CN115029385B - Method for extracting tea saponin from tea seed cake by microbial fermentation - Google Patents

Method for extracting tea saponin from tea seed cake by microbial fermentation Download PDF

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CN115029385B
CN115029385B CN202210875635.0A CN202210875635A CN115029385B CN 115029385 B CN115029385 B CN 115029385B CN 202210875635 A CN202210875635 A CN 202210875635A CN 115029385 B CN115029385 B CN 115029385B
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fermentation
tea
tea saponin
lactobacillus plantarum
lactobacillus casei
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程建华
韩帅鹏
华涛
蓝秀权
周心慧
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South China University of Technology SCUT
South China Institute of Collaborative Innovation
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Abstract

The invention belongs to the field of biological fermentation, and discloses a method for extracting tea saponin from tea-oil camellia 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 culture medium; (2) Inoculating lactobacillus plantarum (Lactobacillus plantarum) and lactobacillus casei (Lactobacillus casei) into a fermentation medium, and performing mixed bacteria liquid fermentation to obtain fermentation stock solution; (3) And (3) centrifugally filtering the fermentation stock solution to obtain clarified tea saponin fermentation liquor, and purifying to obtain tea saponin. The invention only needs one-time inoculation liquid state fermentation, has simple and controllable process and 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

Method for extracting tea saponin from tea seed cake by microbial fermentation
Technical Field
The invention belongs to the field of biological fermentation, and particularly relates to a method for extracting tea saponin from tea seed cake by utilizing microbial fermentation.
Background
The oil tea is a world 4 large woody oil crop, and the total planting scale of the oil tea in China reaches 446.67 ten thousand hectares. A large amount of oil tea cakes are generated after oil tea seed oil extraction, and the oil tea cakes 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 formed by sapogenin (i.e. ligand), sugar body and organic acid. 7 sapogenins were isolated from tea saponin altogether. Tea saponin was first isolated from tea tree seeds by the Japanese scholars Qingshan Xinjiang in 1931. The purified tea saponin is milk white or light yellow solid amorphous powder, has hygroscopicity, bitter and spicy taste, and has the characteristic of stimulating nasal mucosa. Has good activity in the aspects of emulsification, dispersion, wetting, foaming, foam stabilization, decontamination and the like, and is a natural surfactant. Tea saponin has not only surface activity but also biological activity. Has remarkable effects in resisting bacteria, sterilizing, resisting permeation, diminishing inflammation, eliminating phlegm, relieving cough, dispelling alcohol and the like. So the tea saponin has wide application. Can be used as shampoo and bath agent in daily use and chemical industry; the foaming stabilizer can be used in the building material industry; biological pesticides which can be used for preventing and controlling plant diseases and insect pests in agriculture; the culture can be used for clearing Chi Ji, clearing the enemy fishes in the shrimp pond, and also can be used for immersing and killing the adult snails, young snails and snail eggs of oncomelania, and the like.
The oil tea cake contains 10-15% of 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 current extraction process of tea saponin mainly adopts a water extraction method, and researches on extracting tea saponin by a microbial fermentation method are freshly reported. However, the water extraction and microorganism extraction of the prior art face the problem of low extraction rate and purity of tea saponin.
CN104357527a discloses a method for extracting tea saponin by solid state fermentation of large-scale edible fungi. The method takes tea seed cake as a main culture medium, is matched with bran and other auxiliary materials, is inoculated with microorganisms, carries out solid-state fermentation, realizes efficient exudation of tea saponin, and carries out ethanol leaching on the exuded tea saponin and then carries out membrane separation to obtain a tea saponin product. CN101880307a discloses a method for extracting tea saponin from tea seed cake by fermenting aspergillus niger, xanthomonas chrysogena, saccharomycetes and bacillus subtilis at one time, the method uses crushed dry tea seed cake as raw material, water as solvent, adding proper amount of growth factors, and sequentially adding aspergillus niger, xanthomonas chrysogena, saccharomycetes and bacillus subtilis as fermentation medium, and obtaining crude extract of tea saponin through multiple fermentation treatment, and finally purifying the crude extract by macroporous resin method, thus obtaining tea saponin product. CN101880307a discloses a method for extracting tea saponin by mixing and fermenting exogenously added high starch substances with complex enzyme and alcohol yeast. The starch in the exogenous high-starch substances is fully utilized by alcohol yeast to generate alcohol so as to form mash, then a certain amount of crushed oil tea cake meal is soaked into the fermented mash, mixed and cultured uniformly, after a few days, solid-liquid separation is carried out, and the supernatant fluid of the separated liquid is concentrated and dried to obtain the final product tea saponin.
In the prior art, tea seed cake solid state fermentation, multi-strain multi-time liquid state fermentation or fermentation mainly takes water extract and enzymolysis liquid as substrates, and tea saponin products are extracted from the obtained fermentation liquid by macroporous resin adsorption, membrane separation or direct drying of the fermentation liquid. The time of the large fungi required for solid state fermentation is 5-10 days, the period is long, tea seed meal stacked inside cannot be fully decomposed, and the efficiency of membrane separation is too low to be suitable for industrial production. The multi-strain multi-fermentation is subjected to multiple sterilization and inoculation processes, the process is complicated, and the separation process adopts macroporous resin to adsorb ethanol for eluting, so that the efficiency is greatly reduced, and a large amount of organic solvent is consumed. The zymolysis liquid is taken as a substrate, and a plurality of enzyme preparations are firstly added, so that the production cost is greatly improved, and the purity of the product obtained by directly concentrating and drying the fermentation liquid is very low and can not meet the use requirement.
The water extract is directly used as a fermentation substrate, the treatment process is simple and short, but the tea seed cake is discarded in the form of waste residue after being treated by the simple process in the whole fermentation process, the tea seed cake cannot participate in the subsequent microbial activities, the effective components of the tea seed cake cannot be fully extracted, the complete fermentation process in a plurality of patents usually lasts for tens of days, and the core reason is that the concentration of fermentation raw materials is low, so that the process has huge energy consumption and cannot be used for large-scale production.
Disclosure of Invention
The invention provides a method for extracting tea saponin from tea seed cake by utilizing microbial fermentation. The use of metabolites such as enzymes, organic acids and the like generated in the growth process of microorganisms is beneficial to promoting the leaching of tea saponin in plant cells, and consumes a part of protein sugar as a growth factor, so that the obtained high-concentration tea saponin fermentation liquor can be directly applied to the fields of daily chemicals and the like. Aims at exploring a green and environment-friendly tea saponin extraction production process with lower cost, complementing short plates faced by a water extraction method and a microbial fermentation method, providing theoretical reference for a tea-oil cake resource development mechanism, and having important reference significance for industrial production of extracting tea saponin by the fermentation method.
The extraction method of the tea saponin comprises the following steps:
a method for extracting tea saponin from tea seed cake 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 culture medium;
(2) Inoculating lactobacillus plantarum (Lactobacillus plantarum) and lactobacillus casei (Lactobacillus casei) into a fermentation medium, and performing mixed fermentation to obtain a tea seed cake fermentation stock solution;
(3) And (3) centrifugally filtering the fermentation stock solution to obtain clarified high-concentration tea saponin fermentation liquor, and purifying to obtain tea saponin.
Preferably, the components of the fermentation medium are: 8 to 12 weight percent of oil tea meal, 0.2 to 1.5 weight percent of nitrogen source, 0.5 to 3 weight percent of carbon source, 0.1 to 0.5 weight percent 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-glucose syrup, and the addition amount is 1.0+/-0.2 wt%.
Preferably the inorganic salt is K 2HPO4、KH2PO4、MgSO4 or more, added in an amount of 0.2.+ -. 0.05wt%.
Preferably, the oil tea meal is crushed to 60-80 meshes.
Preferably, the oil tea meal is 10+/-0.5 wt%.
Preferably, the fermentation temperature is 30-42 ℃, preferably 36-38 ℃, the natural pH is 80-200 r/min, preferably 120r/min, and the fermentation time is 24-72 h, preferably 48h.
Preferably, the inoculation amount of the lactobacillus plantarum and the lactobacillus casei is 0.5% -3%, preferably 1%, the strain concentration of the lactobacillus plantarum seed solution is 10 7～1010 CFU/mL, and the strain concentration of the lactobacillus casei seed solution is 10 7～1010 CFU/mL.
Preferably, the rotational 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 suction filter, and the clarified fermentation broth is obtained by filtering through a 0.45 μm water-based membrane.
Preferably, in the step (3), the fermentation liquor is mixed with water saturated n-butanol and then is kept stand for 5 to 12 hours, after the extraction is finished, the n-butanol is removed by vacuum rotary evaporation, and the residual liquid after rotary evaporation is dried in vacuum.
Preferably, the temperature of the vacuum rotary steaming is 40-70 ℃, preferably 60 ℃, and the rotating speed is 60-120 r/min, preferably 100r/min. Maintaining for 20-40 min, preferably 30min; the temperature of the vacuum drying is 60-80 ℃, preferably 70 ℃, and the drying time is 6-12 h, preferably 8h.
Summarizing the tea saponin extraction method: crushing tea-oil camellia meal, performing mixed fermentation, performing centrifugal filtration on the fermentation liquor to obtain high-concentration tea saponin fermentation liquor, and performing purification to obtain a tea saponin standard.
The tea saponin extraction method comprises the following steps:
(1) Pretreatment of oil tea meal
The high-speed pulverizing machine is used for processing the dried oil tea meal, after the oil tea meal is pulverized, cell walls are broken, and effective components in cells are exposed, but the pulverizing fineness influences the mixing uniformity and the subsequent processing steps, and the finer the raw materials, the more beneficial the dissolution of the effective components in cells. However, the finer the raw materials, the more difficult it is to uniformly disperse in the fermentation medium, so that the fermentation effect is poor, the subsequent centrifugation and filtration steps are also difficult to carry out, and finally the extraction rate of tea saponin is low, and the purity of tea saponin is low.
(2) Preparation of seed liquid
The fermentation strain is Lactobacillus plantarum and Lactobacillus casei (optimal ratio is 1:1)
Lactobacillus plantarum (Lactobacillus plantarum GIM1.648, LP) and Lactobacillus casei (Lactobacillus casei GIM.204, LC) were each dissolved in sterile water to form a bacterial suspension. And respectively transferring 5-20 mu L of lactobacillus plantarum suspension and 5-20 mu L of lactobacillus casei suspension to the surface of the MRS agar culture medium for streaking and activating culture. The temperature of the culture is 28-40 ℃, preferably 37 ℃, and the time of the culture is 24-50 hours, preferably 48 hours. Seed solutions were prepared by inoculating Lactobacillus plantarum and Lactobacillus casei on plates with an inoculating loop, respectively, to MRS broth. The temperature of the culture is 28-40 ℃, preferably 37 ℃, and the time of the culture is 8-24 hours, preferably 15 hours.
Lactobacillus plantarum (Lactobacillus plantarum GIM1.648, LP) and Lactobacillus casei (Lactobacillus casei GIM.204, LC) were purchased from Xiannong Biotechnology (Shanghai) Inc.
(3) Preparation of fermentation Medium
Adding oil tea meal, nitrogen source, carbon source, inorganic salt and water into a conical flask in proportion, uniformly mixing, sterilizing at 110-130 ℃ for 10-30 min, and storing for later use, preferably sterilizing at 121 ℃ for 15min.
(4) Preparation, sterilization and clarification of fermentation broths
Inoculating lactobacillus plantarum seed solution and lactobacillus casei seed solution into a fermentation medium in proportion for fermentation to obtain fermentation liquor.
And sterilizing and clarifying the obtained fermentation broth. Wherein the sterilization condition is 60-90 ℃, preferably 80 ℃, and the heat preservation is carried out for 15-30 min, preferably 20min. Clarification is achieved by centrifugation and filtration.
(5) Purification and post-treatment of tea saponin
Mixing the fermentation broth with water-saturated n-butanol (water-saturated n-butanol means that n-butanol is uniformly mixed with water in a ratio of 1:1, and then standing for layering, wherein n-butanol is taken for use), and standing for 5-12 h, preferably 8h, in a separating funnel. Shaking vigorously every 1-2 h, and performing vacuum rotary steaming on the extractant after extraction.
Recycling the n-butanol solvent obtained by rotary evaporation and condensation, and then carrying out vacuum drying on the residual liquid after rotary evaporation. The solid obtained by drying is 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 liquid state fermentation, has simple and controllable process and 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 showing the concentration of tea saponin in fermentation broth or extract.
FIG. 2 is a graph showing the extraction rate of tea saponin.
FIG. 3 is a graph showing purity of tea saponin.
FIG. 4 is a graph showing the OH-scavenging effect, wherein the concentration of tea saponin was 0.25mg/mL.
FIG. 5 shows OH-scavenging effect wherein tea saponin concentration was 2mg/mL.
FIG. 6-OH scavenging effect, wherein tea saponin concentration was 4mg/mL.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but embodiments of the present invention are not limited thereto, and may be performed with reference to conventional techniques for process parameters that are not specifically noted.
Raw material information: tea-oil cake (supplied by the biological technology limited company of gold, churn, hunan); soytone, tryptone, MRS broth (guangdong cyca, microbiology limited); glucose, sucrose, K2HPO4, KH2PO4, mgSO4, (Tianjin metallocene chemical reagent factory); ammonium sulfate, fructose syrup, maltose, n-butanol (Guangzhou chemical reagent plant)
Example 1
A method for extracting tea saponin from oil tea meal by microbial mixed fermentation is prepared by the following steps:
1) Pretreatment of oil tea meal
Crushing the oil tea meal by a high-speed powder crusher, and selecting 60-80 mesh oil tea meal powder 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. And respectively transferring 10 mu L of lactobacillus plantarum suspension and lactobacillus casei suspension, streaking on the surfaces of different MRS agar culture mediums, and carrying out activation culture at 37 ℃ for 48 hours to obtain activated lactobacillus plantarum and lactobacillus casei.
The activated lactobacillus plantarum and lactobacillus casei are respectively inoculated into different MRS broth through inoculating loops for culture, the culture temperature is 37 ℃, and the incubator is used for static culture for 15 hours. The strain concentration of the lactobacillus plantarum and lactobacillus casei reaches more than 10 7 CFU/mL.
3) Preparation of fermentation Medium
Adding 10% of oil tea meal, 0.5% of soybean peptone, 1% of glucose and 0.2% of K 2HPO4 into a conical flask, adding the rest water into the conical flask, uniformly mixing, sterilizing at 121 ℃ for 15min, and storing for later use.
4) Preparation, sterilization and clarification of fermentation broths
Inoculating 1% lactobacillus plantarum seed solution and 1% lactobacillus casei seed solution into a fermentation medium, and fermenting for 48 hours at 37 ℃ under the condition that the specific rotation speed of a stirring paddle is 120r/min at natural pH to obtain fermentation liquor.
And (3) sterilization treatment: and (3) inactivating microorganisms in the fermentation broth by using a high-temperature sterilization method, wherein the sterilization temperature is 80 ℃, and the treatment time is 20min.
And (3) clarifying: centrifuging at 6000r/min for 10min, 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 for 8 hours in a pear-shaped separating funnel, shaking vigorously every 2 hours, carrying out vacuum rotary evaporation on an extractant at 60 ℃ and maintaining at 100r/min for 15min after extraction, drying the rotary evaporated liquid at 70 ℃ for 8 hours under vacuum condition, and drying the dried solid to obtain the tea saponin sample.
Example 2
A method for extracting tea saponin from oil tea meal by microbial mixed fermentation is prepared by the following steps:
1) Pretreatment of oil tea meal
The 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. And respectively transferring 20 mu L of lactobacillus plantarum suspension and lactobacillus casei suspension, streaking on the surfaces of different MRS agar culture mediums, and carrying out activation culture at 37 ℃ for 40 hours to obtain activated lactobacillus plantarum and lactobacillus casei.
The activated lactobacillus plantarum and lactobacillus casei are respectively inoculated into different MRS broth through inoculating loops for culture, the culture temperature is 37 ℃, and the incubator is used for static culture for 20 hours. The strain concentration of the lactobacillus plantarum and lactobacillus casei reaches more than 10 8 CFU/mL.
3) Preparation of fermentation Medium
Adding oil tea meal 12%, tryptone 0.5%, sucrose 1.5% and KH 2PO4 into a conical flask, adding water, mixing, sterilizing at 121deg.C for 15min, and storing.
4) Preparation, sterilization and clarification of fermentation broths
Inoculating 2% lactobacillus plantarum seed solution and 1% lactobacillus casei seed solution into a fermentation medium, and fermenting for 48 hours at 37 ℃ under the condition that the specific rotation speed of a stirring paddle is 120r/min at natural pH to obtain fermentation liquor.
And (3) sterilization treatment: and (3) inactivating microorganisms in the fermentation broth by a high-temperature sterilization method, wherein the sterilization temperature is 85 ℃, and the treatment time is 18min.
And (3) clarifying: centrifuging at 8000r/min for 8min, 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 for 8 hours in a pear-shaped separating funnel, shaking vigorously every 2 hours, carrying out vacuum rotary steaming on an extractant at 65 ℃ and 100r/min for 12min after extraction, drying the rotary steamed liquid at 70 ℃ for 8 hours under vacuum condition, and drying the dried solid to obtain the tea saponin sample.
Example 3
A method for extracting tea saponin from oil tea meal by microbial mixed fermentation is prepared by the following steps:
1) Pretreatment of oil tea meal
The 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. And respectively transferring 15 mu L of lactobacillus plantarum suspension and lactobacillus casei suspension, streaking on the surfaces of different MRS agar culture mediums, and carrying out activation culture at 37 ℃ for 48 hours to obtain activated lactobacillus plantarum and lactobacillus casei.
The activated lactobacillus plantarum and lactobacillus casei are respectively inoculated into different MRS broth through inoculating loops for culture, the culture temperature is 37 ℃, and the incubator is used for static culture for 15 hours. The strain concentration of the lactobacillus plantarum and lactobacillus casei reaches more than 10 7 CFU/mL.
3) Preparation of fermentation Medium
Adding 8% of oil tea meal, 1% of ammonium sulfate, 2% of maltose and 0.2% of K 2HPO4 into a conical flask, adding the rest water into the conical flask, uniformly mixing, sterilizing at 121 ℃ for 15min, and storing for later use.
4) Preparation, sterilization and clarification of fermentation broths
Inoculating 1% lactobacillus plantarum seed solution and 2% lactobacillus casei seed solution into a fermentation medium, and fermenting for 48 hours at 37 ℃ under the condition that the specific rotation speed of a stirring paddle is 120r/min at natural pH to obtain fermentation liquor.
And (3) sterilization treatment: and (3) inactivating microorganisms in the fermentation broth by using a high-temperature sterilization method, wherein the sterilization temperature is 90 ℃, and the treatment time is 18min.
And (3) clarifying: centrifuging at 5000r/min for 12min, 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 for 8 hours in a pear-shaped separating funnel, shaking vigorously every 2 hours, carrying out vacuum rotary evaporation on an extractant at 50 ℃ for 30 minutes at 150r/min after extraction, drying the rotary evaporated liquid at 75 ℃ for 6 hours under vacuum condition, and drying the dried solid to obtain the tea saponin sample.
Example 4
A method for extracting tea saponin from oil tea meal by microbial mixed fermentation is prepared by the following steps:
1) Pretreatment of oil tea meal
The 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. And respectively transferring 10 mu L of lactobacillus plantarum suspension and lactobacillus casei suspension, streaking on the surfaces of different MRS agar culture mediums, and carrying out activation culture at 37 ℃ for 48 hours to obtain activated lactobacillus plantarum and lactobacillus casei.
The activated lactobacillus plantarum and lactobacillus casei are respectively inoculated into different MRS broth through inoculating loops for culture, the culture temperature is 37 ℃, and the incubator is used for static culture for 15 hours. The strain concentration of the lactobacillus plantarum and lactobacillus casei reaches more than 10 7 CFU/mL.
3) Preparation of fermentation Medium
Adding 11% of oil tea meal, 0.5% of soybean peptone, 1% of high fructose syrup and 0.3% of KH 2PO4 into a conical flask, adding the rest water into the conical flask, uniformly mixing, sterilizing at 121 ℃ for 15min, and storing for later use.
4) Preparation, sterilization and clarification of fermentation broths
Inoculating 1.3% lactobacillus plantarum seed solution and 1% lactobacillus casei seed solution into a fermentation medium, and fermenting for 52 hours under the condition that the temperature is 37 ℃ and the natural pH value is 100r/min and the special rotation speed of a stirring paddle to obtain fermentation liquid.
And (3) sterilization treatment: and (3) inactivating microorganisms in the fermentation broth by using a high-temperature sterilization method, wherein the sterilization temperature is 70 ℃, and the treatment time is 30min.
And (3) clarifying: centrifuging at 4000r/min for 20min, 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 for 8 hours in a pear-shaped separating funnel, shaking vigorously every 2 hours, carrying out vacuum rotary steaming on an extractant at 55 ℃ and maintaining at 100r/min for 25 minutes after extraction, drying the rotary steamed liquid at 70 ℃ for 8 hours under vacuum condition, and drying the dried solid to obtain the tea saponin sample.
Example 5
A method for extracting tea saponin from oil tea meal by microbial mixed fermentation is prepared by the following steps:
1) Pretreatment of oil tea meal
The 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. And respectively transferring 10 mu L of lactobacillus plantarum suspension and lactobacillus casei suspension, streaking on the surfaces of different MRS agar culture mediums, and carrying out activation culture at 37 ℃ for 48 hours to obtain activated lactobacillus plantarum and lactobacillus casei.
The activated lactobacillus plantarum and lactobacillus casei are respectively inoculated into different MRS broth through inoculating loops for culture, the culture temperature is 37 ℃, and the incubator is used for static culture for 24 hours. The strain concentration of the lactobacillus plantarum and lactobacillus casei reaches more than 10 9 CFU/mL.
3) Preparation of fermentation Medium
Adding 9% of oil tea meal, 0.5% of tryptone, 1% of glucose and 0.2% of MgSO 4 into a conical flask, adding the rest water into the conical flask, uniformly mixing, sterilizing at 121 ℃ for 15min, and storing for later use.
4) Preparation, sterilization and clarification of fermentation broths
Inoculating 0.5% lactobacillus plantarum seed solution and 0.5% lactobacillus casei seed solution into a fermentation medium, and fermenting for 50h under the condition that the temperature is 37 ℃ and the natural pH value is that the special rotating speed of a stirring paddle is 200r/min to obtain fermentation liquor.
And (3) sterilization treatment: and (3) inactivating microorganisms in the fermentation broth by using a high-temperature sterilization method, wherein the sterilization temperature is 80 ℃, and the treatment time is 20min.
And (3) clarifying: centrifuging at 7000r/min for 8min, 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 for 8 hours in a pear-shaped separating funnel, shaking vigorously every 2 hours, carrying out vacuum rotary evaporation on an extractant at 60 ℃ and 100r/min for 15min after extraction, drying the rotary evaporated liquid at 65 ℃ for 9 hours under vacuum condition, and drying the dried solid to obtain the tea saponin sample.
Comparative example 1
Extracting tea saponin from oil tea meal by water extraction method
1) Pretreatment of oil tea meal
The same as in example 1
2) Preparation and clarification of aqueous extracts
Adding 10wt% of the oil tea meal crushed in the step 1) into water, and extracting for 6 hours at the temperature of 80 ℃ and the rotation speed of a stirring paddle of 120r/min to obtain an aqueous extract.
And (3) clarifying: centrifuging at 6000r/min for 10min, and filtering the supernatant with 0.45 μm water system membrane to obtain water extract supernatant.
3) Purification and post-treatment of tea saponin
The same as in example 1.
Comparative example 2
Comparative example 2 was carried out by selecting only lactobacillus casei for fermentation, the inoculum size was 2%, and the other steps were the same as in example 1.
Comparative example 3
Comparative example 3 was carried out by selecting only lactobacillus plantarum for fermentation, the inoculum size was 2%, and the other steps were the same as in example 1.
Comparative example 4
Comparative example 4 the lactobacillus plantarum of example 1 was replaced with saccharomyces cerevisiae with an inoculum size of 2%, and the rest of the procedure was the same as in example 1.
Comparative example 5
The procedure of example 1 was repeated except that the amount of tea seed cake added in step 3) of example 1 was changed from 10% to 7%.
Comparative example 6
The procedure of example 1 was repeated except that the amount of tea seed cake added in step 3) of example 1 was changed from 10% to 13%.
Comparative example 7
The 60-80 mesh oil tea meal powder in the step 1) of the embodiment 1 is changed into 40-50 mesh oil tea meal powder, and the rest steps are the same as the embodiment 1.
Comparative example 8
The 60-80 mesh oil tea meal powder in the step 1) of the embodiment 1 is changed into 90-100 mesh oil tea meal powder, and the rest steps are the same as the embodiment 1.
Test example 1 tea saponin extraction yield, purity
Reference (Jiang Xun, yang Xuejun, deng Xiaogong, zhou Feng, yang Ting. Research on high performance liquid chromatography of tea saponin in tea seed powder [ J ]. Brand and standardization, 2022, (02): 28-31), measuring concentration of tea saponin in fermentation broth by using Shimadzu high performance liquid chromatography, and optimizing and improving the method under the chromatographic condition of Cholester Packed Column C column (150 mm x 4.6 mm); the column temperature is 30 ℃; the ultraviolet detection wavelength is 276nm;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 0.5mL/min; the sample loading was 5uL. The 2 main chromatographic peaks are respectively obtained at about 10min and 14min, and the total area sum of the two chromatographic peaks is calculated. The standard curve regression equation is drawn as y=1656x-35428, and r 2 =0.9997.
The purity and extraction rate of the tea saponin are that the prepared crude product of the tea saponin with the mass of G is dissolved in water with the volume of V, the concentration of the crude product of the tea saponin is C after ultrasonic dissolution by using high performance liquid chromatography of commercial tenant, and the calculation formulas of the purity and the extraction rate are as follows:
Wherein: x is the purity of tea saponin; y is the extraction rate of tea saponin; m is the addition amount of tea seed meal powder, mg; n is the mass of crude tea saponin obtained by extraction and mg; g is the mass of the crude tea saponin product used for HPLC detection and mg; c is the 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 to 3 and Table 1, the extraction rate and purity of the tea saponin in examples 1 to 5 are higher than those in comparative example 1, which shows that the extraction rate and purity of the tea saponin are higher by the microbial method than by the water extraction method. As is clear from the results of comparative examples 1 to 5 and comparative examples 2 to 4, the fermentation effect of Lactobacillus plantarum and Lactobacillus casei was better. As is clear from the results of comparative examples 1 to 5 and comparative examples 5 to 6, in the present invention, the addition amount of the oil-tea camellia meal was suitably controlled to 8 to 12%, and the fermentation effect was poor when exceeding this range. As is clear from the results of comparative examples 1 to 5 and comparative examples 7 to 8, the fermentation effect was best when 60 to 80 mesh oil tea meal was selected.
Test example 2-hydroxy radical (. OH) scavenging ability
The tea saponin samples prepared in the examples and the comparative examples were prepared into 0.25-4 mg/mL samples with 75% ethanol, and the radical scavenging ability of the tea saponin extracted by the three methods was measured, respectively.
Referring to Li Xiaojiao (Li Xiaojiao, wang Yujie, yang Lihua, etc. the method for influencing physicochemical properties and antioxidant activity of small coffee pericarp pectin by different extraction modes and modification [ J/OL ]. Food and fermentation industries: 1-14), 2.0mL of tea saponin solution with different mass concentrations is taken, 0.5mL of FeSO 4 solution with 6mmol/L and 0.5mL of H 2O2 solution with 6mmol/L are sequentially added, and after full mixing, the mixture is reacted at room temperature for 10min, 0.5mL of 6mmol/L salicylic acid is respectively added and mixed uniformly, and the reaction is carried out at room temperature for 30min, wherein the absorbance at 510nm is measured as A 1. In the reaction system, absolute ethyl alcohol is used for replacing salicylic acid, absorbance is measured to be A 2, distilled water is used for replacing a sample solution, absorbance is measured to be A 0, and 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 effects of the tea saponins of examples 1-5 on scavenging hydroxy radicals are all better than those of comparative example 1, which shows that the microbial method of the invention has better effect on scavenging hydroxy radicals on tea saponins 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 after co-fermentation with Lactobacillus plantarum and Lactobacillus casei was better. As is clear from the results of comparative examples 1 to 5 and comparative examples 5 to 6, in the present invention, the amount of the oil tea meal added was suitably controlled to 8 to 12%, and the prepared grass saponin sample exceeding this range had a poor effect of scavenging hydroxyl radicals. As can be seen from the results of comparative examples 1 to 5 and comparative examples 7 to 8, the effect of scavenging hydroxyl radicals was better for the samples of tea saponin obtained by selecting 60-80 mesh oil tea meal.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (6)

1. A method for extracting tea saponin from tea seed cake by utilizing microbial fermentation is characterized by comprising the following steps:
(1) Uniformly mixing the oil tea meal crushed to 60-80 meshes with a nitrogen source, a carbon source, inorganic salt and water, and sterilizing to prepare a fermentation culture medium; the nitrogen source is one or more of soybean peptone, tryptone and ammonium sulfate, the carbon source is one or more of glucose, sucrose, maltose and fructose syrup, and the inorganic salt is one or more of K 2HPO4、KH2PO4、MgSO4;
(2) Inoculating lactobacillus plantarum (Lactobacillus plantarum) GIM1.648 and lactobacillus casei (Lactobacillus casei) GIM 1.204 into a fermentation medium, and performing mixed fermentation to obtain tea seed cake fermentation stock solution;
(3) Centrifugally filtering the fermentation stock solution to obtain clarified high-concentration tea saponin fermentation liquor, and purifying to obtain tea saponin;
The components of the fermentation medium are as follows: 8 to 12 weight percent of oil tea meal, 0.2 to 1.5 weight percent of nitrogen source, 0.5 to 3 weight percent of carbon source, 0.1 to 0.5 weight percent of inorganic salt and the balance of water; the inoculation amount of the lactobacillus plantarum and the lactobacillus casei is 0.5% -3%, the strain concentration of the lactobacillus plantarum is 10 7～1010 CFU/mL, and the strain concentration of the lactobacillus casei is 10 7～1010 CFU/mL;
And (3) mixing the fermentation liquor with water saturated n-butanol, standing for 5-12 h, performing vacuum rotary evaporation on the extractant to recover n-butanol after extraction, and performing vacuum drying on the residual liquid after rotary evaporation.
2. The method according to claim 1, wherein the nitrogen source is added in an amount of 0.5±0.1wt%; the addition amount of the carbon source is 1.0+/-0.2 weight percent; the addition amount of the inorganic salt is 0.2 plus or minus 0.05 weight percent.
3. The method according to claim 1 or 2, wherein the oil tea meal is 10±0.5wt%.
4. The method according to claim 1 or 2, wherein the fermentation temperature is 30-42 ℃ and the fermentation time is 24-72 hours.
5. The method according to claim 1 or 2, wherein the rotational speed of the centrifugation in step (3) is 4000-8000 r/min, the centrifugation time is 5-15 min, and the clarified fermentation broth is obtained by filtration through a 0.45 μm aqueous membrane.
6. The method according to claim 1 or 2, wherein the vacuum rotary steaming temperature is 40-70 ℃, the rotation speed is 60-120 r/min, and the vacuum rotary steaming time is kept for 20-40 min; the temperature of the vacuum drying is 60-80 ℃ and the drying time is 6-12 h.
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