Enterococcus faecalis capable of producing high-yield digestive enzyme through ARTP mutagenesis and application

Abstract

The enterococcus faecalis is preserved in the China General Microbiological Culture Collection Center on January 6, 2025, the address of the China General Microbiological Culture Collection Center is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.33308. The invention further discloses a preparation method of the enterococcus faecalis. According to the EF-448, EF-798 and EF-804 strains obtained through ARTP mutagenesis, the activity of alpha-amylase is improved by 160% or above compared with that of original strains, the activity of lipase is improved by 30% or above, the activity of neutral protease is improved by 40% or above, carbohydrates, protein and fat in feed can be efficiently degraded, and the digestion and absorption efficiency of nutrient substances is improved. The self-coacervation capacity is larger than or equal to 91%, the cell surface hydrophobicity is larger than or equal to 93%, a biological membrane is formed on the intestinal tract surface, digestive juice scouring is resisted, and it is ensured that strains stably survive and play a role in the intestinal tract environment. The enterococcus faecalis is used in the low-fish-meal daily ration of the macrobrachium rosenbergii, so that the growth performance, digestive enzyme activity and antioxidant capacity of the macrobrachium rosenbergii can be improved, and the effects of reducing the cost and improving the effect are achieved.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to an ARTP-mutated Enterococcus faecalis that produces high levels of digestive enzymes and its applications. Background Technology

[0002] With the rapid development of aquaculture and the increasing scarcity of fishmeal resources worldwide, the proportion of plant protein sources in feed is gradually increasing. However, compared with fishmeal, plant proteins are limited in their application by factors such as anti-nutritional factors and unbalanced amino acid distribution. In recent years, probiotics, as functional feed additives, have shown significant effects in improving the growth performance of aquatic animals, enhancing their immunity, and regulating the balance of intestinal flora.

[0003] *Enterococcus faecalis* is a Gram-positive coccus belonging to the genus *Enterococcus*. Widely distributed in the intestines of humans and animals, it has been shown to participate in the digestion, absorption, and metabolism of nutrients, playing a crucial role in maintaining intestinal microecological balance. Recent studies have revealed that this strain exhibits significant enzyme-producing characteristics, particularly in amylase and protease. Research shows that, after optimization using response surface methodology, the *Enterococcus faecalis* mercadA7 strain can produce an amylase yield of up to 1022 U / mL, and this enzyme maintains stable activity even under extreme environmental conditions (such as high temperature, alkalinity, high toluene concentration, or high salt concentration). Furthermore, the purified *Enterococcus faecalis* K-1 strain exhibits a specific amylase activity of 332.4 U / mg, and when cultured in a medium containing soluble starch, it can simultaneously produce amylopectin and cyclodextrin hydrolase. Rohmatussolihat et al. optimized the culture conditions using response surface methodology, increasing the protease activity of *Enterococcus faecalis* strain InaCCB745 to 60.64 U / mL, a 57.3% increase compared to before optimization. These results fully demonstrate the important application value of *Enterococcus faecalis* as a highly efficient digestive enzyme-producing strain. In aquaculture, *Enterococcus faecalis* is a popular probiotic preparation. In production practice, *Enterococcus faecalis* can promote the growth of aquatic animals such as carp, giant freshwater prawn, and whiteleg shrimp, enhance digestion and immunity, and improve the intestinal microecological environment. Li et al. found that adding 1.0 × 10⁻⁶ U / mL to the diet... 8The addition of CFU / g of Enterococcus faecalis or in combination with Lactobacillus lactis upregulates the expression of pro-inflammatory factors (IL-6, IL-1β, TNF-α, IFN-γ, HSP70, HSP90) and anti-inflammatory factors (IL-10 and TGF-β) in the spleen, head kidney, gills, liver, and intestines of crucian carp. Furthermore, Enterococcus faecalis can stably colonize the host intestine, increasing the activity of proteases, amylases, and lipases in the digestive tract by secreting metabolites such as propionic acid and butyrate, thereby enhancing the efficiency of nutrient digestion and absorption. Simultaneously, Enterococcus faecalis can form a protective biofilm on the surface of intestinal epithelial cells. This physical barrier not only resists the invasion of harmful substances but also enhances the host's antioxidant capacity and immune response level by stimulating immune cells to release immune factors. In addition, Enterococcus faecalis can enhance the resistance of aquatic animals to Aeromonas hydrophila, purify aquaculture water quality, and improve the aquaculture environment.

[0004] Atmospheric and room temperature plasma (ARTP) is a novel microbial mutagenesis technique based on a radio frequency driven atmospheric pressure glow discharge plasma system. The mutagenesis mechanism relies primarily on high-energy reactive particles (including ions, electrons, and free radicals) generated by the plasma. These particles interact with the cell surface, altering cell membrane structure and permeability, leading to DNA damage and forcing the cell to activate the SOS repair mechanism, resulting in various mutant strains. These reactive particles attack the OH, OP, and NC glycosidic bonds in the DNA strand structure, disrupting the DNA molecular structure and causing single-strand DNA breaks. When the breakpoints in different single-strand regions approach each other, double-strand DNA breaks occur. After mutagenesis, the cell exhibits increased tolerance to culture medium components, increased cell growth and biomass, enhanced enzyme activity, and increased production of various chemical substances.

[0005] Currently, enzyme preparations such as cellulase, xylanase, protease, lipase, amylase, and glucanase are widely used in feed fermentation. Studies have shown that mutagenesis can significantly enhance the production of chitosan oligosaccharides, glucanase, alkaline amylase, and phospholipase by Bacillus cereus, Arthrobacter, Bacillus subtilis, and Streptomyces ceshiroshimensis. Zhang et al. used ARTP technology to screen and obtain the mutant strain mut80, finding that the amylase and protease activities of Bacillus licheniformis XS-4 were increased by 90.54% and 143.10%, respectively. These research results indicate that mutagenesis can effectively enhance the enzyme production capacity of microorganisms, providing important technical support for enzyme preparation production in feed fermentation and other related fields.

[0006] Currently, natural bacterial strains often have limitations such as insignificant probiotic properties. Atmospheric pressure room temperature plasma (ARTP) technology has been widely used to screen for highly efficient mutant probiotics. Therefore, this study used *Enterococcus faecalis* as the research object, employed ARTP mutagenesis to directionally select *Enterococcus faecalis* mutant strains with high digestive enzyme production, evaluated their safety performance, and applied them to a low-fishmeal diet for *Macrobrachium rosenbergii*. The mitigation effect on damage caused by the low-fishmeal diet was evaluated using indicators such as growth, digestive enzymes, and antioxidant enzymes. Summary of the Invention

[0007] The present invention aims to solve the above-mentioned technical problems by providing a high-yield digestive enzyme-producing Enterococcus faecalis and its applications.

[0008] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0009] A high-yield digestive enzyme-producing Enterococcus faecalis, which was deposited on January 6, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNO.33308.

[0010] Preferably, the Enterococcus faecalis strains EF-448, EF-798, or EF-804 obtained by ARTP screening using ambient pressure room temperature plasma mutagenesis technology have α-amylase activity increased by more than 160%, lipase activity increased by more than 30%, and neutral protease activity increased by more than 40% compared with the original strain.

[0011] Preferably, the EF-448, EF-798, or EF-804 strains have a self-aggregation capacity of ≥91% and a cell surface hydrophobicity of ≥93%.

[0012] Preferably, the survival rate of the EF-448, EF-798 or EF-804 strains in simulated artificial intestinal fluid is ≥50% after 6 hours.

[0013] Preferably, the EF-448, EF-798, or EF-804 strains have no hemolytic activity and can be injected with 1×10⁻⁶ bacteria into giant freshwater prawns. 8 Up to 1×10 10 The mortality rate was 0 when the bacterial culture concentration was CFU / mL.

[0014] Application of a high-yield digestive enzyme-producing Enterococcus faecalis in the preparation of probiotic formulations for aquaculture.

[0015] Preferably, the probiotic preparation is used in low-fishmeal diets for giant freshwater prawns to improve their growth performance, digestive enzyme activity, and antioxidant capacity.

[0016] Preferably, the improvement in growth performance is manifested in increased weight gain rate and specific growth rate, and decreased feed conversion ratio; the improvement in digestive enzyme activity is manifested in increased activity of α-amylase, lipase and neutral protease in the intestine; and the improvement in antioxidant capacity is manifested in increased total antioxidant capacity and hydroxyl radical scavenging capacity of the liver and pancreas, and decreased malondialdehyde content.

[0017] By employing the above method, the present invention has the following advantages:

[0018] The EF-448, EF-798, and EF-804 strains obtained by ARTP mutagenesis in this invention have α-amylase activity increased by more than 160%, lipase activity increased by more than 30%, and neutral protease activity increased by more than 40% compared with the original strains. They can efficiently degrade starch, protein, and fat in feed and enhance the digestibility and absorption of nutrients.

[0019] With a self-aggregation capacity of ≥91% and a cell surface hydrophobicity of ≥93%, it is conducive to the formation of a biofilm on the intestinal surface to resist the scouring of digestive juices; the survival rate in simulated artificial intestinal fluid for 6 hours is ≥50% (85.71% and 82.32% for EF-448 and EF-798, respectively), ensuring that the strain can survive stably and function in the intestinal environment and alleviate liver damage; at the same time, it enhances the activity of intestinal lipase and neutral protease, thereby enhancing the digestion capacity of nutrients.

[0020] No hemolytic activity; inject 1×10⁻⁶ tbsp. into Macrobrachium rosenbergii. 8 Up to 1×10 10 The mortality rate is 0 at CFU / mL bacterial solution, with no toxic risk, meeting the safety requirements for probiotics in aquaculture. It reduces dependence on fishmeal in feed and reduces the impact of anti-nutritional factors, providing technical support for the low-carbon and efficient development of aquaculture.

[0021] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application 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 this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a growth curve diagram of Enterococcus faecalis according to the present invention;

[0024] Figure 2This is a graph showing the effect of ARTP treatment time on the mortality rate of Enterococcus faecalis according to the present invention;

[0025] Figure 3 This is a graph showing the α-amylase activity of the mutant strains of the present invention (the black dots in the graph represent the α-amylase activity of each strain, and the red reference line indicates the screening threshold of the top 10% of strains in terms of activity).

[0026] Figure 4 This is a genetic stability diagram of the mutant strain of the present invention, where (A) represents α-amylase, (B) represents lipase, and (C) represents neutral protease. Different letters represent significant differences (P<0.05).

[0027] Figure 5 The diagram shows the self-aggregation ability and cell surface hydrophobicity of different strains of the present invention, wherein (A) is the self-aggregation ability and (B) is the cell surface hydrophobicity.

[0028] Figure 6 This is a diagram illustrating the intestinal fluid tolerance of the screening strains of this invention;

[0029] Figure 7 This is a graph showing the antioxidant capacity of the mutant strain of the present invention; where (A) is the total antioxidant capacity and (B) is the DPPH free radical scavenging capacity.

[0030] Figure 8 This is the hemolytic activity diagram of the present invention;

[0031] Figure 9 The figure shows the effect of supplementing low fishmeal diets with ARTP Enterococcus faecalis on the activity of intestinal digestive enzymes in giant freshwater prawns, where (A) α-amylase activity (AMS), (B) lipase activity (LPS), and (C) neutral protease activity (NP).

[0032] Figure 10 This is a graph showing the effect of supplementing a low-fishmeal diet with ARTP (Enterococcus faecalis) on brush border enzymes in Macrobrachium rosenbergii, where (A) sodium / potassium ATP transporter (Na... + / K + (A) ATPase, (B) alkaline phosphatase activity (AKP), (C) creatine kinase activity (CK);

[0033] Figure 11 This is a graph showing the effect of supplementing low fishmeal diets with ARTP (Enterococcus faecalis) on the antioxidant activity of the hepatopancreas of Macrobrachium rosenbergii, where (A) total antioxidant capacity (T-AOC), (B) hydroxyl radical scavenging capacity (OH·-), and (C) malondialdehyde content (MDA). Detailed Implementation

[0034] Specific embodiments of the invention will now be described in detail. Although the invention is described in conjunction with these specific embodiments, it should be understood that the invention is not intended to be limited to these specific embodiments. Rather, these embodiments are intended to cover alternative, modified, or equivalent embodiments that may be included within the spirit and scope of the invention as defined by the claims. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. The invention may be practiced without some or all of these specific details. In other instances, well-known processes have not been described in detail so as not to unnecessarily obscure the invention.

[0035] When used in conjunction with the terms "comprising," "method comprising," or similar language in this specification and appended claims, the singular forms "a," "some," and "the" include plural references unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] The present invention will now be described in further detail with reference to the full text.

[0037] Combined with attachment Figure 1-11 The source of the bacterial strain was confirmed to be Enterococcus faecalis FFRCYM01, which was collected from the intestines of Macrobrachium rosenbergii at the Yixing Dapu Base of the Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences.

[0038] Strain characteristics: *Enterococcus faecalis* is a Gram-positive coccus belonging to the genus *Enterococcus*, widely distributed in the intestines of humans and animals. Strain number: FFRC YM 01. Strain preservation number: CGMCC No. 33308.

[0039] Technical Results: Three mutant strains (EF-448, EF-798, and EF-804) with significantly increased digestive enzyme production and stable heritability were screened using ARTP technology. Their α-amylase, lipase, and neutral protease activities were increased by more than 160%, 30%, and 40%, respectively. The three mutant strains exhibited high self-aggregation ability (91%) and cell surface hydrophobicity (93%), maintaining a high survival rate (50%) in simulated intestinal fluid for 6 hours. None of the three strains showed hemolytic activity, and they were effective against hemolytic enzymes in *Macrobrachium rosenbergii* when injected with 1×10⁻⁶ spores. 8 and 1×10 10 CFU / mL bacterial solution did not cause shrimp mortality.

[0040] In aquaculture applications, low-fishmeal diets reduced the growth performance of Macrobrachium rosenbergii, increased the feed conversion ratio, increased serum AST levels, caused some liver damage, and reduced intestinal digestive enzyme activity and the scavenging capacity of hydroxyl radicals in the hepatopancreas. Supplementation with the low-fishmeal diet containing the ARTP mutant strain EF-804 significantly increased the weight gain and specific growth rate of Macrobrachium rosenbergii, while supplementation with both ARTP mutant strains EF-448 and EF-804 significantly reduced the feed conversion ratio. ARTP mutant supplementation significantly reduced serum AST and ALT levels, alleviating liver damage. ARTP mutant strain EF-804 supplementation significantly increased the activity of intestinal lipases and neutral proteases, significantly reduced AKP enzyme activity, and significantly increased the activity of the body's total antioxidant capacity.

[0041] Isolation method: Enterococcus faecalis FFRC YM 01 was isolated from the intestines of Macrobrachium rosenbergii at the Yixing Dapu Base of the Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences.

[0042] Culture conditions: Culture medium formula (peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 mL / L), temperature (37℃), pH (5.7±0.2).

[0043] The Enterococcus faecalis strains EF-448, EF-798, and EF-804 provided by this invention were all isolated from the intestines of Macrobrachium rosenbergii and obtained through ARTP mutagenesis screening. The characteristics of these strains are as follows:

[0044] Taxonomic characteristics: Gram-positive cocci, belonging to the genus Enterococcus, species number FFRC YM01, preservation number CGMCC No.33308.

[0045] Enzyme activity characteristics: α-amylase activity increased by more than 160% compared with the original strain, lipase activity increased by more than 30%, and neutral protease activity increased by more than 40%.

[0046] Probiotic properties: Self-aggregation ability ≥91%, cell surface hydrophobicity ≥93%, survival rate ≥50% in simulated artificial intestinal fluid for 6 hours, no hemolytic activity, and effective in treating giant freshwater prawns by injection of 1×10⁻⁶ mg / L. 8 Up to 1×10 10 The mortality rate was 0 when the bacterial culture concentration was CFU / mL.

[0047] The technical effects are as follows:

[0048] 1. Enhanced enzyme activity: The digestive enzyme activity of the mutant strain is significantly increased, which can effectively degrade macromolecular nutrients in feed.

[0049] 2. Improved growth performance: Adding mutant strains to low fishmeal diets of giant freshwater prawns can increase weight gain and specific growth rates, and reduce the feed conversion ratio.

[0050] 3. Liver protection: Significantly reduces serum AST and ALT levels, alleviating liver damage caused by low fishmeal diets.

[0051] 4. Enhanced digestion and antioxidant capacity: Increases the activity of intestinal lipase and neutral protease, enhances the total antioxidant capacity and hydroxyl radical scavenging capacity of the liver and pancreas, and reduces malondialdehyde content.

[0052] The screening method for the Enterococcus faecalis mutant strain includes the following steps:

[0053] (1) The original strain of Enterococcus faecalis was isolated from the intestine of Macrobrachium rosenbergii;

[0054] (2) The original strain was subjected to mutagenesis using ARTP technology for 40-60 seconds, with a lethality rate of 85-95%.

[0055] (3) Initial screening: Screening for single colonies with α-amylase activity that is more than 160% higher than that of the original strain;

[0056] (4) Secondary screening: The lipase and neutral protease activities of the strains screened in the primary screening were measured, and the comprehensive enzyme activity score was calculated. Mutants with a comprehensive score significantly higher than that of the original strains were screened.

[0057] (5) Genetic stability analysis: Screening mutants whose enzyme activity did not change significantly during the passage process.

[0058] The conditions for ARTP mutagenesis treatment are: ambient pressure room temperature plasma system, radio frequency driven ambient pressure glow discharge, generating high-energy active particles containing ions, electrons and free radicals.

[0059] The comprehensive enzyme activity score is calculated as follows: Comprehensive score (%) = (α-amylase activity / maximum α-amylase activity) × 100 × 0.4 + (neutral protease activity / maximum neutral protease activity) × 100 × 0.4 + (lipase activity / maximum lipase activity) × 100 × 0.2.

[0060] A high-yield digestive enzyme-producing Enterococcus faecalis, which was deposited on January 6, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNO.33308.

[0061] The Enterococcus faecalis strains EF-448, EF-798, or EF-804, obtained by screening using atmospheric pressure and room temperature plasma mutagenesis technology, exhibit α-amylase activity increased by more than 160%, lipase activity increased by more than 30%, and neutral protease activity increased by more than 40% compared to the original strain.

[0062] The EF-448, EF-798, or EF-804 strains have a self-aggregation capacity of ≥91% and a cell surface hydrophobicity of ≥93%.

[0063] The survival rate of the EF-448, EF-798, or EF-804 strains in simulated artificial intestinal fluid was ≥50% after 6 hours.

[0064] The EF-448, EF-798, or EF-804 strains showed no hemolytic activity and, when injected with 1×10⁻⁶ molluscs, were effective against giant freshwater prawns. 8 Up to 1×10 10 The mortality rate was 0 when the bacterial culture concentration was CFU / mL.

[0065] Application of a high-yield digestive enzyme-producing Enterococcus faecalis in the preparation of probiotic formulations for aquaculture.

[0066] The probiotic preparation is used in low-fishmeal diets for giant freshwater prawns to improve their growth performance, digestive enzyme activity, and antioxidant capacity.

[0067] The improved growth performance is manifested in increased weight gain and specific growth rate, and decreased feed conversion ratio; the improved digestive enzyme activity is manifested in increased activity of α-amylase, lipase and neutral protease in the intestine; the improved antioxidant capacity is manifested in increased total antioxidant capacity and hydroxyl radical scavenging capacity of the liver and pancreas, and decreased malondialdehyde content.

[0068] In specific implementations, the invention is applied in the following ways:

[0069] 1. Growth curve of Enterococcus faecalis:

[0070] like Figure 1 As shown, Enterococcus faecalis exhibits a rapid growth trend within 0-18 hours, then enters a plateau phase, and finally declines after 24 hours. Therefore, this experiment selected 18 hours as the optimal fermentation time for the seed culture.

[0071] 2. ARTP-induced mutagenic lethality curve:

[0072] like Figure 2As shown, the lethality of Enterococcus faecalis was positively correlated with the ARTP mutagenesis treatment time. After 10 seconds of treatment, the mortality rate of the strain reached 53.78%, and when the treatment time exceeded 80 seconds, the lethality rate reached 99%, essentially killing all cells. Therefore, the suitable mutagenesis times selected in this experiment were 40 seconds, 50 seconds, and 60 seconds, with a lethality rate of 85-95%, as the screening criteria for mutant strains.

[0073] 3. Initial screening:

[0074] A total of 833 single colonies were obtained in this experiment. Figure 3 The strains were numbered EF-1 to EF-833. α-amylase activity assays showed that 698 strains exhibited significantly increased activity compared to the original strains. Therefore, the top 10% of strains with the most significant activity increases (a total of 84 strains) were selected for further screening. The α-amylase activity of the selected strains was more than 160% higher than that of the original strains.

[0075] 4. Secondary screening:

[0076] Lipase and neutral protease activities were measured after secondary screening, and the overall enzyme activity score was calculated by combining the lipase and α-amylase activities (Table 1). The results showed that the overall digestive enzyme scores of 72 mutant strains were significantly higher than those of the original strains. Based on the enzyme activity measurement results, six high-yield digestive enzyme strains (EF-513, EF-804, EF-494, EF-798, EF-783, and EF-448) were screened.

[0077] Table 1 Results of secondary screening for potential probiotic enzyme activity

[0078]

[0079]

[0080]

[0081]

[0082] Note: Overall score (%) = (α-amylase activity / maximum α-amylase activity) × 100 × 0.4 + (neutral protease activity / maximum neutral protease activity) × 100 × 0.4 + (lipase activity / maximum lipase activity) × 100 × 0.2. "-" indicates not detected.

[0083] 5. Genetic stability analysis:

[0084] The α-amylase activity of strain EF-783 and the lipase activity of strains EF-494, EF-513 and EF-783 showed significant differences during the 1st to 9th generations. Figure 4AB). The neutral protease activities of strains EF-513 and EF-783 also showed significant changes during passage ( ). Figure 4 C). The α-amylase, lipase, and neutral protease activities of strains EF-448, EF-798, and EF-804 did not show significant differences and remained stable. Based on these results, strains EF-448, EF-798, and EF-804, which exhibited good genetic stability, were selected for further performance evaluation in this experiment.

[0085] 6. Evaluation of the probiotic properties of the mutagenic strains, 6.1 Self-aggregation ability and cell surface hydrophobicity of the mutagenic strains:

[0086] like Figure 5 As shown, the self-aggregation abilities of the original Enterococcus faecalis strain and the mutant strains EF-448, EF-798, and EF-804 exhibited similar trends at 2, 4, and 24 hours. The self-aggregation rate of each strain gradually increased over time, reaching over 91% at 24 hours, but there was no significant difference in self-aggregation ability among different strains (P>0.05). Strains EF-448, EF-798, and EF-804 showed relatively strong hydrophobicity (93%), but there was no significant difference in self-aggregation ability among different strains.

[0087] 6.2 Mutagenic strains mimicking artificial intestinal fluid tolerance:

[0088] Tolerance analysis showed that the survival rate of the screened strains in simulated intestinal fluid decreased with prolonged treatment time. Among them, strains EF-448 and EF-798 maintained high survival rates at 6 hours, at 85.71% and 82.32%, respectively. In contrast, strain EF-804 had a relatively low survival rate, remaining around 50% at all time points. Figure 6 ).

[0089] 6.3 Determination of antioxidant capacity of mutant strains

[0090] No significant differences were observed between the original Enterococcus faecalis strains and the mutant strains EF-448, EF-798, and EF-804 in terms of total antioxidant capacity and DPPH free radical scavenging capacity. Figure 7 ).

[0091] 7. Security

[0092] 7.1 Hemolytic activity:

[0093] The hemolysis test results showed that, except for the positive control Aeromonas hydrophila which exhibited β-hemolysis, EF-448, EF-798, and EF-804 did not show hemolytic activity. Figure 8 ).

[0094] 7.2 In vivo safety experiment of giant freshwater prawn

[0095] The results showed that no deaths occurred in the experimental group compared to the control group (Table 2).

[0096] Table 2 Safety tests of mutant strains EF-448, EF-798 and EF-804

[0097]

[0098]

[0099] 8. Applications in aquaculture, 8.1 Effects of supplementing low-fishmeal diets with ARTP (Enterococcus faecalis) on the growth performance of giant freshwater prawns.

[0100] As shown in Table 3, after 56 days of rearing, compared with the control group, the weight gain rate and specific growth rate of the low fishmeal group were significantly lower, while the feed conversion ratio was significantly higher; the weight gain rate and specific growth rate of the group supplemented with EF-804 were significantly higher, while the feed conversion ratio of the groups supplemented with EF-448 and EF-804 was significantly lower. There were no significant differences in final average weight and survival rate among the groups.

[0101] Table 3. Effects of low-fishmeal diets supplemented with ARTP (Enterococcus faecalis) on growth performance of giant freshwater prawns.

[0102]

[0103] Note: "*" indicates a significant difference between the LF and CT groups (P < 0.05), "**" indicates an extremely significant difference (P < 0.01); "#" indicates a significant difference between the LF+EF group and the LF group (P < 0.05), and "##" indicates an extremely significant difference between the LF+EF group and the LF group (P < 0.01).

[0104] 8.2 Effects of ARTP-lactamase supplementation on low-fishmeal diets on serum biochemistry of giant freshwater prawns:

[0105] As shown in Table 4, compared with the control group, the AST level in the low fishmeal group was significantly increased; compared with the low fishmeal group, the AST level in the groups with added mutant bacteria EF-448, EF-798 and EF-804 was significantly decreased, the ALT level in the groups with added mutant bacteria EF-798 and EF-804 was significantly decreased, the GLU level in the group with added mutant bacteria EF-448 was significantly decreased, and the TC, TG and ALB levels in the groups with added mutant bacteria EF-448, EF-798 and EF-804 were increased.

[0106] Table 4. Effects of low-fishmeal diets supplemented with ARTP (Enterococcus faecalis) on serum biochemistry in giant freshwater prawns.

[0107]

[0108] 8.3 Effects of ARTP (Enterococcus faecalis) supplementation in low-fishmeal diets on intestinal digestive enzyme activity in giant freshwater prawns:

[0109] like Figure 9 As shown, compared with the control group, the low fishmeal group showed a significant decrease in α-amylase activity, a decrease in lipase activity, and an increase in neutral protease activity; compared with the low fishmeal group, the group with added mutant bacteria EF-804 showed an increase in α-amylase, lipase, and neutral protease activity.

[0110] Figure 9 The effects of supplementing low-fishmeal diets with ARTP (Enterococcus faecalis) on the activity of intestinal digestive enzymes in Macrobrachium rosenbergii were investigated. The results showed (A) α-amylase activity (AMS), (B) lipase activity (LPS), and (C) neutral protease activity (NP). "*" indicates a significant difference between the LF and CT groups (P < 0.05), "**" indicates an extremely significant difference (P < 0.01); "#" indicates a significant difference between the LF+EF group and the LF group (P < 0.05), and "##" indicates an extremely significant difference between the LF+EF group and the LF group (P < 0.01).

[0111] 8.4 Effects of ARTP-containing Enterococcus faecalis supplementation on brush border enzymes in low-fishmeal diets:

[0112] like Figure 10 As shown, compared with the control group, the low fishmeal group showed a significant decrease in Na+ / K+-ATP activity and a significant increase in AKP enzyme activity; compared with the low fishmeal group, the group with added mutant bacteria EF-804 showed an increase in Na+ / K+-ATP activity, the groups with added mutant bacteria EF-448, EF-798 and EF-804 showed a very significant decrease in AKP enzyme activity, the group with added mutant bacteria EF-448 showed an increase in CK enzyme activity, and the groups with added mutant bacteria EF-798 and EF-804 showed a decrease in CK enzyme activity.

[0113] 8.5 Effects of low-fishmeal diets supplemented with ARTP (Enterococcus faecalis) on antioxidant activity of the hepatopancreas in giant freshwater prawns:

[0114] like Figure 11 As shown, compared with the control group, the low fishmeal group showed a significant decrease in OH·- activity, a decrease in T-AOC activity, and an increase in MDA activity; compared with the LF group, the addition of mutagenic Enterococcus faecalis increased T-AOC and OH·- activities and decreased MDA activity, with the EF-804 group showing a significant increase in T-AOC.

[0115] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown throughout are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A type of Enterococcus faecalis that produces high levels of digestive enzymes through ARTP mutagenesis, characterized in that, The Enterococcus faecalis was deposited on January 6, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNO.33308.

2. The Enterococcus faecalis with high digestive enzyme production induced by ARTP according to claim 1, characterized in that: The Enterococcus faecalis strains EF-448, EF-798, or EF-804, obtained by screening using atmospheric pressure and room temperature plasma mutagenesis technology, exhibit α-amylase activity increased by more than 160%, lipase activity increased by more than 30%, and neutral protease activity increased by more than 40% compared to the original strain.

3. The Enterococcus faecalis with high digestive enzyme production induced by ARTP mutagenesis according to claim 2, characterized in that: The EF-448, EF-798, or EF-804 strains have a self-aggregation capacity of ≥91% and a cell surface hydrophobicity of ≥93%.

4. The Enterococcus faecalis with high digestive enzyme production induced by ARTP mutagenesis according to claim 2, characterized in that: The survival rate of the EF-448, EF-798, or EF-804 strains in simulated artificial intestinal fluid was ≥50% after 6 hours.

5. The Enterococcus faecalis progenitor with high digestive enzyme production via ARTP mutagenesis according to claim 2, characterized in that: The EF-448, EF-798, or EF-804 strains showed no hemolytic activity and, when injected with 1×10⁻⁶ molluscs, were effective against giant freshwater prawns. 8 Up to 1×10 10 The mortality rate was 0 when the bacterial culture concentration was CFU / mL.

6. The application of Enterococcus faecalis with high digestive enzyme production induced by ARTP according to any one of claims 2-5 in the preparation of probiotic preparations for aquaculture.

7. The application according to claim 6, characterized in that: The probiotic preparation is used in low-fishmeal diets for giant freshwater prawns to improve their growth performance, digestive enzyme activity, and antioxidant capacity.

8. The application according to claim 7, characterized in that: The improved growth performance is manifested in increased weight gain and specific growth rate, and decreased feed conversion ratio; the improved digestive enzyme activity is manifested in increased activity of α-amylase, lipase and neutral protease in the intestine; the improved antioxidant capacity is manifested in increased total antioxidant capacity and hydroxyl radical scavenging capacity of the liver and pancreas, and decreased malondialdehyde content.

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