Glutathione-phenyllactic acid composition and application thereof in resisting aquatic pathogenic bacteria and preparing feed additive

Abstract

The invention discloses a glutathione-phenyllactic acid composition and application thereof in resisting aquatic pathogenic bacteria and preparing a feed additive, and belongs to the field of aquaculture. The invention provides application of glutathione in inhibition of aeromonas veronii, and experimental results show that the minimum inhibitory concentration of glutathione to aeromonas veronii is 0.391 mg / mL. According to the invention, glutathione and phenyllactic acid are further compounded into the composition, experimental results show that composite use of glutathione and phenyllactic acid shows a significantly enhanced synergistic bacteriostatic effect, and feeding experiments show that the composition significantly improves the growth performance and disease resistance of carps, improves the structural integrity of intestinal tracts, and improves the immunity of carps. And the intestinal flora is effectively regulated. The glutathione composition provided by the invention has good biological safety and antibiotic replacement potential, provides a feasible scheme for antibiotic-free healthy culture of aquatic animals, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of aquaculture, in particular to a glutathione-phenyllactic acid composition and its application in resisting aquatic pathogens and preparing feed additives. Background Art

[0002] Common pathogens of aquatic animals include bacteria, viruses, and parasites. Bacterial diseases are the most prevalent and serious, with typical pathogens including Aeromonas viridis, Aeromonas hydrophila, Vibrio harveyi, and Vibrio parahaemolyticus. These pathogens pose a widespread threat to farmed fish, especially in intensive farming environments characterized by high density and environmental degradation. They can easily lead to decreased immunity and frequent disease outbreaks, severely impacting aquaculture profitability.

[0003] The fish intestine is not only a crucial site for nutrient absorption and immune regulation, but also a crucial line of defense against external pathogens. The integrity of the intestinal morphology and the stability of the intestinal microbiome complement each other to maintain intestinal health. An intact epithelial cell structure and well-defined mucosal folds promote the colonization and function of probiotics, while a stable microbial community secretes metabolites that enhance mucosal repair and barrier function, significantly improving nutrient utilization and disease resistance in fish.

[0004] Phenyllactic acid is an organic acid with broad-spectrum antibacterial activity. It occurs naturally in honey, kimchi, and lactic acid fermentation products. It can also be obtained through microbial fermentation. It boasts high safety, good thermal stability, and a broad antibacterial spectrum. Currently, phenyllactic acid is used in livestock and poultry feed to improve animal growth performance and enhance immunity.

[0005] Glutathione, a tripeptide antioxidant, is widely found in plants and animals and serves as the primary non-enzymatic antioxidant in cells. Glutathione has multiple functions, including scavenging free radicals, enhancing immunity, and maintaining cellular redox balance. In recent years, its use in aquaculture has gradually emerged as a promising pathway to improving fish health and disease resistance by increasing antioxidant enzyme activity, improving tissue structure, and regulating immune gene expression. However, the antibacterial activity of GSH has not yet been reported.

[0006] Currently, the prevention and control of bacterial diseases in aquatic products primarily relies on antibiotic intervention. While these interventions can effectively control pathogens, their long-term use leads to increasingly prominent problems, including the proliferation of drug-resistant strains, water pollution, and drug residues in aquatic products. These issues have become a significant constraint on the healthy development of the aquaculture industry. While some natural antibacterial substances have been proposed as alternatives to antibiotics, research has primarily focused on their in vitro antibacterial activity, lacking systematic and in-depth evaluation of their biological functions. This has resulted in limited success in improving the overall health of fish.

[0007] Although phenyllactic acid and glutathione have been used separately in livestock, poultry, and some aquatic animals, demonstrating certain antioxidant or growth-promoting effects, current research has largely focused on the use of individual ingredients, lacking research on their combined applications. Therefore, as aquaculture transitions toward antibiotic-free, environmentally friendly, and high-quality practices, there is an urgent need to develop a safe, multifunctional, and systematically modulate fish health. Summary of the Invention

[0008] The purpose of the present invention is to provide a glutathione-phenyllactic acid composition and its application in resisting aquatic pathogens and preparing feed additives, so as to solve the problems existing in the above-mentioned prior art. The present invention provides the application of glutathione in inhibiting aquatic pathogens for the first time, and innovatively proposes a composite application strategy of phenyllactic acid and glutathione, systematically investigating their synergistic effects in in vitro antibacterial effect, in vivo growth promotion, intestinal structure protection and microecological regulation, etc., to provide a scalable and applicable technical support for the construction of green breeding of aquatic animals.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The present invention provides an application of glutathione in any of the following:

[0011] (1) Application in inhibiting aquatic pathogens;

[0012] (2) Application in the preparation of antibacterial agents for aquatic pathogens;

[0013] (3) Use in the preparation of drugs for preventing and / or treating aquatic pathogen infections.

[0014] Preferably, the aquatic pathogen is Aeromonas vermiformis.

[0015] The present invention also provides a composition comprising glutathione and phenyllactic acid.

[0016] Preferably, the mass ratio of the glutathione to the phenyllactic acid is (0.195-0.781):(0.05-0.2).

[0017] The present invention also provides a composition, and an application of the above composition in any of the following:

[0018] (1) Application in inhibiting aquatic pathogens;

[0019] (2) Application in the preparation of antibacterial agents for aquatic pathogens;

[0020] (3) Use in the preparation of drugs for preventing and / or treating aquatic pathogen infections;

[0021] (4) Application in the preparation of feed additives;

[0022] The feed additive has at least one of the functions of promoting the growth of young fish, improving intestinal structure, regulating flora and enhancing disease resistance.

[0023] Preferably, the aquatic pathogen is Aeromonas vermiformis.

[0024] The present invention also provides a composition, an Aeromonas vernix antibacterial agent, which uses the above composition as a main active ingredient.

[0025] The present invention also provides a composition, a medicine for preventing and / or treating Aeromonas vernix infection, wherein the medicine has the above composition as a main active ingredient.

[0026] The present invention also provides a composition and a feed additive, wherein the feed additive has the above composition as a main active ingredient.

[0027] Preferably, the feed additive further comprises adjuvants acceptable to feed additives.

[0028] The present invention discloses the following technical effects:

[0029] The present invention utilizes two naturally active substances, phenyllactic acid and glutathione, through a synergistic combination to create a composite feed additive that combines in vitro antibacterial and in vivo health-regulating effects. Through measurements of minimum inhibitory concentration, swarming and swimming ability, and biofilm formation, the present invention demonstrates that phenyllactic acid (PLA) and glutathione (GSH) can effectively inhibit bacterial activity when used alone, and their combined use exhibits a stronger synergistic antibacterial effect in vitro. In in vivo feeding experiments, the present invention found that the addition of PLA (25.6 mg / kg), GSH (400 mg / kg), and their combination (25.6 mg / kg PLA + 400 mg / kg GSH) to carp feed significantly improved growth performance and disease resistance, with the combination providing the most effective results. Histological analysis showed that the addition of the compound significantly increased the height and number of villi in the fore, mid, and hind intestines, thickened the intestinal muscular layer, and improved intestinal structural integrity. Further 16S rRNA high-throughput sequencing revealed that the PLA+GSH combination can significantly enhance the α-diversity of intestinal flora, increase the relative abundance of beneficial bacteria, and reduce the proportion of potential pathogens.

[0030] In summary, the present invention achieves multiple functions such as antibacterial, growth promotion, intestinal protection, and bacterial flora stabilization through the synergistic effect of natural composite active substances. It has good biosafety and potential to replace antibiotics, providing a feasible solution for antibiotic-free and healthy breeding of aquatic animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only 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.

[0032] Figure 1 This is a statistical graph of the minimum inhibitory concentration (MIC) of GSH against Aeromonas vernix; different lowercase letters indicate statistically significant differences;

[0033] Figure 2 This is a statistical graph of the minimum inhibitory concentration (MIC) of PLA against Aeromonas vickers; different lowercase letters indicate statistically significant differences;

[0034] Figure 3 This is a statistical graph of the antibacterial effect of PLA and GSH combined treatment on Aeromonas vernix; different lowercase letters indicate statistically significant differences;

[0035] Figure 4 The microscopic observations of the effect of GSH on the biofilm structure of Aeromonas vernix; the concentration of GSH in A is 0 mg / mL; the concentration of GSH in B is 0.195 mg / mL; the concentration of GSH in C is 0.391 mg / mL; and the concentration of GSH in D is 0.781 mg / mL.

[0036] Figure 5 These are microscopic observations of the effect of PLA on the biofilm structure of Aeromonas vernix; the concentration of PLA in A is 0 mg / mL; the concentration of PLA in B is 0.025 mg / mL; the concentration of PLA in C is 0.05 mg / mL; the concentration of PLA in D is 0.1 mg / mL; the concentration of PLA in E is 0.2 mg / mL; and the concentration of PLA in F is 0.4 mg / mL.

[0037] Figure 6Figure 2 is a microscopic observation of the effect of PLA and GSH composite treatment on the biofilm structure of Aeromonas vickers. In which, the concentration of PLA in 0 is 0 mg / mL, and the concentration of GSH is 0 mg / mL; the concentration of PLA in A is 0.05 mg / mL, and the concentration of GSH is 0.195 mg / mL; the concentration of PLA in B is 0.05 mg / mL, and the concentration of GSH is 0.391 mg / mL; the concentration of PLA in C is 0.05 mg / mL, and the concentration of GSH is 0.781 mg / mL; the concentration of PLA in D is 0.1 mg / mL, and the concentration of GSH is 0. The concentration of SH was 0.195 mg / mL; the concentration of PLA in E was 0.1 mg / mL, and the concentration of GSH was 0.391 mg / mL; the concentration of PLA in F was 0.1 mg / mL, and the concentration of GSH was 0.781 mg / mL; the concentration of PLA in G was 0.2 mg / mL, and the concentration of GSH was 0.195 mg / mL; the concentration of PLA in H was 0.2 mg / mL, and the concentration of GSH was 0.391 mg / mL; the concentration of PLA in I was 0.2 mg / mL, and the concentration of GSH was 0.781 mg / mL;

[0038] Figure 7 This is a statistical diagram of the effect of GSH on the biofilm formation of Aeromonas veseri; different lowercase letters indicate statistically significant differences;

[0039] Figure 8 This is a statistical graph showing the effect of PLA on the biofilm formation of Aeromonas viridis; different lowercase letters indicate statistically significant differences;

[0040] Figure 9 This is a statistical graph showing the effect of GSH and PLA combined treatment on the biofilm formation of Aeromonas vernix; different lowercase letters indicate statistically significant differences;

[0041] Figure 10 Observation diagram of the effect of GSH on the clustering and swimming ability of Aeromonas veseri;

[0042] Figure 11 Observation diagram of the effect of PLA on the swarming and swimming ability of Aeromonas vermiformis;

[0043] Figure 12The observation diagram of the effect of GSH and PLA composite treatment on the clustering ability of Aeromonas vickers; among them, the concentration of PLA in 0 is 0 mg / mL, and the concentration of GSH is 0 mg / mL; the concentration of PLA in A is 0.05 mg / mL, and the concentration of GSH is 0.195 mg / mL; the concentration of PLA in B is 0.05 mg / mL, and the concentration of GSH is 0.391 mg / mL; the concentration of PLA in C is 0.05 mg / mL, and the concentration of GSH is 0.781 mg / mL; the concentration of PLA in D is 0.1 mg / mL, and the concentration of GSH is The concentration of PLA in E was 0.1 mg / mL and the concentration of GSH was 0.391 mg / mL; the concentration of PLA in F was 0.1 mg / mL and the concentration of GSH was 0.781 mg / mL; the concentration of PLA in G was 0.2 mg / mL and the concentration of GSH was 0.195 mg / mL; the concentration of PLA in H was 0.2 mg / mL and the concentration of GSH was 0.391 mg / mL; the concentration of PLA in I was 0.2 mg / mL and the concentration of GSH was 0.781 mg / mL;

[0044] Figure 13 The observation diagram of the effect of GSH and PLA composite treatment on the swimming ability of Aeromonas vickers; among them, the concentration of PLA in 0 is 0 mg / mL, and the concentration of GSH is 0 mg / mL; the concentration of PLA in A is 0.05 mg / mL, and the concentration of GSH is 0.195 mg / mL; the concentration of PLA in B is 0.05 mg / mL, and the concentration of GSH is 0.391 mg / mL; the concentration of PLA in C is 0.05 mg / mL, and the concentration of GSH is 0.781 mg / mL; the concentration of PLA in D is 0.1 mg / mL, and the concentration of GSH is The concentration of PLA in E was 0.1 mg / mL and the concentration of GSH was 0.391 mg / mL; the concentration of PLA in F was 0.1 mg / mL and the concentration of GSH was 0.781 mg / mL; the concentration of PLA in G was 0.2 mg / mL and the concentration of GSH was 0.195 mg / mL; the concentration of PLA in H was 0.2 mg / mL and the concentration of GSH was 0.391 mg / mL; the concentration of PLA in I was 0.2 mg / mL and the concentration of GSH was 0.781 mg / mL;

[0045] Figure 14 Figure 2 shows the microscopic observation of the effects of GSH and PLA alone and in combination on the foregut structure of carp; A is the control group; B is the PLA+GSH combination group; H represents the height of intestinal villi, T represents the thickness of the muscle layer, and W represents the depth of the crypts; C represents the GSH group; and D represents the PLA group.

[0046] Figure 15The microscopic observations of the effects of GSH and PLA alone and in combination on the midgut tissue structure of carp; A is the control group; B is the PLA+GSH combination group; C is the GSH group; and D is the PLA group.

[0047] Figure 16 The microscopic observations of the effects of GSH and PLA alone and in combination on the hindgut tissue structure of carp; A is the control group; B is the PLA+GSH combination group; C is the GSH group; D is the PLA group;

[0048] Figure 17 This is a statistical graph showing the effects of GSH and PLA alone and in combination on the ability of carp to resist infection with Aeromonas vernix; different lowercase letters indicate statistically significant differences;

[0049] Figure 18 is the Venn diagram of the number of OTUs;

[0050] Figure 19 Statistical chart of the α-diversity index of intestinal flora; A is the Chao1 index; B is the Simpson index;

[0051] Figure 20 Statistical graphs of intestinal flora β-diversity analysis; A is the NMDS analysis graph; B is the UPGMA clustering graph;

[0052] Figure 21 The figure shows the relative abundance analysis results of intestinal flora species composition; A is the phylum level; B is the genus level. DETAILED DESCRIPTION

[0053] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0055] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0056] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0057] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0058] Unless otherwise specified, the glutathione in the present invention is reduced glutathione (GSH), and the glutathione in the present invention (purity ≥98%) was purchased from Yacoin Wuhan Biotechnology Co., Ltd.

[0059] The Aeromonas verticillii strain used in the present invention is from the Tianjin Key Laboratory of Production Ecology and Aquaculture, College of Fisheries, Tianjin Agricultural College, and has been published in "Pei Lishuo, Liu Bin, Han Zhuoran, et al. Isolation, Identification and Biological Characteristics Analysis of Pathogenic Aeromonas verticillii of Carp [J]. Journal of Southern Agriculture, 2023, 54(07): 2184-2194." The applicant promises to distribute the above-mentioned biological materials to the public within 20 years from the filing date of this invention.

[0060] The culture method of Aeromonas vernix in the present invention is as follows: the Aeromonas vernix strain is inoculated into LB liquid culture medium, cultured in a constant temperature shaker at 30°C and 150 rpm for 16 h, the concentration of the cultured bacterial suspension is measured using an ultraviolet spectrophotometer, and the bacterial suspension concentration is adjusted to 1×10 7 cfu / mL was used for the experiments.

[0061] Example 1

[0062] 1.1 Preparation of bacterial suspension

[0063] Aeromonas veronii was inoculated into sterile LB liquid medium and cultured in a constant temperature shaker at 28°C (180 rpm) until the logarithmic growth phase; then, the bacterial solution was concentrated to 1×10 6CFU / mL is reserved.

[0064] 1.2 Minimum inhibitory concentration (MIC) determination

[0065] The present invention uses LB liquid culture medium to prepare reduced glutathione (GSH, 500 mg / mL) and phenyllactic acid (PLA, 64 mg / mL) stock solutions, and obtains a series of concentration gradient working solutions by the serial dilution method. The experiment sets up a GSH treatment group and a PLA treatment group, wherein each group contains different concentration gradients (see Table 1 for details). At the same time, a sterile LB liquid culture medium is set as a control, and 3 biological replicates are set for each concentration gradient. The culture is kept at 28°C for 24 hours, and the optical density value (OD) at a wavelength of 600nm is measured by a microplate reader. 600 ), and the minimum inhibitory concentration (MIC) of GSH and PLA against A. veronii was determined.

[0066] Based on the single-factor MIC values, an orthogonal experimental design was used to construct a GSH-PLA combined treatment group. This combined treatment group included nine concentration combinations (see Table 1), with GSH and PLA concentrations ranging from 0.5 MIC to 1 MIC to 2 MIC, to systematically evaluate their synergistic antibacterial effect. All experiments were performed with three biological replicates, using the same culture conditions and detection methods as the single-factor experiments.

[0067] Table 1 Concentration ratios of phenyllactic acid and glutathione in single and combined use

[0068]

[0069] Depend on Figure 1 and Figure 2 After 24 hours of incubation, the MICs of GSH and PLA against A. veronii were 0.391 mg / mL and 0.1 mg / mL, respectively (P < 0.05). Furthermore, all combined treatments significantly inhibited the growth of A. veronii (P < 0.001), with Group A (GSH 0.195 mg / mL + PLA 0.05 mg / mL) being the lowest effective concentration combination to produce a significant inhibitory effect.

[0070] In addition, group C (GSH 0.781 mg / mL + PLA 0.05 mg / mL) and group D (GSH 0.195 mg / mL + PLA 0.1 mg / mL) had the most significant antibacterial effects, but the combined concentrations were relatively low ( Figure 3 ); and in groups C and D, the combined use of GSH and PLA showed an antibacterial ability that was superior to the MIC of each of them ( Figure 1-Figure 3 ).

[0071] 1.3 Qualitative and quantitative analysis of biofilms

[0072] In a 24-well culture plate, a sterile cell slide (14 mm) was placed in advance. Subsequently, 950 μL of bacterial suspension and 50 μL of different concentrations of GSH, PLA and GSH-PLA mixed solution were added, so that the final concentrations of GSH were 0, 0.5 MIC, 1 MIC and 2 MIC respectively; the final concentrations of PLA were 0, 0.25, 0.5, 1, 2, 4 MIC; the final concentrations of PLA-GSH are shown in Table 1. Three replicates were set for each concentration and cultured at 28°C for 24 hours. The cell slide was taken out and washed 3 times with PBS, fixed with anhydrous methanol for 15 minutes, and stained with 0.2% crystal violet in the dark for 20 minutes. The crystal violet dye was discarded, and the bacterial biofilm morphology was observed under an optical microscope after washing with PBS. The above steps were repeated in another 96-well microplate (without placing a coverslip). After crystal violet staining, 33% acetic acid was added to dissolve the biofilm, and the OD was measured. 630 The biofilm formation amount was quantitatively evaluated.

[0073] 1.3.1 Microscopic observation of biofilm morphology

[0074] In LB medium, the biofilm formed by A. veronii showed a dense bacterial network structure, with cells tightly aggregated and highly adherent ( Figure 4 A and A in 5). As the concentrations of GSH and PLA increased, the aggregation density of A. veronii and the distance between bacteria gradually decreased ( Figure 4 5 in BD and 5 in BF).

[0075] In a composite group ( Figure 6 ), the biofilms in concentration groups A, B, and D were slightly sparse, and the bacterial aggregation density decreased, but the overall community structure was still relatively complete; the biofilms in concentration groups C and E were more dispersed, and the bacterial density further decreased, but some aggregation could still be observed; the biofilms in concentration groups F, G, H, and I were significantly sparse, the bacterial spacing increased, and the community structure was obviously loose.

[0076] 1.3.2 Quantitative detection of biofilm

[0077] Quantitative analysis showed that compared with the control group, GSH ( Figure 7 ) and 0.5-4MIC PLA ( Figure 8 ) could significantly reduce the amount of biofilm formed by A. veronii (P<0.05). Figure 9), the amount of A. veronii biofilm formation at all GSH-PLA concentration combinations was significantly lower than that in the control group (P < 0.05). Further comparison revealed that the inhibitory effect of compound group BI was significantly greater than that of compound group A; and the inhibitory effects of compound groups C and EI were significantly greater than those of groups B and D (P < 0.05). Group C had a superior effect on A. veronii biofilm formation compared to group D.

[0078] In addition, if Figure 5-Figure 9 As shown in the results, the inhibitory effect of the composite C group on biofilm formation ability was better than that of PLA and GSH at the MIC concentration.

[0079] 1.4 Swarming and swimming ability assays

[0080] The experiment selected bacterial solution in logarithmic growth phase (1×10 6 CFU / mL) were inoculated onto the surface of swarming solid medium (tryptone 1 g / L, sodium chloride 0.5 g / L, agar 0.5 g / L, glucose 0.5 g / L) and swimming solid medium (tryptone 1 g / L, sodium chloride 3 g / L, agar 0.3 g / L) containing various concentrations of GSH (0.5 MIC, 1 MIC, and 2 MIC), PLA (0.5 MIC, 1 MIC, and 2 MIC), and GSH-PLA (concentrations indicated in the AI group). Three replicates were performed for each concentration, with standard swarming or swimming medium serving as controls. After 24 hours of incubation at 28°C, the colony spread diameter (mm) was precisely measured using a vernier caliper, and the average of the three measurements was used as the final result.

[0081] 1.4.1 Effects of GSH on Clustering and Motility

[0082] In the swarming experiment, the diameter of the colony in the 0.5, 1 and 2 MIC GSH treatment groups was significantly reduced by 23.1%-58.9% compared with the control group (10.4±0.18 mm). Figure 10 ; Table 2, P < 0.05); In the swimming test, compared with the control (16 ± 0.05 mm), 0.5-2 MIC GSH significantly inhibited the colony diameter by 18.8%-85.9% ( Figure 10 ; Table 2, P < 0.05).

[0083] Table 2 Inhibition rate of GSH on the clustering and swimming ability of A. veronii

[0084]

[0085] Note: Different lowercase letters indicate statistically significant differences.

[0086] 1.4.2 Effects of PLA on Swarming and Swimming Ability

[0087] In the swarming experiment, 1 and 2 MIC of PLA significantly reduced the movement diameter of A. veronii by 8.24% and 16.94% respectively compared with the control group (0.425±0.025 cm); in the swimming experiment, 0.5, 1 and 2 MIC of PLA reduced the movement diameter of the colony from 0.950±0.05 mm in the control group to 0.783±0.076, 0.633±0.029 and 0.300±0.05 mm respectively, with inhibition rates of 17.58%-68.42% (Table 3, Figure 11 , P<0.05).

[0088] Table 3 Migration diameters of A. veronii swarming and swimming motility under PLA treatment

[0089]

[0090] Note: Different lowercase letters indicate statistically significant differences.

[0091] 1.4.3 Effects of GSH+PLA on Clustering and Motility

[0092] In the cluster experiment, the bacterial movement diameter in the composite group AI was significantly reduced by 16.71-23.86% compared with the control group, and there was no significant difference in the inhibition rate among the AI groups (P>0.05, Table 4, Figure 12 In the swimming experiment, when the concentration of PLA and GSH was AI, the inhibition rates on the swimming migration diameter of A. veronii were 50.84%, 52.63%, 50.84%, 50.84%, 56.11%, 49.16%, 54.42%, 43.89% and 50.84%, respectively; the inhibition rate of group H was significantly lower than that of groups B, D, E and G (P < 0.05, Table 5, Figure 13 ).

[0093] Table 4 Migration diameter of A. veronii swarming under combined treatment of PLA and GSH

[0094]

[0095]

[0096] Note: Different lowercase letters indicate statistically significant differences.

[0097] Table 5 Migration diameter of A. veronii swimming motility under combined treatment of PLA and GSH

[0098]

[0099] Note: Different lowercase letters indicate statistically significant differences.

[0100] The results of this example show that phenyllactic acid and glutathione can significantly inhibit the growth, motility and biofilm formation of Aeromonas vermiformis in vitro; among them, when 0.781 mg / mL GSH and 0.05 mg / mL PLA are used in combination, their antibacterial effect is better than that of phenyllactic acid (0.1 mg / mL) and glutathione (0.391 mg / mL) at the MIC concentration.

[0101] Example 2

[0102] 1. Feed Preparation and Experimental Animal Husbandry

[0103] 1.1 Feed preparation

[0104] The experimental feed was based on the commercially available Tongwei brand compound feed, supplemented with phenyllactic acid (PLA, purity ≥98%) and reduced glutathione (GSH, purity ≥98%), both of which were purchased from Yacoin Wuhan Biotechnology Co., Ltd.

[0105] First, PLA and GSH were dissolved in sterile double-distilled water to prepare stock solutions. Subsequently, the stock solutions were evenly sprayed onto the surface of the base pelleted feed to produce three experimental feeds: a PLA group (25.6 mg / kg), a GSH group (400 mg / kg), and a PLA+GSH combination group (25.6 mg / kg PLA + 400 mg / kg GSH). All feeds were dried in the shade at room temperature and stored sealed at 4°C for subsequent experiments.

[0106] 1.2 Animal husbandry and grouping

[0107] The carp fry used in the experiment were provided by the Tianjin Changsheng Fish Hatchery. After being transported to the laboratory, the fish were temporarily placed in a recirculating aquaculture system for 14 days to acclimate to the environment. During this period, the water temperature was kept constant (22±1°C), and good dissolved oxygen and water quality were maintained. After the temporary aquaculture period, the carp were randomly divided into four groups, with three replicates in each group. Each group was fed a different treated feed: PLA group, GSH group, and compound group; the control group was fed a standard compound feed. The experimental period lasted 28 days, with fish fed twice daily (8:30 am and 5:30 pm) at a feeding amount of 2% of their body weight. After 2 hours of feeding, the remaining bait at the bottom of the tank was collected, dried, and weighed to accurately calculate the actual food intake.

[0108] 2. Growth Performance Determination

[0109] On the 28th day, the carp in each group were weighed at the end (accurate to 0.01 g), and the specific growth rate [SGR = 100 × (ln final body weight - ln initial body weight) / number of experimental days], feed conversion ratio (FCR = total feed intake / total weight gain) and weight gain rate [WG = (final body weight - initial body weight) / initial body weight × 100%] were calculated to evaluate the effect of each treatment on growth performance.

[0110] Compared with the control group, feeding PLA, GSH, and the PLA+GSH complex significantly increased the specific growth rate, feed conversion rate, and weight gain rate of carp (P<0.05; Table 6). However, the complex group had a significantly greater effect on promoting carp growth performance than the PLA and GSH groups (P<0.05).

[0111] Table 6 Effects of adding PLA and GSH to feed on the growth performance of carp juveniles

[0112]

[0113] Note: Different lowercase letters indicate statistically significant differences.

[0114] 3. Sample Collection and Histopathological Studies

[0115] Three carp were randomly selected from each group and anesthetized with hypothermia for sample collection. The surface was disinfected with 75% ethanol and then dissected to obtain intact intestinal tissue. After gently rinsing blood and attached fat with sterile saline, the intestine was divided into foregut, midgut, and hindgut according to anatomical location and fixed in 4% paraformaldehyde for preservation. The remaining individuals were used for subsequent infection experiments. Fixed tissues were dehydrated with graded ethanol, transparentized with xylene, and embedded in paraffin. Sections were sliced to a thickness of 5 μm. The sections were dried, stained with hematoxylin and eosin (H&E), and mounted as usual. Under a light microscope, six fields of view were randomly observed in each group, and the following parameters were measured: intestinal villus height (vertical distance from base to apex), muscle layer thickness (vertical thickness from submucosal layer to serosal layer, including circular and longitudinal muscle layers), and intestinal villus number (number per unit length of cross-section). The obtained data were used for further statistical analysis.

[0116] After 28 days of feeding, no obvious pathological structural damage was observed in the foregut, midgut and hindgut tissue sections of all experimental groups ( Figure 14-16 However, compared with the control group, the PLA, GSH and combined groups all showed varying degrees of improvement in intestinal morphological parameters, mainly reflected in the changes in intestinal villus height and number, muscle layer thickness and crypt depth, as shown in the following:

[0117] 3.1 Foregut

[0118] Compared with the control group, the GSH group, PLA group and compound group significantly increased the height of intestinal villi, the number of intestinal villi and the thickness of the muscular layer in the foregut of carp (P<0.05; Table 7, Figure 14 The height and number of intestinal villi in the composite group were significantly higher than those in the PLA and GSH groups (P<0.05), while there was no significant difference between the PLA and GSH groups (P>0.05).

[0119] Table 7 Effects of PLA and GSH on the foregut histomorphology of carp juveniles

[0120]

[0121] Note: Different lowercase letters indicate statistically significant differences.

[0122] 3.2 Midgut

[0123] Compared with the control group, the intestinal villus height, muscular layer thickness, and crypt depth of the GSH, PLA, and combined groups were significantly increased (P<0.05). Among them, the villus height of the combined group was significantly higher than that of the PLA and GSH groups (P<0.05). In addition, the number of intestinal villi in the combined group was significantly higher than that of the control group and GSH group (P<0.05), but there was no significant difference between the combined group and the PLA group (P>0.05; Table 8, Figure 15 ).

[0124] Table 8 Effects of PLA and GSH on the midgut tissue morphology of carp juveniles

[0125]

[0126] Note: Different lowercase letters indicate statistically significant differences.

[0127] 3.3 Hindgut

[0128] In the hindgut, the height and number of intestinal villi in the composite group were significantly higher than those in the other three groups (P<0.05), while there were no significant differences between the PLA group, GSH group, and control group (P>0.05). In terms of muscularis thickness, the composite group and PLA group were significantly higher than the control group (P<0.05), but there was no significant difference between the two groups (P>0.05; Table 9, Figure 16 ).

[0129] Table 9 Effects of PLA and GSH on the morphology of hindgut intestinal tissues of carp juveniles

[0130]

[0131] Note: Different lowercase letters indicate statistically significant differences.

[0132] 4. Infection Experiment

[0133] After the experiment, a total of 288 carp were randomly selected from each treatment group (control, PLA, GSH and compound group) (72 carp per group, 3 parallels, 24 carp per parallel) for intraperitoneal injection infection experiment. Each group was set up with two treatments: one part of the individuals were intraperitoneally injected with 0.1 mL of A. veronii suspension (concentration 7×10 4 CFU / mL), and another portion was injected with an equal volume of sterile saline as a negative control (36 fish per group, 3 replicates, 12 fish per replicate). The experiment lasted for 14 days, during which mortality was observed and recorded daily. After the experiment, the cumulative survival rate of each group was calculated, and the bacteria of the deceased individuals were re-isolated and morphologically identified, and the pathogen was confirmed using molecular biological methods.

[0134] After 14 days of infection with A. veronii, the survival rates of carp in the GSH, PLA and composite groups were significantly higher than those in the control group (0%), namely, 36.11%, 44.44% and 55.56%, respectively (P<0.05). Among them, the survival rate of the composite group was significantly higher than that of the GSH group (P<0.05), but had no significant difference with that of the PLA group (P>0.05, Figure 17 ).

[0135] The results of this example show that adding GSH (400 mg / kg), PLA (25.6 mg / kg), or a PLA-GSH complex (25.6 mg / kg PLA + 400 mg / kg GSH) to the diet for 28 consecutive days significantly increased the weight gain rate, specific growth rate, and feed conversion efficiency of carp (P < 0.05). It also significantly improved the height and number of villi and the thickness of the muscular layer in the fore, mid, and hindguts, and enhanced their resistance to Aeromonas vernix infection. The PLA-GSH complex group showed the most significant improvements in growth performance, intestinal structure, and disease resistance, outperforming the groups supplemented with GSH or PLA alone.

[0136] Example 3

[0137] 1. Feed Preparation and Experimental Animal Husbandry

[0138] Same as Example 2. The groups fed with feed containing PLA, GSH, PLA+GSH and common commercial feed were named PLA group, GSH group, PLA+GSH group and CG group respectively.

[0139] 2. Sample Collection

[0140] After the experiment, three fish were randomly selected from each replicate, for a total of nine per group. The fish were dissected aseptically, and their intestinal contents were collected. The intestinal contents of the three fish within the same replicate were pooled into a single sample, resulting in three biological replicates per group. All samples were immediately snap-frozen in liquid nitrogen and then stored at -80°C until subsequent DNA extraction and microbiome analysis.

[0141] 3.16S rDNA high-throughput sequencing and data analysis

[0142] Extraction, amplification, and high-throughput sequencing of total intestinal microbial DNA were performed by Beijing Novogene Bioinformatics Technology Co., Ltd. Using total DNA extracted from intestinal contents as a template, PCR amplification was performed using universal primers (targeting the V3-V4 region of the 16S rRNA gene). The amplified product was verified by agarose gel electrophoresis, the target band was recovered, and a sequencing library was constructed. Library quality was assessed using Qubit fluorescence quantitative analysis and qPCR, and qualified libraries were subsequently sequenced. The raw data generated by the sequencing platform underwent quality control, including removal of adapter sequences, low-quality sequences, and chimeric sequences, to obtain high-quality, valid sequences.

[0143] Data analysis was performed using the QIIME 2 (Quantitative Insights Into Microbial Ecology 2) platform. Sequences were clustered into operational taxonomic units (OTUs) at 97% similarity and taxonomic annotation was performed according to the Greengenes or SILVA databases. Alpha and beta diversity analyses were performed. Alpha diversity analysis used the Chao1, Shannon, and Simpson indices, reflecting community richness and evenness, and the Wilcoxon rank sum test was used in R to assess intergroup differences. Beta diversity analysis used the Unweighted UniFrac distance, and cluster trees were constructed using principal coordinates analysis (PCoA) and unweighted group average method (UPGMA). Visualization was performed using R packages such as WGCNA, stats, ggplot2, and vegan. Nonmetric multidimensional scaling (NMDS) was also used to further assess intergroup differences. In addition, the LEfSe (Linear Discriminant Analysis Effect Size) method was used to analyze the significant differences in microbial composition between different groups and screen potential characteristic taxa (biomarkers). The differences in microbial diversity levels between groups were statistically verified using t-tests and Wilcoxon tests in the R language.

[0144] 4. Analysis of intestinal flora diversity

[0145] 4.1 Number of OTUs: After clustering the CG, GSH, PLA, and PLA+GSH groups, a total of 2206 OTUs were obtained (97% similarity), of which 72 were common OTUs and the number of unique OTUs was 143, 281, 859, and 414, respectively ( Figure 18 ).

[0146] 4.2α diversity: such as Figure 19 As shown in the results, the Chao1 and Simpson indexes of the PLA and PLA+GSH groups were higher than those of the CG and GSH groups; while the Simpson index of the GSH group was lower than that of the control group.

[0147] 4.3β diversity: NMDS can be seen ( Figure 20 ), most samples in the control group separated from each other and away from the other three groups; whereas samples in the PLA, GSH, and PLA+GSH groups clustered together. The results of UPGMA cluster analysis were consistent with those of NMDA decomposition.

[0148] 4.4 Analysis of intestinal flora composition: Figure 21 As shown in the figure, the dominant bacterial phyla in the intestinal flora of carp juveniles are mainly composed of Fusobacteriota, Firmicutes, Proteobacteria, Desulfobacterota and Actinobacteria.

[0149] At the genus level, the relative abundance of genera such as Cetobacterium, Shinella, Aeromonas, Ralstonia, Acidovorax, Aurantimicrobium, Microbacterium, Vrbrio, and Mycobacterium dominated across all groups. Among them, Ralstonia, Acidovorax, and Mycobacterium, which are potentially pathogenic and include species that can cause diseases such as diarrhea, wound infections, and sepsis, were found at lower relative abundance in the PLA+GSH group.

[0150] This example shows that adding PLA-GSH complex (25.6 mg / kg PLA + 400 mg / kg GSH) to the feed for 28 consecutive days can improve the diversity of carp intestinal flora, optimize the composition structure of the flora, and promote the maintenance of intestinal health.

[0151] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of glutathione in any of the following: (1) Application in inhibiting aquatic pathogens; (2) Application in the preparation of antibacterial agents for aquatic pathogens; (3) Use in the preparation of drugs for preventing and / or treating aquatic pathogen infections.

2. The use according to claim 1, characterized in that The aquatic pathogen is Aeromonas vermiformis.

3. A composition, characterized in that Includes glutathione and phenyllactic acid.

4. The composition according to claim 3, wherein The mass ratio of the glutathione to the phenyllactic acid is (0.195-0.781):(0.05-0.2).

5. Use of the composition according to claim 3 or 4 in any of the following: (1) Application in inhibiting aquatic pathogens; (2) Application in the preparation of antibacterial agents for aquatic pathogens; (3) Use in the preparation of drugs for preventing and / or treating aquatic pathogen infections; (4) Application in the preparation of feed additives; in, The feed additive has at least one of the functions of promoting the growth of juvenile fish, improving intestinal structure, regulating flora and enhancing disease resistance.

6. The use according to claim 5, characterized in that The aquatic pathogen is Aeromonas vermiformis.

7. An Aeromonas vickers bacteriostatic agent, characterized in that: The composition according to claim 3 or 4 is the main active ingredient.

8. A drug for preventing and / or treating Aeromonas vernix infection, characterized in that: The medicine comprises the composition according to claim 3 or 4 as a main active ingredient.

9. A feed additive, characterized in that The feed additive comprises the composition according to claim 3 or 4 as a main active ingredient.

10. The feed additive according to claim 9, characterized in that It also includes acceptable adjuvants in feed additives.

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