Application of combined use of beta-glucan and klebsiella in relieving low-salt stress of penaeus vannamei

Abstract

The invention discloses an application of combined use of beta-glucan and Klebsiella sp. In relieving low salt stress of penaeus vannamei, the Klebsiella sp. Is Klebsiella sp. E26, and is preserved in the China Center for Type Culture Collection (the address is Wuhan University, Wuhan, China), the preservation number is CCTCC NO: M20241218, and the preservation date is June 14, 2024. According to the invention, 0.1% of beta-glucan and 10 < 9 > CFU / g of strain E26 are jointly added into the feed to comprehensively relieve the negative effect of low-salt stress on prawns.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an application of a combination of beta-glucan and Klebsiella in alleviating low-salt stress of Penaeus vannamei. Background Art

[0002] In the current farming process of Penaeus vannamei, inland low-salt farming has become a trend, but long-term low-salt farming will seriously affect the growth and health of Penaeus vannamei, and even cause a large number of shrimp deaths in severe cases. β-glucan is a commonly used prebiotic in aquaculture, which can improve the growth and health of Penaeus vannamei, but there is currently no report on the combined use of β-glucan and Klebsiella to improve the low-salt stress effect of Penaeus vannamei. Summary of the invention

[0003] In view of the above technical problems, the present invention provides an application of the combined use of β-glucan and Klebsiella in alleviating the low-salt stress of Penaeus vannamei, which comprises adding 0.1% β-glucan and 10 9 CFU / g of the bacterial strain E26 can comprehensively alleviate the negative effects of low salt stress on shrimp.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A strain of Klebsiella is provided, which is Klebsiella sp. E26 and is deposited in China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with a deposit number of CCTCC NO: M20241218 and a deposit date of June 14, 2024.

[0006] Provided is a use of the Klebsiella E26 in preparing a preparation for inhibiting pathogenic bacteria.

[0007] Preferably, the pathogenic bacteria include one or more of Streptococcus iniae, Vibrio parahaemolyticus, and Vibrio harveyi.

[0008] Provided is an application of the Klebsiella E26 in improving the digestion capacity of Penaeus vannamei in a low-salt environment.

[0009] Preferably, the Klebsiella E26 improves the digestibility of Penaeus vannamei by secreting amylase and / or protease.

[0010] Provided is a use of the Klebsiella E26 in improving the resistance of Penaeus vannamei to antibiotics.

[0011] Provided is an application of the combined use of beta-glucan and the above-mentioned Klebsiella E26 in improving the growth performance of Penaeus vannamei in a low-salt environment.

[0012] Preferably, the growth performance includes one or more of weight gain rate, survival rate, and hepatosomatic index.

[0013] Provided is an application of the combined use of beta-glucan and the above-mentioned Klebsiella E26 in improving the immunity of Penaeus vannamei in a low-salt environment.

[0014] Preferably, the immune capacity index includes respiratory burst activity and / or hemocyanin content and / or lytic activity.

[0015] Provided is an application of the combined use of beta-glucan and the above-mentioned Klebsiella E26 in improving the digestion and absorption capacity of Penaeus vannamei in a low-salt environment.

[0016] Preferably, the digestion and absorption capacity of Penaeus vannamei in a low-salt environment is improved by increasing the activity of trypsin and / or lipase and / or amylase in the hepatopancreas tissue of Penaeus vannamei.

[0017] Provided is an application of the combined use of beta-glucan and the above-mentioned Klebsiella E26 in regulating the intestinal microbial flora structure of Penaeus vannamei in a low-salt environment.

[0018] The present invention has at least the following beneficial effects:

[0019] In the present invention, 0.1% β-glucan and 10 9 CFU / g of the bacterial strain E26 can significantly improve the weight gain rate, survival rate, nonspecific immunity, trypsin activity and intestinal flora abundance of Penaeus vannamei under low-salt conditions, and comprehensively alleviate the negative effects of low-salt stress on shrimp. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0021] Figure 1 The enzyme production activity, antibacterial activity and hemolytic activity of the strains, including (A) Streptococcus iniae, (B) Vibrio harveyi, (C) Vibrio parahaemolyticus, (D) protease positive, (E) amylase positive, (F) hemolytic positive;

[0022] Figure 2The survival rates of the five strains after 2 hours in artificial gastric juice and 4 hours in artificial intestinal juice, where (A) is artificial gastric juice; (B) is artificial intestinal juice;

[0023] Figure 3 The cell surface hydrophobicity and self-agglutination rate of five strains in three hydrocarbons, including (A) xylene; (B) chloroform; (C) ethyl acetate; (D) self-agglutination rate;

[0024] Figure 4 The phylogenetic tree is constructed based on the 16S rRNA sequencing information of strain E26;

[0025] Figure 5 Effects of different feed types on the growth performance of Penaeus vannamei under low-salinity conditions, including (A) weight gain rate, (B) survival rate, (C) liver-body index, (D) fatness, * indicates significant difference among groups (P<0.05);

[0026] Figure 6 Effects of different feed types on the immune performance of Penaeus vannamei under low-salt conditions, including (A) blood cell count, (B) hemocyanin content, (C) respiratory burst activity, (D) lytic activity, different lowercase letters represent significant differences (P<0.05);

[0027] Figure 7 The effects of different feed types on the digestive enzyme activities of Penaeus vannamei under low-salt conditions, including (A) trypsin, (B) lipase, (C) amylase, and different lowercase letters represent significant differences (P<0.05);

[0028] Figure 8 Figure 2. The composition of the intestinal microbial community of Penaeus vannamei after feeding different diets under low-salinity conditions, including (A) Venn diagram showing the number of unique and shared OTUs, (B) principal coordinate analysis (PCoA), (C) relative abundance of bacterial communities at the phylum level, and (D) relative abundance of bacterial communities at the genus level.

[0029] Fig. 9 Effects of different feed types on the α-diversity of intestinal microorganisms of Penaeus vannamei under low-salinity conditions, including (A) Chao 1 index, (B) observed index, (C) Shannon index, and (D) Simpson index;

[0030] Fig.10 The KEGG three-level function prediction and microbial interaction heat map of the intestinal microorganisms of Penaeus vannamei after feeding different feeds under low-salt conditions, including (A) the proportion of the top five dominant bacterial phyla groups, (B) the proportion of the top five dominant bacterial genera, (C) KEGG intestinal microbial three-level function prediction, and (D) microbial interaction heat map. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] Embodiment 1:

[0033] This embodiment provides an application of the combined use of β-glucan and Klebsiella in alleviating low-salt stress of Penaeus vannamei, which specifically includes the following contents:

[0034] 1. Application of β-glucan and Klebsiella in improving the growth performance of Penaeus vannamei in low-salt environment

[0035] The “low-salt environment” refers to that the salinity of the culture water of Penaeus vannamei is 1-10. In addition, the Klebsiella is Klebsiella sp. E26 (hereinafter referred to as “strain E26”), which is deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with a deposit number of CCTCC NO: M20241218 and a deposit date of June 14, 2024.

[0036] 1.1 Breeding experiment design

[0037] The breeding experiment was divided into 4 treatment groups: (1) low-salt control group: this treatment group was fed with basic feed; (2) Klebsiella group: this treatment group was fed with strain E26 with a content of 10 9 CFU / g of basal feed; (3) β-glucan group: This treatment group was fed with basal feed containing 0.1% (by weight) β-glucan; (4) combined use group: This treatment group was fed with 0.1% (by weight) β-glucan and 10 9 CFU / g of basal feed. Four biological parallels were set for each treatment group. The specific experimental design is shown in Table 1.

[0038] Table 1 Design of breeding experiment

[0039]

[0040] 1.2 Basic feed formula

[0041] The specific formula of the above basic feed and the basic feed added with 0.1% β-glucan is shown in Table 2.

[0042] Table 2 Feed formula and composition

[0043]

[0044]

[0045] After obtaining the basic feed and the basic feed supplemented with β-glucan, strain E26 was revived and activated, then inoculated in LB medium and cultured with shaking for 24 h, then centrifuged at 5000 g for 15 min, the supernatant was removed, the precipitate was washed twice with PBS, the bacterial precipitate was resuspended with sterile PBS, the OD600 value of the bacterial suspension was measured, and the concentration of the bacterial suspension was adjusted to 10 10 CFU / ml, and then the bacterial suspension was evenly sprayed on the surface of the feed with the basic feed and the basic feed added with 0.1% β-glucan at a ratio of 1:10 (weight ratio), and dried at 37℃ for 4-8h to obtain a strain E26 content of 10 9 CFU / g of basal feed, and β-glucan content of 0.1% (by weight), and strain E26 content of 10 9 CFU / g of basic feed. In order to maintain the vitality of the strain, the above feed is prepared every 3 days.

[0046] Furthermore, obtaining the above strain E26 includes the following steps:

[0047] 1.2.1 Shrimp farming

[0048] 25 juvenile shrimps with an initial body weight of 0.1±0.005 g were randomly placed in a culture tank for culture. The salinity of the culture water was 3. The culture lasted for 8 weeks. During the culture period, the shrimps were fed four times a day at 7, 12, 18, and 23 o'clock, respectively. The feed was the basic feed with a β-glucan content of 0.1% (by weight). The daily water exchange rate was 55%, and the ammonia nitrogen and nitrite concentrations of the culture water were regularly tested to ensure that the water quality was maintained at an appropriate level.

[0049] 1.2.2 Strain isolation and purification

[0050] After the breeding experiment, the body surface of the Vannamei shrimp was wiped and disinfected with 75% alcohol by volume, and then placed in a clean bench. The intestines of the shrimp were picked up with sterile tweezers, weighed, and placed in a homogenizer. 9 times the volume of PBS was added to fully homogenize, and then the homogenate was homogenized with PBS for 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 For the gradient dilution, select 50 μL of 10 -3 Up to 10 -8The dilutions were spread in quadruplicate on 2216E plates, MRS plates and LB plates prepared in advance (each plate was spread with 6 dilutions of different concentrations), and cultured in a 30°C constant temperature incubator for 24 h. Single colonies with clear outlines and appropriate numbers on each plate were selected for streak purification. The purified strains were numbered in sequence and inoculated into the corresponding liquid culture medium, and cultured on a constant temperature shaker at 28°C and 200 rpm for 24 h. Then the bacterial solution was mixed with sterilization preservation solution (distilled water: glycerol = 1:1, volume ratio) in equal proportions and stored in a -80°C refrigerator.

[0051] 1.2.3 Hemolytic activity assay

[0052] The preserved strain (i.e. the purified strain mentioned above) was activated, and was spotted on the blood plate with a sterilized toothpick in the clean bench. After numbering, it was placed in a 30°C constant temperature incubator for 24 hours to observe whether there was hemolysis around the colonies. Strains with hemolytic rings were directly discarded. Strains without hemolytic rings were proved to be non-pathogenic and could be used as potential probiotics for the next test.

[0053] 1.2.4 Screening of enzyme-producing strains

[0054] First, prepare the amylase plate culture medium and the protease plate culture medium, and the preparation methods are as follows: (1) Amylase plate: add soluble starch to 2216E, MRS and LB agar culture medium at a ratio of 1% (weight ratio), heat to dissolve, and sterilize at 115°C. Pour the plate after cooling to 60°C, and seal and store in a refrigerator at 4°C after complete solidification. (2) Protease plate: add skim milk powder to 2216E, MRS and LB agar culture medium at a ratio of 1%, and the remaining steps are the same as those of the amylase plate.

[0055] After the plates are prepared, the preserved strains without hemolytic activity are revived and activated, and spotted on the corresponding protease and amylase plates with sterilized toothpicks. After numbering, they are placed in a 30°C constant temperature incubator for 24 hours. After the incubation, 1 mL of 1% Lugol's iodine solution is added to the amylase plate, and the appearance of a transparent circle is observed after 10 minutes. The presence of a transparent circle proves that the strain has the ability to produce amylase; after incubation on the protease plate, the appearance of a transparent circle can be directly observed. The presence of a transparent circle proves that the strain has the ability to produce protease.

[0056] As shown in Table 3, in a low-salt environment, after feeding a basic feed with a β-glucan content of 0.1%, a total of 155 bacterial strains were screened from the intestinal tract of Penaeus vannamei, of which 80 strains (E1-E80) were screened from the 2216E medium, 32 strains (B1-B32) were screened from the MRS medium, and 43 strains (L1-L43) were screened from the LB medium. Among them, there were 7 strains with hemolysis positive (marked in bold in Table 3, Figure 1There were 41 strains with the ability to produce both protease and amylase ( Figure 1 The specific enzyme production capacity is shown in Table 3 (+ indicates enzyme production capacity, blank indicates no enzyme production capacity).

[0057] Table 3 Protease and amylase production capacity of strains

[0058]

[0059]

[0060]

[0061] 1.2.5 Screening of antibacterial strains

[0062] The 41 strains with the ability to produce both protease and amylase obtained in Section 1.2.4 were tested for enzyme production again, and the transparent zone diameter and colony diameter were recorded. The ratio of the transparent zone diameter to the colony diameter was used as the standard for enzyme activity. Finally, 14 strains with strong enzyme production ability were obtained (as shown in Table 4).

[0063] Three aquatic pathogenic bacteria, Vibrio Parahaemolyticus, Vibrio harveyi and Streptococcus iniae, were used as pathogen indicator strains. The above pathogens were activated in 2216E liquid culture medium, and then 50 μL of each was applied on the plate, and a sterile hole puncher with a diameter of 6 mm was used to punch holes on the plate. Then, the above 14 strains with strong enzyme production ability were activated, and 50 μL of the activated bacterial solution was added to each hole. After numbering, they were placed in a 28°C constant temperature incubator and cultured for 24 hours. After the culture was completed, it was observed whether there was an inhibition zone around the hole, and the diameter of the inhibition zone was measured. The appearance of the inhibition zone indicated that the strain had an antagonistic effect on the corresponding pathogen. The strains with antagonistic effects on multiple pathogens were selected for subsequent experiments.

[0064] As can be seen from Table 4, the number of strains that can antagonize 1 pathogen, 2 pathogens, and 3 pathogens are 5, 6, and 2, respectively.

[0065] Table 4 Digestive enzyme production and antagonistic ability of 14 strains

[0066]

[0067]

[0068] Note: Enzyme activity is expressed as the ratio of transparent zone diameter to colony diameter. + represents <1.5, ++ represents 1.5-2.0, +++ represents >2.0, and - represents no enzyme activity.

[0069] 1.2.6 Evaluation of strain tolerance to artificial gastrointestinal fluid

[0070] 0.5% NaCl (by weight) and 0.3 mg / mL pepsin were added to PBS buffer (by volume), and the mixture was sterilized using a filter membrane to prepare artificial gastric juice. 0.1 mg / mL trypsin and 0.3% ox bile salt (by weight) were added to PBS buffer (by volume), and the mixture was sterilized using a filter membrane to prepare artificial intestinal juice.

[0071] The strains with the best enzyme-producing antibacterial effect (E4, E26, E57, B4, B32) screened in Section 1.2.5 were revived and activated, and then centrifuged to obtain the corresponding bacterial precipitates. The bacterial precipitates were then added to the prepared artificial gastric juice and artificial intestinal juice and resuspended (the pH of the artificial gastric juice was adjusted to 3, and the pH of the artificial intestinal juice was adjusted to 6.8) to obtain artificial gastric juice suspension bacteria solution and artificial intestinal juice suspension bacteria solution. The artificial gastric juice suspension bacteria solution and artificial intestinal juice suspension bacteria solution were then diluted by the same multiples, and the dilutions were spread on plates at 0h, 2h, and 4h, respectively, and counted. The survival rates of the five strains after 2h in artificial gastric juice and 4h in artificial intestinal juice were calculated.

[0072] like Figure 2 As shown, the survival rates of E26 and B32 strains after artificial simulated gastrointestinal fluid were significantly higher than those of the other three strains (P<0.05).

[0073] 1.2.7 Evaluation of surface hydrophobicity and self-agglutination rate of strains

[0074] Surface hydrophobicity: The surface hydrophobicity of the strains (E4, E26, E57, B4, B32) with the best enzyme-producing antibacterial effect screened in Section 3.4 was determined using three organic solvents: xylene, chloroform, and ethyl acetate. The liquid culture medium after strain activation was centrifuged at 4000r / min for 10min to collect the strain sediment, which was washed twice with sterile PBS and resuspended to obtain a bacterial suspension. The initial absorbance value A was measured using a spectrophotometer at 600nm. Equal volumes of the bacterial suspension were fully mixed with the three organic solvents, respectively, and allowed to stand at room temperature for 1h to absorb the aqueous phase, and the absorbance value A1 at 600nm was measured. The surface hydrophobicity calculation formula is:

[0075] Surface hydrophobicity (%) = (A-A1) / A×100%.

[0076] Self-agglutination rate: The operation steps are the same as above to obtain a bacterial suspension, which is allowed to stand at room temperature. The absorbance values ​​of the bacterial suspension at 0h (A) and 4h (A1) are measured using a 600nm spectrophotometer. The formula for calculating the self-agglutination rate is:

[0077] Self-aggregation rate (%) = (A-A1) / A×100%.

[0078] like Figure 3 As shown in the results, the above five strains have different hydrophobicity to the three hydrocarbons. In xylene, the surface hydrophobicity of strain B4 is significantly higher than that of the other four strains (P<0.05); in chloroform and ethyl acetate, E26 and B32 are significantly higher than the other three strains (P<0.05). The self-agglutination rate of strain B32 is significantly higher than that of the other four strains (P<0.05), and there is no significant difference between the other four strains.

[0079] 1.2.8 Evaluation of antibiotic sensitivity of strains

[0080] The sensitivity of the strains (E4, E26, E57, B4, B32) with the best enzyme-producing antibacterial effect screened in Section 1.2.5 to antibiotics was determined by the paper agar diffusion method. 50 μL of the activated bacterial solution of each strain was applied to the plate, and various antibiotic paper discs were attached to the plate. The plate was placed in a 28°C constant temperature incubator for 24 hours. Whether there was a transparent circle around the paper disc and the diameter of the transparent circle was recorded. The presence of a transparent circle indicated that the strain was sensitive to the antibiotic. As shown in Table 5, strains E4, E26, and B4 were highly resistant to most antibiotics.

[0081] Table 5 Drug sensitivity results of five strains to 22 antibiotics

[0082]

[0083]

[0084] 1.2.9 Strain identification

[0085] Based on the comprehensive consideration of digestive enzyme production ability, pathogen antagonism ability, adhesion and colonization ability, and safety adaptability, strain E26 was finally selected as the target probiotic.

[0086] The strain E26 was activated and cultured for 24 h, and the bacterial solution was sent to Haikou Nanshan Gene Biotechnology Co., Ltd. for 16S rDNA gene sequencing. Blast sequence alignment was performed in the GenBank database, and the strain phylogenetic tree was constructed using MEGA11 ( Figure 4), it was found that strain E26 and Klebsiella michiganensis were clustered into a branch with a similarity of 99%. Therefore, the strain E26 is a strain of Klebsiella, and the Klebsiella is Klebsiella sp. E26, and it is deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China), with a deposit number of: CCTCC NO: M20241218, and a deposit date of June 14, 2024.

[0087] 1.3 Breeding management

[0088] Vannamei shrimp fry were purchased from a shrimp fry factory in Wenchang, Hainan. Twenty-five young shrimps with an initial body weight of 0.15±0.006 g were randomly placed in the four groups of culture tanks (each group was equipped with four parallel tanks (60×30×36 cm)) of "low-salt control group", "Klebsiella group", "β-glucan group" and "combined use group" in Table 1. The salinity of the culture water in each tank was 3. The culture experiment lasted for 8 weeks. During the culture period, the corresponding feed was fed four times a day at 7, 12, 18 and 23 o'clock according to the design in Table 1. The daily water exchange rate was 50%, and the ammonia nitrogen and nitrite concentrations of the culture water were regularly tested to ensure that the water quality was maintained at an appropriate level.

[0089] 1.4 Growth index determination

[0090] After the breeding experiment in Section 1.3, the growth index of shrimp was calculated using the following formula:

[0091] (1) Survival rate (%) = (initial number of shrimp / final number of shrimp) × 100%

[0092] (2) Weight gain (%) = (final shrimp weight - initial shrimp weight) / initial shrimp weight × 100%

[0093] (3) Hepatosomatic index (%) = (hepatopancreas weight / shrimp weight) × 100%

[0094] (4) Condition factor, g / cm 3 ) = shrimp weight / shrimp length 3 ×100%.

[0095] 1.5 Growth performance analysis

[0096] The breeding data were expressed as mean ± standard error (mean ± SE). SPSS25.0 analysis software, one-way analysis of variance and Duncan multiple comparisons were used to test the differences in various indicators under different feed conditions. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0097] like Figure 5 As shown, the survival rate, weight gain rate and liver-body index of the combined use group (i.e., "β-glucan+Probiotic") were significantly higher than those of the other groups (P<0.05), especially the survival rate exceeded 85%. The survival rates of the β-glucan group (i.e., "β-glucan") and the Klebsiella group (i.e., "Probiotic") were also significantly higher than those of the control group (i.e., "Control") (P<0.05).

[0098] It can be seen that the combined addition of 0.1% β-glucan and 10 9 The probiotic E26 with a CFU / g content can significantly increase the weight gain rate, survival rate and liver-body index of Penaeus vannamei under low-salt conditions, thereby improving the growth performance of shrimp under low-salt conditions.

[0099] 2. The application of β-glucan and Klebsiella in improving the immunity of Penaeus vannamei in low-salt environment

[0100] 2.1 Sample collection

[0101] After the breeding experiment, the shrimp were fasted for 24 hours. Then, after the shrimp surface was disinfected, hemolymph was extracted from the pericardial sinus with a disposable syringe, placed at 4°C overnight and centrifuged the next day, and serum was collected and stored at -80°C. After hemolymph was extracted, the shrimp were dissected, and the hepatopancreas, intestines, and muscles were quickly separated and stored in sterilized cryopreservation tubes, placed in liquid nitrogen, and stored in a -80°C refrigerator after quick freezing with liquid nitrogen for enzyme activity determination and intestinal microbial sequencing.

[0102] 2.2 Determination of immune indicators

[0103] Total hemocyte counts: Pipette 50 μL of freshly extracted shrimp hemolymph and immediately place it in a hemocyte counting plate. Count under a microscope and calculate the number of shrimp hemolymph per mL according to the formula.

[0104] Respiratory burst activity: The OD of the sample was measured by reducing formazan with nitro blue tetrazolium chloride (NBT). 630nm , and calculated the respiratory burst activity.

[0105] Hemocyanin concentration: Mix 30 μL hemolymph with 270 μL double distilled water, place on a 96-well plate, and read the OD using an ELISA reader. 350nm The calculation formula is: Hemocyanin content (mg / mL) = 2.3 × OD 335nm ;

[0106] Lytic activity (LZM): Determined by referring to the method of Wang Yiyan et al. (Wang Yiyan, Sun Hushan, Li Guangyou. Effect of compound immune drugs on the antibacterial and lytic activity of Chinese shrimp hemolymph [J]. Progress in Marine Science, 2004, (1): 69-72).

[0107] 2.3 Analysis of immune indicators

[0108] The indicator data analysis method is the same as Section 1.5 “Growth Performance Analysis” and will not be repeated here.

[0109] like Figure 6 As shown, the respiratory burst activity, complete blood cell count and hemocyanin content of the combined use group were significantly higher than those of the other three groups (P<0.05), and the hemocyanin content and respiratory burst activity of the β-glucan group were significantly higher than those of the Klebsiella group and the control group (P<0.05).

[0110] Hemolymph plays a very important role in the immune system of crustaceans. The number of blood cells, respiratory burst activity, hemocyanin content and lytic activity are all commonly used immune indicators in aquatic animals. Respiratory burst is one of the bactericidal pathways of oxygen-dependent phagocytes and is often used as one of the important indicators to measure the activity of phagocytes. Hemocyanin is a respiratory protein present in the hemolymph of many arthropods. This protein is related to many physiological functions such as energy storage, maintenance of osmotic pressure and regulation of molting process. At the same time, hemocyanin can also produce antimicrobial peptides through self-cleavage, thereby inhibiting the growth and reproduction of certain microorganisms, and has certain antibacterial functions.

[0111] In the examples, the respiratory burst activity, blood cell count, hemocyanin content and lytic activity of the combined use group were significantly increased, indicating that the combined addition of β-glucan and strain E26 can effectively improve the nonspecific immunity of Penaeus vannamei under low-salt conditions, and the prebiotic function is better than that of β-glucan or strain E26 alone.

[0112] 3. Application of β-glucan and Klebsiella in improving the digestion and absorption capacity of Penaeus vannamei in low-salt environment

[0113] 3.1 Determination of digestive enzyme index

[0114] The hepatopancreas samples obtained in Section 2.1 were weighed and thoroughly homogenized with physiological saline at a ratio of 1:9 (weight ratio), and the digestive enzyme activities were determined using amylase, trypsin and lipase kits purchased from Nanjing Jiancheng Biological Co., Ltd.

[0115] 3.2 Analysis of digestive enzyme indicators

[0116] The main digestive organs of Penaeus vannamei are the hepatopancreas and intestines, so measuring the activity of digestive enzymes in them can reflect the body's ability to digest and absorb nutrients. Figure 7 It can be seen that the activities of trypsase, lipase and amylase in the hepatopancreas tissue of the combined use group were significantly higher than those of the other three groups (P<0.05). The reason is that strain E26 can secrete digestive enzymes, thereby increasing the activity of digestive enzymes in the shrimp, thereby improving the growth performance of the shrimp.

[0117] 4. Application of β-glucan and Klebsiella in regulating the intestinal microbial flora structure of Penaeus vannamei in low-salt environment

[0118] 4.1 Gut microbial sequencing

[0119] The intestinal tissues of every four shrimps (obtained in Section 2.1) were mixed into one sample, and four samples were selected from each treatment group and transported to Shanghai Baiqu Medical Technology Co., Ltd. on dry ice. DNA extraction, library construction, and bacterial species sequencing of the samples were all completed by the company. After sequencing, the α diversity, β diversity, bacterial species changes between phyla and genera, and interspecies interactions between the groups were analyzed on the platform to comprehensively and systematically analyze the changes in the intestinal flora between the groups.

[0120] 4.2 Analysis of intestinal flora

[0121] pass Figure 8 The Venn diagram shows that a total of 3126 OTUs were obtained after sequencing, of which the combined use group had the most OTUs, and there were 167 OTUs shared by the four groups ( Figure 8 At the phylum level, Proteobacteria, Planctomycetota, and Firmicutes were the top three dominant phyla. Proteobacteria in the three treatment groups increased compared with the control group, while Firmicutes decreased. The most obvious change was in the combined use group ( Figure 8 in Section C).

[0122] At the genus level, the dominant genus in each group was different. The most common genus in the combined use group and the β-glucan group was Rhodobacter, the most common genus in the Klebsiella group was Lysobacter, and the most common genus in the control group was Halomonas. Compared with the control group, the proportions of Rhodobacter and Lysobacter in the β-glucan group increased, while the proportion of Halomonas decreased ( Figure 8 in Section D).

[0123] The relative abundance of Firmicutes was quite different among the groups, with the control group accounting for more than 20% and the Klebsiella group accounting for less than 5%. The results of principal coordinate analysis (PCoA) showed that in a low-salt environment, the addition of β-glucan and strain E26 to feed could change the intestinal microbial flora structure of Penaeus vannamei ( Figure 8 The intestinal flora structure of the combined use group was more densely clustered compared with the control group.

[0124] Further, from Fig. 9 The α-diversity analysis showed that the Chao 1 index and observed index of the combined use group were significantly higher than those of the other three groups ( Fig. 9 The Chao1 index of the combined use group was more than twice that of the other groups. There was no significant difference in the Shannon index and Simpson index among the groups ( Fig. 9 Part C and D of the .

[0125] Fig.10 The KEGG secondary function prediction analysis showed that compared with the control group, the combined use group was significantly enriched in the amino acid metabolism and cell growth and apoptosis pathways; the KEGG tertiary function prediction analysis showed that the combined use group was significantly enriched in the histidine metabolism pathway, and the β-glucan group was significantly enriched in the phosphorylation transproteinase system pathway ( Fig.10 in Section C).

[0126] As the main digestive organ of shrimp, the intestine mainly realizes its various functions through intestinal microbial metabolism. The structure and composition of intestinal flora are of great significance to the host's nutrient absorption, nonspecific immunity and energy balance.

[0127] The intestinal flora results in this example show that the addition of strain E26, β-glucan, and the combination of β-glucan and strain E26 in the feed can change the intestinal microbial flora structure of Penaeus vannamei.

[0128] Among them, the Chao 1 index and observed index of the combined use group were significantly higher than those of the control group, indicating that the simultaneous addition of strain E26 and β-glucan in the feed can improve the richness of the intestinal flora of Penaeus vannamei under low-salt conditions, and higher α-diversity also represents the improvement of the resistance of shrimp to pathogenic microorganisms and environmental adaptability, which is also one of the reasons for improving the survival rate of Penaeus vannamei under low-salt conditions.

[0129] At the phylum level, Proteobacteria, Firmicutes and Planctomyces were the top three dominant phyla. Compared with the control group, the proportion of Firmicutes in each treatment group decreased, but the overall change was not significant. In addition, the relative abundance of Planctomyces in the combined use group and Klebsiella group was significantly higher than that in the control group. It is speculated that adding strain E26 to the feed may increase the growth and colonization of Planctomyces in the intestine of Penaeus vannamei.

[0130] At the genus level, the dominant genus in each group was different. Rhodobacter accounted for the largest proportion in the combined use group and the β-glucan group, Lysobacter accounted for the largest proportion in the Klebsiella group, and Halomonas accounted for the largest proportion in the control group. Compared with the control group, the proportions of Rhodobacter and Lysobacter increased, while Vibrio decreased. Vibrio is mostly aquatic pathogens. If the number is too large, it will cause intestinal dysfunction and threaten the health of the body. Lysobacter is a Gram-negative bacterium with strong antibacterial activity. It can produce antibacterial substances such as lysozyme and lipopeptides. In addition, Lysobacter can promote the absorption of nutrients and thus promote the growth of Penaeus vannamei. At the same time, Pseudoalteromonas, a genus similar to this bacterium, can significantly improve the immune response of Penaeus vannamei and its resistance to Vibrio harveyi.

[0131] Therefore, adding strain E26 and β-glucan to the feed can inhibit the growth and reproduction of harmful bacteria in the shrimp intestine, thereby improving the health of the body. In addition, the intestinal flora KEGG secondary function prediction analysis showed that the combined use group was significantly enriched in the amino acid metabolism pathway compared with the control group, indicating that adding strain E26 and β-glucan to the feed can improve the absorption and metabolism of nutrients by intestinal microorganisms of Vannamei shrimp under low-salt stress, providing more energy for shrimp to cope with the low-salt environment.

[0132] In summary, in the present invention, by adding 0.1% β-glucan and 10 9 CFU / g of the bacterial strain E26 can significantly improve the weight gain rate, survival rate, nonspecific immunity, trypsin activity and intestinal flora abundance of Penaeus vannamei under low-salt conditions, and comprehensively alleviate the negative effects of low-salt stress on shrimp.

[0133] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A strain of Klebsiella, characterized in that The Klebsiella is Klebsiella sp. E26, and is deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with a deposit number of CCTCC NO: M20241218 and a deposit date of June 14, 2024.

2. Use of the Klebsiella E26 according to claim 1 in preparing a preparation for inhibiting pathogenic bacteria.

3. Use of the Klebsiella E26 according to claim 1 in improving the digestion capacity of Penaeus vannamei in a low-salt environment.

4. Use of Klebsiella E26 according to claim 1 in improving the resistance of Penaeus vannamei to antibiotics.

5. Use of β-glucan and the Klebsiella E26 described in claim 1 in combination to improve the growth performance of Penaeus vannamei in a low-salt environment.

6. Use of β-glucan and the Klebsiella E26 described in claim 1 in combination to improve the immunity of Penaeus vannamei in a low-salt environment.

7. The use according to claim 6, characterized in that The indicators of the immune capacity include respiratory burst activity and / or hemocyanin content and / or bacteriolytic activity.

8. Use of β-glucan and the Klebsiella E26 described in claim 1 in combination to improve the digestion and absorption capacity of Penaeus vannamei in a low-salt environment.

9. The use according to claim 8, characterized in that The digestion and absorption capacity of Penaeus vannamei in a low-salt environment is improved by increasing the activity of trypsin and / or lipase and / or amylase in the hepatopancreas tissue of Penaeus vannamei.

10. Use of β-glucan and the Klebsiella E26 described in claim 1 in combination to regulate the intestinal microbial flora structure of Penaeus vannamei in a low-salt environment.

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