Immunity-enhanced expanded feed additive for leiocassis longirostris and preparation method thereof

Abstract

The invention belongs to the technical field of aquaculture feed, and particularly relates to an immune-enhanced expanded feed additive for leiocassis longirostris and a preparation method of the immune-enhanced expanded feed additive. The additive is prepared from the following components in percentage by mass: 30 to 50 percent of yeast beta-glucan, 15 to 30 percent of astragalus polysaccharide, 10 to 25 percent of bacillus subtilis subjected to sodium alginate-chitosan double-layer coating treatment, 5 to 15 percent of vitamin C ester and the balance of modified zeolite powder treated by 0.1 mol / L citric acid. Wherein the coating treatment comprises the following steps: mixing bacterial sludge with a 3wt% sodium alginate solution according to a ratio of 1: 3, dropwise adding a 2wt% CaCl2 solution, curing for 5 minutes, and immersing into a 1wt% chitosan solution for 10 minutes. During preparation, all the components are mixed at the temperature of 35-40 DEG C in a nitrogen atmosphere, and the rotating speed is 150-250 rpm. According to the additive, through double-layer coating, the survival rate of bacillus subtilis at 140 DEG C per 30 seconds reaches 92%; when the mass ratio of the beta-glucan to the astragalus polysaccharide is (1.8-2.2): 1, the activity of lysozyme in the leiocassis longirostris serum is improved to 22.7 U / mg; 0.5-1.5% of the feed additive is added into the feed, the challenge survival rate is increased to 87%, the intestinal sIgA content reaches 89.6 mu g / mL, and the problem of immune ingredient inactivation caused by high-temperature processing is effectively solved.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture feed, and particularly relates to an immune-enhancing expanded feed additive for catfish and a preparation method thereof. Background Art

[0002] As an important freshwater economic fish species, intensive aquaculture of catfish faces an increasingly serious threat from diseases, such as septicemia and enteritis caused by Aeromonas hydrophila. To control disease, farmers often add antibiotics to feed, but long-term use can lead to drug residues, pathogen resistance, and ecological damage. Therefore, enhancing the disease resistance of catfish through the use of immune enhancers has become a core research direction in green aquaculture.

[0003] Currently, aquatic feeds are generally processed using extrusion technology. Although the high temperature and high pressure conditions (120℃ to 150℃, lasting 30 seconds to 2 minutes) can improve feed digestibility and water stability, they cause serious damage to immune active components. For example:

[0004] 1. Probiotic inactivation problem: The survival rate of probiotics such as Bacillus subtilis drops below 50% within 30 seconds when the temperature exceeds 100°C.

[0005] 2. Degradation of heat-sensitive substances: After Astragalus polysaccharide was treated at 120°C for 1 minute, the loss of immune active components exceeded 40%;

[0006] 3. Vitamin oxidation loss: The decomposition rate of vitamin C ester accelerates at high temperatures, and its bioavailability is reduced by more than 30%.

[0007] To reduce thermal damage, existing technologies mainly use coating or microencapsulation, but there are obvious defects:

[0008] The effect of single-layer coating is limited: after sodium alginate-coated Bacillus subtilis is expanded at 120℃, the survival rate is only about 60%;

[0009] Risk of chemical embedding agent residues: Some synthetic polymer packaging materials may release toxic monomers that do not meet aquatic feed safety standards;

[0010] Insufficient adsorption capacity of the carrier: Ordinary zeolite powder has a low sustained-release rate of active ingredients and is prone to sudden release reactions at high temperatures.

[0011] Furthermore, existing immune supplements often rely on a single component (such as β-glucan or Chinese herbal extracts) and lack synergistic design. Actual aquaculture data shows that single-component supplements only increase catfish survival by less than 10% and increase lysozyme activity by less than 15 U / mg, making them difficult to meet the disease prevention and control needs of high-density aquaculture.

[0012] In summary, developing an immune enhancer for catfish that is resistant to high-temperature processing, has synergistic components, and is safe and efficient is a key technical direction for solving the industry's pain points. Summary of the Invention

[0013] The purpose of the present invention is to provide an immune-enhancing expanded feed additive for catfish and a preparation method thereof.

[0014] In order to achieve the above object, the present invention provides the following technical solutions:

[0015] An immune-enhancing expanded feed additive for catfish, comprising the following components in percentage by mass:

[0016] Yeast beta-glucan 30-50%,

[0017] Astragalus polysaccharide 15-30%,

[0018] 10-25% of Bacillus subtilis treated with sodium alginate-chitosan double-layer coating,

[0019] Vitamin C ester 5-15%,

[0020] The remainder of modified zeolite powder treated with 0.1 mol / L citric acid;

[0021] The coating treatment includes: mixing Bacillus subtilis sludge with 3wt% sodium alginate solution, dropping into 2wt% CaCl2 solution for solidification for 5 minutes, and then immersing in 1wt% chitosan solution for 10 minutes;

[0022] The specific surface area of ​​the modified zeolite powder is 400-450m 2 / g, pore size is 0.5-0.7nm, modified zeolite powder and citric acid solution are mixed at a solid-liquid ratio of 1:5 and soaked for 2h.

[0023] Furthermore, the mass ratio of the yeast β-glucan to astragalus polysaccharide is (1.8-2.2):1.

[0024] A method for preparing an immune-enhancing expanded feed additive for catfish comprises the following steps:

[0025] (a) Bacillus subtilis sludge was mixed with 3 wt% sodium alginate solution in a mass ratio of 1:3, dropped into 2 wt% CaCl2 solution for curing for 5 min, removed and immersed in 1 wt% chitosan solution for 10 min, and freeze-dried;

[0026] (b) mixing yeast β-glucan, astragalus polysaccharide, vitamin C ester and modified zeolite powder at 35-40° C. for 15-20 minutes;

[0027] (c) Add the coated bacterial powder obtained in step (a), mix at 150-250 rpm for 10 min, and then pass through an 80-mesh sieve.

[0028] Furthermore, in step (a), the concentration of the sodium alginate solution is 3wt%, the mass ratio of the sodium alginate solution to the bacterial sludge is 3:1, the concentration of the CaCl2 solution is 2wt%, the concentration of the chitosan solution is 1wt%, and the temperature is 25±2°C.

[0029] Furthermore, the mixing temperature in step (b) is 38±2°C.

[0030] Furthermore, the mixing speed in step (c) is 200±50 rpm.

[0031] Furthermore, step (b) and step (c) are carried out under a nitrogen gas atmosphere with a nitrogen flow rate of 0.5-1 l / min.

[0032] The invention discloses an expanded feed, comprising an immunity-enhancing expanded feed additive for catfish, wherein the additive is added in an amount of 0.5-1.5 wt % in the feed.

[0033] Furthermore, the invention is composed of the following components in percentage by mass: 40% yeast beta-glucan, 22% astragalus polysaccharide, 18% enveloped Bacillus subtilis, 10% vitamin C ester, and 10% modified zeolite powder.

[0034] The beneficial effects of the present invention are:

[0035] 1. Significantly improve high temperature resistance

[0036] By combining a double-layer coating of sodium alginate and chitosan with a specific curing process (the mass ratio of bacterial mud to sodium alginate solution is 1:3, CaCl2 solution is dripped in for curing for 5 minutes, and chitosan solution is immersed for 10 minutes), the survival rate of Bacillus subtilis after puffing treatment at 140℃ / 30s reaches more than 92%, which is 30 percentage points higher than that of single-layer coating technology.

[0037] 2. Optimize the sustained release efficiency of active ingredients

[0038] The modified zeolite powder treated with 0.1 mol / L citric acid is used as the carrier, and its specific surface area is 400-450m 2 / g, 0.5-0.7nm, which can extend the sustained release time of immune active ingredients in the intestine to more than 6 hours, avoiding sudden release response caused by high temperature processing.

[0039] 3. Achieve synergistic efficiency of components

[0040] When the mass ratio of yeast β-glucan to astragalus polysaccharide was 1.8-2.2:1, the serum lysozyme activity increased to 22.7U / mg, which was more than 50% higher than the effect of adding a single component, significantly enhancing the nonspecific immune response of catfish.

[0041] 4. Ensure the stability of the processing process

[0042] The mixing process is carried out under a nitrogen atmosphere (gas flow rate 0.5-1 L / min), with a mixing temperature of 35-40° C. and a rotation speed of 150-250 rpm, so that the retention rate of vitamin C ester in the additive preparation process reaches more than 98%.

[0043] 5. Improve the survival rate of breeding

[0044] After 30 days of feeding with 0.5-1.5% of the additive in the expanded feed, the survival rate of catfish in the Aeromonas hydrophila challenge experiment reached 87%, which was 35 percentage points higher than that of the blank control group.

[0045] 6. Reduce intestinal oxidative damage

[0046] The modified zeolite powder carrier has an efficiency of adsorbing mycotoxins in feed of over 90%. Combined with the antioxidant properties of vitamin C ester, it increases the content of secretory immunoglobulin in the intestine of catfish to 89.6 μg / mL, which is 1.5 times higher than that of the ordinary feed group. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the structure of enveloped Bacillus subtilis.

[0048] Figure 2 This is a diagram showing the distribution of additives in extruded feed. DETAILED DESCRIPTION

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention; it is obvious that the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0050] Example 1

[0051] Additive composition

[0052] The additive involved in this embodiment is composed of a variety of functional ingredients in a scientific ratio. Among them, yeast β-glucan accounts for 40%. As a polysaccharide substance with multiple physiological activities, yeast β-glucan plays an important basic role in the additive system and is of key significance to improving the overall performance. The content of astragalus polysaccharide is 22%. As a natural active ingredient extracted from astragalus, astragalus polysaccharide has unique biological activity and can give specific functions to additives. Encapsulated Bacillus subtilis accounts for 18%. Bacillus subtilis that has undergone special coating treatment can better adapt to specific environments and exert its beneficial effects. The content of vitamin C ester is 10%. As a derivative of vitamin C, vitamin C ester has good stability and bioavailability, adding indispensable functional properties to the additive. Modified zeolite powder also accounts for 10%. The physical and chemical properties of the specially modified zeolite powder are optimized, which has a positive impact on the performance of the additive.

[0053] Preparation method

[0054] 1. Bacteria coating treatment: First, select Bacillus subtilis fermentation liquid, the number of viable bacteria in the fermentation liquid must be ensured to be ≥1×10 10 CFU / g, which is an important prerequisite for ensuring subsequent effects. The selected fermentation broth is placed in a centrifuge, the speed is set to 4000rpm, and the centrifugation time is 10min. In this way, the bacterial mud is effectively collected. Then, the collected bacterial mud is thoroughly mixed with a sodium alginate solution with a mass fraction of 3wt% in a mass ratio of 1:3. Subsequently, the mixed solution is slowly dripped into a CaCl2 solution with a mass fraction of 2wt%. During this process, the mixed solution gradually solidifies, and the solidification time is set to 5min. After the solidification is completed, the gel beads are carefully taken out and immersed in a chitosan solution with a mass fraction of 1wt%. The temperature of this chitosan solution needs to be maintained at 25°C, and the immersion time is 10min. Finally, the gel beads treated as above are dried by vacuum freeze-drying to obtain coated bacterial powder.

[0055] 2. Carrier modification: For the modification of zeolite powder, first mix the zeolite powder with 0.1mol / L citric acid solution at a solid-liquid ratio of 1:5, and then soak it for 2 hours. After soaking, rinse it repeatedly with deionized water until the rinsed solution is neutral. After that, place the rinsed zeolite powder in an environment of 105℃ for drying. After a series of testing methods, the specific surface area of ​​the modified zeolite powder was measured to be 420m 2 / g, and a pore size of 0.6nm. These optimized physical parameters lay the foundation for its role in additives.

[0056] 3. Mixing process: Place yeast β-glucan, astragalus polysaccharide, vitamin C ester and modified zeolite powder together in a mixer. During the mixing process, strictly control the environmental conditions, set the temperature to 38°C, introduce nitrogen at the same time, maintain the nitrogen flow rate at 0.8L / min, and continue mixing for 18 minutes to ensure that these ingredients are initially evenly mixed. Subsequently, add the previously prepared coated bacterial powder, adjust the mixer speed to 200rpm, and mix at a low speed for 10 minutes. After mixing, screen the mixture through an 80-mesh sieve to ensure that the particle size meets the requirements. Finally, use nitrogen-filled packaging to package the screened additives to prevent them from deteriorating due to contact with air.

[0057] Example 2

[0058] Additive composition

[0059] The additives of this embodiment are also composed of multiple active ingredients. The proportion of yeast β-glucan is adjusted to 30%, which is lower than that of Example 1. The proportion of astragalus polysaccharide is increased to 16.5%. The content of coated Bacillus subtilis is 25%, which still plays an important role in the additive system. The proportion of vitamin C ester is 8.5%, and the modified zeolite powder accounts for 20%. The physical parameters of the modified zeolite powder are marked in detail, and its specific surface area is 400m 2 / g, with a pore size of 0.5nm, zeolite powder was mixed with 0.1mol / L citric acid solution at a solid-liquid ratio of 1:5 and soaked for 2h. In this embodiment, the ratio of β-glucan to astragalus polysaccharide was 1.8:1.

[0060] Preparation method

[0061] The preparation method of this embodiment is consistent with that of Example 1. In this way, while ensuring the same preparation process, only the ratio of each component of the additive is changed, so that the effect of the change in component ratio on the performance of the additive can be studied more accurately.

[0062] Example 3

[0063] Additive composition

[0064] In the additive formula of this embodiment, the proportion of yeast β-glucan is further increased to 50%, becoming the largest component. Astragalus polysaccharide accounts for 22.7%, coated Bacillus subtilis accounts for 10%, vitamin C ester accounts for 7.3%, and modified zeolite powder accounts for 10%, and the specific surface area of ​​the modified zeolite powder is 450m 2 / g, and a pore size of 0.7nm. It is worth noting that the ratio of β-glucan to astragalus polysaccharide in this example is changed to 2.2:1. This large ratio difference will provide richer data for studying the changes in the performance of additives.

[0065] Preparation method

[0066] The preparation method also follows the steps of Example 1. By changing the ratio of ingredients under the same preparation process, it is helpful to gain a deeper understanding of the intrinsic relationship between the ratio of each ingredient and the performance of the additive.

[0067] Comparative Example 1

[0068] Components and preparation methods

[0069] The components of this comparative example are exactly the same as those of Example 1, but there is a difference in the coating treatment of Bacillus subtilis. In this comparative example, Bacillus subtilis is only coated with a single layer of sodium alginate and is not immersed in a chitosan solution for further treatment. The specific preparation method is: after mixing the bacterial sludge with a 3wt% sodium alginate solution, it is directly dripped into a CaCl2 solution for solidification, and then directly freeze-dried, omitting the immersion in chitosan solution step in Example 1. Through this comparison, the impact of chitosan treatment on the coating effect of Bacillus subtilis and the performance of the final additive can be clearly seen.

[0070] Comparative Example 2

[0071] Component Description

[0072] The additive composition of this comparative example is relatively simple, containing only 40% yeast β-glucan, with the remainder replaced by an equal amount of ordinary zeolite powder, which has not undergone any modification. This simple formulation is intended to highlight the importance of yeast β-glucan and zeolite modification in the overall additive system. By comparing it with the examples, it is possible to more intuitively observe the impact of the missing other ingredients or the unmodified zeolite powder on the additive's performance.

[0073] Comparative Example 3

[0074] Differences in composition and preparation

[0075] The components of this comparative example are consistent with those of Example 1, but the mixing process differs significantly from that of Example 1. The mixing process in this comparative example was carried out under a normal air atmosphere, rather than the nitrogen atmosphere used in Example 1. This comparative setup allows for the study of the effects of different mixing environments on additive performance and clarifies the role and importance of nitrogen protection in the mixing process.

[0076] Comparative Example 4

[0077] Yeast beta-glucan: 50%,

[0078] Astragalus polysaccharide: 15% (ratio 50:15 = 3.33:1, exceeding 1.8-2.2:1),

[0079] The other components were the same as those in Example 1. The serum lysozyme activity was reduced to 16.2±0.8 U / mg, a decrease of 28.6% compared with Example 1.

[0080] Schematic diagram of the structure of enveloped Bacillus subtilis Figure 1 The distribution state of additives in extruded feed is shown in Figure 2 .

[0081] Table 1: High temperature survival rate test (140℃ / 30s treatment)

[0082] Group Bacillus subtilis survival rate Vitamin C ester retention rate Example 1 92.3±1.5％ 97.8±0.8％ Comparative Example 1 70.2±2.1％ - Comparative Example 3 - 85.6±1.2％

[0083] Table 2: Catfish feeding experiment (30 days, addition amount 1.0%)

[0084] Group Serum lysozyme (U / mg) Survival rate of challenge (Aeromonas hydrophila) Blank feed 8.2±0.3 52％ Example 1 22.7±1.1 87％ Example 2 18.5±0.9 78％ Example 3 19.1±1.0 80％ Comparative Example 1 15.3±0.7 65％ Comparative Example 2 12.5±0.5 60％

[0085] The above description is merely a preferred embodiment of the present invention; however, the scope of protection of the present invention is not limited thereto. Any person skilled in the art who, within the technical scope disclosed by the present invention, makes equivalent substitutions or modifications based on the technical solutions and improved concepts of the present invention shall be covered by the scope of protection of the present invention.

Claims

1. An immune-enhancing expanded feed additive for catfish, characterized in that: It is composed of the following components in percentage by mass: Yeast beta-glucan 30-50%, Astragalus polysaccharide 15-30%, 10-25% of Bacillus subtilis treated with sodium alginate-chitosan double-layer coating, Vitamin C ester 5-15%, The balance is modified zeolite powder treated with 0.1 mol / L citric acid. The coating treatment includes: mixing Bacillus subtilis sludge with 3wt% sodium alginate solution, dropping into 2wt% CaCl2 solution for solidification for 5 minutes, and then immersing in 1wt% chitosan solution for 10 minutes; The specific surface area of ​​the modified zeolite powder is 400-450m 2 / g, pore size is 0.5-0.7nm, modified zeolite powder and citric acid solution are mixed at a solid-liquid ratio of 1:5 and soaked for 2h.

2. The immune-enhancing expanded feed additive for catfish according to claim 1, wherein: The mass ratio of the yeast β-glucan to astragalus polysaccharide is (1.8-2.2):

1.

3. A method for preparing the immune-enhancing expanded feed additive for catfish according to claim 1, characterized in that: Including steps: (a) Bacillus subtilis sludge was mixed with 3 wt% sodium alginate solution in a mass ratio of 1:3, dropped into 2 wt% CaCl2 solution for curing for 5 min, removed and immersed in 1 wt% chitosan solution for 10 min, and freeze-dried; (b) mixing yeast β-glucan, astragalus polysaccharide, vitamin C ester and modified zeolite powder at 35-40° C. for 15-20 minutes; (c) Add the coated bacterial powder obtained in step (a), mix at 150-250 rpm for 10 min, and then pass through an 80-mesh sieve.

4. The method for preparing the immune-enhancing expanded feed additive for catfish according to claim 3, wherein: In step (a), the concentration of sodium alginate solution is 3wt%, the mass ratio of sodium alginate solution to bacterial mud is 3:1, the concentration of CaCl2 solution is 2wt%, the concentration of chitosan solution is 1wt%, and the temperature is 25±2°C.

5. The method for preparing the immune-enhancing expanded feed additive for catfish according to claim 3, wherein: The mixing temperature in step (b) is 38±2°C.

6. The method for preparing the immune-enhancing expanded feed additive for catfish according to claim 3, wherein: The mixing speed in step (c) is 200±50 rpm.

7. The method for preparing the immune-enhancing expanded feed additive for catfish according to claim 3, wherein: Step (b) and step (c) are carried out under a nitrogen gas atmosphere with a nitrogen flow rate of 0.5-1 l / min.

8. An expanded feed, characterized in that: The catfish immunity-enhancing expanded feed additive according to claim 1 is contained, and the amount of the additive added to the feed is 0.5-1.5wt%.

9. The immune-enhancing expanded feed additive for catfish according to claim 1, characterized in that: The invention is composed of the following components in percentage by mass: 40% yeast beta-glucan, 22% astragalus polysaccharide, 18% enveloped Bacillus subtilis, 10% vitamin C ester and 10% modified zeolite powder.

Images