Use of beta-glucan and klebsiella combination in relieving low salt stress in vannamei shrimp

Abstract

The application discloses application of a combination of beta-glucan and Klebsiella in relieving low-salt stress of Litopenaeus vannamei, wherein the Klebsiella is Klebsiella sp. E26, is preserved in China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), and has a preservation number of CCTCC NO: M20241218 and a preservation date of June 14, 2024. 9 The application can comprehensively relieve negative effects of low-salt stress on Litopenaeus vannamei by adding 0.1% beta-glucan and 10 CFU / g of the bacterial strain E26 in feed.

Description

Technical Field

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

[0002] Low-salinity aquaculture is currently a trend in inland Penaeus vannamei farming. However, long-term low-salinity aquaculture can severely impact the growth and health of Penaeus vannamei, and in severe cases, can even cause mass mortality. β-glucan, a commonly used prebiotic in aquaculture, can improve the growth and health of Penaeus vannamei. However, there are currently no reports on the combined use of β-glucan and Klebsiella to alleviate the effects of low-salinity stress in 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 is achieved by adding 0.1% β-glucan and 10 9 CFU / g of the bacterial strain E26 to 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] Provided is a Klebsiella strain, which 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.

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

[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 ability 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 a 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 a 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 bacteriolytic activity.

[0015] Provided is an application of a 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 a 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 Vannamei shrimp 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 following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 The enzyme production activity, antibacterial activity and hemolytic activity of the strains are shown in Figure 2, including (A) Streptococcus iniae, (B) Vibrio harveyi, (C) Vibrio parahaemolyticus, (D) protease positive, (E) amylase positive, and (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 are shown in Figure 1, where (A) is artificial gastric juice; (B) is artificial intestinal juice;

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

[0024] Figure 4 This is a phylogenetic tree 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-salt conditions, including (A) weight gain rate, (B) survival rate, (C) hepatosomatic index, and (D) fatness. * indicates significant differences among the 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, and (D) lytic activity. Different lowercase letters represent significant differences (P<0.05).

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

[0028] Figure 8 Figure 3. 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] Figure 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] Figure 10 Figure 2. KEGG three-level function prediction and microbial interaction heat map of 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 three-level function prediction of intestinal microorganisms, and (D) microbial interaction heat map. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to 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] Example 1:

[0033] This embodiment provides an application of the combined use of β-glucan and Klebsiella in alleviating low-salt stress in 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 a salinity of 1-10 in the aquaculture water of Penaeus vannamei. 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: the treatment group was fed with basic feed; (2) Klebsiella group: the 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 basal feed. Four biological replicates were set up for each treatment group. The specific experimental design is shown in Table 1.

[0038] Table 1 Breeding experiment design

[0039]

[0040] 1.2 Basic feed formula

[0041] The specific formulas of the above-mentioned basic feed and the basic feed supplemented with 0.1% β-glucan are 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 into LB medium and cultured with shaking for 24 h. The culture was then centrifuged at 5000 g for 15 min, the supernatant was removed, the precipitate was washed twice with PBS, and the bacterial precipitate was resuspended in 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 with 0.1% β-glucan added 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. To maintain the vitality of the strain, the above feed was prepared every 3 days.

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

[0047] 1.2.1 Shrimp farming

[0048] Twenty-five juvenile shrimp 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, and the culture lasted for 8 weeks. During the culture period, the shrimp were fed four times a day at 7:00, 12:00, 18:00, and 23:00, respectively, with the above-mentioned basal feed containing 0.1% (by weight) β-glucan. 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 surface of the shrimp was wiped and disinfected with 75% alcohol, and then placed in a clean bench. The intestine of the shrimp was picked 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 minutes. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 50 μL of 10 -3 to 10 -8The dilutions were spread in quadruplicate on 2216E plates, MRS plates and LB plates prepared in advance (six concentrations of dilutions were spread on each plate), and cultured in a 30℃ 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. The culture was cultured in a constant temperature shaker at 28℃ and 200 rpm for 24 h. The bacterial solution was then mixed with sterilization preservation solution (distilled water: glycerol = 1:1, volume ratio) in equal proportions and stored in a -80℃ refrigerator.

[0051] 1.2.3 Hemolytic activity assay

[0052] The preserved strain (i.e. the purified strain mentioned above) was activated and spotted on a blood plate with a sterilized toothpick in a clean bench. After numbering, the plate 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 found 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 as follows: (1) Amylase plate: Add soluble starch at a ratio of 1% (by weight) to 2216E, MRS, and LB agar culture media, respectively. Heat to dissolve, then sterilize at 115°C. Cool to 60°C, pour into plates, and after complete solidification, seal and store in a refrigerator at 4°C. (2) Protease plate: Add skim milk powder at a ratio of 1% to 2216E, MRS, and LB agar culture media, respectively. The remaining steps are the same as those for the amylase plate.

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

[0056] As shown in Table 3, in a low-salt environment, after feeding a basic diet containing 0.1% β-glucan, a total of 155 bacterial strains were screened from the intestinal tract of Penaeus vannamei. Among them, 80 strains (E1 to E80) were screened from 2216E medium, 32 strains (B1 to B32) were screened from MRS medium, and 43 strains (L1 to L43) were screened from LB medium. Among them, 7 strains were hemolytically 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 abilities 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 pathogens, 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 aspirated and spread on a plate. The plate was punched with a sterile puncher with a diameter of 6 mm. Then, the above 14 strains with strong enzyme production ability were all activated, and 50 μL of the activated bacterial solution was aspirated and added to each hole. After numbering, they were placed in a constant temperature incubator at 28°C and cultured for 24 hours. After the incubation period, whether there was an inhibition zone around the hole was observed, and the diameter of the inhibition zone was measured. The appearance of an inhibition zone indicated that the strain had an antagonistic effect on the corresponding pathogen. 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 one pathogen, two pathogens, and three pathogens are 5, 6, and 2 strains, respectively.

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

[0066]

[0067]

[0068] Note: Enzyme activity is expressed as the ratio of the clear zone diameter to the 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 with 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 by filtration to prepare artificial intestinal juice.

[0071] The strains with the best enzyme-producing and antibacterial effects (E4, E26, E57, B4, B32) screened in Section 1.2.5 were revived and activated respectively, 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 in the figure, 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-aggregation rate of strains

[0074] Surface hydrophobicity: The surface hydrophobicity of the strains (E4, E26, E57, B4, and B32) with the best enzyme-producing antibacterial effects screened in Section 3.4 was determined using three organic solvents: xylene, chloroform, and ethyl acetate. The activated liquid culture medium was centrifuged at 4000 rpm for 10 min to collect the strain precipitate, which was washed twice with sterile PBS and resuspended to obtain a bacterial suspension. The initial absorbance A was measured using a spectrophotometer at 600 nm. Equal volumes of the bacterial suspension were thoroughly mixed with the three organic solvents, allowed to stand at room temperature for 1 h, the aqueous phase was absorbed, and the absorbance A1 at 600 nm was measured. The surface hydrophobicity was calculated as follows:

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

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

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

[0078] like Figure 3 As shown, the five strains exhibited varying hydrophobicity toward the three hydrocarbons. In xylene, strain B4 exhibited significantly higher surface hydrophobicity than the other four strains (P < 0.05). In chloroform and ethyl acetate, strains E26 and B32 exhibited significantly higher surface hydrophobicity than the other three strains (P < 0.05). The autoagglutination rate of strain B32 was significantly higher than that of the other four strains (P < 0.05), while no significant differences were observed among the other four strains.

[0079] 1.2.8 Antibiotic sensitivity evaluation of strains

[0080] The antibiotic susceptibility of the strains screened in Section 1.2.5 (E4, E26, E57, B4, and B32) with the best enzyme-producing antibacterial activity was determined using the disc agar diffusion method. 50 μL of the activated bacterial suspension of each strain was spread onto a plate. A disc of each antibiotic was attached to the plate and incubated in a 28°C incubator for 24 hours. The disc was observed for the formation of a clear ring around the ring and its diameter was recorded. The presence of a clear ring indicated that the strain was sensitive to the antibiotic. As shown in Table 5, strains E4, E26, and B4 exhibited strong resistance to most antibiotics.

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

[0082]

[0083]

[0084] 1.2.9 Strain identification

[0085] Based on its digestive enzyme production capacity, pathogen antagonism capacity, adhesion and colonization capacity, 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 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 the deposit number: CCTCC NO: M20241218, and the deposit date is June 14, 2024.

[0087] 1.3 Breeding management

[0088] Vannamei shrimp fry were purchased from a shrimp fry factory in Wenchang, Hainan. Twenty-five juvenile shrimp with an initial 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 / prawn weight) × 100%

[0094] (4) Fullness (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). SPSS 25.0 analysis software was used, and one-way analysis of variance and Duncan's multiple comparison 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 in the results, the survival rate, weight gain rate and hepatosomal 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. The serum was collected and stored at -80°C. After hemolymph extraction, the shrimp were dissected, and the hepatopancreas, intestine, and muscle were quickly separated and stored in sterilized cryopreservation tubes, placed in liquid nitrogen, and quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for enzyme activity determination and intestinal microbial sequencing.

[0102] 2.2 Immune index determination

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

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

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

[0106] Lytic activity (LZM): determined according to the method of Wang Yiyan et al. (Wang Yiyan, Sun Hushan, Li Guangyou. Effects 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 Immune index analysis

[0108] The indicator data analysis method is the same as that in 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 crucial role in the immune system of crustaceans. Hemocyte count, respiratory burst activity, hemocyanin content, and lytic activity are all commonly measured immune indicators in aquatic animals. The respiratory burst is one of the oxygen-dependent bactericidal pathways of phagocytes and is often used as a key indicator of phagocyte activity. Hemocyanin is a respiratory protein present in the hemolymph of many arthropods. It is involved in a variety of physiological functions, including energy storage, osmotic pressure maintenance, and regulation of molting. Furthermore, hemocyanin can produce antimicrobial peptides through self-cleavage, thereby inhibiting the growth and reproduction of certain microorganisms and possessing certain antimicrobial properties.

[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 using β-glucan or strain E26 alone.

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

[0113] 3.1 Determination of digestive enzyme indicators

[0114] The hepatopancreas samples obtained in Section 2.1 were weighed and thoroughly homogenized with normal saline at a ratio of 1:9 (weight ratio). 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 body and thus improving the growth performance of the shrimp.

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

[0118] 4.1 Gut Microbiome Sequencing

[0119] Intestinal tissues from four shrimp (obtained in Section 2.1) were pooled into one sample, and four samples from each treatment group were shipped on dry ice to Shanghai Baiqu Medical Technology Co., Ltd. DNA extraction, library construction, and bacterial sequencing were performed by the company. Following sequencing, α-diversity, β-diversity, inter-phylum and inter-genus bacterial species variation, and interspecies interactions were analyzed on the platform to comprehensively and systematically analyze the changes in the intestinal microbiota between the groups.

[0120] 4.2 Intestinal flora analysis

[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, Planctomycetes, and Firmicutes were the top three dominant phyla. Proteobacteria increased in all three treatment groups compared to the control group, while Firmicutes decreased. The most significant change was observed in the combined use group ( Figure 8 C in the .

[0122] At the genus level, the dominant genera in each group were 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 (Section D in the .

[0123] The relative abundance of Firmicutes varied significantly between 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 the addition of β-glucan and strain E26 to feed could change the intestinal microbial flora structure of Penaeus vannamei under low-salt conditions ( Figure 8 Compared with the control group, the intestinal flora structure of the combined use group was more densely clustered.

[0124] Further, from Figure 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 ( Figure 9 Part A and Part B of the study), 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 ( Figure 9 C and D in the .

[0125] Figure 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 ( Figure 10 C in the .

[0126] The intestine, the primary digestive organ of shrimp, primarily performs its various functions through intestinal microbial metabolism. The structure and composition of the intestinal microbiota are crucial for host 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 to the feed can all 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 to 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 shrimp's resistance 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 overall. 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 the Klebsiella group was significantly higher than that in the control group. It is speculated that the addition of 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 genera varied between groups. Rhodobacter was the most abundant genus in the combined use and β-glucan groups, Lysobacter was the most abundant in the Klebsiella group, and Halomonas was the most abundant in the control group. Compared to the control group, the proportions of Rhodobacter and Lysobacter increased, while Vibrio decreased. Vibrio is mostly aquatic pathogenic bacteria, and excessive numbers can cause intestinal dysfunction and threaten health. Lysobacter is a Gram-negative bacterium with strong antimicrobial activity, producing antimicrobial substances such as lysozyme and lipopeptides. Furthermore, Lysobacter can promote nutrient absorption and thus promote the growth of Penaeus vannamei. Furthermore, the similar genus Pseudoalteromonas significantly enhanced the immune response and resistance of Penaeus vannamei to Vibrio harveyi.

[0131] Therefore, the simultaneous addition of strain E26 and β-glucan to the feed can inhibit the growth and reproduction of harmful bacteria in the shrimp's intestines, thereby improving their health. Furthermore, KEGG secondary function prediction analysis of the intestinal flora showed that the combined group was significantly enriched in the amino acid metabolism pathway compared to the control group, indicating that the simultaneous addition of strain E26 and β-glucan to the feed can improve the absorption and metabolism of nutrients by the intestinal microorganisms of Penaeus vannamei under low-salinity stress, providing the shrimp with more energy to cope with the low-salinity 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 Vannamei shrimp 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 merely illustrate the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the appended 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 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, wherein: The pathogenic bacteria include any one of Staphylococcus aureus, Vibrio parahaemolyticus and Vibrio harveyi.

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

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

5. The use according to claim 4, characterized in that The indicators of immune capacity include respiratory burst activity and / or hemocyanin content and / or bacteriolytic activity.

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

7. The use according to claim 6, 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.

8. Use of β-glucan in combination with the Klebsiella E26 of claim 1 in regulating the intestinal microbial flora structure of Penaeus vannamei in a low-salt environment.

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