Composition as well as preparation method and application thereof

Abstract

The invention discloses a composition as well as a preparation method and application thereof, and relates to the technical field of compound bacterium extract compositions. The compound bacteria comprise schizochytrium limacinum, acremonium terricola and gordonia alcalivorans; the mass ratio of the compound bacterium extract to the antioxidant is 100: (0.5-5). The preparation method of the composition comprises the following steps: adding the antioxidant into the compound bacterium extract, mixing, and drying to obtain the compound bacterium extract. The compound bacterium extract disclosed by the invention is rich in lipid and other components, and when the compound bacterium extract is applied to a laying hen feed, the cholesterol content in eggs can be effectively reduced on the premise of safety, no toxicity, no side effect and no influence on the production performance of laying hens, so that the low-cholesterol eggs are produced.

Description

Technical Field

[0001] The invention belongs to the technical field of composite bacteria extract compositions, and specifically relates to a composition and a preparation method and application thereof. Background Art

[0002] Low cholesterol eggs refer to eggs with low cholesterol content in the egg yolk. Low cholesterol eggs not only reduce the cholesterol content, but also retain the original nutritional value of eggs, such as high protein, lecithin, vitamin E, etc., so they are suitable for people who need to control cholesterol intake. The production of such eggs is usually achieved through genetic selection and changing the feed and feeding methods of chickens. For example, by adding specific plant extracts, Chinese herbal medicine formulas, probiotics, etc. to chicken feed, the cholesterol content in the egg yolk can be effectively reduced. However, the methods in the prior art have the problems of relatively few resources, high costs and poor cholesterol-lowering effects.

[0003] Schizochytrium is rich in ω-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA). Acremonium culture (ATC) can enhance the antioxidant capacity and immune function of laying hens. In terms of antioxidant, the addition of ATC significantly increased the activity of glutathione peroxidase (GSH-Px) in serum and reduced the content of serum malondialdehyde (MDA), which shows that ATC can improve the body's antioxidant capacity. In terms of immunity, the addition of ATC increased the content of immunoglobulin G (IgG) and immunoglobulin M (IgM) in serum, which helps to enhance the immunity of laying hens. Gordonia albicans has an inhibitory effect on pathogens, and can also enhance the antioxidant capacity of laying hens, increase the activity of antioxidant enzymes in serum, and reduce the level of oxidative stress markers, which is of great significance for improving the health of laying hens and extending the egg-laying cycle.

[0004] The composition of the present invention contains a composite bacteria extract, which is obtained by extracting and purifying the effective components of the composite bacteria and applying the extract to laying hen feed, aiming at producing low-cholesterol eggs. Summary of the invention

[0005] In order to overcome the above technical problems, the present invention provides a composition and a preparation method and application thereof. The composite bacteria extract of the present invention can be added to laying hen feed to effectively reduce the cholesterol content of eggs.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The present invention discloses a composition comprising a composite bacterial extract and an antioxidant;

[0008] The composite bacteria include Schizochytrium, Acremonium and Gordonia alkalivorans;

[0009] The mass ratio of the composite bacteria extract to the antioxidant is 100:0.5-5, preferably 100:0.5-1.

[0010] In the present invention, the antioxidant is at least one of pyrroloquinoline quinone (CAS No.: 72909-34-3), ethoxyquinoline (CAS No.: 91-53-2), ascorbic acid (CAS No.: 50-81-7) and tea polyphenols (CAS No.: 84650-60-2); preferably, the antioxidant is pyrroloquinoline quinone.

[0011] In the present invention, the mass ratio of Schizochytrium:Acremonium:Gordonella alkalivorans in the composite bacteria is (4-10):(1-3):1, preferably (4-8):(1-3):1, for example 5:2:1.

[0012] In the present invention, the preparation method of the composite bacterial extract comprises the following steps:

[0013] S1. extracting the composite bacteria with an organic solvent and collecting the solid phase;

[0014] S2. The solid phase is subjected to solid phase extraction and concentration to obtain a composite bacterial extract.

[0015] In S1, the composite bacteria further comprises a pretreatment step, wherein the pretreatment process comprises drying, crushing and screening the composite bacteria; wherein the sieved particle size is 0.2 to 2.5 mm.

[0016] In S1, the organic solvent is petroleum ether and diethyl ether.

[0017] Further in S1, the volume ratio of petroleum ether to diethyl ether in the organic solvent is 0.5-1.5:1, for example, 1:1.

[0018] In S1, the solid-liquid ratio of the composite bacteria to the organic solvent is 1: (15-35) g / mL.

[0019] In S1, the extraction method is ultrasonic extraction.

[0020] In S1, the extraction time is 3 to 8 hours.

[0021] In S1, the extracted frequency is 30-60 kHz, for example, 40 kHz.

[0022] In S1, the extraction further includes the steps of centrifugation, dilution with water and collection of the lower solid phase; wherein the purpose of dilution with water is to remove water-soluble impurities.

[0023] S2, the concentration method is reduced pressure concentration.

[0024] In S2, the concentration pressure is 0.04-0.1 MPa, preferably 0.04-0.06 MPa.

[0025] In S2, the concentration temperature is 35-70°C, preferably 35-50°C.

[0026] In S2, the concentration needs to be to a constant weight.

[0027] In S2, the solid phase extraction comprises the following steps:

[0028] S2.1. Sample preparation: The solid phase is dissolved in a methanol-n-hexane mixture, and then filtered to remove the insoluble solid to obtain a sample solution;

[0029] S2.2. Loading: The sample solution passes through an amino solid phase extraction column;

[0030] S2.3. Elution: First, use a methanol-water mixture to remove impurities, then elute with acetonitrile and collect the acetonitrile eluate;

[0031] In S2.1, the volume ratio of methanol to normal hexane in the methanol-normal hexane mixture is 1:1-5, preferably 1:1-3, for example 1:1.

[0032] In S2.3, the volume ratio of methanol to water in the methanol-water mixture is 0.8 to 1.5:1; for example, 1:1.

[0033] In the present invention, the components of the composite bacteria extract include ergosterol, cordycepin, docosahexaenoic acid, triglyceride and phospholipid.

[0034] In the present invention, the components of the composite bacteria extract include ergosterol, docosahexaenoic acid, triglyceride and phospholipid.

[0035] The present invention also discloses a method for preparing the above-mentioned composition, comprising the following steps:

[0036] Antioxidants are added to the composite bacteria extract, mixed, and dried to obtain the composite bacteria extract.

[0037] In the present invention, the mixing temperature is 2-8°C, preferably 4-6°C.

[0038] In the present invention, the pH of the mixture is 7 to 8, preferably 7 to 7.5.

[0039] In the present invention, the drying method is a vacuum drying method.

[0040] In the present invention, the drying temperature is 40-60°C.

[0041] In the present invention, the drying time is 12 to 18 hours.

[0042] In the present invention, the drying pressure is 10 to 30 MPa.

[0043] The invention also provides application of the above composition in laying hen feed.

[0044] In the present invention, the composition is a laying hen feed additive.

[0045] In the present invention, the composition is added to the laying hen diet in an amount of 0.1 to 5 wt%, preferably 0.1 to 2 wt%, such as 0.5 wt%.

[0046] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1. The composition of the present invention comprises a composite bacterial extract of Schizochytrium, Acremonium and Gordonia alkalivorans. The preparation process of the composite bacterial extract involves removing impurities and enriching beneficial substances to ensure a high content of functional ingredients in the extract. In addition, the extract can reduce the immune response of animals, reduce the risk of allergies, and improve nutrient absorption.

[0049] 2. The present invention extracts the composite bacteria and purifies them using solid phase extraction technology to enrich the key effective components in the composite bacteria. The obtained extract is rich in lipids, including lipid substances such as ergosterol, docosahexaenoic acid, triglycerides and phospholipids.

[0050] 2. Further research found that adding the composition to laying hen feed can effectively reduce the cholesterol content in eggs, thereby producing low-cholesterol eggs, while being safe, non-toxic, and without side effects, and without affecting the production performance of laying hens. This discovery provides a new feed additive option for the laying hen farming industry, helping to meet the market demand for healthy food. DETAILED DESCRIPTION

[0051] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and carefully in combination with preferred embodiments below, but the protection scope of the present invention is not limited to the following specific embodiments.

[0052] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0053] The "range" disclosed in the present invention is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of a particular range. The range defined in this way can be inclusive or exclusive of end values, and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60-120 and 80-110 is listed for a specific parameter, it is understood that the range of 60-110 and 80-120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present invention, unless otherwise specified, the numerical range "ab" represents an abbreviation of any real number combination between a and b, wherein a and b are real numbers. For example, the numerical range "0-5" represents that all real numbers between "0-5" have been fully listed herein, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0054] If not otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution.

[0055] Unless otherwise specified, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution.

[0056] If not otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0057] If there is no special explanation, the "include" and "comprising" mentioned in the present invention represent open-ended or closed-ended expressions. For example, the "include" and "comprising" may represent that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0058] If not specifically stated, in the present invention, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0059] The Schizochytrium used in the following examples and comparative examples was purchased from the American Type Culture Collection (ATCC), ATCC No. 20888.

[0060] Acremonium implicatum was purchased from Guangdong Province Microbial Culture Collection Center, GDMCC NO.: 3.680; Chinese name: Acremonium implicatum; Latin name: Acremonium implicatum.

[0061] Gordonia alkanivorans was purchased from Hunan Weicao Biotechnology Co., Ltd.; the bacterium was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.26361; Latin name: Gordonia alkanivorans.

[0062] Example 1

[0063] The preparation method of the composition of this embodiment comprises the following steps:

[0064] (1) Pretreatment: The composite bacteria are prepared in a mass ratio of 5:2:1 of Schizochytrium:Acremonium:Gordonella alkalivorans, and the composite bacteria are dried, crushed, and sieved to a particle size of 1 mm;

[0065] (2) The pretreated composite bacteria were subjected to ultrasonic extraction for 5 h using an organic solvent (petroleum ether: ether volume ratio of 1:1) at a solid-liquid ratio of 1:20 g / mL, and the ultrasonic extraction conditions were 240 W and 40 kHz; then diluted with water to remove water-soluble impurities, centrifuged, and the solid phase was removed;

[0066] (3) washing the lower solid phase with petroleum ether, performing solid phase extraction, and then concentrating under reduced pressure at 40° C. and 0.06 MPa to constant weight to obtain a composite bacterial extract;

[0067] Wherein, solid phase extraction comprises the following steps:

[0068] S2.1. Sample preparation: The solid phase was dissolved in a mixture of methanol and n-hexane (volume ratio 1:1), and then filtered to remove the insoluble solid to obtain a sample solution;

[0069] S2.2. Loading: The sample solution was passed through an amino solid phase extraction column (1000 mg, 6 ml);

[0070] S2.3. Elution: firstly elute with a mixture of methanol and water (volume ratio 1:1) to remove impurities, then elute with acetonitrile and collect the acetonitrile eluate, and then concentrate the acetonitrile eluate under reduced pressure;

[0071] (4) According to the mass ratio of composite bacterial extract to antioxidant (pyrroloquinoline quinone) of 100:1, pyrroloquinoline quinone is added to the extract, and then mixed at 5°C, and the mixed pH is 7; the composite bacterial extract can be obtained; wherein, sodium carbonate can be used to adjust the pH during the mixing process.

[0072] Example 2

[0073] The difference between this embodiment and embodiment 1 is that:

[0074] The mixing temperature was 2° C., and the mixing pH was 7.5; the other steps and parameters were the same as in Example 1.

[0075] Example 3

[0076] The difference between this embodiment and embodiment 1 is that:

[0077] The particle size of the sieved particles was 0.5 mm, and the other steps and parameters were the same as those in Example 1.

[0078] Example 4

[0079] The difference between this embodiment and embodiment 1 is that:

[0080] The solid-liquid ratio during extraction was 1:15 g / mL, and the other steps and parameters were the same as in Example 1.

[0081] Example 5

[0082] The difference between this embodiment and embodiment 1 is that:

[0083] The solid-liquid ratio during extraction was 1:35 g / mL, and the other steps and parameters were the same as in Example 1.

[0084] Comparative Example 1

[0085] The difference between this comparative example and Example 1 is:

[0086] The composite bacteria in this comparative example does not contain Schizochytrium, and the mass ratio of Acremonium truncatum to Gordonia alkalivorans in the composite bacteria is 2:1.

[0087] Comparative Example 2

[0088] The difference between this comparative example and Example 1 is:

[0089] The composite bacteria in this comparative example does not contain Acremonium truncatum, and the mass ratio of Schizochytrium to Gordonia alkalivorans in the composite bacteria is 5:1.

[0090] Comparative Example 3

[0091] The difference between this comparative example and Example 1 is:

[0092] The composite bacteria in this comparative example does not contain Gordonia alkalivorans, and the mass ratio of Schizochytrium to Acremonium in the composite bacteria is 5:2.

[0093] Comparative Example 4

[0094] The difference between this comparative example and Example 1 is:

[0095] In the mixing step of Comparative Example S4, the temperature was 25° C. and the pH was 6, and the other steps and parameters were the same as those in Example 1.

[0096] Comparative Example 5

[0097] The difference between this comparative example and Example 1 is:

[0098] No antioxidant was added to the purified product in this comparative example, and other steps and parameters were the same as those in Example 1.

[0099] Application Examples

[0100] In order to conduct the feeding experiment, a total of 5,400 Hy-Line brown laying hens with similar health status, weight and age were selected and divided into 9 groups, each containing 6 replicates, each replicate containing 100 chickens. All groups were fed with a basic diet.

[0101] In the 9 test groups, composite bacterial extracts were added to the basal diet as feed additives, and these additives were composite bacterial extracts prepared in the above control examples and examples, and the addition ratio was 0.5wt%. No additives were added to the blank control group.

[0102] The experiment was divided into a 7-day pre-feeding period and a 60-day test period. In both phases, feeding management and immunization measures were carried out as usual. The total egg production rate of laying hens was counted during the test period, and at the end of the test period, 30 eggs were randomly selected from each group to measure the cholesterol content in the eggs (test method refers to GB / T 37077); specific data are shown in Table 1.

[0103] Table 1 Production performance of laying hens and cholesterol content of eggs

[0104]

[0105] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods. The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A composition, characterized in that Includes complex bacterial extracts and antioxidants; The composite bacteria include Schizochytrium, Acremonium and Gordonia alkalivorans; The mass ratio of the composite bacteria extract to the antioxidant is 100:0.5-5.

2. The composition according to claim 1, characterized in that Satisfy at least one of the following conditions ①~②: ① The antioxidant is at least one of pyrroloquinoline quinone, ethoxyquinoline, ascorbic acid and tea polyphenols; ② The mass ratio of Schizochytrium:Acremonium:Gordonella alkalivorans in the composite bacteria is 4-8:1-3:

1.

3. The composition according to claim 1, characterized in that The preparation method of the composite bacterial extract comprises the following steps: S1. extracting the composite bacteria with an organic solvent and collecting the solid phase; S2. The solid phase is subjected to solid phase extraction and concentration to obtain a composite bacterial extract.

4. The composition according to claim 3, characterized in that Satisfy at least one of the following conditions ① to ③: ① The composite bacteria also includes a pretreatment step, wherein the pretreatment process is drying, crushing, and screening the composite bacteria; wherein the screening particle size is 0.2 to 2.5 mm; ② The organic solvent is petroleum ether and ether; ③The extraction method is ultrasonic extraction.

5. The composition according to claim 4, characterized in that Satisfy at least one of the following conditions ① to ④: ① The volume ratio of petroleum ether to ether in the organic solvent is 0.5-1.5:1; ② The solid-liquid ratio of the composite bacteria to the organic solvent is 1: (15-35) g / mL; ③ The extraction time is 3 to 8 hours; ④ The extraction frequency is 30 to 60 kHz.

6. The composition according to claim 3, characterized in that Satisfy at least one of the following conditions ① to ③: ① The concentration method is reduced pressure concentration; ② The concentration pressure is 0.04-0.06MPa; ③ The concentration temperature is 35-70°C.

7. The composition according to claim 3, characterized in that The solid phase extraction comprises the following steps: S2.

1. Sample preparation: The solid phase is dissolved in a methanol-n-hexane mixture, and then filtered to remove the insoluble solid to obtain a sample solution; S2.

2. Loading: The sample solution passes through an amino solid phase extraction column; S2.

3. Elution: First use a methanol-water mixture to remove impurities, then elute with acetonitrile and collect the acetonitrile eluate.

8. The composition according to claim 7, characterized in that The volume ratio of methanol to normal hexane in the methanol-normal hexane mixture is 1:1-5; And / or, the volume ratio of methanol:water in the methanol-water mixture is 0.8-1.5:

1.

9. The method for preparing the composition according to any one of claims 1 to 8, characterized in that: It includes the following steps: adding an antioxidant to the composite bacterial extract, mixing, and drying to obtain the composite bacterial extract; Wherein, the mixing temperature is 2-8°C; the mixing pH is 7-8.

10. Use of the composition according to any one of claims 1 to 8 in laying hen feed, characterized in that: The composition is added to laying hen diets at a ratio of 0.1 to 5 wt%.

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