CN109247581B - Application of cordyceps sinensis extracellular polysaccharide in probiotic health food and/or probiotic traditional Chinese medicine - Google Patents
Application of cordyceps sinensis extracellular polysaccharide in probiotic health food and/or probiotic traditional Chinese medicine Download PDFInfo
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- CN109247581B CN109247581B CN201710576108.9A CN201710576108A CN109247581B CN 109247581 B CN109247581 B CN 109247581B CN 201710576108 A CN201710576108 A CN 201710576108A CN 109247581 B CN109247581 B CN 109247581B
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 37
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- 230000000529 probiotic effect Effects 0.000 title claims abstract description 23
- 239000003814 drug Substances 0.000 title claims abstract description 8
- 235000013402 health food Nutrition 0.000 title claims abstract description 5
- 239000000463 material Substances 0.000 claims abstract description 8
- 230000001737 promoting effect Effects 0.000 claims abstract description 6
- 230000035899 viability Effects 0.000 claims abstract description 6
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 15
- 238000009630 liquid culture Methods 0.000 claims description 14
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- 238000005119 centrifugation Methods 0.000 claims description 11
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- 238000004108 freeze drying Methods 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
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- 239000007788 liquid Substances 0.000 claims description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 241000186660 Lactobacillus Species 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 229940039696 lactobacillus Drugs 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
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- 241000186000 Bifidobacterium Species 0.000 description 8
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
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- 241000233866 Fungi Species 0.000 description 2
- 230000009858 acid secretion Effects 0.000 description 2
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- 239000002609 medium Substances 0.000 description 2
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- 238000004321 preservation Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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- 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
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- 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
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention belongs to the technical field of polysaccharide, and particularly relates to application of cordyceps sinensis extracellular polysaccharide as an auxiliary material for promoting growth of probiotics and/or an auxiliary material for protecting viability of the probiotics in probiotics health food and/or probiotics traditional Chinese medicine. Experiments prove that the cordyceps sinensis extracellular polysaccharide can promote the growth of probiotics and protect the survival activity of the probiotics, so that the cordyceps sinensis extracellular polysaccharide has wide application in the production fields of probiotic traditional Chinese medicines, probiotic health-care foods and the like, and the use value of the probiotics can be obviously improved.
Description
Technical Field
The invention belongs to the technical field of polysaccharide, and particularly relates to application of cordyceps sinensis extracellular polysaccharide in probiotic health-care food and/or traditional Chinese medicine.
Background
Cordyceps sinensis (Berk.) Sace is a medicinal fungus of Clavicipiraceae (Clavicipiraceae) Cordyceps (Cordyceps), is a famous and precious Chinese medicinal material and a traditional nourishing medicine. Cordyceps sinensis contains abundant physiologically active substances, and has unique medicinal value and wide pharmacological action, wherein polysaccharide is one of the main active ingredients.
Because of strict requirements on parasitic and special ecological environments of natural cordyceps sinensis, the resources of the natural cordyceps sinensis are extremely limited, the price of the natural cordyceps sinensis is high, in recent years, with the success of cordyceps sinensis anamorph research and the continuous development of artificial fermentation culture, research on cordyceps sinensis polysaccharide is gradually increased, but documents and patent reports on ultrasonic degradation of cordyceps sinensis extracellular polysaccharide and application of the cordyceps sinensis extracellular polysaccharide in promoting growth of probiotics are not provided.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides the application of cordyceps sinensis extracellular polysaccharide as an auxiliary material for promoting the growth of probiotics and/or an auxiliary material for protecting the survival activity of the probiotics in probiotic health-care food and/or probiotic traditional Chinese medicines, and aims to solve the technical problem that the application of the existing cordyceps sinensis extracellular polysaccharide is limited.
In order to achieve the purpose, the invention adopts the following technical scheme:
application of Cordyceps sinensis extracellular polysaccharide as adjuvant for promoting growth of probiotic bacteria and/or protecting viability of probiotic bacteria in probiotic bacteria health food and/or probiotic bacteria traditional Chinese medicine is provided.
Experiments prove that the cordyceps sinensis extracellular polysaccharide can promote the growth of probiotics and protect the survival activity of the probiotics, so that the cordyceps sinensis extracellular polysaccharide has wide application in the production fields of probiotic traditional Chinese medicines, probiotic health-care foods and the like, and the use value of the probiotics can be obviously improved.
Drawings
Fig. 1 shows the effect of different carbon sources on the growth of b. adolescentis in example 2 of the present invention;
fig. 2 shows the effect of different carbon sources on the growth of b.bifidum in example 2 of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides application of cordyceps sinensis extracellular polysaccharide as an auxiliary material for promoting growth of probiotics and/or an auxiliary material for protecting viability of the probiotics in probiotics health-care food and/or probiotics traditional Chinese medicine.
Experiments prove that the cordyceps sinensis extracellular polysaccharide can promote the growth of probiotics and protect the survival activity of the probiotics, so that the cordyceps sinensis extracellular polysaccharide has wide application in the production fields of probiotic traditional Chinese medicines, probiotic health-care foods and the like, and the value of the probiotics can be obviously improved.
Further, in the above use, the probiotic is a bifidobacterium and/or a lactobacillus. In one embodiment of the invention, the bifidobacterium may be b.adolescentis, b.bifidum, b.breve, b.infarnntis and b.longum.
Further, in the above application, the preparation process of the cordyceps sinensis exopolysaccharide comprises the following steps:
s01: providing cordyceps sinensis mycelia and an autoclaved mycelia liquid culture medium;
s02: fermenting and culturing the cordyceps sinensis mycelia with a mycelium liquid culture medium to obtain a fermentation liquid;
s03: performing first centrifugation treatment on the fermentation liquor, collecting supernatant, and sequentially performing first alcohol precipitation treatment and second centrifugation treatment on the supernatant to obtain a precipitate;
s04: freeze drying the precipitate to obtain Cordyceps extracellular polysaccharide.
Further, in step S01, the mycelium liquid culture medium includes the following components: 30-40g/L of glucose, 5g/L of peptone, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate and 10g/L of yeast extract. Among these, glucose is more preferably 40 g/L. The mycelium culture medium is suitable for fermenting Cordyceps sinensis mycelium.
Further, in step S02, the fermentation process includes: inoculating Cordyceps sinensis mycelia in 50mL of mycelia liquid culture medium, shaking at 20-25 deg.C and 100-200rpm for 5-7d, adding into 200mL of mycelia liquid culture medium, and shaking at 20-25 deg.C and 100-200rpm for 5-7 d. The optimized fermentation process can be more effectively beneficial to the fermentation of cordyceps sinensis mycelia to produce cordyceps sinensis exopolysaccharides with high yield.
Further, in step S03, the conditions of the first centrifugation processing are: the centrifugal force is 4000-; the conditions of the second centrifugation treatment were: the centrifugal force is 10000-. The two times of centrifugal treatment are more beneficial to the separation and precipitation of cordyceps sinensis extracellular polysaccharide.
Further, in step S03, the first alcohol precipitation process includes: mixing the supernatant with 90-95 vol% ethanol solution at a volume ratio of 1 (4-5), and standing at 4 deg.C for 12-24 hr. The alcohol precipitation treatment process is more beneficial to removing impurities from the cordyceps sinensis extracellular polysaccharide precipitate and improving the purity of the product.
Further, step S04, before freeze-drying, further comprises the following steps: dissolving the precipitate in water to obtain polysaccharide solution with concentration of 8-12g/L, performing ultrasonic treatment for 20-60min, and sequentially performing second precipitation treatment and third centrifugation treatment. The ultrasonic treatment can break the covalent bond in the cordyceps sinensis exopolysaccharide, so that the cordyceps sinensis exopolysaccharide is degraded, and the degradation effect is obvious. Tests prove that the degraded cordyceps sinensis extracellular polysaccharide obtained by ultrasonic treatment can more obviously promote the growth of probiotics and/or more obviously protect the viability of the probiotics. In one embodiment of the invention, the ultrasonic treatment time is preferably 30 min.
Further, the ultrasonic treatment conditions are as follows: in a 750W ultrasonic disruptor, the amplitude is 30-80%. Under the condition, the ultrasonic treatment effect is optimal.
Further, the second elution process includes: mixing the polysaccharide solution after ultrasonic treatment with an ethanol solution with the volume fraction of 90-95% according to the volume ratio of 1:5, and standing for 12-24h at the temperature of 4 ℃; the conditions of the third centrifugation treatment were: the centrifugal force is 10000-. And after the third alcohol precipitation treatment and the third centrifugal treatment, obtaining pure cordyceps sinensis extracellular polysaccharide precipitate from the polysaccharide solution after the ultrasonic treatment.
The invention is described in further detail with reference to a part of the test results, which are described in detail below with reference to specific examples.
Example 1
Collecting fresh wild Cordyceps at Cordyceps producing area (Sichuan Tibetan plateau) at the beginning of 6 months, washing, sterilizing, separating from fruiting body, purifying to obtain fungus, and identifying to Cordyceps related bacteria by morphology and gene. The strain separation and mycelium preservation are carried out by adopting PDA solid culture medium at the temperature of 20 ℃ (long-term preservation temperature is 4 ℃).
Cordyceps sinensis strain preserved in solid PDA culture medium obtained from wild Cordyceps sinensis is inoculated into liquid culture medium for mycelium fermentation culture. The liquid culture medium comprises the following components: 40g/L glucose, 5g/L peptone, 1g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate and 10g/L yeast extract; packaging into 250mL triangular bottles, sterilizing at high pressure (121 deg.C, 20min) 50mL each bottle, cooling to room temperature, inoculating solid cultured Cordyceps mycelium, placing the culture solution in shaking table, culturing at 20 deg.C and 150rpm, and fermenting for 7 days. Then inoculating the fermented Cordyceps sinensis mycelia into 1000mL triangular flask (containing 200mL culture medium), and continuously fermenting and culturing for 7 days at 20 deg.C and 200rpm shaking table. And after fermentation is finished, centrifuging the fermentation liquor (6000g, 10min), collecting supernatant, adding 5 times of ethanol (volume fraction 95%) for precipitation, standing at 4 ℃ overnight, centrifuging (10000g, 10min), collecting precipitate, and freeze-drying to obtain the cordyceps sinensis Extracellular Polysaccharide (EPS). Weighing a certain amount of cordyceps sinensis extracellular polysaccharide, dissolving, and treating by using a 750W ultrasonic crusher, wherein the ultrasonic intensity is fixed at 30% of amplitude, and the ultrasonic time is 30 min. Adding 5 times volume of ethanol solution into the polysaccharide solution after ultrasonic treatment, standing at 4 deg.C for 24 hr, centrifuging to collect precipitate, and freeze drying to obtain Cordyceps extracellular polysaccharide (EPS-US) after ultrasonic treatment.
Example 2
EPS-US instead of glucose (Glc) or galacto-oligosaccharide (GOS) is added into a liquid culture medium of enhanced clostridium-RCM with the concentration of 5g/L, autoclaved (121 ℃, 20min), inoculated into fermentation liquor of bifidobacterium B.adolescents and bifidobacterium B.bifidum cultured in RCM for 48 hours according to the volume fraction of 1 percent, cultured for 48 hours under the conditions of 37 ℃, anaerobic environment and 200rpm, and then the fermentation liquor is subjected to test analysis. The results of increasing the OD concentration (600nm) of bacteria in the EPS-US group compared to the control group without a carbon source (control) are shown in FIGS. 1 and 2. In fig. 1 and 2, Control group: control without any carbon source added; glc group: taking glucose Glc as a carbon source; group GOS: using galactooligosaccharide GOS as a carbon source; EPS-US group: EPS-US is taken as a carbon source; compared with the control group, the data indicates significant difference, and P is less than 0.05. As can be seen from fig. 1 and 2, the OD value of the bacterial concentration was increased in the EPS-US group, which promoted the growth of bifidobacterium b.
The pH and acetic acid secretion results for bifidobacterium b. adolescentis broth and bifidobacterium b. bifidum broth are shown in table 1. In table 1, Control group: control without any carbon source; glc group: taking glucose as a carbon source; group GOS: galactose oligosaccharide is used as a carbon source; EPS-US group uses Cordyceps sinensis extracellular polysaccharide degraded by ultrasound as carbon source. And the initial pH value of the fermentation liquor is 6.6-6.8; significant differences (P <0.05) and very significant differences (P <0.01) were indicated compared to the control group. As is clear from Table 1, the pH of the fermentation broth was decreased and the acetic acid secretion was increased in the EPS-US group as compared with the control group (control group) having no carbon source.
TABLE 1
Example 3
Adding EPS-US instead of glucose as carbon source into reinforced Clostridium-RCM liquid culture medium at concentration of 5g/L, autoclaving (121 deg.C, 20min), inoculating 1% by volume of Bacillus bifidus B.adolescents, B.bifidum, B.breve, B.infarnitis and B.longum fermentation broth cultured in RCM for 48 hr, culturing at 37 deg.C, anaerobic environment, 200rpm for 20 hr, 48 hr or 6 days, taking out the fermentation broth, and diluting to different concentrations (10 deg.C, 200 rpm)-1,10-2,10-3…10-10) And inoculated on RCM solid medium and cultured for 2 days, and the results are shown in Table 2. Table 2 shows the effect of Cordyceps sinensis exopolysaccharide and other carbon sources on Bifidobacterium solid culture Colony Forming Unit (CFU) (. times.10)8mL, n ═ 3), where, Control group: control without any carbon source; glc group: taking glucose as a carbon source; group GOS: galactose oligosaccharide is used as a carbon source; EPS-US group: the cordyceps sinensis extracellular polysaccharide subjected to ultrasonic degradation is used as a carbon source.
As can be seen from Table 2, the colony Forming Unit CFU values of the EPS-US group were significantly higher than those of the Control group and the Glc and GOS groups without carbon source for the 5 bifidobacteria tested during all three incubation periods. More importantly, bifidobacteria from the control and other carbon-source groups were transferred to solid medium after 48h of culture and essentially no longer grew (CFU value trended towards 0); and the bifidobacterium using EPS-US as a carbon source still has colony forming CFU (circulating fluid Unit) of more than 0 in the other four strains except B.breve strain even after the liquid fermentation time is 6 days. This experiment shows that EPS-US can promote the growth of probiotics and protect the viability of probiotics.
TABLE 2
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. Application of Cordyceps sinensis extracellular polysaccharide in probiotic health food and/or probiotic traditional Chinese medicine as auxiliary material for promoting growth of probiotic and protecting viability of probiotic, wherein the probiotic is Bacillus bifidus and Lactobacillus.
2. The use of claim 1, wherein the preparation process of the cordyceps sinensis exopolysaccharide comprises the following steps:
providing cordyceps sinensis mycelia and an autoclaved mycelia liquid culture medium;
fermenting and culturing the cordyceps sinensis mycelia with the mycelium liquid culture medium to obtain a fermentation liquid;
performing first centrifugation treatment on the fermentation liquor, collecting supernatant, and sequentially performing first alcohol precipitation treatment and second centrifugation treatment on the supernatant to obtain a precipitate;
and freeze-drying the precipitate to obtain the cordyceps sinensis exopolysaccharide.
3. The use according to claim 2, wherein the mycelium liquid culture medium comprises the following components: 30-40g/L of glucose, 5g/L of peptone, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate and 10g/L of yeast extract.
4. The use according to claim 3, wherein the fermentation culture is carried out by: inoculating the cordyceps sinensis mycelia in 50mL of the mycelia liquid culture medium, shaking for 5-7d under the conditions that the temperature is 20-25 ℃ and the rotating speed is 100-200rpm, then adding the cordyceps sinensis mycelia into 200mL of the mycelia liquid culture medium, and shaking for 5-7d under the conditions that the temperature is 20-25 ℃ and the rotating speed is 100-200 rpm.
5. Use according to claim 2, wherein the conditions of the first centrifugation treatment are: the centrifugal force is 4000-; and/or
The conditions of the second centrifugation treatment are as follows: the centrifugal force is 10000-.
6. The use according to claim 2, wherein the first alcohol precipitation treatment is carried out by: mixing the supernatant with 90-95% ethanol solution by volume ratio of 1 (4-5), and standing at 4 deg.C for 12-24 h.
7. The use according to any one of claims 2 to 6, further comprising, prior to said freeze-drying, the steps of: dissolving the precipitate in water to obtain polysaccharide solution with concentration of 8-12g/L, performing ultrasonic treatment for 20-60min, and sequentially performing second chromatography treatment and third centrifugation treatment.
8. The use of claim 7, wherein the sonication conditions are: in a 750W ultrasonic disruptor, the amplitude is 30-80%.
9. The use of claim 7, wherein the second elution process is carried out by: mixing the polysaccharide solution after ultrasonic treatment with an ethanol solution with the volume fraction of 90-95% according to the volume ratio of 1:5, and standing for 12-24h at the temperature of 4 ℃; and/or
The conditions of the third centrifugal treatment are as follows: the centrifugal force is 10000-.
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