CN115886152A - Novel eucommia ulmoides fermentation liquor and application thereof in large yellow croaker culture - Google Patents

Abstract

The invention discloses a novel eucommia fermented liquid, which is obtained by inoculating activated Bacillus subtilis into a culture medium containing eucommia leaf powder and fermenting after expanding culture, and the culture medium containing eucommia leaf powder mainly includes eucommia ulmoides Composed of leaf powder and water, the mass fraction of eucommia leaf powder is 25‑30%. The invention also discloses the application of the eucommia fermented liquid, especially the application as an additive in preparing large yellow croaker feed. Tests have proved that the novel Eucommia ulmoides fermented liquid provided by the invention can not only significantly promote the growth of large yellow croaker, but also significantly improve the body color and muscle quality of large yellow croaker.

Description

New Eucommia fermented liquid and its application in large yellow croaker culture

technical field

The invention relates to an aquaculture additive and its application, in particular to a novel eucommia fermented liquid. At the same time, the invention also relates to the application of Eucommia ulmoides fermented liquid in large yellow croaker cultivation.

Background technique

Large yellow croaker (Larimichthys crocea) belongs to the order Perciformes, Perciformes, and the genus Larimichthys. Because of its delicious meat and high nutritional value, it is favored by consumers. After breaking through the technical bottleneck of seed cultivation, the large yellow croaker industry has developed rapidly, and the aquaculture output has increased year by year. In 2021, it has exceeded 240,000 tons, ranking first in marine fish aquaculture output. Economic Value.

However, with the rapid development of the large yellow croaker farming industry, problems such as insufficient growth-promoting performance of feed and the decline in the quality of farmed fish have become increasingly prominent; at the same time, consumers are paying more and more attention to the quality of large yellow croakers. Always in short supply. In order to shorten the breeding cycle and improve the quality of farmed fish, farmers have to use chilled fish for beating and feeding, and even use illegal drugs, which seriously wastes fishery resources and endangers food safety.

Eucommia ulmoides Oliv, also known as wood cotton, Sizhong, and silk cotton wood, is a species of Eucommia ulmoides in the genus Eucommia. It is one of the unique economic forest tree species in my country, the only one in the world. It is native to the mountainous areas of Guizhou, Sichuan, Hunan and other places in my country. After introduction and cultivation, it can grow normally in the northern regions with an average annual temperature of 11°C to 18°C and a minimum temperature of -20°C. Traditionally, the use of Eucommia resources in my country is limited to the development and utilization of Eucommia bark for medical care. Further research has found that the leaves of Eucommia ulmoides with the largest yield contain high levels of alkaloids, polysaccharides, aucubin, flavonoids and chlorogenic acid, etc. Active substances have great potential development and utilization value. A number of studies have shown that Eucommia leaves have significant growth-promoting, anti-inflammatory, antibacterial, obesity-inhibiting and meat-improving effects, but the efficiency of direct addition to feed is low.

Fermentation of Chinese herbal medicine can not only reduce or remove toxic and harmful substances by using microorganisms, but also use enzymes produced by microorganisms to promote the release of active ingredients and increase the content of related active substances. As a by-product of Eucommia ulmoides planting, because it becomes viscous when mixed with water, when the mass fraction exceeds 5%, it is difficult to maintain stirring and ventilation stably and block a variety of pure liquid fermenters. The method of solid-state fermentation after mixing with feed ingredients is less efficient. The liquid state fermentation technology of Chinese herbal medicine can effectively improve the release efficiency of active ingredients, and the fermentation process is more controllable. Therefore, in order to promote the efficient utilization of Eucommia ulmoides leaves and improve the growth and quality of cultured large yellow croaker, it is of great significance to develop a new type of Eucommia ulmoides fermentation liquid by using liquid fermentation technology to promote the healthy development of the industry.

Contents of the invention

One of the objectives of the present invention is to provide a novel Eucommia fermented liquid that can effectively improve the growth and muscle quality of artificially cultured large yellow croaker.

The object of the present invention is achieved through the following technical solutions: the novel eucommia fermented liquid is obtained by inoculating the activated Bacillus subtilis into a culture medium containing eucommia leaf powder and fermenting after expanding the culture, and the culture medium containing eucommia leaf powder The base mainly consists of eucommia leaf powder and water, and the mass fraction of the eucommia leaf powder is 25-30%.

The fermentation conditions of the present invention are preferably: rotating speed 200-300r, ventilation volume 6-8vv, fermentation 30-40h.

As an embodiment of the present invention, in a 100L fermentation tank, according to Eucommia leaf powder 25kg, purified water 45kg, 25L of bacterial liquid was pipetted in a 50L tank, the rotation speed was 250r, and the ventilation rate was 7vv, and fermentation was carried out for 36h.

Activation of Bacillus subtilis: Bacillus subtilis powder was inoculated into the complete medium, shaken at 200r for 6h for activation.

Further, the complete medium used for the activation of the strains contains 6.25g of tryptone, 6.25g of hydrolyzed casein, 10g of beef extract, 5g of dry yeast powder, 2g of diammonium hydrogen citrate, 1.53g of K ₂ HPO ₄ , 5g of NaAc, MgSO ₄ · 7H ₂ O 0.58g, MnSO ₄ 0.19g, Tween-80 1ml, distilled water 1L, pH 6.5-7.0, sterilized by high pressure steam.

Expanded cultivation of Bacillus subtilis: Inoculate 1L of the cultured bacterial solution into a 50L fermenter, and the fermenter medium is 25L. The rotation speed is 355r, the temperature is 3730.5°C, the dissolved oxygen is >40%, and the pH is adjusted to 6.5-7.5 with ammonia water. Continuously cultivated for 8h.

Furthermore, the medium preparation method for strain expansion culture is as follows: peptone 10g, corn starch 10g, glucose 10g, molasses mother liquor 10g, yeast extract 20g, Na ₂ HPO ₄ 12H ₂ O 7.5g, (NH ₄ ) ₂ SO ₄ 3.2g, CaCO ₃ 1g, defoamer 0.05g, sterile water to make up 1L; put the above materials except glucose into the fermenter, then steam, sterilize at 120°C for 30min; adjust the pH to 7.430.1 with NaOH; glucose After single digestion at 110°C for 30 minutes, add to the fermenter; the fermentation temperature is 36±1°C; the fermentation pH is 7.030.1, adjusted with ammonia water.

In the present invention, after the fermentation is finished, the eucommia ulmoides fermented liquid is obtained by press-filtering under aseptic conditions for 20-30 min at 80° C. for heat preservation.

Another object of the present invention is to provide a method for preparing the above-mentioned novel Eucommia fermented liquid.

Specifically, it is obtained by inoculating activated Bacillus subtilis into a medium containing Eucommia leaf powder and fermenting after expanding the culture. The medium containing Eucommia leaf powder is mainly composed of Eucommia leaf powder and water. Eucommia leaf The powder mass fraction is 25-30%.

The fermentation conditions of the present invention are preferably: rotating speed 200-300r, ventilation volume 6-8vv, fermentation 30-40h.

As an embodiment of the present invention, in a 100L fermentation tank, according to Eucommia leaf powder 25kg, purified water 45kg, 25L of bacterial liquid was pipetted in a 50L tank, the rotation speed was 250r, and the ventilation rate was 7vv, and fermentation was carried out for 36h.

Activation of Bacillus subtilis: Bacillus subtilis powder was inoculated into the complete medium, shaken at 200r for 6h for activation.

Further, the complete medium used for the activation of the strains contains 6.25g of tryptone, 6.25g of hydrolyzed casein, 10g of beef extract, 5g of dry yeast powder, 2g of diammonium hydrogen citrate, 1.53g of K ₂ HPO ₄ , 5g of NaAc, MgSO ₄ · 7H ₂ O 0.58g, MnSO ₄ 0.19g, Tween-80 1ml, distilled water 1L, pH 6.5-7.0, sterilized by high pressure steam.

Expanded cultivation of Bacillus subtilis: Inoculate 1L of the cultured bacterial solution into a 50L fermenter, and the fermenter medium is 25L. The rotation speed is 355r, the temperature is 3730.5°C, the dissolved oxygen is >40%, and the pH is adjusted to 6.5-7.5 with ammonia water. Continuously cultivated for 8h.

Furthermore, the medium preparation method for strain expansion culture is as follows: peptone 10g, corn starch 10g, glucose 10g, molasses mother liquor 10g, yeast extract 20g, Na ₂ HPO ₄ 12H ₂ O 7.5g, (NH ₄ ) ₂ SO ₄ 3.2g, CaCO ₃ 1g, defoamer 0.05g, sterile water to make up 1L; put the above materials except glucose into the fermenter, then steam, sterilize at 120°C for 30min; adjust the pH to 7.430.1 with NaOH; glucose After single digestion at 110°C for 30 minutes, add to the fermenter; the fermentation temperature is 36±1°C; the fermentation pH is 7.030.1, adjusted with ammonia water.

In the present invention, after the fermentation is finished, the eucommia ulmoides fermented liquid is obtained by press-filtering under aseptic conditions for 20-30 min at 80° C. for heat preservation.

Another object of the present invention is to provide the application of the above-mentioned Eucommia fermented liquid.

Specifically, it relates to the application of the above-mentioned novel Eucommia fermented liquid as an additive in the preparation of large yellow croaker feed.

Another object of the present invention is to provide an aquaculture feed comprising the above-mentioned eucommia fermented liquid.

Specifically, the aquaculture feed includes basic feed, fish oil and the above-mentioned Eucommia fermentation liquid; wherein, the Eucommia fermentation liquid accounts for 4% of the total mass, and fish oil accounts for 5.5-6.5%.

The basic feed includes white fish meal, krill meal, soybean meal, soybean protein concentrate, high-gluten flour, choline chloride, soybean lecithin powder, Vc phosphate, calcium dihydrogen phosphate, vitamin premix, mineral premix materials, food attractants, antifungal agents and antioxidants, etc.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The invention provides a novel Eucommia ulmoides fermented liquid obtained by a liquid fermentation method, and using a liquid medium containing more than 30% solid matter, and the filtrate obtained after the fermentation is completed can be directly applied to feed without the need for concentration and purification.

(2) The novel Eucommia fermented liquid provided by the present invention can not only significantly improve the growth of large yellow croaker and the muscle quality of large yellow croaker, but the addition of novel Eucommia fermented liquid in the feed can significantly reduce the side brightness and yellowness of juvenile large yellow croaker. Significantly improved the redness value of the side, and the red and yellow values of the abdomen showed a rising trend from the test results, which is conducive to improving the economic value of large yellow croaker.

Description of drawings

Figure 1 shows the effect of the new Eucommia fermented liquid on the survival rate of large yellow croaker juveniles, "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P <0.01, "***" means P<0.001.

Figure 2 shows the effect of the new Eucommia ulmoides fermented liquid on the final body weight of large yellow croaker juveniles. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P<0.01, "***" means P<0.001.

Figure 3 shows the effect of the new Eucommia fermented liquid on the weight gain rate of large yellow croaker juveniles. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P<0.01, "***" means P<0.001.

Figure 4 shows the effect of the new Eucommia fermented liquid on the specific growth rate of large yellow croaker juveniles, "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P<0.01, "***" means P<0.001.

Figure 5 shows the effect of the new Eucommia fermented liquid on the body length of large yellow croaker juveniles, "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P <0.01, "***" means P<0.001.

Figure 6 shows the effect of the new Eucommia fermented liquid on the fatness of large yellow croaker juveniles. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P<0.01, "***" means P<0.001.

Figure 7 shows the effect of the new Eucommia fermented liquid on the lateral brightness (L*) of large yellow croaker juveniles, "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, " **" means P<0.01, and "***" means P<0.001.

Figure 8 shows the effect of the new Eucommia ulmoides fermented liquid on the red value (a*) of the juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, and "*" indicates P<0.05, "**" indicates P<0.01, and "***" indicates P<0.001.

Figure 9 shows the effect of the new Eucommia ulmoides fermented liquid on the yellow value (b*) of the juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, and "*" indicates P<0.05, "**" indicates P<0.01, and "***" indicates P<0.001.

Figure 10 shows the effect of the new Eucommia fermented liquid on the abdomen brightness (L*) of juvenile large yellow croaker, "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, "* *" means P<0.01, and "***" means P<0.001.

Figure 11 shows the effect of the new Eucommia ulmoides fermented liquid on the redness value (a*) of the belly of juvenile large yellow croaker, "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, " **" means P<0.01, and "***" means P<0.001.

Figure 12 shows the effect of the new Eucommia ulmoides fermented liquid on the yellow value (b*) of the juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, and "*" indicates P<0.05, "**" indicates P<0.01, and "***" indicates P<0.001.

Figure 13 shows the effect of the new Eucommia fermented liquid on the muscle hardness of juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P <0.01, "***" means P<0.001.

Figure 14 shows the effect of the new Eucommia ulmoides fermented liquid on the muscle viscosity of juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P<0.01, "***" means P<0.001.

Figure 15 shows the effect of the new Eucommia fermented liquid on the muscle elasticity of juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P <0.01, "***" means P<0.001.

Figure 16 shows the effect of the new Eucommia fermented liquid on the muscle cohesion of juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" Indicates P<0.01, "***" indicates P<0.001.

Figure 17 shows the effect of the new Eucommia ulmoides fermented liquid on the muscle cohesion of juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P<0.01, "***" means P<0.001.

Figure 18 shows the effect of the new Eucommia ulmoides fermented liquid on the muscle chewiness of juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P<0.01, "***" means P<0.001.

Figure 19 shows the effect of the new Eucommia ulmoides fermented liquid on the muscle recovery of juvenile large yellow croaker. "*", "**" and "***" all indicate significant differences, "*" indicates P<0.05, and "**" indicates P<0.01, "***" means P<0.001.

Detailed ways

The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments, but this does not constitute any limitation to the present invention.

Unless otherwise specified, the reagents used in the present invention can be purchased from commercial sources.

1. Screening of Eucommia leaf powder fermentation conditions

1.1 Small test experiment

1.1.1 Single factor experiment

Under the conditions of fermentation temperature 36°C, fermentation time 48h, inoculum size 10%, rotation speed 180r and pH value 6.0, the effect of water content on the release content of effective components in Eucommia leaves was studied. The results are shown in Table 1.

Table 1 Effect of water content on the release content of active ingredients in Eucommia leaves

Table 1 Effect of water content on the release content of active ingredients in Eucommia leaves

1.1.2 Orthogonal test

In order to further determine the optimal process parameters for the release of active ingredients in Eucommia leaves by fermentation, the fermentation temperature (A) is selected as 30°C, 33°C, and 36°C; the inoculum size (B) is 2%, 4%, 6%, and 8%. , 10%, 12%; Fermentation time (C) is 24h, 36h, 48h, 72h, 96h as factor and level, carried out 3 factor orthogonal designs, altogether 30 are processed, and the results are shown in Table 2 and Table 3.

Table 2 Orthogonal experiment chlorogenic acid content results

Table 2 Orthogonal experiment chlorogenic acid content results

Table 2 Orthogonal experiment chlorogenic acid content results

Table 3 Orthogonal Experiment All Active Component Content Results

Table 3 Orthogonal Experiment All Active Component Content Results

1.1.3 Optimal combination conditions

From the results of the single factor experiment, it can be seen that when the water content is 70-75%, the difference between the chlorogenic acid release content of the fermented Eucommia leaf powder and the chlorogenic acid release content of the unfermented Eucommia leaf powder is the largest. It shows that under the condition of this moisture content, the increase of chlorogenic acid release content is due to the function of Bacillus. In addition, the reduction of moisture and eucommia polysaccharide content will significantly reduce the cost of spray drying, the most expensive part of actual production.

According to the variance analysis of the orthogonal experiment, the optimal combination is A2B4C4, that is, the fermentation temperature is 33°C, the inoculum size is 8%, the fermentation time is 72h, the rotation speed is 180r, and the pH value is 6.0. Under the condition of the optimal level combination of each factor, the verification test of chlorogenic acid released by fermented Eucommia leaf powder was carried out, and the OD value of chlorogenic acid was measured to be 630, which was higher than any treatment in the orthogonal test, which proved that the test results were credible. This result is also close to the chlorogenic acid release of unfermented Eucommia ulmoides leaf powder with a water content of 90%, indicating that when the content is reduced, it does not rely on the osmotic effect of water on the chlorogenic acid in the cell wall, but depends on the Bacillus The method of enzymatic wall-breaking effect and promoting the synthesis of chlorogenic acid precursors can increase the release of chlorogenic acid, improve the release efficiency and reduce the actual production cost.

Two, the preparation of Eucommia fermented liquid

According to the optimal conditions obtained from the triangular flask experiment: the fermentation temperature is 33°C, the inoculum size is 8%, the fermentation time is 72h, the rotation speed is 180r, and the pH value is 6.0. Combined with the actual conditions of the fermentation equipment in the pilot fermentation, after optimizing the combination Carry out pilot scale production.

2.1 Strain activation

Under aseptic conditions, directly inoculate 10 g of Bacillus subtilis powder into 1 L of complete medium, and vibrate at 200 r for 6 h for activation.

The preparation method of the strain activation medium is as follows: tryptone 6.25g, hydrolyzed casein peptone 6.25g, beef extract 10g, yeast dry powder 5g, diammonium hydrogen citrate 2g, K2HPO4 1.53g, NaAc 5g, MgSO4 7H2O 0.58g, MnSO4 0.19 g. Tween-80 1ml, dilute to 1L distilled water, adjust pH to 6.5-7.0. Autoclaved.

2.2 Expanded culture of fermentation tank strains

Inoculate 1L of the cultured bacterial solution into a 50L fermenter, and the fermenter medium is 25L. The rotation speed is 355r, the temperature is 3730.5°C, the dissolved oxygen is >40%, and the pH is adjusted to 6.5-7.5 with ammonia water. Continuously cultivated for 8h.

The preparation method of the fermentation tank strain expansion medium is as follows: peptone 10g, corn starch 10g, glucose 10g, molasses mother liquor 10g, yeast extract 20g, Na ₂ HPO ₄ 12H ₂ O 7.5g, (NH ₄ ) ₂ SO ₄ 3.2g, CaCO ₃ 1g, antifoaming agent 0.05g, sterile water to make up 1L. Add steam to the fermenter, sterilize at 120°C for 30 minutes; adjust the pH to 7.430.1 with NaOH; adjust.

2.3 Fermentation tank experiment

In a 100L fermentation tank, according to 20kg of eucommia leaf powder, 45kg of purified water, 25L of bacterial liquid was pipetted in a 50L tank, the rotation speed was 250r, and the ventilation rate was 7vv, and fermentation was carried out for 36h. After the fermentation is completed, pasteurization is performed by incubating at 80° C. for 20 to 30 minutes, and pressure filtration is carried out under aseptic conditions to obtain the eucommia fermented liquid.

3. Application of new eucommia fermented liquid

The basic feed formula for juvenile large yellow croaker is: white fish meal 40%, krill meal 3%, peeled soybean meal 10%, high-gluten flour 24.65%, soybean protein concentrate 6%, choline chloride 0.2%, soybean lecithin powder 2 %, Vc phosphate 0.05%, calcium dihydrogen phosphate 2%, vitamin premix 1%, mineral premix 0.5%, food attractant 0.5%, antifungal agent 0.05% and ethoxyquin 0.05%.

The preparation process of juvenile large yellow croaker feed: mix white fish meal, krill meal, peeled soybean meal, high-gluten flour and soybean protein concentrate, and then ultrafinely pulverize it to 120 mesh to obtain mixture 1; mix choline chloride, soybean lecithin Powder, Vc phosphate ester, calcium dihydrogen phosphate, vitamin premix, mineral premix, food attractant, antifungal agent and ethoxyquine are mixed uniformly to obtain mixture 2; mix mixture 1, mixture 2 and fish oil After homogenization, add 10-15% water for granulation.

Comparative example feed 1

The basic feed for juvenile large yellow croaker, the oil source is fish oil, accounting for 6% of the total weight of the feed, and the new eucommia fermentation broth is not added, and 4% microcrystalline cellulose is used instead.

Example Feed 2

Fish oil and new-type Eucommia fermented liquid were added to the basic feed for juvenile large yellow croaker. The added amount of new-type Eucommia fermented liquid was 4% of the total mass of the basic feed for juvenile large yellow croaker, and fish oil accounted for 6% of the total mass of the feed.

Following table 3 is the formula of above-mentioned embodiment and comparative example:

The feed formula of table 1 embodiment and comparative example

Among them, vitamin premix (IU or mg kg ^-1 dry weight): vitamin A palmitate, 3000000IU; vitamin D3, 1200000IU; DL-α-tocopherol 40.0mg kg ^-1 ; menadione, 8.0mg kg ^{- 1} ; Thiamine-HCl, 5.0mg kg- ¹ ; Riboflavin, 5.0mg kg-1; Calcium D-pantothenate, 16.0mg kg-1; Pyridoxine-HCl, 4.0mg kg-1; Inositol, 200.0 mg kg ^-1 ; D-biotin, 8.0mg kg ^-1 ; folic acid, 1.5mg kg ^-1 ; p-aminobenzoic acid, 5.0mg kg ^-1 ; niacin, 20.0mg kg ^-1 ; vitamin B12, 0.01mg kg ^-1 ; Vitamin C (content 35%), 2000.0mg kg ^-1 . Mineral premix (mg kg ^-1 dry weight) Ca(H ₂ PO ₄ )·H ₂ O, 675.0; CoSO ₄ ·H ₂ O, 0.15; CuSO ₄ ·H ₂ O, 5.0; FeSO ₄ ·7H ₂ O, 50.0; KCl, 0.1; MnSO4-2H2O _{, 101.7; MnSO4-2H2O, 18.0; NaCl, 80.0; NaSeO3-H2O , 0.05 ; ZnSO4-7H2O , 20.0 .} The food attractant is prepared by mixing 1% betaine and 3% glycine with fillers (all provided by Qingdao Master Biotechnology Co., Ltd.)

Experimental example Effect of adding a new type of Eucommia fermented liquid on the growth and body color of juvenile large yellow croaker

240 large yellow croakers with an initial body weight of 12.930.02 g were selected as juvenile fish for the experiment, and they were randomly divided into 6 groups, with three repetitions in each group, and 40 fish in each repetition, and a 63-day feeding and growth experiment was carried out; the breeding water temperature was 18-22 ℃.

The group fed with feed 1 was the CON group, and the group fed with feed 2 was the DZ group.

Growth and body shape were directly measured by electronic balance and vernier caliper; muscle texture was measured by texture analyzer.

Statistical and analytical methods:

Survival rate (SR,%)=Wa/Wb×100%

Weight gain (WG,%)=(Na-Nb)/Nb×100%

Fullness (CFR, g/cm ³ %)=Na/(La) ³ ×100%

Specific growth rate (SGR, %/day)=(lnNa-lnNb)/experimental period×100%

Among them, Wa is the number of large yellow croaker juveniles in each tank after breeding, Wb is the initial number of juveniles in each tank; La is the final body length; Na and Nb are the final body weight and initial body weight, respectively.

The experimental data will be statistically analyzed using one-way analysis of variance in SPSS 20.0, and Tukey’s multiple comparisons will be performed after significant differences, and the significance level is P<0.05. The experimental data obtained are represented by mean 3 standard errors (means 3S.E.M., n=3).

1) Growth performance

As shown in Figure 1-4, the addition of the new Eucommia ulmoides fermentation broth in the feed did not affect the survival rate of juvenile large yellow croaker, but significantly increased the final body weight, weight gain rate and specific growth rate of juvenile large yellow croaker. This shows that adding 4% new Eucommia ulmoides fermentation broth to the basic feed can significantly promote the growth of large yellow croaker juveniles without affecting the survival rate and effectively shorten the breeding cycle.

2) body type

As shown in Figure 5-6, the addition of the new Eucommia ulmoides fermentation liquid in the feed did not affect the body length and fatness of large yellow croaker juveniles, and the addition showed a trend of increasing body length and reducing fatness. This indicated that the addition of 4% new Eucommia ulmoides fermented liquid in the basal feed would not lead to abnormal body size and obesity of large yellow croaker juveniles.

3) Body color

As shown in Figure 7-12, although the addition of the new Eucommia fermented liquid in the feed significantly reduced the brightness and yellowness of the sides of juvenile large yellow croakers, it significantly increased the redness of the sides; the redness and yellowness of the abdomen showed an upward trend. This indicated that adding 4% new Eucommia fermented liquid in the basic feed would significantly improve the body color of juvenile large yellow croaker.

4) Muscle texture

As shown in Figure 13-19, the addition of the new Eucommia ulmoides fermented liquid in the feed did not affect the muscle hardness, viscosity, elasticity, cohesiveness, glueiness and recovery of large yellow croaker, but significantly improved the muscle chewiness. This indicated that the addition of 4% new Eucommia fermented liquid in the basal feed would significantly improve the muscle quality of large yellow croaker juveniles.

Claims (10)

1. novel eucommia fermented liquid, it is characterized in that, after the Bacillus subtilis after the activation is enlarged culture, inoculate in the culture medium that contains Eucommia leaf powder and obtain by fermentation, and described culture medium that contains Eucommia leaf powder is mainly made of Eucommia ulmoides leaf powder The leaf powder and water are composed, and the mass fraction of the eucommia leaf powder is 25-30%. 2. according to the described novel eucommia fermented liquid of claim 1, it is characterized in that, described fermentation condition is rotating speed 200-300r, ventilation rate 6-8vv, fermentation 30-40h. 3. according to the described novel Eucommia ulmoides fermented liquid of claim 1, it is characterized in that, the activation of described Bacillus subtilis: Bacillus subtilis bacterium powder is inoculated in the complete culture medium, and 200r shaking culture 6h is activated in 37 ℃ of constant temperature shakers; The complete medium used for the activation of the strains contains tryptone 6.25g, hydrolyzed casein peptone 6.25g, beef extract 10g, yeast dry powder 5g, diammonium hydrogen citrate 2g, K ₂ HPO ₄ 1.53g, NaAc 5g, MgSO ₄ . 7H ₂ O 0.58g, MnSO ₄ 0.19g, Tween-80 1ml, distilled water 1L, pH 6.5-7.0, sterilized by high pressure steam. 4. novel Eucommia ulmoides fermented liquid according to claim 1, it is characterized in that, the expansion culture of described bacillus subtilis: inoculate in the 50L fermentor with the 1L bacterium liquid that cultivates, and fermentor culture medium is 25L; Fermentation condition: The rotation speed is 355r, the temperature is 3730.5°C, the dissolved oxygen is >40%, the pH is adjusted to 6.5-7.5 with ammonia water, and the culture is continued for 8 hours. 5. according to the described novel Eucommia fermented liquid of claim 4, it is characterized in that, the substratum preparation method of described Bacillus subtilis expanded culture is, peptone 10g, cornstarch 10g, glucose 10g, molasses mother liquor 10g, yeast extract 20g, Na ₂ HPO ₄ 12H ₂ O 7.5g, (NH ₄ ) ₂ SO ₄ 3.2g, CaCO ₃ 1g, defoamer 0.05g, make up 1L of sterile water; put the above materials except glucose into the fermenter, and then steam , sterilized at 120°C for 30 minutes; pH was adjusted to 7.430.1 with NaOH; glucose was single-eliminated at 110°C for 30 minutes, then added to the fermenter; the culture temperature for strain expansion was 36±1°C; the pH of fermentation was 7.030.1, adjusted with ammonia water . 6. according to the novel eucommia fermented liquid described in any one of claim 1-5, it is characterized in that, in 100L fermentation tank, according to eucommia leaf powder 25kg, purified water 45kg, pipette bacterium liquid 25L in 50L tank, rotating speed 250r, ventilate The amount is 7vv, and fermentation is carried out for 36h. 7. The preparation method of the novel eucommia fermented liquid is characterized in that, after the activated Bacillus subtilis is expanded and cultivated, it is inoculated into a medium containing eucommia leaf powder and fermented, and the culture medium containing eucommia leaf powder It is mainly composed of eucommia leaf powder and water, and the mass fraction of the eucommia leaf powder is 25-30%. 8. The application of the Eucommia ulmoides fermented liquid described in any one of claims 1-6 as an additive in the preparation of large yellow croaker feed. 9. Aquaculture feed comprising eucommia fermented liquid, comprising basal feed and fish oil, characterized in that it contains the eucommia fermented liquid described in any one of claims 1-7. 10 . The aquaculture feed comprising eucommia fermented liquid according to claim 9 , wherein the eucommia fermented liquid accounts for 4% of the total mass, and fish oil accounts for 5.5-6.5%.

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