Method for synergistically preparing tea saponin and microbial flocculant

Abstract

The invention discloses a method for synergistically preparing tea saponin and a microbial flocculant, which comprises the following steps: preparing tea saponin crude extract and oil-tea cake extract from oil-tea cake, preparing the extract into an oil-tea cake culture medium, inoculating a flocculation functional bacteria seed solution for culturing, and preparing a pure microbial flocculant product. Preparing the tea saponin into a solution, mixing the solution with a tea saponin crude extract after oil removal and a coagulant aid solution for flocculation, and standing to obtain liquid tea saponin; and carrying out primary concentration, extraction, decoloration and secondary concentration on the liquid tea saponin to obtain the solid tea saponin. According to the method for synergistically preparing the tea saponin and the microbial flocculant, the microbial flocculant and the tea saponin which are high in yield and purity can be prepared at the same time, circulating sharing of water and an organic solvent can be achieved, and resource utilization of the oil tea cakes is achieved. The method also has the advantages of simple process, convenience in operation, low cost, environmental friendliness and the like, and is high in use value and good in application prospect.

Description

Technical Field

[0001] The invention relates to a method for synergistically preparing tea saponin and microbial flocculant. Background Art

[0002] Camellia oleifera cake is the main by-product in the production of camellia oleifera. It contains rich nutrients. In addition to lignocellulose, it also contains bioactive ingredients such as protein, polysaccharide, polyphenol, tea saponin, etc. It is a potential material for making a microbial alternative culture medium. However, in the prior art, when the camellia oleifera cake extract from which tea saponin has been extracted is used as a culture medium, the flocculation rate of the prepared microbial flocculant on kaolin suspension is up to 86.1%, and there is still a defect of low flocculation rate. In addition, the above-mentioned method of preparing microbial flocculants using camellia oleifera cake extracts also requires additional addition of sucrose, yeast extract and inorganic salts, which also makes it difficult to effectively reduce the production cost. Therefore, how to use camellia oleifera cake to prepare a microbial flocculant with high yield and high flocculation rate is of great significance for reducing the production cost of microbial flocculants and realizing the high-value utilization of camellia oleifera cake.

[0003] Tea cakes are rich in tea saponins, and tea saponins, as a natural nonionic surfactant, have the advantages of biodegradability, good biocompatibility, and low toxicity. They can be widely used in agriculture, daily chemicals, medicine, food and other fields. Therefore, it is worth studying to extract high-value tea saponins from tea cakes. At present, the industrial extraction methods of tea saponins are mainly water extraction and organic solvent extraction. Among them, water extraction is the traditional method for extracting tea saponins. It mainly uses the property of tea saponins being easily soluble in hot water to extract tea saponins with hot water as a solvent. This method has simple process, low investment and low cost, but long-term hot water extraction causes tea saponins to hydrolyze and a large amount of water-soluble impurities are dissolved. Organic solvent extraction generally uses a certain proportion of methanol or ethanol as a solvent, and uses the trace solubility characteristics of impurity components in alcohol solvents to extract tea saponins. This method has low energy consumption, but large solvent consumption, complex production process and high equipment requirements. The crude tea saponin products obtained by extraction often contain a large amount of impurities such as proteins, polysaccharides, tannins, pigments, etc., with high viscosity and dark color, and are easy to absorb moisture and condense in the air. When there are requirements for product purity, the crude product is generally further purified through flocculation and impurity removal, chemical precipitation, recrystallization, membrane separation, resin adsorption, gel chromatography and other methods. Among them, flocculation and impurity removal can be connected to different types of extraction schemes upward, and is compatible with different levels of purification paths downward. It is at the node position of the extraction and preparation process of plant functional components 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, and have 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, if tea cake can be used to simultaneously prepare high-yield and high-purity microbial flocculants and tea saponin, it will be beneficial to maximize the value potential of tea cake.

[0004] In addition, most microbial flocculants need to be extracted and purified with organic solvents before use in order to effectively remove the excess chemical components in the flocculants and fermentation broth that do not have a flocculating effect. However, this will consume a large amount of organic solvents, which not only increases the production cost, but also poses potential hazards to the environment and human health. At the same time, the extraction process of tea saponin also requires a large amount of organic solvents, generally using methanol or ethanol as solvents, and extracting tea saponin by utilizing the trace solubility characteristics of impurities in alcohol solvents. Therefore, if the organic solvents recovered from the tea saponin extraction process can be applied to the preparation of microbial flocculants, it can not only achieve the recycling of organic solvents, but also reduce the cost of the process while protecting the environment.

[0005] Therefore, it is very necessary to develop a method for the synergistic preparation of tea saponin and microbial flocculants with simple process, convenient operation, low cost and environmental friendliness. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for the synergistic preparation of tea saponin and microbial flocculant with simple process, convenient operation, low cost and environmental friendliness in view of the deficiencies in the prior art. Not only tea saponin with high yield and high purity can be prepared, but also microbial flocculant with high yield and high purity can be prepared.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] A method for synergistically preparing tea saponin and microbial flocculant comprises the following steps:

[0009] S1. Mixing tea oil cake with water, ultrasonicating, standing, filtering, and obtaining a tea saponin crude extract and residue;

[0010] S2, mixing the tea saponin crude extract and the residue, performing hydrothermal treatment, filtering, and obtaining a tea cake extract;

[0011] S3, preparing a tea cake medium with the tea cake extract obtained in step S2, inoculating the flocculating functional bacteria seed solution for cultivation, and centrifuging to obtain a crude microbial flocculant;

[0012] S4, mixing the crude microbial flocculant obtained in step S3 with an organic solvent, allowing to stand, centrifuging, and drying to obtain a pure microbial flocculant;

[0013] S5, mixing the tea saponin crude extract obtained in step S1 with water, stirring, and standing to separate the oil and water, and retaining the aqueous solution;

[0014] S6, preparing a microbial flocculant solution from the pure microbial flocculant obtained in step S4, mixing the solution with the aqueous solution and the coagulant aid solution obtained in step S5 for flocculation, allowing the solution to stand, filtering, and obtaining liquid tea saponin;

[0015] S7, concentrating the liquid tea saponin obtained in step S5, recovering water, and obtaining a concentrate;

[0016] S8, mixing the concentrate obtained in step S7 with an organic solvent for extraction, and collecting the upper liquid;

[0017] S9, decolorizing and re-concentrating the upper liquid collected in step S8, recovering the organic solvent, and obtaining solid tea saponin.

[0018] The above method is further improved, in step S7, the recovered water is returned to step S1, or returned to step S5.

[0019] The above method is further improved, in step S9, the recovered organic solvent is returned to step S4, or returned to step S8.

[0020] 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.

[0021] The above method is further improved, in step S1, the ultrasonic treatment is performed at a temperature of 40°C to 60°C; and the ultrasonic treatment time is 20min to 50min.

[0022] The above method is further improved, in step S1, the ratio of the oil-tea camellia cake to water is 50g-150g:1L.

[0023] The above method is further improved, in step S2, 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.

[0024] The above method is further improved, in step S2, the polysaccharide content in the oil-tea camellia cake extract is 800mg / L-15000mg / L, the protein content is 1500mg / L-24000mg / L, and the SCOD content is 8000mg / L-110000mg / L.

[0025] The above method is further improved, in step S3, the method for preparing the camellia oleifera cake culture medium is: mixing camellia oleifera cake extract, potassium dihydrogen phosphate and dipotassium hydrogen phosphate to obtain the camellia oleifera cake culture medium.

[0026] The above method is further improved, in step S3, the concentration of potassium dihydrogen phosphate in the camellia oleifera cake culture medium is 1.0g / L-2.0g / L, and the concentration of dipotassium hydrogen phosphate is 3.0g / L-5.0g / L.

[0027] The above method is further improved in that in step S3, the pH value of the tea oil cake culture medium is 5-9.

[0028] 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.

[0029] 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.

[0030] The above method is further improved, in step S3, the centrifugal speed is 8000rpm~10000rpm; the centrifugal time is 8min~10min;

[0031] The above method is further improved, in step S3, the method for preparing the flocculation functional bacteria seed solution comprises the following steps:

[0032] (1) Inoculating a flocculating functional bacterial strain into a solid activation culture 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 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 bacterial strain is one of Bacillus polymyxa, lactic acid bacteria, Clostridium butyricum and Halomonas; the Bacillus polymyxa is GA1;

[0033] (2) inoculating the primary seed colony obtained in step (1) 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.

[0034] The above method is further improved, in step S4, the volume ratio of the crude microbial flocculant to the organic solvent is 1-2:1-3.

[0035] The above method is further improved, in step S4, 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 to 24h.

[0036] The above method is further improved, in step S4, the standing temperature is 4°C; the standing time is 24h to 48h; the centrifugal speed is 8000rpm to 10000rpm; the centrifugal time is 8min to 10min.

[0037] The above method is further improved, in step S5, the volume ratio of the tea saponin crude extract to water is 2-4:1; the stirring is carried out on a shaker; the stirring time is 2h-5h; and the standing time is 5h-12h.

[0038] The above method is further improved, in step S6, the microbial flocculant solution is prepared by mixing pure microbial flocculant with water; the mass ratio of the pure microbial flocculant to water is 1:100-200.

[0039] The above method is further improved, in step S6, the volume ratio of the aqueous solution to the microbial flocculant solution is 100:1-2.

[0040] The above method is further improved, in step S6, the volume ratio of the aqueous solution to the coagulant aid solution is 100:2-3.

[0041] The above method is further improved, in step S6, 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% to 10%.

[0042] The above method is further improved, in step S6, 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.

[0043] The above method is further improved, in step S6, the standing time is 10 minutes to 30 minutes.

[0044] The above method is further improved, in step S8, the organic solvent is acetone and / or ethanol.

[0045] The above method is further improved in that in step S9, activated carbon or hydrogen peroxide is used to decolorize the upper liquid collected in step S8.

[0046] Compared with the prior art, the advantages of the present invention are:

[0047] (1) The present invention provides a method for the synergistic preparation of tea saponin and microbial flocculants, wherein tea cake is used as a raw material to prepare a tea saponin crude extract and a tea cake extract, respectively. On the one hand, the tea cake extract is used to prepare a tea cake culture medium, and flocculation functional bacteria are inoculated for cultivation. After the cultivation is completed, the bacteria are removed by centrifugation, and the microbial flocculant is purified under the action of an organic solvent, thereby effectively removing excess chemical components in the fermentation broth that have no flocculation effect, thereby obtaining a high-yield and high-purity microbial flocculant; on the other hand, the tea saponin crude extract is mixed with water to remove residual oil in the tea saponin crude extract, thereby eliminating the effect of oil on subsequent flocculation. The adverse effects of the purification process can be eliminated, and the oil content in the tea saponin product can be reduced. On this basis, the prepared high-purity microbial flocculant and coagulant are used to flocculate the crude tea saponin extract after oil removal. Under the joint action of the coagulant solution and the microbial flocculant, not only can the suspended particles be removed by flocculation, but also soluble impurities such as polysaccharides, proteins, brown pigments and tannins can be adsorbed and removed. At the same time, the tea saponin can be retained to the greatest extent, and high-purity liquid tea saponin can be obtained while ensuring high yield. Finally, through concentration, extraction and decolorization, impurities can be further removed, the quality of tea saponin can be improved, and higher-purity solid tea saponin can be obtained. In addition, water and organic solvents can be recycled, and the recycling of water and organic solvents can be realized, which can protect the environment while reducing process costs. In the present invention, the pure microbial flocculant needs to be first prepared into a microbial flocculant solution and then mixed with a coagulant aid solution, which is conducive to improving the flocculation effect and further ensuring the effective removal of various impurities in the system. This is because if they are directly mixed, a chemical reaction will occur between the coagulant aid and the microbial flocculant, causing their chemical properties to change, which is not conducive to improving the flocculation effect. Moreover, direct mixing will cause the mixed substances to be unable to be evenly dispersed in the water, resulting in excessive concentrations of flocculants or coagulants in certain areas, while the concentrations in other areas are insufficient. The existence of these defects is not conducive to improving the flocculation effect. Compared with conventional methods, the method of the present invention for the synergistic preparation of tea saponin and microbial flocculant has the following advantages: (a) less amount of organic solvent. In order to increase the yield of microbial flocculant, in the conventional purification method of microbial flocculant, the amount of organic solvent used is usually 2 to 3 times the volume of the fermentation broth. In the present invention, the amount of organic solvent used can be as low as 50% of the volume of the crude microbial flocculant. At this time, the yield of the microbial flocculant is 6.21 g / L, reaching 95.9% of the extraction amount of 2 times the volume of organic solvent, which greatly reduces the amount of organic solvent used in the extraction process. More importantly, in the present invention, the extraction of microbial flocculant and tea saponin shares a set of organic solvent recycling system, which can realize the recycling and sharing of organic solvent, thereby further reducing the amount of organic solvent used; (b) the yield of microbial flocculant is high and the purity is high. For example, the molecular weight of the extracellular polymer in the prepared microbial flocculant is concentrated at 4.4×105 -4.6×10 5 Da, compared with the molecular weight of the flocculant produced by traditional fermentation medium, it has higher consistency, which is conducive to the centralized extraction of a single proportion of organic solvent. At the same time, when the volume of organic solvent is 50% of the crude microbial flocculant, the average molecular weight of the extracted microbial flocculant reaches the highest value of 4.6×10 5 Da, has a more concentrated molecular weight distribution range and a stronger peak level of flocculation active functional groups, showing a more excellent flocculation ability. Specifically, the flocculation rate of the microbial flocculant for kaolin suspension can reach 99.1%, and can also reach 98.9% without the auxiliary effect of phosphate. More importantly, the prepared microbial flocculant has a suitable molecular weight level and distribution range, which is conducive to the synchronous separation of flocculant components during the flocculation process, reduces the residual risk of flocculants in the supernatant, and reduces the impurity interference of subsequent tea saponin products; (c) The yield of tea saponin products is high and the quality is high, among which the yield of high-purity tea saponin products is as high as 89.93%. In addition, the removal rates of polysaccharides, proteins, tannins and brown pigments are as high as 58.4%, 53.8%, 54.1% and 51.1% respectively. The method for synergistically preparing tea saponin and microbial flocculants of the present invention also has the advantages of simple process, convenient operation, low cost, environmental friendliness, high use value and good application prospects.

[0048] (2) In the present invention, by optimizing the volume ratio of the crude tea saponin 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 flocculants 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 the 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 reagents 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. By optimizing the volume ratio of crude microbial flocculant to organic solvent to 1-2:1-3, the consumption of organic solvent can be reduced while harvesting high-yield and high-purity microbial flocculants. In particular, if the dosage of organic solvent is too high, the process cost will increase, and it will bring potential harm to the environment and health; if the dosage of organic solvent is too low, the microbial flocculant cannot be effectively extracted, and the product obtained will have low yield and low purity.

[0049] (3) In the present invention, by optimizing the ultrasonic conditions, specifically, ultrasonicating at a temperature of 40°C to 60°C for 20min to 50min, while ensuring energy saving, the thermal effect and cavitation effect can be utilized to promote the dissolution of tea saponin in the tea oil cake as much as possible in a shorter time, thereby helping to increase the yield of tea saponin. At the same time, by subjecting the mixture of the tea saponin crude extract and the residue to hydrothermal treatment, a tea oil cake extract with rich nutrients and good adaptability of various nutrients can be prepared.

[0050] (4) 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 high-purity microbial flocculant 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.

[0051] (5) 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. The microorganisms grow rapidly in the first stage of fermentation, and a large amount of floccules are produced in the second stage of fermentation. Thus, the fermentation time can be reduced while obtaining a high-yield and high-purity microbial flocculant, which is conducive to large-scale preparation and convenient for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] 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 accompanying drawings in the embodiments of the present invention.

[0053] Figure 1 This is a process flow chart for the coordinated preparation of tea saponin and microbial flocculant in Example 1 of the present invention.

[0054] Figure 2 It is a comparison chart of the yield of pure microbial flocculants in Examples 1-8 of the present invention. DETAILED DESCRIPTION

[0055] 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.

[0056] The materials and instruments used in the following examples are all commercially available.

[0057] Embodiment 1:

[0058] A method for synergistically preparing tea saponin and microbial flocculant, the process flow chart of which is as follows Figure 1 As shown, the following steps are included:

[0059] (1) Preparation of tea saponin crude extract and residue

[0060] (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.

[0061] (1.2) 50 g of sieved tea oil cake particles are mixed with 1000 mL of water. The tea oil cake particles are mixed with water, placed in an ultrasonic device, ultrasonicated at 40° C. for 30 min, allowed to stand, and filtered to obtain a tea saponin crude extract and residue.

[0062] (2) Preparation of tea cake extract

[0063] The tea saponin crude extract obtained in step (1) is divided into ten parts, three parts of the tea saponin crude extract are mixed with the residue, and then placed in a high-pressure steam sterilizer, hydrothermal at 121° C. for 30 minutes, and the hydrothermal solution is vacuum filtered using a 300-500 mesh filter cloth to obtain a filtrate that is a tea oil cake extract. The tea oil cake extract has a polysaccharide content of 1000 mg / L, a protein content of 1800 mg / L, and a SCOD content of 10000 mg / L.

[0064] In this step, the waste residue obtained after vacuum filtration can be used to prepare molded fuel and organic fertilizer.

[0065] (3) Preparation of tea cake culture medium

[0066] Take the camellia oleifera cake extract prepared in step (2), 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 a camellia oleifera cake culture medium.

[0067] (4) Preparation of microbial flocculants

[0068] (4.1) 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.

[0069] 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.

[0070] (4.2) 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 24.0 h to activate the bacterial strain and form a primary seed colony.

[0071] (4.3) 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.

[0072] (4.4) The inoculation amount of seed liquid is 5% of the volume of the camellia oleifera cake culture medium, and the above-mentioned Bacillus polymyxa GA1 seed liquid is added to the camellia oleifera cake culture medium, and the culture is placed in a shaking incubator, first cultured at a temperature of 30°C and a rotation speed of 150 rpm for 24.0 h, and then cultured at a temperature of 25°C and a rotation speed of 120 rpm for 48.0 h. The fermentation product obtained after the culture is placed in a centrifuge and centrifuged at a rotation speed of 10000 rpm for 10 min to remove the bacteria, thereby obtaining a fermentation product without the bacteria, which is a crude microbial flocculant.

[0073] (4.5) After the cultivation is completed, the crude microbial flocculant is purified, including the following treatment: taking the crude microbial flocculant obtained after the cultivation at a volume ratio of 2:1, adding acetone (before the use of acetone, the following treatment is also included: pre-cooling at 4°C for 24 hours), placing it in a 4°C refrigerator for 24.0 hours, and then placing it in a centrifuge, centrifuging it at a speed of 10000 rpm for 10.0 minutes, drying it, collecting acetone, and the obtained solid is the pure microbial flocculant.

[0074] (5) According to the volume ratio of tea saponin crude extract to pure water of 4:1, take the remaining seven parts of tea saponin crude extract in step (1) and mix with water, put it into a shaking table and mix it thoroughly for 2 hours (i.e., stir it for 2 hours), pour it into a separating 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.

[0075] (6) According to the mass ratio of pure microbial flocculant to water of 1:150, the pure microbial flocculant obtained in step (4) is mixed with water to prepare a microbial flocculant solution, and mixed with 100 mL of the aqueous solution obtained in step (5) and 2.0 mL of calcium chloride solution (the mass concentration of calcium chloride in the solution is 10%), stirred at a speed of 200 r / min for 5 min, stirred at a speed of 30 r / min for 5 min, left to stand for 10 minutes, filtered, and the supernatant is retained, which is liquid tea saponin. The waste residue obtained by filtration and separation in this step can be used to prepare molded fuel and organic fertilizer.

[0076] (7) The liquid tea saponin obtained in step (6) is concentrated once, and water is collected to obtain a concentrate. In this step, the collected water can be returned to steps (1), (5), and (6), and can also be reused in other steps if necessary.

[0077] (8) The concentrate obtained in step (7) is mixed with an organic solvent (acetone) for extraction, and the upper layer liquid is collected.

[0078] (9) Decolorizing the upper layer liquid collected in step (8) with activated carbon, filtering, and concentrating the decolorized upper layer liquid for a second time, collecting the organic solvent (acetone), and obtaining solid tea saponin.

[0079] In this embodiment, the organic solvent (acetone) collected in steps (4) and (9) can be returned to steps (4) and (8) for reuse.

[0080] Example 2

[0081] A method for synergistically preparing tea saponin and microbial flocculant is basically the same as Example 1, except that in step (4.5), the volume ratio of the crude microbial flocculant to acetone is 1:1.

[0082] Example 3

[0083] A method for synergistically preparing tea saponin and microbial flocculant is basically the same as Example 1, except that in step (4.5), the volume ratio of the crude microbial flocculant to acetone is 2:3.

[0084] Example 4

[0085] A method for synergistically preparing tea saponin and microbial flocculant is basically the same as Example 1, except that in step (4.5), the volume ratio of the crude microbial flocculant to acetone is 1:2.

[0086] Example 5

[0087] A method for synergistically preparing tea saponin and microbial flocculant is basically the same as Example 1, except that the organic solvent used is recycled ethanol, and the volume ratio of the crude microbial flocculant to ethanol in step (4.5) is 2:1.

[0088] Example 6

[0089] A method for synergistically preparing tea saponin and microbial flocculant is basically the same as Example 1, except that the organic solvent used is recycled ethanol, and the volume ratio of the crude microbial flocculant to ethanol in step (4.5) is 1:1.

[0090] Example 7

[0091] A method for synergistically preparing tea saponin and microbial flocculant is basically the same as Example 1, except that the organic solvent used is recycled ethanol, and the volume ratio of the crude microbial flocculant to ethanol in step (4.5) is 2:3.

[0092] Example 8

[0093] A method for synergistically preparing tea saponin and microbial flocculant is basically the same as Example 1, except that the organic solvent used is recycled ethanol, and the volume ratio of the crude microbial flocculant to ethanol in step (4.5) is 1:2.

[0094] The flocculation rate detection method of the microbial flocculant prepared by the present invention is as follows: 500 mL of 4 g / L kaolin suspension is prepared, 10 mL of 10% calcium chloride solution is added, 10 mL of pure microbial flocculant is added, and the pH value of the solution is adjusted to 8-8.5. The kaolin suspension without flocculant is used as a control, and the mixture is stirred rapidly for 3 minutes, then stirred slowly for 3 minutes, and then allowed to settle for 5 minutes. The liquid at 1 cm above the liquid surface is aspirated, and the absorbance value is measured at a wavelength of 550 nm using an ultraviolet spectrophotometer, and the flocculation rate of the microbial flocculant on the kaolin suspension is calculated. The detection results are shown in Table 1.

[0095] Flocculation rate calculation formula:

[0096] Flocculation rate / % = (AB) / A*100%

[0097] Where: A——OD500 of original kaolin suspension;

[0098] B——OD500 of kaolin suspension after adding flocculant.

[0099] Table 1 Flocculation rate of pure microbial flocculants prepared in Examples 1-8

[0100] Test items Example 1 Example 2 Example 3 Example 4 Flocculation rate 93.68％ 92.64％ 92.86％ 93.62％ Test items Example 5 Example 6 Example 7 Example 8 Flocculation rate 93.29％ 86.31％ 88.49％ 92.09％

[0101] It can be seen from the test data in Table 1 above that the microbial flocculants extracted by purifying the organic solvent (recovered ethanol) recovered by acetone and tea saponin process have a high flocculation rate, among which the flocculant rate extracted by acetone is higher, which shows that the microbial flocculant prepared by the present invention has a higher purity. By comparing Examples 1-4 and Examples 5-8, when the volume ratio of the crude microbial flocculant to the organic solvent is 2:1 and 1:2, the flocculation rate of the obtained microbial flocculant is higher, and the microbial flocculants extracted by purifying the organic solvent recovered by the tea saponin process also have a good flocculation effect, and have a wide range of application prospects.

[0102] Figure 2 It is a comparison chart of the yield of pure microbial flocculants in Examples 1-8 of the present invention. Figure 2It shows that the microbial flocculants extracted by acetone and tea saponin process recovery of organic solvents have high yields, and there is almost no difference in the yield of microbial flocculants extracted by the two solvents between Examples 1-4 and 5-8. In addition, Examples 4 and 8 show that when the volume ratio of crude bioflocculant to organic solvent is 1:2, the yield of pure microbial flocculant obtained is the highest.

[0103] Table 2 Yield data of solid tea saponin prepared in Examples 1-8

[0104] Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Tea saponin 89.93％ 87.71％ 88.06％ 89.77％ 89.40％ 87.13％ 87.26％ 88.50％

[0105] It can be seen from the test data in Table 2 above that the efficiency of tea saponin harvesting by the microbial flocculants purified and extracted using acetone and organic solvents (recovered ethanol) recovered from the tea saponin process is relatively high, among which the efficiency of the flocculant extracted by acetone is slightly higher. By comparing Examples 1-4 and 5-8, when the volume ratio of the crude microbial flocculant to the organic solvent is 2:1, the yield of tea saponin is higher, and when the volume ratio of the crude microbial flocculant to acetone is 2:1, the yield of tea saponin is as high as 89.77%. It can be seen that in the present invention, the tea saponin can be purified and extracted using organic solvents recovered from the tea saponin process, which can also improve the yield of tea saponin and has a wide range of application prospects.

[0106] It can be seen from the above results that, compared with the conventional method, the method of the present invention for the synergistic preparation of tea saponin and microbial flocculant has the following advantages: (a) less amount of organic solvent. In order to increase the yield of microbial flocculant, in the conventional microbial flocculant purification method, the amount of organic solvent used is usually 2 to 3 times the volume of the fermentation broth. In the present invention, the amount of organic solvent used can be as low as 50% of the volume of the crude microbial flocculant. At this time, the yield of the microbial flocculant is 6.21 g / L, reaching 95.9% of the 2-fold volume of organic solvent extraction, which greatly reduces the amount of organic solvent used in the extraction process. More importantly, in the present invention, the extraction of microbial flocculant and tea saponin shares a set of organic solvent recycling system, which can realize the recycling and sharing of organic solvent, thereby further reducing the amount of organic solvent used; (b) the yield of microbial flocculant is high and the purity is high. For example, the molecular weight of the extracellular polymers in the prepared microbial flocculant is concentrated at 4.4×10 5 -4.6×10 5 Da, compared with the molecular weight of the flocculant produced by traditional fermentation medium, it has higher consistency, which is conducive to the centralized extraction of a single proportion of organic solvent. At the same time, when the volume of organic solvent is 50% of the crude microbial flocculant, the average molecular weight of the extracted microbial flocculant reaches the highest value of 4.6×10 5Da, has a more concentrated molecular weight distribution range and a stronger peak level of flocculation active functional groups, showing a more excellent flocculation ability. Specifically, the flocculation rate of the microbial flocculant for kaolin suspension can reach 99.1%, and can also reach 98.9% without the assistance of phosphate. More importantly, the prepared microbial flocculant has a suitable molecular weight level and distribution range, which is conducive to the synchronous separation of flocculant components during the flocculation process, reduces the risk of flocculant residue in the supernatant, and reduces the impurity interference with the subsequent tea saponin product; (c) the tea saponin product has a high yield and high quality, among which the yield of high-purity tea saponin product is as high as 89.93%. In addition, the removal rates of polysaccharides, proteins, tannins and brown pigments are as high as 58.4%, 53.8%, 54.1% and 51.1%, respectively. Therefore, the method for the synergistic preparation of tea saponin and microbial flocculant of the present invention can not only simultaneously prepare microbial flocculant and tea saponin with high yield and high purity, but also realize the recycling of water and organic solvents. It also has the advantages of simple process, convenient operation, low cost, environmental friendliness, etc., has high use value and good application prospects.

[0107] 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 synergistically preparing tea saponin and microbial flocculant, characterized in that: The following steps are involved: S1. Mixing tea oil cake with water, ultrasonicating, standing, filtering, and obtaining a tea saponin crude extract and residue; S2, mixing the tea saponin crude extract and the residue, performing hydrothermal treatment, filtering, and obtaining a tea cake extract; S3, preparing a tea cake medium with the tea cake extract obtained in step S2, inoculating the flocculating functional bacteria seed solution for cultivation, and centrifuging to obtain a crude microbial flocculant; S4, mixing the crude microbial flocculant obtained in step S3 with an organic solvent, allowing to stand, centrifuging, and drying to obtain a pure microbial flocculant; S5, mixing the tea saponin crude extract obtained in step S1 with water, stirring, and standing to separate the oil and water, and retaining the aqueous solution; S6, preparing a microbial flocculant solution from the pure microbial flocculant obtained in step S4, mixing the solution with the aqueous solution and the coagulant aid solution obtained in step S5 for flocculation, allowing the solution to stand, filtering, and obtaining liquid tea saponin; S7, concentrating the liquid tea saponin obtained in step S5, recovering water, and obtaining a concentrate; S8, mixing the concentrate obtained in step S7 with an organic solvent for extraction, and collecting the upper liquid; S9, decolorizing and re-concentrating the upper liquid collected in step S8, recovering the organic solvent, and obtaining solid tea saponin.

2. The method according to claim 1, characterized in that In step S7, the recovered water is returned to step S1, or returned to step S5; In step S9, the recovered organic solvent is returned to step S4, or returned to step S8.

3. The method according to claim 1 or 2, 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 to 60-mesh sieve to obtain camellia oleifera cake particles; the ultrasonication is performed at a temperature of 40°C-60°C; and the ultrasonication time is 20min-50min.

4. The method according to claim 3, characterized in that The ratio of the oil-tea cake to water is 50g-150g:1L.

5. The method according to claim 1 or 2, characterized in that: In step S2, the hydrothermal treatment is carried out at a temperature of 111°C to 126°C; the time of the hydrothermal treatment is 10min to 50min; the content of polysaccharides in the oil-tea camellia cake extract is 800mg / L to 15000mg / L, the content of protein is 1500mg / L to 24000mg / L, and the content of SCOD is 8000mg / L to 110000mg / L.

6. The method according to claim 1 or 2, characterized in that: In step S3, the preparation method of the camellia oleifera cake culture medium is as follows: the camellia oleifera cake extract, potassium dihydrogen phosphate and dipotassium hydrogen phosphate are mixed 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-2.0 g / L, and the concentration of dipotassium hydrogen phosphate is 3.0 g / L-5.0 g / L; the pH value of the camellia oleifera cake culture medium is 5-9; the inoculation amount of the flocculation functional bacteria seed liquid is 4%-5% of the volume of the camellia oleifera cake culture medium; the culturing is as follows: the culturing is performed at a temperature of 30°C-35°C and a rotation speed of 130rpm-150rpm for 12.0h-24.0h, and the culturing is performed at a temperature of 20°C-25°C and a rotation speed of 100rpm-120rpm for 24.0h-72.0h; the centrifugal rotation speed is 8000rpm-10000rpm; the centrifugal time is 8min-10min; The method for preparing the flocculation functional bacteria seed solution comprises the following steps: (1) Inoculating a flocculating functional bacterial strain into a solid activation culture 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 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 bacterial strain is one of Bacillus polymyxa, lactic acid bacteria, Clostridium butyricum and Halomonas; the Bacillus polymyxa is GA1; (2) inoculating the primary seed colony obtained in step (1) 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.

7. The method according to claim 1 or 2, characterized in that: In step S4, the volume ratio of the crude 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 treatments before use: precooling at a temperature of 4°C for 12h to 24h; the standing temperature is 4°C; the standing time is 24h to 48h; the centrifugal speed is 8000rpm to 10000rpm; the centrifugal time is 8min to 10min.

8. The method according to claim 1 or 2, characterized in that: In step S5, the volume ratio of the tea saponin crude extract to water is 2-4:1; the stirring is performed on a shaking table; the stirring time is 2h-5h; and the standing time is 5h-12h.

9. The method according to claim 1 or 2, characterized in that: In step S6, the microbial flocculant solution is prepared by mixing pure microbial flocculant with water; the mass ratio of the pure microbial flocculant to water is 1:100-200; the volume ratio of the aqueous solution to the microbial flocculant solution is 100:1-2; the volume ratio of the aqueous solution to the coagulant aid solution is 100:2-3; the coagulant in the coagulant aid solution is at least one of calcium chloride and lime; the mass concentration of the coagulant in the coagulant aid solution is 8%-10%; the flocculation is: stirring for 3min-5min at a rotation speed of 200r / min-300r / min, and stirring for 3min-5min at a rotation speed of 30r / min-60r / min; the standing time is 10min-30min.

10. The method according to claim 1 or 2, characterized in that: In step S8, the organic solvent is acetone and / or ethanol; In step S9, activated carbon or hydrogen peroxide is used to decolorize the upper liquid collected in step S8.

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