CN110558256B

CN110558256B - Nutrition method for regulating and controlling bile acid secretion of takifugu rubripes in programmed mode

Info

Publication number: CN110558256B
Application number: CN201910951631.4A
Authority: CN
Inventors: 徐后国; 梁萌青; 廖章斌; 卫育良
Original assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Current assignee: WEIFANG KENENG BIOTECHNOLOGY Co.,Ltd.
Priority date: 2019-10-09
Filing date: 2019-10-09
Publication date: 2020-06-26
Anticipated expiration: 2039-10-09
Also published as: CN110558256A

Abstract

A nutrition method for regulating and controlling bile acid secretion of takifugu rubripes in a programmed mode belongs to the field of aquatic product nutrition, and aims at feeds with different fat contents, the bile acid secretion of takifugu rubripes is regulated and controlled in a programmed mode by changing the composition and proportion of bile acid metabolism regulation and control functional substances in the feeds. The method can realize programmed regulation and control of the bile acid secretion of the takifugu rubripes under different nutritional conditions, promote bile secretion, improve the digestion and utilization of fat, prevent excessive accumulation of fat in the liver, maintain proper liver-body ratio and improve growth performance; the technology is based on the blending of functional nutrients in the takifugu rubripes feed, and has strong operability; and the cost is in a controllable range, and the economy is high.

Description

Nutrition method for regulating and controlling bile acid secretion of takifugu rubripes in programmed mode

Technical Field

The invention belongs to the field of aquatic product nutrition, and particularly relates to a nutriology method for regulating and controlling bile acid secretion of takifugurbripes.

Background

The takifugu rubripes is an important high-end economically cultured fish in China. The fish meat is delicious and plays an important role in the traditional food culture of China. For a long time, the market circulation and consumption of the tetrodotoxin special for puffer fish are strictly controlled. Although China is a big country for culturing puffer fish, products mainly export Japan and Korea in the past. In recent years, the circulation and consumption of puffer fish in China are rapidly increased, and the puffer fish breeding industry is driven to rapidly develop.

Fugu rubripes has a unique fat storage profile, the fish has no abdominal adipose tissue and very low muscle fat content (fresh weight between 0.5-1.0%), so the fish stores almost all fat in the liver. However, since the liver is the central organ of the whole body's metabolism, excessive fat accumulation in the liver of fugu rubripes will inevitably impair the normal physiological functions of the liver. The maintenance of the normal fat metabolism in the liver of the takifugu rubripes has important significance for maintaining the normal fish body physiological metabolism of the takifugu rubripes.

Bile acids are converted from cholesterol in hepatocytes and play an important role in the absorption, transport and metabolism of fats. In addition, bile acid can be used as a signal molecule to activate the expression of nuclear receptors so as to regulate the metabolism of fat and cholesterol, and has important physiological functions. It is emphasized that the development of the regulation of bile acid lipid metabolism in animals is closely related to the size of the animal and the fat content of the feed, and these two factors have important influence on the fat accumulation process and amount of the animals and the tissue distribution of fat, thereby influencing the interaction process of bile acid and lipid substances.

In the takifugu rubripes individuals with abnormal fat metabolism, bile stasis and bile acid metabolism disorder are typical symptoms, so that the regulation of fat metabolism by regulating bile acid has high potential application value. However, in current feed nutrition practices and feed production, means for regulating liver fat metabolism in fish by regulating bile acids are still very lacking. Particularly, in the current aquaculture in China, the use of high-fat feed is a common problem, farmers generally tend to use high-fat and high-energy feed in order to seek high growth speed, and the feeding of the high-fat and high-energy feed to the normal lipid metabolism of many fishes causes great pressure, which causes a series of problems of fatty liver and the like. In the case of fugu rubripes, because they store most of their fat in the liver, the problems caused by high-fat high-energy feeds on fugu rubripes are particularly significant, and are generally manifested by a very large liver-body ratio, a low anti-stress ability and a low disease resistance. Although the aquaculture in China is in the transition period from 'yield oriented' to 'quality oriented', the feeding habit of high-fat high-energy feed is difficult to change in a short time, and the industry urgently needs a method for specifically relieving or solving the problems generated by the feeding mode.

Disclosure of Invention

The invention aims to solve the technical problem of providing a nutriology method for regulating and controlling the secretion of takifugu rubripes bile acid in a programming mode according to fish body size and feed fat content. The intake of the bile acid regulating substance through the nutriology way is closely related to the fish body size and the feed fat content, thereby realizing the programmed, accurate and dynamic regulation of the bile acid secretion of the takifugu rubripes and making up the defects of the prior art in the field.

The invention is realized by the following technical scheme:

a nutriology method for regulating and controlling the secretion of bile acid of takifugu rubripes by programming comprises the following steps:

(1) for juvenile fish of takifugu rubripes with weight of 20-250g, when the fat content of the feed is 8-12% (the fat content is the percentage of dry matter of the feed and is recorded as DL), adding DL/20 taurine, DL/18 cysteine, DL/1800 chenodeoxycholic acid and DL/3600 hyocholic acid into the feed;

(2) for juvenile fish of takifugu rubripes with the weight of 20-250g, when the fat content of the feed is 13-18%, adding DL/15 taurine, DL/12 cysteine, DL/1200 chenodeoxycholic acid and DL/2400 hyocholic acid into the feed;

(3) for juvenile fish of takifugu rubripes with the weight of 250-;

taurine and cysteine are both L-type, and the purity is 99%; hyodeoxycholic acid, chenodeoxycholic acid and hyocholic acid are pure products; "DL/20 taurine" means that the taurine content of the feed is 20 times of the fat content of the feed; the representation method of the content of cysteine, hyodeoxycholic acid, chenodeoxycholic acid and hyocholic acid is the same as that of taurine.

Compared with the prior art, the invention has the beneficial effects that:

(1) the method can realize accurate regulation and control of secretion of the takifugu rubripes bile acid under the condition of different feed fat contents, promote secretion of the bile acid (the improvement range is more than 64 percent and average 119 percent), reduce the liver body ratio (average reduction is 34 percent), maintain the liver body ratio in a reasonable range (accounting for 3-10 percent of the weight), reduce the liver fat content (average reduction is 29 percent), further maintain the steady state of liver fat metabolism, and improve the survival rate of the takifugu rubripes in high temperature and transportation stress (average improvement is 51 percent and 76 percent respectively). (2) The technology is based on the management of functional nutrient substances in the takifugu rubripes feed, and has very strong operability. (3) The cost is in a controllable range, and the economy is high.

Drawings

FIG. 1 shows liver and gallbladder morphology of Fugu rubripes juvenile fish under normal physiological conditions.

Fig. 2, example 1, changes in the basic bile acid levels (total bile acid) of takifugu rubripes serum after applying the method to feed simulating a common commercial formulation. Data are expressed as mean ± sem (n ═ 6); there were significant differences between data columns that did not contain the same letter (P < 0.05).

Fig. 3, example 1, changes in liver ratio of takifugu rubripes after applying the method to feed simulating common commercial formula. Data are expressed as mean ± sem (n ═ 6); there were significant differences between data columns that did not contain the same letter (P < 0.05).

Fig. 4, example 1, changes in liver fat content of takifugu rubripes after applying the method to feed simulating common commercial formula. Data are expressed as mean ± sem (n ═ 6); there were significant differences between data columns that did not contain the same letter (P < 0.05). WW, fresh weight.

Fig. 5, example 1, the change of survival rate of fugu rubripes under high temperature stress after applying the method to feed simulating common commercial formula. Data are expressed as mean ± sem (n ═ 6); there were significant differences between data columns that did not contain the same letter (P < 0.05).

Fig. 6, example 1, change in survival rate of takifugu rubripes under stress of long-term transportation (6 hours) after applying the method to feed simulating common commercial formula. Data are expressed as mean ± sem (n ═ 6); there were significant differences between data columns that did not contain the same letter (P < 0.05).

Fig. 7, example 2, changes in the basic bile acid levels (total bile acid) of takifugu rubripes serum after applying the method to a feed simulating a common commercial formulation. Data are expressed as mean ± sem (n ═ 6); there were significant differences between data columns that did not contain the same letter (P < 0.05).

Fig. 8, example 2, changes in liver ratio of takifugu rubripes after applying the method to feed simulating common commercial formula. Data are expressed as mean ± sem (n ═ 6); there were significant differences between data columns that did not contain the same letter (P < 0.05).

Fig. 9, example 2, changes in liver fat content of takifugu rubripes after applying the method to a feed simulating a common commercial formula. Data are expressed as mean ± sem (n ═ 6); there were significant differences between data columns that did not contain the same letter (P < 0.05). WW, fresh weight.

Fig. 10, example 2, the change of survival rate of fugu rubripes under high temperature stress after applying the method to feed simulating common commercial formula. Data are expressed as mean ± sem (n ═ 6); there were significant differences between data columns that did not contain the same letter (P < 0.05).

Fig. 11, example 2, change in survival rate of fugu rubripes under stress of long-term transport (6 hours) after applying the method to feed simulating common commercial formula. Data are expressed as mean ± sem (n ═ 6); there were significant differences between data columns that did not contain the same letter (P < 0.05).

Detailed Description

The technical features of the present invention are further explained below by way of examples, but the scope of the present invention is not limited in any way by the examples.

Example 1 Effect evaluation test Using the method in juvenile Fugu rubripes having an initial average weight of 35g

1. Experimental design and experimental feed formula (the basic feed formula is a commercial feed formula which is used for simulating common use, is not limited to the protection scope of the invention, and the effect of the invention can be achieved by implementing the dynamic addition scheme of the invention under the condition that the normal growth of the takifugu rubripes can be met)

This example is a method for programming and controlling the secretion of bile acid in takifugu rubripes, which comprises the following steps (as shown in table 1):

TABLE 1 flow sheet of the process of the invention

(3) for juvenile fish of takifugu rubripes with the weight of 250-;

The control group feed is prepared by taking fish meal, yeast, krill meal, millet meal, wheat gluten, bean pulp and other raw materials as main protein sources and taking fish oil and soybean lecithin as main fat sources. In the control group of feeds, the addition amounts of functional substances such as taurine (L type, purity 99%), cysteine (L type, purity 99%), chenodeoxycholic acid (pure amount, the amount of the composite product can be calculated according to the effective components of the composite product), hyodeoxycholic acid (pure amount, the amount of the composite product can be calculated according to the effective components of the composite product) and the like are dynamically adjusted according to the growth stage of fish and the fat content of the feeds as shown in Table 2, and the experimental feeds of the test group applying the method are prepared.

Table 2 feed formulation and coarse content (% dry matter) of experimental feeds

2. Fish and culture management for experiment

The experiment adopts experimental fish with the initial weight of 35g, different feeds are fed in a feeding management process according to physiological characteristics of the fish in a segmented mode, the feed with the protein content of 47.5 percent and the fat content of 9 percent is fed in a stage of 35-120g of average weight, the feed with the protein content of 46.5 percent and the fat content of 15 percent is fed in a stage of 120-250 g of average weight, and the feed with the protein content of 46.5 percent and the fat content of 12 percent is fed in a stage of 250-1000g of average weight. And (3) sampling in sections, evaluating the secretion of bile acid (expressed by serum basal level), liver body ratio and liver fat content of the liver, and evaluating the anti-stress capability of the fish by high-temperature and stress experiments. Before formal test, experimental fish is temporarily cultured in a cement pond (25 square meters) for 7 days to adapt to the culture environment condition. Before the start of the experiment, the fish were randomly distributed into 6 glass fiber reinforced plastic barrels (diameter: 230cm, height: 100cm) and 6 replicates in each group were used. Every barrel of 100 fishes is cultivated by indoor running water, the water flow is 50L/min, and the fishes are fed three times per day after being fed with food. The culture experiment is carried out in the breeding base of flounder and flounder in the research institute of yellow sea aquatic products of Chinese academy of aquatic scienceThe culture period is 280 days. The method is carried out under the natural photoperiod and the environmental temperature (N36 degrees, 41 degrees and E121 degrees, 07 degrees) of the Shandong province Haiyang city in China, and in the experimental process, the water temperature ranges from 18 ℃ to 24 ℃; salinity of 30-31; pH of 7.2-8.5; 6-7 mg L of dissolved oxygen^-1. And (4) cleaning residual feed and excrement after half an hour after the ingestion every day.

3. Sample collection and index analysis

Sampling is respectively carried out at 3 stages of feed conversion, the sampling time is respectively 90 days (average end weight is 125g), 200 days (average end weight is 254g) and 280 days (average end weight is 343g), 5 tails are sampled from each barrel, the basic bile acid level, the liver body ratio and the liver fat content of serum are respectively measured, the bile acid is measured by a Nanjing constructed kit according to a manufacturer method, the unit is mmol/L, the liver body ratio (%) -fresh weight of liver/whole fish weight × 100, and the liver fat content is measured by a fresh liver sample according to an AOAC method by a Soxhlet extraction method.

After sampling, 12 fishes are taken out from each barrel for high-temperature stress experiment in 3 of 6 repetitions of treatment. The fish is placed in a 300L experiment barrel independently, the temperature is raised to 28 ℃ by adopting an electric heating rod, and the survival rate of the fish is counted after one day and night (the stress method is determined by a pre-experiment).

Long distance transport stress experiments were performed on 12 fish per barrel for 3 more replicates out of 6 replicates per treatment. The oxygen bag is placed in the trunk of a bus by adopting a conventional method, transported to a smoke platform from a Qingdao and then transported to a laboratory from a station, and the total transportation time is about 6 hours (the stress method is determined by a pre-experiment). And counting the survival rate after reaching the laboratory.

4. Experimental statistical method

Statistics of experimental data were performed using the T-test analysis method using SPSS 16.0. Data are presented as mean ± sem (n ═ 6). Significant differences were expressed as P < 0.5.

5. Results of the experiment

In the first phase of the experiment, during which the young fish were fed with low-fat feed, the liver and gall bladder of the test fish of the test group using the method showed normal color and morphology (fig. 1). Compared with the control group, the serum bile acid level is increased by 87 percent (figure 2, P is less than 0.5), the liver body ratio is reduced by 36 percent (figure 3, P is less than 0.5), the liver fat content is reduced by 25 percent (figure 4, P is less than 0.5), the survival rate under high-temperature stress is improved by 30 percent (figure 5, P is less than 0.5), and the survival rate under transportation stress is improved by 45 percent (figure 6, P is less than 0.5).

In the second phase of the experiment, namely the phase of feeding high-fat feed to juvenile fish, compared with the control group, the serum bile acid level is increased by 108 percent (figure 2, P is less than 0.5), the liver-to-body ratio is reduced by 34 percent (figure 3, P is less than 0.5), the liver fat content is reduced by 23 percent (figure 4, P is less than 0.5), the survival rate under high-temperature stress is increased by 63 percent (figure 5, P is less than 0.5), and the survival rate under transportation stress is increased by 78 percent (figure 6, P is less than 0.5).

In the third stage of the experiment, namely the stage of feeding the fish with the uniform fat feed, compared with the control group, the method has the advantages that the serum bile acid level is increased by 178 percent (figure 2, P is less than 0.5), the liver body ratio is reduced by 41 percent (figure 3, P is less than 0.5), the liver fat content is reduced by 38 percent (figure 4, P is less than 0.5), the survival rate under high-temperature stress is increased by 91 percent (figure 5, P is less than 0.5), and the survival rate under transportation stress is increased by 122 percent (figure 6, P is less than 0.5). The application effect of the method in the embodiment is better in adult fish.

Example 2 Effect evaluation test of Using the method in Fugu rubripes juvenile Fish having an initial average weight of 35g (different from the feed feeding sequence strategy of "example 1")

(3) for juvenile fish of takifugu rubripes with the weight of 250-;

The control group feed is prepared by taking fish meal, yeast, krill meal, millet meal, wheat gluten, bean pulp and other raw materials as main protein sources and taking fish oil and soybean lecithin as main fat sources. In the control group of feeds, the addition amounts of functional substances such as taurine (L type, purity 99%), cysteine (L type, purity 99%), chenodeoxycholic acid (pure amount, the amount of the composite product can be calculated according to the effective components of the composite product), hyodeoxycholic acid (pure amount, the amount of the composite product can be calculated according to the effective components of the composite product) and the like are dynamically adjusted according to the growth stage of fish and the fat content of the feeds as shown in Table 3, and the experimental feeds of the test group applying the method are prepared.

Table 3 feed formulation and coarse content (% dry matter) of experimental feeds

2. Fish and culture management for experiment

The experiment adopts experimental fish with the initial weight of 35g, different feeds are fed in a feeding management process according to physiological characteristics of the fish in a segmented mode, the feed with the protein content of 46.5 percent and the fat content of 15 percent is fed in a stage of about 35-120g of the average weight, the feed with the protein content of 47.5 percent and the fat content of 9 percent is fed in a stage of about 120-250 g of the average weight, and the feed with the protein content of 46.5 percent and the fat content of 12 percent is fed in a stage of 250-1000g of the average weight. And (3) sampling in sections, evaluating the secretion of bile acid (expressed by serum basal level), liver body ratio and liver fat content of the liver, and evaluating the anti-stress capability of the fish by high-temperature and stress experiments. The experimental procedure and conditions were as in "example 1". The only difference between this example and "example 1" is the order of low fat and high fat feed feeding during the juvenile stage, "example 1" feeding low fat feed first followed by high fat feed (some breeders tend to keep the fish survival higher with low fat feed first and then grow faster with high fat feed); in this embodiment, the fish is fed with the high fat feed first and then the low fat feed in the young fish stage (some farmers tend to feed the high fat feed first to accelerate growth so as to reach a larger size for selling seedlings earlier).

3. Sample collection and index analysis

Sample collection and index analysis as in "example 1" was performed at 3 stages of feed conversion for 90 days (average end weight of 149g), 200 days (average end weight of 263g) and 280 days (average end weight of 361 g). 5 samples were taken from each barrel and serum basic bile acid level, liver body ratio, liver fat content were measured separately. The method for measuring and calculating the index was the same as in "example 1".

After sampling, 12 fishes are taken out from each barrel for high-temperature stress experiment in 3 of 6 repetitions of treatment. Long distance transport stress experiments were performed on 12 fish per barrel for 3 more replicates out of 6 replicates per treatment. The stress test method was the same as in "example 1".

4. Experimental statistical method

The statistics of the experimental data are the same as in "example 1".

5. Results of the experiment

In the first stage of the experiment, i.e. the stage of feeding juvenile fish with high-fat feed, compared with the control group, the group adopting the method has the advantages that the serum bile acid level is increased by 64 percent (figure 7, P is less than 0.5), the liver body ratio is reduced by 29 percent (figure 8, P is less than 0.5), the liver fat content is reduced by 24 percent (figure 9, P is less than 0.5), the survival rate under high-temperature stress is increased by 40 percent (figure 10, P is less than 0.5), and the survival rate under transportation stress is increased by 56 percent (figure 11, P is less than 0.5).

In the second phase of the experiment, namely the phase of feeding juvenile fish with low-fat feed, compared with the control group, the group adopting the method has the advantages that the serum bile acid level is increased by 80 percent (figure 7, P is less than 0.5), the liver-to-body ratio is reduced by 25 percent (figure 8, P is less than 0.5), the liver fat content is reduced by 20 percent (figure 9, P is less than 0.5), the survival rate under high-temperature stress is increased by 38 percent (figure 10, P is less than 0.5), and the survival rate under transportation stress is increased by 67 percent (figure 11, P is less than 0.5).

In the third stage of the experiment, namely the stage of feeding the fish with the uniform fat feed, compared with the control group, the method has the advantages that the serum bile acid level is increased by 192 percent (figure 7, P is less than 0.5), the liver body ratio is reduced by 39 percent (figure 8, P is less than 0.5), the liver fat content is reduced by 40 percent (figure 9, P is less than 0.5), the survival rate under high-temperature stress is improved by 45 percent (figure 10, P is less than 0.5), and the survival rate under transportation stress is improved by 83 percent (figure 11, P is less than 0.5). The same results in this example show that the application effect of the method is better in adult fish.

Example 3 Effect evaluation in production Pilot scale Link under cage culture conditions by applying the method

1. Carrying out the process

In the actual cultivation production process of the takifugu rubripes, 2 net cages of farmers located in the tobacco platform sesame 32600600island are rented, each net cage is 3m in specification and is 400 g in weight, and each net cage is filled with the takifugu rubripes, and the weight is 265 g. One net cage feeds commercial feed with protein content of 50% and fat content of 13%, and the other net cage feeds the commercial feed by crushing, adding the functional components according to the dosage of the table 1, and re-granulating. Feeding for 2 times per day. The breeding experiments continued from month 5 to month 9. During the cultivation, the number of dead fish was recorded daily.

2. Evaluation of Effect

After 4 months of culture experiments, 30 samples are randomly sampled in each net cage, and the basic bile acid content, the liver body ratio and the liver fat content of the serum are calculated according to the method. The results show that the content of serum bile acid in the net cage applying the method is improved by 110 percent compared with the net cage of a control group, the liver body ratio is reduced by 41 percent, and the liver fat content is reduced by 20 percent. In the whole culture process (suffering from a high temperature period in summer and a parasite disease outbreak period at the beginning of 9 months), the survival rate is improved by 30 percent (69 dead animals in the whole culture process of the group by applying the method and 144 dead animals in the whole culture process of the control group).

Claims

1. A nutriology method for programmed regulation and control of bile acid secretion of takifugu rubripes is characterized by comprising the following steps:

(1) for juvenile fish of takifugu rubripes with the weight of 20-250g, when the fat content of the feed is 8-12%, adding DL/20 taurine, DL/18 cysteine, DL/1800 chenodeoxycholic acid and DL/3600 hyocholic acid into the feed; the fat content is the percentage of the dry matter of the feed and is marked as DL;

(3) for the juvenile fish of takifugu rubripes with the weight of 250-;

taurine and cysteine are both L-type, and the purity is 99%; hyodeoxycholic acid, chenodeoxycholic acid and hyocholic acid are pure products; DL/20 taurine means that the taurine content of the feed is 20 times of the fat content of the feed; the representation method of the content of cysteine, hyodeoxycholic acid, chenodeoxycholic acid and hyocholic acid is the same as that of taurine.