Application of protocatechuic acid in regulating intestinal flora of procambarus clarkia and flavor amino acid composition in muscle of procambarus clarkia

Abstract

The invention provides application of protocatechuic acid in regulating intestinal flora of procambarus clarkii and flavor amino acid composition in muscle of the procambarus clarkii, and belongs to the technical field of development of aquatic feed additives. It is found that the protocatechuic acid can reduce the abundance of Vibrionaceae in the intestinal tract level of the procambarus clarkii, improve the abundance of Rhodobacteroceae, Lactobacillus and Bacillus, reduce the abundance of Vibrio in the intestinal tract level of the procambarus clarkii, and improve the abundance of Halocynthiibacter and Ruegeria, so that the composition of flavor amino acids in the muscles of the procambarus clarkii is improved. The invention further provides a procambarus clarkii feed and application thereof in procambarus clarkii culture, the content of flavor amino acids in procambarus clarkii can be effectively increased, the muscle quality of procambarus clarkii is improved, the cost is low, and the culture method is simple.

Description

Technical Field

[0001] This invention belongs to the field of aquatic feed additive development technology, specifically involving the application of protocatechuic acid in regulating the intestinal flora of Procambarus clarkii and the flavor amino acid composition of its muscle. Background Technology

[0002] The red swamp crayfish (Procambarus clarkii) belongs to the phylum Arthropoda, class Crustacea, order Decapoda, family Procambaridae, and genus Procambarus. Native to North America, it is an important economic crustacean in freshwater aquaculture with a high degree of industrialization and rapidly growing consumer demand. The red swamp crayfish has tender, uniquely flavorful muscle, rich in high-quality protein and various amino acids, giving it high edible and processing value. Its commercial value depends not only on yield and size but also heavily on muscle quality, especially flavor characteristics. Quality control of red swamp crayfish mainly focuses on farming methods, feed protein sources, and aquaculture environment management. In actual production, significant differences in product flavor occur under different farming methods and feed systems, and quality fluctuations have become one of the key issues restricting the high-quality development of the industry. Although adjusting feed protein levels, optimizing plant protein substitution ratios, or adopting integrated rice-crayfish farming models can improve its growth performance and basic nutritional composition, these measures often primarily aim to increase yield or reduce costs, with limited ability to specifically regulate muscle flavor.

[0003] The flavor of red swamp crayfish muscle is primarily determined by water-soluble flavor compounds such as flavor amino acids, whose content and ratio directly affect the intensity of umami and sweetness. The content and ratio of flavor amino acids are not only influenced by the amino acid supply in the feed but are also closely related to digestibility, intestinal barrier status, stress levels, and metabolic processes such as amino acid decomposition and transport. In actual production, the composition of flavor amino acids in muscle is prone to fluctuation due to factors such as a monotonous feed structure, high-density farming, and environmental volatility, leading to unstable commercial quality. Current methods for regulating flavor amino acids mostly rely on directly increasing feed protein or specific amino acid levels, which is not only costly but also suffers from low utilization efficiency and increased environmental burden. Summary of the Invention

[0004] To address the problems existing in the prior art, the primary objective of this invention is to provide the application of protocatechuic acid in the preparation of products that regulate the intestinal flora of Procambarus clarkii and increase the content of flavor amino acids in the muscle of Procambarus clarkii.

[0005] A second objective of this invention is to provide a feed for the red swamp crayfish and its application in red swamp crayfish farming.

[0006] A third objective of this invention is to provide a method for farming Procambarus clarkii.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of protocatechuic acid in the preparation of products that regulate the intestinal flora of Procambarus clarkii.

[0008] Preferably, protocatechuic acid reduces the abundance of Vibrionaceae in the intestine of Procambarus clarkii and increases the abundance of Rhodobacteraceae, Lactobacillaceae, and Bacillaceae in the intestine of Procambarus clarkii.

[0009] Preferably, protocatechuic acid reduces the levels of protocatechuic acid in the intestines of red swamp crayfish. Vibrio Increase the abundance of [unclear] in the intestines of Procambarus clarkii. Halocynthiibacter and Ruegeria The abundance of.

[0010] This invention also provides the application of protocatechuic acid in the preparation of products that increase the content of flavor amino acids in the muscle of Procambarus clarkii.

[0011] Preferably, the flavor amino acids include glycine, alanine, tyrosine, phenylalanine, aspartic acid, and glutamic acid.

[0012] The present invention also provides a feed for Procambarus clarkii, wherein the feed contains 3-15 g / kg of protocatechuic acid by weight of feed.

[0013] Preferably, the feed consists of the following components: fish meal, soybean meal, cottonseed protein, rapeseed meal, peanut meal, corn DDGS, flour, sodium alginate, calcium dihydrogen phosphate, spray-dried yeast powder, soybean oil, fish oil, bentonite, soybean lecithin oil, cholesterol, multivitamins and minerals, sodium chloride, choline chloride, vitamin C, chitosan, L-Thr, L-Lys, Met and protocatechuic acid.

[0014] Preferably, the feed, by weight parts, comprises the following components: 4-6 parts fishmeal, 12-18 parts soybean meal, 5-7 parts cottonseed protein, 12-15 parts rapeseed meal, 8-12 parts peanut meal, 4-6 parts corn DDGS, 25-30 parts wheat flour, 1-3 parts sodium alginate, 2-3 parts calcium dihydrogen phosphate, 1-3 parts spray-dried yeast powder, 1-3 parts soybean oil, 1-2 parts fish oil, 2-5 parts bentonite, 1-2 parts soybean lecithin oil, 0.2-0.5 parts cholesterol, 0.5-1.5 parts multivitamins and minerals, 0.1-0.3 parts sodium chloride, 0.2-0.3 parts choline chloride, 0.2-0.3 parts vitamin C, 0.05-0.15 parts chitosan, 0.2-0.5 parts L-Thr, 0.2-0.5 parts L-Lys, and 0.5 parts Met. 0.05~0.15 parts and protocatechuic acid 0.3~1.5 parts.

[0015] This invention also provides the application of the above-mentioned Procambarus clarkii feed in Procambarus clarkii farming.

[0016] The present invention also provides a method for farming red swamp crayfish, comprising the following steps: feeding the above-mentioned red swamp crayfish feed.

[0017] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention significantly improves the α and β diversity of the gut microbiota of *Procambarus clarkii* by adding protocatechuic acid to its feed. At the family level, it significantly reduces the relative abundance of Vibrionaceae and significantly increases the relative abundance of Rhodobacteraceae, Lactobacillaceae, and Bacillaceae. At the genus level, it significantly reduces... Vibrio Abundance, significantly improved Halocynthiibacter and Ruegeria Abundance. Furthermore, by altering the gut microbiota and influencing amino acid metabolism and host nutrient utilization efficiency, this invention increases the content of flavor amino acids in the muscle of the red swamp crayfish, thereby improving muscle quality. The feed and farming costs of this invention are low, and the solution is simple and feasible. Attached Figure Description

[0018] Figure 1 Effects of protocatechuic acid on the content of single umami amino acids in the muscle of Procambarus clarkii; Figure 2 Effect of protocatechuic acid on the content of single sweet amino acids in the muscle of Procambarus clarkii; Figure 3 Effect of protocatechuic acid on the total amount of flavor amino acids in the muscle of Procambarus clarkii; Figure 4 : Fitting curves of flavor amino acids and protocatechuic acid addition amounts; Figure 5 : Gut microbiota β diversity; Figure 6 The effect of protocatechuic acid on the relative abundance of gut microbiota families in Procambarus clarkii; Figure 7 The correlation between flavor amino acids and gut microbiota levels; Figure 8 Effects of protocatechuic acid on the relative abundance of gut microbiota in Procambarus clarkii; Figure 9 Correlation between flavor amino acids and gut microbiota levels. Detailed Implementation

[0019] This invention provides the application of protocatechuic acid in the preparation of products that regulate the intestinal flora of Procambarus clarkii.

[0020] At the scientific level, protocatechuic acid significantly reduced the abundance of Vibrionaceae (Vibrioceae) in the intestine of *Procambarus clarkii*, and significantly increased the abundance of Rhodobacteraceae, Lactobacillaceae, and Bacillaceae (Bacillusaceae) in the intestine of *Procambarus clarkii*. Several members of Vibrionaceae can cause disease in aquatic animals; several members of Rhodobacteraceae can efficiently utilize various organic substances; and several members of Lactobacillaceae and Bacillaceae are associated with the synthesis of various amino acids.

[0021] At the subordinate level, the protocatechuic acid can significantly reduce the levels of [unclear text - possibly related to a specific substance or ingredient] in the intestines of *Procambarus clarkii*. Vibrio The abundance of Vibrio species increases the amount of bacteria in the intestines of Procambarus clarkii. Halocynthiibacter (Spp. of the genus *Oryctospora*) and Ruegeria Abundance of (Rugeriella) species. Among them, Vibrio It can cause diseases in aquatic animals, such as enteritis and septicemia; Halocynthiibacter Participates in metabolism, Ruegeria They participate in the degradation of organic matter and maintain the stability of the bacterial community, thus acting as beneficial symbiotic bacteria.

[0022] The types and abundance of gut microbiota affect nutrient absorption, metabolic regulation, and tissue nutrient deposition in *Procambarus clarkii* (claw crayfish). Changes in the gut microecological state can influence amino acid metabolism and host nutrient utilization efficiency in *Procambarus clarkii*. In this invention, protocatechuic acid significantly improves the flavor amino acid composition of *Procambarus clarkii* muscle by regulating the abundance of gut microbiota, and provides its application in preparing products that enhance the flavor amino acid content of *Procambarus clarkii* muscle.

[0023] Preferably, the flavor amino acids in this invention include umami amino acids and sweet amino acids. This invention significantly increases the individual content and total amount of umami amino acids and sweet amino acids in the muscle of *Procambarus clarkii* by adding protocatechuic acid to the feed. In this invention, the umami amino acids include glycine, alanine, tyrosine, and phenylalanine; the sweet amino acids include aspartic acid and glutamic acid.

[0024] The present invention also provides a feed for Procambarus clarkii, wherein the feed contains 3-15 g / kg (0.3-1.5 wt%) of protocatechuic acid by weight of feed, and the content of protocatechuic acid is preferably 4 g / kg, 5 g / kg, 6 g / kg, 7 g / kg, 8 g / kg, 9 g / kg, 10 g / kg, 11 g / kg, 12 g / kg, 13 g / kg or 14 g / kg.

[0025] Preferably, the feed of the present invention is composed of the following components: fish meal, soybean meal, cottonseed protein, rapeseed meal, peanut meal, corn DDGS, flour, sodium alginate, calcium dihydrogen phosphate, spray-dried yeast powder, soybean oil, fish oil, bentonite, soybean lecithin oil, cholesterol, multivitamins and minerals, sodium chloride, choline chloride, vitamin C, chitosan, L-Thr (L-threonine), L-Lys (L-lysine), Met (methionine / methionine), and protocatechuic acid.

[0026] Preferably, the feed of the present invention, by weight parts, comprises the following components: 4-6 parts fish meal, 12-18 parts soybean meal, 5-7 parts cottonseed protein, 12-15 parts rapeseed meal, 8-12 parts peanut meal, 4-6 parts corn DDGS, 25-30 parts wheat flour, 1-3 parts sodium alginate, 2-3 parts calcium dihydrogen phosphate, 1-3 parts spray-dried yeast powder, 1-3 parts soybean oil, 1-2 parts fish oil, 2-5 parts bentonite, 1-2 parts soybean lecithin oil, 0.2-0.5 parts cholesterol, 0.5-1.5 parts multivitamins and minerals, 0.1-0.3 parts sodium chloride, 0.2-0.3 parts choline chloride, 0.2-0.3 parts vitamin C, 0.05-0.15 parts chitosan, 0.2-0.5 parts L-Thr, and L-Lys. 0.2-0.5 parts, Met 0.05-0.15 parts, and protocatechuic acid 0.3-1.5 parts. More preferably: 5 parts fish meal, 15 parts soybean meal, 6 parts cottonseed protein, 13 parts rapeseed meal, 10 parts peanut meal, 5 parts corn DDGS, 28 parts flour, 2 parts sodium alginate, 2.3 parts calcium dihydrogen phosphate, 2 parts spray-dried yeast powder, 2 parts soybean oil, 1.5 parts fish oil, 2.2-3.4 parts bentonite, 1.5 parts soybean lecithin oil, 1.45 parts cholesterol, 1 part multivitamin and mineral, 0.2 parts sodium chloride, 0.25 parts choline chloride, 0.25 parts vitamin C, 0.1 parts chitosan, 0.35 parts L-Thr, 0.3 parts L-Lys, 0.1 parts Met, and 0.6-0.9 parts protocatechuic acid.

[0027] In this invention, the multivitamin and mineral premix refers to a premix of vitamins and minerals including: vitamin A acetate, vitamin D, dl-α-tocopherol acetate, menadione, thiamine nitrate, riboflavin (vitamin B2), pyridoxine hydrochloride, cyanocobalamin, calcium D-hexanoate, nicotinamide, folic acid, D-biotin, L-ascorbic acid-2-phosphate, inositol, magnesium, iron, zinc, manganese, copper, cobalt, selenium, and iodine. All raw material components of the feed described in this invention are conventional commercially available products.

[0028] The present invention also provides the application of the above-mentioned Procambarus clarkii feed in Procambarus clarkii farming, which can change the intestinal microecological state of Procambarus clarkii, thereby affecting the amino acid metabolism and host nutrient utilization efficiency of Procambarus clarkii, increasing the content of flavor amino acids in Procambarus clarkii muscle, improving muscle quality, and effectively reducing feed costs and farming costs.

[0029] This invention also provides a method for cultivating *Procambarus clarkii*, comprising the following steps: feeding the *Procambarus clarkii* feed described above. As an optional embodiment, the feed is preferably fed twice daily, at 7:00 AM and 7:00 PM, with a daily feeding amount of 4-6% of the *Procambarus clarkii*'s weight, preferably 5%. The specific feeding amount can be adjusted according to the *Procambarus clarkii*'s feeding behavior and weather conditions.

[0030] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] In a specific embodiment of the present invention, the multivitamin and mineral premix refers to a premix of vitamins and minerals (per kg of feed) comprising the following: Vitamin A acetate, 450,000 IU; Vitamin D, 1,000,000 IU; dl-α-tocopherol acetate, 5 g; menadione, 0.5 g; thiamine nitrate, 0.5 g; riboflavin (vitamin B2), 0.7 g; pyridoxine hydrochloride, 0.6 g; cyanocobalamin, 0.002 g; calcium D-hexanoate, 2 g; nicotinamide, 3.5 g; folic acid, 0.15 g; D-biotin, 0.006 g; L-ascorbic acid-2-phosphate (calculated as L-ascorbic acid), 10 g; inositol, 8 g; magnesium, 20 g; iron, 2 g; zinc, 7.5 g; manganese, 2 g; copper, 1.5 g; cobalt, 0.08 g; selenium, 0.01 g; iodine, 0.1 g.

[0032] In a specific embodiment of the present invention, protocatechuic acid was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0033] Unless otherwise specified, the following embodiments are all conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] Example 1 A feed for *Procambarus clarkii* (by weight percentage): fishmeal 5%, soybean meal 15%, cottonseed protein 6%, rapeseed meal 13%, peanut meal 10%, corn DDGS 5%, wheat flour 28%, sodium alginate 2%, calcium dihydrogen phosphate 2.3%, spray-dried yeast powder 2%, soybean oil 2%, fish oil 1.5%, bentonite 3.4%, soybean lecithin oil 1.5%, cholesterol 0.45%, multivitamins and minerals 1%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.25%, chitosan 0.1%, L-Thr 0.35%, L-Lys 0.3%, Met 0.1%, protocatechuic acid 0.3%. This is designated PA3.

[0036] Preparation method: Crush all feed ingredients, pass them through an 80-mesh sieve, add them one by one in order of increasing formula ratio and mix them evenly. Then use a feed machine to make them into pellets with a diameter of 2mm, dry them at 60℃, seal them, and store them in a -20℃ refrigerator for later use.

[0037] Example 2 A feed for *Procambarus clarkii* (by weight percentage): fishmeal 5%, soybean meal 15%, cottonseed protein 6%, rapeseed meal 13%, peanut meal 10%, corn DDGS 5%, wheat flour 28%, sodium alginate 2%, calcium dihydrogen phosphate 2.3%, spray-dried yeast powder 2%, soybean oil 2%, fish oil 1.5%, bentonite 3.1%, soybean lecithin oil 1.5%, cholesterol 0.45%, multivitamins and minerals 1%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.25%, chitosan 0.1%, L-Thr 0.35%, L-Lys 0.3%, Met 0.1%, protocatechuic acid 0.6%. This is designated PA6.

[0038] Preparation method: Same as in Example 1.

[0039] Example 3 A feed for *Procambarus clarkii* (by weight percentage): fishmeal 5%, soybean meal 15%, cottonseed protein 6%, rapeseed meal 13%, peanut meal 10%, corn DDGS 5%, wheat flour 28%, sodium alginate 2%, calcium dihydrogen phosphate 2.3%, spray-dried yeast powder 2%, soybean oil 2%, fish oil 1.5%, bentonite 2.8%, soybean lecithin oil 1.5%, cholesterol 0.45%, multivitamins and minerals 1%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.25%, chitosan 0.1%, L-Thr 0.35%, L-Lys 0.3%, Met 0.1%, protocatechuic acid 0.9%. This is designated PA9.

[0040] Preparation method: Same as in Example 1.

[0041] Example 4 A feed for *Procambarus clarkii* (by weight percentage): fishmeal 5%, soybean meal 15%, cottonseed protein 6%, rapeseed meal 13%, peanut meal 10%, corn DDGS 5%, wheat flour 28%, sodium alginate 2%, calcium dihydrogen phosphate 2.3%, spray-dried yeast powder 2%, soybean oil 2%, fish oil 1.5%, bentonite 2.5%, soybean lecithin oil 1.5%, cholesterol 0.45%, multivitamins and minerals 1%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.25%, chitosan 0.1%, L-Thr 0.35%, L-Lys 0.3%, Met 0.1%, protocatechuic acid 1.2%. Designated PA12.

[0042] Preparation method: Same as in Example 1.

[0043] Example 5 A feed for *Procambarus clarkii* (by weight percentage): fishmeal 5%, soybean meal 15%, cottonseed protein 6%, rapeseed meal 13%, peanut meal 10%, corn DDGS 5%, wheat flour 28%, sodium alginate 2%, calcium dihydrogen phosphate 2.3%, spray-dried yeast powder 2%, soybean oil 2%, fish oil 1.5%, bentonite 2.2%, soybean lecithin oil 1.5%, cholesterol 0.45%, multivitamins and minerals 1%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.25%, chitosan 0.1%, L-Thr 0.35%, L-Lys 0.3%, Met 0.1%, protocatechuic acid 1.5%. This is designated PA15.

[0044] Preparation method: Same as in Example 1.

[0045] Comparative Example 1 A feed for *Procambarus clarkii* (by weight percentage): fishmeal 5%, soybean meal 15%, cottonseed protein 6%, rapeseed meal 13%, peanut meal 10%, corn DDGS 5%, wheat flour 28%, sodium alginate 2%, calcium dihydrogen phosphate 2.3%, spray-dried yeast powder 2%, soybean oil 2%, fish oil 1.5%, bentonite 3.7%, soybean lecithin oil 1.5%, cholesterol 0.45%, multivitamins and minerals 1%, sodium chloride 0.2%, choline chloride 0.25%, vitamin C 0.25%, chitosan 0.1%, L-Thr 0.35%, L-Lys 0.3%, Met 0.1%, protocatechuic acid 0%. This is denoted as PA0.

[0046] Preparation method: Same as in Example 1.

[0047] Experimental Example 1 Aquaculture trials were conducted using the feed formulations from Examples 1 to 5 and Comparative Example 1, respectively.

[0048] The crude protein and crude fat content of each feed were tested, and the results are shown in the table below.

[0049] Table 1. Nutritional levels (%) of each feed group

[0050] The results showed that the crude protein and crude fat contents of the feeds in each group were similar, belonging to isonitrogenous and isolithic feeds.

[0051] Red swamp crayfish were fed different diets for 6 weeks, twice daily at 7:00 AM and 7:00 PM, with a daily feed amount of 5% of the crayfish's weight. Each group had three replicates, with 30 crayfish per replicate. Initial weight, final weight, survival rate, weight gain, specific growth rate, and feed conversion ratio were recorded for each group. After 6 weeks of rearing, the crayfish were fasted for 24 hours. Muscle tissue was collected for amino acid composition analysis, and intestinal tissue was collected for gut microbiota structure analysis.

[0052] The initial weight, final weight, survival rate, weight gain rate, specific growth rate, and feed conversion ratio of each group of red swamp crayfish were statistically analyzed, and the results are shown in the table below.

[0053] Table 2 Growth performance of each group

[0054] The results showed that, compared with the control group, Example 1 (PA3 group), Example 2 (PA6 group), and Example 3 (PA9 group) could significantly increase the final weight of Procambarus clarkii, Example 2 (PA6 group) and Example 3 (PA9 group) could significantly increase the weight gain rate and specific growth rate of Procambarus clarkii, and Example 1 (PA3 group), Example 2 (PA6 group), and Example 3 (PA9 group) could significantly increase the feed conversion ratio.

[0055] The effect of protocatechuic acid on the amino acid composition of the muscle of Procambarus clarkii is shown in the table below.

[0056] Table 3. Amino acid composition results of each group of feeds

[0057] Note: # represents umami amino acids, and * represents sweet amino acids.

[0058] The effect of protocatechuic acid on the content of single umami amino acids in the muscle of Procambarus clarkii, such as Figure 1 As shown, the effect of protocatechuic acid on the content of single sweet amino acids in the muscle of Procambarus clarkii is as follows: Figure 2 As shown, the effect of protocatechuic acid on the total amount of flavor amino acids in the muscle of Procambarus clarkii is as follows: Figure 3 As shown, the fitting curves of the total amount of flavor amino acids and the amount of protocatechuic acid added are as follows: Figure 4As shown in the figure. The results showed that adding protocatechuic acid to the feed could effectively increase the content of flavor amino acids in the muscle of Procambarus clarkii, thereby improving muscle quality. Specifically, compared with the control group, the contents of glycine, tyrosine, phenylalanine, aspartic acid, and glutamic acid, as well as the total amount of umami amino acids, sweet amino acids, and flavor amino acids in the muscle of Procambarus clarkii in each protocatechuic acid-added group were significantly increased. Using the total amount of flavor amino acids as the criterion, the optimal addition amount of protocatechuic acid in the feed was determined to be 9 g / kg.

[0059] The gut microbiota of *Procambarus clarkii* from Example 3 (PA9) and the control group (Comparative Example 1, PA0) were compared and analyzed. The α-diversity of gut microbiota is shown in the table below, and the β-diversity of gut microbiota is shown in the table below. Figure 5 As shown.

[0060] Table 4. α-diversity of gut microbiota in Procambarus clarkii

[0061] The results showed that the α diversity of the intestinal flora of Procambarus clarkii was significantly increased in the protocatechuic acid-added group, as reflected in the fact that the values ​​of Sobs, Chao1, ACE and Pd in ​​the PA9 group were significantly higher than those in the control group (PA0 group); the β diversity of the intestinal flora of Procambarus clarkii was significantly increased in the protocatechuic acid-added group.

[0062] The effect of protocatechuic acid on the relative abundance of gut microbiota families in Procambarus clarkii, such as Figure 6 As shown in the figure, (A) represents the relative abundance at the family level, and (B) represents the comparison of the relative abundance of Vibrionaceae, Rhodobacteraceae, Lactobacillaceae, and Bacillaceae. The results showed that, compared with the control group (Comparative Example 1), the relative abundance of Vibrionaceae was significantly decreased, while the relative abundance of Rhodobacteraceae, Lactobacillaceae, and Bacillaceae was significantly increased at the family level in the gut microbiota of *Procambarus clarkii* in the protocatechuic acid-added group.

[0063] The correlation between flavor amino acids and gut microbiota levels, such as Figure 7 As shown in the figure. Correlation analysis showed that the content of flavor amino acids in the muscle of Procambarus clarkii was significantly correlated with changes in the abundance of Vibrionaceae, Rhodobacteraceae, and Bacillaceae at the gut microbiota level, suggesting that protocatechuic acid significantly improved the flavor amino acid composition of Procambarus clarkii muscle by regulating the abundance of gut microbiota.

[0064] The effect of protocatechuic acid on the relative abundance of gut microbiota in Procambarus clarkii, such as Figure 8As shown in the figure, (A) represents the relative abundance at the genus level, and (B) represents... Vibrio , Halocynthiibacter and Ruegeria The relative abundance results were compared. The results showed that, compared with the control group (Comparative Example 1), the level of gut microbiota in the protocatechuic acid-added group of *Procambarus clarkii* was significantly higher. Vibrio The relative abundance decreased significantly. Halocynthiibacter and Ruegeria The relative abundance increased significantly.

[0065] The correlation between flavor amino acids and gut microbiota levels, such as Figure 9 As shown in the figure. Correlation analysis indicates that the content of flavor amino acids in the muscle of *Procambarus clarkii* is related to the level of gut microbiota. Vibrio , Halocynthiibacter and Ruegeria The changes in abundance were significantly correlated, suggesting that protocatechuic acid significantly improved the amino acid composition of the muscle flavor of Protoctomyces clarkii by regulating the abundance of gut microbiota.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of protocatechuic acid in the preparation of products that regulate the intestinal flora of Procambarus clarkii.

2. Use according to claim 1, characterized in that, Protocatechuic acid reduces the abundance of Vibrionaceae in the intestine of Procambarus clarkii, while increasing the abundance of Rhodobacteraceae, Lactobacillaceae, and Bacillaceae in the intestine of Procambarus clarkii.

3. The application according to claim 1, characterized in that, Protocatechuic acid reduces the abundance of Vibrio in the gut of Procambarus clarkii and increases the abundance of Halocynthiibacter and Ruegeria in the gut of Procambarus clarkii.

4. Application of protocatechuic acid in the preparation of products that increase the content of flavor amino acids in the muscle of Procambarus clarkii.

5. The application according to claim 4, characterized in that, The flavor amino acids include glycine, alanine, tyrosine, phenylalanine, aspartic acid, and glutamic acid.

6. A feed for the red swamp crayfish, characterized in that, The feed contains 3-15 g / kg of protocatechuic acid by feed weight.

7. The Procambarus clarkii feed according to claim 6, characterized in that, The feed consists of the following components: fish meal, soybean meal, cottonseed protein, rapeseed meal, peanut meal, corn DDGS, flour, sodium alginate, calcium dihydrogen phosphate, spray-dried yeast powder, soybean oil, fish oil, bentonite, soybean lecithin oil, cholesterol, multivitamins and minerals, sodium chloride, choline chloride, vitamin C, chitosan, L-Thr, L-Lys, Met and protocatechuic acid.

8. The Procambarus clarkii feed according to claim 7, characterized in that, By weight, the feed comprises the following components: 4-6 parts fishmeal, 12-18 parts soybean meal, 5-7 parts cottonseed protein, 12-15 parts rapeseed meal, 8-12 parts peanut meal, 4-6 parts corn DDGS, 25-30 parts wheat flour, 1-3 parts sodium alginate, 2-3 parts calcium dihydrogen phosphate, 1-3 parts spray-dried yeast powder, 1-3 parts soybean oil, 1-2 parts fish oil, 2-5 parts bentonite, 1-2 parts soybean lecithin oil, 0.2-0.5 parts cholesterol, 0.5-1.5 parts multivitamins and minerals, 0.1-0.3 parts sodium chloride, 0.2-0.3 parts choline chloride, 0.2-0.3 parts vitamin C, 0.05-0.15 parts chitosan, 0.2-0.5 parts L-Thr, 0.2-0.5 parts L-Lys, and 0.5 parts Met. 0.05~0.15 parts and protocatechuic acid 0.3~1.5 parts.

9. The application of the Procambarus clarkii feed according to any one of claims 6 to 8 in the cultivation of Procambarus clarkii.

10. A method for farming Procambarus clarkii, characterized in that, Includes the following steps: Feeding the red swamp crayfish with the feed described in any one of claims 6 to 8.

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