Oligosaccharide compositions and uses thereof

Abstract

The present application relates to oligosaccharide composition and its use. The present application particularly relates to the use of oligosaccharide composition in the preparation of products for preventing and / or treating aquaculture diseases caused by Vibrio parahaemolyticus, Vibrio harveyi and Streptococcus agalactiae, wherein the oligosaccharide composition is obtained by degrading polygalacturonan or pectin by a polygalacturonase as shown in SEQ ID No: 1, under the conditions of a polygalacturonase concentration of 0.045 mg / mL to 0.055 mg / mL, a polygalacturonan concentration of 7% to 9%, and a degradation time of 12 hours to 20 hours. The present application also relates to the use of oligosaccharide composition in aquaculture. The oligosaccharide composition of the present application has good bacteriostatic effect and disease prevention and control effect in aquaculture.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of enzyme engineering, and particularly relates to the use of oligosaccharide composition in bacteriostasis and prevention and / or treatment of aquaculture diseases. BACKGROUND

[0002] Pectin is the most common structural polysaccharide in the middle lamella and primary cell wall of plants, and is one of the most complex polysaccharides, mainly composed of homogalacturonan (HG), rhamnogalacturonan-I and rhamnogalacturonan-II. In some structural characterization of pectin, xylogalacturonan and apio-galacturonan were also found.

[0003] Pectic oligosaccharides are low molecular weight fragments extracted or degraded from natural pectin, rich in galacturonic acid, and have diverse structures. Pectic oligosaccharides have significant prebiotic effects and immunomodulatory functions, and have broad application prospects in the fields of functional foods, health products, feed additives, etc.

[0004] Enzymatic degradation is the main means to obtain oligosaccharides with specific structures. Pectinase is a general term for a class of enzymes that specifically hydrolyze pectin substrates, and is widely used in food, beverage, feed, papermaking and textile industries. Polygalacturonase (PG) is one of the most widely studied, most clearly characterized and most widely used commercial pectinases, which can cleave the alpha-1, 4-glycosidic bonds of pectin backbone (i.e. HG) by hydrolysis to obtain pectic oligosaccharides.

[0005] Currently, it has been reported that pectic oligosaccharides can directly inhibit specific pathogenic bacteria such as Staphylococcus aureus and Listeria monocytogenes through their structural characteristics (such as acidic groups, side chain structures, etc.).

[0006] However, most of the existing researches report that pectic oligosaccharides have good inhibitory effect on Gram-positive bacteria such as Staphylococcus aureus, and there are few reports on the inhibition of pectic oligosaccharides on Vibrio parahaemolyticus, Vibrio harveyi and other aquaculture pathogens.

[0007] Vibrio parahaemolyticus is a Gram-negative bacterium that usually carries TDH (thermostable direct hemolysin) or TRH (thermostable related hemolysin) gene, has strong pathogenicity, and can cause great harm to shrimp, shellfish, fish and other aquatic animals. It can survive in sediment and plankton for a long time and is difficult to completely eliminate.

[0008] Vibrio harveyi is a Gram-negative bacterium, and its extracellular proteases and phospholipases are strong virulence factors. The main hosts of Vibrio harveyi are shrimps, fish, cephalopods, etc., which can secrete proteases to destroy host tissues, induce systemic infection, and also co-infect with other Vibrio to aggravate the disease.

[0009] Streptococcus agalactiae is a devastating pathogen of aquaculture (especially tilapia), and its capsule anti-phagocytosis, hemolysin destroys tissue and environmental persistence constitute the core virulence. It can survive in the sediment for 120 days, and conventional disinfection cannot completely remove it.

[0010] At present, for the prevention and treatment of the above-mentioned pathogenic bacteria, environmental management (such as measures to control density, increase oxygen, and regularly dredge) and precise use of antibiotics are mostly used. However, with the continuous change of the environment and the evolution of pathogenic microorganisms, the prevention and treatment work is facing severe challenges.

[0011] Therefore, the field of aquaculture urgently needs a substitute means that can effectively inhibit the growth of pathogenic microorganisms and better achieve the prevention and treatment effect without relying on antibiotics. SUMMARY

[0012] The technical problem to be solved by the present application is how to efficiently prevent and treat the growth of pathogenic bacteria such as Vibrio parahaemolyticus, Vibrio harveyi and Streptococcus agalactiae and the diseases caused by these pathogenic bacteria in aquaculture. Accordingly, the inventors have unexpectedly found that an oligosaccharide composition prepared from the polygalacturonase shown in SEQ ID No: 1 under specific enzymatic conditions can simultaneously inhibit the growth of Vibrio parahaemolyticus, Vibrio harveyi and Streptococcus agalactiae, thereby completing the present application.

[0013] Accordingly, the technical solution adopted by the present application to solve its technical problem is as follows.

[0014] The present application provides, in a first aspect, a use of an oligosaccharide composition in aquaculture, the oligosaccharide composition being obtained by degrading polygalacturonan with a polygalacturonase as shown in SEQ ID No: 1, the degrading conditions being a concentration of the polygalacturonase of 0.045 mg / mL to 0.055 mg / mL, a concentration of the polygalacturonan of 7% to 9%, and a degrading time of 12 hours to 20 hours.

[0015] In some embodiments, the use is a use for inhibiting the growth of Vibrio parahaemolyticus, Vibrio harveyi and Streptococcus agalactiae in aquaculture.

[0016] In some embodiments, the concentration of the polygalacturonase is 0.048 mg / mL to 0.052 mg / mL.

[0017] In some embodiments, the concentration of the polygalacturonan is 7.5% to 8.5%.

[0018] In some embodiments, the polygalacturonan is present in pectin.

[0019] In some implementations, the degradation time is 14 to 18 hours.

[0020] In some embodiments, the concentration of the oligosaccharide composition is from 100 mg / mL to 500 mg / mL.

[0021] In some embodiments, the concentration of the oligosaccharide composition is from 250 mg / mL to 350 mg / mL.

[0022] A second aspect of the present invention provides the use of an oligosaccharide composition in the preparation of products for the prevention and / or treatment of aquaculture diseases caused by Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae, the oligosaccharide composition being obtained by degradation of polygalacturonic acid by polygalacturonase as shown in SEQ ID No:1, wherein the degradation conditions are a polygalacturonase concentration of 0.045 mg / mL to 0.055 mg / mL, a polygalacturonic acid concentration of 7% to 9%, and a degradation time of 12 hours to 20 hours.

[0023] In some implementations, the concentration of polygalacturonase is from 0.048 mg / mL to 0.052 mg / mL.

[0024] In some implementations, the concentration of polygalacturonic acid is 7.5% to 8.5%.

[0025] In some implementations, polygalacturonic acid is present in pectin.

[0026] In some implementations, the degradation time is 14 to 18 hours.

[0027] In some embodiments, the concentration of the oligosaccharide composition is from 100 mg / L to 500 mg / L.

[0028] In some embodiments, the concentration of the oligosaccharide composition is from 250 mg / L to 350 mg / L.

[0029] A third aspect of the present invention provides an oligosaccharide composition obtained by degrading polygalacturonic acid with polygalacturonase as shown in SEQ ID No:1, wherein the degradation conditions are that the concentration of polygalacturonase is 0.045 mg / mL to 0.055 mg / mL, the concentration of polygalacturonic acid is 7% to 9%, and the degradation time is 12 hours to 20 hours.

[0030] The beneficial effects of this invention are that the oligosaccharide composition obtained by degrading polygalacturonic acid with the polygalacturonase shown in SEQ ID No:1 under specific enzymatic hydrolysis conditions successfully inhibits the growth of Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae, with significantly better inhibitory effects than commercially available enzymes. The oligosaccharide composition of this invention can prevent and treat aquaculture diseases caused by Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae, providing the aquaculture industry with an efficient and convenient means to inhibit the growth of pathogenic microorganisms and prevent and control diseases. Attached Figure Description

[0031] The accompanying drawings, which are provided to further illustrate embodiments of the invention and constitute a part of this invention, are not intended to limit the scope of the invention.

[0032] Figure 1 The figure shows the results of single-factor experiments on the degradation conditions of polygalacturonic acid; where A represents the single-factor result of substrate concentration; B represents the single-factor result of time; and C represents the single-factor result of enzyme concentration.

[0033] Figure 2 The response surface plot and contour plot are for substrate concentration and time.

[0034] Figure 3 The response surface plot and contour plot are for substrate concentration and enzyme concentration.

[0035] Figure 4 The output shows the response surface plot and contour plot for enzyme concentration and time.

[0036] Figure 5 This is a mass spectrometry result of the oligosaccharide composition in the first group of Example 3.

[0037] Figure 6 This is a mass spectrometry result of the oligosaccharide composition in the second group of Example 3.

[0038] Figure 7 This is a mass spectrometry result of the oligosaccharide composition in the third group of Example 3.

[0039] Figure 8 This is a mass spectrometry result of the oligosaccharide composition in the fourth group of Example 3.

[0040] Figure 9 This is a diagram showing the inhibition zone results of the experimental group in Example 5.

[0041] Figure 10 This is a diagram showing the inhibition zone results of control group 1 in Example 5.

[0042] Figure 11 This is a diagram showing the inhibition zone results of control group 2 in Example 5.

[0043] Figure 12 This is a diagram showing the inhibition zone results of control group 3 in Example 5.

[0044] Figure 13 OD of Vibrio harveyi 0-72h 600 Change curve graph.

[0045] Figure 14 OD of Vibrio parahaemolyticus 0-72h 600 Change curve graph.

[0046] Figure 15 OD of Streptococcus agalactiae for 0-72 hours 600 Change curve graph.

[0047] Figure 16 This is a graph showing the growth of Vibrio harveyi in each group after 0 hours of culture in Example 6.

[0048] Figure 17 This is a diagram showing the growth of Vibrio harveyi in each group after 12 hours of culture in Example 6.

[0049] Figure 18 This is a diagram showing the growth of Vibrio harveyi in each group after 24 hours of culture in Example 6.

[0050] Figure 19 This is a diagram showing the growth of Vibrio harveyi in each group after 36 hours of culture in Example 6.

[0051] Figure 20 This is a diagram showing the growth of Vibrio harveyi in each group after 72 hours of culture in Example 6.

[0052] Figure 21 This is a graph showing the growth of Vibrio parahaemolyticus in each group after 0 hours of culture in Example 6.

[0053] Figure 22 This is a graph showing the growth of Vibrio parahaemolyticus in each group after 12 hours of culture in Example 6.

[0054] Figure 23 This is a graph showing the growth of Vibrio parahaemolyticus in each group after 24 hours of culture in Example 6.

[0055] Figure 24 This is a graph showing the growth of Vibrio parahaemolyticus in each group after 36 hours of culture in Example 6.

[0056] Figure 25 This is a graph showing the growth of Vibrio parahaemolyticus in each group after 72 hours of culture in Example 6.

[0057] Figure 26 This is a graph showing the growth of agalactia-streptococci in each group after 0 hours of culture in Example 6.

[0058] Figure 27 This is a graph showing the growth of agalactia-streptococci in each group after 12 hours of culture in Example 6.

[0059] Figure 28 This is a graph showing the growth of agalactia-streptococci in each group after 24 hours of culture in Example 6.

[0060] Figure 29 This is a graph showing the growth of agalactia-streptococci in each group after 36 hours of culture in Example 6.

[0061] Figure 30 This is a graph showing the growth of agalactia-streptococci in each group after 72 hours of culture in Example 6. Detailed Implementation

[0062] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood in the art to which this invention pertains. The following definitions are supplementary to those definitions in the art and relate to this application, but are not extrapolated to any relevant or unrelated circumstances, such as any conventionally used patent or application. While any methods and materials similar to or equivalent to those described herein may be used in the practical testing of this valve, the materials and methods described herein are preferred. Therefore, the terminology used herein is intended to describe specific embodiments only and is not intended to limit the invention.

[0064] The terms "comprising," "including," and "having" in this invention are open-ended descriptions, encompassing the specified steps described, as well as other steps that do not materially affect them, and are optional and not excluded. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of said sequence, or may have additional amino acids or nucleotides at one or both ends of said protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0065] The term "about" in this invention is used to indicate the standard deviation allowed by the numerical value and the apparatus or method for determining the numerical value.

[0066] The first aspect of the present invention provides the use of an oligosaccharide composition in aquaculture, the oligosaccharide composition being obtained by degrading polygalacturonic acid with polygalacturonase as shown in SEQ ID No:1, wherein the degradation conditions are that the concentration of polygalacturonase is 0.045 mg / mL to 0.055 mg / mL, the concentration of polygalacturonic acid is 7% to 9%, and the degradation time is 12 hours to 20 hours.

[0067] The amino acid sequence of the polygalacturonase (also known as MlPG288A) of the present invention is shown in SEQ ID No:1:

[0068] TTCTVAKDASDDATTITAAFNACKNGGTVVFTKGQTYNLKSLVSVSGLKNVNVQFYGTVNLPAYNTKFDGESSYFLIKGDNIHWDGNNVGGFVGGGQDWWNAQDKKAPSVLRITATHSSFINFKISQSPRAHLGVTSSDDVLLQHITLHSVSSNSNLPKNTDALDISNSKNIVVQNS DFTVGDDCLAINGNVSNVTLSDVTCTTNGHGFSVGSLGKGGETDVVKDITVQNSACINCQNGVRIKTWPGGKGSVSNVKFKNVNLPSVENAVLITTHYCDNNQMSYCNGKDDASLTISDVNISGLTGSMSGSNPMVNINCSTNTPCSGFSLSGITISKNSKTKANVCTNLNGASSISYC

[0069] In this document, the term "oligosaccharide composition" refers to the enzymatic hydrolysis product obtained by degrading polygalacturonic acid using polygalacturonase as shown in SEQ ID No:1.

[0070] In this article, polygalacturonic acid can exist alone or in a mixed system with other substances (e.g., pectin).

[0071] In some specific implementations, polygalacturonic acid is present in pectin.

[0072] In some implementations, the use is for inhibiting the growth of Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae in aquaculture.

[0073] In some embodiments, the concentration of polygalacturonase is from 0.045 mg / mL to 0.055 mg / mL. For example, the concentration of polygalacturonase can be 0.045 mg / mL, 0.046 mg / mL, 0.047 mg / mL, 0.048 mg / mL, 0.049 mg / mL, 0.05 mg / mL, 0.051 mg / mL, 0.052 mg / mL, 0.053 mg / mL, 0.054 mg / mL, or 0.055 mg / mL.

[0074] In some embodiments, the concentration of polygalacturonase is from 0.047 mg / mL to 0.053 mg / mL. In some embodiments, the concentration of polygalacturonase is from 0.049 mg / mL to 0.051 mg / mL. In one specific embodiment, the concentration of polygalacturonase is 0.05 mg / mL.

[0075] In some embodiments, the concentration of polygalacturonic acid is 7% to 9%. For example, the concentration of polygalacturonic acid can be 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.1%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, or 9%.

[0076] In some embodiments, the concentration of polygalacturonic acid is 7.5% to 8.5%. In some embodiments, the concentration of polygalacturonic acid is 7.9% to 8.1%. In one specific embodiment, the concentration of polygalacturonic acid is 8%.

[0077] In some implementations, the degradation time is 12 to 20 hours. For example, the degradation time can be 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours.

[0078] In some embodiments, the degradation time is 14 to 18 hours. In some embodiments, the degradation time is 15 to 17 hours. In one specific embodiment, the degradation time is 16 hours.

[0079] By controlling the degradation conditions within the above range, the resulting oligosaccharide composition can effectively inhibit the growth of Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae.

[0080] In one specific implementation, the concentration of polygalacturonase is 0.05 mg / mL, the concentration of polygalacturonic acid is 8%, and the degradation time is 16 hours.

[0081] In some embodiments, the oligosaccharide composition is used at concentrations ranging from 100 mg / mL to 500 mg / mL to inhibit the growth of Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae. For example, the concentrations of the oligosaccharide composition can be 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, 200 mg / mL, 220 mg / mL, 240 mg / mL, 260 mg / mL, 280 mg / mL, 300 mg / mL, 320 mg / mL, 340 mg / mL, 360 mg / mL, 380 mg / mL, 400 mg / mL, 420 mg / mL, 440 mg / mL, 460 mg / mL, 480 mg / mL, and 500 mg / mL.

[0082] When the concentration of the oligosaccharide composition is within the above range, it can fully exert its inhibitory effect on the growth of Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae, effectively inhibiting the growth of these three pathogenic bacteria.

[0083] In some embodiments, the concentration of the oligosaccharide composition is from 200 mg / mL to 400 mg / mL. In some embodiments, the concentration of the oligosaccharide composition is from 250 mg / mL to 350 mg / mL. In one specific embodiment, the concentration of the oligosaccharide composition is 300 mg / mL.

[0084] The present invention also provides the use of oligosaccharide compositions in the preparation of products for the prevention and / or treatment of aquaculture diseases caused by Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae, wherein the oligosaccharide compositions are obtained by degradation of polygalacturonic acid by polygalacturonase as shown in SEQ ID No:1, wherein the degradation conditions are a polygalacturonase concentration of 0.045 mg / mL to 0.055 mg / mL, a polygalacturonic acid concentration of 7% to 9%, and a degradation time of 12 hours to 20 hours.

[0085] In some implementations, the product may be a drug for the prevention and / or treatment of aquaculture diseases caused by Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae.

[0086] In alternative implementations, the product may be other products capable of preventing and / or treating aquaculture diseases caused by Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae, for example, aquaculture feed.

[0087] In some implementation plans, aquaculture is marine aquaculture.

[0088] In the specific implementation plan, marine aquaculture is seahorse farming.

[0089] In some implementation plans, aquaculture is defined as freshwater aquaculture.

[0090] In the specific implementation plan, freshwater aquaculture is carried out through willow root cultivation.

[0091] In some embodiments, the concentration of polygalacturonase is from 0.045 mg / mL to 0.055 mg / mL. For example, the concentration of polygalacturonase can be 0.045 mg / mL, 0.046 mg / mL, 0.047 mg / mL, 0.048 mg / mL, 0.049 mg / mL, 0.05 mg / mL, 0.051 mg / mL, 0.052 mg / mL, 0.053 mg / mL, 0.054 mg / mL, or 0.055 mg / mL.

[0092] In some embodiments, the concentration of polygalacturonase is from 0.047 mg / mL to 0.053 mg / mL. In some embodiments, the concentration of polygalacturonase is from 0.049 mg / mL to 0.051 mg / mL. In one specific embodiment, the concentration of polygalacturonase is 0.05 mg / mL.

[0093] In some embodiments, the concentration of polygalacturonic acid is 7% to 9%. For example, the concentration of polygalacturonic acid can be 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.1%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, or 9%.

[0094] In some embodiments, the concentration of polygalacturonic acid is 7.5% to 8.5%. In some embodiments, the concentration of polygalacturonic acid is 7.9% to 8.1%. In one specific embodiment, the concentration of polygalacturonic acid is 8%.

[0095] In some implementations, the degradation time is 12 to 20 hours. For example, the degradation time can be 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours.

[0096] In some embodiments, the degradation time is 14 to 18 hours. In some embodiments, the degradation time is 15 to 17 hours. In one specific embodiment, the degradation time is 16 hours.

[0097] By controlling the degradation conditions within the above range, the resulting oligosaccharide composition can achieve good preventive and therapeutic effects against infections in aquaculture seawater caused by Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae.

[0098] In one specific implementation, the concentration of polygalacturonase is 0.05 mg / mL, the concentration of polygalacturonic acid is 8%, and the degradation time is 16 hours.

[0099] In some embodiments, the concentration of the oligosaccharide composition in the aquaculture environment is from 100 mg / L to 500 mg / L. For example, the concentration of the oligosaccharide composition can be 100 mg / L, 120 mg / L, 140 mg / L, 160 mg / L, 180 mg / L, 200 mg / L, 220 mg / L, 240 mg / L, 260 mg / L, 280 mg / L, 300 mg / L, 320 mg / L, 340 mg / L, 360 mg / L, 380 mg / L, 400 mg / L, 420 mg / L, 440 mg / L, 460 mg / L, 480 mg / L, or 500 mg / L.

[0100] When the concentration of the oligosaccharide composition is within the above range, it can fully exert the preventive and therapeutic effects of the oligosaccharide composition on infections in aquaculture seawater, and effectively prevent and treat infections in aquaculture seawater caused by Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae.

[0101] In some embodiments, the concentration of the oligosaccharide composition is from 200 mg / L to 400 mg / L. In some embodiments, the concentration of the oligosaccharide composition is from 250 mg / L to 350 mg / L. In one specific embodiment, the concentration of the oligosaccharide composition is 300 mg / L.

[0102] The present invention also provides an oligosaccharide composition obtained by degradation of polygalacturonic acid by polygalacturonase as shown in SEQ ID No:1, wherein the concentration of polygalacturonase is from 0.045 mg / mL to 0.055 mg / mL, the concentration of polygalacturonic acid is from 7% to 9%, and the degradation time is from 12 hours to 20 hours.

[0103] In some embodiments, the oligosaccharide compositions of the present invention are used for non-therapeutic purposes.

[0104] In some embodiments, the oligosaccharide compositions of the present invention can be used to prepare products for inhibiting the growth of Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae.

[0105] In some embodiments, the oligosaccharide compositions of the present invention can be used to prepare products for the prevention and / or treatment of infections in aquaculture seawater caused by Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae.

[0106] The present invention also provides a method for inhibiting the growth of Vibrio parahaemolyticus, Vibrio harveyi and Streptococcus agalactiae, comprising applying the oligosaccharide composition of the present invention at a concentration of 100 mg / mL to 500 mg / mL.

[0107] The present invention also provides a method for preventing and / or treating infections in aquaculture seawater caused by Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae, comprising applying the oligosaccharide composition of the present invention to the aquaculture seawater at a concentration of 100 mg / L to 500 mg / L.

[0108] The following describes preferred embodiments of the present invention, but the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products.

[0109] Example 1: Single-factor experiment on the degradation conditions of polygalacturonic acid

[0110] Single-factor preliminary experiments were conducted to investigate the substrate concentration, time, and enzyme concentration in the degradation process of polygalacturonic acid. The specific reaction conditions are as follows:

[0111] (1) Keep the enzyme concentration and time constant, and adjust the substrate concentration to 5%, 6%, 7%, 8%, 9%, and 10%, respectively;

[0112] (2) Keep the enzyme concentration and substrate concentration constant, and adjust the time to 4, 8, 12, 16, 20 and 24 hours respectively;

[0113] (3) Keeping the substrate concentration and time constant, the enzyme concentration was adjusted to 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, and 0.08 mg / g, respectively.

[0114] After the reaction was completed, the reducing sugar yield of each group was determined by the (3,5-dinitrosalicylic acid (DNS) method), and the change curves were plotted.

[0115] result:

[0116] The change curves of the three single-factor experiments are as follows: Figure 1 As shown in A, B, and C, based on the results of the single-factor experiment, the substrate concentration range of 6% to 10%, the time range of 8 hours to 24 hours, and the enzyme concentration range of 0.04 mg / g to 0.06 mg / g were selected for subsequent experiments.

[0117] Example 2: Response surface methodology optimization experiment for polygalacturonic acid degradation conditions

[0118] Based on the single-factor experimental results in Example 1, response surface methodology was used to optimize the degradation of polygalacturonic acid using polygalacturonase (MlPG28A) as shown in SEQ ID No:1. The key experimental conditions and their corresponding levels are shown in Table 1.

[0119] Table 1

[0120]

[0121] Using Box-Behnken design (BBD), 17 experimental combinations were generated to meet the three-level optimization requirements of the three sets of key conditions mentioned above, as shown in Table 2, including 12 boundary condition experiments and 5 center point repetition experiments.

[0122] In each experiment, reducing sugar yield was used as the primary response variable to quantitatively evaluate the reaction efficiency under different parameter combinations. For the three key conditions mentioned above, pairwise factor analyses were performed, and response surface plots and contour plots were generated.

[0123] Table 2

[0124]

[0125]

[0126] Results: The experimental data were analyzed by multiple regression using Design-Expert 11 software. A mathematical model was established between reducing sugar yield (Y) and key process parameters (substrate concentration A, time B, and enzyme concentration). Analysis of variance (ANOVA) was also performed. The results are shown in Table 3 below.

[0127] Table 3

[0128]

[0129]

[0130] As shown in Table 3, the regression model is highly statistically significant (F = 77.96, P < 0.0001), effectively explaining the changes in reducing sugar content. The model fit index shows that the coefficient of determination R0 is high. 2 =0.9901 indicates that the model can explain 99.01% of the response variation, while the adjusted coefficient of determination Adj R 2 The p-value of 0.9774 further confirms the reliability of the model. The goodness-of-fit test results (P = 0.1141 > 0.05) indicate that there is no significant underfit, confirming that the selected quadratic model accurately reflects the relationship between each factor and the response.

[0131] The results further indicated that the importance of the influencing factors was in the following order: reaction time (B) > enzyme concentration (C) > substrate concentration (A), with time having a highly significant effect on reducing sugar yield (P < 0.0001). Interaction analysis showed a highly significant synergistic effect between time and enzyme concentration (BC) (P < 0.0001), while the interaction between substrate concentration and enzyme concentration (AC) was relatively significant (P = 0.0191). In contrast, the interaction between substrate concentration and time (AB) was less significant (P = 0.8039).

[0132] The response surface methodology results for the 17 groups of experiments in Table 2 are as follows: response surface plots and contour plots for substrate concentration and time are shown below. Figure 2 As shown; the response surface plot and contour plot of substrate concentration and enzyme concentration are as follows. Figure 3 As shown; the response surface plot and contour plot of enzyme concentration and time are as follows. Figure 4 As shown in the figure. By measuring the content of reducing sugars produced, it was found that the highest content of reducing sugars was produced under the conditions of 0.05 mg / mL MlPG28A enzyme solution, 8% polygalacturonic acid concentration, and 16 hours of enzymatic hydrolysis; the highest content of reducing sugars was produced under the conditions of 0.04 mg / mL MlPG28A enzyme solution, 8% polygalacturonic acid concentration, and 24 hours of enzymatic hydrolysis; and the lowest content of reducing sugars was produced under the conditions of 0.04 mg / mL MlPG28A enzyme solution, 8% polygalacturonic acid concentration, and 8 hours of enzymatic hydrolysis.

[0133] Based on the significance and response surface methodology results, oligosaccharide compositions were prepared under the conditions corresponding to the three groups mentioned above, and subsequent experiments were completed.

[0134] Example 3: Preparation of Oligosaccharide Composition

[0135] Enzyme solution was added to 100 mL of 10 mg / mL polygalacturonic acid (PGA) at pH 5.0 according to the following concentrations, and the enzymatic hydrolysis reaction was carried out at 30 °C under the following conditions. After the reaction was completed, the enzyme was inactivated by heating at 95 °C. Then, undegraded proteins and substrates were removed using a 0.22 μm hydrophilic filter membrane, and then the mixture was freeze-dried for two days to obtain the corresponding oligosaccharide composition powder.

[0136] Group 1: 0.05 mg / mL MlPG28A enzyme solution, polygalacturonic acid concentration of 8%, enzymatic hydrolysis time of 16 hours;

[0137] Group 2: 0.04 mg / mL MlPG28A enzyme solution, polygalacturonic acid concentration of 8%, enzymatic hydrolysis time of 24 hours;

[0138] Group 3: 0.04 mg / mL MlPG28A enzyme solution, polygalacturonic acid concentration of 8%, enzymatic hydrolysis time of 8 hours;

[0139] Group 4: 0.05 mg / mL Jiahe Xuri pectinase solution, polygalacturonic acid concentration of 8%, and enzymatic hydrolysis time of 16 hours.

[0140] Example 4: Mass spectrometry characterization of oligosaccharide compositions

[0141] The oligosaccharide composition powders obtained in the four groups in Example 3 were dissolved in water, filtered through a 0.22 μm aqueous filter membrane, and then analyzed by mass spectrometry (ESI-MS). The ESI-MS mass spectrometry conditions were: anion mode; scan range of 100 to 2000 m / z.

[0142] Results: The mass spectrometry results of the oligosaccharide compositions in groups one through four are as follows: Figures 5 to 8 As shown, the mass spectrum of the first group (optimized conditions: enzyme concentration 0.05 mg / mL, substrate concentration 8%, 16 hours) is as follows: Figure 5 The mass spectrometry results showed typical characteristic peaks of galacturonic acid oligosaccharides, with the main peaks at m / z 369.1 (dimer) and m / z 545.1 (trimer), exhibiting regular 176 Da intervals (monomer molecular weight). The high molecular weight components (>pentamers) showed weak signals, indicating thorough enzymatic digestion and homogeneous products. The second group (optimized conditions: enzyme concentration 0.04 mg / mL, substrate concentration 8%, 24 hours) mass spectrometry... Figure 6 The mass spectrometry of the third group (optimized conditions: enzyme concentration 0.04 mg / mL, substrate concentration 8%, 8 hours) showed a broader peak distribution (m / z 150-800), a decrease in peak intensity of approximately 40% at m / z 353.1, and the appearance of highly polymerized components such as m / z 897.2 (pentamer), reflecting incomplete degradation due to insufficient enzyme concentration. Figure 7 The first group (commercially available enzymes) exhibited abnormal peak shapes, with a raised baseline and the m / z 353.1 peak almost disappearing, indicating incomplete degradation due to short-duration enzymatic digestion and the presence of a large amount of undegraded substrate. The fourth group (commercially available enzymes) showed the following mass spectrometry results: Figure 8 The results were significantly different, with the main peak shifting to m / z 369.1 and lacking regular 176Da interval peak groups, indicating that the commercial enzyme may contain other glycosidase activities, producing modified products and complex impurities.

[0143] Example 5: Antibacterial experiment of oligosaccharide composition

[0144] Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae were selected as target strains. After being retrieved from the glycerol bacterial library, the strains were streaked onto LB agar plates and incubated at 37°C for 18 hours to revive the bacterial culture. Single colonies were picked and inoculated into 5 mL of LB liquid medium and cultured at 37°C with shaking until the logarithmic growth phase. The bacterial culture was then diluted to 10⁻⁶ with physiological saline. 6 CFU / mL available for use.

[0145] Prepare LB agar medium and pour it into a 90 mm sterile petri dish to cool and solidify. Take 100 μL of LB agar. 6 A CFU / mL bacterial suspension was evenly spread on the surface of the culture medium, followed by the even placement of 8 mm sterile filter paper discs on the plate surface. 34 μL of the experimental group and each control group solution were added to the filter paper discs. After incubating the plates at 37°C for 12 hours, the inhibition zones around the filter paper discs were observed and measured. The antibacterial activity of the oligosaccharide composition was evaluated by comparing the size of the inhibition zones between the experimental and control groups.

[0146] The solution compositions of the experimental group and the control group are as follows:

[0147] Experimental group: 300 mg / mL oligosaccharide composition solution prepared in the first group of Example 3;

[0148] Control group 1: 300 mg / mL oligosaccharide composition solution prepared in the second group of Example 3;

[0149] Control group 2: 300 mg / mL oligosaccharide composition solution prepared in the third group of Example 3;

[0150] Control group 3: 300 mg / mL oligosaccharide composition solution prepared in the fourth group of Example 3;

[0151] In each group, 1 mg / mL ampicillin solution was added separately as a positive control and sterile deionized water as a negative control.

[0152] Results: The inhibition zone results of the experimental group, control group 1, control group 2, and control group 3 are as follows: Figures 9 to 12 As shown in the figure, the oligosaccharide composition in the experimental group exhibits excellent antibacterial effects against Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae, while the oligosaccharide compositions in control groups 2 and 4 show only moderate antibacterial effects against the three bacteria, and control group 3 shows no antibacterial effect against any of the three bacteria. Therefore, it can be concluded that the oligosaccharide composition in the experimental group can effectively inhibit the growth of Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae.

[0153] The differences in the antibacterial effects mentioned above can be explained by the mass spectrometry differences of the compositions in Example 4. The homogeneous dimer / trimer combination in the experimental group product has the best antibacterial activity, while the modified product of the commercially available enzyme in control group 3 may destroy the free carboxyl structure necessary for activity, resulting in a significant reduction in antibacterial effect.

[0154] Example 6: Experiment on the prevention and treatment of aquaculture waterborne infections using oligosaccharide compositions

[0155] (1) First, weigh 0.3g of the oligosaccharide composition prepared in the first group of Example 3, then add 1mL of preheated (40℃) sterile water, vortex and sonicate, and then filter with a 0.22μm PVDF filter membrane to obtain a 300mg / mL oligosaccharide composition mother liquor.

[0156] (2) The seahorse culture water (when selecting the culture water, the number of Vibrio obtained from the blank group culture was used as the reference standard for this experiment) was used as the experimental water for Vibrio parahaemolyticus and Vibrio harveyi. The willow root culture water (when selecting the culture water, the number of colonies obtained from the blank group culture was used as the reference standard for this experiment) was used as the experimental water for Streptococcus agalactiae.

[0157] (3) The three pathogens were cultured to OD 600 =0.6, then adjust the bacterial culture to 1×10 using PBS. 7 CFU / mL available;

[0158] (4) Process according to the following groups:

[0159] Prevention group: 100 mL water + 100 μL oligosaccharide composition stock solution (final concentration 300 mg / L), shake to mix, let stand at 28℃ for 30 minutes, then add 1 mL bacterial solution (final concentration 1×10⁻⁶). 5 (CFU / mL), start timing 0h.

[0160] Treatment group: 100mL water + 1mL bacterial solution, pre-cultured at 28℃ for 2 hours (simulated infection), then 100μL oligosaccharide stock solution was added, and the timer was reset to 0h.

[0161] Positive control: No oligosaccharide composition added, 100 mL water + 1 mL bacterial solution.

[0162] Control group: No oligosaccharide composition or bacterial solution added, 100mL water + 1mL sterile water.

[0163] (5) The mixtures of each group were incubated at 37℃ and 180 rpm with shaking. OD values ​​of each group were measured at 0h, 6h, 12h, 18h, 24h, 36h, 48h, 60h and 72h. 600Additionally, 100 μL of samples were taken from samples collected at 0h, 12h, 24h, 36h, and 72h and spread onto solid plates (TCBS Vibrio parahaemolyticus and Vibrio harveyi were cultured on LCBS medium, and LB medium was used for Streptococcus agalactiae). The plates were incubated at 37℃ for 12 hours, and the samples were observed and photographed after the incubation period.

[0164] result:

[0165] OD of Vibrio harveyi 0-72h 600 Changes as follows Figure 13 As shown; OD of Vibrio parahaemolyticus 0-72h 600 Changes as follows Figure 14 As shown; OD of Streptococcus agalactiae from 0-72h 600 Changes as follows Figure 15 As shown.

[0166] It can be seen that, compared with the positive control group without the oligosaccharide composition, the biomass of Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae in the prevention and treatment groups with the oligosaccharide composition was significantly reduced. This indicates that the oligosaccharide composition in both the prevention and treatment groups can effectively inhibit the proliferation of Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae, and achieve good preventive and therapeutic effects.

[0167] The growth of Vibrio harveyi in samples from 0h, 12h, 24h, 36h, and 72h after 12 hours of culture is shown below. Figures 16 to 20 As shown; the growth of Vibrio parahaemolyticus in samples from 0h, 12h, 24h, 36h, and 72h after 12 hours of culture in each group are as follows. Figures 21 to 25 As shown; the growth of agalactia-resistant streptococci in samples from 0h, 12h, 24h, 36h, and 72h after 12 hours of culture is shown in the figures below. Figures 26 to 30 As shown.

[0168] It can be seen that, compared with the positive control group, no colonies of Vibrio parahaemolyticus and Vibrio harveyi were produced on the plates of the prevention and treatment groups. Only a small number of colonies appeared on the plates of Streptococcus agalactiae in the prevention and treatment groups, but the number of colonies was significantly less than that in the positive control group. This also indicates that the oligosaccharide composition of the experimental group can effectively inhibit the growth of Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae, and thus can effectively prevent and treat marine infections caused by Vibrio parahaemolyticus, Vibrio harveyi, and Streptococcus agalactiae. The colony aggregation on the plates of the blank group after 12 hours of incubation at 0h and 12h may be due to the presence of some environmental microorganisms other than the above three bacteria in the aquaculture water. These microorganisms gradually died out under subsequent incubation conditions due to incompatibility with high temperature, nutrient deficiency, or inhibition by selective culture media (such as TCBS), and thus no longer appeared on the plates after 12 hours of incubation at 24h, 36h, and 72h.

[0169] This document uses specific embodiments to illustrate the principles and implementation methods of the present invention. The descriptions of these embodiments are merely illustrative of the method and its central idea, and are not intended to limit the process. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall under the protection of the claims of the present invention.

Claims

1. Oligosaccharide compositions in the preparation of inhibitors of Vibrio parahaemolyticus in aquaculture environments ( Vibrio parahaemolyticus ), Vibrio harveyi ( Vibrio harveyi ) and agalactococci ( Streptococcus agalactiae The uses of the products grown from ) are characterized by, The oligosaccharide composition is obtained by degrading polygalacturonic acid with polygalacturonase as shown in SEQ ID No:

1. The degradation conditions are that the concentration of the polygalacturonase is 0.045 mg / mL to 0.055 mg / mL, the concentration of the polygalacturonic acid is 7% to 9%, and the degradation time is 12 hours to 20 hours.

2. The use as described in claim 1, characterized in that, The concentration of the polygalacturonase is from 0.048 mg / mL to 0.052 mg / mL, the concentration of the polygalacturonic acid is from 7.5% to 8.5%, and the degradation time is from 14 hours to 18 hours.

3. The use as described in claim 1, characterized in that, The polygalacturonic acid is present in pectin.

4. The use as described in claim 1, characterized in that, The concentration of the oligosaccharide composition is from 100 mg / mL to 500 mg / mL.

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