 106CFU/mL。
Preferably, the strain in the bacterial liquid is a high-yield protease strain.
Further preferably, the strain is at least one of bacillus amyloliquefaciens, bacillus pumilus, bacillus licheniformis and bacillus subtilis.
Preferably, the fermentation condition is 30-40 ℃ for 20-25 h.
Further preferably, the fermentation condition is 35-40 ℃ for 22-25 h.
More preferably, the fermentation condition is 35-37 ℃ for 22-24 h.
Preferably, the pH of the fermentation is 6-8.
Further preferably, the pH of the fermentation is 6-6.5.
Preferably, the carbon source in the medium is sterilized separately.
Further preferably, the sterilization condition is 115-121 ℃ sterilization for 15-30 min.
Preferably, the preparation process of the phycocyanin fermentation liquid further comprises the following steps: centrifuging, filtering and heating.
More preferably, the centrifugation condition is 3000-5000 rpm centrifugation for 8-15 min.
More preferably, the centrifugation condition is 4000-5000 rpm for 8-10 min.
Further preferably, the filtration is ceramic membrane filtration.
Even more preferably, the filtration is 200nm to 230nm ceramic membrane filtration.
Further preferably, the heating treatment is carried out in a water bath at 90-100 ℃ for 5-15 min.
More preferably, the heating treatment is carried out in a water bath at 90-95 ℃ for 10-15 min.
In a third aspect of the present invention, a phycocyanin fermentation broth is provided, which is prepared by the preparation method of the second aspect of the present invention.
In a fourth aspect of the invention, there is provided the use of the phycocyanin fermentation broth of the third aspect of the invention in the preparation of a product;
the product is used for:
(1) relieving skin irritation; and/or
(2) Oxidation resistance; and/or
(3) Improving immunity.
Preferably, the products include food, pharmaceuticals and cosmetics.
In a fifth aspect of the invention, there is provided a product comprising the phycocyanin fermented liquid of the third aspect of the invention.
Preferably, the product is used for:
(1) relieving skin irritation; and/or
(2) Oxidation resistance; and/or
(3) Improving immunity.
Preferably, the products include food, pharmaceuticals and cosmetics.
The invention has the beneficial effects that: 1. the invention discloses the application of bacillus in improving the antioxidant stability of phycocyanin for the first time, and the bacillus and phycocyanin extracting solution are mixed and fermented to obtain the phycocyanin; not only can effectively improve the oxidation resistance of the phycocyanin, but also can effectively improve the oxidation resistance stability of the phycocyanin; 2. the invention provides a preparation method of phycocyanin fermentation liquor, the antioxidant activity of phycocyanin obtained after fermentation is obviously higher than that of unfermented phycocyanin, and the antioxidant stability of the phycocyanin is improved by 4.7 times compared with that of the unfermented phycocyanin, therefore, the preparation method provided by the invention not only can effectively improve the antioxidant activity of the phycocyanin, but also can effectively improve the antioxidant stability of the phycocyanin; 3. the phycocyanin fermentation liquor obtained by the fermentation method provided by the invention has a good relieving effect, can effectively eliminate or relieve skin irritation caused by a surfactant in cosmetics, and can be used as a relieving component in the cosmetics; 4. the preparation method of the phycocyanin fermentation liquid provided by the invention has the advantages of short process flow, simplicity in operation, easiness in control and low cost.
Drawings
FIG. 1 is a graph showing the antioxidant stability of the phycocyanin fermented liquid and the phycocyanin extracted liquid obtained in example 1.
FIG. 2 is a graph showing the relative antioxidant stability of the phycocyanin fermented solution and the phycocyanin extracted solution prepared in example 1.
FIG. 3 is a diagram showing the results of the blood vessel test of the chick embryo chorioallantoic membrane using the phycocyanin fermentation broth obtained in example 1.
Detailed Description
The present invention will now be described in detail with reference to specific examples, but the scope of the present invention is not limited thereto.
The materials, reagents and the like used in the present examples are commercially available materials and reagents unless otherwise specified. Wherein the spirulina powder is purchased from Shanghai plain biological science and technology Limited company with the cargo number GY-AP-01-S; bacillus amyloliquefaciens AS1.892 is purchased from China agricultural microorganism culture Collection center with the number of ACCC 10149; the bacillus pumilus FRI 2007044B 208 is purchased from China agricultural microbial culture collection center with the number of ACCC 01671; the bacillus subtilis KC969077 is purchased from China general microbiological culture collection center with the serial number of CGMCC 1.14985; bacillus licheniformis SDZHYB-12 is purchased from China agricultural microorganism culture Collection with the number of ACCC 19744.
Example 1
A preparation method of phycocyanin fermentation liquid comprises the following steps:
(1) weighing 40g of spirulina powder, dissolving in 500mL of water, recording the concentration (8%), uniformly mixing, crushing by using a high-pressure homogenizer (700bar, cycle times are 8 times), destroying the spirulina wall, and releasing phycocyanin contained in the spirulina powder; centrifuging the crushed spirulina solution at 10000rpm for 10 min; filtering the upper layer blue solution with 0.22 μm filter membrane for sterilization to obtain phycocyanin extractive solution, and storing at 4 deg.C; all operations in this step are carried out on ice or at 4 ℃;
(2) adding sterilized (sterilized at 121 ℃ for 20min) 10 wt% glucose into the phycocyanin extracting solution obtained in the step (1) to finish the preparation of a culture medium, wherein the final concentration of the glucose in the culture medium is 1 wt%, and the final concentration of the phycocyanin is 1 wt%;
(3) inoculating bacillus amyloliquefaciens AS1.892 bacterial liquid into the culture medium, wherein the inoculation amount of the bacterial liquid is 5 v/v% of the culture medium, and the number of the live bacteria in the bacterial liquid is about 106CFU/mL, fermenting for 24h at 37 ℃, and controlling the pH to be 6.0-6.5;
(4) and after the fermentation is finished, centrifuging the fermentation liquor at 4000rpm for 10min, removing thalli, filtering supernatant by using a 220nm ceramic membrane, putting filtrate in water bath at 95 ℃ for 10min, stopping the activity of residual enzyme in the fermentation liquor, and simultaneously killing possible residual viable bacteria in the fermentation liquor to obtain the phycocyanin fermentation liquor.
The preparation method of the bacillus amyloliquefaciens AS1.892 bacterial liquid comprises the following steps: streaking and separating a single colony of the bacillus amyloliquefaciens AS1.892 on a plate containing an LB culture medium, and culturing for 16h at 37 ℃; and (3) selecting a single colony, transferring the single colony into 5mL LB culture medium, and culturing overnight at 37 ℃ at a speed of 200rpm/min to obtain a bacillus amyloliquefaciens AS1.892 bacterial liquid.
Example 2
A preparation method of phycocyanin fermentation liquid comprises the following steps:
(1) weighing 50g of spirulina powder, dissolving in 500mL of water, recording the concentration (10%), uniformly mixing, crushing by using a high-pressure homogenizer (700bar, cycle times are 8 times), destroying the spirulina wall, and releasing phycocyanin contained in the spirulina powder; centrifuging the crushed spirulina solution at 10000rpm for 10 min; filtering the upper layer blue solution with 0.22 μm filter membrane for sterilization to obtain phycocyanin extract, and storing at 4 deg.C; all operations in this step are carried out on ice or at 4 ℃;
(2) adding sterilized (sterilized at 121 ℃ for 20min) 10 wt% glucose into the phycocyanin extracting solution obtained in the step (1) to finish the preparation of a culture medium, wherein the final concentration of the glucose in the culture medium is 1 wt%, and the final concentration of the phycocyanin is 5 wt%;
(3) inoculating bacillus pumilus FRI 2007044B 208 bacterial liquid into the culture medium, wherein the inoculation amount of the bacterial liquid is 5 v/v% of the culture medium, and the number of the viable bacteria in the bacterial liquid is about 106CFU/mL, fermenting for 24h at 37 ℃, and controlling the pH to be 6.0-6.5;
(4) and after the fermentation is finished, centrifuging the fermentation liquor at 4000rpm for 10min, removing thalli, filtering supernatant by using a 220nm ceramic membrane, putting filtrate in water bath at 95 ℃ for 10min, stopping the activity of residual enzyme in the fermentation liquor, and simultaneously killing possible residual viable bacteria in the fermentation liquor to obtain the phycocyanin fermentation liquor.
The preparation method of the bacillus pumilus FRI 2007044B 208 bacterial liquid comprises the following steps: streaking the Bacillus pumilus FRI 2007044B 208 on a plate containing an LB culture medium to separate a single colony, and culturing for 16h at 37 ℃; and (3) selecting a single colony, transferring the single colony into 5mL LB culture medium, and culturing at 37 ℃ at 200rpm/min overnight to obtain the Bacillus pumilus FRI 2007044B 208 bacterial liquid.
Example 3
A preparation method of phycocyanin fermentation liquid comprises the following steps:
(1) weighing 80g of spirulina powder, dissolving in 500mL of water, recording the concentration (16%), uniformly mixing, crushing by using a high-pressure homogenizer (700bar, cycle times are 8 times), destroying the spirulina wall, and releasing phycocyanin contained in the spirulina powder; centrifuging the crushed spirulina solution at 10000rpm for 10 min; filtering the upper layer blue solution with 0.22 μm filter membrane for sterilization to obtain phycocyanin extractive solution, and storing at 4 deg.C; all operations in this step are carried out on ice or at 4 ℃;
(2) adding sterilized (sterilized at 121 ℃ for 20min) 10 wt% glucose into the phycocyanin extracting solution obtained in the step (1) to finish the preparation of a culture medium, wherein the final concentration of the glucose in the culture medium is 3 wt%, and the final concentration of the phycocyanin is 1 wt%;
(3) inoculating bacillus subtilis KC969077 to the culture medium, wherein the inoculation amount of the bacillus subtilis is 2 v/v% of the culture medium, and the number of viable bacteria in the bacillus subtilis is about 106CFU/mL, fermenting for 24h at 37 ℃, and controlling the pH to be 6.0-6.5;
(4) and after the fermentation is finished, centrifuging the fermentation liquor at 4000rpm for 10min, removing thalli, filtering supernatant by using a 220nm ceramic membrane, putting filtrate in water bath at 95 ℃ for 10min, stopping the activity of residual enzyme in the fermentation liquor, and simultaneously killing possible residual viable bacteria in the fermentation liquor to obtain the phycocyanin fermentation liquor.
The preparation method of the bacillus subtilis KC969077 bacterial liquid comprises the following steps: streaking the bacillus subtilis KC969077 on a plate containing an LB culture medium to separate a single colony, and culturing at 37 ℃ for 16 h; and (3) selecting a single colony, transferring the single colony into 5mL LB culture medium, and culturing overnight at 37 ℃ at 200rpm/min to obtain the bacillus subtilis KC969077 bacterial liquid.
Example 4
A preparation method of phycocyanin fermentation liquid comprises the following steps:
(1) weighing 60g of spirulina powder, dissolving in 500mL of water, recording the concentration (12%), uniformly mixing, crushing by using a high-pressure homogenizer (700bar, cycle times are 8 times), destroying the spirulina wall, and releasing phycocyanin contained in the spirulina powder; centrifuging the crushed spirulina solution at 10000rpm for 10 min; filtering the upper layer blue solution with 0.22 μm filter membrane for sterilization to obtain phycocyanin extractive solution, and storing at 4 deg.C; all operations in this step are carried out on ice or at 4 ℃;
(2) adding sterilized (sterilized at 121 ℃ for 20min) 10 wt% glucose into the phycocyanin extracting solution obtained in the step (1) to finish the preparation of a culture medium, wherein the final concentration of the glucose in the culture medium is 3 wt%, and the final concentration of the phycocyanin is 5 wt%;
(3) inoculating Bacillus licheniformis SDZHYB-12 bacterial liquid into the culture medium, wherein the inoculation amount of the bacterial liquid2 v/v% of the culture medium, the number of viable bacteria in the bacterial liquid is about 106CFU/mL, fermenting for 24h at 37 ℃, and controlling the pH to be 6.0-6.5;
(4) and after the fermentation is finished, centrifuging the fermentation liquor at 4000rpm for 10min, removing thalli, filtering supernatant by using a 220nm ceramic membrane, putting filtrate in water bath at 95 ℃ for 10min, stopping the activity of residual enzyme in the fermentation liquor, and simultaneously killing possible residual viable bacteria in the fermentation liquor to obtain the phycocyanin fermentation liquor.
The preparation method of the bacillus licheniformis SDZHYB-12 bacterial liquid comprises the following steps: streaking Bacillus licheniformis SDZHYB-12 on a plate containing LB culture medium to separate single colony, and culturing at 37 deg.C for 16 h; and (3) selecting a single colony, transferring the single colony into 5mL of LB culture medium, and culturing overnight at 37 ℃ at a speed of 200rpm/min to obtain the bacillus licheniformis SDZHYB-12 bacterial liquid.
Effects of the embodiment
1. Stability testing (characterization of antioxidant Activity by DPPH free radical scavenging)
Diluting the phycocyanin fermentation liquid obtained in example 1 and the phycocyanin extracting solution obtained in step (1) in example 1 to a phycocyanin concentration of 4 wt%, and storing at 4 ℃; the DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) free radical clearance rate of the phycocyanin fermentation liquid and the phycocyanin extracting solution in 0, 3, 5, 12 and 21 days is sequentially detected, and the specific experimental steps are as follows: weighing DPPH, dissolving in absolute methanol, and preparing a DPPH solution with the concentration of 0.3 mmol/L; transferring 100 mu L of a sample to be detected (10 times dilution of phycocyanin fermentation liquid with phycocyanin concentration of 4% and phycocyanin extracting solution) into a 96-hole plate, measuring an absorbance Ab at 517nm by using an enzyme-labeling instrument, then adding 100 mu L of a PPH solution into each hole, uniformly mixing and reacting for 30min, and measuring an absorbance As at 517nm by using the enzyme-labeling instrument; replacing a sample with distilled water as a control group, wherein the blank absorbance value and the absorbance value after reaction are respectively Acb and Ac; the clearance of DPPH was calculated using the following formula: DPPH radical clearance ═ 1- (As-Ab)/(Ac-Acb) ] × 100%; and calibrating the antioxidant activity of the phycocyanin in the corresponding days 3, 5, 12 and 21 by taking the antioxidant activity measured on the day 0 as1 to obtain a phycocyanin relative stability curve.
Characterizing the antioxidant activity by DPPH free radical scavenging rate, monitoring the antioxidant activity of the phycocyanobilin fermentation liquor and the phycocyanobilin extracting solution after storage for 0, 3, 5, 12 and 21 days, wherein the result is shown in figure 1, and the antioxidant activity of the phycocyanobilin fermentation liquor is obviously higher than that of the phycocyanobilin extracting solution during the whole monitoring period; along with the increase of the storage time, the antioxidant activity of the phycocyanin extracting solution is sharply reduced, when the storage time is up to 21 days, the antioxidant activity of the phycocyanin extracting solution is almost 0, the antioxidant activity of the phycocyanin fermenting solution is relatively slowly reduced, when the storage time is up to 21 days, the phycocyanin fermenting solution still keeps the DPPH free radical clearance rate of more than 40%, and the antioxidant activity of the phycocyanin fermenting solution is still higher than the antioxidant activity of the phycocyanin extracting solution at 0 day. Comparing the DPPH free radical relative removal capacity of the phycocyanobilin fermentation liquid and the phycocyanobilin extracting solution, the result is shown in figure 2, along with the increase of the storage time, the antioxidant activity of the phycocyanobilin fermentation liquid is reduced slowly, the original activity is still maintained by 69% when the phycocyanobilin fermentation liquid is stored for 21 days, the antioxidant activity of the phycocyanobilin extracting solution is rapidly reduced along with the increase of the storage time, and the antioxidant activity is lost at 21 days, which indicates that the phycocyanobilin fermentation liquid obtained by fermentation can effectively improve the antioxidant stability of the phycocyanobilin. The storage time of the phycocyanin fermentation liquid and the phycocyanin extracting solution, which are 33 days (33-50/((100-69)/21)) and 7 days respectively, is estimated to reduce the antioxidant activity to half of the initial activity, so that the antioxidant stability of the phycocyanin treated by fermentation is improved by 4.7 times.
The results of detecting the antioxidant activity and stability of phycocyanin in phycocyanin fermentation liquid obtained in examples 2 to 4 are basically the same as those of example 1.
2. Chick embryo chorioallantoic membrane blood vessel experiment
Incubating the normally fertilized eggs in a constant temperature box at 37 ℃, taking the eggs on the 10 th day, sterilizing the eggs by using 75% ethanol, removing eggshells at the top ends of air chambers, and carefully peeling eggshell membranes by using bent dental tweezers to expose chorioallantoic membranes; visually observing the integrity of the allantoic membrane and the existence of bleeding points, and removing the allantoic membrane if the allantoic membrane is damaged; 100 mul of the sample to be tested (negative control group: 0.9% NaCl; experimental group: 1% sodium dodecyl sulfate and 2% phycocyanin fermentation liquid (prepared according to the method of example 1), and positive control group: 1% sodium dodecyl sulfate) was added to the chorioallantoic membrane, incubated at 37 ℃ for 30min, and the vascular damage of the chorioallantoic membrane of each group was observed.
As shown in FIG. 3, there was no significant change in the blood vessels of the chorioallantoic membrane after 0.9% NaCl treatment; sodium Dodecyl Sulfate (SDS) is an anionic surfactant, is commonly used in cosmetics, and the main blood vessels of chorioallantoic membrane after SDS treatment are gradually blurred, and partial fine blood vessels are completely eliminated; after SDS and phycocyanin fermentation liquor are jointly treated, blood vessels have no obvious change; the results show that: the phycocyanin fermentation broth prepared in example 1 can effectively eliminate or relieve the stimulation of chick chorioallantoic membrane caused by SDS, and can be used as a soothing component in cosmetics to eliminate or relieve the stimulation caused by surfactants.
The results of experiments on the blood vessels of the chick embryo chorioallantoic membrane of the phycocyanin fermentation broth obtained in examples 2 to 4 are substantially the same as the results of the experiments on the blood vessels of the chick embryo chorioallantoic membrane of the phycocyanin fermentation broth obtained in example 1.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. Application of bacillus in improving the antioxidant stability of phycocyanin.
2. A preparation method of phycocyanin fermentation liquid is characterized by comprising the following steps: mixing the culture medium and the bacterial liquid, and fermenting to obtain phycocyanin fermentation liquid; the culture medium contains phycocyanin extracting solution;
preferably, the medium further comprises a carbon source.
3. The method according to claim 2, wherein the final concentration of phycocyanin in the culture medium is 1 to 10 wt%;
preferably, the final concentration of the carbon source in the culture medium is 1 wt% to 8 wt%.
4. The method according to claim 2, wherein the inoculum size of the bacterial liquid is 1 v/v% to 8 v/v% of the culture medium;
preferably, the number of viable bacteria in the bacterial liquid is (1-9) multiplied by 106CFU/mL；
Preferably, the strain in the bacterial liquid is a high-yield protease strain;
preferably, the strain is at least one of bacillus amyloliquefaciens, bacillus pumilus, bacillus licheniformis and bacillus subtilis.
5. The preparation method according to any one of claims 2 to 4, wherein the fermentation is carried out at 30 to 40 ℃ for 20 to 25 hours.
6. The method according to any one of claims 2 to 4, further comprising the steps of: centrifuging, filtering and heating.
7. The method according to any one of claims 2 to 4, wherein the method for preparing the phycocyanin extract comprises: mixing spirulina with water, and crushing to obtain phycocyanin extractive solution;
preferably, the mass ratio of the spirulina to the water is (3-10): 50;
preferably, the preparation method of the phycocyanin extracting solution further comprises the following steps: and (4) centrifuging and sterilizing.
8. A phycocyanin fermentation broth, characterized by being produced by the production method according to any one of claims 2 to 7.
9. Use of the phycocyanin fermentation broth of claim 8 in a product;
the product is used for:
(1) relieving skin irritation; and/or
(2) Oxidation resistance; and/or
(3) Improving immunity.
10. A product comprising the phycocyanin fermented liquid of claim 8.
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