Feed additive for improving availability of micropterus salmoides to plant protein source

Abstract

The invention relates to the technical field of low-carbon aquaculture, in particular to a feed additive for improving the availability of micropterus salmoides to a plant protein source. The additive comprises the following components in parts by mass: 5-10 parts of butyric acid, 10-20 parts of lactic acid, 10-20 parts of citric acid, 1-2 parts of chenodeoxycholic acid, 3-6 parts of taurocholic acid and 46-50 parts of a filling agent. The additive can improve the utilization performance of the micropterus salmoides on the plant protein source in the daily ration and reduce the feed carbon footprint of unit aquatic products, and the carbon footprint is improved along with the increase of the dosage of the plant protein source.

Description

Technical Field

[0001] The invention belongs to the technical field of low-carbon aquaculture, and in particular relates to a feed additive for improving the utilization of plant protein sources (PPS) by largemouth bass. Background Art

[0002] In recent years, the aquaculture of largemouth bass has grown rapidly. This fish, lacking intermuscular spines, is considered an alternative to traditional carp species such as crucian carp. Largemouth bass are typically carnivorous, and traditionally farmed primarily using frozen fish. In recent years, the use of formulated feeds has shifted, with a consequent decline in growth rate and an increase in disease. Therefore, it is necessary to optimize their formulated feeds.

[0003] Due to the limited availability and high cost of fish meal, PPS is widely used as a substitute for fish meal in fish feed formulation. PPS has a low carbon footprint, making its use in place of animal protein sources like fish meal an important development direction for low-carbon feeds. PPS is known to be rich in dietary fiber (DFs). DFs are not digestible and absorbable, and they also affect the digestibility and absorption of other nutrients, significantly limiting feed efficiency and increasing the carbon footprint of each unit of fish feed. DFs also have the ability to bind and shield bile acids, which is the mechanism by which DFs exert their cholesterol-lowering effects. However, bile acids not only promote fat digestion and absorption but are also ligands for the farnesoid X receptor (FXR). When DFs are consumed in large quantities, some bile acids in the intestine are blocked by DFs, thereby inhibiting FXR activity. FXR has a negative feedback regulatory function on bile acid synthesis and is involved in regulating bile acid circulation and inflammatory responses. FXR inhibition leads to excessive bile acid synthesis and cholestasis, resulting in a dramatic increase in serum bile acid levels (up to 70-fold). Bile acids are cytotoxic, causing tissue damage and disease, increasing risks in aquaculture. Therefore, inhibiting the interference of DFs on bile acid homeostasis is an important approach to improve the utilization of PPS.

[0004] DFs are fermentable and thus affect the homeostasis of the intestinal flora, which in turn determines the size and composition of the bile acid pool and directly affects the inflammatory response through its metabolites.

[0005] Numerous studies have examined the limiting factors for replacing fish meal with PPS in largemouth bass diets, but few have examined the potential interference of DFs contained in PPS with bile acid and intestinal bacterial homeostasis. This present invention, building on this understanding, provides a feed additive that works by weakening the effects of DFs on bile acid shielding, inhibiting the fermentation of DFs by intestinal flora and its impact on the balance of fermentation products, thereby improving largemouth bass's ability to utilize PPS. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a feed additive that improves the utilization of plant protein sources by largemouth bass, wherein the feed additive has the effects of promoting growth, reducing feed conversion ratio and carbon footprint of feed per ton of fish products, and improving the health of largemouth bass.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a feed additive for improving the utilization of plant protein sources by largemouth bass, which is prepared from the following components in parts by weight:

[0009] 5-10 parts of butyric acid, 10-20 parts of lactic acid, 10-20 parts of citric acid, 1-2 parts of chenodeoxycholic acid, 3-6 parts of taurocholic acid, and 46-50 parts of filler.

[0010] Preferably, the butyric acid, lactic acid, citric acid, chenodeoxycholic acid and taurocholic acid are all salts, more preferably soluble salts, and more preferably sodium salts, calcium salts or potassium salts.

[0011] Preferably, the filler is one or more of corn flour, defatted rice sugar, zeolite powder, and bentonite.

[0012] Preferably, the added amount of the feed additive is 0.5% to 1.5%.

[0013] Preferably, the higher the amount of the plant protein source in the largemouth bass formula feed, the higher the amount of the feed additive.

[0014] Preferably, when the plant protein source in the largemouth bass feed is cottonseed meal or a plant protein source that has been fermented, the added amount of the feed additive is 0.5%.

[0015] Contains at least the following beneficial technical effects:

[0016] 1. Alleviate the damage of PPS to the health of largemouth bass

[0017] The present invention is based on the mechanism by which PPS induces enteritis and fatty liver: (1) By adding taurocholic acid to the feed, the binding shielding effect of DFs in PPS on bile acids is offset; the bile salts released after taurocholic acid decoupling have a strong activating effect on FXR; and taurine can alleviate the damage induced by PPS; (2) by adding chenodeoxycholic acid, FXR is activated, improving FXR's ability to regulate bile acid homeostasis and inflammatory response; (3) by adding short-chain fatty acids, DFs' fermentation in the intestine is inhibited (possibly also improving the balance of short-chain fatty acids) and its interference with intestinal flora homeostasis. The above measures rescue the interference of DFs on bile acid homeostasis and ultimately alleviate PPS-induced liver and intestinal damage.

[0018] 2. Improve the utilization of PPS by largemouth bass

[0019] DFs are indigestible and indigestible, and they also hinder the absorption of other nutrients. The additive described herein contains taurocholic acid and chenodeoxycholic acid, both of which emulsify fat and promote its absorption and utilization. Short-chain fatty acids nourish the intestines and improve their absorption function. This improves the largemouth bass's ability to utilize nutrients in the feed, thereby promoting growth.

[0020] 3. Reduce the carbon footprint of each ton of fish feed

[0021] The additive of the present invention significantly reduces the feed coefficient, thereby significantly reducing the feed carbon footprint per ton of fish products. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a linear regression analysis of the effects of different levels of PPS on the weight gain rate (WGR) and feed conversion ratio (FCR) of largemouth bass in Example 1, where A is the weight gain rate and B is the feed conversion ratio;

[0023] Figure 2 The bile acid profiles in the bile of largemouth bass of each group in Example 1 are shown in FIG. 1 , wherein A represents the bile acid content, B represents the percentage of bile acid, and different English letters in the same series indicate significant differences (P<0.05).

[0024] Figure 3 The differences in intestinal flora structure between the 20% PPS group and the 58.2% PPS group in Example 1; wherein A represents the difference in abundance of bacteria involved in bile acid deconjugation, B represents the difference in abundance of bacteria involved in hydroxyl group oxidation and epimerization, C represents the difference in abundance of bacteria involved in bile acid esterification, D represents the difference in abundance of bacteria involved in 7α dehydroxylation, E represents the difference in abundance of bacteria involved in desulfurization, and F represents the difference in abundance of bacteria involved in bile acid hydrolase secretion; the vertical axis represents "relative abundance";

[0025] Figure 4 The effects of each group of additives in Example 1 on the histology of liver, hindgut and muscle of largemouth bass;

[0026] Figure 5 The improvement of the additive in Example 3 on the utilization of PPS by largemouth bass; where A is weight gain rate, B is feed coefficient, C is liver index, D is liver fat content, E is collagen content, and F is carbon footprint of feed per ton of fish. Different letters above the columns indicate significant differences (P<0.05);

[0027] Figure 6 The figure shows the rescue effect of the additive in Example 3 on PPS-induced bile acid disorder; wherein, A is the bile acid content, B is the percentage of bile acid, and different English letters in the same series indicate significant differences (P<0.05). DETAILED DESCRIPTION

[0028] Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative work shall fall within the scope of protection of the present invention. The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0029] Example 1

[0030] Effects of plant protein source (PPS) on growth, health, and bile acid homeostasis in largemouth bass

[0031] In this example, soybean meal, rapeseed meal, and cottonseed meal were mixed in a ratio of 4:3:3. PPS was added to the experimental diets at 20%, 30%, 40%, 50%, and 58.2%, respectively. The amount of fish meal added was reduced (see Table 1). Three tanks of fish were fed each diet twice daily at apparent satiation, and samples were analyzed after 56 days.

[0032] Table 1 Experimental diet formula and nutrient levels (air-dry basis, g / kg)

[0033]

[0034]

[0035] Note: Multivitamin premix contains (mg / kg): Iron 10,000, Copper 250, Zinc 800, Manganese 1200, Selenium 10, Vitamin E 5280, Vitamin K 3400, Vitamin B1 1000, Vitamin B2 1160, Vitamin B6 600, Nicotinamide 3000, Pantothenic Acid 2500; Vitamin A 445 KIU, Vitamin D3 80 KIU.

[0036] The experimental results showed that as the PPS level in the diet increased, the growth performance of largemouth bass decreased and the FCR increased (P < 0.05, see Figure 1 ), suggesting that the amount of PPS added to largemouth bass feed should be controlled below 20%.

[0037] Table 2 shows that PPS significantly affects bile acid levels in tissues. Compared with the 20% PSS group, total bile acid (TBA) levels in bile initially increased and then decreased significantly. TBA levels in the intestine decreased significantly, while TBA in the liver initially decreased significantly and then increased. TBA levels in whole fish initially increased and then decreased. Previous studies have shown that bile acid concentrations slightly above physiological concentrations can induce inflammatory responses. These results suggest that PPS may cause tissue damage by interfering with bile acid homeostasis.

[0038] Table 2 Effects of different levels of plant protein sources on total bile acid (TBA) concentrations in serum, bile, liver, intestine, and whole fish of largemouth bass

[0039]

[0040]

[0041] The present invention also detects the content of various bile acids in the bile of largemouth bass. Figure 2 It can be seen that taurocholic acid (TCA) has the highest content in bile, accounting for more than 70%, followed by taurochenodeoxycholic acid (TCDCA). As PPS increases, the content of various bile acids in bile generally decreases (see Figure 2 A), but the proportion of TCDCA did not decrease (see Figure 2 B).

[0042] FXR has the function of inhibiting bile acid synthesis and inflammatory response, and CDCA and CA are its main activators. The content of CDCA in bile is very low, and the content of CA decreases with the increase of PPS (see Figure 2 B), suggesting that the activity of FXR is affected.

[0043] The present invention also analyzed the differences in the intestinal flora structure between the 20% PPS group and the 58.2% PPS group. Figure 3 It can be seen that the relative abundance of bacteria related to bile acid deconjugation, hydroxyl group oxidation and epimerization, 7α dehydroxylation, bile esterification, desulfurization and regulation of bile salt hydrolase activity increased in the 58.2% PPS group, suggesting that the biotransformation activity of intestinal flora on bile acids was enhanced. Figure 4 As shown in the data, as the PPS level increased, liver fibrosis worsened, and 58.2% of PPS also caused intestinal tissue damage.

[0044] It can be seen from this that as the PPS content in the diet increases, the growth performance of largemouth bass decreases and liver fibrosis worsens. The lower the PPS content in the diet, the better, and the appropriate addition content is less than 20%.

[0045] PPS induces bile acid metabolism disturbances, which may be a key contributor to PPS-induced tissue damage. Regarding bile acid composition, TCA and CA levels decreased in bile after PPS exposure, while TCDCA levels increased. This suggests that alternative pathways for bile acid synthesis may be upregulated in largemouth bass under these experimental conditions. Furthermore, active bile acid metabolism by the intestinal microbiota may exacerbate bile acid homeostasis disturbances. These results suggest that improving bile acid metabolic homeostasis may be an important pathway for improving PPS utilization in largemouth bass.

[0046] Example 2

[0047] Effects of bile acid and short-chain fatty acids supplemented in high-meal diets on growth and liver collagen content in largemouth bass

[0048] According to the experimental study of Example 1, the present invention attempts to add sodium taurocholate (TCA) to the feed to improve the utilization of PPS by largemouth bass, on the one hand to compensate for the decrease in TCA content caused by dietary PPS (see Figure 2 ), on the other hand, TCA will decouple into CA and taurine. Many studies have shown that adding taurine to PPS feed can improve its utilization, and CA has a strong activation effect on FXR. The present invention has found in other studies that PPS can cause a decrease in FXR activity in the intestine, and FXR has the function of regulating bile acid homeostasis and inflammatory response.

[0049] The present invention also adds sodium chenodeoxycholate (CDCA) to the feed, because CDCA is the most efficient FXR activator among endogenous bile acids.

[0050] In previous studies, the present invention was the first to discover that fatty degeneration of fish liver is not necessarily due to excessive supply of dietary fat. On the contrary, high levels of dietary fat can alleviate fatty liver under specific conditions. The present invention also found that enhanced liver bile acid synthesis or increased tissue levels are not due to excessive supply of exogenous bile acid. On the contrary, supplementing bile acid in the diet can inhibit bile acid synthesis and reduce tissue bile acid levels. Therefore, although PPS causes an increase in TCDCA, the present invention still attempts to add CDCA to the feed. For the same reason, the present invention also adds short-chain fatty acids (formic acid, acetic acid, propionic acid, butyric acid, lactic acid and citric acid, all sodium salts) produced by dietary fiber fermentation to the feed, aiming to inhibit the fermentation of DFs by intestinal flora and explore its potential improvement effect on the utilization of PPS by largemouth bass.

[0051] The present invention adopts an orthogonal design, and the addition levels of various additives are shown in Table 3. The basic feed formula is the 40% PPS group in Table 1. Largemouth bass with an average tail weight of 6.2 g were raised for 8 weeks with 2 replicates.

[0052] Table 3 Addition levels of eight additives (%) and their effects on weight gain, liver fat and collagen content of largemouth bass (%)

[0053]

[0054]

[0055] As shown in Table 3, formic, acetic, and propionic acids had a smaller effect on improving PPS utilization in largemouth bass, while butyric, lactic, citric, CDCA, and TCA had a greater effect. Treatments 1, 8, and 12 showed higher weight gain rates, lower liver fat content in treatments 1, 8, and 11, and lower liver collagen content in treatments 1, 8, and 10. In summary, treatments 1 and 8 significantly improved PPS utilization, promoting growth and alleviating liver damage.

[0056] Example 3

[0057] Effects of optimized additives on the improvement of PPS utilization by largemouth bass and the carbon footprint of feed per ton of fish products

[0058] Example 2 shows that formic acid, acetic acid, and propionic acid have a smaller effect on improving PPS utilization in largemouth bass, while butyric acid, lactic acid, citric acid, CDCA, and TCA have a greater effect. Based on the results of Example 2 (Treatment 8), this experiment designed an additive formulation to verify its effectiveness and explore its dosage. The additive formulation is shown in Table 4.

[0059] Table 4 Additive formula (%)

[0060]

[0061] The crude protein contents of fish meal, chicken meal, yeast extract, soybean meal, and cottonseed protein used in the experimental feed formula were 67%, 58%, 35%, 43%, and 60%, respectively. The designed protein content of the control feed was 43.92%, while the experimental feeds were designed to contain 44.25%. The feed formula and measured protein levels are shown in Table 5.

[0062] Table 5 Basic feed formula and its carbon footprint

[0063]

[0064]

[0065] The above-prepared diet was used to feed largemouth bass with an average initial weight of 4.4 g for 12 weeks. Figures 5-6 As shown in the figure, the higher the PPS level in the feed, the lower the largemouth bass weight gain rate and the higher the feed conversion rate. These additives can improve growth performance and reduce the feed conversion rate. Based on the weight gain rate and feed conversion rate, the higher the PPS content, the higher the addition level should be, with an optimal addition level of less than 1.0%.

[0066] There were no significant differences in liver index or liver fat content among the groups. Hepatic collagen content increased with increasing PPS levels, with both the PPS1 and PPS2 groups showing higher collagen content than the control group (P < 0.05). This further demonstrates that high levels of PPS cause liver damage in largemouth bass, while the additive reduces collagen content, suggesting that liver damage is alleviated. Based on collagen content, the optimal addition level is 1.5%.

[0067] Because the additive reduces FCR, the carbon footprint per ton of fish feed also decreases significantly. The higher the PPS content, the higher the addition level should be, with an optimal addition level of less than 1.0%.

[0068] From the bile acid spectrum, the additive rescued the interference of PPS on bile acid homeostasis, and the appropriate addition amount was 1%.

[0069] In conclusion, the optimal additive can improve the utilization of PPS by largemouth bass. The higher the PPS content in the diet, the higher the addition amount should be. The addition range is 0.5% to 1.5%. In general, it does not need to exceed 1%.

[0070] One of the mechanisms of action of this additive is to counteract the binding and shielding effect of DFs in PPS on bile acids, thereby inhibiting the disruption of intestinal flora homeostasis caused by DFs fermentation. Therefore, if the PPS used in feed is primarily non-fermentable PPS, such as cottonseed meal or fermented PPS, the recommended addition level should be controlled at around 0.5%.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A feed additive for improving the utilization of plant protein sources by largemouth bass, characterized in that: It is prepared from the following components in parts by weight: 5-10 parts of butyric acid, 10-20 parts of lactic acid, 10-20 parts of citric acid, 1-2 parts of chenodeoxycholic acid, 3-6 parts of taurocholic acid, and 46-50 parts of filler.

2. The feed additive according to claim 1, characterized in that The butyric acid, lactic acid, citric acid, chenodeoxycholic acid and taurocholic acid are all salts.

3. The feed additive according to claim 1, characterized in that The filler is one or more of corn flour, defatted rice sugar, zeolite powder and bentonite.

4. The feed additive according to claim 1, characterized in that The added amount of the feed additive is 0.5% to 1.5%.

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