CN111088298B - Method for improving content of active ingredients of anoectochilus formosanus by utilizing probiotic fermentation - Google Patents
Method for improving content of active ingredients of anoectochilus formosanus by utilizing probiotic fermentation Download PDFInfo
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Abstract
The invention discloses a method for improving the content of active ingredients of anoectochilus formosanus by utilizing probiotic fermentation, which comprises the following steps: (1) preparing a seed solution: respectively inoculating the activated bacillus coagulans and the activated lactic acid bacteria to corresponding liquid culture media, and culturing under appropriate conditions to prepare seed liquid; (2) raw material treatment: firstly, carrying out low-temperature vacuum drying treatment on the anoectochilus formosanus medicinal material, and then carrying out superfine grinding treatment; (3) and (3) performing segmented fermentation on the composite strains: preparing a fermentation medium containing the anoectochilus roxburghii superfine powder, inoculating bacillus coagulans for fermentation, and inoculating lactobacillus for fermentation; (4) after the fermentation is completed, the obtained fermentation product is freeze-dried. The invention comprehensively utilizes the superfine grinding technology and the microbial fermentation technology, utilizes the bacillus coagulans and the lactobacillus to perform segmented fermentation to release the main active ingredients in the anoectochilus formosanus to a greater extent, and obviously improves the contents of the glycoside, flavone and polysaccharide of the anoectochilus formosanus after fermentation.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for improving the content of active ingredients of anoectochilus formosanus by utilizing probiotic fermentation.
Background
There are about 23 Orchidaceae orceae labiate (Anoectochilus BI.) in China, and some herbs are used as medicinal herbs in folk, which are rare and rare Chinese herbal medicines known as Anoectochilus roxburghii, also known as Anoectochilus roxburghii, black ginseng, etc. Anoectochilus roxburghii (Wall.) Lindl, Arctochilus formosanus Hayata, Arctochilus isodesmus Hayata and Anoectochilus yunnanensis Hayata are used as main primordial plants of Anoectochilus roxburghii (A. roxburghii) Lindl, and Anoectochilus formosanus Hayata and Anoectochilus yunnanensis (A. chapatensis) as medicinal materials of Anoectochilus roxburghii. Anoectochilus roxburghii is distributed in tropical and subtropical regions of Asia, mainly China, Japan, India, Srilanka, Nepal and southeast Asia, and is mainly distributed in Fujian, Taiwan, Zhejiang, Guangdong, Guangxi, Hainan and the like in China. Anoectochilus roxburghii has reputations of king and golden grass in folk, and the drug effect of Anoectochilus roxburghii is recorded in a plurality of traditional documents, for example, the Anoectochilus roxburghii is sweet in flavor and mild in nature, has the effects of clearing heat, cooling blood, removing dampness and detoxifying, and is used for treating symptoms such as pulmonary tuberculosis hemoptysis, diabetes, nephritis, cystitis, myasthenia gravis, rheumatic and rheumatoid arthritis, venomous snake bite and the like; zhenan materia medica new edition carries that the medicine has light taste, mild nature, wind-damp expelling, tendon relaxing and collateral relaxing and is used for treating the rheumatic arthritis; anoectochilus formosanus from Fujian Yaozhi journal has effects of clearing heat, cooling blood, dispelling pathogenic wind and promoting diuresis, and can be used for treating hemoptysis, bronchitis, nephritis, cystitis, diabetes, chyluria, hematuria, rheumarthritis, infantile acute infantile convulsion, venomous snake bite, etc. Anoectochilus roxburghii is also a common Chinese medicinal material used as both medicine and food in China, and long-term eating experience proves that Anoectochilus roxburghii has no toxic or side effect, and the health-care effect of Anoectochilus roxburghii is more and more favored by people. Modern researches show that the anoectochilus formosanus contains chemical components such as polysaccharide, anoectochilus formosanus glycoside, flavone, organic acid, steroid compounds, alkaloid, various trace elements and the like, and has the effects of reducing blood sugar, reducing blood pressure, resisting fat, resisting inflammation, resisting viruses, protecting liver, protecting kidney, regulating immunity, resisting convulsion, calming, resisting tumors and the like in the aspect of biological activity.
Traditional Chinese medicine fermentation, also called traditional Chinese medicine microbial transformation or biocatalysis, refers to that under certain environmental conditions (such as temperature, humidity, air, moisture and the like), the original performance of the medicine is changed, new efficacy is enhanced or generated, and the medicine variety is expanded through the fermentation process by means of the action of enzyme and microorganism so as to adapt to the needs of clinical medication. The traditional Chinese medicine fermentation has outstanding advantages which are mainly reflected in the following points: first, the decomposition of microorganisms may decompose toxic substances in the Chinese medicine, thereby reducing the toxic side effects of the medicine. Second, the extraction rate of the effective components is improved. The ingredients and the structure of the traditional Chinese medicinal materials are complex, so that active substances are wrapped, the medicinal properties cannot be completely released, the traditional Chinese medicinal materials are decomposed by various enzymes metabolized by microorganisms in the growth and metabolism process, the intercellular substance is enlarged, the resistance of cell walls and cells to the dissolution of the active ingredients is reduced, and the active ingredients can be more quickly and better dissolved. Thirdly, the absorption and utilization of the active ingredients are improved. During the fermentation process, the microbes can decompose large molecular active substances which can not be directly absorbed and utilized by human bodies into small molecular active substances, so that the fermented traditional Chinese medicine can be absorbed in the human bodies more quickly and completely, and the treatment effect is better. Fourth, a new active species is generated. Some Chinese medicines can generate new active substances after being fermented by microorganisms, so that the Chinese medicines have new health-care, prevention or treatment functions. Fifthly, the traditional Chinese medicine fermentation preparation can well save medicinal material resources, and is beneficial to the conservation and sustainable development of the treasure resources.
Fungi are mostly selected as fermentation strains in traditional Chinese medicine fermentation, and for example, the fungi fermentation traditional Chinese medicines still used clinically at present comprise pinellia ternate koji, red yeast rice, medicated leaven, fermented soybean and the like. Compared with fungi, the bacteria have the characteristics of huge variety, short breeding cycle, various metabolic pathways and the like, so the bacteria have greater advantages as fermentation strains. In view of the bacterial fermentation strains, probiotics are the most preferred. Probiotic refers to a live microbial preparation that is beneficial to the health of the host (human or animal) when taken in the proper dosage. The probiotics are mainly divided into lactic acid bacteria (lactobacillus, streptococcus, leuconostoc, pediococcus, bifidobacterium, listeria and the like) and non-lactic acid bacteria (part of saccharomycetes, bacillus and the like). The probiotics have the effects of enhancing human immunity, adjusting the balance of intestinal flora, maintaining the integrity of intestinal mucosa and improving the digestive absorption function of intestinal tracts, the optimized human probiotics are used as fermentation strains to ferment the traditional Chinese medicine, the active ingredients in the traditional Chinese medicine are subjected to biotransformation, and macromolecular substances of the traditional Chinese medicine are converted into micromolecular ingredients which can be directly absorbed by the intestinal tracts of human bodies through the microorganisms, so that the traditional Chinese medicine ingredients can be absorbed more quickly and better. Therefore, the probiotic fermentation is used for preparing the medicine, so that the medicine cost is reduced, the effect is improved, and a way is provided for developing new medicines.
Because the processing and application of the anoectochilus formosanus does not reach the extraction stage of biotechnical components, the anoectochilus formosanus is mainly eaten fresh or processed into tea bags and medicinal liquor simply at present. The folk medicine for anoectochilus roxburghii is mainly to be dried for use or decocted with other medicinal materials for oral administration. The methods cause that most of the functional components in the anoectochilus formosanus are difficult to absorb by a human body, the bioavailability is low, and the serious waste of medicine resources is caused. At present, no study on the fermentation of anoectochilus formosanus by utilizing probiotics is reported, so that the exploration of the probiotic fermentation process of the anoectochilus formosanus is necessary and has wide prospects.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention provides a method for fermenting Anoectochilus roxburghii with probiotics, so as to improve the extraction rate and bioavailability of the main active ingredients of Anoectochilus roxburghii.
The purpose of the invention is realized by the following technical scheme:
a method for improving the content of active ingredients of anoectochilus formosanus by utilizing probiotic fermentation is characterized by comprising the following steps:
(1) preparing a seed solution: inoculating the activated bacillus coagulans into a nutrient broth culture medium, and performing shaking culture on a shaker for 20-28 h at 37 ℃ and 80r/min to prepare bacillus coagulans seed liquid; inoculating activated lactobacillus rhamnosus and bifidobacterium longum to an improved MRS liquid culture medium together in a ratio of 1:1, and performing static culture at 37 ℃ for 20-28 h to prepare lactobacillus seed liquid;
(2) raw material treatment: cleaning fresh herba Anoectochili Roxburghii, vacuum drying at low temperature to reduce water content to below 5%, and pulverizing step by step to obtain herba Anoectochili Roxburghii micropowder;
(3) and (3) performing segmented fermentation on the composite strains: preparing a fermentation culture medium containing anoectochilus roxburghii superfine powder, sterilizing at 115 ℃ for 20min, cooling to room temperature, adjusting the initial pH to 6.5-7.0, inoculating bacillus coagulans seed liquid, fermenting and culturing for 40-50 h at the temperature of 35-40 ℃ and the rotating speed of 80-120 r/min, inoculating lactobacillus seed liquid, and continuing fermenting and culturing for 65-75 h at the temperature of 35-40 ℃ and standing;
(4) after fermentation, pre-freezing the fermentation product at-20 ℃, freeze-drying and crushing the fermentation product under the conditions that the vacuum degree is 0.05-0.1 MPa and the freezing temperature is-60 to-70 ℃, and preparing freeze-dried powder.
In the step (1), the activating method of the bacillus coagulans comprises the following steps: recovering the strain glycerol freezing tube at room temperature, picking a small amount of strain with a sterile inoculating loop, streaking and inoculating the strain in a nutrient agar culture medium, and performing inverted culture at 37 ℃ for 24 h.
In the step (1), the activation method of lactobacillus rhamnosus and bifidobacterium longum comprises the following steps: the strain glycerol frozen tube is revived at room temperature, inoculated in MRS liquid culture medium with the inoculation amount of 2 percent, kept stand and cultured for 12h at 37 ℃, and thus passaged for 2 times.
In the step (1), the formula of the improved MRS liquid culture medium is as follows: 10.0g/L of peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract, 5.0g/L of glucose, 5.0g/L of sodium acetate, 2.0g/L of diammonium citrate, 801.0 g/L of tween-K 2 HPO 4 2.0g/L，MgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 O 0.05g/L，CaCO 3 2.0g/L。
In the step (2), the low-temperature vacuum drying temperature is 40-45 ℃, and the vacuum degree is 200-300 Pa.
In the step (2), the specific method for stepwise crushing comprises the following steps: firstly, coarsely crushing the dried anoectochilus formosanus by using a crusher, and sieving the crushed anoectochilus formosanus by using a 100-mesh sieve; and then, secondarily crushing the anoectochilus formosanus coarse powder by using an airflow type superfine crusher to enable the particle size to reach the level of 1-75 microns.
In the step (3), the fermentation medium comprises the following components: 100-200g/L anoectochilus roxburghii submicron powder, 10-20g/L sucrose, 20-30g/L black bean powder, 0.8-1.2g/L NaCl, K 2 HPO 4 1.6-2.4g/L，CaCO 3 0.07-0.13g/L, 0.8-1.2g/L sodium succinate, and 3.0-5.0ml/L vitamin solution.
In the step (3), the inoculation amount of the bacillus coagulans seed liquid is 1% -5%, and the inoculation amount of the lactobacillus seed liquid is 6% -10%.
The vitamin solution comprises the following components: biotin 2.0mg/L, folic acid 2.0mg/L, vitamin B 6 10.0mg/L, thiamine hydrochloride 5.0mg/L, riboflavin 5.0mg/L, nicotinic acid 5.0mg/L, calcium D-pantothenate 5.0mg/L, vitamin B 12 0.10mg/L and lipoic acid 5.0 mg/L.
The implementation of the scheme of the invention has at least the following advantages:
1) according to the invention, the anoectochilus formosanus medicinal material is prepared into superfine powder by adopting a superfine grinding technology, so that the active ingredients in the anoectochilus formosanus medicinal material are dissolved out at an accelerated speed, the fermentation and enzymolysis reaction speed of the medicinal material can be accelerated, the medicinal material dosage can be reduced, and the medicinal material resource can be saved.
2) The invention utilizes bacillus coagulans and lactobacillus (lactobacillus rhamnosus and bifidobacterium longum) to carry out segmented composite fermentation on the anoectochilus roxburghii, and aims to exert the synergistic effect of multiple strains, thereby enhancing the biotransformation effect of probiotics on the anoectochilus roxburghii.
3) The invention utilizes the probiotic fermentation to greatly improve the contents of main active ingredients of the anoectochilus formosanus, such as the anoectochilus formosanus glycoside, flavone and polysaccharide, particularly, the content of the anoectochilus formosanus glycoside is improved most obviously and is about 2.5 before fermentation, thereby laying a foundation for the development of high-efficiency utilization of the anoectochilus formosanus and further-processed products of the anoectochilus formosanus.
Detailed Description
The following are specific embodiments of the present invention and further description of the technical solutions of the present invention, but the scope of the present invention is not limited to these embodiments, and all changes or equivalent substitutions that do not depart from the spirit of the present invention are included in the scope of the present invention. The test methods used in the following examples are, unless otherwise specified, all conventional methods, and the raw materials, reagents and the like used are, unless otherwise specified, all commercially available raw materials and reagents from conventional commercial sources and the like.
Example 1
1. The materials used
The strain Bacillus coagulans (A)Bacillus coagulans) Accession number BNCC 337209; lactobacillus rhamnosus (A), (B), (C)Lactobacillus rhamnosus) Accession number BNCC 134266; bifidobacterium longum (b)Bifidobacterium longum) The preservation number BNCC 337084 is purchased from Beijing Beinanna institute of Biotechnology.
Fresh anoectochilus roxburghii as a medicinal material raw material is purchased from agriculture development limited company Zhangzhou city of Fujian province.
Culture medium and reagent nutrient agar culture medium: 10.0g/L of peptone, 3.0g/L of beef extract, 5.0g/L of NaCl and 15.0g/L of agar; nutrient broth culture medium: no agar was added.
MRS liquid medium: 10.0g/L of peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract, 20.0g/L of glucose, 5.0g/L of sodium acetate, 2.0g/L of diammonium citrate, 801.0 ml/L of tween-K 2 HPO 4 2.0g/L，MgSO 4 ·7H 2 O 0.58g/L，MnSO 4 ·H 2 O 0.25g/L。
Improving MRS liquid culture medium: 10.0g/L of peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract, 5.0g/L of glucose, 5.0g/L of sodium acetate, 2.0g/L of diammonium citrate, 801.0 ml/L of tween-K 2 HPO 4 2.0g/L，MgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 O 0.05g/L，CaCO 3 2.0g/L。
Fermentation medium: 150g/L of anoectochilus roxburghii submicron powder, 15g/L of cane sugar, 20g/L of black bean powder, 1.0g/L of NaCl and K 2 HPO 4 2.0g/L，CaCO 3 0.1g/L, 1.0g/L sodium succinate and 4.0ml/L vitamin solution.
Vitamin solution: biotin 2.0mg/L, folic acid 2.0mg/L, vitamin B 6 10.0mg/L, thiamine hydrochloride 5.0mg/L, riboflavin 5.0mg/L, nicotinic acid 5.0mg/L, calcium D-pantothenate 5.0mg/L, vitamin B 12 0.10mg/L and lipoic acid 5.0 mg/L.
2. Method of producing a composite material
(1) Preparing a seed solution: placing a glycerol cryopreservation tube of bacillus coagulans at room temperature, after thawing, picking a small amount of strains by using an aseptic inoculating loop, streaking and inoculating the strains to a nutrient agar culture medium, and carrying out inverted culture at 37 ℃ for 24 hours; inoculating the activated Bacillus coagulans into nutrient broth culture medium, and shake-culturing at 37 deg.C and 80r/min for 24 hr to obtain Bacillus coagulans seed solution. Resuscitating glycerol cryopreservation tubes of lactobacillus rhamnosus and bifidobacterium longum at room temperature, respectively inoculating the glycerol cryopreservation tubes into MRS liquid culture medium in an inoculum size of 2%, and performing static culture at 37 ℃ for 12h, so as to perform passage for 2 times; inoculating activated Lactobacillus rhamnosus and Bifidobacterium longum at a ratio of 1:1 into improved MRS liquid culture medium, and standing at 37 deg.C for 24 hr to obtain lactobacillus seed solution.
(2) Raw material treatment: cleaning fresh anoectochilus roxburghii whole plants, carrying out low-temperature vacuum drying at 40-45 ℃ and under the vacuum degree of 200-300 Pa to reduce the water content to below 5%, coarsely crushing the dried anoectochilus roxburghii, and sieving the crushed anoectochilus roxburghii with a 100-mesh sieve; and (3) carrying out secondary crushing on the anoectochilus formosanus coarse powder by using an airflow type superfine crusher to enable the particle size to reach the level of 1-75 microns, and preparing the anoectochilus formosanus superfine powder.
(3) And (3) performing segmented fermentation on the composite strains: preparing a fermentation culture medium, sterilizing at 115 ℃ for 20min, cooling to room temperature, adjusting the initial pH to 6.8, inoculating bacillus coagulans seed liquid with 3% of inoculum size, performing fermentation culture for 45h at 37 ℃ and 100r/min of rotation speed, inoculating lactobacillus seed liquid with 8% of inoculum size, and continuing the fermentation culture for 70h at 37 ℃ and standing.
(4) After fermentation, pre-freezing the fermentation product at-20 ℃, freeze-drying and crushing the fermentation product under the conditions of vacuum degree of 0.05-0.1 MPa and freezing temperature of-60 to-70 ℃ to prepare freeze-dried powder.
Example 2
(1) Preparing a seed solution: placing a glycerol cryopreservation tube of bacillus coagulans at room temperature, picking a small amount of strains by using an aseptic inoculating loop after melting, streaking and inoculating the strains to a nutrient agar culture medium, and carrying out inverted culture at 37 ℃ for 24 hours; inoculating the activated bacillus coagulans into a nutrient broth culture medium, and performing shake culture for 24h at 37 ℃ and 80r/min to prepare bacillus coagulans seed liquid. Resuscitating glycerol cryopreservation tubes of lactobacillus rhamnosus and bifidobacterium longum at room temperature, respectively inoculating the glycerol cryopreservation tubes into an MRS culture medium in an inoculum size of 2%, and carrying out standing culture at 37 ℃ for 12h, so as to carry out passage for 2 times; inoculating activated Lactobacillus rhamnosus and Bifidobacterium longum in an improved MRS liquid culture medium at a ratio of 1:1, and standing at 37 deg.C for 24 hr to obtain lactobacillus seed solution.
(2) Raw material treatment: cleaning fresh anoectochilus roxburghii whole plants, carrying out low-temperature vacuum drying at 40-45 ℃ and under the vacuum degree of 200-300 Pa to reduce the water content to below 5%, coarsely crushing the dried anoectochilus roxburghii, and sieving the crushed anoectochilus roxburghii with a 100-mesh sieve; and (3) carrying out secondary crushing on the anoectochilus roxburghii coarse powder by using an airflow type superfine crusher to enable the particle size to reach 1-75 mu m, and preparing the anoectochilus roxburghii superfine powder.
(3) And (3) performing segmented fermentation on the composite strains: preparing a fermentation culture medium, sterilizing at 115 ℃ for 20min, cooling to room temperature, adjusting the initial pH to 6.8, inoculating bacillus coagulans seed liquid with an inoculum size of 5%, fermenting and culturing for 45h at 37 ℃ and a rotation speed of 100r/min, inoculating lactobacillus seed liquid with an inoculum size of 10%, and continuously fermenting and culturing for 70h at 37 ℃ and standing.
(4) After fermentation, pre-freezing the fermentation product at-20 ℃, freeze-drying and crushing the fermentation product under the conditions that the vacuum degree is 0.05-0.1 MPa and the freezing temperature is-60 to-70 ℃, and preparing freeze-dried powder.
Example 3
(1) Preparing a seed solution: placing a glycerol cryopreservation tube of bacillus coagulans at room temperature, after thawing, picking a small amount of strains by using an aseptic inoculating loop, streaking and inoculating the strains to a nutrient agar culture medium, and carrying out inverted culture at 37 ℃ for 24 hours; inoculating the activated bacillus coagulans into a nutrient broth culture medium, and performing shake culture for 24h at 37 ℃ and 80r/min to prepare bacillus coagulans seed liquid. Resuscitating glycerol cryopreservation tubes of lactobacillus rhamnosus and bifidobacterium longum at room temperature, respectively inoculating the glycerol cryopreservation tubes into an MRS culture medium in an inoculum size of 2%, and carrying out standing culture at 37 ℃ for 12h, so as to carry out passage for 2 times; inoculating activated Lactobacillus rhamnosus and Bifidobacterium longum in an improved MRS liquid culture medium at a ratio of 1:1, and standing at 37 deg.C for 24 hr to obtain lactobacillus seed solution.
(2) Raw material treatment: cleaning fresh anoectochilus roxburghii whole plants, carrying out low-temperature vacuum drying at 40-45 ℃ and under the vacuum degree of 200-300 Pa to reduce the water content to below 5%, coarsely crushing the dried anoectochilus roxburghii, and sieving with a 100-mesh sieve; and (3) secondarily grinding the anoectochilus roxburghii coarse powder by using an airflow type superfine grinder to enable the particle size to reach 1-75 mu m, and preparing the anoectochilus roxburghii superfine powder.
(3) And (3) performing segmented fermentation on the composite strain: preparing a fermentation culture medium, sterilizing at 115 ℃ for 20min, cooling to room temperature, adjusting the initial pH to 7.0, inoculating bacillus coagulans seed liquid with 3% of inoculum size, fermenting and culturing for 50h at 37 ℃ and 100r/min of rotation speed, inoculating lactobacillus seed liquid with 8% of inoculum size, and continuing to ferment and culture for 75h at 37 ℃ and standing.
(4) After fermentation, pre-freezing the fermentation product at-20 ℃, freeze-drying and crushing the fermentation product under the conditions of vacuum degree of 0.05-0.1 MPa and freezing temperature of-60 to-70 ℃ to prepare freeze-dried powder.
Comparative example 1
This comparative example differs from example 1 in that: in the raw material treatment, the anoectochilus formosanus is subjected to conventional crushing treatment.
(1) Preparing a seed solution: placing a glycerol cryopreservation tube of bacillus coagulans at room temperature, after thawing, picking a small amount of strains by using an aseptic inoculating loop, streaking and inoculating the strains to a nutrient agar culture medium, and carrying out inverted culture at 37 ℃ for 24 hours; inoculating the activated bacillus coagulans into a nutrient broth culture medium, and performing shake culture for 24h at 37 ℃ and 80r/min to prepare bacillus coagulans seed liquid. Resuscitating glycerol cryopreservation tubes of lactobacillus rhamnosus and bifidobacterium longum at room temperature, respectively inoculating the glycerol cryopreservation tubes into MRS liquid culture medium in an inoculum size of 2%, and performing static culture at 37 ℃ for 12h, so as to perform passage for 2 times; inoculating activated Lactobacillus rhamnosus and Bifidobacterium longum in an improved MRS liquid culture medium at a ratio of 1:1, and standing at 37 deg.C for 24 hr to obtain lactobacillus seed solution.
(2) Treating raw materials: cleaning fresh anoectochilus roxburghii whole plants, then carrying out low-temperature vacuum drying at the temperature of 40-45 ℃ and the vacuum degree of 200-300 Pa to reduce the water content to below 5%, and then crushing and sieving with a 60-mesh sieve.
(3) And (3) performing segmented fermentation on the composite strains: preparing a fermentation culture medium, sterilizing at 115 ℃ for 20min, cooling to room temperature, adjusting the initial pH to 6.8, inoculating the bacillus coagulans seed liquid with 3% of inoculum size, performing fermentation culture for 45h at 37 ℃ and 100r/min of rotation speed, inoculating the lactobacillus seed liquid with 8% of inoculum size, and continuing the fermentation culture for 70h at 37 ℃ and standing.
(4) After fermentation, pre-freezing the fermentation product at-20 ℃, freeze-drying and crushing the fermentation product under the conditions of vacuum degree of 0.05-0.1 MPa and freezing temperature of-60 to-70 ℃ to prepare freeze-dried powder.
Comparative example 2
This comparative example differs from example 1 in that: only bacillus coagulans is adopted to ferment the anoectochilus roxburghii.
(1) Preparing a seed solution: placing a glycerol cryopreservation tube of bacillus coagulans at room temperature, after thawing, picking a small amount of strains by using an aseptic inoculating loop, streaking and inoculating the strains to a nutrient agar culture medium, and carrying out inverted culture at 37 ℃ for 24 hours; inoculating the activated bacillus coagulans into a nutrient broth culture medium, and performing shake culture for 24h at 37 ℃ and 80r/min to prepare bacillus coagulans seed liquid.
(2) Raw material treatment: cleaning fresh anoectochilus roxburghii whole plants, carrying out low-temperature vacuum drying at 40-45 ℃ and under the vacuum degree of 200-300 Pa to reduce the water content to below 5%, coarsely crushing the dried anoectochilus roxburghii, and sieving the crushed anoectochilus roxburghii with a 100-mesh sieve; and (3) carrying out secondary crushing on the anoectochilus roxburghii coarse powder by using an airflow type superfine crusher to enable the particle size to reach 1-75 mu m, and preparing the anoectochilus roxburghii superfine powder.
(3) Fermenting strains: preparing a fermentation culture medium, adjusting the initial pH to 6.8, inoculating 3% of the inoculum size into the bacillus coagulans seed solution, and performing fermentation culture for 50h at the temperature of 37 ℃ and the rotating speed of 100 r/min.
(4) After fermentation, pre-freezing the fermentation product at-20 ℃, freeze-drying and crushing the fermentation product under the conditions of vacuum degree of 0.05-0.1 MPa and freezing temperature of-60 to-70 ℃ to prepare freeze-dried powder.
Comparative example 3
This comparative example differs from example 1 in that: fermenting anoectochilus roxburghii only by using lactic acid bacteria, and adjusting the initial pH of a fermentation medium to be within a proper range of the lactic acid bacteria.
(1) Preparing a seed solution: resuscitating glycerol cryopreservation tubes of lactobacillus rhamnosus and bifidobacterium longum at room temperature, respectively inoculating the glycerol cryopreservation tubes into MRS liquid culture medium in an inoculum size of 2%, and performing static culture at 37 ℃ for 12h, so as to perform passage for 2 times; inoculating activated Lactobacillus rhamnosus and Bifidobacterium longum at a ratio of 1:1 into improved MRS liquid culture medium, and standing at 37 deg.C for 24 hr to obtain lactobacillus seed solution.
(2) Raw material treatment: cleaning fresh anoectochilus roxburghii whole plants, carrying out low-temperature vacuum drying at 40-45 ℃ and under the vacuum degree of 200-300 Pa to reduce the water content to below 5%, coarsely crushing the dried anoectochilus roxburghii, and sieving the crushed anoectochilus roxburghii with a 100-mesh sieve; and (3) secondarily grinding the anoectochilus roxburghii coarse powder by using an airflow type superfine grinder to enable the particle size to reach the level of 1-75 mu m, and preparing the anoectochilus roxburghii superfine powder.
(3) And (3) fermenting strains: preparing a fermentation culture medium, sterilizing at 115 ℃ for 20min, cooling to room temperature, adjusting the initial pH to 6.0, inoculating 8% of the inoculum size into the lactobacillus seed solution, and performing fermentation culture for 70h at 37 ℃ under the standing condition.
(4) After fermentation, pre-freezing the fermentation product at-20 ℃, freeze-drying and crushing the fermentation product under the conditions of vacuum degree of 0.05-0.1 MPa and freezing temperature of-60 to-70 ℃ to prepare freeze-dried powder.
Test example 1
Taking the freeze-dried powder prepared in the examples 1-3 and the comparative examples 1-3 as a sample of the anoectochilus roxburghii after fermentation, drying fresh anoectochilus roxburghii to constant weight, crushing the dried fresh anoectochilus roxburghii to obtain a sample of the anoectochilus roxburghii before fermentation, and measuring the content of active ingredients according to the following method.
1. Determination of content of Anoectochilus roxburghii glycoside
Reference is made to the method in "high performance liquid chromatography for determining the content of Anoectochilus roxburghii glycoside in Anoectochilus roxburghii".
Chromatographic conditions
Mobile phase: methanol-acetonitrile-water (5: 5: 90); flow rate: 1.0 mL/min; column temperature: room temperature; detection wavelength: 215 nm.
Preparation of control solutions
Precisely weighing 103.5mg of Anoectochilus roxburghii glycoside reference substance dried at 105 deg.C to constant weight, placing in a 10ml volumetric flask, adding purified water to dissolve and dilute to scale, shaking up, and making into reference substance solution.
Preparation of sample solution
Precisely weighing 1.0g of a Clematis chinensis sample, adding 15ml of methanol, carrying out ultrasonic treatment (power of 250W and frequency of 50 kHz) for 60min, carrying out cold soaking at room temperature overnight, filtering to obtain a methanol extracting solution, evaporating to dryness, dissolving with about 4ml of water, extracting with a proper amount of chloroform for degreasing, separating a water layer, adding water to a constant volume of 5ml, filtering with a 0.22 mu m filter membrane, discarding a primary filtrate, and taking a subsequent filtrate for diluting by 10 times to obtain a sample solution.
Standard curve and linear range
Precisely weighing the reference substance of the kinsenoside to prepare a standard series solution, injecting 20 mu L of the solution, drawing a standard curve by taking the peak area mean value of the compound as the ordinate and the concentration of the reference substance of the kinsenoside as the abscissa, and obtaining a regression equation.
Determination of sample content
A sample solution is injected with 20 mu L of sample solution, the peak area is measured, and the content of the sample solution is calculated according to a standard curve.
2. Determination of Total Flavonoids content
Drawing a standard curve: precisely weighing 10mg of rutin control substance dried at 105 deg.C to constant weight, placing in a 50ml volumetric flask, adding appropriate amount of methanol, ultrasonically dissolving, adding methanol to scale, and shaking to obtain rutin standard solution (rutin content is 0.2 mg/ml). Precisely measuring 0, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0ml of rutin standard solution, respectively putting into 25ml volumetric flasks, and adding water to 6.0 ml; adding 1.0ml of 5% sodium nitrite solution, shaking, and standing for 6 min; adding 1.0ml of 10% aluminum nitrate solution, and standing for 6 min; adding 10.0ml of 4% sodium hydroxide solution, adding distilled water to scale, shaking, standing for 15min, measuring absorbance values at 510nm wavelength, drawing a standard curve, and solving a regression equation.
Sample treatment: precisely weighing 2.0g of clematis chinensis sample, adding 95% ethanol according to the material-liquid ratio of 1:20, reflux-extracting for 3 times, each time for 90min, filtering, combining filtrates, concentrating under reduced pressure at 45 ℃ to dryness, adding a proper amount of anhydrous methanol to dissolve, and fixing the volume to 10ml to obtain a sample solution.
And (3) total flavone content determination: precisely sucking 1.0ml to 25ml of sample solution into a volumetric flask, and adding water to 6.0 ml; adding 1.0ml of 5% sodium nitrite solution, shaking, and standing for 6 min; adding 1.0ml of 10% aluminum nitrate solution, and standing for 6 min; adding 4% sodium hydroxide solution 10.0ml, adding distilled water to scale, shaking, standing for 15min, measuring absorbance value at 510nm wavelength, and calculating total flavone content according to standard curve.
3. Determination of polysaccharide content
Drawing a standard curve: precisely weighing 25mg of glucose dried to constant weight at 105 ℃, placing the glucose in a 250ml volumetric flask, adding distilled water to constant volume to scale, shaking up, and preparing a glucose standard solution (the glucose content is 0. lmg/ml). Accurately measuring 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and l.0ml of glucose standard solution, placing the glucose standard solution into a test tube, adding distilled water to l.0ml, and adding 1.0 of distilled water into another test tube as a blank control. Adding 1.0ml of 5% phenol into each test tube, shaking, adding 5.0ml of concentrated sulfuric acid vertically, shaking, standing at room temperature for 10min, boiling in water bath for 15min, and cooling. And (4) respectively measuring the absorbance values at 490nm wavelength, drawing a standard curve, and solving a regression equation.
Sample treatment: precisely weighing 1.0g of clematis chinensis sample, adding ultrapure water according to the material-liquid ratio of 1:35, ultrasonically extracting for 30min at 60 ℃, centrifuging for l5min at 4000r/min, and collecting supernatant. Extracting residue for 3 times, mixing the supernatants for 3 times, vacuum rotary evaporating to 10ml, adding 40ml anhydrous ethanol, precipitating at 4 deg.C overnight, centrifuging, collecting precipitate, and dissolving in 40ml ultrapure water to obtain sample solution.
And (3) polysaccharide content determination: sucking sample solution 0.1ml, adding distilled water to l.0ml, then adding 5% phenol 1.0ml, shaking, adding concentrated sulfuric acid 5.0ml, shaking, standing at room temperature for 10min, boiling water bath for 15min, and cooling. The absorbance values were measured at 490nm and the polysaccharide content was calculated from the standard curve.
As can be seen from Table 1, the content of active ingredients in the fermentation products of Anoectochilus roxburghii prepared in the embodiments of the invention is higher than each proportion, and the content of the active ingredients is obviously different from that in the samples before fermentation, wherein the content of Anoectochilus roxburghii glycosides is 2.51-2.60 times of that before fermentation, the content of total flavonoids is 1.68-1.71 times of that before fermentation, and the content of polysaccharides is 1.64-1.71 times of that before fermentation. The method can obviously improve the content of the main active ingredients of the anoectochilus formosanus, especially the content of the anoectochilus formosanus glycoside, and lays a foundation for the efficient utilization and further deep processing of the anoectochilus formosanus.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A method for improving the content of active ingredients of anoectochilus formosanus by utilizing probiotic fermentation is characterized by comprising the following steps:
(1) preparing a seed solution: inoculating the activated bacillus coagulans to a nutrient broth culture medium, and performing shaking culture on a shaker for 20-28 h at 37 ℃ and 80r/min to prepare bacillus coagulans seed liquid; inoculating activated lactobacillus rhamnosus and bifidobacterium longum to an improved MRS liquid culture medium together in a ratio of 1:1, and performing static culture at 37 ℃ for 20-28 h to prepare lactobacillus seed liquid;
(2) raw material treatment: cleaning fresh anoectochilus roxburghii whole plants, performing low-temperature vacuum drying to reduce the water content to below 5%, and preparing into anoectochilus roxburghii superfine powder by adopting a step-by-step crushing mode;
(3) and (3) performing segmented fermentation on the composite strains: preparing a fermentation culture medium containing anoectochilus roxburghii superfine powder, sterilizing at 115 ℃ for 20min, cooling to room temperature, adjusting the initial pH to 6.5-7.0, inoculating a bacillus coagulans seed solution, performing fermentation culture for 40-50 h at the temperature of 35-40 ℃ and the rotation speed of 80-120 r/min, inoculating a lactobacillus seed solution, and continuing the fermentation culture for 65-75 h at the temperature of 35-40 ℃ and standing;
(4) after fermentation, pre-freezing the fermentation product at-20 ℃, freeze-drying and crushing the fermentation product under the conditions that the vacuum degree is 0.05-0.1 MPa and the freezing temperature is-60 to-70 ℃, and preparing freeze-dried powder.
2. The method for improving the content of the active ingredients in the anoectochilus formosanus by utilizing the probiotic fermentation as claimed in claim 1, wherein in the step (1), the activating method of the bacillus coagulans is as follows: recovering the strain glycerol freezing tube at room temperature, picking a small amount of strain with a sterile inoculating loop, streaking and inoculating the strain in a nutrient agar culture medium, and performing inverted culture at 37 ℃ for 24 h.
3. The method for improving the content of the active ingredients in the anoectochilus formosanus by utilizing the probiotic fermentation according to claim 1, wherein in the step (1), the activation method of the lactobacillus rhamnosus and the bifidobacterium longum comprises the following steps: the strain glycerol frozen tube is revived at room temperature, inoculated in MRS liquid culture medium with the inoculation amount of 2 percent, kept stand and cultured for 12h at 37 ℃, and thus passaged for 2 times.
4. The method for improving the content of the active ingredients of anoectochilus formosanus by utilizing probiotic fermentation in the step (1), wherein the formula of the modified MRS liquid medium in the step (1) is as follows: 10.0g/L of peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract, 5.0g/L of glucose, 5.0g/L of sodium acetate, 2.0g/L of diammonium citrate, 801.0 g/L of tween-801.0, K 2 HPO 4 2.0g/L，MgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 O 0.05g/L，CaCO 3 2.0g/L。
5. The method for improving the content of the active ingredients in the anoectochilus formosanus by utilizing the probiotic fermentation as claimed in claim 1, wherein in the step (2), the temperature of the low-temperature vacuum drying is 40-45 ℃ and the vacuum degree is 200-300 Pa.
6. The method for improving the content of the active ingredients in the anoectochilus formosanus by utilizing the probiotic fermentation as claimed in claim 1, wherein in the step (2), the step-by-step crushing method comprises the following specific steps: firstly, coarsely crushing the dried anoectochilus formosanus by using a crusher, and sieving the crushed anoectochilus formosanus by using a 100-mesh sieve; and then, secondarily crushing the anoectochilus formosanus coarse powder by using an airflow type superfine crusher to enable the particle size to reach the level of 1-75 microns.
7. The method for improving the content of the active ingredients in the anoectochilus formosanus by utilizing the probiotic fermentation in the step (3) is characterized in that the fermentation medium comprises the following components: 100-200g/L anoectochilus roxburghii submicron powder, 10-20g/L sucrose, 20-30g/L black bean powder, 0.8-1.2g/L NaCl, K 2 HPO 4 1.6-2.4g/L，CaCO 3 0.07-0.13g/L, 0.8-1.2g/L sodium succinate, and 3.0-5.0ml/L vitamin solution.
8. The method for improving the content of the active ingredients in the anoectochilus formosanus by utilizing the probiotic fermentation according to claim 1, wherein in the step (3), the inoculation amount of the bacillus coagulans seed solution is 1% -5%, and the inoculation amount of the lactobacillus seed solution is 6% -10%.
9. The method for improving the content of the active ingredients in the anoectochilus formosanus by utilizing the probiotic fermentation as claimed in claim 7, wherein the vitamin solution comprises the following components: biotin 2.0mg/L, folic acid 2.0mg/L, vitamin B 6 10.0mg/L, thiamine hydrochloride 5.0mg/L, riboflavin 5.0mg/L, nicotinic acid 5.0mg/L, calcium D-pantothenate 5.0mg/L, vitamin B 12 0.10mg/L and lipoic acid 5.0 mg/L.
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