Method for inhibiting growth of pseudomonas by using phloroglucinol and application thereof

Abstract

The application discloses a method for inhibiting growth of pseudomonas by using fucoidan and application thereof, and is characterized in that the fucoidan is a degradation product of natural seaweed (sodium alginate), and is a linear oligosaccharide with a polymerization degree of 2-25 and composed of guluronic acid and mannuronic acid, and has the advantages of safety, health and no toxic side effects; in combination with the research of the inventors that the fucoidan can target and inhibit growth of pseudomonas in vitro and in intestinal flora, the fucoidan can meet the demand of practical application, can be used for delaying corruption and deterioration of fresh food caused by pseudomonas, and can be used for inhibiting growth of pseudomonas on the surface or in the body of aquatic products or fresh food, and has good application prospect and safety.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for inhibiting the growth of Pseudomonas aeruginosa using brown algae oligosaccharides and its application. Background Technology

[0002] Alginate oligosaccharides (AOS) are linear oligosaccharides composed of α-L-guluronic acid and β-D-mannuronic acid, with a degree of polymerization (DP) of 2-25. They are mostly derived from natural seaweed, such as degradation products of kelp alginate. They are safe, healthy, and have no toxic side effects. Studies have found that they possess various biological activities, including anti-inflammatory and immunomodulatory effects, and are currently widely used in the pharmaceutical, health care, skincare, and animal feed industries. The inventors of this case discovered that alginate oligosaccharides can specifically inhibit the growth of *Pseudomonas* in vitro and in the gut microbiota. *Pseudomonas* are bacteria belonging to the genus *Pseudomonas* of the family Pseudomonadaceae, including *Pseudomonas proteus*, *Pseudomonas fructus*, *Pseudomonas putida*, *Pseudomonas fluorescens*, *Pseudomonas aeruginosa*, *Pseudomonas lundii*, and *Pseudomonas chicory*. Pseudomonas is ubiquitous in soil, freshwater, and seawater environments. It is an obligate aerobic, Gram-negative, non-spore-forming, capsule-like, terminally flagellated, motile bacillus. The bacteria are slightly curved or rod-shaped, and the optimal growth temperature for most strains is 24-28℃.

[0003] *Pseudomonas proteus* is a major pathogen of visceral white spot disease in fish farming. It has been reported in large yellow croaker, sea bass, snakehead, and shad, with the greatest losses occurring in large yellow croaker farming. Large yellow croaker (Larimichthys crocea) was domesticated in the 1990s and is one of my country's largest-scale farmed marine fish and a leading export-oriented aquatic product. Currently, Fujian Province is the main production area for large yellow croaker in my country, accounting for over 80% of the total production. With increasing stocking densities and deteriorating aquatic environments, visceral white spot disease has become a major disease in large yellow croaker farming. *Pseudomonas proteus* is a resident bacterium in the fish gut; under normal conditions, the anaerobic environment in the gut is unfavorable for the growth of obligate aerobic *Pseudomonas*. During spring and winter, large yellow croakers have a decreased feeding capacity, resulting in empty intestines and increased dissolved oxygen levels. This increases the growth of *Pseudomonas proteus*. When this bacterium reaches a certain abundance, it can lead to intestinal flora imbalance, thereby damaging the intestinal immune defense and barrier function. This can cause *Pseudomonas proteus* to invade the host and cause bacteremia. It can then enter organs such as the spleen, kidneys, and liver through blood circulation, triggering a series of clinical symptoms and leading to the death of the host.

[0004] In addition, *Pseudomonas* are also important microorganisms causing spoilage of fresh food. *Pseudomonas berrieseri*, *Pseudomonas putida*, and *Pseudomonas fluorescens*, among others, exhibit strong metabolic decomposition capabilities and are major microorganisms causing undesirable sensory and quality deterioration in meats such as chicken, pork, and beef. The quantity of these psychrophilic bacteria and the proteolytic and lipolytic enzymes they produce also affect the color and flavor of milk, and are a major cause of spoilage and reduced shelf life of chilled cheese. Simultaneously, *Pseudomonas* are also important spoilage bacteria causing fruit and vegetable spoilage, mainly through the production of nitrites and the consumption of nutrients, leading to the spoilage and deterioration of broccoli. Furthermore, *Pseudomonas cichorii* is a key pathogen causing tomato necrosis and lettuce leaf rot.

[0005] Inhibiting Pseudomonas can reduce the occurrence of white spot disease in fish viscera and delay the spoilage of fresh food. In aquaculture, antibiotics can inhibit or kill Pseudomonas proteoglycans in the intestines, reducing the occurrence of white spot disease. However, the prevalence of white spot disease in large yellow croaker is long, lasting from December to May of the following year. Furthermore, the pathogen is widespread in various environments, making long-term preventative medication difficult. Antibiotic administration can also disrupt the intestinal flora, promoting other diseases, slowing growth, accelerating the emergence and spread of drug-resistant strains, and causing long-term adverse effects on the aquatic ecosystem. In the preservation of fresh food, physical sterilization methods such as ozone, ultraviolet light, and steam are commonly used, along with chemical methods such as spraying disinfectants and antibacterial chemicals to inhibit bacteria. In the preservation of fresh food, physical sterilization methods are mostly temporary and cannot completely kill all Pseudomonas bacteria, making it difficult to achieve long-term preservation. Disinfectants and antibacterial chemicals have broad-spectrum bactericidal and antibacterial abilities, and spraying these chemicals may introduce new hazards, especially having a lasting adverse effect on the health of human intestinal flora.

[0006] Therefore, using harmless natural products to target and inhibit the growth of Pseudomonas aeruginosa can reduce the abundance of Pseudomonas proteoglycans in the gut, thereby reducing the occurrence of visceral white spot disease. In fresh food, it can delay spoilage and deterioration without adversely affecting the health of human gut microbiota. Meanwhile, fucoidan, a degradation product of natural seaweed, is a linear oligosaccharide with a degree of polymerization of 2-25, composed of guluronic acid and mannulic acid. It has the advantages of being safe, healthy, and free of toxic side effects. The inventors' research found that fucoidan can target and inhibit the growth of Pseudomonas aeruginosa in vitro and in the gut microbiota, meeting the needs of such practical applications. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a reliable, safe, and effective method for inhibiting the growth of Pseudomonas aeruginosa using fucoidan, and its application.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by this invention is as follows:

[0009] Application of brown alginic oligosaccharides in inhibiting the growth of Pseudomonas aeruginosa.

[0010] As one possible implementation, further, in this application, the alginic oligosaccharide is an alginic oligosaccharide with a degree of polymerization of 2-25 formed by the degradation treatment of sodium alginate.

[0011] As a preferred implementation option, the sodium alginate described in this scheme has an oligosaccharide content of ≥60%, a particle size of ≥100 mesh, a moisture content of ≤10%, an insoluble matter content of ≤1%, and a pH value of 4.0–7.0.

[0012] As a preferred implementation option, the brown algae oligosaccharide described in this scheme is preferably added to nutrients to form a culture medium or used as a component in feed to inhibit the growth of Pseudomonas aeruginosa in aquatic products.

[0013] As a preferred implementation option, the Pseudomonas species mentioned in this scheme are preferably Pseudomonas proteans, Pseudomonas fruticosa, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas lundii, or Pseudomonas chicoryensis.

[0014] Based on the above, the present invention also provides a method for inhibiting the growth of Pseudomonas aeruginosa using fucoidan oligosaccharides, which includes the above-described applications and further includes:

[0015] The brown algae oligosaccharide is dispersed in the growth environment of the subjects or used as a feed component in the feeding of the subjects to inhibit the growth of Pseudomonas aeruginosa on the body surface or in the intestines of the subjects.

[0016] As a preferred implementation option, the implementation object of this solution is preferably aquatic products or fresh food, wherein aquatic products include fish.

[0017] As a preferred implementation option, the target species described in this scheme are large yellow croaker, perch, snakehead, or shad.

[0018] Based on the above, the present invention also provides the application of brown algae oligosaccharides in the farming of large yellow croaker, which includes the method described above.

[0019] Compared with the prior art, the present invention, using the above-mentioned technical solution, has the following beneficial effects: This solution ingeniously proposes the application of fucoidan in inhibiting the growth of Pseudomonas aeruginosa. Utilizing the characteristic that fucoidan is a degradation product derived from natural seaweed (sodium alginate), it is a linear oligosaccharide with a degree of polymerization of 2-25, composed of guluronic acid and mannouronic acid. It has the advantages of being safe, healthy, and free of toxic side effects. Combined with the inventors' research finding that fucoidan can target and inhibit the growth of Pseudomonas aeruginosa in vitro and in the gut microbiota, it meets the needs of practical applications and can be used to delay the spoilage of fresh food caused by Pseudomonas aeruginosa. Furthermore, when used as a feed additive to prevent white spot disease in fish viscera, fucoidan does not have a harmful effect on the health of the fish's gut microbiota. It can be used continuously for a long time without leading to bacterial resistance or water pollution. Therefore, its application in inhibiting the growth of Pseudomonas aeruginosa on or inside aquatic products or fresh seafood has good application prospects and safety. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a comparative experimental diagram showing the inhibition of Pseudomonas proteus growth by alginate oligosaccharide (AOS) in Example 1 of this scheme, where CONT is the control group and AOS is the experimental group;

[0022] Figure 2 This is a comparative experimental diagram showing the inhibition of the growth of *Pseudomonas berries* by alginate oligosaccharides (AOS) in Example 2 of this scheme. CONT represents the control group, and AOS represents the experimental group.

[0023] Figure 3 This is a comparative experimental diagram showing the inhibition of the growth of *Pseudomonas putida* by alginate oligosaccharides (AOS) in Example 3 of this scheme. CONT represents the control group, and AOS represents the experimental group.

[0024] Figure 4 This is a PCoA analysis diagram of the gut microbiota composition of large yellow croaker after in vitro culture under different culture conditions in Example 4 of this scheme;

[0025] Figure 5 This is a comparison chart of the relative abundance of intestinal flora at the genus level after in vitro culture of four large yellow croakers in Example 4 of this scheme;

[0026] Figure 6 This is a heatmap of species abundance at the genus level after in vitro culture of the gut microbiota of large yellow croaker in Example 4 of this scheme;

[0027] Figure 7 This is a comparison chart of the absolute and relative abundance of Pseudomonas bacteria in the intestinal flora of large yellow croaker reduced by brown algae oligosaccharides in Example 4 of this scheme;

[0028] Figure 8 This is a comparison chart of the absolute and relative abundance of Pseudomonas bacteria after in vitro culture of the four major yellow croaker in this program. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be particularly noted that the following embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the following embodiments are only some, not all, embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] To demonstrate the application effect of the present invention, the specific implementation scheme is illustrated by the following examples. In these examples, the fucoidan used is an oligosaccharide derived from seaweed-derived alginate (sodium) degraded into a polymerization degree of 2-25. The technical specifications of the fucoidan are as follows:

[0031]

[0032]

[0033] When inhibiting the growth of Pseudomonas aeruginosa in vitro, this method can be used to spray a liquid containing 0.01%-10% fucoidan onto the surface of the object to inhibit the growth of Pseudomonas aeruginosa.

[0034] When inhibiting the growth of Pseudomonas in the gut, this protocol can use 0.01%-10% of fucoidan to reduce the abundance of Pseudomonas in the gut microbiota of large yellow croaker.

[0035] Example 1

[0036] This embodiment provides the implementation details of in vitro inhibition of Pseudomonas proteus growth by algal oligosaccharides, and the experimental content includes:

[0037] Strains: *Pseudomonas proteus* isolated from cases of white spot disease in large yellow croaker viscera. After streaking culture, single colonies were picked and added to sterile physiological saline, thoroughly mixed, and adjusted to approximately 1.0 × 10⁻⁶. 9 cfu / mL was used as the inoculum.

[0038] Culture media: nutrient broth (control group) and nutrient broth supplemented with 0.5% fucoidan (experimental group).

[0039] The nutritional broth consists of: 10.0 g / L peptone, 3.0 g / L beef extract, and 5.0 g / L sodium chloride, adjusted to a pH of 7.2 ± 0.2.

[0040] Culture conditions: aerobic environment, 25℃, 100rpm.

[0041] The process is as follows:

[0042] (1) Nutrient broth culture medium and nutrient broth culture medium with added 0.5% brown algae oligosaccharide were used as the control group and the experimental group, respectively. Both were pressure sterilized at 115℃ for 20 min and then dispensed into 50mL sterile test tubes, with a specification of 5mL / tube.

[0043] (2) 20 μL of bacterial culture was inoculated into the positive control group and the experimental group, and 20 μL of sterile physiological saline was inoculated into the negative control group. Positive control group and negative control group: 3 test tubes of nutrient broth each; experimental group: 3 test tubes of nutrient broth supplemented with 0.5% fucoidan.

[0044] (3) Culture conditions: Aerobic environment, 25℃, 100rpm culture for 24h;

[0045] (4) Growth detection: Growth was detected in the positive control group and experimental group when the negative control group was sterile. OD values ​​were measured at 15, 20, and 24 hours after culture. Three samples were collected from each tube, and 200 μL of each sample was added to a 96-well plate for detection at a wavelength of 600 nm. The above experiment was repeated three times.

[0046] (5) Calculation: Take the average of three wells for each sample, calculate the average of three samples in the same group, calculate the average of three repeated experiments, calculate the standard error, and use the t test to calculate the significance of the difference between different groups at the same time point. If p < 0.05, mark it as *, and if p < 0.01, mark it as **.

[0047] (6) Results Comparison

[0048] Depend on Figure 1 As shown in the control curve, compared with the control group (CONT, nutrient broth), the experimental group (AOS, nutrient broth medium supplemented with 0.5% alginate oligosaccharide) contained alginate oligosaccharide (AOS), which significantly inhibited the growth of Pseudomonas proteoglycans relative to the control group.

[0049] Example 2

[0050] This embodiment provides the implementation details of in vitro inhibition of *Pseudomonas berries* growth by fucoidan oligosaccharides. The experimental content includes:

[0051] Strains: *Pseudomonas berries* isolated from the surface of spoiled meat, were cultured by streak plating, and single colonies were picked and added to sterile physiological saline, which was then thoroughly mixed to adjust the concentration to approximately 1.0 × 10⁻⁶. 9 cfu / mL was used as the inoculum.

[0052] Culture medium: nutrient broth (control group) and nutrient broth with 0.5% added brown algae oligosaccharides (experimental group), the composition of the nutrient broth was the same as in the example.

[0053] Culture conditions: aerobic environment, 25℃, 100rpm.

[0054] The process is as follows:

[0055] (1) Nutrient broth culture medium (control group) and nutrient broth culture medium with 0.5% brown algae oligosaccharide added (experimental group) were pressure sterilized at 115℃ for 20 min and then dispensed into 50mL sterile test tubes, with a specification of 5mL / tube.

[0056] (2) 20 μL of bacterial culture was inoculated into the positive control group and the experimental group, and 20 μL of sterile physiological saline was inoculated into the negative control group. Positive control group and negative control group: 3 test tubes of nutrient broth each; experimental group: 3 test tubes of nutrient broth supplemented with 0.5% fucoidan.

[0057] (3) Culture conditions: Aerobic environment, 25℃, 100rpm culture for 24h;

[0058] (4) Growth detection: Growth was detected in the positive control group and experimental group when the negative control group was sterile. OD values ​​were measured at 15, 20, and 24 hours after culture. Three samples were collected from each tube, and 200 μL of each sample was added to a 96-well plate for detection at a wavelength of 600 nm. The above experiment was repeated three times.

[0059] (5) Calculation: Take the average of three wells for each sample, calculate the average of three samples in the same group, calculate the average of three repeated experiments, calculate the standard error, and use the t test to calculate the significance of the difference between different groups at the same time point. If p < 0.05, mark it as *, and if p < 0.01, mark it as **.

[0060] (6) Results Comparison

[0061] Depend on Figure 2 As shown in the control curve, compared with the control group (CONT, nutrient broth), the experimental group (AOS, nutrient broth medium supplemented with 0.5% alginate oligosaccharide) contains alginate oligosaccharide (AOS), which significantly inhibits the growth of Pseudomonas berries relative to the control group.

[0062] Example 3

[0063] This embodiment provides the implementation details of the in vitro inhibition of *Pseudomonas putida* growth by fucoidan oligosaccharides, and the experimental content includes:

[0064] Strain: *Pseudomonas putida*. After streaking culture, single colonies were picked and added to sterile physiological saline, thoroughly mixed, and adjusted to approximately 1.0 × 10⁻⁶. 9 cfu / mL was used as the inoculum.

[0065] Culture medium: nutrient broth (control group) and nutrient broth with 0.5% added brown algae oligosaccharides (experimental group), the composition of the nutrient broth was the same as in the example.

[0066] Culture conditions: aerobic environment, 18℃, 100rpm.

[0067] The process is as follows:

[0068] (1) Nutrient broth culture medium (control group) and nutrient broth culture medium with 0.5% brown algae oligosaccharide added (experimental group) were pressure sterilized at 115℃ for 20 min and then dispensed into 50mL sterile test tubes, with a specification of 5mL / tube.

[0069] (2) 20 μL of bacterial culture was inoculated into the positive control group and the experimental group, and 20 μL of sterile physiological saline was inoculated into the negative control group. Positive control group and negative control group: 3 test tubes of nutrient broth each; experimental group: 3 test tubes of nutrient broth supplemented with 0.5% fucoidan.

[0070] (3) Culture conditions: aerobic environment, 18℃, 100rpm for 24h.

[0071] (4) Growth detection: Growth was detected in the positive control group and the experimental group while the negative control group was sterile. OD values ​​were measured at 15, 20, and 24 hours after culture. Three samples were collected from each tube, and 200 μL of each sample was added to a 96-well plate for detection at a wavelength of 600 nm. The above experiment was repeated three times.

[0072] (5) Calculation: Take the average of three wells for each sample, calculate the average of three samples in the same group, calculate the average of three repeated experiments, calculate the standard error, and use the t test to calculate the significance of the difference between different groups at the same time point. If p < 0.05, mark it as *, and if p < 0.01, mark it as **.

[0073] (6) Results Comparison

[0074] Depend on Figure 3 As shown in the control curve, compared with the control group (CONT, nutrient broth), the experimental group (AOS, nutrient broth medium supplemented with 0.5% alginate oligosaccharide) contains alginate oligosaccharide (AOS), which significantly inhibits the growth of *Pseudomonas putida* relative to the control group.

[0075] Example 4

[0076] This embodiment provides the implementation details of reducing the abundance of Pseudomonas in the gut microbiota of large yellow croaker using brown algae oligosaccharides. The experimental content includes:

[0077] 1.1 Culture medium

[0078] (1) Anaerobic culture medium (GAM) (Qingdao High-tech Park Haibo Biotechnology Co., Ltd.), each L contains: Prepare the following ingredients according to the instructions: 10g peptone, 3g soybean peptone, 5g yeast extract, 2.2g beef powder, 13.5g digestive serum powder, 1.2g beef liver extract, 3g glucose, 2.5g potassium dihydrogen phosphate (KH2PO4), 5g soluble starch, 0.3g L-cysteine ​​salt, and 0.3g sodium thioglycolate. Adjust the pH to 6.8.

[0079] (2) The laboratory-made brown algae oligosaccharide meets the aforementioned brown algae oligosaccharide index standards.

[0080] (3) A compound seaweed enzymatic hydrolysate was used. The raw materials consisted of 29% laver, 24% sea lettuce, and 47% kelp. After enzymatic hydrolysis, the content of alginate oligosaccharides was 43.4 mg / g, which did not meet the standard for alginate oligosaccharides. The preparation process is as follows:

[0081] 1) Dry and pulverize laver, Ulva lactuca, and kelp;

[0082] 2) Thoroughly combine the crushed laver, Ulva lactuca, and kelp;

[0083] 3) Add the compounded seaweed powder to drinking water and stir to mix, thus obtaining a compound seaweed suspension;

[0084] 4) Add citric acid and hydrochloric acid to the compound seaweed suspension to make the final concentrations 0.01M and 0.10M respectively. At the same time, heat the compound seaweed suspension to 40-50℃ and stir at ≥100rpm for ≥30min. During this period, use hydrochloric acid to adjust the pH value to stabilize it at pH 4.5-5.0.

[0085] 5) Add cellulase and pectinase to make the enzyme / substrate mass ≥0.50×10~2U / g, and stir for 2-6 hours at pH 4.5-5.0, temperature 40-50℃, and speed ≥150rpm.

[0086] 6) Add NaOH and Na2HPO4 to the compound seaweed hydrolysate to a final concentration of 0.05M. Stir at ≥30rpm for ≥30min. During this time, use HCl and NaOH to adjust the acidity to stabilize it at pH 7.0-8.0.

[0087] 7) Add neutral protease, aminopeptidase and alginate lyase to make the enzyme / substrate mass ≥3000U / g, ≥300U / g and ≥1000U / g respectively, and stir for 2-6 hours at pH 7.0-8.0, temperature 40-50℃ and speed ≥150rpm.

[0088] 8) Inactivate the enzyme by heating;

[0089] 9) The product is dried and pulverized.

[0090] 1.2 Experimental Methods

[0091] 1.2.1 Collection and processing of gut microbiota in large yellow croaker

[0092] Four healthy, one-year-old large yellow croakers (weighing 150-200g and measuring 20-24cm in length) were collected from the Ningde sea area of ​​Fujian Province. They were cultured in net cages and fed with fresh mixed fish paste. No antibiotics were used in the past three months.

[0093] Under sterile conditions, the surface of live large yellow croakers was wiped with 75% alcohol, dissected, and the digestive tract was extracted. The contents of the small intestine and rectum of each fish were collected, mixed, and intestinal bacterial solutions YC1, YC2, YC3, and YC4 were prepared using sterilized GAM culture medium at a ratio of 1g:9mL and refrigerated for later use.

[0094] 1.2.2 In vitro culture of gut microbiota

[0095] Anaerobic culture was performed using GAM medium, and the specific culture method is as follows:

[0096] (1) The experiment was first divided into three groups: BC: GAM culture; TA: GAM with 0.5% (W / V) compound seaweed enzymatic hydrolysate added; TD: GAM with 0.5% (W / V) brown algae oligosaccharide added; each group was prepared with 4 large glass test tubes, each tube containing 9 mL of culture medium and 3-5 g of hard wax added. At the same time, 3 tubes of contamination control group (GAM culture medium) were prepared and sterilized at 115℃ for 20 min by autoclaving.

[0097] (2) After cooling to room temperature, add filtered and sterilized deoxyheme and vitamin K to a final concentration of 12.5 mg / L and 2.5 mg / L, respectively. Then add 1 mL (10% addition ratio) of the prepared intestinal bacterial culture YC1, YC2, YC3, and YC4 to each group's test tube, seal with wax, and mix thoroughly by inverting. The contamination control group uses sterilized GAM culture medium for parallel operation.

[0098] (3) The culture conditions are 25℃ and 100rpm for 24h. Under the premise that the pollution control group is clear and free of turbidity, the experimental group is sampled. After each test tube is inverted and mixed, 3mL is taken from it and stored at -80℃.

[0099] 1.2.3 Gut microbiota 16S sequencing

[0100] 16S rDNA amplicon sequencing and analysis of gut microbiota cultures were commissioned to Wuhan Maiwei Metabolism Co., Ltd. 16S rDNA amplicon sequencing was performed using the NovaSeq PE250 protocol. After read assembly filtering, OTU (Operational Taxonomic Unit) clustering, species annotation and abundance analysis, as well as alpha diversity and beta diversity analyses, were conducted.

[0101] 1.3 Statistical Analysis

[0102] Data are expressed as mean ± standard deviation. Analysis of variance or other analyses were performed using SPSS 23 software. Significant differences (P<0.05) were marked with *, and highly significant differences (P<0.01) were marked with **.

[0103] 2 Results

[0104] 2.1 Characteristics of Gut Microbiota Structure

[0105] 2.2.1 Group Difference Analysis

[0106] After 16S rDNA amplicon sequencing, in vitro cultures of gut microbiota were first analyzed using PCoA (Principal Coordinates Analysis) based on unweighted unifrac distance. Closer sample distances indicate more similar species composition; therefore, samples with high community structural similarity tend to cluster together, while samples with significant community differences tend to separate. Combined with... Figure 4 The PCoA analysis showed that the TA group samples were clustered in relatively independent communities, far from the TD and BC groups; while there was distance between the TD and BC group communities, the distance was not significant, indicating that the addition of fucoidan had no significant effect on the gut microbiota composition. Figure 5 and Figure 6 It can be seen that the composition of each dominant bacterial genera also shows that alginic oligosaccharides have no significant effect on the composition of the gut microbiota.

[0107] Among them, based on the PCoA analysis of the unweighted Unifrac distance, ANOVA: p = 0.004. Figure 4Each point in the diagram represents a sample. Samples within the same group are represented by the same color, and the color region represents a confidence interval. BC (BC.1, BC.2, BC.3, BC.4), blank group: anaerobic medium (GAM); TD (TD.1, TD.2, TD.3, TD.4), fucoidan-supplemented group: anaerobic medium (GAM) + 0.5% fucoidan; TA (TA.1, TA.2, TA.3, TA.4), compound seaweed enzymatic hydrolysate-supplemented group: anaerobic medium (GAM) + 0.5% compound seaweed enzymatic hydrolysate.

[0108] Figure 5 In the table, each label represents: BC, blank group: anaerobic medium (GAM); TD, fucoidan-supplemented group: anaerobic medium (GAM) + 0.5% fucoidan; TA, compound seaweed enzymatic hydrolysate-supplemented group: anaerobic medium (GAM) + 0.5% compound seaweed enzymatic hydrolysate. Others indicate... Figure 6 The sum of the relative abundance of all phyla other than these 10 genera.

[0109] Figure 6 In the figure, each label is represented as follows: BC (BC.1, BC.2, BC.3, BC.4), blank group: anaerobic medium (GAM); TD (TD.1, TD.2, TD.3, TD.4), fucoidan-supplemented group: anaerobic medium (GAM) + 0.5% fucoidan; TA (TA.1, TA.2, TA.3, TA.4), compound seaweed enzymatic hydrolysate-supplemented group: anaerobic medium (GAM) + 0.5% compound seaweed enzymatic hydrolysate.

[0110] In addition, combined Figure 7 It was found that the addition of 0.5% fucoidan decreased both the absolute number and relative abundance of Pseudomonas bacteria in the gut microbiota; combined with Figure 8 It can be seen that the abundance of Pseudomonas bacteria in the brown algae oligosaccharide-added group was significantly lower than that in the control group and the compound seaweed hydrolysate group.

[0111] Figure 7 In the figure, each label is represented as follows: BC (LC.1, LC.2, LC.3, LC.4), blank group: anaerobic medium (GAM); TD (LC.1, LC.2, LC.3, LC.4), fucoidan-supplemented group: anaerobic medium (GAM) + 0.5% fucoidan; TA (LC.1, LC.2, LC.3, LC.4), compound seaweed enzymatic hydrolysate-supplemented group: anaerobic medium (GAM) + 0.5% compound seaweed enzymatic hydrolysate.

[0112] Figure 8In the figure, each label represents: BC, blank group: anaerobic medium (GAM); TD, brown algae oligosaccharide-added group: anaerobic medium (GAM) + 0.5% brown algae oligosaccharide; TA, compound seaweed enzymatic hydrolysate-added group: anaerobic medium (GAM) + 0.5% compound seaweed enzymatic hydrolysate.

[0113] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made based on the content of the present invention specification and drawings, or direct or indirect application in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. Use of phloro-oligosaccharides for the preparation of a composition for inhibiting the growth of Pseudomonas syringae, characterized in that: The brown algae oligosaccharide is used as the only active ingredient; the brown algae oligosaccharide is a brown algae oligosaccharide with a degree of polymerization of 2-25 and a content of greater than or equal to 60% formed by degradation treatment of sodium alginate; the brown algae oligosaccharide has a particle size of greater than or equal to 100 mesh, a moisture content of less than or equal to 10%, an insoluble content of less than or equal to 1%, and a pH value of 4.0-7.

0.

2. Use according to claim 1, wherein The brown algae oligosaccharide is added to nutrients to form a culture medium for growth inhibition of Pseudomonas syringae.

3. A method for preventing the deterioration of fresh food products for non- therapeutic purposes, characterized in that: A composition containing brown algae oligosaccharide is applied to the surface of the fresh food to inhibit the growth of Pseudomonas syringae on the surface of the fresh food, thereby prolonging the shelf life of the fresh food, wherein the brown algae oligosaccharide is used as the only active ingredient; the brown algae oligosaccharide is a brown algae oligosaccharide with a degree of polymerization of 2-25 and a content of greater than or equal to 60% formed by degradation treatment of sodium alginate; the brown algae oligosaccharide has a particle size of greater than or equal to 100 mesh, a moisture content of less than or equal to 10%, an insoluble content of less than or equal to 1%, and a pH value of 4.0-7.0.

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