A method for purifying tea saponins by flocculation using microbial flocculants
By combining microbial flocculants and coagulants, the extraction process of tea saponin was optimized, solving the problems of low yield, low purity, and incomplete removal of impurities during the extraction process. This resulted in efficient and safe purification of tea saponin, which is suitable for the pharmaceutical, food, and daily chemical industries.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-14
- Publication Date
- 2026-03-13
AI Technical Summary
Existing methods for extracting tea saponins suffer from problems such as complex processes, low yields, low purity, and insufficient safety. In particular, the removal of impurities is ineffective, which hinders its high-value utilization in the fields of medicine, food, and daily chemicals.
By using microbial flocculants combined with coagulants, and through steps of stirring, settling, and filtration, the ratio of crude tea saponin extract to water and the amount of flocculant were optimized to achieve efficient purification of tea saponin, remove oils and soluble impurities, and improve the yield and purity of tea saponin.
It significantly improves the yield and purity of tea saponins, with an oil content of less than 0.1% and a soluble impurity removal rate of over 50%, making it suitable for large-scale industrial applications. It also reduces product toxicity and is applicable to the pharmaceutical, food, and daily chemical industries.
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Figure CN119409759B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of tea saponin preparation technology, and relates to a method for purifying tea saponin by flocculation using microbial flocculants. Background Technology
[0002] Tea saponins are a mixture of oleanane-type pentacyclic triterpenoid saponins, composed of sapogenins (glycosidic aglycones), sugars (glycosyl groups), and organic acids. They are widely found in the roots, stems, leaves, flowers, and seeds of Camellia plants. They possess hemolytic and piscitoxic effects, insecticidal and bactericidal properties, as well as anti-inflammatory, expectorant, antitussive, and analgesic pharmacological functions, and promote plant growth. Due to structural differences, various tea saponins exhibit certain activity variations. Simultaneously, tea saponins are also a natural nonionic surfactant with advantages such as biodegradability, good biocompatibility, and low toxicity, making them widely applicable in agriculture, daily chemicals, medicine, and food industries.
[0003] As a plant of the Camellia genus, the content of tea seed saponins in camellia seeds initially increases and then decreases during the ripening process, eventually stabilizing in the later stages of maturity. The distribution of tea seed saponin content in different parts of the seed is: kernel > pericarp > seed coat, with the tea saponin content in camellia seed cake reaching 10-14 wt%. Therefore, camellia seed cake is a major source of tea saponins.
[0004] Currently, the main industrial extraction methods for tea saponin are water extraction and organic solvent extraction. Water extraction is the traditional method, utilizing the high solubility of tea saponin in hot water. While simple and low-cost, prolonged hot water extraction can lead to saponin hydrolysis and the leaching of large amounts of water-soluble impurities, making it difficult to obtain high yields and high-quality tea saponin. Organic solvent extraction typically uses methanol or ethanol as a solvent, leveraging the trace solubility of impurities in alcoholic solvents to extract tea saponin. This method has low energy consumption but high solvent consumption, complex production processes, and demanding equipment. Furthermore, the crude tea saponin obtained from these extraction methods often contains impurities such as oils, proteins, polysaccharides, tannins, and pigments. It is viscous, dark in color, and easily absorbs moisture and condenses in the air. Therefore, when high purity is required, further purification of the crude product is generally necessary through methods such as flocculation, chemical precipitation, recrystallization, membrane separation, resin adsorption, and gel chromatography. Among these methods, flocculation and impurity removal can be integrated with different types of extraction schemes and is compatible with different levels of purification pathways. Positioned at a crucial point in the extraction and preparation process of plant functional components such as tea saponins, it demonstrates broad applicability and effectiveness across various processes. Compared to chemical flocculants, microbial flocculants offer advantages such as safety, non-toxicity, and biodegradability. Furthermore, the flocculated precipitate does not contain harmful substances, offering potential advantages for the subsequent high-value utilization of tea saponins. Therefore, developing a versatile, safe, and efficient tea saponin purification method, addressing the complex flocculation conditions in the extraction and purification process of tea saponins and meeting multiple objective requirements such as product yield and purity, is of great significance for maximizing the value potential of tea saponins. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a safe and efficient method for purifying tea saponins by flocculation using microbial flocculants, which not only improves the yield of tea saponins but also improves the purity of tea saponins.
[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0007] A method for purifying tea saponins by flocculation using microbial flocculants includes the following steps:
[0008] (1) Mix the crude tea saponin extract with water, stir, let stand, and let the oil and water separate into layers, retaining the aqueous solution;
[0009] (2) Mix the aqueous solution, coagulant solution and microbial flocculant solution obtained in step (1) for flocculation, let stand, filter, and complete the flocculation and purification of tea saponin.
[0010] In a further improvement to the above method, in step (1), the volume ratio of the crude tea saponin extract to water is 2 to 4:1.
[0011] In a further improvement to the above method, in step (1), the crude tea saponin extract is prepared from camellia seed cake. For example, the crude tea saponin extract is prepared by mixing camellia seed cake and water and then ultrasonically treating the mixture, wherein the temperature of the water is 30℃~80℃. However, the preparation method of the crude tea saponin extract is not limited to this.
[0012] In a further improvement to the above method, in step (1), the stirring is carried out on a shaker; the stirring time is 2h to 5h.
[0013] In a further improvement to the above method, in step (1), the settling time is 5h to 12h.
[0014] The above method is further improved in step (2), where the flocculation is carried out by stirring for 3 min to 5 min at a speed of 200 r / min to 300 r / min, and stirring for 3 min to 5 min at a speed of 30 r / min to 60 r / min.
[0015] In a further improvement to the above method, in step (2), the settling time is 10 min to 30 min.
[0016] In a further improvement to the above method, in step (2), the volume ratio of the aqueous solution to the coagulant solution is 100:2 to 3.
[0017] In a further improvement to the above method, in step (2), the coagulant in the coagulant solution is at least one of calcium chloride and lime.
[0018] In a further improvement to the above method, in step (2), the mass concentration of the coagulant in the coagulant solution is 8% to 10%.
[0019] In a further improvement to the above method, in step (2), the volume ratio of the aqueous solution to the microbial flocculant solution is 100:1 to 2.
[0020] In a further improvement to the above method, in step (2), the microbial flocculant solution is prepared by mixing microbial flocculant with water; the mass ratio of the microbial flocculant to water is 1:100 to 200.
[0021] A further improvement to the above method is the preparation method of the microbial flocculant, which includes the following steps:
[0022] S1. Mix camellia seed cake with water, sonicate, hydrothermally treat, filter, collect the filtrate to obtain camellia seed cake extract;
[0023] S2. Prepare a camellia oil cake extract obtained in step S1 into a camellia oil cake culture medium.
[0024] S3. Inoculate the seed liquid of flocculating bacteria into the tea oil cake medium obtained in step S2 and culture it to obtain microbial flocculant.
[0025] In a further improvement to the above method, in step S1, the ratio of camellia oil cake meal to water is 30g-200g:1L; the camellia oil cake meal also includes the following treatment before use: drying the camellia oil cake meal, crushing it, and passing it through a 40-60 mesh sieve to obtain camellia oil cake meal granules.
[0026] In a further improvement to the above method, in step S1, the ultrasound is performed at a temperature of 30℃ to 40℃; and the ultrasound duration is 20 min to 50 min.
[0027] In a further improvement to the above method, in step S1, the hydrothermal treatment is performed at a temperature of 111℃ to 126℃; and the hydrothermal treatment time is 10 min to 50 min.
[0028] In a further improvement to the above method, in step S1, the polysaccharide content in the camellia oil cake extract is 1800 mg / L to 15000 mg / L, the protein content is 3300 mg / L to 24000 mg / L, and the SCOD content is 18000 mg / L to 110000 mg / L.
[0029] In a further improvement to the above method, in step S1, the ratio of camellia seed cake to water is 50g to 150g: 1L.
[0030] In a further improvement to the above method, in step S2, the preparation method of the camellia oil cake meal culture medium is as follows: Camellia oil cake meal extract, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate are mixed to obtain the camellia oil cake meal culture medium; the concentration of potassium dihydrogen phosphate in the camellia oil cake meal 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 oil cake meal culture medium is 5 to 9.
[0031] In a further improvement to the above method, in step S3, the inoculation amount of the flocculating functional bacteria seed liquid is 4% to 5% of the volume of the camellia oil cake culture medium.
[0032] In a further improvement to the above method, in step S3, the cultivation is carried out as follows: 12.0h to 24.0h at a temperature of 30℃ to 35℃ and a rotation speed of 130rpm to 150rpm, and 24.0h to 72.0h at a temperature of 20℃ to 25℃ and a rotation speed of 100rpm to 120rpm.
[0033] In a further improvement to the above method, step S3, after the culture is completed, includes the following treatment: centrifuging the fermentation product obtained after culture at a speed of 8000 rpm to 10000 rpm for 8 min to 10 min to remove the bacterial cells and obtain the microbial flocculant.
[0034] In a further improvement to the above method, step S3 further includes the following treatment of the microbial flocculant: mixing the microbial flocculant with an organic solvent, allowing it to stand, centrifuging it at 8000 rpm to 10000 rpm for 8 min to 10 min, removing the supernatant, and obtaining 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 pre-cooling it at 4°C for 12 h to 24 h; the standing is carried out at 4°C; the standing time is 24 h to 48 h.
[0035] A further improvement to the above method, in step S3, the preparation method of the flocculating functional bacteria seed solution includes the following steps:
[0036] (a) The flocculating functional bacteria strain was inoculated into a solid activation medium and cultured at 30℃~35℃ for 12.0h~24.0h to obtain primary seed colonies; the solid activation medium had the following formula: 10.0g / L peptone, 3.0g / L beef extract, 5.0g / L sodium chloride, 20.0g / L agar powder, and a pH of 7~8; the flocculating functional bacteria was one of Bacillus polymyxa, lactic acid bacteria, Clostridium butyricum, and Halomonas; the Bacillus polymyxa was GA1;
[0037] (b) The primary seed colonies obtained in step (a) are inoculated into liquid proliferation medium and cultured at a temperature of 25℃~30℃ and a rotation speed of 130rpm~150rpm for 12.0h~24.0h to obtain flocculation functional bacteria seed liquid; the formula of the liquid proliferation medium is: 10.0g / L peptone, 3.0g / L beef extract, 5.0g / L sodium chloride, and pH value of 7~8.
[0038] Compared with the prior art, the advantages of the present invention are as follows:
[0039] (1) To address the shortcomings of existing tea saponin purification methods, such as complex processes, large tea saponin losses, and difficulty in effectively removing impurities, resulting in low tea saponin yield, low purity, and low safety, this invention creatively proposes a method for purifying tea saponin using microbial flocculants. First, the crude tea saponin extract is mixed with water, stirred, and allowed to stand to effectively remove oil from the crude extract. This not only eliminates the adverse effects of oil on the subsequent flocculation and purification process but also reduces the oil content in the tea saponin product. Based on this, a coagulant aid solution and a microbial flocculant solution are added to the aqueous solution obtained after oil-water separation for flocculation treatment. The coagulant aid can neutralize the charge of suspended particles and colloidal substances in the water, reducing their surface charge and making them easier to aggregate into larger flocs. Simultaneously, the microbial flocculant, based on the flocculation and separation of suspended particles, can adsorb and remove impurities such as proteins, polysaccharides, tannins, and pigments from the crude extract using its abundant functional groups. Furthermore, by optimizing flocculation conditions, the synergistic removal of particulate matter and macromolecular substances can be effectively achieved. Specifically, rapid stirring is performed first to allow the microbial flocculant and coagulant aid to disperse quickly and evenly in the water. This avoids uneven dispersion of the agents, which could lead to excessively high local concentrations. Rapid stirring also promotes the rapid collision and aggregation of suspended matter in the water. When flocs begin to form, the stirring speed and intensity are reduced. Under these slow stirring conditions, it is helpful for the flocs to further form larger particles, facilitating subsequent sedimentation or filtration. Using these flocs as a matrix, dissolved impurities are adsorbed during the mixing process, achieving the best flocculation and impurity removal effect. Finally, after settling and filtration, the resulting supernatant is liquid tea saponin, which has a high yield and high purity. Compared with conventional flocculation purification methods for tea saponin, the method of purifying tea saponin using microbial flocculants in this invention has the following advantages: (a) Low oil content: By combining water removal and microbial flocculant removal, the oil content in the tea saponin product can be effectively removed, with the oil content in the tea saponin product being less than 0.1%. (b) High purity: By combining water removal, coagulant aid, and microbial flocculant, not only suspended particles and soluble macromolecules can be effectively removed, but also soluble impurities such as polysaccharides, proteins, brown pigments, and tannins can be effectively removed, with the removal rate of these soluble impurities reaching over 50%. (c) High yield: By combining water removal, microbial flocculant, and coagulant aid, tea saponin can be retained to the greatest extent possible while effectively removing impurities, with the tea saponin retention rate reaching as high as 93.3%. (d) Low toxicity. On the one hand, the microbial flocculant used has low toxicity and easy biodegradability, which is conducive to reducing the toxicity of tea saponin products. On the other hand, the low residual amount of coagulant and microbial flocculant is also conducive to reducing the toxicity of tea saponin products. Therefore, tea saponin can be used in the fields of medicine, food, and daily chemicals.Meanwhile, the method of purifying tea saponin by flocculation using microbial flocculants has the advantages of being simple, convenient, safe, and environmentally friendly, making it suitable for large-scale preparation of tea saponin and facilitating its industrial application.
[0040] (2) In this invention, by optimizing the volume ratio of crude tea saponin extract to water to 2-4:1, the volume of the aqueous solution to be treated is reduced while ensuring the oil removal effect, thereby reducing the processing difficulty and energy consumption of subsequent processes. In particular, if the amount of water is too high, it will increase the volume of the sample to be treated, increasing the process difficulty and energy consumption; if the amount of water is too low, it will reduce the oil removal effect of the pretreatment and reduce the content of tea saponin extracted from the water. By optimizing the volume ratio of aqueous solution to microbial flocculant solution to 100:1-2, the stability and efficiency of the flocculation process can be ensured. At this time, the formation and settling speed of flocs is faster, which can shorten the treatment time and improve the treatment efficiency. At the same time, it can also avoid the waste of flocculant caused by excessive addition and effectively avoid 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 solids can be accelerated, forming large and dense flocs, which is beneficial for subsequent sedimentation or filtration processes. Simultaneously, precise control of the coagulant dosage can avoid unnecessary waste and reduce reagent costs. In particular, excessive coagulant dosage can cause coagulation of colloidal particles in the solution, forming too many fine particles that are difficult to settle, thus negatively impacting the flocculation effect.
[0041] (3) In this invention, the microbial flocculant used is prepared by culturing flocculating functional bacteria using camellia seed cake extract as the culture medium raw material. It 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 the synergistic removal of soluble macromolecular impurities through adsorption, which has higher safety and impurity removal efficiency, and can prepare tea saponin products with higher purity.
[0042] (4) In the preparation method of the microbial flocculant used in this invention, by optimizing the extraction conditions, specifically by ultrasonication for 20 min to 50 min at a temperature of 30℃~40℃, and under the combined effect of hydrothermal treatment, it is possible to prepare an extract of camellia seed cake with rich nutrients and good compatibility of various nutrients, which is more conducive to preparing a microbial flocculant with high yield and high purity.
[0043] (5) In this invention, by optimizing the ratio of camellia seed cake to water, specifically optimizing the ratio of the two to 30g~200g∶1L, and especially 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 flocculating bacteria, it is not only conducive to the formation of microbial flocculants with high flocculation rate as the main component of extracellular polymers, but also significantly increases the unit yield of microbial flocculants, which is conducive to improving the utilization rate of camellia seed cake and making the preparation cost lower.
[0044] (6) In this invention, by optimizing the culture conditions, specifically by inoculating the seed liquid of flocculating functional bacteria into the culture medium of camellia oil cake, first culturing it at a temperature of 30℃~35℃ and a rotation speed of 130rpm~150rpm for 12.0h~24.0h, and then culturing it at a temperature of 20℃~25℃ and a rotation speed of 100rpm~120rpm for 24.0h~72.0h, the microorganisms grow rapidly in the first stage of fermentation and produce a large amount of flocs in the second stage of fermentation. Thus, while reducing the fermentation time, a high-yield and high-flocculation-rate microbial flocculant can be obtained, which is conducive to large-scale preparation and convenient for industrial application. Attached Figure Description
[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.
[0046] Figure 1 This is a process flow diagram of the purification of tea saponin by flocculation using microbial flocculants in Example 1 of the present invention. Detailed Implementation
[0047] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention.
[0048] The materials and instruments used in the following examples are all commercially available.
[0049] Example 1:
[0050] A method for purifying tea saponins using microbial flocculants is illustrated in the process flow diagram below. Figure 1 As shown, it includes the following steps:
[0051] S1. Prepare a crude extract of tea saponin from camellia oil cake. For example, mix camellia oil cake with hot water at 40°C and sonicate for 30 minutes, filter, collect the filtrate, and obtain a crude extract of tea saponin.
[0052] S2. According to the volume ratio of crude tea saponin extract to pure water of 4:1, mix the crude tea saponin extract with water, put it in a shaker and mix thoroughly for 2 hours, pour it into a separatory funnel and let it stand for 5 hours to fully separate the oil phase and the water phase. Remove the upper oil phase and retain the middle aqueous solution.
[0053] S3. Mix 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, stir at 200 r / min for 5 min, stir at 30 r / min for 5 min, let stand for 10 minutes, filter, and retain the supernatant, which is liquid tea saponin, thus completing the flocculation and purification of tea saponin.
[0054] In this embodiment, the microbial flocculant solution is prepared by mixing microbial flocculant and water, with a mass ratio of microbial flocculant to water of 1:150. The preparation method of the microbial flocculant includes the following steps:
[0055] (1) Preparation of camellia oil cake and pulp extract:
[0056] (1.1) Place the camellia oil cake and dry it continuously at 60℃ for 5.0h until constant weight. Then crush it with a pulverizer and use a steel screen to take 40-60 mesh camellia oil cake particles for later use.
[0057] (1.2) Mix 50g of sieved camellia oil cake particles with water per 1000mL of water. First, place the mixture in an ultrasonic device and sonicate at 40℃ for 30min. Then, place it in a high-pressure steam sterilizer and hydrothermally heat it at 121℃ for 30min. Vacuum filter the hydrothermally heated solution using a 300-500 mesh filter cloth. The resulting filtrate is the camellia oil cake extract. The polysaccharide content in this camellia oil cake extract is 1800mg / L, the protein content is 3300mg / L, and the SCOD content is 18000mg / L.
[0058] If the ultrasonic treatment is omitted in this step, 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. This is something that needs to be overcome in this invention.
[0059] (2) Preparation of Camellia oleifera cake meal culture medium
[0060] Take the extract of camellia seed cake, add potassium dihydrogen phosphate and dipotassium hydrogen phosphate solution to make the concentration of potassium dihydrogen phosphate in the mixed solution 2.0 g / L and the concentration of dipotassium hydrogen phosphate 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℃ for 30 min to obtain camellia seed 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 to 7. Put the prepared culture medium into autoclave and sterilize at 121°C for 30 min. After sterilization, pour the culture medium into a clean culture dish in a sterile operating table, wait for the culture medium to cool and solidify, and then invert it to obtain a solid activated culture medium.
[0063] Weigh out 10.0 g / L peptone, 3.0 g / L beef extract and 5.0 g / L sodium chloride, and adjust the pH to 7. Place the prepared culture medium in an autoclave and sterilize at 121°C for 30 min to obtain liquid proliferation culture medium.
[0064] (3.2) Take the bacterial strain (Bacillus polymyxa GA1) stored in glycerol tubes and inoculate it into solid activation medium. Incubate at 35℃ for 24.0h to activate the bacterial strain and form primary seed colonies.
[0065] (3.3) The primary seed colonies were inoculated into liquid proliferation medium and cultured in a shaker incubator at 30℃ and 150rpm for 24.0h to proliferate the microorganisms and obtain the secondary seed liquid, which was named Bacillus polymyxa GA1 seed liquid.
[0066] (3.4) The above-mentioned Bacillus polymyxa GA1 seed liquid was added to the Camellia oleifera cake medium at an inoculation amount of 5% of the volume of the Camellia oleifera cake medium and transferred to a shaker incubator. It was first cultured at 30℃ and 150 rpm for 24.0 h, and then cultured at 25℃ and 120 rpm for 48.0 h. The fermentation product obtained after culture was placed in a centrifuge and centrifuged at 10000 rpm for 10 min to remove the bacterial cells. The fermentation product with the bacterial cells removed was the microbial flocculant.
[0067] (3.5) After the culture is completed, the microbial flocculant is purified, including the following treatment: take the fermentation product obtained after culture at a volume ratio of 2:1, add acetone (before using acetone, the following treatment is also included: pre-cool at 4℃ for 24h), put it in a 4℃ refrigerator and let it stand for 24.0h, then put it in a centrifuge and centrifuge at 10000rpm for 10.0min, remove the supernatant, and obtain the precipitate, which can be used as the purified microbial flocculant.
[0068] Example 2:
[0069] A method for purifying tea saponins by flocculation using microbial flocculants includes the following steps:
[0070] (1) The tea saponin crude extract was prepared from the tea oil cake residue, the same as in Example 1.
[0071] (2) According to the volume ratio of crude tea saponin extract to pure water of 4:1, the crude tea saponin extract is mixed with water and placed in a shaker to mix thoroughly for 2 hours. Then, it is poured into a separatory funnel and left to stand for 5 hours to fully separate the oil phase and the water phase. The upper oil phase is removed and the middle aqueous solution is retained.
[0072] (3) Mix 100 mL of the aqueous solution obtained in step (2), 3.0 mL of 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. Stir for 5 min at a speed of 200 r / min, then stir for 5 min at a speed of 30 r / min. Let stand for 10 minutes, filter, and retain the supernatant, which is the liquid tea saponin, thus completing the flocculation and purification of tea saponin.
[0073] Example 3:
[0074] A method for purifying tea saponins by flocculation using microbial flocculants includes the following steps:
[0075] (1) The tea saponin crude extract was prepared from the tea oil cake residue, the same as in Example 1.
[0076] (2) According to the volume ratio of crude tea saponin extract to pure water of 2:1, the crude tea saponin extract is mixed with water, and then mixed thoroughly in a shaker for 2 hours. The mixture is then poured into a separatory funnel and left to stand for 5 hours to fully separate the oil phase and the water phase. The upper oil phase is removed, and the middle aqueous solution is retained.
[0077] (3) Mix 100 mL of the aqueous solution obtained in step (2), 3.0 mL of 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. Stir for 5 min at a speed of 200 r / min, then stir for 5 min at a speed of 30 r / min. Let stand for 10 minutes, filter, and retain the supernatant, which is the liquid tea saponin, thus completing the flocculation and purification of tea saponin.
[0078] Example 4:
[0079] A method for purifying tea saponins by flocculation using microbial flocculants includes the following steps:
[0080] (1) The tea saponin crude extract was prepared from the tea oil cake residue, the same as in Example 1.
[0081] (2) According to the volume ratio of crude tea saponin extract to pure water of 2:1, the crude tea saponin extract is mixed with water, and then mixed thoroughly in a shaker for 2 hours. The mixture is then poured into a separatory funnel and left to stand for 5 hours to fully separate the oil phase and the water phase. The upper oil phase is removed, and the middle aqueous solution is retained.
[0082] (3) Mix 100 mL of the aqueous solution obtained in step (2), 2.0 mL of 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. Stir for 5 min at a speed of 200 r / min, then stir for 5 min at a speed of 30 r / min. Let stand for 10 minutes, filter, and retain the supernatant, which is liquid tea saponin, thus completing the flocculation and purification of tea saponin.
[0083] Example 5:
[0084] A method for purifying tea saponins by flocculation using microbial flocculants includes the following steps:
[0085] (1) The tea saponin crude extract was prepared from the tea oil cake residue, the same as in Example 1.
[0086] (2) According to the volume ratio of crude tea saponin extract to pure water of 2:1, the crude tea saponin extract is mixed with water, and then mixed thoroughly in a shaker for 2 hours. The mixture is then poured into a separatory funnel and left to stand for 5 hours to fully separate the oil phase and the water phase. The upper oil phase is removed, and the middle aqueous solution is retained.
[0087] (3) Mix 100 mL of the aqueous solution obtained in step (2), 2.0 mL of 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. Stir for 5 min at a speed of 200 r / min and then stir for 5 min at a speed of 30 r / min. Let stand for 10 minutes, filter, and retain the supernatant, which is the liquid tea saponin, thus completing the flocculation and purification of tea saponin.
[0088] The supernatant (liquid tea saponin) prepared in Examples 1-5 was used to detect the brown pigment, tannin, polysaccharide, protein and tea saponin in the supernatant according to the following methods. The detection results are shown in Table 1 and Table 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 tungstic acid test solution, and make up to 50.0 mL. Develop color in a water bath at 25℃ 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, and quickly add 5.0 mL of concentrated sulfuric acid. Shake the sample thoroughly on a vortex mixer, develop the color in a water bath at 25℃ 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-Ciocalteu solution A. Mix well and let stand at room temperature for 10 min. Then add 0.5 mL of Folin-Ciocalteu solution B and mix immediately. Develop color in a water bath at 25℃ 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 Venusil C18 Plus column (4.6 mm × 250 mm, 5 μm); mobile phase was methanol; flow rate was 0.5 mL / min; injection volume was 10 μL; and detection wavelength was 282 nm.
[0094] Table 1. Removal rates of brown pigments, tannins, polysaccharides, and proteins in the liquid tea saponins 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 saponins prepared in Examples 1-5
[0097] Example 1 Example 2 Example 3 Example 4 Example 5 Tea saponins 89.77％ 90.39％ 93.30％ 79.28％ 80.24％
[0098] Table 1 shows the removal rates of impurities under different treatment conditions. When the ratio of crude tea saponin extract to water was 4:1, the removal rates of brown pigments, tannins, polysaccharides, and proteins were relatively high; when the ratio was 2:1, the removal rates of brown pigments, tannins, polysaccharides, and proteins were no 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 was the highest, at 93.30%. Examples 1 and 2 show that the yield of tea saponin can also reach approximately 90% when the ratio of crude tea saponin extract to water is 4:1. Considering both the removal rate of impurities and the yield of tea saponin, the flocculation and purification effect was best when the ratio of crude tea saponin extract to water was 4:1, and 3.0 mL of calcium chloride solution and 2 mL of microbial flocculant solution were mixed.
[0099] In addition, the oil content of 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 was tested, and the results are shown in Table 3.
[0100] Table 3. Oil removal rate data of liquid tea saponins prepared in Examples 1-4 in the intermediate aqueous phase.
[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 oil removal effect under different treatment conditions. Water extraction of the camellia seed cake extract removed over 94% of the oil from the sample. Further treatment of the intermediate aqueous phase with a microbial flocculant removed over 99% of the oil content from the sample.
[0103] The results above show that, compared with conventional methods for flocculating and purifying tea saponin, the method of purifying tea saponin using microbial flocculants in this invention has the following advantages: (a) Low oil content: By combining water removal and microbial flocculant removal, the oil content in the tea saponin product can be effectively removed, with the oil content in the tea saponin product being less than 0.1%. (b) High purity: By combining water removal, coagulant aid, and microbial flocculant, not only suspended particles and soluble macromolecules can be effectively removed, but also soluble impurities such as polysaccharides, proteins, brown pigments, and tannins can be effectively removed, with the removal rate of these soluble impurities reaching over 50%. (c) High yield: By combining water removal, microbial flocculant, and coagulant aid, tea saponin can be retained to the greatest extent possible while effectively removing impurities, with the retention rate of tea saponin reaching as high as 93.3%. (d) Low toxicity: On the one hand, the microbial flocculant used has low toxicity and is easily biodegradable, which helps reduce the toxicity of tea saponin products. On the other hand, the low residue levels of the coagulant aid and microbial flocculant also help reduce the toxicity of tea saponin products. Therefore, high-value utilization of tea saponin can be achieved in the fields of medicine, food, and daily chemicals. Furthermore, the method for purifying tea saponin using microbial flocculants in this invention also has advantages such as simple and convenient operation, safety, and environmental friendliness, making it suitable for large-scale preparation of tea saponin and facilitating its industrial application.
[0104] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.
Claims
1. A method for purifying tea saponins by flocculation using microbial flocculants, characterized in that, Includes the following steps: (1) Mix the crude tea saponin extract with water, stir for 2 h to 5 h, let stand for 5 h to 12 h to separate the oil and water, and retain the aqueous solution; the volume ratio of the crude tea saponin extract to water is 2 to 4:
1. (2) The aqueous solution, coagulant solution and microbial flocculant solution obtained in step (1) are mixed for flocculation, allowed to stand, and filtered to complete the flocculation and purification of tea saponin; the volume ratio of the aqueous solution to the coagulant solution is 100:2-3; 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 flocculation is carried out by stirring for 3-5 minutes at a speed of 200 r / min-300 r / min and stirring for 3-5 minutes at a speed of 30 r / min-60 r / min; the coagulant in the coagulant solution is calcium chloride; the microbial flocculant solution is prepared by mixing microbial flocculant with water; the mass ratio of the microbial flocculant to water is 1:100-200; the preparation method of the microbial flocculant includes the following steps: S1. Mix camellia seed cake with water, sonicate, hydrothermally treat, filter, collect the filtrate to obtain camellia seed cake extract; S2. Prepare a camellia oil cake extract obtained in step S1 into a camellia oil cake culture medium. S3. The seed liquid of flocculating functional bacteria is inoculated into the tea oil cake medium obtained in step S2 and cultured to obtain a microbial flocculant; the strain in the seed liquid of flocculating functional bacteria is Bacillus polymyxa; the Bacillus polymyxa is GA1.
2. The method according to claim 1, characterized in that, In step (1), the crude tea saponin extract is prepared from camellia oil cake residue; the stirring is carried out on a shaker.
3. The method according to claim 1 or 2, characterized in that, In step (2), the settling time is 10 min to 30 min.
4. The method according to claim 1 or 2, characterized in that, In step S1, the ratio of camellia oil cake to water is 30 g to 200 g: 1 L. Before use, the camellia oil cake undergoes the following treatment: drying, pulverizing, and passing through a 40-60 mesh sieve to obtain camellia oil cake granules. The ultrasonic treatment is performed at a temperature of 30 ℃ to 40 ℃ for 20 min to 50 min. The hydrothermal treatment is performed at a temperature of 111 ℃ to 126 ℃ for 10 min to 50 min. The polysaccharide content in the camellia oil cake extract is 1800 mg / L to 15000 mg / L, the protein content is 3300 mg / L to 24000 mg / L, and the SCOD content is 18000 mg / L to 110000 mg / L.
5. The method according to claim 4, characterized in that, The ratio of camellia seed cake to water is 50 g to 150 g: 1 L.
6. The method according to claim 1 or 2, characterized in that, In step S2, the preparation method of the camellia oil cake meal culture medium is as follows: the camellia oil cake meal extract, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate are mixed to obtain the camellia oil cake meal culture medium; the concentration of potassium dihydrogen phosphate in the camellia oil cake meal 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 oil cake meal culture medium is 5 to 9.
7. The method according to claim 1 or 2, characterized in that, In step S3, the inoculation amount of the flocculating functional bacteria seed liquid is 4%–5% of the volume of the camellia oil cake culture medium; the culture is as follows: cultured at 30 ℃–35 ℃ and 130 rpm–150 rpm for 12.0 h–24.0 h, and cultured at 20 ℃–25 ℃ and 100 rpm–120 rpm for 24.0 h–72.0 h; after the culture is completed, the following treatment is also included: centrifuging the fermentation product obtained after culture at 8000 rpm–10000 rpm for 8 min–10 min to remove the bacterial cells and obtain the microbial flocculant; the microbial flocculant is further treated as follows: mixing the microbial flocculant with an organic solvent, letting it stand, and centrifuging at 8000 rpm–10000 rpm for 8 min–10 min. After min, the supernatant was removed to obtain purified microbial flocculant; the volume ratio of the microbial flocculant to the organic solvent was 1-2:1-3; the organic solvent was acetone and / or ethanol; the organic solvent was further treated before use as follows: pre-cooled at 4 ℃ for 12 h-24 h; the standing was carried out at 4 ℃; the standing time was 24 h-48 h.
8. The method according to claim 7, characterized in that, In step S3, the preparation method of the flocculating functional bacteria seed liquid includes the following steps: (a) The flocculating functional bacteria strain was inoculated into a solid activation medium and cultured at 30 ℃~35 ℃ for 12.0 h~24.0 h to obtain primary seed colonies; the formula of the solid activation medium is as follows: 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, 20.0 g / L agar powder, pH 7–8; (b) The primary seed colonies obtained in step (a) are inoculated into liquid proliferation medium and cultured at a temperature of 25 ℃~30℃ and a rotation speed of 130 rpm~150 rpm for 12.0 h~24.0 h to obtain flocculation functional bacteria seed liquid; the formula of the liquid proliferation medium is: 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, and pH value of 7~8.