CN115029385A - Method for extracting tea saponin from tea seed meal by microbial fermentation - Google Patents
Method for extracting tea saponin from tea seed meal by microbial fermentation Download PDFInfo
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- CN115029385A CN115029385A CN202210875635.0A CN202210875635A CN115029385A CN 115029385 A CN115029385 A CN 115029385A CN 202210875635 A CN202210875635 A CN 202210875635A CN 115029385 A CN115029385 A CN 115029385A
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- tea saponin
- meal
- lactobacillus plantarum
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/245—Lactobacillus casei
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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Abstract
The invention belongs to the field of biological fermentation, and discloses a method for extracting tea saponin from oil tea meal by utilizing microbial fermentation, which comprises the following steps: (1) uniformly mixing the crushed oil tea meal with a nitrogen source, a carbon source, inorganic salt and water, and sterilizing to prepare a fermentation medium; (2) inoculating Lactobacillus plantarum (Lactobacillus plantarum) and Lactobacillus casei (Lactobacillus casei) into a fermentation culture medium, and performing mixed liquid fermentation to obtain a fermentation stock solution; (3) and (4) carrying out centrifugal filtration on the fermentation stock solution to obtain clarified tea saponin fermentation liquor, and then purifying to obtain the tea saponin. The invention only needs one-time inoculation of liquid fermentation, has simple and controllable process, easy operation, can realize industrial production, and has green and environment-friendly production process; the obtained product has high purity and high extraction rate.
Description
Technical Field
The invention belongs to the field of biological fermentation, and particularly relates to a method for extracting tea saponin from tea seed meal by utilizing microbial fermentation.
Background
The camellia oleifera is 4 woody oil crops in the world, and the total planting scale of the camellia oleifera in China reaches 446.67 ten thousand hectares. After oil extraction, a large amount of oil-tea cake dregs can be generated, and the oil-tea cake dregs contain various effective components such as tea saponin, polysaccharide, protein, polyphenol and the like.
Tea saponin is a pentacyclic triterpene saponin, and is a complex mixture of sapogenin (i.e. aglycone), saccharide and organic acid. A total of 7 sapogenins were isolated from tea saponin. Tea saponin was first isolated from tea plant seeds in 1931 by the Japanese scholar Qingshan Xinxianlang. The purified tea saponin is milky white or light yellow solid amorphous powder, and has the characteristics of hygroscopicity, bitter and spicy taste and irritation to nasal mucosa. Has good activity in the aspects of emulsification, dispersion, wetting, foaming, foam stabilization, decontamination and the like, and is a natural surfactant. The tea saponin has surface activity and bioactivity. Has obvious effects in multiple aspects of antibiosis, sterilization, permeation resistance, inflammation diminishing, phlegm eliminating, cough relieving, alcohol effect dispelling and the like. Therefore, the tea saponin has wide application. Can be used as shampoo and bath agent in daily chemical industry; the building material can be used as a foaming foam stabilizer in the building material industry; biological pesticides for controlling plant diseases and insect pests in agriculture; the culture medium can be used as a pond cleaning agent for cleaning enemy fishes in a shrimp pond, and can also be used for soaking and killing adult snails, young snails and snail eggs of oncomelania snails and the like.
The oil tea cake contains 10-15% natural nonionic surfactant tea saponin. In order to reduce the use of organic solvents, improve the safety of tea saponin and protect the environment, the existing extraction process of tea saponin mainly adopts a water extraction method, and the research on extracting tea saponin by a microbial fermentation method is rarely reported. However, the water extraction method and the microbial extraction method in the prior art face the problem of low extraction rate and purity of the tea saponin.
CN104357527A discloses a method for extracting tea saponin by solid state fermentation of large edible fungi. The method comprises the steps of taking tea seed meal as a main culture medium, adding bran and other auxiliary materials, inoculating microorganisms, carrying out solid state fermentation to realize efficient exudation of tea saponin, and carrying out ethanol extraction on the exuded tea saponin and then carrying out membrane separation to obtain a tea saponin product. CN101880307A discloses a method for extracting tea saponin from tea seed meal by one-time fermentation of aspergillus niger, cellulomonas flavigena, saccharomycetes and bacillus subtilis, which takes crushed dry tea seed meal as a raw material, water as a solvent, a proper amount of growth factors are added, the aspergillus niger, cellulomonas flavigena, saccharomycetes and bacillus subtilis which are added in sequence are taken as fermentation media, a crude tea saponin extract is prepared by multiple fermentation treatments, and finally the crude tea saponin extract is purified by a macroporous resin method to obtain a tea saponin product. CN101880307A discloses a method for extracting tea saponin by mixing and fermenting exogenously added high-starch substances, complex enzyme and alcohol yeast. The method comprises the steps of fully utilizing starch in exogenous high-starch substances by alcohol yeast to produce alcohol to form mash, soaking a certain amount of crushed oil-tea camellia cake meal into the fermented mash, uniformly mixing and culturing, carrying out solid-liquid separation after a few days, and concentrating and drying supernatant liquid of the separated liquid to obtain the final product tea saponin.
In the prior art, tea seed meal solid-state fermentation, multi-strain multi-time liquid-state fermentation or fermentation with water extract and enzymolysis liquid as substrates are mainly used, and a tea saponin product is extracted from obtained fermentation liquor by a method of macroporous resin adsorption, membrane separation or direct drying of the fermentation liquor. The large fungus time required by solid state fermentation needs 5-10 days, the period is long, the tea seed cake stacked inside cannot be fully decomposed, and the membrane separation efficiency is too low to be suitable for industrial production. Multiple times of sterilization and inoculation processes are required for multiple times of multi-strain fermentation, the process is complicated, macroporous resin is adopted for ethanol adsorption and elution in the separation process, the efficiency is greatly reduced, and a large amount of organic solvent is consumed. The enzymolysis liquid is used as a substrate, various enzyme preparations are added firstly, so that the production cost is greatly improved, and the product obtained by directly concentrating and drying the fermentation liquid has low purity and cannot meet the use requirement.
The method has the advantages that the water extract is directly used as a fermentation substrate, the treatment process is simple, the time is short, but the tea seed meal is discarded in the form of waste residues after being treated by the simple process in the whole fermentation process, cannot participate in subsequent microbial activities, cannot fully extract effective components of the tea seed meal, the complete fermentation process in a plurality of patents usually lasts for tens of days, and the key reason is that the concentration of fermentation raw materials is low, so that the process has huge energy consumption and cannot be produced in a large scale.
Disclosure of Invention
The invention provides a method for extracting tea saponin from tea seed meal by microbial fermentation. The utilization of metabolic products such as enzyme, organic acid and the like generated in the microbial growth process is beneficial to promoting the leaching of the tea saponin in plant cells, and a part of protein and sugar are consumed as growth factors, so that the obtained high-concentration tea saponin fermentation liquor can be directly applied in the fields of daily chemicals and the like. Aims to explore a green and environment-friendly tea saponin extraction production process with low cost, complement short plates faced by a water extraction method and a microbial fermentation method, provide theoretical reference for a resource development mechanism of oil-tea camellia meal, and have important reference significance for industrial production of extracting tea saponin by a fermentation method.
The extraction method of the tea saponin comprises the following steps:
a method for extracting tea saponin from tea seed meal by microbial fermentation comprises the following steps:
(1) uniformly mixing the crushed oil tea meal with a nitrogen source, a carbon source, inorganic salt and water, and sterilizing to prepare a fermentation medium;
(2) inoculating Lactobacillus plantarum (Lactobacillus plantarum) and Lactobacillus casei (Lactobacillus casei) into a fermentation culture medium, and performing mixed fermentation to obtain a tea seed meal fermentation stock solution;
(3) and carrying out centrifugal filtration on the fermentation stock solution to obtain clarified high-concentration tea saponin fermentation liquor, and then purifying to obtain the tea saponin.
Preferably, the components of the fermentation medium are: 8-12 wt% of oil tea meal, 0.2-1.5 wt% of nitrogen source, 0.5-3 wt% of carbon source, 0.1-0.5 wt% of inorganic salt and the balance of water.
Preferably, the nitrogen source is one or more of soybean peptone, tryptone and ammonium sulfate, and the addition amount is 0.5 +/-0.1 wt%.
Preferably, the carbon source is one or more of glucose, sucrose, maltose and fructose syrup, and the addition amount is 1.0 +/-0.2 wt%.
Preferably the inorganic salt is K 2 HPO 4 、KH 2 PO 4 、MgSO 4 One or more of them, the addition amount is 0.2 +/-0.05 wt%.
Preferably, the camellia oleifera meal is crushed to 60-80 meshes.
Preferably, the oil tea meal is 10 ± 0.5 wt%.
Preferably, the fermentation temperature is 30-42 ℃, the fermentation temperature is preferably 36-38 ℃, the natural pH value is realized, the rotating speed is 80-200 r/min, the fermentation time is preferably 120r/min, and the fermentation time is 24-72 h, preferably 48 h.
Preferably, the inoculation amount of the lactobacillus plantarum and the lactobacillus casei is 0.5-3%, preferably 1%, and the strain concentration of the lactobacillus plantarum seed solution is 10 7 ～10 10 CFU/mL, the strain concentration of the lactobacillus casei seed liquid is 10 7 ～10 10 CFU/mL。
Preferably, the rotation speed of the centrifugation in the step (3) is 4000-8000 r/min, preferably 6000r/min, the centrifugation time is 5-15 min, preferably 10min, the fermentation residues are removed by a vacuum filter, and the clarified fermentation liquor is obtained by filtering through a 0.45-micron water system membrane.
Preferably, the fermentation liquor is mixed with water saturated n-butyl alcohol and then stands for 5-12 h after the purification in the step (3), after the extraction is finished, vacuum rotary evaporation is carried out to remove the n-butyl alcohol, and the residual liquid after the rotary evaporation is subjected to vacuum drying.
Preferably, the temperature of the vacuum rotary evaporation is 40-70 ℃, preferably 60 ℃, and the rotating speed is 60-120 r/min, preferably 100 r/min. Keeping for 20-40 min, preferably for 30 min; the temperature of the vacuum drying is 60-80 ℃, the preferred temperature is 70 ℃, and the drying time is 6-12 h, the preferred time is 8 h.
Summary of the tea saponin extraction method: crushing the oil tea meal, performing mixed fermentation, performing centrifugal filtration on the fermentation liquor to obtain high-concentration tea saponin fermentation liquor, and then performing a purification process to obtain a tea saponin standard product.
The detailed steps of tea saponin extraction:
(1) oil tea meal pretreatment
The dried oil-tea camellia meal is processed by a high-speed powder beater, after the oil-tea camellia meal is crushed, cell walls are crushed, active ingredients in cells are exposed, but the crushing fineness influences the mixing uniformity and the subsequent processing steps, and the thinner the raw material is, the more the active ingredients in the cells are dissolved out. However, the fine raw materials are difficult to uniformly disperse in the fermentation substrate, so that the fermentation effect is poor, and the subsequent steps of centrifugation and filtration are difficult to perform, and finally the extraction rate of the tea saponin is low, and the purity of the tea saponin is low.
(2) Preparation of seed liquid
The fermentation strain is Lactobacillus plantarum and Lactobacillus casei (the optimal ratio is 1:1)
Lactobacillus plantarum GIM1.648 (LP) and Lactobacillus casei GIM 1.204 (LC) were dissolved in sterile water to prepare bacterial suspensions, respectively. Respectively transferring 5-20 mul of lactobacillus plantarum suspension and 5-20 mul of lactobacillus casei suspension to the surface of the MRS agar culture medium for lineation and activated culture. The culture temperature is 28-40 ℃, the preferable temperature is 37 ℃, and the culture time is 24-50 h, the preferable time is 48 h. The lactobacillus plantarum and lactobacillus casei on the plate are respectively inoculated to MRS broth culture medium by using an inoculating loop to prepare seed liquid. The culture temperature is 28-40 ℃, the preferable temperature is 37 ℃, and the culture time is 8-24 h, and the preferable time is 15 h.
Lactobacillus plantarum GIM1.648 (LP), Lactobacillus casei GIM 1.204 (LC) were purchased from Xiannong Biotech (Shanghai) Co., Ltd.
(3) Preparation of fermentation Medium
Adding oil tea meal, nitrogen source, carbon source, inorganic salt and water into a conical flask according to a certain proportion, uniformly mixing, sterilizing at 110-130 deg.C for 10-30 min, and storing for later use, preferably sterilizing at 121 deg.C for 15 min.
(4) Preparation, sterilization and clarification of fermentation liquor
Inoculating lactobacillus plantarum seed liquid and lactobacillus casei seed liquid into a fermentation culture medium in proportion for fermentation to obtain fermentation liquid.
And sterilizing and clarifying the obtained fermentation liquor. Wherein the sterilization condition is 60-90 ℃, preferably 80 ℃, and the heat preservation time is 15-30 min, preferably 20 min. The clarification treatment is achieved by centrifugation and filtration.
(5) Purification and post-treatment of tea saponin
And (2) mixing the fermentation liquor with water-saturated n-butyl alcohol (the water-saturated n-butyl alcohol means that the n-butyl alcohol and water are uniformly mixed in a ratio of 1:1, then standing for layering, and taking the n-butyl alcohol for use), and then standing for 5-12 h, preferably 8h, in a separating funnel. During the period, the extraction agent is violently shaken every 1-2 h, and after the extraction is finished, the extraction agent is subjected to vacuum rotary evaporation.
And recycling the n-butanol solvent obtained by rotary evaporation and condensation, and then carrying out vacuum drying on the residual liquid obtained by rotary evaporation. The dried solid is a tea saponin product, the purity of the tea saponin is above 70%, and the extraction rate of the tea saponin is 13-15%.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention only needs one-time inoculation of liquid fermentation, has simple and controllable process, easy operation, can realize industrial production, and has green and environment-friendly production process.
(2) The product obtained by the invention has high purity and high extraction rate.
Drawings
FIG. 1 is a graph of the concentration of tea saponin in a fermentation broth or an extract.
FIG. 2 is a graph of the extraction rate of tea saponin.
FIG. 3 is a diagram showing the purity of tea saponin.
FIG. 4 is a graph showing OH scavenging effect, wherein the concentration of tea saponin is 0.25 mg/mL.
FIG. 5 is a graph showing OH scavenging effect, wherein the concentration of tea saponin is 2 mg/mL.
FIG. 6. OH scavenging effect, wherein the tea saponin concentration was 4 mg/mL.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.
Raw material information: oil tea meal (provided by Jinchang biotechnology, Inc. of Hunan); soy peptone, tryptone, MRS broth (guangdong cyclokay microbiological technologies ltd); glucose, sucrose, K2HPO4, KH2PO4, MgSO4, (majol chemical reagent works, tianjin); ammonium sulfate, fructose syrup, maltose, n-butanol (Guangzhou chemical reagent factory)
Example 1
A method for extracting tea saponin from oil tea meal by using mixed microorganism fermentation is prepared by the following steps:
1) oil tea meal pretreatment
And (3) crushing the camellia oleifera abel dregs by using a high-speed powder mill, and selecting 60-80 meshes of camellia oleifera abel dregs as a fermentation raw material.
2) Preparation of seed liquid
The lactobacillus plantarum and the lactobacillus casei are respectively dissolved in sterile water to prepare lactobacillus plantarum suspension and lactobacillus casei suspension. Respectively transferring 10 mu L of lactobacillus plantarum suspension and lactobacillus casei suspension on the surfaces of different MRS agar culture media, and carrying out activated culture at the culture temperature of 37 ℃ for 48h to obtain activated lactobacillus plantarum and lactobacillus casei.
Inoculating activated lactobacillus plantarum and lactobacillus casei to different MRS broth through inoculating loops respectively for culturing at 37 ℃, and statically culturing for 15h in an incubator. The concentration of the strains of the lactobacillus plantarum and the lactobacillus casei reaches 10 7 CFU/mL or more.
3) Preparation of fermentation Medium
Mixing oil-tea camellia meal 10%, soybean peptone 0.5%, glucose 1%, and K0.2% 2 HPO 4 Adding into a conical flask, adding the rest water into the conical flask, mixing well, sterilizing at 121 deg.C for 15min, and storing for use.
4) Preparation, sterilization and clarification of fermentation liquor
Inoculating 1% of lactobacillus plantarum seed liquid and 1% of lactobacillus casei seed liquid into a fermentation culture medium, and fermenting for 48 hours at 37 ℃ under natural pH and the exclusive rotation speed of a stirring paddle of 120r/min to obtain fermentation liquor.
And (3) sterilization treatment: inactivating the microorganisms in the fermentation liquor by high temperature sterilization at 80 deg.C for 20 min.
Clarification treatment: centrifuging at 6000r/min for 10min, and filtering the supernatant with 0.45 μm water system membrane to obtain clear liquid of fermentation liquid.
5) Purification and post-treatment of tea saponin
Mixing the clear liquid of the fermentation liquor with water saturated n-butanol, standing in a pear-shaped separating funnel for 8h, shaking vigorously every 2h, carrying out vacuum rotary evaporation on the extractant at 60 ℃ after the extraction is finished, keeping the temperature at 100r/min for 15min, drying the liquid after the rotary evaporation at 70 ℃ for 8h under a vacuum condition, and drying the solid after the drying to obtain the tea saponin sample.
Example 2
A method for extracting tea saponin from oil tea meal by utilizing microbial mixed fermentation is prepared by the following steps:
1) oil tea meal pretreatment
Same as in example 1.
2) Preparation of seed liquid
The lactobacillus plantarum and the lactobacillus casei are respectively dissolved in sterile water to prepare lactobacillus plantarum suspension and lactobacillus casei suspension. Respectively transferring 20 mu L of lactobacillus plantarum suspension and lactobacillus casei suspension on the surfaces of different MRS agar culture media, and carrying out activated culture at the culture temperature of 37 ℃ for 40h to obtain activated lactobacillus plantarum and lactobacillus casei.
Inoculating activated lactobacillus plantarum and lactobacillus casei to different MRS broth through inoculating loops respectively for culturing at 37 ℃, and statically culturing for 20h in an incubator. The concentration of the strains of the lactobacillus plantarum and the lactobacillus casei reaches 10 8 CFU/mL or more.
3) Preparation of fermentation Medium
Mixing oil tea cake 12%, tryptone 0.5%, sucrose 1.5%, and KH 0.4% 2 PO 4 Adding into a conical flask, adding the rest water into the conical flask, mixing well, sterilizing at 121 deg.C for 15min, and storing for use.
4) Preparation, sterilization and clarification of fermentation liquor
Inoculating 2% of lactobacillus plantarum seed liquid and 1% of lactobacillus casei seed liquid into a fermentation medium, and fermenting for 48h at 37 ℃ and natural pH under the condition that the special rotating speed of a stirring paddle is 120r/min to obtain fermentation liquid.
And (3) sterilization treatment: inactivating the microorganisms in the fermentation liquor by high temperature sterilization at 85 deg.C for 18 min.
Clarification treatment: centrifuging at 8000r/min for 8min with high speed centrifuge, and filtering the supernatant with 0.45 μm water system membrane to obtain clear liquid of fermentation liquid.
5) Purification and post-treatment of tea saponin
Mixing the clear liquid of the fermentation liquor with water saturated n-butanol, standing in a pear-shaped separating funnel for 8h, shaking vigorously every 2h, performing vacuum rotary evaporation on the extractant at 65 ℃ after extraction is finished, keeping the temperature at 100r/min for 12min, drying the liquid after rotary evaporation at 70 ℃ for 8h under a vacuum condition, and drying the solid after drying to obtain the tea saponin sample.
Example 3
A method for extracting tea saponin from oil tea meal by using mixed microorganism fermentation is prepared by the following steps:
1) oil tea meal pretreatment
Same as in example 1.
2) Preparation of seed liquid
The lactobacillus plantarum and the lactobacillus casei are respectively dissolved in sterile water to prepare lactobacillus plantarum suspension and lactobacillus casei suspension. Respectively transferring 15 mu L of lactobacillus plantarum suspension and lactobacillus casei suspension on the surfaces of different MRS agar culture media, and carrying out activated culture at the culture temperature of 37 ℃ for 48h to obtain activated lactobacillus plantarum and lactobacillus casei.
Inoculating the activated lactobacillus plantarum and lactobacillus casei into different MRS broth respectively through inoculating loops for culturing at 37 ℃, and standing and culturing in an incubator for 15 h. The concentration of the strains of the lactobacillus plantarum and the lactobacillus casei reaches 10 7 CFU/mL or more.
3) Preparation of fermentation Medium
Mixing 8% of oilTea seed cake, 1% ammonium sulfate, 2% maltose, 0.2% K 2 HPO 4 Adding into a conical flask, adding the rest water into the conical flask, mixing, sterilizing at 121 deg.C for 15min, and storing.
4) Preparation, sterilization and clarification treatment of fermentation liquor
Inoculating 1% of lactobacillus plantarum seed liquid and 2% of lactobacillus casei seed liquid into a fermentation medium, and fermenting for 48h at 37 ℃ and natural pH under the condition that the special rotating speed of a stirring paddle is 120r/min to obtain fermentation liquid.
And (3) sterilization treatment: inactivating the microorganisms in the fermentation liquor by high temperature sterilization at 90 deg.C for 18 min.
Clarification treatment: centrifuging at 5000r/min for 12min with high speed centrifuge, and filtering the supernatant with 0.45 μm water system membrane to obtain fermentation broth supernatant.
5) Purification and post-treatment of tea saponin
Mixing the clear liquid of the fermentation liquor with water saturated n-butanol, standing in a pear-shaped separating funnel for 8h, shaking vigorously every 2h, performing vacuum rotary evaporation on the extractant at 50 ℃ after extraction is finished, keeping the temperature at 150r/min for 30min, drying the liquid after rotary evaporation at 75 ℃ for 6h under a vacuum condition, and drying the solid after drying to obtain the tea saponin sample.
Example 4
A method for extracting tea saponin from oil tea meal by using mixed microorganism fermentation is prepared by the following steps:
1) oil tea meal pretreatment
Same as in example 1.
2) Preparation of seed liquid
The lactobacillus plantarum and the lactobacillus casei are respectively dissolved in sterile water to prepare lactobacillus plantarum suspension and lactobacillus casei suspension. Respectively transferring 10 mu L of lactobacillus plantarum suspension and lactobacillus casei suspension on the surfaces of different MRS agar culture media, and carrying out activated culture at the culture temperature of 37 ℃ for 48h to obtain activated lactobacillus plantarum and lactobacillus casei.
Respectively passing activated Lactobacillus plantarum and Lactobacillus casei through inoculating loopInoculating into different MRS broth, culturing at 37 deg.C, and standing in incubator for 15 h. The concentration of the strains of the lactobacillus plantarum and the lactobacillus casei reaches 10 7 CFU/mL or more.
3) Preparation of fermentation Medium
Mixing 11% of oil-tea meal, 0.5% of soybean peptone, 1% of fructose-glucose syrup, and 0.3% of KH 2 PO 4 Adding into a conical flask, adding the rest water into the conical flask, mixing well, sterilizing at 121 deg.C for 15min, and storing for use.
4) Preparation, sterilization and clarification treatment of fermentation liquor
Inoculating 1.3% of lactobacillus plantarum seed liquid and 1% of lactobacillus casei seed liquid into a fermentation culture medium, and fermenting for 52h at 37 ℃ and natural pH under the condition that the special rotating speed of a stirring paddle is 100r/min to obtain fermentation liquid.
And (3) sterilization treatment: inactivating the microorganisms in the fermentation liquor by high temperature sterilization at 70 deg.C for 30 min.
Clarification treatment: centrifuging at 4000r/min for 20min by a high-speed centrifuge, and filtering the supernatant with 0.45 μm water system membrane to obtain the clear liquid of the fermentation liquid.
5) Purification and post-treatment of tea saponin
Mixing the clear liquid of the fermentation liquid with water saturated n-butyl alcohol, standing in a pear-shaped separating funnel for 8h, shaking violently once every 2h, carrying out vacuum rotary evaporation on an extracting agent at 55 ℃ after extraction is finished, keeping at 100r/min for 25min, drying the liquid after rotary evaporation at 70 ℃ for 8h under a vacuum condition, and drying the solid after drying to obtain a tea saponin sample.
Example 5
A method for extracting tea saponin from oil tea meal by using mixed microorganism fermentation is prepared by the following steps:
1) oil tea meal pretreatment
Same as in example 1.
2) Preparation of seed liquid
The lactobacillus plantarum and the lactobacillus casei are respectively dissolved in sterile water to prepare lactobacillus plantarum suspension and lactobacillus casei suspension. Respectively transferring 10 mu L of lactobacillus plantarum suspension and lactobacillus casei suspension on the surfaces of different MRS agar culture media, and carrying out activated culture at the culture temperature of 37 ℃ for 48h to obtain activated lactobacillus plantarum and lactobacillus casei.
Inoculating activated lactobacillus plantarum and lactobacillus casei to different MRS broth through inoculating loops respectively for culturing at 37 ℃, and statically culturing for 24h in an incubator. The concentration of the strains of the lactobacillus plantarum and the lactobacillus casei reaches 10 9 CFU/mL or more.
3) Preparation of fermentation Medium
Mixing oil tea meal 9%, tryptone 0.5%, glucose 1%, and MgSO 0.2% 4 Adding into a conical flask, adding the rest water into the conical flask, mixing well, sterilizing at 121 deg.C for 15min, and storing for use.
4) Preparation, sterilization and clarification of fermentation liquor
Inoculating 0.5% lactobacillus plantarum seed solution and 0.5% lactobacillus casei seed solution into a fermentation culture medium, and fermenting at 37 deg.C and natural pH for 50h with the exclusive rotation speed of the stirring paddle of 200r/min to obtain fermentation liquid.
And (3) sterilization treatment: inactivating the microorganisms in the fermentation broth by high temperature sterilization at 80 deg.C for 20 min.
Clarification treatment: centrifuging at 7000r/min for 8min by high speed centrifuge, and filtering the supernatant with 0.45 μm water system membrane to obtain fermentation liquid supernatant.
5) Purification and post-treatment of tea saponin
Mixing the clear liquid of the fermentation liquor with water saturated n-butanol, standing in a pear-shaped separating funnel for 8h, shaking vigorously every 2h, performing vacuum rotary evaporation on the extractant at 60 ℃ after extraction is finished, keeping the temperature at 100r/min for 15min, drying the liquid after rotary evaporation at 65 ℃ for 9h under vacuum, and drying the solid after drying to obtain the tea saponin sample.
Comparative example 1
Extracting tea saponin from oil tea cake by water extraction method
1) Oil tea meal pretreatment
Same as in example 1
2) Preparation and clarification treatment of water extract
Adding 10 wt% of the oil-tea camellia meal crushed in the step 1) into water, and extracting for 6 hours at 80 ℃ and the rotating speed of a stirring paddle of 120r/min to obtain water extract.
Clarification treatment: centrifuging at 6000r/min for 10min, and filtering the supernatant with 0.45 μm water system membrane to obtain water extractive solution.
3) Purification and post-treatment of tea saponin
Same as in example 1.
Comparative example 2
Comparative example 2 Lactobacillus casei alone was used for fermentation with an inoculum size of 2%, and the procedure was the same as in example 1.
Comparative example 3
Comparative example 3 fermentation was carried out using only lactobacillus plantarum, the inoculum size was 2%, and the remaining procedure was the same as in example 1.
Comparative example 4
Comparative example 4 the lactobacillus plantarum used in example 1 was replaced with saccharomyces cerevisiae with an inoculum size of 2%, and the remaining steps were the same as in example 1.
Comparative example 5
The addition amount of the tea seed cake in the step 3) of the example 1 is changed from 10 percent to 7 percent, and the rest steps are the same as the example 1.
Comparative example 6
The addition amount of the tea seed cake in the step 3) of the example 1 is changed from 10 percent to 13 percent, and the rest steps are the same as the example 1.
Comparative example 7
The method comprises the steps of changing the 60-80-mesh oil tea meal powder in the step 1) of the embodiment 1 into 40-50-mesh oil tea meal powder, and the rest steps are the same as the step 1 of the embodiment.
Comparative example 8
The method comprises the steps of changing the 60-80-mesh oil tea meal powder in the step 1) of the embodiment 1 into 90-100-mesh oil tea meal powder, and the rest steps are the same as the step 1 of the embodiment.
Test example 1-tea saponin extraction yield, purity
Reference (gazang, Yang scholar, Deng Xiaohong, Zhongfeng, Yangting, Camellia seed powder tea saponin high performance liquid color)Study of Spectrometry method [ J]2022, (02):28-31), adopting Shimadzu high performance liquid chromatography to measure the tea saponin concentration in the fermentation liquor, and optimizing and improving the method, wherein the chromatographic condition is Cholester Packed Column C18 Column (150mm x 4.6 mm); the column temperature is 30 ℃; the ultraviolet detection wavelength is 276 nm; 0.1% TFA as mobile phase, chromatographic grade methanol as organic phase, V (0.1% TFA water) V (methanol) 60: 40; the flow rate is 0.5 mL/min; the sample size was 5 uL. The 2 main chromatographic peaks respectively appear at about 10min and 14min, and the total area sum of the two chromatographic peaks is used for calculation. The regression equation of the standard curve is drawn as y 1656x-35428, R 2 ＝0.9997。
Dissolving the prepared crude product of the tea saponin with the mass of G in water with the volume of V, ultrasonically dissolving the crude product of the tea saponin, and measuring the concentration of C by using a high performance liquid chromatography of a merchant, wherein the calculation formulas of the purity and the extraction rate are as follows:
in the formula: x is the purity of tea saponin; y is the extraction rate of tea saponin; m is the addition amount of the tea seed meal powder, mg; n is the weight of the crude product of the tea saponin obtained by extraction, mg; g is the mass of the crude product of the tea saponin used for HPLC sample measurement, mg; c is concentration measured by HPLC, mg/ml; v is the volume used for the measurement, ml.
TABLE 1
As can be seen from the results of FIGS. 1-3 and Table 1, the extraction rate and purity of tea saponin in examples 1-5 are higher than those in comparative example 1, which shows that the extraction rate and purity of tea saponin are higher by the microbiological method of the present invention than by the aqueous extraction method. As is clear from the results of comparative examples 1 to 5 and comparative examples 2 to 4, the fermentation effect was better when Lactobacillus plantarum and Lactobacillus casei were used. Comparing the results of examples 1-5 and comparative examples 5-6, it can be seen that the addition amount of the oil tea meal is properly controlled to be 8-12%, and the fermentation effect beyond the range is poor. As is clear from the results of comparative examples 1 to 5 and comparative examples 7 to 8, the best fermentation effect was obtained by using 60 to 80 mesh of oil tea meal.
Test example 2-hydroxy radical (. OH) scavenging ability
The tea saponin samples prepared in the examples and the comparative examples are prepared into sample liquid of 0.25-4 mg/mL by using 75% ethanol, and the free radical scavenging capacity of the tea saponin extracted by the three methods is respectively measured.
Reference is made to the influence of different extraction modes and modifications on physicochemical properties and antioxidant activity of pericarp pectin of coffea arabica [ J/OL]Food and fermentation industry 1-14), taking 2.0mL of tea saponin solution with different mass concentrations, and sequentially adding 0.5mL of 6mmol/L FeSO 4 Solution and 0.5mL of 6mmol/L H 2 O 2 The solution is fully and evenly mixed and reacted for 10min at room temperature, then 0.5mL of salicylic acid with the concentration of 6mmol/L is respectively added and evenly mixed, the reaction is carried out for 30min at room temperature, and the absorbance A is measured at 510nm 1 . In the above reaction system, salicylic acid was replaced with absolute ethanol, and the absorbance was measured as A 2 Replacing the sample solution with distilled water, and measuring the absorbance as A 0 The hydroxyl radical clearance is calculated according to the following formula:
TABLE 2
As can be seen from the results of FIGS. 4-6 and Table 2, the effect of scavenging hydroxyl radicals of the tea saponin of examples 1-5 is better than that of comparative example 1, which shows that the microbial method of the present invention has better effect of scavenging hydroxyl radicals of the tea saponin than the water extraction method. As is clear from the results of comparative examples 1 to 5 and comparative examples 2 to 4, the effect of scavenging hydroxyl radicals is better when Lactobacillus plantarum and Lactobacillus casei are used for co-fermentation. Comparing the results of examples 1-5 and comparative examples 5-6, it is known that the addition amount of the camellia oleifera cake powder is properly controlled to be 8-12%, and the effect of removing hydroxyl radicals of the prepared saponin sample beyond the range is poor. Comparing the results of examples 1-5 and comparative examples 7-8, it can be seen that the hydroxyl radical scavenging effect of the sample of tea saponin obtained by using 60-80 mesh oil tea meal is better.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for extracting tea saponin from tea seed meal by microbial fermentation is characterized by comprising the following steps:
(1) uniformly mixing the crushed oil tea meal with a nitrogen source, a carbon source, inorganic salt and water, and sterilizing to prepare a fermentation medium;
(2) inoculating Lactobacillus plantarum (Lactobacillus plantarum) and Lactobacillus casei (Lactobacillus casei) into a fermentation culture medium, and performing mixed fermentation to obtain a tea seed meal fermentation stock solution;
(3) and carrying out centrifugal filtration on the fermentation stock solution to obtain clarified high-concentration tea saponin fermentation liquor, and then purifying to obtain the tea saponin.
2. The method of claim 1, wherein the fermentation medium comprises: 8-12 wt% of oil tea meal, 0.2-1.5 wt% of nitrogen source, 0.5-3 wt% of carbon source, 0.1-0.5 wt% of inorganic salt and the balance of water.
3. The method of claim 2, wherein the nitrogen source is one or more of soybean peptone, tryptone, and ammonium sulfate, and the addition amount is 0.5 ± 0.1 wt%;
the carbon source is one or more of glucose, sucrose, maltose and fructose-glucose syrup, and the addition amount is 1.0 +/-0.2 wt%;
the inorganic salt is K 2 HPO 4 、KH 2 PO 4 、MgSO 4 One or more of them, the addition amount is 0.2 +/-0.05 wt%.
4. The method according to claim 1, 2 or 3, characterized in that the camellia oleifera cake is crushed to 60-80 mesh.
5. The method according to claim 4, wherein the oil tea meal is 10 ± 0.5 wt%.
6. The method according to claim 4, wherein the fermentation temperature is 30-42 ℃ and the fermentation time is 24-72 h.
7. The method as claimed in claim 6, wherein the inoculation amount of Lactobacillus plantarum and Lactobacillus casei is 0.5% -3%, and the strain concentration of Lactobacillus plantarum is 10 7 ～10 10 CFU/mL, the concentration of the strain of lactobacillus casei is 10 7 ～10 10 CFU/mL。
8. The method according to claim 1, 2 or 3, wherein the rotation speed of the centrifugation in the step (3) is 4000-8000 r/min, the centrifugation time is 5-15 min, and the clarified fermentation liquor is obtained by filtering through a 0.45-micron water system membrane.
9. The method according to claim 1, 2 or 3, characterized in that in the purification in the step (3), the fermentation liquor is mixed with water saturated n-butanol and then is kept stand for 5-12 h, after the extraction is finished, the extractant is subjected to vacuum rotary evaporation to recover the n-butanol, and the residual liquid after the rotary evaporation is subjected to vacuum drying.
10. The method according to claim 1, 2 or 3, wherein the temperature of the vacuum rotary evaporation is 40-70 ℃, the rotating speed is 60-120 r/min, and the temperature is kept for 20-40 min; the drying time of the vacuum drying is 6-12 h at the temperature of 60-80 ℃.
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