Feed additive suitable for young foxes and preparation method thereof

Abstract

The invention belongs to the field of feed additives, and particularly relates to a feed additive suitable for young foxes and a preparation method thereof, and the preparation method comprises the following steps: step 1, preparing 50kDa beta-glucan; 2, beta-glucan modified montmorillonite is prepared, and the specific preparation process is as follows: raw montmorillonite and a NaCl solution with the molar concentration of 1 M are subjected to sodium modification pretreatment to obtain sodium modified montmorillonite, and the sodium modified montmorillonite and the beta-glucan with the molecular weight of 50 kDa are subjected to loading treatment to obtain the beta-glucan modified montmorillonite; and step 3, performing cross-linking reaction on the beta-glucan modified montmorillonite and a gallic acid modified genipin solution with the mass fraction of 0.3%, and performing centrifugal drying treatment to obtain the feed additive suitable for young foxes. The young fox feed additive with the functions of accurate detoxification, nutrition loss reduction and immunity enhancement is prepared through an enzyme digestion beta-glucan accurate loading and genipin biological cross-linking technology.

Description

Technical Field

[0001] The invention belongs to the field of feed additives, in particular to a feed additive suitable for young foxes and a preparation method thereof. Background Art

[0002] Fox cubs face problems from weaning to rearing, such as intestinal toxin accumulation, fragile immune barriers, and nutritional malabsorption. Traditional solutions have significant drawbacks:

[0003] Montmorillonite adsorbents: Although they can adsorb toxins, their broad-spectrum properties lead to the loss of key nutrients such as vitamins and amino acids, and aluminum dissolution poses a risk of neurotoxicity.

[0004] β-glucan additive: Its large molecular weight makes it difficult to penetrate the intestinal mucosa, its immune activation efficiency is insufficient, and its high viscosity inhibits fat digestion. Summary of the Invention

[0005] In order to make up for the shortcomings of the existing technology and solve at least one technical problem raised in the background technology, the present invention uses the "enzymatic β-glucan precise loading and genipin bio-cross-linking" technology to prepare a fox feed additive with the functions of "precise detoxification, nutritional loss reduction, and immune enhancement".

[0006] The technical solution adopted by the present invention to solve the technical problem is: a method for preparing a feed additive suitable for young foxes according to the present invention comprises the following steps:

[0007] Step 1: Preparation of 50 kDa β-glucan;

[0008] Step 2: Preparation of β-glucan modified montmorillonite. The specific preparation process is as follows:

[0009] The montmorillonite raw soil was subjected to sodium pretreatment with a 1M NaCl solution to obtain sodium montmorillonite, and the sodium montmorillonite was loaded with the above-mentioned 50 kDa β-glucan to obtain β-glucan-modified montmorillonite;

[0010] Step 3: cross-linking the β-glucan-modified montmorillonite with a 0.3% by mass solution of gallic acid-modified genipin, followed by centrifugal drying to obtain a feed additive suitable for fox cubs;

[0011] The preparation of gallic acid-modified genipin comprises the following steps:

[0012] Genipin, gallic acid, and immobilized CALB were added to a reactor injected with liquid CO2 at a temperature of 45°C and a pressure of 15 MPa to carry out an enzyme-catalyzed esterification reaction to obtain gallic acid-modified genipin.

[0013] The parameters of the enzyme-catalyzed esterification reaction are: reaction time is 4h.

[0014] As a further technical solution of the present invention, the preparation process of 50kDa β-glucan is as follows:

[0015] A 1% mass fraction of β-glucan solution was added to a 0.1M molar concentration of acetate buffer, the pH was controlled at 6.8, and then β-1,3-glucanase with an enzyme activity unit of 100U / g was added to carry out enzymatic hydrolysis reaction. After the reaction for 2 hours, 50kDa β-glucan was obtained.

[0016] As a further technical solution of the present invention: the parameters of the enzymatic hydrolysis reaction are: the enzymatic hydrolysis temperature is 50°C.

[0017] As a further technical solution of the present invention: the parameters of the sodium pretreatment are: pretreatment temperature is 60° C., and pretreatment time is 2 h.

[0018] As a further technical solution of the present invention: the parameters of the loading treatment are: the pH of the loading treatment is 5.0, the temperature of the loading treatment is 40° C., and the time of the loading treatment is 6 hours.

[0019] As a further technical solution of the present invention: the mass ratio of montmorillonite to 1M NaCl solution is 1:10-20; the mass ratio of sodium montmorillonite to 50kDa β-glucan is 1:15-17.

[0020] As a further technical solution of the present invention: the parameters of the cross-linking reaction are: the pH of the cross-linking reaction is 7.5, the temperature of the cross-linking reaction is 45° C., and the time of the cross-linking reaction is 8 hours.

[0021] As a further technical solution of the present invention, the parameters of centrifugal drying are: centrifugation time is 10-20 minutes, and the precipitate obtained by centrifugation is washed three times with deionized water; the drying parameters are: vacuum drying at a temperature of 40° C. to a moisture content of ≤8%.

[0022] As a further technical solution of the present invention: the mass ratio of the β-glucan-montmorillonite complex to the gallic acid-modified genipin is 10-12:1.

[0023] A feed additive suitable for young foxes is prepared by the above method and is prepared by cross-linking beta-glucan and montmorillonite through modified genipin.

[0024] The beneficial effects of the present invention are as follows:

[0025] The 50kDa β-glucan of the present invention is precisely inserted into the interlayers of montmorillonite (the interlayer spacing ranges from 1.2nm to 2.2nm), forming a three-dimensional molecular sieve structure, which can effectively improve the adsorption rate of aflatoxin B1. The negative charge on the surface of β-glucan repels negatively charged nutrients (such as lysine and vitamin B1), which can effectively reduce the adsorption rate of nutrients.

[0026] The pyrogallol groups in the gallic acid-modified genipin cross-linked network specifically capture vomitoxin through hydrophobic interactions, effectively increasing the adsorption rate of vomitoxin. Genipin-gallate slowly releases genipin acid, promoting goblet cell proliferation and raising intestinal villus height to normal levels.

[0027] More specifically, the primary hydroxyl group (-OH) of genipin undergoes an esterification reaction with the carboxyl group of gallic acid, and the gallic acid is used to modify genipin into a green antioxidant, thereby conferring strong antioxidant activity while retaining its cross-linking function.

[0028] After the ester ring structure of genipin is opened, it undergoes nucleophilic attack with the primary amino group (-NH2) of β-glucan to form a blue Schiff base (C=N) and a covalent bond with a secondary amine (CN), forming a covalent bridge between the β-glucan chains, which has better resistance to hydrolysis. The cross-linked product releases trace amounts of genipin, which can effectively increase the diameter of the Escherichia coli inhibition zone. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific implementation methods.

[0030] Example 1

[0031] The embodiment of the present invention provides a feed additive suitable for fox cubs, wherein the feed additive is formed by cross-linking β-glucan and montmorillonite through modified genipin;

[0032] The present invention also provides a method for preparing a feed additive suitable for fox cubs, comprising the following steps:

[0033] Step 1: Prepare 50kDa β-glucan. The specific preparation process is as follows:

[0034] S11, adding a 1% mass fraction β-glucan solution to a 0.1 M molar concentration of acetate buffer, controlling the pH to 6.8, and then adding 100 U / g β-1,3-glucanase to carry out enzymatic hydrolysis. After reacting for 2 h, the solution was inactivated and purified by ultrafiltration using a 50 kDa MWCO (molecular weight cutoff) hollow fiber membrane. The purified filtrate was freeze-dried to obtain 50 kDa β-glucan;

[0035] The parameters of the enzymatic hydrolysis reaction are as follows: the enzymatic hydrolysis temperature is 50°C;

[0036] The parameters of the inactivation treatment were as follows: inactivation temperature 85°C, maintained for 15 min, and then rapidly cooled to 4°C;

[0037] The parameters of ultrafiltration purification were as follows: ultrafiltration pressure: 0.5 MPa, flow rate 10 L / min;

[0038] Step 2: Preparation of β-glucan modified montmorillonite. The specific preparation process is as follows:

[0039] S21, pre-treating the montmorillonite with a 1M NaCl solution to obtain sodium-modified montmorillonite, and loading the sodium-modified montmorillonite with the 50 kDa β-glucan to obtain β-glucan-modified montmorillonite;

[0040] The parameters of sodium pretreatment are as follows: pretreatment temperature is 60°C, pretreatment time is 2h;

[0041] The parameters of the loading treatment were as follows: pH of the loading treatment was 5.0, temperature of the loading treatment was 40°C, and time of the loading treatment was 6 h;

[0042] Also, the mass ratio of montmorillonite to 1M NaCl solution is 1:10; the mass ratio of sodium montmorillonite to 50kDa β-glucan is 1:15;

[0043] Step 3: cross-linking the β-glucan-modified montmorillonite with a 0.3% by mass solution of gallic acid-modified genipin, followed by centrifugal drying to obtain a feed additive suitable for fox cubs;

[0044] The parameters of the cross-linking reaction are as follows: pH of the cross-linking reaction is 7.5, the temperature of the cross-linking reaction is 45° C., and the time of the cross-linking reaction is 8 h;

[0045] The parameters of centrifugal drying are: centrifugation time is 10 minutes, and the precipitate obtained by centrifugation is washed three times with deionized water; the drying parameters are: vacuum drying at a temperature of 40°C to a moisture content of ≤8%;

[0046] The mass ratio of β-glucan-montmorillonite complex to gallic acid-modified genipin is 10:1;

[0047] More specifically, the preparation of gallic acid-modified genipin comprises the following steps:

[0048] Genipin, gallic acid, and immobilized CALB were added to a reactor injected with liquid CO2 at a temperature of 45°C and a pressure of 15 MPa to carry out an enzyme-catalyzed esterification reaction to obtain gallic acid-modified genipin.

[0049] The parameters of the enzyme-catalyzed esterification reaction are as follows: reaction time is 4 h;

[0050] The post-treatment of the enzyme-catalyzed esterification reaction is as follows: releasing the pressure to normal pressure at a rate of 1 MPa / min, centrifuging the reaction mixture, molecularly distilling the crude genipin-gallate in the middle layer, and collecting the heavy components to obtain genipin-gallate; wherein the parameters of the molecular distillation treatment are: distillation temperature of 120° C. and vacuum degree of 0.1 MPa;

[0051] The mass ratio of genipin, gallic acid and immobilized CALB was 1:1.2:0.4.

[0052] Example 2

[0053] The embodiment of the present invention provides a feed additive suitable for fox cubs, wherein the feed additive is formed by cross-linking β-glucan and montmorillonite through modified genipin;

[0054] The present invention also provides a method for preparing a feed additive suitable for fox cubs, comprising the following steps:

[0055] Step 1: Prepare 50kDa β-glucan. The specific preparation process is as follows:

[0056] S11, adding a 1% mass fraction β-glucan solution to a 0.1 M molar concentration of acetate buffer, controlling the pH to 6.8, and then adding 100 U / g β-1,3-glucanase to carry out enzymatic hydrolysis. After reacting for 2 h, the solution was inactivated and purified by ultrafiltration using a 50 kDa MWCO (molecular weight cutoff) hollow fiber membrane. The purified filtrate was freeze-dried to obtain 50 kDa β-glucan;

[0057] The parameters of the enzymatic hydrolysis reaction are as follows: the enzymatic hydrolysis temperature is 50°C;

[0058] The parameters of the inactivation treatment were as follows: inactivation temperature 85°C, maintained for 15 min, and then rapidly cooled to 4°C;

[0059] The parameters of ultrafiltration purification were as follows: ultrafiltration pressure: 0.5 MPa, flow rate 10 L / min;

[0060] Step 2: Preparation of β-glucan modified montmorillonite. The specific preparation process is as follows:

[0061] S21, pre-treating the montmorillonite with a 1M NaCl solution to obtain sodium-modified montmorillonite, and loading the sodium-modified montmorillonite with the 50 kDa β-glucan to obtain β-glucan-modified montmorillonite;

[0062] The parameters of sodium pretreatment are as follows: pretreatment temperature is 60°C, pretreatment time is 2h;

[0063] The parameters of the loading treatment were as follows: pH of the loading treatment was 5.0, temperature of the loading treatment was 40°C, and time of the loading treatment was 6 h;

[0064] Also, the mass ratio of montmorillonite to 1M NaCl solution is 1:15; the mass ratio of sodium montmorillonite to 50kDa β-glucan is 1:16;

[0065] Step 3: cross-linking the β-glucan-modified montmorillonite with a 0.3% by mass solution of gallic acid-modified genipin, followed by centrifugal drying to obtain a feed additive suitable for fox cubs;

[0066] The parameters of the cross-linking reaction are as follows: pH of the cross-linking reaction is 7.5, the temperature of the cross-linking reaction is 45° C., and the time of the cross-linking reaction is 8 h;

[0067] The parameters of centrifugal drying are: centrifugation time is 15 minutes, and the precipitate obtained by centrifugation is washed three times with deionized water; the drying parameters are: vacuum drying at a temperature of 40°C to a moisture content of ≤8%;

[0068] The mass ratio of β-glucan-montmorillonite complex to gallic acid-modified genipin is 11:1;

[0069] More specifically, the preparation of gallic acid-modified genipin comprises the following steps:

[0070] Genipin, gallic acid, and immobilized CALB were added to a reactor injected with liquid CO2 at a temperature of 45°C and a pressure of 15 MPa to carry out an enzyme-catalyzed esterification reaction to obtain gallic acid-modified genipin.

[0071] The parameters of the enzyme-catalyzed esterification reaction are as follows: reaction time is 4 h;

[0072] The post-treatment of the enzyme-catalyzed esterification reaction is as follows: releasing the pressure to normal pressure at a rate of 1 MPa / min, centrifuging the reaction mixture, molecularly distilling the crude genipin-gallate in the middle layer, and collecting the heavy components to obtain genipin-gallate; wherein the parameters of the molecular distillation treatment are: distillation temperature of 120° C. and vacuum degree of 0.1 MPa;

[0073] The mass ratio of genipin, gallic acid and immobilized CALB was 1:1.2:0.5.

[0074] Example 3

[0075] The embodiment of the present invention provides a feed additive suitable for fox cubs, wherein the feed additive is formed by cross-linking β-glucan and montmorillonite through modified genipin;

[0076] The present invention also provides a method for preparing a feed additive suitable for fox cubs, comprising the following steps:

[0077] Step 1: Prepare 50kDa β-glucan. The specific preparation process is as follows:

[0078] S11, adding a 1% mass fraction β-glucan solution to a 0.1 M molar concentration of acetate buffer, controlling the pH to 6.8, and then adding 100 U / g β-1,3-glucanase to carry out enzymatic hydrolysis. After reacting for 2 h, the solution was inactivated and purified by ultrafiltration using a 50 kDa MWCO (molecular weight cutoff) hollow fiber membrane. The purified filtrate was freeze-dried to obtain 50 kDa β-glucan;

[0079] The parameters of the enzymatic hydrolysis reaction are as follows: the enzymatic hydrolysis temperature is 50°C;

[0080] The parameters of the inactivation treatment were as follows: inactivation temperature 85°C, maintained for 15 min, and then rapidly cooled to 4°C;

[0081] The parameters of ultrafiltration purification were as follows: ultrafiltration pressure: 0.5 MPa, flow rate 10 L / min;

[0082] Step 2: Preparation of β-glucan modified montmorillonite. The specific preparation process is as follows:

[0083] S21, pre-treating the montmorillonite with a 1M NaCl solution to obtain sodium-modified montmorillonite, and loading the sodium-modified montmorillonite with the 50 kDa β-glucan to obtain β-glucan-modified montmorillonite;

[0084] The parameters of sodium pretreatment are as follows: pretreatment temperature is 60°C, pretreatment time is 2h;

[0085] The parameters of the loading treatment were as follows: pH of the loading treatment was 5.0, temperature of the loading treatment was 40°C, and time of the loading treatment was 6 h;

[0086] Also, the mass ratio of montmorillonite to 1M NaCl solution is 1:20; the mass ratio of sodium montmorillonite to 50kDa β-glucan is 1:17;

[0087] Step 3: cross-linking the β-glucan-modified montmorillonite with a 0.3% by mass solution of gallic acid-modified genipin, followed by centrifugal drying to obtain a feed additive suitable for fox cubs;

[0088] The parameters of the cross-linking reaction are as follows: pH of the cross-linking reaction is 7.5, the temperature of the cross-linking reaction is 45° C., and the time of the cross-linking reaction is 8 h;

[0089] The parameters of centrifugal drying are: centrifugation time is 20 minutes, and the precipitate obtained by centrifugation is washed three times with deionized water; the drying parameters are: vacuum drying at a temperature of 40°C to a moisture content of ≤8%;

[0090] The mass ratio of β-glucan-montmorillonite complex to gallic acid-modified genipin is 12:1;

[0091] More specifically, the preparation of gallic acid-modified genipin comprises the following steps:

[0092] Genipin, gallic acid, and immobilized CALB were added to a reactor injected with liquid CO2 at a temperature of 45°C and a pressure of 15 MPa to carry out an enzyme-catalyzed esterification reaction to obtain gallic acid-modified genipin.

[0093] The parameters of the enzyme-catalyzed esterification reaction are as follows: reaction time is 4 h;

[0094] The post-treatment of the enzyme-catalyzed esterification reaction is as follows: releasing the pressure to normal pressure at a rate of 1 MPa / min, centrifuging the reaction mixture, molecularly distilling the crude genipin-gallate in the middle layer, and collecting the heavy components to obtain genipin-gallate; wherein the parameters of the molecular distillation treatment are: distillation temperature of 120° C. and vacuum degree of 0.1 MPa;

[0095] The mass ratio of genipin, gallic acid and immobilized CALB was 1:1.2:0.6.

[0096] Comparative Example 1

[0097] An embodiment of the present invention provides a feed additive suitable for young foxes, wherein the feed additive is montmorillonite.

[0098] Performance Testing

[0099] The feed additives suitable for fox cubs prepared in Examples 1, 2, 3 and Comparative Example 1 were subjected to the following performance tests: aflatoxin B1 adsorption rate (test conditions: toxin concentration: 500 ppb, pH environment 6.5, temperature 37 ° C, time 30 min, detection method HPLC-FLD), vomitoxin adsorption rate (test conditions: toxin concentration: 1 ppm, pH environment 6.8, temperature 37 ° C, time 60 min, detection method UPLC-MS / MS), lysine adsorption rate (test conditions: addition amount: 0.1%, simulated intestinal fluid (containing 0.5% bile salts) ), pH 6.5, detection method amino acid analyzer), vitamin B1 adsorption rate (test conditions: additive amount: 0.1%, simulated intestinal fluid (containing 0.5% bile salts), pH 6.5, detection method amino acid analyzer), Escherichia coli inhibition zone diameter (using Mueller-Hinton agar, additive content 1% (w / v), temperature 37°C, time 24 h, measured by vernier caliper) and intestinal villus height (35-day-old weaned fox pups, testing time 28 days, sampling site is the mid-jejunum, villus height: vertical distance from base to tip (Image Pro Plus 6.0 measurement ≥10 villi / sample));

[0100]

[0101] It can be seen from the above table that the feed additive prepared by the present invention has a higher adsorption rate for aflatoxin B1 and vomitoxin than traditional montmorillonite, and has a lower adsorption rate for lysine and vitamin B1 than traditional montmorillonite;

[0102] Furthermore, the feed additive prepared by the present invention has a greater effect on improving the diameter of the Escherichia coli inhibition zone at intestinal villus height than traditional montmorillonite.

[0103] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a feed additive suitable for young foxes, characterized in that: The following steps are involved: Step 1: Preparation of 50 kDa β-glucan; Step 2: Preparation of β-glucan modified montmorillonite. The specific preparation process is as follows: The montmorillonite raw soil was subjected to sodium pretreatment with a 1M NaCl solution to obtain sodium montmorillonite, and the sodium montmorillonite was loaded with the above-mentioned 50 kDa β-glucan to obtain β-glucan-modified montmorillonite; Step 3: cross-linking the β-glucan-modified montmorillonite with a 0.3% by mass solution of gallic acid-modified genipin, followed by centrifugal drying to obtain a feed additive suitable for fox cubs; The preparation of gallic acid-modified genipin comprises the following steps: Genipin, gallic acid, and immobilized CALB were added to a reactor injected with liquid CO2 at a temperature of 45°C and a pressure of 15 MPa to carry out an enzyme-catalyzed esterification reaction to obtain gallic acid-modified genipin. The parameters of the enzyme-catalyzed esterification reaction are: reaction time is 4h.

2. a preparation method of a feed additive applicable to young foxes according to claim 1, characterized in that: The preparation process of 50kDa β-glucan is as follows: A 1% mass fraction of β-glucan solution was added to a 0.1M molar concentration of acetate buffer, the pH was controlled at 6.8, and then β-1,3-glucanase with an enzyme activity unit of 100U / g was added to carry out enzymatic hydrolysis reaction. After the reaction for 2 hours, 50kDa β-glucan was obtained.

3. A method for preparing a feed additive suitable for young foxes according to claim 1, wherein: The parameters of the enzymatic hydrolysis reaction are: the enzymatic hydrolysis temperature is 50°C.

4. A method for preparing a feed additive suitable for young foxes according to claim 1, wherein: The parameters of sodium pretreatment are: pretreatment temperature is 60℃, and pretreatment time is 2h.

5. A method for preparing a feed additive suitable for young foxes according to claim 1, wherein: The parameters of the loading treatment are as follows: pH of the loading treatment is 5.0, the temperature of the loading treatment is 40° C., and the time of the loading treatment is 6 h.

6. A method for preparing a feed additive suitable for young foxes according to claim 1, characterized in that: The mass ratio of the original montmorillonite to the 1M NaCl solution is 1:10-20; the mass ratio of the sodium montmorillonite to the 50kDa beta-glucan is 1:15-17.

7. A method for preparing a feed additive suitable for young foxes according to claim 1, characterized in that: The parameters of the cross-linking reaction are as follows: pH of the cross-linking reaction is 7.5, the temperature of the cross-linking reaction is 45° C., and the time of the cross-linking reaction is 8 h.

8. A method for preparing a feed additive suitable for young foxes according to claim 1, characterized in that: The parameters of centrifugal drying are: centrifugal time is 10-20 minutes, and the precipitate obtained by centrifugation is washed 3 times with deionized water; the parameters of drying are: vacuum drying at a temperature of 40° C. until the moisture content is ≤8%.

9. A method for preparing a feed additive suitable for young foxes according to claim 1, characterized in that: The mass ratio of β-glucan-montmorillonite complex to gallic acid modified genipin is 10-12:

1.

10. A feed additive suitable for fox cubs, characterized in that: The feed additive is prepared by the method according to any one of claims 1 to 9, and is formed by cross-linking beta-glucan and montmorillonite through modified genipin.

Images