Method for flocculating and purifying tea saponin by using microbial flocculant

Abstract

The invention discloses a method for flocculating and purifying tea saponin by using a microbial flocculant, which comprises the following steps: mixing a tea saponin crude extract with water, stirring, standing, removing grease, retaining an aqueous solution, mixing with a coagulant aid solution and a microbial flocculant solution, flocculating, standing, retaining a supernatant, and completing the flocculation and purification of the tea saponin. According to the method disclosed by the invention, the residual oil in the tea saponin crude extract is removed, so that the subsequent flocculation purification effect can be improved, the grease content in the tea saponin product can be reduced, and other impurities in the tea saponin crude extract can be quickly and greatly removed by optimizing the flocculation process under the combined action of the coagulant aid and the microbial flocculant; and flocculant residues and toxicity risks are reduced, so that a liquid tea saponin product with high yield and high purity is obtained. The method also has the advantages of simple and convenient operation, safety, environmental protection and the like, is suitable for large-scale preparation of tea saponin, and is convenient for industrial application.

Description

Technical Field

[0001] The invention belongs to the technical field of tea saponin preparation and relates to a method for flocculating and purifying tea saponin by utilizing a microbial flocculant. Background Art

[0002] Tea saponin is a mixture of oleanane-type pentacyclic triterpenoid saponins, composed of sapogenins (aglycones), sugars (sugar groups) and organic acids. It is widely found in the roots, stems, leaves, flowers and seeds of Camellia plants. It has hemolytic and fish poisoning effects, anti-insect and bactericidal effects, as well as pharmacological functions such as anti-osmotic and anti-inflammatory, expectorant and antitussive, analgesic, and has the effect of promoting plant growth. Due to structural differences, various types of tea saponins show certain differences in activity. At the same time, tea saponin is also a natural non-ionic surfactant with the advantages of biodegradability, good biocompatibility and low toxicity, and can be widely used in agriculture, daily chemicals, medicine, food and other fields.

[0003] Camellia oleifera is a plant of the genus Camellia. During the maturation process of its seeds, the content of tea saponin increases first and then decreases, and tends to be stable in the late maturation period. The distribution of tea saponin content in different parts of the seeds is: grain > fruit shell > seed shell. Among them, the content of tea saponin in camellia oleifera cake reaches 10-14wt%. Therefore, camellia oleifera cake is the main raw material source of tea saponin.

[0004] At present, the industrial extraction methods of tea saponin are mainly water extraction and organic solvent extraction. Among them, water extraction is the traditional method of tea saponin extraction, which mainly utilizes the property of tea saponin being easily soluble in hot water, and uses hot water as solvent to extract tea saponin. This method has simple process, low investment and low cost, but long-term hot water extraction is easy to cause tea saponin hydrolysis, and a large amount of water-soluble impurities are easily dissolved, making it difficult to obtain high yield and high-quality tea saponin. Organic solvent extraction generally uses a certain proportion of methanol or ethanol as a solvent, and utilizes the trace solubility characteristics of impurity components in alcohol solvents to extract tea saponin. This method has low energy consumption, but large solvent consumption, complex production process and high equipment requirements. In addition, in the above-mentioned extraction method, the tea saponin crude product obtained by extraction often contains certain impurities such as grease, protein, polysaccharide, tannin, pigment, etc., has high viscosity and dark color, and is easy to absorb moisture and condense in the air. Therefore, when there is a requirement for product purity, it is generally necessary to further purify the crude product by methods such as flocculation and impurity removal, chemical precipitation, recrystallization, membrane separation, resin adsorption, gel chromatography, etc. Among them, flocculation and impurity removal treatment can be connected to different types of extraction schemes upward and compatible with different levels of purification paths downward. It is at the node position of the extraction and preparation process of plant functional ingredients such as tea saponin, and has shown wide applicability and effectiveness for various processes. Compared with chemical flocculants, microbial flocculants have the advantages of safety, non-toxicity, and biodegradability. At the same time, the flocculated precipitates do not contain harmful substances, which has potential advantages in the subsequent high-value utilization of tea saponin. Therefore, in view of the complex flocculation conditions in the extraction and purification process of tea saponin, as well as multiple target requirements such as product yield and purity, obtaining a tea saponin purification method that is highly versatile, safe and efficient is of great significance for realizing the value potential of tea saponin to a greater extent. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a safe and efficient method for flocculating and purifying tea saponin by using a microbial flocculant in view of the deficiencies in the prior art, which can not only improve the yield of tea saponin, but also improve the purity of tea saponin.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A method for flocculating and purifying tea saponin using a microbial flocculant comprises the following steps:

[0008] (1) mixing the tea saponin crude extract with water, stirring, and standing to separate the oil and water, and retaining the aqueous solution;

[0009] (2) The aqueous solution, coagulant aid solution and microbial flocculant solution obtained in step (1) are mixed for flocculation, allowed to stand, and filtered to complete the flocculation purification of tea saponin.

[0010] The above method is further improved in that in step (1), the volume ratio of the crude tea saponin extract to water is 2 to 4:1.

[0011] The above method is further improved, in step (1), the tea saponin crude extract is prepared from tea oil cake, for example, the tea saponin crude extract is prepared by mixing tea oil cake and water and then ultrasonically treating the mixture, wherein the water temperature is 30°C to 80°C, but the preparation method of the tea saponin crude extract is not limited to this.

[0012] The above method is further improved in that in step (1), the stirring is carried out on a shaking table; and the stirring time is 2h to 5h.

[0013] The above method is further improved in that in step (1), the standing time is 5h to 12h.

[0014] The above method is further improved, in step (2), the flocculation is: stirring for 3min to 5min at a rotation speed of 200r / min to 300r / min, and stirring for 3min to 5min at a rotation speed of 30r / min to 60r / min.

[0015] The above method is further improved in that in step (2), the standing time is 10 min to 30 min.

[0016] The above method is further improved in that in step (2), the volume ratio of the aqueous solution to the coagulant aid solution is 100:2-3.

[0017] The above method is further improved in that in step (2), the coagulant in the coagulant solution is at least one of calcium chloride and lime.

[0018] The above method is further improved in that in step (2), the mass concentration of the coagulant in the coagulant solution is 8% to 10%.

[0019] The above method is further improved in that in step (2), the volume ratio of the aqueous solution to the microbial flocculant solution is 100:1-2.

[0020] The above method is further improved, in step (2), the microbial flocculant solution is prepared by mixing a microbial flocculant with water; the mass ratio of the microbial flocculant to water is 1:100-200.

[0021] The above method is further improved, and the preparation method of the microbial flocculant comprises the following steps:

[0022] S1, mixing camellia oleifera cake with water, ultrasonicating, hydrothermally treating, filtering, collecting the filtrate, and obtaining a camellia oleifera cake extract;

[0023] S2, preparing a tea cake culture medium by using the tea cake extract obtained in step S1;

[0024] S3, inoculating the flocculating functional bacteria seed solution into the camellia oleifera cake culture medium obtained in step S2 for culturing to obtain a microbial flocculant.

[0025] The above method is further improved, in step S1, the ratio of the camellia oleifera cake to water is 30g-200g:1L; the camellia oleifera cake also includes the following treatment before use: drying the camellia oleifera cake, crushing it, passing it through a 40-mesh to 60-mesh sieve, and obtaining camellia oleifera cake particles.

[0026] The above method is further improved, in step S1, the ultrasonic treatment is performed at a temperature of 30°C to 40°C; and the ultrasonic treatment time is 20min to 50min.

[0027] The above method is further improved, in step S1, the hydrothermal treatment is carried out at a temperature of 111° C. to 126° C.; the time of the hydrothermal treatment is 10 min to 50 min.

[0028] The above method is further improved, in step S1, the polysaccharide content in the oil-tea camellia cake extract is 1800mg / L-15000mg / L, the protein content is 3300mg / L-24000mg / L, and the SCOD content is 18000mg / L-110000mg / L.

[0029] The above method is further improved, in step S1, the ratio of the oil-tea camellia cake to water is 50g-150g:1L.

[0030] The above method is further improved, in step S2, the preparation method of the camellia oleifera cake culture medium is: mixing the camellia oleifera cake extract, potassium dihydrogen phosphate and dipotassium hydrogen phosphate to obtain the camellia oleifera cake culture medium; the concentration of potassium dihydrogen phosphate in the camellia oleifera cake culture medium is 1.0 g / L to 2.0 g / L, and the concentration of dipotassium hydrogen phosphate is 3.0 g / L to 5.0 g / L; the pH value of the camellia oleifera cake culture medium is 5 to 9.

[0031] The above method is further improved, in which in step S3, the inoculation amount of the flocculation functional bacteria seed solution is 4% to 5% of the volume of the camellia oleifera cake culture medium.

[0032] The above method is further improved, in step S3, the culturing is: culturing for 12.0h to 24.0h at a temperature of 30°C to 35°C and a rotation speed of 130rpm to 150rpm, and culturing for 24.0h to 72.0h at a temperature of 20°C to 25°C and a rotation speed of 100rpm to 120rpm.

[0033] The above method is further improved, in step S3, after the cultivation is completed, the following treatment is also included: the fermentation product obtained after the cultivation is centrifuged at a rotation speed of 8000rpm to 10000rpm for 8min to 10min to remove the bacteria and obtain a microbial flocculant.

[0034] The above method is further improved, in step S3, the microbial flocculant also includes the following treatment: mixing the microbial flocculant with the organic solvent, standing, centrifuging at a rotation speed of 8000rpm~10000rpm for 8min~10min, removing the supernatant to obtain a purified microbial flocculant; the volume ratio of the microbial flocculant to the organic solvent is 1~2:1~3; the organic solvent is acetone and / or ethanol; the organic solvent also includes the following treatment before use: precooling at a temperature of 4°C for 12h~24h; the standing is carried out at a temperature of 4°C; the standing time is 24h~48h.

[0035] The above method is further improved, in step S3, the method for preparing the flocculation functional bacteria seed solution comprises the following steps:

[0036] (a) inoculating a flocculating functional bacteria strain into a solid activation culture medium, and culturing at a temperature of 30° C. to 35° C. for 12.0 h to 24.0 h to obtain a primary seed colony; the formula of the solid activation culture medium is: 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, 20.0 g / L agar powder, and the pH value is 7 to 8; the flocculating functional bacteria is one of Bacillus polymyxa, lactic acid bacteria, Clostridium butyricum and Halomonas; the Bacillus polymyxa is GA1;

[0037] (b) inoculating the primary seed colony obtained in step (a) into a liquid proliferation culture medium, culturing for 12.0 h to 24.0 h at a temperature of 25° C. to 30° C. and a rotation speed of 130 rpm to 150 rpm to obtain a flocculation functional bacteria seed solution; the formula of the liquid proliferation culture medium is: 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, and a pH value of 7 to 8.

[0038] Compared with the prior art, the advantages of the present invention are:

[0039] (1) In view of the shortcomings of the existing tea saponin purification methods, such as complex process, large tea saponin loss, and difficulty in effectively removing impurities, as well as the resulting defects of low tea saponin yield, low purity, and low safety, the present invention creatively proposes a method for flocculating and purifying tea saponin using a microbial flocculant. First, a tea saponin crude extract is mixed with water, and the oil in the tea saponin crude extract is effectively removed by stirring and standing. This can not only eliminate the adverse effects of oil on the subsequent flocculation purification process, but also reduce the oil content in the tea saponin product. On this basis, a coagulant solution and a microbial flocculant solution are added to the aqueous solution obtained after oil-water separation for flocculation treatment, wherein the coagulant can react with suspended particles, colloidal substances, etc. in the water to generate electrical neutralization, reduce their surface charge, and make it easier to aggregate to form larger flocs. At the same time, the microbial flocculant can achieve adsorption and removal of impurities such as proteins, polysaccharides, tannins, and pigments in the crude extract based on the flocculation separation of suspended particles by means of its rich functional groups. Furthermore, by optimizing the flocculation conditions, the synergistic removal of particulate matter and macromolecular substances can be effectively achieved. Specifically, firstly, the microbial flocculant and coagulant aid can be quickly and evenly dispersed in the water, thereby avoiding uneven dispersion of the reagents and causing excessive local concentration. Moreover, the rapid stirring can also promote the rapid collision and aggregation of suspended matter in the water. When the floccules begin to form, the stirring speed is reduced and the stirring intensity is reduced. Under this slow stirring condition, the floccules are further helped to form larger particles, which is convenient for subsequent precipitation or filtration. The floccules are used as the matrix to achieve the adsorption of soluble impurities in the mixing process, and the best flocculation and impurity removal effect is achieved. Finally, by standing and filtering, the obtained supernatant is liquid tea saponin, which has high yield and purity. Compared with the conventional tea saponin flocculation purification method, the method of flocculation purification of tea saponin by microbial flocculant of the present invention has the following advantages: (a) low oil content, the combination of water degreasing and microbial flocculant degreasing can effectively remove oil in the tea saponin product, wherein the oil content in the tea saponin product is less than 0.1%. (b) high purity, the combination of water degreasing, coagulant and microbial flocculant can not only effectively remove suspended particles and soluble macromolecules, but also effectively remove soluble impurities such as polysaccharides, proteins, brown pigments and tannins, and the removal rate of these soluble impurities is more than 50%. (c) high yield, the combination of water degreasing, microbial flocculant and coagulant can effectively remove impurities while retaining tea saponin as much as possible, wherein the retention rate of tea saponin is as high as 93.3%. (d) Low toxicity. On the one hand, the microbial flocculant used has low toxicity and is easily biodegradable, which is beneficial to reducing the toxicity of the tea saponin product. On the other hand, the residual amount of the coagulant and the microbial flocculant is low, which is also beneficial to reducing the toxicity of the tea saponin product. Therefore, the high-value utilization of tea saponin in the fields of medicine, food, daily chemicals, etc. can be achieved.At the same time, the method of flocculating and purifying tea saponin by using a microbial flocculant in the present invention also has the advantages of simple and convenient operation, safety, environmental protection, etc., is suitable for large-scale preparation of tea saponin, and is convenient for realizing the industrial application of tea saponin.

[0040] (2) In the present invention, by optimizing the volume ratio of the tea saponin crude extract to water to 2 to 4:1, the volume of the aqueous solution to be treated is reduced while ensuring the oil removal effect, thereby reducing the difficulty and energy consumption of the subsequent process. In particular, if the amount of water used is too high, the volume of the sample to be treated will increase, increasing the difficulty and energy consumption of the process; if the amount of water used is too low, the oil removal effect of the pretreatment will be reduced, and the tea saponin content extracted from the water will be reduced. By optimizing the volume ratio of the aqueous solution to the microbial flocculant solution to 100:1 to 2, the stability and efficiency of the flocculation process can be ensured. At this time, the formation and sedimentation rate of the flocs are faster, which can shorten the treatment time and improve the treatment efficiency. At the same time, it can also avoid the waste of flocculants caused by excessive addition, and can effectively avoid the defects such as poor flocculation effect caused by insufficient addition. By optimizing the volume ratio of aqueous solution to coagulant solution to 100:2-3, the coagulation of suspended matter can be accelerated to form large and dense flocs, which is beneficial to the subsequent precipitation or filtration process. At the same time, by accurately controlling the dosage of coagulant, unnecessary waste can be avoided and the cost of the agent can be reduced. In particular, if the dosage of coagulant is too large, the colloidal particles in the solution will coagulate and form too many fine particles. These particles are not easy to settle, but will affect the flocculation effect.

[0041] (3) In the present invention, the microbial flocculant used is prepared by culturing flocculating functional bacteria using oil tea cake extract as the culture medium raw material, and has the advantages of low production cost, high production efficiency, high yield and high quality. Therefore, when it is used to remove impurities in the system, it can efficiently remove suspended particles in the system through flocculation. In particular, it can achieve coordinated removal of soluble macromolecular impurities through adsorption, has higher safety and impurity removal efficiency, and can prepare a tea saponin product with higher purity.

[0042] (4) In the present invention, in the preparation method of the microbial flocculant, by optimizing the extraction conditions, specifically ultrasonication at a temperature of 30°C to 40°C for 20min to 50min, and under the combined action of hydrothermal treatment, an oil-tea cake extract with rich nutrients and good adaptability of various nutrients can be prepared, which is more conducive to the preparation of a microbial flocculant with high yield and high purity.

[0043] (5) In the present invention, by optimizing the ratio of camellia oleifera cake to water, specifically optimizing the ratio of the two to 30g-200g:1L, and in particular optimizing the ratio of the two to 50g-150g:1L, the content of nutrients in the extract can be effectively increased. When it is used to cultivate flocculation functional bacteria, it is not only conducive to the formation of a microbial flocculant with a high flocculation rate with extracellular polymers as the main component, but also can significantly increase the unit yield of the microbial flocculant, which is conducive to improving the utilization rate of camellia oleifera cake and making the preparation cost lower.

[0044] (6) In the present invention, by optimizing the culture conditions, specifically, the flocculation functional bacteria seed liquid is inoculated into the oil-tea cake culture medium, firstly cultured at a temperature of 30°C to 35°C and a rotation speed of 130rpm to 150rpm for 12.0h to 24.0h, and then cultured at a temperature of 20°C to 25°C and a rotation speed of 100rpm to 120rpm for 24.0h to 72.0h. In the fermentation process of the former stage, the microorganisms grow rapidly, and in the fermentation process of the latter stage, a large amount of floccules are produced. Thus, a microbial flocculant with high yield and high flocculation rate can be obtained while reducing the fermentation time, which is conducive to large-scale preparation and convenient for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention.

[0046] Figure 1 This is a process flow chart for flocculating and purifying tea saponin using a microbial flocculant in Example 1 of the present invention. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0048] The materials and instruments used in the following examples are all commercially available.

[0049] Embodiment 1:

[0050] A method for flocculating and purifying tea saponin using a microbial flocculant, the process flow chart is as follows Figure 1 As shown, the following steps are included:

[0051] S1. Prepare tea saponin crude extract from tea oil cake, for example, mix tea oil cake with 40° C. hot water, perform ultrasonic treatment for 30 minutes, filter, collect the filtrate, and obtain tea saponin crude extract.

[0052] S2. Mix the crude tea saponin extract with water in a volume ratio of 4:1, put it into a shaker and mix it thoroughly for 2 hours, pour it into a separatory funnel and let it stand for 5 hours to allow the oil phase and the water phase to be fully separated, remove the upper oil phase, and retain the middle aqueous solution.

[0053] S3, 100 mL of the aqueous solution obtained in step S2, 2.0 mL of calcium chloride solution (the mass concentration of calcium chloride in the solution is 10%) and 1 mL of microbial flocculant solution are mixed, stirred at a speed of 200 r / min for 5 min, stirred at a speed of 30 r / min for 5 min, allowed to stand for 10 minutes, filtered, and the supernatant is retained, which is liquid tea saponin, thereby completing the flocculation purification of tea saponin.

[0054] In this embodiment, the microbial flocculant solution is prepared by mixing a microbial flocculant with water, wherein the mass ratio of the microbial flocculant to water is 1:150. The preparation method of the microbial flocculant comprises the following steps:

[0055] (1) preparing tea cake extract, specifically:

[0056] (1.1) Place the camellia oleifera cake in an oven and dry it at 60°C for 5.0 hours until it reaches a constant weight. Then, crush it in a pulverizer and use a steel sieve to select the camellia oleifera cake particles of 40-60 mesh for later use.

[0057] (1.2) 50 g of sieved camellia oleifera cake particles were mixed into 1000 mL of water, and the camellia oleifera cake particles were mixed with water, first placed in an ultrasonic device, ultrasonicated at 40°C for 30 min, then placed in a high-pressure steam sterilizer, hydrothermally heated at 121°C for 30 min, and the hydrothermally heated solution was vacuum filtered using a 300-500 mesh filter cloth. The filtrate obtained was the camellia oleifera cake extract. The camellia oleifera cake extract had a polysaccharide content of 1800 mg / L, a protein content of 3300 mg / L, and a SCOD content of 18000 mg / L.

[0058] In this step, if the ultrasonic treatment is omitted, the content of nutrients dissolved in the extract will be significantly reduced, making it difficult to meet the growth requirements. If other nutrients are added, the processing cost will inevitably increase, which is a problem that needs to be overcome in the present invention.

[0059] (2) Preparation of tea cake culture medium

[0060] Take the camellia oleifera cake extract, add potassium dihydrogen phosphate and dipotassium hydrogen phosphate solutions to make the concentration of potassium dihydrogen phosphate in the mixed solution be 2.0 g / L, and the concentration of dipotassium hydrogen phosphate be 5.0 g / L, and adjust the pH value to 8, then place it in a high-pressure steam sterilizer and sterilize it at 121°C for 30 minutes to obtain the camellia oleifera cake culture medium.

[0061] (3) Preparation of microbial flocculants

[0062] (3.1) Weigh 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride and 20.0 g / L agar powder, and adjust the pH value to 7. Sterilize the prepared culture medium in a high-pressure steam autoclave at 121°C for 30 min. Pour the sterilized culture medium into a clean culture dish on a sterile operating table, wait for the culture medium to cool and solidify, and then invert it to prepare a solid activated culture medium.

[0063] Weigh 10.0 g / L peptone, 3.0 g / L beef extract and 5.0 g / L sodium chloride, and adjust the pH value to 7. Place the prepared culture medium in a high-pressure steam sterilizer and sterilize at 121° C. for 30 minutes to prepare a liquid proliferation medium.

[0064] (3.2) The bacterial strain (Bacillus polymyxa GA1) stored in the glycerol tube was inoculated into a solid activation culture medium and cultured at 35°C for 24 hours to activate the bacterial strain and form a primary seed colony.

[0065] (3.3) The primary seed colony was inoculated into a liquid proliferation medium and cultured in a shaking incubator at a temperature of 30° C. and a rotation speed of 150 rpm for 24.0 h to proliferate the microorganisms and prepare a secondary seed solution, which was recorded as Bacillus polymyxa GA1 seed solution.

[0066] (3.4) The seed solution of Bacillus polymyxa1 was added to the camellia oil cake culture medium at an inoculation rate of 5% of the volume of the camellia oil cake culture medium, and the mixture was placed in a shaking incubator and cultured at 30°C and 150 rpm for 24 h, and then cultured at 25°C and 120 rpm for 48 h. The fermentation product obtained after the culture was placed in a centrifuge and centrifuged at 10,000 rpm for 10 min to remove the bacteria, thereby obtaining a fermentation product with the bacteria removed, which is a microbial flocculant.

[0067] (3.5) After the cultivation is completed, the microbial flocculant is purified, including the following treatment: taking the fermentation product obtained after the cultivation at a volume ratio of 2:1, adding acetone (before the use of acetone, the following treatment is also included: precooling at 4°C for 24 hours), placing it in a 4°C refrigerator and standing it for 24.0 hours, then placing it in a centrifuge and centrifuging it at a speed of 10000 rpm for 10.0 minutes, removing the supernatant, and obtaining the precipitated substance, which can be used as the purified microbial flocculant.

[0068] Embodiment 2:

[0069] A method for flocculating and purifying tea saponin using a microbial flocculant comprises the following steps:

[0070] (1) The tea saponin crude extract was prepared from the tea cake in the same manner as in Example 1.

[0071] (2) Mix the crude tea saponin extract with water in a volume ratio of 4:1, place in a shaker and mix thoroughly for 2 hours, pour into a separatory funnel and let stand for 5 hours to allow the oil phase and the water phase to be fully separated, remove the upper oil phase, and retain the middle aqueous solution.

[0072] (3) 100 mL of the aqueous solution obtained in step (2), 3.0 mL of the calcium chloride solution (the mass concentration of calcium chloride in the solution is 10%) and 1 mL of the microbial flocculant solution prepared in Example 1 were mixed, stirred at a speed of 200 r / min for 5 min, stirred at a speed of 30 r / min for 5 min, allowed to stand for 10 minutes, filtered, and the supernatant was retained, which was liquid tea saponin, thereby completing the flocculation purification of tea saponin.

[0073] Embodiment 3:

[0074] A method for flocculating and purifying tea saponin using a microbial flocculant comprises the following steps:

[0075] (1) The tea saponin crude extract was prepared from the tea cake in the same manner as in Example 1.

[0076] (2) Mix the tea saponin crude extract with water in a volume ratio of 2:1, place in a shaker and mix thoroughly for 2 hours, pour into a separatory funnel and let stand for 5 hours to allow the oil phase and the water phase to be fully separated, remove the upper oil phase, and retain the middle aqueous solution.

[0077] (3) 100 mL of the aqueous solution obtained in step (2), 3.0 mL of the calcium chloride solution (the mass concentration of calcium chloride in the solution is 10%) and 1 mL of the microbial flocculant solution prepared in Example 1 were mixed, stirred at a speed of 200 r / min for 5 min, stirred at a speed of 30 r / min for 5 min, allowed to stand for 10 minutes, filtered, and the supernatant was retained, which was liquid tea saponin, thereby completing the flocculation purification of tea saponin.

[0078] Embodiment 4:

[0079] A method for flocculating and purifying tea saponin using a microbial flocculant comprises the following steps:

[0080] (1) The tea saponin crude extract was prepared from the tea cake in the same manner as in Example 1.

[0081] (2) Mix the tea saponin crude extract with water in a volume ratio of 2:1, place in a shaker and mix thoroughly for 2 hours, pour into a separatory funnel and let stand for 5 hours to allow the oil phase and the water phase to be fully separated, remove the upper oil phase, and retain the middle aqueous solution.

[0082] (3) 100 mL of the aqueous solution obtained in step (2), 2.0 mL of the calcium chloride solution (the mass concentration of calcium chloride in the solution is 10%) and 1 mL of the microbial flocculant solution prepared in Example 1 were mixed, stirred at a speed of 200 r / min for 5 min, stirred at a speed of 30 r / min for 5 min, allowed to stand for 10 minutes, filtered, and the supernatant was retained, which was liquid tea saponin, thereby completing the flocculation purification of tea saponin.

[0083] Embodiment 5:

[0084] A method for flocculating and purifying tea saponin using a microbial flocculant comprises the following steps:

[0085] (1) The tea saponin crude extract was prepared from the tea cake in the same manner as in Example 1.

[0086] (2) Mix the tea saponin crude extract with water in a volume ratio of 2:1, place in a shaker and mix thoroughly for 2 hours, pour into a separatory funnel and let stand for 5 hours to allow the oil phase and the water phase to be fully separated, remove the upper oil phase, and retain the middle aqueous solution.

[0087] (3) 100 mL of the aqueous solution obtained in step (2), 2.0 mL of the calcium chloride solution (the mass concentration of calcium chloride in the solution is 10%) and 2 mL of the microbial flocculant solution prepared in Example 1 were mixed, stirred at a speed of 200 r / min for 5 min, stirred at a speed of 30 r / min for 5 min, allowed to stand for 10 minutes, filtered, and the supernatant was retained, which was liquid tea saponin, thereby completing the flocculation purification of tea saponin.

[0088] The supernatant (liquid tea saponin) prepared in Examples 1-5 was taken, and the brown pigment, tannin, polysaccharide, protein and tea saponin in the supernatant were detected according to the following methods. The detection results are shown in Tables 1 and 2.

[0089] (1) Detection method of brown pigment: measure the absorbance of the sample at 278 nm.

[0090] (2) Detection method of tannin: Take 1.0 mL of sample, add 5.0 mL of 1 mol / L saturated Na2CO3, then add 2.5 mL of phosphomolybdic acid test solution, make up to 50.0 mL, develop color in a 25°C water bath for 30 minutes, and measure the absorbance of the sample at 500 nm.

[0091] (3) Detection method of polysaccharides: Take 1.0 mL of sample, add 1.0 mL of distilled water, then add 1.0 mL of 6% phenol, quickly add 5.0 mL of concentrated sulfuric acid, shake on a vortex to mix thoroughly, color in a 25°C water bath for 30 minutes, and measure the absorbance of the sample at 490 nm.

[0092] (4) Protein detection method: Take 1.0 mL of sample and add 5.0 mL of Folin phenol A solution. Mix well and place at room temperature for 10 min, then add 0.5 mL of Folin phenol B solution and mix well immediately. Color develop in a 25°C water bath for 30 min, and measure the absorbance of the sample at 650 nm.

[0093] (5) Detection method of tea saponin: liquid chromatography was used with a Venusil C18 Plus column (4.6 mm × 250 mm, 5 μm); the mobile phase was methanol; the flow rate was 0.5 mL / min; the injection volume was 10 μL; and the measurement wavelength was 282 nm.

[0094] Table 1 Removal rate data of brown pigment, tannin, polysaccharide and protein in liquid tea saponin prepared in Examples 1-5

[0095] Example 1 Example 2 Example 3 Example 4 Example 5 Tannin 48.65％ 54.05％ 47.72％ 50.00％ 46.59％ Brown pigment 50.56％ 51.09％ 29.02％ 30.90％ 30.80％ Polysaccharides 58.41％ 57.00％ 36.62％ 32.86％ 32.53％ protein 52.33％ 53.79％ 48.06％ 40.07％ 48.26％

[0096] Table 2 Yield data of tea saponin in liquid tea saponin obtained in Examples 1-5

[0097] Example 1 Example 2 Example 3 Example 4 Example 5 Tea saponin 89.77％ 90.39％ 93.30％ 79.28％ 80.24％

[0098] Table 1 shows the removal rate of impurities under different treatment conditions. When the ratio of tea saponin crude extract to water is 4:1, the removal rate of brown pigment, tannin, polysaccharide and protein is high; when the ratio of tea saponin crude extract to water is 2:1, the removal rate of brown pigment, tannin, polysaccharide and protein is not higher than 50%. Table 2 shows the yield of tea saponin under different treatment conditions. Under the conditions of Example 3, the yield of tea saponin is the highest, which is 93.30%. When the ratio of tea saponin crude extract to water is 4:1, Examples 1 and 2 show that the yield of tea saponin can also reach about 90%. Considering the removal rate of impurities and the yield of tea saponin, the flocculation purification effect is best when the ratio of tea saponin crude extract to water is 4:1, 3.0mL calcium chloride solution and 2mL microbial flocculant solution are mixed.

[0099] In addition, the middle aqueous phase (aqueous solution) extracted in Example 1, the supernatant (liquid tea saponin) prepared in Example 2, the middle aqueous phase (aqueous solution) extracted in Example 3, and the supernatant (liquid tea saponin) prepared in Example 4 were taken to detect the oil content. The results are shown in Table 3.

[0100] Table 3 Oil removal rate data of liquid tea saponin and middle water phase prepared in Examples 1-4

[0101] Example 1 Example 2 Example 3 Example 4 Oil removal rate 94.48％ 99.13％ 99.07％ 99.94％

[0102] Table 3 shows the removal effect of oil under different treatment conditions. Extracting the tea cake extract with water can remove more than 94% of the oil in the sample. Further treating the middle water phase with a microbial flocculant can remove more than 99% of the oil content in the sample.

[0103] It can be seen from the above results that, compared with the conventional tea saponin flocculation purification method, the method of flocculating and purifying tea saponin by microbial flocculant of the present invention has the following advantages: (a) Low oil content. The combination of water degreasing and microbial flocculant degreasing can effectively remove oil from the tea saponin product, wherein the oil content in the tea saponin product is less than 0.1%. (b) High purity. The combination of water degreasing, coagulant and microbial flocculant can effectively remove not only suspended particles and soluble macromolecules, but also soluble impurities such as polysaccharides, proteins, brown pigments and tannins, and the removal rate of these soluble impurities is more than 50%. (c) High yield. The combination of water degreasing, microbial flocculant and coagulant can effectively remove impurities while retaining tea saponin as much as possible, wherein the retention rate of tea saponin is as high as 93.3%. (d) Low toxicity. On the one hand, the microbial flocculant used has low toxicity and is easily biodegradable, which is beneficial to reducing the toxicity of the tea saponin product. On the other hand, the residual amount of the coagulant and the microbial flocculant is low, which is also beneficial to reducing the toxicity of the tea saponin product, thereby realizing the high-value utilization of tea saponin in the fields of medicine, food, daily chemicals, etc. At the same time, the method of flocculating and purifying tea saponin using a microbial flocculant of the present invention also has the advantages of simple and convenient operation, safety, and environmental protection, and is suitable for large-scale preparation of tea saponin, and is convenient for realizing the industrial application of tea saponin.

[0104] The above embodiments are only preferred implementations of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for flocculating and purifying tea saponin using a microbial flocculant, characterized in that: The following steps are involved: (1) mixing the tea saponin crude extract with water, stirring, and standing to separate the oil and water, and retaining the aqueous solution; (2) The aqueous solution, coagulant aid solution and microbial flocculant solution obtained in step (1) are mixed for flocculation, allowed to stand, and filtered to complete the flocculation purification of tea saponin.

2. The method according to claim 1, characterized in that In step (1), the volume ratio of the tea saponin crude extract to water is 2-4:1; the tea saponin crude extract is prepared from camellia oleifera cake; the stirring is carried out on a shaking table; the stirring time is 2h-5h; and the standing time is 5h-12h.

3. The method according to claim 1 or 2, characterized in that: In step (2), the flocculation is: stirring for 3 min to 5 min at a rotation speed of 200 r / min to 300 r / min, stirring for 3 min to 5 min at a rotation speed of 30 r / min to 60 r / min; and the standing time is 10 min to 30 min.

4. The method according to claim 1 or 2, characterized in that: In step (2), the volume ratio of the aqueous solution to the coagulant solution is 100:2-3; the coagulant in the coagulant solution is at least one of calcium chloride and lime; the mass concentration of the coagulant in the coagulant solution is 8%-10%; the volume ratio of the aqueous solution to the microbial flocculant solution is 100:1-2; the microbial flocculant solution is prepared by mixing a microbial flocculant with water; the mass ratio of the microbial flocculant to water is 1:100-200.

5. The method according to claim 4, characterized in that The preparation method of the microbial flocculant comprises the following steps: S1, mixing camellia oleifera cake with water, ultrasonicating, hydrothermally treating, filtering, collecting the filtrate, and obtaining a camellia oleifera cake extract; S2, preparing a tea cake culture medium by using the tea cake extract obtained in step S1; S3, inoculating the flocculating functional bacteria seed solution into the camellia oleifera cake culture medium obtained in step S2 for culturing to obtain a microbial flocculant.

6. The method according to claim 5, characterized in that In step S1, the ratio of the camellia oleifera cake to water is 30g-200g:1L; the camellia oleifera cake also includes the following treatments before use: drying the camellia oleifera cake, crushing it, and passing it through a 40-mesh sieve to a 60-mesh sieve to obtain camellia oleifera cake particles; the ultrasonic treatment is performed at a temperature of 30°C-40°C; the ultrasonic treatment lasts for 20min-50min; the hydrothermal treatment is performed at a temperature of 111°C-126°C; the hydrothermal treatment lasts for 10min-50min; the polysaccharide content in the camellia oleifera cake extract is 1800mg / L-15000mg / L, the protein content is 3300mg / L-24000mg / L, and the SCOD content is 18000mg / L-110000mg / L.

7. The method according to claim 6, characterized in that The ratio of the oil-tea cake to water is 50g-150g:1L.

8. The method according to claim 5, characterized in that In step S2, the preparation method of the camellia oleifera cake culture medium is: mixing camellia oleifera cake extract, potassium dihydrogen phosphate and dipotassium hydrogen phosphate to obtain a camellia oleifera cake culture medium; the concentration of potassium dihydrogen phosphate in the camellia oleifera cake culture medium is 1.0 g / L to 2.0 g / L, and the concentration of dipotassium hydrogen phosphate is 3.0 g / L to 5.0 g / L; the pH value of the camellia oleifera cake culture medium is 5 to 9.

9. The method according to claim 5, characterized in that In step S3, the inoculation amount of the flocculation functional bacteria seed liquid is 4% to 5% of the volume of the oil-tea cake culture medium; the cultivation is: culturing for 12.0h to 24.0h at a temperature of 30°C to 35°C and a rotation speed of 130rpm to 150rpm, and culturing for 24.0h to 72.0h at a temperature of 20°C to 25°C and a rotation speed of 100rpm to 120rpm; after the cultivation is completed, the following treatment is also included: centrifuging the fermentation product obtained after the cultivation at a rotation speed of 8000rpm to 10000rpm for 8min to 10min to remove The microbial flocculant is obtained by mixing the microbial flocculant with an organic solvent, allowing the mixture to stand, centrifuging the mixture at a speed of 8000 rpm to 10000 rpm for 8 to 10 minutes, removing the supernatant, and obtaining a purified microbial flocculant. The volume ratio of the microbial flocculant to the organic solvent is 1 to 2: 1 to 3. The organic solvent is acetone and / or ethanol. The organic solvent is further treated before use by precooling the mixture at 4°C for 12 to 24 hours. The standing still is performed at 4°C. The standing still time is 24 to 48 hours.

10. The method according to claim 9, characterized in that in step S3, the method for preparing the flocculation functional bacteria seed solution comprises the following steps: (a) inoculating the flocculating functional bacteria into a solid activation medium, culturing at a temperature of 30° C. to 35° C. for 12.0 h to 24.0 h to obtain a primary seed colony; the formula of the solid activation medium is: 10.0 g / L of peptone, 3.0 g / L of beef extract, 5.0 g / L of sodium chloride, 20.0 g / L of agar powder, and a pH value of 7-8; the flocculating functional bacteria is one of Paenibacillus polymyxa, lactic acid bacteria, Clostridium butyricum and Halomonas; the Paenibacillus polymyxa is GA1; (b) inoculating the primary seed colony obtained in step (a) into a liquid proliferation culture medium, culturing for 12.0 h to 24.0 h at a temperature of 25° C. to 30° C. and a rotation speed of 130 rpm to 150 rpm to obtain a flocculation functional bacteria seed solution; the formula of the liquid proliferation culture medium is: 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, and a pH value of 7 to 8.

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