Application of beta-glucan in relieving low-salt stress negative effect of penaeus vannamei

Abstract

The invention discloses an application of beta-glucan in relieving low-salt stress negative effects of penaeus vannamei, which can improve the growth performance and immunity of the penaeus vannamei in a low-salt environment through the beta-glucan, and can significantly improve the relative abundance of a probiotic strain E26 in the intestinal tract of the penaeus vannamei. The negative effect of low salt stress of the penaeus vannamei is comprehensively relieved.

Description

Technical Field

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

[0002] In the current farming process of Penaeus vannamei, commonly used probiotics include Bacillus, lactic acid bacteria, etc., which can promote the growth of Penaeus vannamei, activate the immune system, improve disease resistance, inhibit the growth of Vibrio in the surrounding environment, and increase the specific growth rate and apparent digestibility of Penaeus vannamei. However, there are few reports on alleviating the low-salt stress of shrimp. Summary of the invention

[0003] In view of the above technical problems, the present invention provides an application of β-glucan in alleviating the negative effects of low-salt stress on Penaeus vannamei. The β-glucan improves the growth performance and immune ability of Penaeus vannamei in a low-salt environment, and can significantly increase the relative abundance of probiotic strain E26 in the shrimp intestine, so as to comprehensively alleviate the negative effects of low-salt stress on Penaeus vannamei.

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

[0005] Provided is an application of beta-glucan in improving the growth performance of Penaeus vannamei in a low-salt environment.

[0006] Preferably, the growth performance includes one or more of weight gain rate, survival rate, and hepatosomatic index.

[0007] Provided is an application of beta-glucan in improving the immunity of Penaeus vannamei in a low-salt environment.

[0008] Preferably, the immune capacity index includes respiratory burst activity and / or phenol oxidase activity.

[0009] Provided is an application of β-glucan in increasing the abundance of Klebsiella E26 in Penaeus vannamei under a low-salt environment. The Klebsiella E26 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.

[0010] Provided is a Klebsiella E26 obtained by the above application, wherein the Klebsiella E26 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.

[0011] Provided is a use of the Klebsiella E26 in preparing a preparation for inhibiting pathogenic bacteria.

[0012] Preferably, the pathogenic bacteria include one or more of Streptococcus iniae, Vibrio parahaemolyticus, and Vibrio harveyi.

[0013] Provided is an application of the Klebsiella E26 in improving the digestion capacity of Penaeus vannamei in a low-salt environment.

[0014] Preferably, the Klebsiella E26 improves the digestibility of Penaeus vannamei by secreting amylase and / or protease.

[0015] Provided is a use of the Klebsiella E26 in improving the resistance of Penaeus vannamei to antibiotics.

[0016] The present invention has at least the following beneficial effects:

[0017] In the present invention, by adding 1‰ of β-glucan to the feed, the growth performance and immune ability of Penaeus vannamei in a low-salt environment can be significantly improved, and the relative abundance of the probiotic strain E26 in the shrimp intestine can be significantly increased. The strain E26 has good enzyme production and antibacterial effect, high biosafety, strong adhesion and colonization ability, etc., so as to comprehensively alleviate the negative effects of low-salt stress on Penaeus vannamei. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 Effects of different water salinity and β-glucan content on the growth performance of Penaeus vannamei, including (A) weight gain rate, (B) survival rate, (C) liver and body index, (D) fatness, * indicates significant difference among groups (P<0.05);

[0020] Figure 2Effects of different water salinity and β-Glucan content on immune parameters of Penaeus vannamei, including (A) blood cell count, (B) respiratory burst activity, (C) phenoloxidase activity, * indicates significant difference among groups (P<0.05), ** indicates extremely significant difference (P<0.05);

[0021] Figure 3 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 4 The 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 5 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 6 The phylogenetic tree is constructed based on the 16S rRNA sequencing information of strain E26;

[0025] Figure 7 The relative abundance of strain E26 in the shrimp intestine under different groups. DETAILED DESCRIPTION

[0026] 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.

[0027] Embodiment 1:

[0028] This embodiment provides an application of β-glucan in alleviating the negative effects of low-salt stress on Penaeus vannamei, which specifically includes the following contents:

[0029] 1. Application of β-glucan in improving the growth performance of Penaeus vannamei in low-salt environment

[0030] The "low-salinity environment" refers to the salinity of the water body for breeding Penaeus vannamei being 1-10.

[0031] 1.1 Breeding experiment design

[0032] The aquaculture experiment was divided into 4 treatment groups: low-salt control group, β-glucan low-salt group, β-glucan seawater group and seawater control group. Each treatment group had 4 biological parallels. The specific grouping is shown in Table 1.

[0033] Table 1 Design of breeding experiment

[0034]

[0035] 1.2 Experimental feed formulation and preparation

[0036] In this embodiment, two kinds of feeds with equal nitrogen and lipid content were prepared, namely "basic feed" and "feed of the present invention" added with β-glucan (containing 1‰ β-glucan by weight). The specific formulas are shown in Table 2.

[0037] Table 2 Feed formula and composition

[0038]

[0039]

[0040] 1.3 Breeding management

[0041] Vannamei shrimp fry were purchased from a shrimp fry factory in Wenchang, Hainan. Twenty-five young shrimps with an initial body weight of 0.1±0.005 g were randomly placed in the four groups of culture tanks in Table 1.1, namely, “low-salt control group”, “β-glucan low-salt group”, “β-glucan seawater group” and “seawater control group” (each group was equipped with four parallel tanks (60×30×36 cm)). The culture experiment lasted for 8 weeks. During the culture period, the shrimps were fed four times a day at 7:00, 12:00, 18:00 and 23:00, respectively. 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.

[0042] 1.4 Growth index determination

[0043] After the breeding experiment in Section 1.3, the growth index of shrimp was calculated using the following formula:

[0044] (1) Survival rate (%) = (initial number of shrimp / final number of shrimp) × 100%

[0045] (2) Weight gain (%) = (final shrimp weight - initial shrimp weight) / initial shrimp weight × 100%

[0046] (3) Hepatosomatic index (%) = (hepatopancreas weight / shrimp weight) × 100%

[0047] (4) Condition factor, g / cm 3 ) = shrimp weight / shrimp length 3 ×100%.

[0048] 1.5 Growth performance analysis

[0049] The breeding data were expressed as mean ± SE, and SPSS25.0 analysis software was used to perform independent sample T tests for the same water salinity group and the same β-glucan addition group. In addition, two-way ANOVA was used to test the effects of water salinity ("Salinity"), β-glucan addition ("β-Glucan"), and the interaction between the two ("Salinity×β-Glucan") on the experimental results. P<0.05 indicated a significant difference, and P<0.01 indicated an extremely significant difference.

[0050] like Figure 1 As shown in Figure 2, the weight gain rate of shrimp was significantly affected by the amount of β-glucan added (P<0.05). At salinity of 3 and 25, the weight gain rate of the group fed with the feed of the present invention was significantly higher than that of the control group (P<0.05) ( Figure 1 The salinity of the water body had a significant effect on the survival rate of Penaeus vannamei (P<0.05). Under the same feed conditions, the survival rate of the 3-salinity group was significantly lower than that of the 25-salinity control group (P<0.05). However, compared with the control group, feeding the feed of the present invention could still improve the survival rate of the shrimp to a certain extent ( Figure 1 The hepatic index was significantly affected by the amount of β-glucan added, and the hepatic index of the β-glucan low-salt group was significantly lower than that of the β-glucan seawater group (P<0.05) ( Figure 1 Water salinity, β-glucan addition, and their interaction had no significant effect on fatness, and there was no significant difference in fatness among the treatment groups (P<0.05) ( Figure 1 in Section D).

[0051] It can be seen that by adding β-glucan to the feed, the weight gain rate, survival rate, and liver-body index of shrimp in a low-salinity environment can be significantly increased, thereby improving the growth performance of shrimp.

[0052] 2. Application of β-glucan in improving the immunity of Penaeus vannamei in low-salt environment

[0053] 2.1 Sample collection

[0054] After the breeding experiment, the shrimps were fasted for 24 h. 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 shrimps 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 by liquid nitrogen for subsequent determination of immune, antioxidant and other enzyme activities.

[0055] 2.2 Determination of immune indicators

[0056] 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.

[0057] 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.

[0058] Phenol oxidase activity: Prepare 0.3% catechol (L-DOPA) with sterile water as the substrate of the enzyme, mix 10 μL of the substrate solution with an equal volume of serum, then add 80 μL of normal saline, place at room temperature for 30 min, and measure the absorbance at a wavelength of 490 nm on a spectrophotometer.

[0059] 2.3 Analysis of immune indicators

[0060] The indicator data analysis method is the same as Section 1.5 “Growth Performance Analysis” and will not be repeated here.

[0061] like Figure 2 As shown in the figure, when the amount of β-glucan added was the same, the complete blood cell count and respiratory burst activity of the 3-salinity group were significantly higher than those of the 25-salinity control group (P<0.05) ( Figure 2 The phenoloxidase activity in the low-salt group of β-glucan was significantly higher than that in the seawater group of β-glucan (P<0.05) ( Figure 2 Section C of the Regulations);

[0062] Moreover, under the same salinity conditions, the respiratory burst activity and phenoloxidase activity of the β-glucan group were significantly higher than those of the control group (P<0.05) ( Figure 2 The difference in respiratory burst activity was extremely significant (P<0.01) ( Figure 2 in Part B).

[0063] Respiratory burst activity and phenoloxidase activity are both common immune indicators of crustaceans. Respiratory burst is one of the oxygen-dependent phagocyte bactericidal pathways and is often used as one of the important indicators for measuring phagocyte activity. The humoral immunity of crustaceans includes the prophenoloxidase activation system, various lectins and antimicrobial peptides. β-glucan can activate the prophenoloxidase system in the body, promote the conversion of prophenoloxidase into phenoloxidase (PO), and produce 76KD protein and α-macroglobulin. The short-term oxidation products (monophenol and phenol) of this reaction chain can kill foreign pathogens and enhance specific immunity. As mentioned above, under low-salt conditions, adding 1‰ of β-glucan to the feed can significantly increase the respiratory burst activity and phenoloxidase activity of shrimp, and at a salinity of 25, the respiratory burst of the β-glucan group is also significantly higher than that of the control group. It can be concluded that adding 1‰ of β-glucan to the feed can significantly enhance the nonspecific immunity of Vannamei shrimp under low-salt conditions.

[0064] 3. Application of β-glucan in increasing the abundance of Klebsiella E26 in shrimp Penaeus vannamei under low salt conditions

[0065] 3.1 Isolation and purification of strains

[0066] After the breeding experiment, the body surface of the Vannamei shrimp in the β-glucan low-salt group 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 -8 The 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.

[0067] 3.2 Hemolytic activity assay

[0068] 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.

[0069] 3.3 Screening of enzyme-producing strains

[0070] 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.

[0071] 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.

[0072] As shown in Table 3, in a low-salt environment, after feeding the feed of the present invention, a total of 155 bacterial strains were screened in the intestine of Penaeus vannamei, of which 80 strains (E1-E80) were screened out by 2216E medium, 32 strains (B1-B32) were screened out by MRS medium, and 43 strains (L1-L43) were screened out by LB medium. Among them, there were 7 strains with hemolysis positive (marked in bold in Table 3, Figure 3 There were 41 strains with the ability to produce both protease and amylase ( Figure 3 The specific enzyme production capacity is shown in Table 3 (+ indicates enzyme production capacity, blank indicates no enzyme production capacity).

[0073] Table 3 Protease and amylase production capacity of strains

[0074]

[0075]

[0076] 3.4 Screening of antibacterial strains

[0077] The 41 strains with the ability to produce both protease and amylase obtained in Section 3.4 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).

[0078] 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.

[0079] 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.

[0080] Table 4 Digestive enzyme production and antagonistic ability of 14 strains

[0081]

[0082] 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.

[0083] 3.5 Evaluation of strain tolerance to artificial gastrointestinal fluid

[0084] 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.

[0085] The strains with the best enzyme-producing antibacterial effect (E4, E26, E57, B4, B32) screened in Section 3.4 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.

[0086] like Figure 4 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).

[0087] 3.6 Evaluation of surface hydrophobicity and self-agglutination rate of strains

[0088] 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:

[0089] Surface hydrophobicity (%) = (A-A1) / A×100%.

[0090] 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:

[0091] Self-aggregation rate (%) = (A-A1) / A×100%.

[0092] like Figure 5 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.

[0093] 3.7 Evaluation of antibiotic sensitivity of strains

[0094] The sensitivity of the strains (E4, E26, E57, B4, B32) screened in Section 3.4 with the best enzyme-producing antibacterial effect 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.

[0095] Table 5 Drug sensitivity results of five strains to 22 antibiotics

[0096]

[0097]

[0098] 3.8 Strain identification

[0099] 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.

[0100] 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 6 ), 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.

[0101] 3.9 Abundance calculation

[0102] The relative abundance of strain E26 obtained in Section 3.8 in the shrimp intestine was calculated based on 16s rRNA sequencing data.

[0103] Repeat the operations in Sections 3.1-3.9 to separate, purify and identify the strains in the shrimp intestines of the low-salt control group, β-glucan seawater group, and seawater control group. Finally, it was found that strain E26 existed in the shrimp intestines of the low-salt control group, β-glucan seawater group, and seawater control group. The relative abundance of strain E26 in the shrimp intestines of the low-salt control group, β-glucan seawater group, and seawater control group was further calculated. The results are shown in the figure. Figure 7 shown.

[0104] from Figure 7 It can be concluded that at salinity 3 and 25, after feeding the feed of the present invention, the relative abundance of strain E26 in the intestine of shrimp was significantly higher than that of the control group (P<0.05), and the relative abundance in the salinity environment of 3 was higher than that in the salinity environment of 25. This shows that adding 1‰ β-glucan to the feed can increase the abundance of Klebsiella E26 in the body of Penaeus vannamei in a low-salt environment.

[0105] Through the above-mentioned enzyme production and antibacterial experiments, it was found that strain E26 can secrete amylase and protease at the same time and has a good enzyme production effect. It has a significant inhibitory effect on three common aquatic pathogens (Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio harveyi), indicating that strain E26 has the potential to be used as an aquatic probiotic.

[0106] At the same time, if probiotics want to truly play a probiotic role in aquatic animals, they also need to tolerate the harsh physiological environment in the gastrointestinal tract. Therefore, this example simulates the gastric juice and intestinal juice of the digestive tract of Penaeus vannamei. The results show that strain E26 has a strong tolerance to both simulated gastric juice and intestinal juice. The reason is that Klebsiella has a strong ability to adapt to the environment and can survive in many harsh environments.

[0107] In addition, the adhesion and colonization ability of the strain in the intestine is also one of the important criteria for determining whether it can be used as a probiotic, and the surface hydrophobicity and self-aggregation ability of the strain are common indicators for judging the adhesion and colonization ability, wherein the cell surface hydrophobicity of the microorganism is one of the important factors that determine its non-specific adhesion to various biological and non-biological surfaces and interfaces, and is also one of the main factors that affect the absorption and degradation of hydrophobic organic substances by microorganisms. This embodiment found that the surface hydrophobicity of strain E26 in three hydrocarbons (xylene, chloroform, ethyl acetate) was relatively high, wherein the hydrophobicity to chloroform exceeded 80%. It was found through antibiotic resistance detection that strain E26 was sensitive to most antibiotics and had high safety. This shows that strain E26 has good in vitro probiotic properties and has the potential to be a probiotic for Penaeus vannamei.

[0108] In summary, in the present invention, by adding 1‰ β-glucan to the feed, the growth performance and immune ability of Penaeus vannamei in a low-salt environment can be significantly improved, and the relative abundance of the probiotic strain E26 in the shrimp intestine can be significantly increased. The strain E26 has good enzyme production and antibacterial effect, high biosafety, and strong adhesion and colonization ability, so as to comprehensively alleviate the negative effects of low-salt stress on Penaeus vannamei.

[0109] 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. Application of β-glucan in improving the growth performance of Penaeus vannamei in a low-salt environment.

2. The use according to claim 1, characterized in that The growth performance includes one or more of weight gain rate, survival rate, and hepatosomatic index.

3. Application of β-glucan in improving the immunity of Penaeus vannamei in a low-salt environment.

4. The use according to claim 3, characterized in that The indicators of immune capacity include respiratory burst activity and / or phenol oxidase activity.

5. An application of β-glucan in increasing the abundance of Klebsiella E26 in Penaeus vannamei under a low-salt environment, wherein the Klebsiella E26 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.

6. A Klebsiella E26 obtained by the use of claim 5, wherein the Klebsiella E26 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.

7. Use of the Klebsiella E26 according to claim 6 in preparing a preparation for inhibiting pathogenic bacteria.

8. The use according to claim 7, characterized in that The pathogenic bacteria include one or more of Streptococcus iniae, Vibrio parahaemolyticus and Vibrio harveyi.

9. Use of the Klebsiella E26 according to claim 6 in improving the digestion ability of Penaeus vannamei in a low-salt environment.

10. Use of the Klebsiella E26 according to claim 6 in improving the resistance of Penaeus vannamei to antibiotics.

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