CN117462612A - Ginseng and cassia seed compound fermentation liquor and preparation process thereof - Google Patents
Ginseng and cassia seed compound fermentation liquor and preparation process thereof Download PDFInfo
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- CN117462612A CN117462612A CN202311424892.3A CN202311424892A CN117462612A CN 117462612 A CN117462612 A CN 117462612A CN 202311424892 A CN202311424892 A CN 202311424892A CN 117462612 A CN117462612 A CN 117462612A
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- ginseng
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- fermentation
- cassia seed
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Abstract
The invention belongs to the technical field of traditional Chinese medicine development, and particularly relates to a ginseng and cassia seed compound fermentation broth and a preparation process thereof. The ginseng and cassia seed compound fermentation liquor is prepared by taking a ginseng and cassia seed compound composition as a raw material and adopting strain fermentation; the raw materials of the ginseng and cassia seed compound composition comprise, by weight, 4 parts of cordyceps militaris, 1 part of ginseng, 4 parts of hawthorn, 4 parts of stir-fried cassia seed, 4 parts of medlar and 2 parts of chrysanthemum. The compound fermentation liquor prepared by the invention has the functions of enhancing immunity, nourishing yin and supplementing blood, and the like, the taste and the nutritional ingredients of the fermentation liquor are obviously improved, the absorption by human bodies is facilitated, and the compound fermentation liquor has important practical significance for developing Chinese medicine resources and improving economic value.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine development, and particularly relates to a ginseng and cassia seed compound fermentation broth and a preparation process thereof.
Background
In recent years, modern people often have accelerated life rhythm and increasingly vigorous competition, and sub-health people are increasingly huge along with the continuous increase of social relations and family pressure. Studies show that about 80% of the current population is in sub-health state. Sub-health status is divided into sub-health status of body, sub-health status of psychology, sub-health status of social interaction, etc. People in sub-health state, though not clear disease, have reduced mental activity and adaptability, if the state can not be corrected in time, the mental disease is very easy to cause.
The sub-health people are more and more, the health care products on the market have certain limitation, side effects can be brought to the body, and the large-multivalent price is expensive, so that additional economic burden is brought to the life of people. Compared with Western medicine, the traditional Chinese medicine has less adverse reaction. The Chinese herbal compound has various chemical components, has the characteristics of multiple targets, multiple layers and multiple curative effects, and can increase the activity of the Chinese herbal compound by correct compatibility. However, the traditional Chinese medicine decoction has large dosage, inconvenient carrying and poor sensory properties, and cannot be produced commercially. Therefore, it is necessary to develop a fermentation broth which is convenient to carry and has better sensory properties, is convenient for people to take for a long time, achieves the effect of enhancing immunity, improves sub-health, and has important practical significance for developing Chinese medicine resources and improving economic value.
Disclosure of Invention
Based on the problems, the invention provides a ginseng and cassia seed compound fermentation broth and a preparation process thereof.
The invention adopts the following technical scheme:
in a first aspect, the invention provides a ginseng and cassia seed compound fermentation liquor, which is prepared by taking a ginseng and cassia seed compound composition as a raw material and adopting strain fermentation; the raw materials of the ginseng and cassia seed compound composition comprise, by weight, 4 parts of cordyceps militaris, 1 part of ginseng, 4 parts of hawthorn, 4 parts of stir-fried cassia seed, 4 parts of medlar and 2 parts of chrysanthemum.
Preferably, the strain is a compound strain, wherein the compound strain comprises lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus bulgaricus, bifidobacterium and lactobacillus acidophilus, and the mass ratio of the lactobacillus plantarum to the lactobacillus rhamnosus is 1:1:1:1:1.
in the invention, ginseng and cordyceps militaris are monarch drugs, the ginseng has the effects of nourishing and strengthening, eliminating fatigue, tonifying qi and strengthening body, and the cordyceps militaris can tonify lung and kidney, tonify essence, invigorate spleen and tranquilize, and the two drugs can be used together to tonify qi and strengthen yang; fructus Lycii as ministerial drug, replenishing blood, nourishing yin, and nourishing liver and kidney; semen Cassiae, flos Chrysanthemi and fructus crataegi are used as adjuvant drugs for suppressing liver yang, loosening bowel to relieve constipation, removing liver heat and improving eyesight. The medicines are combined, qi and blood are concurrently nourished, yin and yang are co-supplemented, and balance is achieved.
In a second aspect, the invention also provides a preparation process of the ginseng and cassia seed compound fermentation broth, which comprises the following specific steps:
weighing dry and crushed compound ginseng and cassia seed composition powder according to a proportion, adding distilled water, adjusting the pH value to be between 5.5 and 6.0, adopting an isomaltooligosaccharide-crystalline fructose mixture to adjust the sugar content, adding a proper amount of peptone, sterilizing and cooling to obtain a fermentation substrate; and inoculating a composite strain into the fermentation substrate, performing anaerobic fermentation, and centrifuging to obtain fermentation liquor.
Preferably, the ginseng and cassia seed compound composition comprises the following components in percentage by mass: distilled water = 1:8.
preferably, the isomaltooligosaccharides, in weight ratio: crystalline fructose = 3:7, preparing a base material; the sugar content was 12%.
The isomaltooligosaccharide is safe and palatable, promotes the development of bifidobacteria in intestinal tracts, and improves the immunity of organisms; neither blood glucose nor blood insulin levels are altered; the crystalline fructose is easy to be absorbed and utilized by the organism, and can improve the gastrointestinal functions and metabolism of human beings. The mixed use of the isomaltooligosaccharide and the crystalline fructose is adopted to adjust the sugar content, so that the defects of relatively large single use amount and relatively high cost of the isomaltooligosaccharide can be avoided; in addition, the crystallized fructose has higher sweetness, and can improve the taste of the peracid of the fermentation liquor and the compliance of patients when being used together with the isomaltooligosaccharide, and can meet the requirements of people suffering from hyperglycemia, diabetes and the like.
Preferably, the volume mass ratio of the composite strain to the fermentation substrate is 3%.
Preferably, the anaerobic fermentation time is 72 hours and the fermentation temperature is 37 ℃.
Preferably, the fermentation broth is centrifuged at 3000r/min for 10min.
The fermentation liquor prepared by the invention can be prepared into drinks or oral liquid according to different technological means, and the description is omitted herein.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention takes six Chinese medicines of cordyceps militaris, ginseng, medlar, hawthorn, cassia seed and chrysanthemum as raw materials, optimizes the compatibility ratio, and tramples the concepts of 'food and medicine dual-purpose' and 'food and medical health preserving', thereby having higher safety; after fermentation, the active ingredients in the compound have synergistic effect, so that the fermentation liquid has the effects of enhancing immunity, resisting fatigue, improving sleep, tonifying qi, dredging collaterals, nourishing yin, supplementing blood, activating blood, removing blood stasis, reducing blood lipid, reducing blood pressure, strengthening spleen, regulating stomach, relaxing bowel, nourishing liver, improving eyesight and the like;
(2) The invention adopts the compound strain to ferment the ginseng and cassia seed compound composition, and has fast fermentation speed and short fermentation time; meanwhile, the components of the ginseng and cassia seed compound are improved, and the identification shows that the glycoside substances in the fermentation liquor are reduced, the macromolecular compounds are converted into micromolecular substances, the molecular weight is reduced, the micromolecular substances are easier to be absorbed by human bodies, and the drug property effect is exerted;
(3) The invention optimizes the conditions of the fermentation process, obviously improves the content of active ingredients and nutritional ingredients in the fermentation liquid after optimization, improves the taste and has wide applicable crowd.
Drawings
FIG. 1 is a flow chart of the compound fermentation of ginseng and cassia seed of the invention;
FIG. 2 is a chromatogram of a cordycepin control;
FIG. 3 is a chart of cordycepin of the unfermented semen Cassiae compound extract;
FIG. 4 is a chart of cordycepin of the compound fermentation broth of ginseng and cassia seed;
FIG. 5 shows the total flavonoids content change of the fermented and unfermented ginseng and cassia seed compound; wherein I is represented as unfermented;
FIG. 6 is a graph showing the change in total phenol content of the fermented and unfermented ginseng and cassia seed compound; wherein I is represented as unfermented;
FIG. 7 shows the change in total saponins content of the compound with fermented and unfermented ginseng and cassia seed; wherein I is represented as unfermented;
FIG. 8 shows the variation of the compound cordycepin content of the fermented and unfermented ginseng and cassia seeds; wherein I is represented as unfermented;
FIG. 9 shows the change in DPPH radical scavenging activity of the fermented and unfermented ginseng and cassia seed compounds; wherein I is represented as unfermented;
FIG. 10 shows the change in the scavenging activity of hydroxyl radicals of the compound ginseng and non-fermented cassia seed; wherein I is represented as unfermented;
FIG. 11 shows the change in the reducing power of the compound iron of fermented and unfermented ginseng cassia seeds; wherein I is represented as unfermented;
FIG. 12 is a TIC diagram of the mixed control solution and fermented and unfermented ginseng and cassia seed in compound positive and negative ion mode; A. c, E: a positive ion mode; B. d, F: a negative ion mode; A. b: mixing the reference substance solution; C. d: unfermented compound extract of ginseng and cassia seed; E. f: compound fermentation liquor of ginseng and cassia seed;
FIG. 13 is a HPLC chromatogram of a combination of the control solution and fermented and unfermented ginseng cassia seed; a: mixing a reference substance solution, B: unfermented compound extract of ginseng and cassia seed, C: a compound fermentation broth of semen Cassiae and radix Ginseng (1: cordycepin, 2: chlorogenic acid, 3: luteolin, 4: luteolin, 5: chrysophanol).
Detailed Description
The invention will be described in further detail with reference to specific embodiments and drawings.
Example 1
The ginseng and cassia seed compound fermentation liquor is prepared by taking a ginseng and cassia seed compound composition as a raw material and adopting strain fermentation; the raw materials of the ginseng and cassia seed compound composition comprise, by weight, 4 parts of cordyceps militaris, 1 part of ginseng, 4 parts of hawthorn, 4 parts of stir-fried cassia seed, 4 parts of medlar and 2 parts of chrysanthemum.
The strain is a compound strain, the compound strain comprises lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus bulgaricus, bifidobacterium and lactobacillus acidophilus, and the weight ratio of the lactobacillus plantarum to the lactobacillus rhamnosus is 1:1:1:1:1.
weighing 100g of dry and crushed ginseng and cassia seed compound composition powder respectively according to a proportion (sieving with a 60-mesh sieve), placing the powder into a 2000mL conical flask, and weighing the powder according to a proportion of 1:8 (M/M) in distilled water, adjusting pH to 5.5, and adjusting sugar content with isomaltooligosaccharide crystalline fructose mixture (isomaltooligosaccharide: crystalline fructose=3:7, W/W)Adding appropriate amount of peptone to 12%, sterilizing with high pressure steam at 121deg.C for 30min, and cooling to room temperature to obtain fermentation substrate; inoculating compound strain into fermentation substrate, fermenting at 37deg.C for 72 hr at 3000 r.min -1 Centrifuging at a speed of 10min, and keeping the supernatant, namely the fermentation broth.
EXAMPLE 2 screening of fermentation conditions
1 preparation of ginseng and cassia seed compound fermentation liquor
Weighing 100g of dried and crushed ginseng and cassia seed compound composition powder respectively according to a proportion (sieving with a 60-mesh sieve), placing the powder into a 2000mL conical flask, and weighing the powder according to a proportion of 1: 6. 1:8 (M/M) adding distilled water, regulating pH to 5.5-6.0, regulating sugar content to 12% and 16% by using a mixture of isomaltooligosaccharide and crystalline fructose, setting a blank group without independently regulating sugar content, adding a proper amount of nitrogen source peptone, sterilizing with high-pressure steam at 121 ℃ for 30min, cooling a conical flask to room temperature, respectively inoculating single strain lactobacillus plantarum and composite strain (lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus bulgaricus, bifidobacterium and lactobacillus acidophilus in a mass ratio of 1:1:1:1:1) according to inoculum size, performing anaerobic fermentation at 37 ℃ for 24h, 48h and 72h, and performing anaerobic fermentation at 3000 r.min -1 Centrifuging at a speed of 10min, and keeping the supernatant, namely the fermentation broth. The fermentation conditions are shown in Table 1, and the fermentation flow chart is shown in FIG. 1.
TABLE 1 fermentation conditions
2 preparation of unfermented ginseng and cassia seed compound extract
Taking 12.5g of ginseng and cassia seed compound composition powder, soaking the powder in a proper amount of distilled water, and performing ultrasonic extraction for 1h at 30 ℃ and the extracting solution is subjected to 3000 r.min -1 Centrifuging at a rotating speed of (2) for 10min, collecting supernatant, and storing at 4deg.C for use.
3 determination of cordycepin content
3.1 chromatographic conditions
Chromatographic column: agilent C18 column (4.6 mm. Times.250 mm,5 μm);a detector: an ultraviolet detector; mobile phase: water: methanol=85: 15, V/V; flow rate: 1 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature: 30 ℃; sample injection amount: 10. Mu.L; detection wavelength: 260nm.
3.2 preparation of control solution
Precisely weighing cordycepin reference substance to obtain a proper amount with concentration of 0.1000mg.mL -1 The control solution was stored at 4℃until use.
3.3 determination of content
Taking the fermentation liquor prepared in the item "1" and the unfermented extract prepared in the item "2" as sample solutions, respectively diluting, filtering with a 0.45 μm microporous filter membrane, taking the subsequent filtrate, and injecting samples. Analyzing cordycepin content in the ginseng and cassia seed compound composition before and after fermentation under different conditions.
3.4 linear relationship investigation
Precisely sucking 0.3mL, 0.5mL, 0.7mL, 0.9mL and 1.1mL of the reference substance solution under the item "3.2", respectively, placing into 5mL volumetric flasks, diluting to scale with methanol, and shaking to obtain a series of reference substance solutions; the assay was performed under the "3.1" term chromatographic conditions. Peak area (A) is taken as the ordinate, concentration (. Mu.g.mL) -1 ) For the abscissa, a regression equation was calculated, which was y= 46.54x-61.26, r=0.9995.
4 determination of total phenol content
Preparing gallic acid reference substance solutions with different concentrations, diluting to 5mL with distilled water, adding 2mL of Fu Lin Fen reagent, 3mL of 15% Na 2 CO 3 The solution reacts for 30min at room temperature in a dark place, the absorbance of the solution is measured at 760nm wavelength, and a standard curve is drawn according to the relation between different concentrations of gallic acid and absorbance values. The regression equation y=0.0385x+0.016 and r=0.9994 was established. The absorbance of the test solution is detected by the same method, and the total phenol content is calculated by using a regression equation.
5 determination of total flavone content
Preparing rutin reference substance solutions with different concentrations, and adding 0.16mL 5% NaNO respectively 2 The solution was reacted for 6min, and 0.16mL of 10% Al (NO) was added to each 3 ) 3 The solution was reacted for 6min, 2mL of 4% NaOH solution was added respectively, and the reaction was carried out for 15min, using 3And (3) determining the volume of 0% ethanol, taking distilled water as a blank, measuring the absorbance at the wavelength of 510nm, and drawing a standard curve according to the relation between different concentrations of rutin and absorbance values. The regression equation y=1.248 x+0.1159, r=0.9995 was established. The absorbance of the test solution is measured by the same method, and the total flavone content is calculated by using a regression equation.
6 determination of Total saponins content
The total saponin content was determined by vanillin color development. Placing ginsenoside Re reference substance solutions with different concentrations at 80 ℃ for volatilizing, respectively adding 0.2mL of 5% vanillin-glacial acetic acid solution and 0.8mL of perchloric acid solution, shaking uniformly, performing water bath reaction at 60 ℃ for 15min, stopping the reaction in an ice water bath, standing to room temperature, adding 5mL of glacial acetic acid solution, shaking uniformly, standing for 15min, taking absolute ethyl alcohol as a blank reference, and measuring the absorbance at 550nm wavelength. And drawing a standard curve according to the relation between different concentrations of ginsenoside Re and absorbance values. Regression equation y=8.8342x+0.0242 was established, r=0.9994. And (3) measuring the sample solution according to the method, and calculating the total saponin content by using a regression equation.
7 antioxidant Activity assay
7.1DPPH radical scavenging Activity
Taking 0.02mL of diluted unfermented ginseng and cassia seed compound extract and 0.2mL of 0.2 mmol.L -1 The DPPH absolute ethanol solution of (2) is uniformly mixed, is subjected to dark treatment at room temperature for 30min, and the absorbance A is measured at 517nm wavelength i The absolute ethyl alcohol is used to replace DPPH absolute ethyl alcohol solution, and the absorbance A is measured j Measuring absorbance A with ethanol instead of the sample c Using VC as a reference, drawing a standard curve according to the relation between different concentrations of VC and clearance, and calculating the DPPH free radical clearance according to the following formula:
regression equations were established for y=5.5943x+0.0473, r= 0.9996. The absorbance of the test solution is measured by the same method, and the DPPH free radical clearance of the test solution is calculated by using a regression equation.
7.2 hydroxy radical scavenging Activity
Taking 6 mmol.L-1 FeSO 4 Solution and 6 mmol.L-1H 2 O 2 Mixing 0.3mL each in a test tube for 10min, adding 0.1mL of diluted unfermented ginseng and cassia seed compound extract, reacting for 10min, and adding 0.3mL of 6mmol.L -1 Is incubated in the dark for 30min, and its absorbance A is measured at a wavelength of 510nm 1 Measuring absorbance A with distilled water instead of salicylic acid solution 2 Measuring absorbance A with distilled water instead of sample 0 Using VC as a control, a standard curve was drawn according to the relationship of different concentrations of VC versus the clearance, and the hydroxyl radical clearance was calculated according to the following formula:
the regression equation y= 5.8688x-0.0299, r=0.9997 was established. The absorbance of the test solution is measured by the same method, and the hydroxyl radical clearance of the test solution is calculated by using a regression equation.
7.3 Total antioxidant Capacity (FRAP)
Taking 0.6mL of FeSO with different concentrations 4 Adding 6mL FAP solution into the reference solution, mixing thoroughly, reacting for 10min, measuring absorbance at 593nm, and measuring FeSO 4 A standard curve is drawn from the relationship between the different concentration-absorbance values. Regression equation y=0.8425x+0.0245, r=0.9994 was established according to the relationship between different concentration-absorbance values of FeSO 4. And measuring the absorbance of the test solution under the same method, and calculating the FRAP value of the test solution by using a regression equation. FRAP value of sample and FeSO per gram of dry sample extracted 4 The mmol numbers are equal.
8 statistical analysis of data
The test results were expressed as mean ± standard deviation [ (m ]n=3) represents. Analysis of significance between quantum sample data using SPSS22.0 statistical software independent sample T-test, significance between multiple sets of sample data for one-way analysis of variance (ANOVA, one-way analysis ofvariance), p<0.05 indicates the presence ofSignificant differences. The content of main active components and the antioxidant activity are used as indexes, and the original data are standardized first and then are subjected to main component analysis.
9 results
Fig. 2 is a cordycepin reference chromatogram, fig. 3 is a cordycepin chromatogram of an unfermented ginseng and cassia seed compound extract, fig. 4 is a cordycepin chromatogram of a ginseng and cassia seed compound fermentation liquid, and the content of main active ingredients and the antioxidant activity of the ginseng and cassia seed compound under different fermentation conditions are shown in fig. 5-11.
10 principal component analysis method for screening optimal fermentation process
The main component analysis is carried out on 7 indexes of total phenol, total flavone, total saponin, cordycepin content, hydroxyl radical scavenging activity, DPPH radical scavenging activity and FRAP iron ion reducing capability of the unfermented compound extract of the ginseng and cassia seed and the compound fermentation liquid of the ginseng and the cassia seed by using SPSS22.0, the results are shown in a table 2, and the main component scores and the comprehensive scores are shown in a table 3.
TABLE 2 component eigenvalues, variance contribution rates, and cumulative variance contribution rates
TABLE 3 principal component score and composite score
Conclusion: 72h-7 fermentation sample, the comprehensive score is highest, and the fermentation effect is best, namely: the feed liquid ratio is 1:8, the sugar degree is 12%, the strain is a composite strain, and the fermentation time is 72h as the optimal fermentation condition.
Example 3 qualitative study of active ingredients before and after Compound fermentation of Ginseng semen Cassiae
1 preparation of ginseng and cassia seed compound fermentation liquor
The experimental procedure is the same as in example 1.
2 preparation of unfermented ginseng and cassia seed compound extract
The experimental procedure is the same as in example 2.
3 preparation of the Mixed control solution
Precisely weighing 1.41mg of chrysophanol, 1.16mg of luteolin, 1.61mg of oleanolic acid, 1.98mg of adenine, 1.30mg of adenosine, 1.34mg of aurantium, 1.71mg of ergosterol, 1.44mg of chlorogenic acid, 1.22mg of betaine and Rg of ginsenoside 1 1.40 mg, ginsenoside Rb 1 1.15 The reference substances of mg, mannitol 1.99mg, (+) -catechin 1.09mg, ginsenoside Re 0.95mg and citric acid (citric acid) 1.93mg are placed in a 25mL volumetric flask, and the mixed reference substance solution is obtained after the reference substances are dissolved completely by ultrasound after chromatographic methanol is fixed to volume to scale.
4 chromatographic conditions
Chromatographic column: agilent Eclipse XDB-C18 column (2.1 mm. Times.150 mm,3.5 μm); 0.03% formic acid aqueous solution (A) -0.03% formic acid methanol (B) as mobile phase according to 0.3 mL.min -1 The flow rate gradient elution of (2) is shown in table 4; sample injection amount: 5. Mu.L; column temperature: 25 ℃; detection wavelength: 200nm, 230nm, 260nm, 290nm and 337nm.
TABLE 4 gradient of mobile phase elution
5 Mass Spectrometry conditions
Electrospray ionization sources (ESI) were detected in positive and negative ion modes, respectively. Capillary voltage: 3500V, atomizing gas pressure: 0.24MPa, dry gas flow rate: 8L min -1 Desolventizing gas temperature: 325 ℃, fragmentation voltage: 175V, mass spectral scan range m/z:50-2000.
6 identification of chemical Components
The chemical components of the existing reference substance are confirmed by comparing with the accurate mass-to-charge ratio, the information of the secondary fragment ions, the retention time and the like of the excimer ions of the reference substance.
Extracting total ion flow diagrams (TICs) collected in positive and negative ion modes according to an established compound chemical composition database of the ginseng and cassia seeds by using Agilent Masshunter Qualitative Analysis analysis software, and determining the molecular formula of the compound under the condition that the deviation delta is less than 10ppm (the deviation delta= (actual value-theoretical value)/theoretical value) according to the comparison of the mass-to-charge ratio of the excimer ions and the theoretical molecular weight; and identifying the compound according to the secondary fragment ion information.
Results 7 results
The total ion flow diagram is shown in figure 12, 176 chemical components are identified in the unfermented compound extract of the ginseng and cassia seed, and 135 chemical components are identified in the compound fermentation liquid of the ginseng and cassia seed. The components of the ginseng and cassia seed compound before and after fermentation are obviously different, and the reduction of glycoside substances in the ginseng and cassia seed compound fermentation liquid can be obviously found, which indicates that the fermentation leads to the cleavage of glycosidic bonds, so that macromolecular compounds in the traditional Chinese medicine are converted into micromolecular substances, and are easier to be absorbed by human bodies, and the drug property is exerted.
Example 4 quantitative investigation of five active ingredients before and after Compound fermentation of Ginseng semen Cassiae
1 preparation of ginseng and cassia seed compound fermentation liquor
The experimental procedure is the same as in example 1.
2 preparation of unfermented ginseng and cassia seed compound extract
The experimental procedure is the same as in example 2.
3 chromatographic conditions
Chromatographic column: agilent ZORBAX Eclipse XDB-C18 (250 mm. Times.4.6 mm,5 μm); acetonitrile (A) -0.2% phosphoric acid aqueous solution (B) is taken as mobile phase according to the ratio of 1.0 mL.min -1 The flow rate gradient elution of (2) is shown in table 5; sample injection amount: 10. Mu.L; column temperature: 25 ℃; detection wavelength: 295nm.
TABLE 5 gradient of mobile phase elution
4 preparation of the Mixed control solution
Accurately weighing cordycepin, chlorogenic acid, luteolin and chrysophanol reference substances respectively, and making into dosage forms with concentration of 0.05 mg/mL respectively -1 、0.05mg·mL -1 、0.05mg·mL -1 、0.025mg·mL -1 、0.0125mg·mL -1 Is stored at 4 ℃ for standby.
5 content determination
Taking the fermentation liquor prepared in the item "1" and the unfermented extract prepared in the item "2" as test sample solutions, filtering with a 0.45 μm microporous filter membrane, taking the subsequent filtrate, and sampling. Analyzing the content of the main active ingredients.
6 linear relationship investigation
Accurately sucking the mixed reference substance solutions 1, 2, 4, 6, 8 and 10mL under item 4 respectively in a 10mL volumetric flask, fixing the volume to the scale with methanol, and shaking uniformly to obtain a series of reference substance solutions. The assay was performed under "3" chromatographic conditions. Peak area (A) is taken as the ordinate, concentration (mg.mL) -1 ) For the abscissa, a regression equation was calculated, and the results are shown in Table 6.
Table 65 linear equations, correlation coefficients and linear ranges of the components
Results 7 results
HPLC chromatograms are shown in FIG. 13 and the measurement results are shown in Table 7. The chemical components in the ginseng and cassia seed compound before and after fermentation have obvious difference, the content of cordycepin and luteolin is obviously increased, and the difference before and after fermentation is 0.0822,0.1273 respectively; the content of luteolin is obviously reduced, and the difference value before and after fermentation is 0.0411. The content of chlorogenic acid is slightly reduced, and the difference value before and after fermentation is 0.0090; the content of chrysophanol has no obvious change, and the difference value between the chrysophanol before and after fermentation is 0.0027. The reduced amount of luteolin and the increased amount of luteolin indicate that fermentation not only causes cleavage of glycosidic bonds in luteolin to produce luteolin, but also causes conversion between compounds. Meanwhile, the content of cordycepin is increased after fermentation, which indicates that a certain conversion relationship exists between cordycepin and adenosine. The content of chlorogenic acid after fermentation is slightly reduced, and it is presumed that chlorogenic acid undergoes an enzymatic hydrolysis reaction in the fermentation process to cause ester bond cleavage, and caffeic acid or quinic acid is generated, and further conversion of caffeic acid occurs to produce 3-hydroxy phenylpropionic acid, p-coumaric acid, ferulic acid, and the like. Because the solubility of the chrysophanol in water is smaller, the chrysophanol content in the solution is smaller, and the content change before and after fermentation is not obvious. Experimental results further show that the fermentation can convert the components in the compound; not only can lead to the cleavage of glycosidic bonds of chemical components, but also can reduce the molecular weight and convert the glycosidic bonds into small molecular substances which are easier to digest and absorb by human bodies.
TABLE 7 determination of the 5 ingredients content of the ginseng and cassia seed Compound (mg.g) -1 ，n＝3)
The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.
Claims (10)
1. The ginseng and cassia seed compound fermentation liquor is characterized by being prepared by taking a ginseng and cassia seed compound composition as a raw material and adopting strain fermentation; the raw materials of the ginseng and cassia seed compound composition comprise, by weight, 4 parts of cordyceps militaris, 1 part of ginseng, 4 parts of hawthorn, 4 parts of stir-fried cassia seed, 4 parts of medlar and 2 parts of chrysanthemum.
2. The compound fermentation broth of the ginseng and cassia seed according to claim 1, wherein the strain is a compound strain, the compound strain comprises lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus bulgaricus, bifidobacterium and lactobacillus acidophilus, and the mass ratio is 1:1:1:1:1.
3. the preparation process of the ginseng and cassia seed compound fermentation broth disclosed in claim 1 is characterized by comprising the following specific steps:
weighing dry and crushed compound ginseng and cassia seed composition powder according to a proportion, adding distilled water, adjusting the pH value to be between 5.5 and 6.0, adopting an isomaltooligosaccharide-crystalline fructose mixture to adjust the sugar content, adding a proper amount of peptone, sterilizing and cooling to obtain a fermentation substrate; and inoculating a composite strain into the fermentation substrate, performing anaerobic fermentation, and centrifuging to obtain fermentation liquor.
4. The process for preparing the compound fermentation broth of the ginseng and the cassia seeds according to claim 3, which is characterized by comprising the following steps of: distilled water = 1:8.
5. the process for preparing the compound fermentation broth of the ginseng and the cassia seeds according to claim 3, which is characterized by comprising the following steps of: crystalline fructose = 3:7, preparing a base material; the sugar content was 12%.
6. The process for preparing the compound fermentation broth of ginseng and cassia seeds according to claim 3, wherein the volume-mass ratio of the compound strain to the fermentation substrate is 3%.
7. The process for preparing the compound fermentation broth of the ginseng and the cassia seeds according to claim 3, wherein the anaerobic fermentation time is 72 hours, and the fermentation temperature is 37 ℃.
8. The fermentation process of the compound fermentation broth of ginseng and cassia seeds according to claim 3, wherein the centrifugation speed is 3000r/min for 10min.
9. The process for compound fermentation of ginseng and cassia seed according to claim 3, further comprising a compound fermentation liquidQualitative detection of the active ingredient in (a); the specific chromatographic conditions are as follows: chromatographic column: agilent Eclipse XDB-C18 column, 2.1 mm. Times.150 mm,3.5 μm; mobile phase a was 0.03% formic acid in water and mobile phase B was 0.03% methanol formate, flow rate: 0.3mL min -1 Binary linear elution; sample injection amount: 5. Mu.L; column temperature: 25 ℃; detection wavelength: 200nm, 230nm, 260nm, 290nm and 337nm; mass spectrometry conditions: electrospray ionization sources for detection in positive and negative ion modes respectively; capillary voltage: 3500V, atomizing gas pressure: 0.24MPa, dry gas flow rate: 8L min -1 Desolventizing gas temperature: 325 ℃, fragmentation voltage: 175V, mass spectral scan range m/z:50-2000.
10. The compound fermentation process of ginseng and cassia seed according to claim 3, further comprising quantitative detection of active ingredients in the compound fermentation liquid, wherein the active ingredients are cordycepin, chlorogenic acid, luteolin and chrysophanol; the specific chromatographic conditions are as follows: chromatographic column: agilent ZORBAX Eclipse XDB-C18, 250 mm. Times.4.6 mm,5 μm; the mobile phase A is acetonitrile, the mobile phase B is 0.2% phosphoric acid aqueous solution, and binary linear elution is carried out; flow rate: 1.0 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Sample injection amount: 10. Mu.L; column temperature: 25 ℃; detection wavelength: 295nm.
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