Enterococcus faecium and bacterial preparation prepared from the same, preparation method and application thereof

Abstract

The present application belongs to the field of microorganisms, and particularly relates to a strain of Enterococcus faecium, a bacterial preparation prepared from the strain, a preparation method of the bacterial preparation and application of the bacterial preparation. The specific technical scheme is as follows: a strain of Enterococcus faecium was preserved in the Guangdong Microbial Culture Collection Center on December 12, 2022, the address of the preservation center is No. 59 Building, 5th Floor, Institute of Guangzhou Martyrs Road, Guangzhou, and the preservation number is GDMCC NO: 62359. The present application provides a new strain of Enterococcus faecium, which is the first discovered Enterococcus faecium with desulfurization and denitrification functions and can be fed. The bacterial preparation prepared by using the Enterococcus faecium has strong resistance to gastric acid and intestinal fluid, can enter the digestive tract through the food channel and survive in large quantities and rapidly colonize in the intestinal tract. After the Enterococcus faecium or the bacterial preparation is ingested by live pigs, the intestinal health degree of the live pigs can be adjusted, the ratio of ammonium nitrogen to total nitrogen, the ratio of sulfur ions to total sulfur and the total content of volatile fatty acids in the intestinal tract are reduced, and the purpose of reducing the emission of foul-smelling gas is achieved from the source.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of Enterococcus faecalis, bacterial preparations made from it, their preparation methods, and applications. Background Technology

[0002] Odor pollution from pig farming is a significant obstacle to the development of this industry. Existing research shows that the odor from pig farming primarily originates from gaseous pollutants produced by the decomposition of organic matter in feces by microorganisms. Ranked by their contribution to odor, these pollutants mainly include volatile nitrogen compounds, volatile sulfur compounds, volatile fatty acids, and aromatic compounds. Due to the wide distribution, low concentration, complex composition, and difficulty in detection of odor sources, pig farming odor treatment is challenging and differs from the treatment of general air pollution.

[0003] Currently, methods for removing malodorous substances mainly include physical, chemical, and biological methods. Physical deodorization involves adsorbing the odor-producing gases onto the surface of a solid or liquid medium, preventing them from easily dissipating into the air; this is generally used as a pretreatment process. Chemical deodorization uses chemicals to kill bacteria or react with odor-producing substances; this method requires high-quality chemicals and is prone to secondary pollution. Biological methods primarily rely on microbial deodorization technology. Microbial deodorization involves preparing microbial agents with deodorizing functions, utilizing the competitive antagonistic and synergistic metabolic effects between microorganisms to inhibit the growth and reproduction of putrefactive microorganisms while simultaneously degrading malodorous components.

[0004] Compared with physical and chemical methods, microbial deodorization technology has advantages such as simple operation, economic and environmental protection, high deodorization efficiency, and convenient maintenance and management. However, existing biological deodorization technologies generally focus on end-point deodorization after odorous substances have been generated, emphasizing the off-site treatment of manure while neglecting the source of odorous substances. At the same time, there is still no effective solution for the negative impact of overall environmental odor in pigsties on the health and growth performance of pigs.

[0005] Developing a new feedable probiotic agent for reducing odors in pigs to achieve large-scale source reduction would have significant practical implications. Summary of the Invention

[0006] The purpose of this invention is to provide a probiotic preparation for pigs, its preparation method, and its application.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this invention is as follows: A strain of Enterococcus faecium DS4-3 was deposited on December 12, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 62359.

[0008] Accordingly, the application of Enterococcus faecalis in deodorization.

[0009] Accordingly, the application of the Enterococcus faecalis in denitrification and / or desulfurization.

[0010] Correspondingly, bacterial preparations are made using the aforementioned Enterococcus faecalis.

[0011] Preferably, the bacterial preparation also includes Bacillus and yeast.

[0012] Preferably, the Bacillus species include Bacillus subtilis and Bacillus licheniformis.

[0013] Preferably, the yeast is Saccharomyces cerevisiae.

[0014] Preferably, in the bacterial preparation, the ratio of Enterococcus faecalis: Bacillus: Yeast is (1-2): (1-2): (1-2).

[0015] Accordingly, the application of the bacterial preparation in deodorization.

[0016] Preferably, the application method is to feed the bacterial preparation to animals.

[0017] The present invention has the following beneficial effects: It provides a novel *Enterococcus faecalis* strain, which is the first discovered feedable *Enterococcus faecalis* strain with desulfurization and denitrification capabilities. The bacterial preparation made using this *Enterococcus faecalis* in combination with other microorganisms exhibits strong resistance to gastric acid and intestinal fluid, and can enter the digestive tract through ingestion, surviving in large quantities and rapidly colonizing the intestines.

[0018] When ingested by pigs, the *Enterococcus faecalis* or its preparation can regulate the intestinal health of pigs, inhibit the growth of pathogenic bacteria such as *Escherichia coli*, *Shigella*, and *Xanthomonas*, and promote the growth of beneficial bacteria such as lactic acid bacteria. Simultaneously, the preparation can reduce the ammonium nitrogen-to-total nitrogen ratio, the sulfide ion-to-total sulfur ratio, and the total content of volatile fatty acids in the intestines, thereby reducing odor emissions at the source. Animal experiments show that feeding pigs with the preparation at a rate of 0.3% in their diet, starting from day 5, can significantly reduce odor indicators such as NH3 content and OU value in the pigpens.

[0019] The bacterial preparation provided by this invention is simple to prepare, has low energy consumption, produces safe products, and causes no secondary pollution. It achieves source reduction of odor emissions and has a promising prospect for widespread application. Attached Figure Description

[0020] Figure 1 The electron micrograph of Enterococcus faecalis provided by this invention;

[0021] Figure 2 The growth curve of Enterococcus faecalis provided by this invention. Detailed Implementation

[0022] This invention provides a novel *Enterococcus faecium* strain DS4-3, which was deposited on December 12, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 62359. Its 16S rDNA sequence is shown in SEQ ID NO: 1.

[0023] This invention provides a microbial preparation based on the aforementioned Enterococcus faecium, which can be used for feeding pigs. A preferred embodiment is that, in addition to Enterococcus faecium, the microbial preparation also includes Bacillus and yeast. A more preferred embodiment is that the Bacillus includes Bacillus subtilis and Bacillus licheniformis; and the yeast is Saccharomyces cerevisiae. An even more preferred embodiment is that the Saccharomyces cerevisiae is Saccharomyces cerevisiae 1421, deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 23, 2021, with accession number CGMCC NO: 22945.

[0024] The preferred embodiment is that the viable bacteria concentration in the bacterial preparation is 1×10⁻⁶. 8 ~1×10 9 CFU / g or 1×10 8 ~1×10 9 CFU / mL. In the bacterial preparation, the ratio of live bacteria to Bacillus subtilis to yeast is (1-2):(1-2):(1-2), more preferably: Enterococcus faecium: Bacillus subtilis: yeast = 1:2:1, and even more preferably: Enterococcus faecium: Bacillus subtilis: Bacillus licheniformis: yeast = 1:1:1:1.

[0025] This invention also provides methods for preparing and using the aforementioned microbial preparation. The preparation method involves culturing each microorganism in an animal-acceptable culture medium to the required viable cell concentration, mixing the cultures in the required proportion, or preparing the cultures as freeze-dried powder or drying them before mixing in the required proportion; the resulting mixture is the microbial preparation. The mixture can be further granulated as needed (care should be taken to control the temperature and added excipients during granulation to avoid microbial inactivation) before use. The usage method involves adding the microbial preparation to the diet at a ratio of 1–5 mL or 1–5 g per kg of daily feed, along with the feed.

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0027] The culture media and reagents involved in this invention are as follows:

[0028] (1) Beef extract peptone medium: 2g yeast powder, 5g beef extract, 10g peptone, 5g sodium chloride, pH 7.0, add water to 1000mL, sterilization conditions: 121℃ for 20min.

[0029] (2) MRS medium: 5g yeast powder, 10g peptone, 8g beef extract, 20g glucose, 5g sodium acetate, 2g diammonium citrate, 1mL Tween 80, 2g K2HPO4, 0.2g MgSO4·7H2O, 16g CaCO3, 0.05g MnSO4·H2O, pH 6.3~6.8, add water to 1000mL, sterilize at 115℃ for 30min.

[0030] (3) YPED medium: 15g peptone, 5g yeast powder, 20g glucose, 2g maltose, 3mL Bengal red, 16g agar, pH 6.5±0.2, add water to 1000mL, sterilization conditions: 115℃ for 30min.

[0031] (4) Artificial gastric juice: Dilute 9.5% to 10.5% hydrochloric acid with water to pH=3.0, then add 1.0g of pepsin to every 100mL of diluted hydrochloric acid solution, mix well, and filter with a 0.22μm sterile filter membrane.

[0032] (5) Artificial intestinal fluid: Dissolve 6.8g of potassium dihydrogen phosphate in 500mL, adjust the pH to 6.8 with 1mol / L sodium hydroxide solution, and dilute with water to 1000mL. Then add 0.3g of porcine bile salts and 1.0g of trypsin to each 100mL of liquid, mix well, and filter with a 0.22μm sterile filter membrane.

[0033] (6) PBS solution: 0.27g potassium dihydrogen phosphate, 1.42g disodium hydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride, add deionized water and stir thoroughly to dissolve, add concentrated hydrochloric acid to adjust pH=7.4, and finally dilute with deionized water to 1L.

[0034] Example 1: Screening of Microorganisms

[0035] 1. Microbial sources: (1) Sample collection for strain screening: The healthy pigs and pig-related samples used in this invention came from Chengdu Tianfu Pig Farm of Juxing Agricultural and Animal Husbandry Group. The contents of the pig colon, fresh feces, sediment in the manure ditch of the breeding site and high-quality fermented feed preferred by piglets were collected respectively, and then sealed and refrigerated in sterile wide-mouth bottles for subsequent strain isolation. (2) Sources of other strains to be screened: Probiotics previously preserved in the laboratory and purchased.

[0036] 2. Initial screening of microorganisms.

[0037] Screening of aerobic spore-forming strains: Take 1.0 g of sample, dilute with sterile water, mix by shaking at a constant temperature, heat in an 85℃ water bath for 5 min, and then take 10 μL of the sample. -3 10 -4 10 -5 Three dilutions of heat-treated bacterial culture, 100 μL each, were evenly spread on LB solid agar plates, with three replicates for each dilution. The plates were incubated at 38°C for 24 h in a biochemical incubator. The morphological characteristics of the colonies were observed and recorded. Single colonies were selected and streaked onto LB solid agar plates for repeated purification until pure colonies were obtained.

[0038] Screening of anaerobic lactic acid-producing bacteria: Take 1.0 g of sample, dilute with sterile water, and shake at a constant temperature for 1 hour, then let stand. Take 10... -5 10 -6 10 -7 Three dilutions of bacterial culture, 100 μL each, were evenly spread on MRS solid medium supplemented with 16 g / L CaCO3. Each dilution was replicated in triplicate. The cultures were anaerobic incubator at 38 °C for 24 h. The morphological characteristics of the colonies were observed and recorded. Single colonies with clear zones were selected in an anaerobic clean bench and streaked onto MRS solid medium for repeated purification until pure colonies were obtained.

[0039] Screening of intestinal probiotics: Take 1.0g of sample, dilute with sterile water, and shake at a constant temperature for 1 hour, then let stand. Take 10... -3 10 -4 10 -5 100 μL of bacterial suspension at each of the three dilutions was evenly spread on YPED solid medium plates, with three replicates for each dilution. The plates were incubated at 37°C for 24 h. The morphological characteristics of the colonies were observed and recorded. Single colonies were streaked into YPED solid medium and repeatedly purified until pure colonies were obtained.

[0040] 16S rRNA PCR of the selected prokaryotes was performed using universal primers 27F and 1492R, while universal primers ITS1F and ITS2R were used for eukaryotes. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The amplification results were sent to Qingke Biotechnology (Beijing) Co., Ltd. for sequencing. Homology comparison analysis was performed on the results using NCBI data, and a phylogenetic tree was constructed using MEGA 7.0 software to identify the bacterial species. A total of over 100 strains from 23 categories were obtained. Based on the "Feed Additives Catalogue (2013)," over 60 strains prohibited for use in feed additives were removed, leaving 38 strains with potential for intestinal beneficial growth. These strains include: *Bacillus coagulans*, *Bacillus subtilis*, *Lactobacillus reuteri*, *Enterococcus faecalis*, *Clostridium butyricum*, *Bacillus licheniformis*, *Saccharomyces cerevisiae*, *Candida utilis*, *Rhodopseudomonas palustris*, *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Pediococcus lactis*, *Bacillus amyloliquefaciens*, and *Streptococcus thermophilus*. Simultaneously, microorganisms of the same genus or species with potentially similar properties were selected from previously preserved and purchased microorganisms in the laboratory for further screening.

[0041] 3. Microbial re-screening. The microorganisms screened in step 2, along with previously preserved / purchased probiotics, were subjected to a digestibility test: 2 mL of each test microorganism's bacterial suspension was transferred to a 10 mL centrifuge tube, centrifuged at 6500 rpm for 2 min, the supernatant was discarded, and the bacterial cells were collected and resuspended in 2 mL of sterile water to OD0.05. 600 (Bacteria) or OD 560 After the (fungal) concentration reached 0.7, 0.5 mL of each solution was added to separate 10 mL EP tubes containing 4.5 mL of sterile water, 4.5 mL of artificial gastric fluid, and 4.5 mL of intestinal fluid, respectively, and labeled as CK group, H group, and DY group. The CK group was immediately serially diluted, and 10 mL of each solution was taken. -4 10 -5 10 -6 100 μL of each dilution solution was prepared, with three replicates for each dilution. Plates were spread and incubated at 38°C. Plate selection: aerobic spore-forming bacteria were cultured on lysozyme broth (LB), anaerobic acid-producing bacteria on lactic acid bacteria medium (MRS), and yeast on yeast extract glucose medium (YPD). Unless otherwise specified, the culture media used for each microorganism are the same as those used here. After incubating groups H and DY at 38°C and 140 rpm for 2 hours using a constant temperature shaker, they were serially diluted, and 10 μL of each solution was used. -3 10 -4 10 -5 100 μL of each dilution solution was prepared, with three replicates for each dilution. After plating, the solutions were incubated at 38°C. Single colonies were counted after they became visible to the naked eye. The survival rate of digestibility was calculated as follows.

[0042]

[0043]

[0044] The strains with the highest survival rates against gastric acid and intestinal fluid were selected as candidates, with two candidates selected for each microorganism, as shown in Table 1.

[0045] Table 1. Candidate Microorganisms and Survival Rates

[0046]

[0047] Among them, Enterococcus faecalis DS4-3 was a newly screened strain, while the others were microorganisms that had been previously preserved or purchased by the laboratory.

[0048] Antagonism test: Each microorganism in Table 1 was spread onto its corresponding suitable solid culture medium and allowed to stand for a short time to infiltrate. A sterilized Oxford cup was then placed on the culture medium, and 200 μL of the other microorganisms in Table 1 was added to the Oxford cup using a sterile pipette. The cups were incubated at 38°C for 24 hours, and the formation of inhibition zones was observed. The test results showed no antagonistic effects between any of the microorganisms in Table 1.

[0049] 4. Physiological and biochemical characteristics of *Enterococcus faecalis* DS4-3. *Enterococcus faecalis* is a Gram-positive bacterium. In MRS medium, its vegetative cells are milky white and opaque, with a spherical, convex shape. The cell surface is moist and smooth, with regular edges, and calcification zones are produced near the colonies. The culture temperature is 30℃~40℃, and the optimal growth temperature is 35~38℃. SEM electron micrographs of *Enterococcus faecalis* are shown below. Figure 1 As shown, the growth curve is as follows Figure 2 As shown.

[0050] Example 2: Demonstration of Odor Reduction Effects in an In Vitro Simulation

[0051] Each Bacillus species listed in Table 1 was aerobically cultured in beef extract peptone broth, while lactic acid bacteria (Enterococcus faecium) were anaerobically cultured in MRS broth, and yeast was aerobically cultured in YPED broth. After centrifugation of the different bacterial cultures, the cells were diluted with PBS to prepare a uniform concentration (bacteria:OD). 600 =0.70; Yeast: OD 560 Standard bacterial solutions (0.70%) were prepared. The bacterial solutions were then compounded according to the combinations shown in Table 2 (where % represents volume concentration). The control group used 0.3% PBS solution instead of the bacterial solutions. It should be noted that the inventors did not only conduct experiments using the combinations shown in Table 2; however, due to space limitations, only a portion of representative combinations were selected for demonstration.

[0052] Table 2. Comparison of strain combinations

[0053]

[0054] Fresh swine colon contents were aseptically aliquoted into sterilized 150mL Erlenmeyer flasks, with 50g of contents in each flask. Each bacterial strain combination according to Table 2 was inoculated with a total inoculum of 0.3% (v / v). The control group was treated with an equal volume of PBS solution. After replacing the gas in the flasks with high-purity nitrogen to create an anaerobic environment, the samples were incubated on a shaker at 120 rpm and 38℃ for 8 hours. Once no gas production was observed, the samples were immediately transferred to a -20℃ freezer for storage. The contents of ammonium nitrogen, sulfide ions, total nitrogen, total sulfur, VFA, and pH were measured.

[0055] Ammonium nitrogen (NH4) in colonic contents + The content of sulfide ions (S⁻N) was determined by potassium chloride extraction-indophenol blue colorimetric method. 2- The content of α-S was determined by iodometric titration, and the content of VFAs was determined by gas chromatography. Total nitrogen content was determined using a Milti N / C2100S total organic carbon and total nitrogen analyzer, and total sulfur content was determined using a KWKLS-300 total sulfur analyzer. pH was determined using an S400-K pH meter. The formulas for calculating the ammonium nitrogen ratio and sulfur ion ratio are as follows. The results are shown in Table 3.

[0056]

[0057]

[0058] Table 3. Demonstration of in vitro simulated deodorization effects

[0059]

[0060] The results showed that the ammonium nitrogen ratio, sulfur ion ratio, and total VFA content of G11 and G16–G19 were significantly lower than those of the control group (CK), while the pH value was not significantly different from that of the control group (CK). Different subspecies of Bacillus licheniformis and Bacillus subtilis had little effect on the results, while different subspecies of Enterococcus faecalis and Saccharomyces cerevisiae had a significant impact on the results.

[0061] Example 3: Demonstration of Animal Experiment Results

[0062] Animal experiments were conducted using the G11 combination that showed the best results in Example 2. Duroc × Landrace × Large White × Large White three-way crossbred piglets, approximately 35 days old and weaned, with similar weights (7.50 ± 1.25 kg) and half male and half female, were selected and the experiments were conducted at the animal testing facility of the Chengdu Institute of Biology, Chinese Academy of Sciences, in Yong'an Town, Shuangliu District.

[0063] Feeding Method: After acclimatizing the piglets for 7 days, they were numbered, vaccinated, dewormed, and given digestive aids. Following the acclimatization period, the formal feeding trial period began, lasting 8 weeks. The feeding and management of all experimental groups were identical. During the trial, the piglets were cared for according to the "NY / T65-2004—Pig Feeding Standards." Each group received ample feeding, ensuring the pigs were well-fed at each meal without restriction. All piglets were housed in uniform cement-floored pens. Each pen was equipped with a single-sided automatic feeder and a nipple drinker, allowing the pigs free access to feed and water throughout the experiment. If weighing was required, feed was withheld the night before weighing, and sufficient feed was immediately added on the day of weighing. During the trial, antibiotics, traditional Chinese medicine, and other probiotics or other feed additives and drugs that might affect the experimental results were not used.

[0064] Feeding control: 96 piglets that successfully adapted to the new environment and whose weight difference was less than 3 kg were selected and randomly divided into 4 groups (half male and half female) by sex. Each group consisted of 3 parallel pens, with 8 piglets per pen. The 4 groups were further divided according to their dietary treatment: control group CK (basal diet), experimental group T1 (basal diet + 0.3% probiotic), experimental group T2 (basal diet + 0.2% probiotic), and experimental group T3 (basal diet + 0.1% probiotic). Commercially available piglet feed was used, which typically resulted in a daily weight gain of approximately 483 grams for weaned piglets. The probiotic referred to the G11 group probiotic. Feeding was conducted for 45 days starting from the end of the adaptation period. Fresh fecal samples were collected weekly, and pen odor and feed intake data were collected daily. Weight gain data were collected every 15 days. The results are shown in Table 3.

[0065] Table 3 Feeding Results

[0066]

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. Enterococcus faecium (E. faecium) strain DS4-3, characterized by: Enterococcus faecium ) a genome comprising a 16S rRNA gene sequence of SEQ ID NO:

1. It was deposited on December 12, 2022 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 62359.

2. The use of the Enterococcus faecalis of claim 1 in removing malodorous gases produced by pigs.

3. The use of the Enterococcus faecalis according to claim 1 in denitrification and / or desulfurization.

4. The bacterial preparation made using *Enterococcus faecium* as described in claim 1, characterized in that: The microbial preparation also includes yeast, specifically Saccharomyces cerevisiae, Saccharomyces cerevisiae 1421, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 23, 2021, with accession number CGMCC NO: 22945.

5. The bacterial preparation according to claim 4, characterized in that: The bacterial preparation also includes Bacillus.

6. The bacterial preparation according to claim 5, characterized in that: The Bacillus species include Bacillus subtilis and Bacillus licheniformis.

7. The bacterial preparation according to claim 5, characterized in that: In the bacterial preparation, the ratio of live bacteria is Enterococcus faecalis: Bacillus: Yeast = (1-2): (1-2): (1-2).

8. The use of the bacterial preparation according to any one of claims 4 to 7 in removing malodorous gases produced by pigs.

9. The application according to claim 8, characterized in that: The application method is as follows: feed the bacterial preparation to pigs.

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