Aquatic feed for improving bioavailability of L-ascorbic acid-2-phosphate and preparation method of aquatic feed

Abstract

The invention discloses an aquatic feed for improving the bioavailability of L-ascorbic acid-2-phosphate and a preparation method of the aquatic feed. The feed comprises L-ascorbic acid-2-phosphate, phytase, a natural high-phytic-acid plant raw material (such as full-fat rice bran, and the phytic acid content is greater than or equal to 1.0%) and an organic acidifying agent (such as citric acid). By controlling the molar ratio of phytic acid to Vc ester to be greater than or equal to 3: 1 and adjusting the pH value of the mixture to 4.0-5.0, advanced hydrolysis of phytase to the Vc ester is inhibited by utilizing the competitive property of phytic acid in the feed puffing processing process, so that the retention rate of the Vc ester is remarkably increased (up to 88%); in in-vitro simulated digestion (a buffer system with the pH value of 5.8), after phytic acid is preferentially degraded, released phytase efficiently hydrolyzes Vc ester, and the Vc release rate reaches 72%. Through comprehensive calculation, the total effective Vc utilization rate reaches up to 63.4% and is increased by 48% compared with a control group. The feed does not need an embedding process, is low in cost, is simple and convenient to operate, and is suitable for aquatic feeds for carps, grass carps, tilapia mossambica, penaeus vannamei and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of aquatic feed additives, and specifically relates to a compound feed containing phytase, L-ascorbic acid-2-phosphate, natural high-phytate plant raw material and organic acidifying agent. Through the double mechanisms of substrate competition inhibition and pH dynamic regulation, the premature enzymatic hydrolysis of Vc ester is prevented during the feed processing, and the efficient hydrolysis and release of free ascorbic acid in the simulated aquatic animal digestion environment are promoted, so that the biological availability potential is significantly improved. BACKGROUND

[0002] L-ascorbic acid (vitamin C) is an essential micronutrient for aquatic animals, which has antioxidant, immunomodulatory and growth-promoting effects. However, it is chemically unstable and easily oxidized and inactivated during feed processing (such as pelleting and puffing) and storage. Therefore, its phosphate ester derivative, L-ascorbic acid-2-phosphate (referred to as Vc ester), is generally used in industry to improve stability.

[0003] Vc ester needs to be hydrolyzed by phosphomonoesterase in the animal body to release free ascorbic acid before it can be absorbed and utilized. Studies have shown that phytase, in addition to hydrolyzing phytic acid, also has broad-spectrum phosphomonoester hydrolysis activity and can effectively catalyze the hydrolysis of Vc ester. However, if phytase and Vc ester are directly mixed in the feed under wet and hot processing conditions (such as 85-110°C and high humidity), even if a heat-resistant phytase is used, premature enzymatic hydrolysis may still occur due to the increase in local water activity, resulting in the generation of free Vc and its rapid degradation, thereby reducing the overall potency.

[0004] Existing solutions rely on physical isolation techniques such as microcapsule embedding and enteric coating, but these methods are costly and complex, making them difficult to apply in ordinary aquatic feeds. In addition, the intestinal pH of most aquatic animals (such as carp and grass carp) is slightly neutral (6.5-7.5), which is outside the optimal pH range (5.0-5.5) for phytase, limiting its hydrolysis efficiency of Vc ester.

[0005] Therefore, there is an urgent need for a low-cost, easy-to-implement technical solution that does not require complex embedding processes, which can both protect the stability of Vc ester during processing and efficiently activate its release during digestion. SUMMARY

[0006] The purpose of the present application is to provide a new type of aquatic feed formula and preparation method, by introducing natural high-phytate plant raw material and organic acidifying agent, to build a dual regulation system of "inhibition during processing + activation during digestion". As follows: (1) Processing stage protection mechanism: Natural high phytate material (e.g. whole rice bran) added in feed provides high concentration of phytic acid (IP6) as a preferential substrate for phytase, reducing its hydrolysis of Vc ester by competitive inhibition; Organic acidifier (e.g. citric acid) reduces the pH of the mixture to 4.0-5.0, further inhibiting the residual activity of phytase in a wet heat environment, effectively preventing the premature degradation of Vc ester.

[0007] (2) Digestive phase activation mechanism: In a simulated aquatic animal intestinal environment, phytic acid is first rapidly degraded; The residual acidifier synergizes with the buffer system to maintain the local pH at 5.0-6.0, within the optimal activity range of phytase, thereby efficiently hydrolyzing Vc ester and releasing free ascorbic acid.

[0008] All components of the present invention are conventional feed raw materials or additives, do not require special equipment or embedding technology, are low in cost, high in safety, and easy to industrialize, and are particularly suitable for mainstream aquaculture species such as Cyprinidae, Tilapia, and South American white prawns. DETAILED DESCRIPTION Example 1: Preparation of extruded grass carp feed and verification of its protection and release performance for L-ascorbic acid-2-phosphate.

[0009] Feed formula (weight percentage) Ingredients Content (%) Fish meal 10.0 Soybean meal 30.0 Whole rice bran 6.0 Corn meal 20.0 Fish oil 5.0 Sodium L-ascorbyl-2-phosphate 0.05 Thermostable phytase (activity ≥ 20,000 FTU / g) 0.02 Citric acid 1.0 Calcium dihydrogen phosphate 1.0 Vitamin / mineral premix 1.0 Others (binder, antifungal agent, etc.) Supplemented to 100.0 Note: The phytic acid content of whole rice bran is about 1.8%, making the molar ratio of phytic acid to Vc ester in the feed about 5:1. Citric acid reduces the pH of the mixture to 4.5.

[0010] Preparation method ① Mix all dry powder raw materials except phytase uniformly; ② Adjust the moisture content to 12% and treat at 85°C for 30 seconds; ③ Extrude with a twin-screw extruder (die temperature 110°C, pressure 3 MPa); ④ Cool to below 40°C and spray phytase solution (diluted with 5% molasses water); ⑤ Air dry to a moisture content of ≤10% to obtain finished pellet feed.

[0011] Control group setup Same formula, but without whole rice bran and citric acid, increase the dosage of calcium dihydrogen phosphate to 2.0%, and the rest of the process is the same.

[0012] I. Processing stability test: determination of Vc ester retention rate after extrusion Method: Take the same batch of raw materials and expanded samples, and determine the L-ascorbic acid-2-phosphate content by HPLC method (reference GB / T 23882-2009 Determination of L-ascorbic acid-2-phosphate in feed - High performance liquid chromatography); Vc ester retention rate (%) = (expanded content / raw material content) x 100%.

[0013] Results: Group Vc ester content before expansion (mg / kg) Vc ester content after expansion (mg / kg) Vc ester retention rate (%) Invention group 500 ± 6 440 ± 5 88 ± 1 Control group 502 ± 5 376 ± 6 75 ± 1 Take three times in parallel.

[0014] II. In vitro digestion experiment: Vc release rate determination Method (simplified buffer system): ①Take 1.0 g of expanded feed and add 50 mL of 0.1 M acetic acid-sodium acetate buffer (pH 5.8); ②Supplement exogenous phytase to the total activity of the reaction system of 1000 FTU; ③30°C water bath oscillation, reaction for 90 min; ④Boiling water bath enzyme inactivation, centrifugation to take supernatant; ⑤HPLC method (detection wavelength 245 nm) to determine the concentration of free ascorbic acid; Vc release rate (%) = (measured free Vc amount / theoretical releasable Vc amount of residual Vc ester in sample) x 100%.

[0015] Results: Group Vc release rate (90 min, %) Invention group 72 ± 2 Control group 57 ± 2 III. Total effective Vc utilization rate calculation Definition: Total effective Vc utilization rate (%) = Vc ester retention rate x Vc release rate Calculation results: Group Vc ester retention rate (%) Vc release rate (%) Total effective Vc utilization rate (%) Invention group 88 72 63.4 Control group 75 57 42.8 The final Vc ester added in the group of the application is converted into absorbable free Vc by 63.4%, which is increased by 48% compared with the control group (42.8%).

[0016] Conclusion The application realizes the stable preservation of Vc ester in processing and efficient release in digestion through the synergistic effect of natural high phytic acid raw materials and organic acidifying agents, and the total effective utilization rate is significantly improved, and without complex process, has outstanding industrial application value.

Claims

1. An aquatic feed for improving the bioavailability of L-ascorbic acid-2-phosphate, characterized in that, It contains the following components: (1) L-ascorbic acid-2-phosphate; (2) Phytase; (3) Natural high phytic acid plant raw materials, wherein the phytic acid content of the raw materials is not less than 1.0%, and the amount added to the feed makes the molar ratio of phytic acid to L-ascorbic acid-2-phosphate in the feed ≥3:1; (4) Organic acidifier, the amount of which is added to make the pH of the feed mixture 4.0–5.

0.

2. The aquatic feed as described in claim 1, characterized in that, The natural high-phytic acid plant raw material is selected from full-fat rice bran, wheat bran or sunflower meal, and the addition amount is 3%–10% of the total feed mass.

3. The aquatic feed as described in claim 1 or 2, characterized in that, The organic acidifier is selected from one or more of citric acid, fumaric acid, and calcium formate, and the amount added is 0.3%–2.0% of the total feed mass.

4. The aquatic feed according to any one of claims 1-3, characterized in that, The phytase is a thermostable phytase, and its activity retention rate is ≥60% after wet heat treatment at 90°C for 30 seconds.

5. The method for preparing aquatic feed according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Mix, condition, puff or granulate the raw materials except for phytase; (2) After cooling, phytase is sprayed onto the surface of the feed pellets in liquid form; (3) Drying to obtain finished feed.