Novel camellia saponin derivative feed additive and preparation method thereof

Abstract

The invention relates to a novel camellia saponin derivative feed additive and a preparation method thereof, and belongs to the technical field of feed additives. The modified chitosan is used in the flocculation and impurity removal process for preparing the camellia oleifera saponin derivative, and the modified chitosan reduces the loss of effective components caused by flocculation and impurity removal while ensuring the purity of the camellia oleifera saponin derivative; soybean lecithin and gelatin are used as capsule walls, the camellia saponin derivative and pyropheophorbide-a are wrapped, pyropheophorbide-a can generate singlet oxygen under excitation of visible light, phospholipid bonds are damaged, the camellia saponin derivative is released, and therefore the slow release effect is achieved; the novel camellia saponin derivative feed additive prepared by the invention has good antibacterial property and timeliness.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, and relates to a novel camellia saponin derivative feed additive and its preparation method. Background Technology

[0002] Camellia oleifera saponins (also known as tea saponins) are pentacyclic triterpenoid compounds extracted from camellia oleifera cake. Their basic structure consists of aglycone, glycosides, and organic acids. They possess excellent emulsifying, foaming, and foam-holding properties, as well as antibacterial, anti-inflammatory, and hemolytic biological activities, making them suitable for use in daily chemical, building material, aquaculture, and pharmaceutical industries. However, the complex composition of camellia oleifera cake, containing not only abundant saponins but also large amounts of proteins, flavonoids, tannins, polysaccharides, and pigments, presents significant challenges to the extraction and purification of saponins. Traditional methods often result in low-purity, dark-colored saponins that readily form a paste when dissolved in water, severely limiting their applications.

[0003] Current research suggests utilizing the binding of chitosan with anions to generate flocculation and sedimentation, which removes impurities through filtration. However, while removing impurities, the adsorption of camellia saponins also occurs, leading to a loss of camellia saponin content and a decrease in performance. Summary of the Invention

[0004] This invention relates to a novel camellia oleifera saponin derivative feed additive and its preparation method, belonging to the field of feed additive technology. The invention utilizes modified chitosan in the flocculation and impurity removal process for preparing camellia oleifera saponin derivatives. The modified chitosan ensures the purity of the camellia oleifera saponin derivatives while reducing the loss of effective components during flocculation and impurity removal. Soybean lecithin and gelatin are used as encapsulation walls to encapsulate the camellia oleifera saponin derivatives and pyrophyllin-a. Pyrophyllin-a, under visible light excitation, generates singlet oxygen, breaking phospholipid bonds and releasing the camellia oleifera saponin derivatives, thus achieving a slow-release effect. The novel camellia oleifera saponin derivative feed additive prepared by this invention exhibits good antibacterial properties and long-lasting effects.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A novel camellia saponin derivative feed additive, comprising the following raw materials in parts by weight: 10-20 parts camellia saponin derivative, 20-30 parts soybean lecithin, 3 parts pyrophosphate chlorophyll-α1-, 8-10 parts gelatin, 4-6 parts emulsifier, and 50-60 parts distilled water.

[0007] Furthermore, the preparation method of the camellia saponin derivative includes the following steps:

[0008] A1: Crush the camellia seed cake, then add distilled water and protease for enzymatic hydrolysis. After enzymatic hydrolysis, filter to obtain filtrate and residue. Extract the residue with ethanol by heating and reflux, and dry to obtain substance a.

[0009] A2: The filtrate is evaporated and concentrated, and dried to obtain substance b. The solid obtained by combining substances a and b is put into deionized water and stirred. Then, modified chitosan is added and stirred. After standing, flocculent flocculation is formed, and then filtered.

[0010] A3: Filtrate a is adsorbed by AB-8 macroporous resin, eluted with anhydrous ethanol, and the eluent is evaporated and dried to obtain camellia saponin derivatives.

[0011] Further, in step A1, the mass ratio of camellia seed cake, distilled water, and protease is 5-8:20-25:0.5-1, where the protease refers to papain. The enzymatic hydrolysis temperature and time are 35-45℃ and 5-10h, respectively. The mass ratio of filter residue to ethanol is 2:3-5, where the ethanol mass fraction is 75%. The heating reflux temperature and time are 80-100℃ and 1-2h, respectively. The drying temperature is 60-70℃.

[0012] Further, in step A2, the water content of substances a and b is 3-6%, the mass ratio of the solid, deionized water and modified chitosan is 8-10:50:3-6, and the modified chitosan is prepared by: first swelling the chitosan with sodium hydroxide solution, then adding it to the modified liquid, heating and stirring, distilling to remove the organic solvent, and drying to obtain the modified chitosan.

[0013] Furthermore, the mass ratio of chitosan, sodium hydroxide solution, and modified liquid is 1:2:4-5, wherein the mass fraction of sodium hydroxide solution is 50-60%, the swelling temperature and time are 40-50℃ and 40-60min, respectively, the modified liquid is composed of epichlorohydrin and trimethylammonium hydrochloride in a mass ratio of 0.9-1:1, and the heating and stirring temperature and time are 60-65℃ and 3-5h, respectively.

[0014] Furthermore, in step A3, the volume ratio of filtrate a to anhydrous ethanol is 1:1-2, and the drying temperature is 60-70℃.

[0015] Furthermore, the preparation method of the novel camellia saponin derivative feed additive includes the following steps:

[0016] (1) Mix gelatin, soybean lecithin and distilled water to form an aqueous phase;

[0017] (2) Emulsifier, camellia saponin derivative and pyromethesin-α are mixed to form an oil phase.

[0018] (3) The aqueous phase and oil phase are mixed, stirred and allowed to stand, filtered and dried to obtain a novel tea oil saponin derivative feed additive.

[0019] Furthermore, the temperature of the aqueous phase in step (1) is 60-75°C, the emulsifier in step (2) is soybean oil, and the temperature of the oil phase is 45-50°C.

[0020] Further, in step (3), the volume ratio between the aqueous phase and the oil phase is 2:1-2, the settling time and temperature are 2-3h and 30-40℃ respectively, and the drying temperature is 50-55℃.

[0021] The beneficial effects of this invention are:

[0022] 1. This invention uses modified chitosan in the flocculation and impurity removal process for preparing camellia saponin derivatives. The modified chitosan ensures the purity of the camellia saponin derivatives while reducing the loss of effective components caused by flocculation and impurity removal. Soybean lecithin and gelatin are used as the capsule wall to encapsulate the camellia saponin derivatives and pyromethesin-a. Pyromethesin-a can generate singlet oxygen under visible light excitation, which breaks the phospholipid bonds and releases the camellia saponin derivatives, thereby achieving a slow-release effect. The novel camellia saponin derivative feed additive prepared by this invention has good antibacterial properties and time-release effect. Detailed Implementation

[0023] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with embodiments, is provided below.

[0024] The chitosan involved in this invention was purchased from Qingdao Yuekang Biotechnology Co., Ltd.

[0025] Example 1

[0026] A novel camellia saponin derivative feed additive comprises the following raw materials in parts by weight: 10 parts camellia saponin derivative, 20 parts soybean lecithin, 1 part pyrophosphate chlorophyll-a, 8 parts gelatin, 4 parts emulsifier, and 50 parts distilled water.

[0027] The preparation method of the camellia saponin derivative includes the following steps:

[0028] A1: Crush the camellia seed cake, then add distilled water and protease for enzymatic hydrolysis. After enzymatic hydrolysis, filter to obtain filtrate and residue. Extract the residue with ethanol by heating and reflux, and dry to obtain substance a.

[0029] A2: The filtrate is evaporated and concentrated, and dried to obtain substance b. The solid obtained by combining substances a and b is put into deionized water and stirred. Then, modified chitosan is added and stirred. After standing, flocculent flocculation is formed, and then filtered.

[0030] A3: Filtrate a is adsorbed by AB-8 macroporous resin, eluted with anhydrous ethanol, and the eluent is evaporated and dried to obtain camellia saponin derivatives.

[0031] In step A1, the mass ratio of camellia seed cake, distilled water, and protease is 5:20:0.5, where the protease refers to papain. The enzymatic hydrolysis temperature and time are 35°C and 5 hours, respectively. The mass ratio of filter residue to ethanol is 2:3, where the ethanol mass fraction is 75%. The heating reflux temperature and time are 80°C and 1 hour, respectively. The drying temperature is 60°C.

[0032] In step A2, the water content of substances a and b is 3%, the mass ratio of the solid, deionized water and modified chitosan is 8:50:3, and the modified chitosan is prepared by: first swelling the chitosan with sodium hydroxide solution, then adding it to the modified liquid, heating and stirring, distilling to remove the organic solvent, and drying to obtain the modified chitosan.

[0033] The mass ratio of chitosan, sodium hydroxide solution, and modified liquid is 1:2:4, wherein the mass fraction of sodium hydroxide solution is 50%, the swelling temperature and time are 40℃ and 40min, respectively, the modified liquid is composed of epichlorohydrin and trimethylammonium hydrochloride in a mass ratio of 0.9:1, and the heating and stirring temperature and time are 60℃ and 3h, respectively.

[0034] In step A3, the volume ratio of filtrate a to anhydrous ethanol is 1:1, and the drying temperature is 60°C.

[0035] The preparation method of the novel camellia saponin derivative feed additive includes the following steps:

[0036] (1) Mix gelatin, soybean lecithin and distilled water to form an aqueous phase;

[0037] (2) Emulsifier, camellia saponin derivative and pyromethesin-α are mixed to form an oil phase.

[0038] (3) The aqueous phase and oil phase are mixed, stirred and allowed to stand, filtered and dried to obtain a novel tea oil saponin derivative feed additive.

[0039] The temperature of the aqueous phase in step (1) is 60°C, the emulsifier in step (2) is soybean oil, and the temperature of the oil phase is 45°C.

[0040] In step (3), the volume ratio between the aqueous phase and the oil phase is 2:1, the settling time and temperature are 2h and 30℃ respectively, and the drying temperature is 50℃.

[0041] Example 2

[0042] A novel camellia saponin derivative feed additive comprises the following raw materials in parts by weight: 15 parts camellia saponin derivative, 25 parts soybean lecithin, 2 parts pyrophosphate chlorophyll-a, 11 parts gelatin, 5 parts emulsifier, and 55 parts distilled water.

[0043] The preparation method of the camellia saponin derivative includes the following steps:

[0044] A1: Crush the camellia seed cake, then add distilled water and protease for enzymatic hydrolysis. After enzymatic hydrolysis, filter to obtain filtrate and residue. Extract the residue with ethanol by heating and reflux, and dry to obtain substance a.

[0045] A2: The filtrate is evaporated and concentrated, and dried to obtain substance b. The solid obtained by combining substances a and b is put into deionized water and stirred. Then, modified chitosan is added and stirred. After standing, flocculent flocculation is formed, and then filtered.

[0046] A3: Filtrate a is adsorbed by AB-8 macroporous resin, eluted with anhydrous ethanol, and the eluent is evaporated and dried to obtain camellia saponin derivatives.

[0047] In step A1, the mass ratio of camellia seed cake, distilled water, and protease is 6.5:22.5:0.8, where the protease refers to papain. The enzymatic hydrolysis temperature and time are 40℃ and 7h, respectively. The mass ratio of filter residue to ethanol is 2:4, where the ethanol mass fraction is 75%. The heating reflux temperature and time are 90℃ and 1.5h, respectively. The drying temperature is 65℃.

[0048] In step A2, the water content of substances a and b is 4.5%, and the mass ratio of the solid, deionized water and modified chitosan is 9:50:4.5. The modified chitosan is prepared by first swelling the chitosan with sodium hydroxide solution, then adding it to the modified liquid, heating and stirring, distilling to remove the organic solvent, and drying to obtain the modified chitosan.

[0049] The mass ratio of chitosan, sodium hydroxide solution, and modified liquid is 1:2:4.5, wherein the mass fraction of sodium hydroxide solution is 55%, the swelling temperature and time are 45℃ and 50min, respectively, the modified liquid is composed of epichlorohydrin and trimethylammonium hydrochloride in a mass ratio of 0.95:1, and the heating and stirring temperature and time are 63℃ and 4h, respectively.

[0050] In step A3, the volume ratio of filtrate a to anhydrous ethanol is 1:1.5, and the drying temperature is 65°C.

[0051] The preparation method of the novel camellia saponin derivative feed additive includes the following steps:

[0052] (1) Mix gelatin, soybean lecithin and distilled water to form an aqueous phase;

[0053] (2) Emulsifier, camellia saponin derivative and pyromethesin-α are mixed to form an oil phase.

[0054] (3) The aqueous phase and oil phase are mixed, stirred and allowed to stand, filtered and dried to obtain a novel tea oil saponin derivative feed additive.

[0055] The temperature of the aqueous phase in step (1) is 68°C, the emulsifier in step (2) is soybean oil, and the temperature of the oil phase is 48°C.

[0056] In step (3), the volume ratio between the aqueous phase and the oil phase is 2:1.5, the settling time and temperature are 2.5h and 35℃ respectively, and the drying temperature is 52℃.

[0057] Example 3

[0058] A novel camellia saponin derivative feed additive comprises the following raw materials in parts by weight: 20 parts camellia saponin derivative, 30 parts soybean lecithin, 3 parts pyrophosphate chlorophyll-a, 10 parts gelatin, 6 parts emulsifier, and 60 parts distilled water.

[0059] The preparation method of the camellia saponin derivative includes the following steps:

[0060] A1: Crush the camellia seed cake, then add distilled water and protease for enzymatic hydrolysis. After enzymatic hydrolysis, filter to obtain filtrate and residue. Extract the residue with ethanol by heating and reflux, and dry to obtain substance a.

[0061] A2: The filtrate is evaporated and concentrated, and dried to obtain substance b. The solid obtained by combining substances a and b is put into deionized water and stirred. Then, modified chitosan is added and stirred. After standing, flocculent flocculation is formed, and then filtered.

[0062] A3: Filtrate a is adsorbed by AB-8 macroporous resin, eluted with anhydrous ethanol, and the eluent is evaporated and dried to obtain camellia saponin derivatives.

[0063] In step A1, the mass ratio of camellia seed cake, distilled water, and protease is 8:25:1, where the protease refers to papain. The enzymatic hydrolysis temperature and time are 45℃ and 10h, respectively. The mass ratio of filter residue to ethanol is 2:5, where the ethanol mass fraction is 75%. The heating reflux temperature and time are 100℃ and 2h, respectively. The drying temperature is 70℃.

[0064] In step A2, the water content of substances a and b is 6%, the mass ratio of the solid, deionized water and modified chitosan is 10:50:6, and the modified chitosan is prepared by first swelling the chitosan with sodium hydroxide solution, then adding it to the modified liquid, heating and stirring, distilling to remove the organic solvent, and drying to obtain the modified chitosan.

[0065] The mass ratio of chitosan, sodium hydroxide solution, and modified liquid is 1:2:5, wherein the mass fraction of sodium hydroxide solution is 60%, the swelling temperature and time are 50℃ and 60min, respectively, the modified liquid is composed of epichlorohydrin and trimethylammonium hydrochloride in a mass ratio of 1:1, and the heating and stirring temperature and time are 65℃ and 5h, respectively.

[0066] In step A3, the volume ratio of filtrate a to anhydrous ethanol is 1:2, and the drying temperature is 70°C.

[0067] The preparation method of the novel camellia saponin derivative feed additive includes the following steps:

[0068] (1) Mix gelatin, soybean lecithin and distilled water to form an aqueous phase;

[0069] (2) Emulsifier, camellia saponin derivative and pyromethesin-α are mixed to form an oil phase.

[0070] (3) The aqueous phase and oil phase are mixed, stirred and allowed to stand, filtered and dried to obtain a novel tea oil saponin derivative feed additive.

[0071] The temperature of the aqueous phase in step (1) is 75°C, the emulsifier in step (2) is soybean oil, and the temperature of the oil phase is 50°C.

[0072] In step (3), the volume ratio between the aqueous phase and the oil phase is 2:2, the settling time and temperature are 3h and 40℃ respectively, and the drying temperature is 55℃.

[0073] Comparative Example 1

[0074] Based on Example 2, the modified chitosan was replaced with an equal mass of chitosan, while other conditions remained the same as in Example 2.

[0075] Comparative Example 2

[0076] Based on Example 2, the enzymatic hydrolysis process in step A1 of preparing camellia saponin derivatives was removed, while other conditions remained the same as in Example 2.

[0077] Comparative Example 3

[0078] Based on Example 2, pyrophyllophosphochlorophyll-a was replaced with an equal mass of azobenzene, while other conditions remained the same as in Example 2.

[0079] Performance testing

[0080] Determination of Camellia oleifera saponin derivative content: The vanillin-concentrated sulfuric acid colorimetric method was used. Accurately weigh 100.0 mg of Camellia oleifera saponin standard, dissolve it in 80% ethanol solution in a 50 mL volumetric flask, dilute to volume, and shake well to obtain the standard solution. Take 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the Camellia oleifera saponin standard solution respectively, dilute to 0.5 mL with methanol, add 0.5 mL of 8% vanillin solution, add 4 mL of 77% sulfuric acid solution in an ice-water bath, use reagent as a blank, shake well, heat at 60℃ for 15 min, cool in an ice-water bath for 10 min, remove and place at room temperature, measure the absorbance at a wavelength of 452 nm, and plot a standard curve based on absorbance versus concentration. Mass determination: Weigh approximately 50.0 mg of the sample, dilute to 25 mL with 80% ethanol, and take 1.0 mL. Measure the absorbance, substitute into the corresponding regression equation, and calculate the saponin mass fraction according to the standard curve. The camellia saponin derivatives prepared in Examples 1-3 and Comparative Examples 1-2 were used as samples. The loss rate of the camellia saponin derivatives was calculated. Specifically, the loss rate % of the camellia saponin derivatives was calculated as follows: (mass of camellia saponins before flocculation - mass of camellia saponins after flocculation) / mass of camellia saponins before flocculation.

[0081] The tea saponin derivative feed additives prepared in Examples 1-3 and Comparative Examples 1-3 were used as samples for antibacterial testing. Experimental bacterial suspensions of *Escherichia coli* and *Staphylococcus aureus* were added to MH broth medium to prepare a concentration of 10 CFU / mL and incubated at 37°C for 6–8 hours. The tea saponin derivative feed additive was prepared as a 2.0 mL solution (concentration 1 mg / mL, solvent: ethanol) and diluted with broth to nine gradient concentrations: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512. Tube 10, containing only the experimental bacterial suspension and no drug, served as a positive control; tube 11, containing only the drug and no bacteria, served as a negative control. Each tube was mixed with 0.1 mL of the three experimental bacterial suspensions and incubated at 37°C for 16–24 hours. The clear, bacteria-free broth with the lowest concentration of tea saponin derivative was transferred to an agar plate. If no bacteria grew, it means that the amount of the drug in that tube was the minimum inhibitory concentration of the tea saponin derivative.

[0082] Test Examples 1-3 and Comparative Example 3 were used as samples. The samples were placed in milk, and ultraviolet scanning was used to compare whether the differences before and after treatment affected the protein. The test results are shown in Table 1.

[0083] Table 1 Test Results

[0084]

[0085] Analysis of the results in Table 1 shows that the tea saponin derivatives in Examples 1-3 are less than those in Comparative Examples 1-2. In Comparative Example 1, chitosan was not modified, resulting in poor adsorption selectivity and causing the tea saponin derivatives to flocculate and be removed. In Comparative Example 2, no enzymatic hydrolysis was performed; however, the addition of protease hydrolysis allowed the proteins in the camellia seed cake to fully dissolve into the solution, reducing flocculation. The minimum inhibitory concentrations (MICs) of Examples 1-3 are all lower than those of Comparative Examples 1-3. The reduced content of tea saponin derivatives in Comparative Examples 1-2 leads to poor antibacterial effects. Comparative Example 3, due to protein isomerization, has a better antibacterial effect, but it severely affects proteins and cells in animals, making the use of azobenzene impractical.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A novel sasanqua saponin derivative feed additive, characterized in that, The novel oil tea saponin derivative feed additive comprises the following raw materials in parts by weight: 10-20 parts of oil tea saponin derivative, 20-30 parts of soybean lecithin, 1-3 parts of pyro-ellagic acid-a, 8-10 parts of gelatin, 4-6 parts of emulsifier, and 50-60 parts of distilled water.

2. The novel tea saponin derivative feed additive according to claim 1, characterized in that, The preparation method of the oil tea saponin derivative comprises the following steps: A1: crushing the oil tea cake, then adding distilled water and protease and placing for enzymolysis, filtering the filtrate and residue after the enzymolysis, heating and refluxing the residue with ethanol for extraction, and drying to obtain substance a; A2: evaporating and concentrating the filtrate, drying to obtain substance b, and stirring and mixing the combined solid of substances a and b in deionized water, then adding modified chitosan for stirring, standing, filtering after flocculation; A3: adsorbing the filtrate a on AB-8 type macroporous resin, eluting with anhydrous ethanol, evaporating the eluate, and drying to obtain the oil tea saponin derivative.

3. The novel tea saponin derivative feed additive according to claim 2, characterized in that, In step A1, the mass ratio of the oil tea cake, distilled water and protease is 5-8:20-25:0.5-1, wherein the protease is papain, the temperature and time of the enzymolysis are 35-45℃ and 5-10h respectively, the mass ratio of the residue and ethanol is 2:3-5, wherein the mass fraction of the ethanol is 75%, the temperature and time of the heating and refluxing are 80-100℃ and 1-2h respectively, and the drying temperature is 60-70℃.

4. The novel tea saponin derivative feed additive according to claim 2, characterized in that, In step A2, the water content of substances a and b is 3-6%, and the mass ratio of the solid, deionized water and modified chitosan is 8-10:50:3-6, wherein the preparation method of the modified chitosan is as follows: swelling the chitosan with sodium hydroxide solution, then adding a modified liquid for heating and stirring, distilling off the organic solvent, and drying to obtain the modified chitosan.

5. The novel tea saponin derivative feed additive according to claim 4, characterized in that, The mass ratio of the chitosan, sodium hydroxide solution and modified liquid is 1:2:4-5, wherein the mass fraction of the sodium hydroxide solution is 50-60%, the temperature and time of the swelling are 40-50℃ and 40-60min respectively, the modified liquid is composed of epoxy chloropropane and trimethylammonium chloride hydrochloride with a mass ratio of 0.9-1:1, and the temperature and time of the heating and stirring are 60-65℃ and 3-5h respectively.

6. The novel tea saponin derivative feed additive according to claim 2, characterized in that, In step A3, the volume ratio of the filtrate a and anhydrous ethanol is 1:1-2, and the drying temperature is 60-70℃.

7. A novel method for the preparation of a feed additive of a saponin derivative of Camellia oleifera according to claim 1, characterized by, The preparation method of the novel oil tea saponin derivative feed additive comprises the following steps: (1) mixing gelatin, soybean lecithin and distilled water to form an aqueous phase; (2) mixing an emulsifier, oil tea saponin derivative and pyro-ellagic acid-a to form an oil phase (3) mixing the aqueous phase and the oil phase, standing after stirring, filtering, and drying to obtain the novel oil tea saponin derivative feed additive.

8. The preparation method of a novel tea saponin derivative feed additive according to claim 6, characterized in that, In step (1), the temperature of the aqueous phase is 60-75℃, and in step (2), the emulsifier is soybean oil, and the temperature of the oil phase is 45-50℃.

9. The preparation method of a novel oil tea saponin derivative feed additive according to claim 6, characterized in that, In step (3), the volume ratio of the aqueous phase and the oil phase is 2:1-2, the standing time and temperature are 2-3h and 30-40℃ respectively, and the drying temperature is 50-55℃.

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