NL2033126B1

NL2033126B1 - Gallnut fermentation method for aquatic animal growth promotion and disease resistance and application

Info

Publication number: NL2033126B1
Application number: NL2033126A
Authority: NL
Inventors: Liu Bo; Lin Yan; Jin Yan; Miao Linghong; Zhou Qunlan; Sun Cunxin; Ge Xianping
Original assignee: Freshwater Fisheries Res Center Cafs
Priority date: 2021-09-26
Filing date: 2022-09-26
Publication date: 2023-06-21
Also published as: NL2033126A

Abstract

The present invention discloses a gallnut fermentation method for aquatic animal growth promotion and disease resistance and an application, including activation of bacterial strains and preparation of bacterial solution, preparation of gallnut powder 5 slurry, fermentation culture and preparation of a fermented gallnut finished product, as well as a culture test and an attack drug test. By a single factor test and an orthogonal test, it is determined that the best fermentation conditions of gallnut powder are that pH is 6, the material—to—water ratio is 2.5%, the time is 10 24 h, and the temperature is 35°C. (+ Fig. l)

Description

GALLNUT FERMENTATION METHOD FOR AQUATIC ANIMAL GROWTH PROMOTION

AND DISEASE RESISTANCE AND APPLICATION

TECHNICAL FIELD

The present invention relates to a fermentation method for gallnut, in particular to a gallnut fermentation method for aquat- ic animal growth promotion and disease resistance and an applica- tion, and belongs to the technical field of fermentation.

BACKGROUND ART

Gallnut, also known as Chinese gall, Galla chinensis, sumac gallnut and the like, is an insect gall formed by some aphids par- asitic on leaves of trees in genus Rhus of Anacardiaceae. The main production areas of the gallnut in my country are Sichuan, Gui- zhou, Yunnan, Hunan, Hubei, and Shaanxi, among which Sichuan has the highest production. The gallnut has antioxidant and antibacte- rial functions, and its main chemical components mainly include tannin, a gallic acid and the like. Herein the gallic acid is a natural polyphenol substance with strong antioxidant, antiviral and bacteriostatic effects. It is found from some researches that the use of an enzyme system produced by microorganisms in the pro- cess of microbial fermentation may effectively degrade some anti- nutritional factors in fermentation raw materials, improve the palatability of fermentation products, produce unknown factors such as vitamins and organic acids, and increase the nutritional value of the fermentation products. Therefore, it may effectively improve animal feed intake and enhance its immunity. In addition, the metabolites of fermented probiotics may effectively degrade organic matters in a water body, and effectively purify the eco- logical environment of the aquaculture water body.

As a natural Chinese herbal medicine, the gallnut has the ad- vantages of pure natural, non-toxic and not easy to generate drug resistance. It is mainly used in aquaculture to prevent and treat aquatic animal diseases, enhance the immunity of the organisms, and promote the growth. The present invention uses beneficial strains to ferment the gallnut, aims to improve the nutritional value of the gallnut, and lays a foundation for developing a new type of an aquatic feed additive.

SUMMARY

A purpose of the present invention is to provide a gallnut fermentation method for aquatic animal growth promotion and dis- ease resistance and an application, the nutritional level and ef- fective components of gallnut are improved by fermentation.

A second purpose of the present invention is to provide an application of the above fermented gallnut powder as a feed addi- tive in an aquatic feed, and it is added to the feed, the animal growth performance may be significantly improved, the feed coeffi- cient is reduced and the antioxidant function is improved.

The present invention solves its technical problem by adopt- ing the following technical schemes: a gallnut fermentation method for aquatic animal growth promotion and disease resistance, in- cluding the following steps:

S1. activation of bacterial strains and preparation of bacte- rial solution inoculating Bacillus amyloliquefaciens (JSSW-LA), Lactobacil- lus plantarum (FF34) and Clostridium butyricum (JSIM-MCB) respec- tively into a Luria-Bertani (LB) solid medium, culturing at 35°C for 24 h, picking an activated single colony in the LB solid medi- um with an inoculating loop and inoculating into an LB liquid me- dium, culturing at 35°C for 24 h, and then measuring the concen- tration of the bacterial solution, diluting the bacterial solution by using sterilized phosphate buffer solution (PBS), to prepare bacterial suspensions of JSSW-LA, FF34 and JSIM-MCB of which the viable count is 1x10’ CFU/mL;

S2. preparation of gallnut powder slurry after crushing gallnut, adding deionized water to form the gallnut powder slurry, adjusting pH of the gallnut powder slurry to 6-7 and sterilizing, cooling to a room temperature after the sterilization is completed, and standing by for use;

S3. fermentation culture inoculating 3 bacterial suspensions of JSSW-LA, FF34 and

JSIM-MCB obtained in the step (2) into a fermentation tank accord- ing to a ratio of 2:1:1, herein the total inoculation quantity is 2~4%, the rotation speed of the fermentation tank is set to 180 r/min, the fermentation temperature is 30~35°C, and the fermenta- tion time is 24~36 h, acquiring gallnut fermentation broth; and

S4. preparation of a fermented gallnut finished product performing ultrafine pulverization after drying the gallnut fermentation broth in the step (3), to obtain fermented gallnut powder, and detecting the total amino acid and gallic acid con- tents of the gallnut before and after fermentation, herein results show that, compared with before the fermentation, the total amino acid content of the gallnut after the fermentation is increased by more than 1 time, and the gallic acid content is increased by more than 1 time.

Preferably, the LB solid medium composition includes 10 g/L of a peptone, 3 g/L of a beef extract, 5 g/L of NaCl and 15~20 g/L of an agar, the LB liquid medium composition includes 10 g/L of the peptone, 3 g/L of the beef extract and 5 g/L of NaCl, the LB solid medium and the LB liquid medium are all volume-fixed and prepared with distilled water, and pH is 7~7.2.

Preferably, in the step S2, after pulverizing the dried gall- nut with a multifunctional pulverizer, sieving with a 40~60-mesh sieve, to obtain the gallnut powder, herein the pulverizer model is CWJZ-30, and it is purchased from Guangzhou Xulang Machinery

Equipment Co., Ltd.

Preferably, in the step S2, weighing the deionized water and the gallnut powder according to the material-to-water ratio of 2.5%~5%, fully stirring uniformly, and preparing to obtain the gallnut slurry.

Preferably, in the step S2, adjusting pH of the gallnut slur- ry to 6-7 by using 1 mol/L of hydrochloric acid solution and 1 mol/L of sodium hydroxide solution respectively, and then auto- claving at 121°C for 30 min.

Preferably, the deposit number of JSSW-LA is CCTCC No: M 2015602; the deposit number of FF34 is CCTCC No: M 20211221; and the deposit number of JSIM-MCB is CGMCC No.1647.

Preferably, the suitable material-to-water ratio, pH, fermen-

tation time and fermentation temperature of the gallnut powder fermentation are determined by a single factor test and an orthog- onal test, and based on results of the single factor test, by de- signing a four-factor three-level L.(3‘) orthogonal experiment, the best fermentation conditions are determined that pH is 6, the ma- terial-to-water ratio is 2.5%, the time is 24 h, and the tempera- ture is 35°C.

Preferably, the drying condition in the step S4 is vacuum- drying under a condition of -20°C~-40°C.

Disclosed is an application of a gallnut fermentation method for aquatic animal growth promotion and disease resistance.

Beneficial effects of the present invention: 1. The present invention effectively reduces or eliminates anti-nutritional factors in the gallnut powder by a method of ben- eficial microorganism fermentation, and improves the nutritional value of the gallnut; 2. after the fermentation process of the gallnut powder of the present invention is optimized, it has the characteristics of short fermentation period, easy control of the fermentation condi- tions, simple operations and the like; 3. the present invention provides a fermentation production process method for gallnut powder, the total amino acid content, the gallic acid content and the protein content in the gallnut af- ter the fermentation are respectively increased by 115.59%, 147.16% and 23.72% compared with those before the fermentation, and the use of the fermented gallnut in freshwater shrimp feed has the significant effects of promoting the growth and improving the immunity; and 4. the addition of 0.5~2% fermented gallnut in the feed may significantly improve the weight gain rate and specific growth rate of freshwater shrimps, and significantly reduce the feed co- efficient, improve the antioxidant activity, reduce the cost of feed input, and improve the economic benefits of breeding; in ad- dition, the addition of 2% fermented gallnut in the feed may sig- nificantly reduce the pathogenic bacteria infection mortality rate of the freshwater shrimps. Therefore, the fermented gallnut pro- vided by the present invention has the significant growth-

promoting and disease-resistant effects, and has a broad applica- tion space in the aquatic feed.

BRIEF DESCRIPTION OF THE DRAWINGS

5 FIG. 1 is an optimization result of a fermentation material- to-water ratio of the present invention.

FIG. 2 is an optimization result of a fermentation time of the present invention.

FIG. 3 is an optimization result of a fermentation tempera- ture of the present invention.

FIG. 4 is a pH optimization result of the present invention.

FIG. 5 is a cumulative mortality rate of freshwater shrimps fed with fermented gallnut of the present invention after chal- lenge of vibrio cholerae.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Technical schemes in embodiments of the present invention are clearly and completely described below with reference to the draw- ings in the embodiments of the present invention. Apparently, the embodiments described are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments ob- tained by those of ordinary skill in the art without creative work shall fall within a scope of protection of the present invention.

Unless otherwise specified, test methods used in the present invention are all conventional methods, and test instruments, ma- terials, reagents and the like used may be obtained from mer- chants.

Bacterial strains used in the present invention are JSSW-LA,

FF34 and JSIM-MCBLB.

JSSW-LA, deposit unit: China Center for Type Culture Collec- tion, CCTCC for short, deposit number: CCTCC No: M 2015602. The deposit date is October 12, 2015, and the deposit period is 30 years. Deposit address: Luojia Mountain, Bayi Road, Wuchang Dis- trict, Wuhan City, Hubei Province, Wuhan University.

FF34, deposit unit: China Center for Type Culture Collection,

CCTCC for short, deposit number: CCTCC No: M 20211221. The deposit date is September 26, 2021, and the deposit period is 30 years.

Deposit address: Lucjia Mountain, Bayi Road, Wuchang District, Wu- han City, Hubei Province, Wuhan University.

Clostridium butyricum is also known as JSIM-MCB 20040312, de- posit unit: China General Microbiological Culture Collection Cen- ter, CGMCC for short. The deposit number is CGMCC No. 1647, the deposit date is March 10, 2006, and the deposit period is 30 years. Deposit address: No. 13, Beiyi, Zhongguancun, Haidian Dis- trict, Beijing, Institute of Microbiology, Chinese Academy of Sci- ences.

All the above strains are in living state.

Gallnut fermentation method for aquatic animal growth promo- tion and disease resistance and application

Embodiment 1: Preparation of fermented gallnut (1) Activation of strains: JSSW-LA, FF34 and JSIM-MCBLB are inoculated into an LB solid medium, and cultured at 35°C for 24 h; and the LB solid medium composition includes 10 g/L of a peptone, 3 g/L of a beef extract, 5 g/L of NaCl, and 15-20 g/L of an agar, it is volume-fixed and prepared with distilled water, and pH is 77.2. (2) Preparation of bacterial suspensions: an activated single colony is picked with an inoculating loop and inoculated into an

LB liquid medium, the concentration of bacterial solution is meas- ured after being cultured at 35°C for 24 h, and the bacterial so- lution is diluted with sterilized PBS, to prepare 3 bacterial sus- pensions of which the viable count is 1x10’ CFU/mL; and the LB liquid medium composition includes 10 g/L of the pep- tone, 3 g/L of the beef extract, and 5 g/L of NaCl, it is volume- fixed and prepared with the distilled water, and pH is 77.2. (3) Fermentation culture: after gallnut is pulverized, it is sieved with a 40-60-mesh sieve to obtain gallnut powder, deionized water is added according to a material-to-water ratio of 2.5%, pH of gallnut powder slurry is adjusted to 7 by using 1 mol/L of hy- drochloric acid and sodium hydroxide solution, the gallnut powder slurry is poured into a fermentation tank and autoclaved at 121°C for 30 min, after the sterilization is completed, it is cooled to a room temperature, 3 bacterial suspensions of JSSW-LA, FF34 and

JSIM-MCB are inoculated into the fermentation tank according to a ratio of 2:1:1, the total inoculation amount is 4% of the gallnut powder slurry, the rotation speed of the fermentation tank is set to 180 r/min, the fermentation temperature is 35 G, and the time is 24 h, gallnut fermentation broth is obtained. (4) Preparation of fermented gallnut finished product: the fermentation product in the step (3) is pre-cooled at -80°C for 2 h, vacuum-dried at -20°C~-40°C, and then ultrafinely pulverized to obtain fermented gallnut powder.

The total amino acid, gallic acid, and total protein contents of the gallnut powder before and after the fermentation are meas- ured respectively, and results are shown in Table 1. The total amino acid content in the fermented gallnut is 1532.88 umol/g, the gallic acid content is 6.58 mg/g, and the protein content is 163.82 mg/g; which are respectively improved by 154.173, 145.28%, and 24.05% compared with the unfermented gallnut. It is indicated from the above results that the nutritional components of the fer- mented gallnut powder are significantly higher than that of the unfermented gallnut powder.

Table 1: Comparison of nutrient substances of gallnut powder before and after fermentation

Before Embodiment | Content increase fermentation |1 %

Total amino acid content

Sample 2 598.00 1459.62 144.08%

In the above embodiment, the pulverizer model is CWJZ-30, and it is purchased from Guangzhou Xulang Machinery Equipment Co.,

Ltd.

Embodiment 2: Condition comparison test of gallnut fermenta- tion process

The suitable material-to-water ratio, pH, fermentation time and fermentation temperature are determined by a single factor test and an orthogonal test. The main operation steps are as fol- lows: 1. Single factor test 1-1. Single factor comparison test of material-to-water ratio

Fermented samples of the gallnut powder to be tested and de- ionized water are weighed according to material-to-water ratios of 2.5%, 5%, 7.5%, 10% and 12.5% respectively, and it is fully stirred and mixed uniformly, pH of obtained gallnut slurry is ad- justed to 7, it is autoclaved at 121°C for 30 min, and cooled to a room temperature after the sterilization is completed, and 3 bac- terial suspensions of JSSW-LA, FF34 and JSIM-MCB are inoculated into a fermentation tank according to a ratio of 2:1:1. The total inoculation amount is 4% of the gallnut powder slurry, the fermen- tation temperature is kept at 35°C, it is fermented in a 180 r/min shaker for 48 h, obtained fermentation broth is centrifuged under conditions of 4°C and 6000 r/min for 10 min after 48 h of the fer- mentation, a supernatant is taken, the total amino acid content of the fermentation broth is measured, and the fermentation material- to-water ratio is determined. Each test is repeated for 3 times, and an average value is taken finally. Results are shown in FIG. 1, and while the material-to-water ratio is 2.5%, the total amino acid content in the fermented gallnut is the highest. 1-2. Single factor comparison test of fermentation time

The fermentation temperature is kept at 35°C, the material- to-water ratio is 10%, pH of the prepared gallnut powder slurry is adjusted to 7, 3 bacterial suspensions of JSSW-LA, FF34 and JSIM-

MCB are inoculated into a fermentation tank according to a ratio of 2:1:1, the total inoculation amount is 4% of the gallnut powder slurry, the fermentation times are 12 h, 24 h, 36 h, 48 h, and 60 h respectively, and the rotation speed of a shaker is 180 r/min.

Obtained fermentation broth is centrifuged under conditions of 4°C and 6000 r/min for 10 min, a supernatant is taken, the total amino acid of the fermentation broth is measured, and the fermentation time is determined. Each test is repeated for 3 times, and an av- erage value is taken finally. Results are shown in FIG. 2, and while the fermentation time is 24 h, the total amino acid content in the fermented gallnut is the highest. 1-3. Single factor comparison test of fermentation tempera- ture

The fermentation time is kept at 48 h, the material-to-water ratio is 10%, pH of the prepared gallnut powder slurry is adjusted to 7, 3 bacterial suspensions of JSSW-LA, FF34 and JSIM-MCB are inoculated into a fermentation tank according to a ratio of 2:1:1, the total inoculation amount is 4% of the gallnut powder slurry, the fermentation temperature is 25°C, 30°C, 35°C, 40°C, and 45°C, and the rotation speed of a shaker is 180 r/min. After 48 h of the fermentation, obtained fermentation broth is centrifuged under conditions of 4°C and 6000 r/min for 10 min, a supernatant is tak- en, the total amino acid content of the fermentation broth is measured, and the fermentation temperature range is determined.

Each test is repeated for 3 times, and an average value is taken finally. Results are shown in FIG. 3, and while the fermentation temperature is 30°C, the total amino acid content in the fermented gallnut is the highest. 1-4. Single factor comparison test of pH

The fermentation temperature is kept at 35°C, the fermenta- tion time is 48 h, and the material-to-water ratio is 103. 3 bac- terial suspensions of JSSW-LA, FF34 and JSIM-MCB are inoculated into a fermentation tank according to a ratio of 2:1:1, the total inoculation amount is 4% of the gallnut powder slurry, the fermen- tation pH is 5, 6, 7, 8, 2, and the rotation speed of a shaker is

180 r/min. After 48 h of the fermentation, obtained fermentation broth is centrifuged under conditions of 4°C and 6000 r/min for 10 min, a supernatant is taken, the total amino acid content of the fermentation broth is measured, and the optimum pH range for the fermentation is determined. Each test is repeated for 3 times, and an average value is taken finally. Results are shown in FIG. 4, and while the fermentation pH is 6, the total amino acid content in the fermented gallnut is the highest. 2. Orthogonal test

Based on the above single factor test results, the optimal fermentation conditions for fermenting the gallnut are further de- termined by designing a four-factor three-level Lo(3°) orthogonal test. The investigated factors are the fermentation material-to- water ratio, the fermentation temperature, the fermentation time and the fermentation pH. The design factors and levels are shown in Table 2, and there are a total of 9 groups of tests. The rota- tion speed of each group of a fermentation shaker is 180 r/min, and after the fermentation is completed, obtained fermentation broth is centrifuged under conditions of 4°C and 6000 r/min for 10 min, a supernatant is taken to be tested. The Ls (3%) orthogonal de- sign and results are shown in Table 3.

Table 2: Level of each factor in orthogonal test of gallnut fermentation

Factor

Level pH Material-to-water ratio % Time (h) Temperature{°C) 1 6 2.50 12 25 2 7 5 24 30 3 8 7.50 36 35

Table 3: Orthogonal test results

B{material-

Test Gallic acid content

A{pH) to-water C(time) D(temperature)} number (mg/g) ratio} 1 1(6) 1(2.5%) 1(12h) 1(30°C) 3.88 2 1(6) 2(5%) 2(24h) 2(35°C) 6.01 3 16) 3(7.5%) 3{36h) 3(40°C) 3.91 4 217} 1(2.5%) 2(24h) 3{40°C} 4.08 2{7) 2(5%) 3(36h) 1{30°C) 1.72 6 2(7) 3(7.5%) 1{12h) 2(35°C} 4.63 7 3(8) 1(2.5%) 3(36h} 2(35°C) 2.58 8 3(8) 2(5%) 1(12h) 3(40°C) 1.35 9 3{8) 3(7.5%) 2(24h) 1{30°C) 1.58

T1 13.8 10.54 9.86 7.18

T2 10.43 9.31 11.67 13.22

T3 5.51 10.12 8.21 9.34 n 3 3 3 3

K1 4.60 3.51 3.29 2.39

K2 3.48 3.10 3.89 441

K3 1.84 3.37 2.74 3.11

R 2.76 0.41 1.15 2.01

Note: T1-T3 in the table is the sum of 1-3 gallic acid con- tents; n is the number of repetitions; Kl1-K3 is T1-T3/n; and R is the maximum value minus the minimum value in T1-T3. 5 According to the results of the orthogonal test, the primary and secondary factors of the influence factors in this test are

A>D>C>B, namely the primary and secondary factors of the influence factors are pH>temperature>time>material-to-water ratio. Combined with data in the table, the average of each factor is estimated to be the highest, and the optimal fermentation conditions are deter- mined as: A1B1C2D2, namely the optimal fermentation conditions are that pH is 6, the material-to-water ratio is 2.5%, the time is 24 h, and the temperature is 35°C.

Fermentation is performed again under the optimal fermenta- tion culture conditions, and the contents of the total amino acid, gallic acid and total protein are measured. Results show that by the implementation of this embodiment, the total amino acid con- tent in the fermented gallnut is 1303.53 umol/g, the gallic acid content is 6.51 mg/g, and the protein content is 129.67 mg/g, which are respectively improved by 115.594, 147.16%, and 23.72% compared with the unfermented gallnut.

Embodiment 3: Application effects of fermented gallnut powder in aquatic animal feed

In this embodiment, 4 groups of experimental compound feeds are designed, fish meal, soybean meal, rapeseed meal and shrimp meal are used as main protein sources, fish oil and soybean oil are used as main fat sources, a basal feed is used as a control group, and a test group is based on this in which 0.25%, 0.5%, 1%, and 2% of the fermented gallnut are added. The test feed formula and main nutrient substances are shown in Table 4. The test object is a freshwater shrimp, and purchased from Dapu Base, Yixing,

Freshwater Fisheries Research Center, Chinese Academy of Fishery

Sciences, the average size is 0.28+0.03 g, and there are 3 repeti- tions in each group, and 50 shrimps in each repetition. The corre- sponding feed is fed every day according to the feed grouping con- ditions. The daily feeding amount is 5% of the shrimp body weight, and the feeding amount is adjusted according to the feeding situa- tion and growth condition of the shrimps. It is advisable to fin- ish feeding after 1 hour, feeding is performed for 3 times (8:00, 12:00 and 18:00) every day, and all are fed on full food.

The diameter of a breeding barrel is 1 m, the water level is kept at 40+5 cm, and the water quality indicators are regularly detected to guarantee that the dissolved oxygen is 26.0 mg/L, pH is 7.3+1.2, ammonia nitrogen is <0.2 mg/L, and nitrite is <0.005mg/L during the test. A sewage pipe is used to discharge sewage every day, and water is changed once a week to guarantee the good water quality. The growth and feeding conditions of the shrimps are observed every day, and the dead shrimps are picked up in time after being found, and weighed and recorded. The breeding test is performed for a total of 56 d.

After the breeding test, 10 freshwater shrimps are selected in parallel to continue to be temporarily raised, and each group of feed is fed regularly. On the 7th day, non-01 wvibrio cholerae {isolated from the diseased freshwater shrimps) is used for an at- tack drug test, and 2% addition group with the better growth per- formance and the control group are selected to perform the attack drug test. 10 shrimps are selected in each test tank, and three repetitions are set in the control group and the experimental group. An LB liquid medium is selected for the activation and cul- tivation of vibrio cholerae, the shaker temperature is 28°C, and it is shake-cultured for 18 h under a condition of 180 r/min of the rotation speed. After the viable count is calculated, the con- centration of bacterial solution is adjusted to a gradient bacte- rial suspension with sterile physiological saline. Before the for- mal experiment, a preliminary test is performed to determine the appropriate injection concentration and injection volume. The in- jection site is a muscle between the second abdominal segment and the third abdominal segment of the shrimp. It is determined by the preliminary test that the appropriate injection concentration is 1x10° CFU/ml and the injection volume is 10 pL. The death situation of the shrimps is observed and recorded at 6 h, 12 h, 24 h and 48 h after the injection, and the cumulative mortality is counted.

Table 4: Test of feed formula and main nutrient substances

Addition amount g/kg item 0 (control 0.25 0.5 1 2 group)

Fish meal 300 300 300 300 300

Soybean meal 220 220 220 220 220

Rapeseed meal 100 100 100 100 100

Shrimp meal 60 60 60 60 60

Squid paste 30 30 30 30 30

Flour 204.3 201.8 199.3 194.3 184.3

Fish oil 15 15 15 15 15

Soybean oil 15 15 15 15 15

Soy lecithin 10 10 10 10 10

Cholesterol 5 5 5 5 5

Calcium dihydrogen 20 20 20 20 20 phosphate

Multi-mineral 5 5 5 5 5

Multi-vitamin 5 5 5 5 5

Ve 5 5 5 5 5

Choline chloride 5 5 5 5 5

Ecdysin 0.2 0.2 0.2 0.2 0.2

Galinut 0 25 5 10 20

DMPT 0.5 0.5 0.5 0.5 0.5

Nutrition level %

Fat content 8.55 8.55 8.55 8.54 8.52

Protein content 40.38 40.35 40.33 40.27 40.15

Table 5: Effects of fermented gallnut on growth performance of freshwater shrimps

Addition amount % 0 0.25 0.5 1 2

Survival 88.6214.12 92.560+5.09 92.60+1.09 94.20+5.12 94.22+4.67 rate/%

Weight gain b b 96.65+10.49° 102.11#5.77% 118.51+10.11° 117.2148.18° 116.25+6.12° rate/%

Specific growth rate 1.30+0.12° 1.33+0.05°% 1.45+0.14° 1.49+0.09° 1.48+0.03°

Id

Feed b b b a a 4.33+0.47 3.6910.27° 3.9610.98° 3.11+0.20 3.27+0.41 coefficient

Note: Data are mean + standard error, different letters in the same row indicate significant differences between pairs (P<0.05).

Table 6: Effects of fermented gallnut on serum antioxidant enzyme activities of freshwater shrimp

Addition amount % 0 0.25 0.5 1 2

Superoxide dis- 26.63+0.69° 56.63+3.38° 55.36:1.69° 54.36:2.88° 36.74+1.89™ mutase/{U/mL}

Malondialdehyd . . ‚ . 4 29.13+0.55 19.12+0.86 23.55+0.49° 14.66+0.88 15.1810.61 e /{nmol/m}}

Catalase 5 b b 23.8311.41 23.9510.04 26.2411.93 44,50+1.87° 43.42+3.56° /{U/mL)

As shown in Table 5-Table 6, by evaluating the growth perfor- mance, the antioxidant capacity and the disease resistance, it is found that the addition of 0.5~2% fermented gallnut in the com- pound feed of the freshwater shrimp may significantly improve the weight gain rate and specific growth rate of the freshwater shrimp, and significantly reduce the feed coefficient, improve the antioxidant activity, reduce the cost of feed input, and improve the economic benefits of breeding; and in addition, 2% fermented gallnut in the feed may significantly reduce the pathogen infec- tion mortality rate of the freshwater shrimp (as shown in FIG. 5).

Therefore, the fermented gallnut provided by the present invention has the significant growth-promoting and disease-resistant ef- fects, and has a broad application space in aquatic feed.

It is apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary em- bodiments, but that the present invention may be embodied in other specific forms in the case without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments are to be regarded in all respects as illustrative and not restrictive, and the scope of the present invention is to be defined by the appended claims rather than the above descriptions.

Therefore, it is intended to include all changes falling within the meaning and scope of equivalent elements of the claims in the present invention. Any reference signs in the claims shall not be construed as limiting the involved claim.

In addition, it should be understood that although this de- scription is described in terms of the embodiments, each embodi- ment does not only include an independent technical scheme, and this description mode in the description is only for the sake of clarity, and those skilled in the art should take the description as a whole, the technical schemes in each embodiment may also be appropriately combined, to form other implementation modes that may be understood by those skilled in the art.

Claims

CONCLUSIESCONCLUSIONS

1. Werkwijze voor het fermenteren van galnoot voor het bevorderen van de groei van waterdieren en ziekteresistentie, met het ken- merk, dat de werkwijze de volgende stappen omvat:A method of fermenting gallnut for promoting aquatic animal growth and disease resistance, characterized in that the method comprises the following steps:

Sl. activering van bacteriestammen en bereiding van bacteriële op- lossing het inoculeren van respectievelijk Bacillus amyloliquefaciens (JSSW-LA), Lactobacillus plantarum (FF34) en Clostridium butyricum (JSIM-MCB) in een vast Luria-Bertani (LB) medium, kweken bij 35 °C gedurende 24 uur, het plukken van een geactiveerde enkele kolonie in het vaste LB medium met een entlus en enten in een vloeibaar LB medium, 24 uur kweken bij 35 ° C, en het vervolgens meten van de concentratie van de bacteriële oplossing, het verdunnen van de bacteriële oplossing met behulp van gesteriliseerde fosfaatbuffer- oplossing (PBS), om bacteriële suspensies te bereiden van JSSW-LA, FF34 en JSIM-MCB waarvan de levensvatbare telling 1 x 107 CFU/ml is;Sl. activation of bacterial strains and preparation of bacterial solution inoculation of Bacillus amyloliquefaciens (JSSW-LA), Lactobacillus plantarum (FF34) and Clostridium butyricum (JSIM-MCB) respectively in a solid Luria-Bertani (LB) medium, culture at 35° C for 24 hours, picking an activated single colony in the solid LB medium with an inoculation loop and inoculation into a liquid LB medium, culturing at 35 °C for 24 hours, and then measuring the concentration of the bacterial solution, diluting of the bacterial solution using sterilized phosphate buffer solution (PBS), to prepare bacterial suspensions of JSSW-LA, FF34 and JSIM-MCB whose viable count is 1 x 10 7 CFU/ml;

S2. bereiding van galnootpoeder suspensie na het pletten van de galnoot, het toevoegen van gedeïoniseerd wa- ter om de galnootpoeder suspensie te vormen, het instellen van de pH van de galnootpoeder suspensie tot 6 ~ 7 en steriliseren, af- koelen tot kamertemperatuur nadat de sterilisatie is voltooid, en laten staan voor gebruik;S2. preparation of gallnut powder suspension after crushing the gallnut, adding deionized water to form gallnut powder suspension, adjusting the pH of gallnut powder suspension to 6~7 and sterilizing, cooling to room temperature after sterilization is completed completed, and left for use;

S3. fermentatiekweek het enten van 3 bacteriële suspensies van JSSW-LA, FF34 en JSIM- MCB verkregen in stap (2) in een fermentatietank volgens een ver- houding van 2:1:1, waarbij de totale inoculatiehoeveelheid 2~4% is, de rotatiesnelheid van de fermentatietank is ingesteld op 180 r / min, de fermentatietemperatuur is 30 ~ 35 ° C en de fermenta- tietijd is 24 ~ 36 uur, waarbij galnootfermentatiebouillon wordt verkregen; enS3. fermentation culture inoculating 3 bacterial suspensions of JSSW-LA, FF34 and JSIM-MCB obtained in step (2) into a fermentation tank at a ratio of 2:1:1, the total inoculation amount being 2~4%, the rotation speed of the fermentation tank is set to 180 r / min, the fermentation temperature is 30 ~ 35 ° C, and the fermentation time is 24 ~ 36 hours, obtaining gall nut fermentation broth; and

S4. bereiding van een eindproduct van gefermenteerde galnoten het uitvoeren van ultrafijne verpulvering na het drogen van de galnootfermentatiebouillon in stap (3), om gefermenteerd galnoot- poeder te verkrijgen, en het detecteren van het totale aminozuur-S4. preparation of fermented gallnut final product performing ultrafine pulverization after drying the gallnut fermentation broth in step (3), to obtain fermented gallnut powder, and detecting the total amino acid

en galluszuurgehalte van de galnoot voor en na de fermentatie, waarbij de resultaten aantonen dat, vergeleken met vóór de fermen- tatie , het totale aminozuurgehalte van de galnoot na de fermenta- tie met meer dan 1 keer wordt verhoogd, en het galluszuurgehalte met meer dan 1 keer wordt verhoogd.and gallic acid content of the gall nut before and after fermentation, the results showing that, compared to before fermentation, the total amino acid content of the gall nut is increased by more than 1 times after fermentation, and the gallic acid content by more than 1 time is increased.

2. Werkwijze voor het fermenteren van galnoot voor het bevorderen van de groei van waterdieren en ziekteresistentie volgens conclu- sie 1, met het kenmerk, dat: de samenstelling van het vaste LB me- dium 10 g/L van een pepton, 3 g/L van een rundvleesextract, 5 g/ L Nacl en 15-20 g/L van een agar omvat, waarbij de samenstelling van het vloeibare LB medium omvat 10 g/L van het pepton, 3 g/L van het rundvleesextract en 5 g/L NaCl omvat, waarbij het vaste LB medium en het LB vloeibaar medium allemaal volumevast zijn en zijn bereid met gedestilleerd water, en waarbij de pH 7 ~ 7,2 is.A method of fermenting gallnut for promoting aquatic animal growth and disease resistance according to claim 1, characterized in that : the composition of the LB solid medium is 10 g/L of a peptone, 3 g/ L of a beef extract, 5 g/L of NaCl and 15-20 g/L of an agar, the composition of the liquid LB medium comprising 10 g/L of the peptone, 3 g/L of the beef extract and 5 g/L L includes NaCl, where the LB solid medium and the LB liquid medium are all volumetric and prepared with distilled water, and the pH is 7 ~ 7.2.

3. Werkwijze voor het fermenteren van galnoot voor het bevorderen van de groei van waterdieren en ziekteresistentie volgens conclu- sie 1, met het kenmerk, dat: in stap S2, na het verpulveren van de gedroogde galnoot met een multifunctionele vergruizer, zeven met een 40 tot 60 mesh zeef wordt uitgevoerd, om het galnootpoeder te verkrijgen, waarbij het model van de vergruizer CWJZ-30 is, en het is gekocht bij Guangzhou Xulang Machinery Equipment Co., Ltd...A method of fermenting gallnut for promoting aquatic animal growth and disease resistance according to claim 1, characterized in that : in step S2, after pulverizing the dried gallnut with a multifunctional crusher, sieving with a 40 up to 60 mesh sieve is run, to obtain the gall nut powder, where the model of the pulverizer is CWJZ-30, and it is purchased from Guangzhou Xulang Machinery Equipment Co., Ltd...

4. Werkwijze voor het fermenteren van galnoot voor het bevorderen van de groei van waterdieren en ziekteresistentie volgens conclu- sie 1, met het kenmerk dat: in stap S2 het gedeïoniseerde water en het galnootpoeder worden gewogen volgens de materiaal- waterverhouding van 2,5% ~ 5% , volledig gelijkmatig roeren en be- reiden om de galnootsuspensie te verkrijgen.A method of fermenting gallnut for promoting aquatic animal growth and disease resistance according to claim 1, characterized in that : in step S2, the deionized water and the gallnut powder are weighed according to the material-to-water ratio of 2.5% ~ 5%, stir and cook completely evenly to obtain the galnut suspension.

5. Werkwijze voor het fermenteren van galnoot voor het bevorderen van de groei van waterdieren en ziekteresistentie volgens conclu- sie 1, met het kenmerk dat: in stap S2 de pH van de galnootsuspen- sie wordt ingesteld op 6 tot 7 door gebruik te maken van respec- tievelijk 1 mol/L zoutzuuroplossing en 1 mol/L natriumhydroxide- oplossing, en vervolgens 30 minuten autoclaveren bij 121 °C.A method of fermenting gallnut for promoting aquatic animal growth and disease resistance according to claim 1, characterized in that : in step S2, the pH of the gallnut suspension is adjusted to 6 to 7 by using 1 mol/L hydrochloric acid solution and 1 mol/L sodium hydroxide solution, respectively, then autoclave at 121°C for 30 minutes.

6. Werkwijze voor het fermenteren van galnoot voor het bevorderen van de groei van waterdieren en ziekteresistentie volgens conclu- sie 1, met het kenmerk, dat: het depotnummer van JSSW-LA CCTCC-The method of fermenting gallnut for promoting aquatic animal growth and disease resistance according to claim 1, characterized in that : the deposit number of JSSW-LA CCTCC-

nr.: M 2015602 is; het depotnummer van FF34 is CCTCC No: M 20211221; en het depotnummer van JSIM-MCB is CGMCC No.1647.no.: M 2015602; the deposit number of FF34 is CCTCC No: M 20211221; and the deposit number of JSIM-MCB is CGMCC No.1647.

7. Werkwijze voor het fermenteren van galnoot voor het bevorderen van de groei van waterdieren en ziekteresistentie volgens conclu- sie 1, met het kenmerk dat: de geschikte materiaal- waterverhouding, pH, fermentatietijd en fermentatietemperatuur van de fermentatie van de galnootpoeder worden bepaald door een enkele factortest en een orthogonale test, en op basis van de resultaten van de enkele factortest, door het ontwerpen van een orthogonaal L9(34)-experiment met vier factoren en drie niveaus, de beste fer- mentatieomstandigheden worden bepaald waarbij de pH 6 is, de mate- riaal-tot-water verhouding 2,5% is, de tijd 24 uur is en de tem- peratuur 35 °C is.A method of fermenting gallnut for promoting aquatic animal growth and disease resistance according to claim 1, characterized in that : the appropriate material-to-water ratio, pH, fermentation time and fermentation temperature of the fermentation of the gallnut powder are determined by a single factor test and an orthogonal test, and based on the results of the single factor test, by designing a four factor, three level orthogonal L9(34) experiment, the best fermentation conditions are determined where the pH is 6, the material-to-water ratio is 2.5%, the time is 24 hours, and the temperature is 35°C.

8. Werkwijze voor het fermenteren van galnoot voor het bevorderen van de groei van waterdieren en ziekteresistentie volgens conclu- sie 1, met het kenmerk dat: de droogconditie in stap S4 vacuümdro- gen is onder een conditie van -20 °C tot -40 °C.A method of fermenting gallnut for promoting aquatic animal growth and disease resistance according to claim 1, characterized in that : the drying condition in step S4 is vacuum drying under a condition of -20°C to -40° C.

9. Toepassing van een werkwijze voor het fermenteren van galnoot voor het bevorderen van de groei van waterdieren en ziekteresis- tentie.9. Use of a method of fermenting gallnut for promoting aquatic animal growth and disease resistance.