Paracoccus versutus strain ky-t1 capable of degrading nitrite and resistant to salt and low temperature, high-density fermentation culture method and application

Abstract

The present application relates to the technical field of biology, and discloses a Parvularcula australis KY-T1 capable of degrading nitrite and resistant to salt and low temperature, a high-density fermentation culture method and application.The Parvularcula australis provided by the present application has a preservation number of CCTCC NO: M2026770, can degrade nitrite and ammonia nitrogen in water, shows high sensitivity to commonly used antibiotics, can reduce the accumulation and spread of drug resistance caused by large-scale use of microbial strains, has a wide adaptation range, is resistant to low temperature, can grow in a salt concentration of 1‰ to 50‰, and has high nitrite nitrogen degradation capacity.Under high-density and high-protein feeding conditions for prawns, sea bass and the like, ammonia nitrogen and nitrite will continuously increase, and C / N will decrease to 3.18, but the strain of the present application can still stably and efficiently degrade nitrite nitrogen, reduce the accumulation of nitrite nitrogen in aquaculture water, and reduce the toxic effect of nitrite nitrogen on aquatic animals.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a strain of Paracoccus KY-T1 that is degradable in nitrite and tolerant to salt and low temperatures, a high-density fermentation culture method, and its application. Background Technology

[0002] The most prominent water quality problems currently faced in aquaculture are excessive levels of ammonia nitrogen and nitrite. Ammonia nitrogen damages the gill tissue of farmed organisms, affecting their respiratory function, leading to decreased feed intake and stunted growth. High concentrations of ammonia nitrogen can also directly cause poisoning and death in farmed organisms. Nitrite, on the other hand, can cause methemoglobinemia in farmed organisms, causing their blood to lose its oxygen-carrying capacity, resulting in hypoxia and stress, which in turn induces diseases such as gill rot and enteritis. Long-term excessive levels of both not only significantly reduce the survival rate of farmed organisms but also affect the quality of aquatic products, causing huge economic losses to aquaculture operators.

[0003] Ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen are the main forms of inorganic nitrogen in aquaculture water. For aquaculture animals, excessive levels of ammonia nitrogen and nitrite nitrogen in the water can lead to poisoning. Excessive ammonia nitrogen (greater than 0.2 mg / L) can damage gill filaments, disrupt osmotic pressure, and cause fish and shrimp to surface for air, lose appetite, and have reduced immunity. Long-term excessive levels can cause gill rot, bleeding, and outbreaks of viral and bacterial diseases. Excessive nitrite nitrogen (greater than 0.05 mg / L) can cause aquaculture animals to lose their oxygen-carrying capacity due to the large amount of nitrite entering the blood and oxidizing hemoglobin into methemoglobin. This can lead to systemic hypoxia, a sharp decrease in feed intake, and acute or chronic death. Nitrate nitrogen is not toxic to the farmed animals themselves, but long-term accumulation can lead to eutrophication of the water and the outbreak of cyanobacteria. At the same time, nitrate in the water can also be reduced back to nitrite, causing nitrite rebound.

[0004] Currently, research on *Paragonimus westermani* mainly focuses on the degradation of ammonia and nitrate nitrogen. Patent CN118146994A discloses a strain of *Paragonimus westermani* YB-1, which achieved a 100% removal rate of nitrate nitrogen and a 53.54% removal rate of total nitrogen over 24 hours. However, a significant accumulation of nitrite nitrogen was detected during the experiment, increasing from 0 mg / L at 0 hours to 359.44 mg / L over 24 hours. Patent CN106834182B discloses a strain of *Paragonimus westermani* 2#, which is salt-intolerant and cannot utilize citrate, but achieves a 93.8% removal rate of hydrogen sulfide from biogas.

[0005] However, in practical applications, environmental factors have a significant impact on the degradation efficiency of ammonia nitrogen and nitrate nitrogen by the strains.

[0006] 1) C / N ratio

[0007] Patent CN110656057A discloses a strain of *Paragonimus westermani* LJ2, which exhibits relatively good degradation effects on nitrite nitrogen under specific conditions. However, its nitrite degradation is dependent on the C / N ratio; a C / N ratio of 15-120 results in better nitrite degradation, while the degradation efficiency significantly decreases when the C / N ratio drops to 8. In aquaculture, during the early stages of rearing (initial stocking), the feed intake is low, and the nitrogen-to-carbon ratio (N / C ratio) is typically 12-20:1. During the middle stages, due to continuous feeding and the accumulation of uneaten feed and feces, the N / C ratio drops to 6-12:1. In the later stages, the N / C ratio further decreases to 3-8:1, especially in high-density, industrialized ponds where it can fall to 2-5:1, leading to severe nitrogen excess and persistently high levels of ammonia nitrogen and nitrite. Therefore, this strain has certain limitations in practical applications.

[0008] 2) Salt tolerance

[0009] Salt tolerance and nitrite degradation are not typical properties of *Paragonimus* bacteria. According to a 2003 report by Maryam I. Daneshvar et al., their phenotypic classification of *Paragonimus* bacteria revealed that 1 or 2 out of 8 *Paragonimus* strains could grow in NB medium with 0% sodium chloride, and only 1 or 2 strains could grow in NB medium with 6% sodium chloride; 1 out of 8 *Paragonimus* strains were nitrite-positive, and 12 out of 13 strains could degrade nitrate, but only 1 strain could utilize nitrate to produce gas.

[0010] 3) Antibiotic resistance

[0011] In practical applications, we should screen for antibiotic-sensitive strains to ensure that there is no risk of drug resistance transmission after use, thus improving safety. However, the antibiotic sensitivity of microorganisms varies from strain to strain. A study by Simon C. Baker et al. reported that in the study of Paracoccus, the antibiotic resistance of the strain needs to be considered. For example, the commonly used Paracoccus Pd1222 is intrinsically resistant to spectinomycin and sensitive to streptomycin, but Paracoccus GB-17 is the complete opposite; GB-17 is sensitive to spectinomycin and resistant to streptomycin.

[0012] 4) Dissolved oxygen tolerance

[0013] Grass carp and other common warm-water fish require dissolved oxygen (DO) greater than 5.0 mg / L for normal growth, shrimp require DO greater than 6.0 mg / L, and cold-water fish such as salmon require DO greater than 7.0 mg / L. However, existing technology CN110656057A suggests that as the rotation speed increases, the DO increases, which can promote the growth and denitrification performance of LJ2. But when the rotation speed increases to a certain level, excessive DO can inhibit the growth and denitrification performance of LJ2. Therefore, paracocci used in aquaculture also need to possess the characteristic of tolerating high dissolved oxygen levels.

[0014] Based on these factors, although some literature reports that certain isolates of *Paragonimus westermani* have the ability to degrade nitrite, the degradation efficiency of nitrite varies greatly among different strains, and their applicable conditions also differ significantly. Therefore, there is an urgent need to develop green and safe biological agents that can effectively degrade nitrite nitrogen under the special environmental conditions of aquaculture. Summary of the Invention

[0015] The purpose of this invention is to provide a strain of Paracoccus versutus KY-T1, which has the accession number CCTCC NO: M2026770.

[0016] Another object of the present invention is to provide the application of Paracoccus versutus KY-T1, the application of which includes its use as a water quality conditioner and modifier in aquaculture water, effectively degrading ammonia nitrogen and nitrite in aquaculture water.

[0017] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0018] Paracoccus versutus KY-T1 was obtained through the following screening steps:

[0019] Soil and water samples were collected from the ecological wetland environment, appropriately diluted, and spread onto NA medium. The samples were incubated at 35°C for 48-72 hours. Single colonies were picked from the plates and repeatedly streaked on NA medium plates for purification, followed by incubation at 35°C to obtain pure colonies. After extensive sorting, physiological and biochemical analysis, and 16S identification, a strain of *Paracoccus versutus* was isolated and screened. The applicant named it *Paracoccus versutus* KY-T1. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on April 22, 2026, with the classification name *Paracoccus versutus* KY-T1, accession number CCTCC NO: M2026770, address: Wuhan University, Wuhan, China.

[0020] Paracoccus versutus KY-T1 has the following biological characteristics:

[0021] *Paracoccus versutus* belongs to the genus *Paracoccus*. It is Gram-negative, catalase-positive, and oxidase-positive. After 18-24 hours of culture, colonies are 0.5-1 mm in diameter; after 24-72 hours, they are 3-4 mm in diameter. It utilizes glycerol, acetate, and pyruvate, and undergoes respiratory metabolism. On NA medium, colonies grow singly, in pairs, or in clusters, appearing milky white to pale yellow, with intact, round edges and a smooth, slightly raised surface. Single cells are 0.5–1.1 μm in diameter. The optimal growth temperature is 30-37℃, but it can grow within the range of 10℃ to 37℃, with an optimal growth pH of 6.0-7.5. It is an aerobic bacterium and does not grow under anaerobic conditions on NA medium. Sequencing of its 16S rRNA gene and BLASTN alignment using NCBI revealed the highest sequence homology (99%) with *Paracoccus versutus* (ON358036.1). Based on physiological and biochemical characteristics and 16S rRNA comparison results, the isolated strain KY-T1 was confirmed to be Paracoccus versutus.

[0022] The scope of protection of this invention also includes:

[0023] The strains described above contain the specific sequence shown in SEQ ID NO.2.

[0024] A compound comprising Paracoccus versutus KY-T1.

[0025] Application of Paracoccus versutus KY-T1 or the above-mentioned compound in the preparation of water quality conditioning and improving agents.

[0026] Application of Paracoccus versutus KY-T1 or the above-mentioned compound in water quality conditioning.

[0027] The above-described applications involve adjusting water quality by reducing the content of ammonia nitrogen and / or nitrite in the water.

[0028] In the above-described applications, the salinity of the water is 0-50‰, and more preferably 15‰-30‰.

[0029] In the above-described applications, the pH value of the water is 5.0-9.0.

[0030] In the above applications, the water temperature is 28-37℃.

[0031] In the above-described applications, the Paracoccus versutus KY-T1 or its compound is pre-activated using a carbon source, preferably brown sugar, molasses, glucose, sodium acetate, and / or sodium citrate.

[0032] A high-density large-scale fermentation method for *Paracoccus versutus* KY-T1 includes inoculating *Paracoccus versutus* KY-T1 into a fermentation medium at an inoculum size of 1% to 10%. The fermentation medium has the following composition: corn starch 10.0 g / L, glucose 5-10.0 g / L, yeast extract 10.0 g / L, fish peptone 20.0 g / L, calcium chloride 0.02 g / L, magnesium sulfate 1.0 g / L, dipotassium hydrogen phosphate 4.0 g / L, potassium dihydrogen phosphate 2.0 g / L, manganese sulfate 0.01 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 2.0 ml / L, pH 7.2 ± 0.1, with the remainder being water. The *Paracoccus versutus* KY-T1 has the accession number CCTCC NO: M2026770.

[0033] The method described above is preferably applicable to fermenters with a volume of 30 to 10,000 cubic meters.

[0034] The method described above, preferably, involves fed-batch fermentation for large-scale fermentation. Feeding is initiated when dissolved oxygen first rises above 60 or residual sugar is <2g / L. During feeding, dissolved oxygen is controlled at 20%-40%, and pH is 7.0-7.2. The feed is prepared by mixing 1L of solution A and 1L of solution B. Solution A is a 500g / L glucose solution, and solution B is a mixture of 100g / L peptone, 100g / L yeast extract, and 5g / L magnesium sulfate.

[0035] The protection method of the present invention also includes the application of the above-mentioned culture method in the fermentation production of Paracoccus KY-T1.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] 1. The Paracoccus variegata provided by this invention can quickly enter the logarithmic growth phase and reach the stable growth phase in 10 hours, with a short fermentation cycle, which significantly reduces the fermentation production cost.

[0038] 2. The *Paragonimus westermani* provided by this invention can grow in environments with a high salt concentration of 50‰ and a pH of 5.0-9.0, and is resistant to low temperatures, exhibiting very strong environmental tolerance.

[0039] 3. The Paracoccus variolidinium provided by this invention is sensitive to antibiotics, does not pose a risk of drug-resistant transmission, and has extremely high safety in use.

[0040] 4. The Paracoccus variegata provided by this invention can not only grow under high salt concentrations, but also effectively degrade nitrite nitrogen under high salt concentrations.

[0041] 5. The *Paragonimus westermani* provided by this invention can efficiently degrade nitrite nitrogen in water under high dissolved oxygen conditions.

[0042] 6. The *Paragonimus westermani* provided by this invention has the ability to degrade nitrite nitrogen under low carbon-nitrogen ratio conditions. It can effectively degrade nitrite nitrogen in the low carbon-nitrogen ratio and high nitrite environment in the middle and late stages of aquaculture, reduce the toxicity of nitrite to aquatic animals, improve aquaculture efficiency, reduce poisoning stress mortality caused by continuous increase in nitrite, and reduce economic losses.

[0043] 7. The Paracoccus variegata provided by this invention can effectively degrade nitrite and ammonia nitrogen in freshwater aquaculture, and can also efficiently degrade nitrite and ammonia nitrogen in high-salinity aquaculture waters such as seawater.

[0044] 8. The carbon source of Paracoccus variegata provided by this invention has a wide range of applications. It can be rapidly activated in carbon sources such as brown sugar, glucose, molasses, sodium acetate, and sodium citrate, thereby improving the efficacy and utilization rate of the preparation.

[0045] 9. The fermentation method for Paracoccus variegata provided by this invention can achieve a viable count of up to 37.5 billion CFU / ml in shake flask fermentation, 62.3 billion CFU / ml in batch fermentation in fermenter, and up to 122.3 billion CFU / ml in fed fermenter, thus saving costs and increasing yield. Attached Figure Description

[0046] Figure 1 The colony morphology of Paracoccus versutus KY-T1 on NA nutrient agar.

[0047] Figure 2 This is a scanning electron microscope (SEM) schematic diagram of Paracoccus versutus KY-T1.

[0048] Figure 3 This is a schematic diagram of the growth curve of Paracoccus versutus KY-T1.

[0049] Figure 4 Phylogenetic tree of Paracoccus versutus KY-T1 16S.

[0050] Figure 5 This data represents the optimal growth temperature for Paracoccus versutus KY-T1.

[0051] Figure 6 The study aimed to determine the degradation efficiency of nitrite nitrogen in aquaculture greenhouse water by Paracoccus versutus KY-T1. Detailed Implementation

[0052] Unless otherwise specified, the experimental methods and conditions in the embodiments of this invention are conventional methods. These embodiments are for illustrative purposes only, and the scope of protection of this invention is not limited by these embodiments. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial sources.

[0053] The culture medium used in this invention is as follows:

[0054] The plate selection medium for nitrite-degrading strains consists of: 2g sodium nitrite, 20g sodium citrate, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 5g peptone, 3g yeast extract, 2g sodium bicarbonate, 20g agar, and 1000ml distilled water. The pH is 7.0-7.5, and the medium is sterilized at 121℃ for 15-20 minutes. Sodium acetate can be used instead of sodium citrate in the selection medium.

[0055] Nitrite medium: glucose 10.0 g / L, sodium nitrite 0.345 g / L, dipotassium hydrogen phosphate 2.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate 0.2 g / L, calcium chloride 0.02 g / L, trace elements 10 ml, pH 7.0-7.5, used after filtration and sterilization.

[0056] The trace element mixture consists of: ferric chloride 0.016 g / L, zinc sulfate 0.1 g / L, manganese chloride 0.3 g / L, and copper sulfate 0.02 g / L.

[0057] Example 1:

[0058] Isolation, screening, and identification of highly efficient nitrite-degrading strains:

[0059] 1. Isolation and screening of nitrite-degrading strains

[0060] Environmental soil and water samples were collected. 10g of soil sample was added to 90ml of sterile single-distilled water and incubated at 16℃ and 200rpm for 30min. Alternatively, 10ml of water sample was added to 90ml of sterile single-distilled water and shaken thoroughly. The prepared soil or water samples were serially diluted 10-fold. Appropriate dilutions were then plated onto a nitrite-degrading strain selection medium and incubated at 37℃ for 48-72h. Single colonies were picked from the plates and streaked multiple times on the nitrite-degrading strain selection medium plates for purification, incubating at 37℃ for 24-48h to obtain pure colonies. The purified strain was inoculated into nitrite medium, ultimately yielding a highly efficient nitrite-degrading strain, named KY-T1. The nitrite nitrogen degradation efficiency of the selected strain is shown in Table 1. Colony morphology and Gram staining of strain KY-T1 were performed. When cultured on NA nutrient agar for 24-48 hours, the strain formed smooth, milky-white to pale yellow, raised, round colonies with a diameter of 2-3 mm. This strain is Gram-negative, round or short-round, and motile. The colony morphology of this strain on NA agar plates is shown in the figure. Figure 1 Scanning electron micrographs of bacterial cells can be found here. Figure 2 The growth curves in NB liquid culture are shown below. Figure 3 .

[0061] Table 1 Isolation and screening of nitrite-degrading strains

[0062] .

[0063] 2. Strain identification

[0064] Physiological and biochemical identification and 16S rRNA identification were performed on the obtained strain KY-T1. Physiological and biochemical experiments were conducted according to Berger's Manual of Bacterial Identification. Micro-biochemical reaction tubes were purchased from Hangzhou Binhe Microbial Reagent Co., Ltd. The results of the physiological and biochemical identification are shown in Table 2. The 16S rRNA gene sequence of strain KY-T1 was amplified and sequenced using published universal 16S primers. The PCR amplification product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The nucleotide sequence of the 16S rRNA of strain KY-T1 is shown in SEQ ID NO.1. The sequenced sequence was aligned to NCBI using BLASTN, and a phylogenetic tree was constructed using MEGA software. (See Table 2). Figure 4Homology comparison results showed that its 16S rRNA had the highest sequence homology with *Paracoccus versutus* (ON358036.1), with a similarity of 99%. Based on physiological and biochemical characteristics and 16S rRNA comparison results, the isolated strain KY-T1 was confirmed as *Paracoccus versutus*. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on April 22, 2026, with the classification name: *Paracoccus versutus* KY-T1, accession number CCTCC NO: M2026770, address: Wuhan University, Wuhan, China.

[0065] Table 2. Physiological and biochemical identification results of Paracoccus versutus KY-T1

[0066] .

[0067] Note: "+" represents a positive reaction; "-" represents a negative reaction.

[0068] 3. Whole genome sequencing information of Paracoccus versutus KY-T1

[0069] The whole genome of Paracoccus versutus KY-T1 was sequenced using the PacBio Revio microbial sequencer, including:

[0070] The whole-genome sequencing results are shown in Tables 3 and 4. The total genome size of Paracoccus versutus KY-T1 is 5,197,297 bp, with a GC content of 67.84%. It contains two circular chromosomes and two circular plasmids, with a sequencing coverage of 100%. It encodes 5,265 genes, accounting for 89.41% of the total number of bases in the genome. It contains 52 tRNAs, 3 sets of rRNAs (5S / 16S / 23S), and 31 other non-coding RNAs; 371 tandem repeats (TRFs) and 54 simple tandem repeats (SSRs); 4 CRISPR sequences, 3 in chr1 and 1 in plasmid1; 21 gene islands with a total length of 335,375 bp and an average gene island length of 15,970.24 bp; and 6 gene clusters for the biosynthesis of secondary metabolites.

[0071] Paracoccus versutus KY-T1 has two chromosomes, two plasmids, and abundant prophages and gene islands. When stored, its nutrition decreases, its metabolism slows down, and multiple replicons are prone to separation failure. High GC bacteria are more likely to have their DNA broken and die when starved or frozen. It is extremely sensitive to temperature fluctuations and is easily inactivated when stored at 37°C.

[0072] Sequence alignment analysis of the whole genome of strain KY-T1 revealed that the strain has a specific coding sequence located on chromosome 1 of the strain, and the specific sequence is shown in SEQ ID NO.2.

[0073] Table 3. Basic genome information of Paracoccus versutus KY-T1

[0074] .

[0075] Table 4. Chromosome and plasmid information of Paracoccus versutus KY-T1

[0076] .

[0077] Example 2:

[0078] Effects of environmental factors on the growth of Paracoccus KY-T1

[0079] 1. The effect of temperature on the growth of *Paragonimus westermani*

[0080] Temperature affects bacterial growth rate, cell concentration, and growth cycle by influencing enzyme activity, cell membrane fluidity, and metabolic rate. In this embodiment, *Paragonimus westermani* KY-T1 was cultured at different temperatures, and the OD600 of the culture medium was measured at different time points to observe the effect of culture temperature on the growth of the strain.

[0081] The specific steps are as follows:

[0082] Preparation of seed culture and seed culture: Take the glycerol KY-T1 strain stored at -80℃, rapidly warm it to 37.0±1.0℃ for 2-3 min, use an inoculation loop to pick up glycerol bacteria and streak it onto NA nutrient agar plates, and incubate at 37℃ for 24-48 h. Pick a single colony from the cultured plate and inoculate it onto NB nutrient broth medium, and incubate at 37℃ and 180-200 rpm for 12-18 h. The cultured fermentation broth is used as the seed culture for later use. The preparation method of the seed culture is the same as described below.

[0083] The prepared seed culture was inoculated into fresh NB medium at a ratio of 1% (V / V) and incubated at 28℃, 30℃, 32℃, 34℃, and 37℃ respectively. Samples were taken every 4 hours to measure the OD600 of the culture medium. Three replicates were performed for each incubation temperature. 600 Values ​​are shown in Table 5 and Figure 5 Within a culture temperature range of 28℃-37℃, *Paragonimus westermani* can grow. With increasing temperature, the growth rate of *Paragonimus westermani* accelerates. The effect of temperature on the final OD value increases with the extension of culture time. 600The impact is gradually decreasing.

[0084] Furthermore, during cultivation and preservation, it was found that the fermentation broth of *Paragonimus westermani* KY-T1 of this invention can grow at 4°C, and the viable cell content increases by 150%-200% after being placed at 4°C for 1-3 days.

[0085] Table 5. Effect of culture temperature on the growth of *Paragonimus westermani*

[0086] .

[0087] 2. Effects of salinity and inoculum size on the growth of *Paragonimus westermani*.

[0088] Salinity is an important environmental factor affecting bacterial growth. In this example, *Paragonimus westermani* KY-T1 was cultured under different salinity conditions, and the OD of the culture medium was measured at different time points. 600 The effect of sodium chloride concentration in the culture medium on the growth of the strain was observed.

[0089] The specific steps are as follows:

[0090] The prepared seed culture was inoculated into NB medium supplemented with sodium chloride at concentrations of 1%, 2%, 5%, 8%, and 10% (V / V) at ratios of 5‰, 10‰, 15‰, 20‰, 25‰, 30‰, 35‰, 40‰, 45‰, and 50‰, respectively. The growth of the strains was monitored using an automated growth curve analyzer. The culture was incubated at 37℃, and the OD of the culture medium was measured every 0.5 hours. 600 Triple replicates were performed for each culture temperature. *Paragonimus westermani* KY-T1 could grow in media supplemented with 5‰–50‰ sodium chloride. At a salinity of 25‰, different inoculum doses showed varying OD values. 600 The growth rates were all higher than other salinity groups, as shown in Table 6. The optimal salinity for most bacteria in nature is 0.5‰–5‰, and they are quite sensitive to salinity stress. Only a few strains or halophilic bacteria can tolerate higher salinities, typically with an optimal salinity of 15‰–30‰. The growth of *Paragonimus westermani* in this invention under different salinity levels indicates that it has high salinity tolerance and a wide range of adaptability, showing good application prospects in both freshwater and marine aquaculture.

[0091] Table 6. Effects of culture medium salinity and inoculum size on the growth of *Paracoccus vulgaris*.

[0092] .

[0093] 3. Effects of pH and inoculum size on the growth of *Paragonimus westermani*

[0094] pH significantly affects the growth rate and fermentation biomass of microorganisms by altering cell surface charge, enzyme activity, and nutrient solubility. In this example, *Paragonimus westermani* KY-T1 was cultured under different pH conditions, and the OD of the culture medium was measured at different time points. 600 The effect of culture medium pH on the growth of the strain was observed.

[0095] The specific steps are as follows:

[0096] The prepared seed culture was inoculated into NB medium with pH values ​​of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 at ratios of 1%, 2%, 5%, 8%, and 10% (V / V), respectively. The growth of the strains was monitored using an automated growth curve analyzer. The culture was incubated at 37℃, and the OD of the culture medium was measured every 0.5 hours. 600 Each treatment was performed in triplicate. The specific growth of *Paragonimus westermani* KY-T1 in different pH media is shown in Tables 7 and 8. At pH 5.0 and 5.5, increasing the inoculum size allowed *Paragonimus westermani* to reach higher fermentation levels. At pH 6.0, 6.5, 7.0, 7.5, and 8.0, lower inoculum sizes were more conducive to *Paragonimus westermani* growth. At pH 8.5, fermentation with a 1% inoculum size was higher than with 2% and 5% inoculum sizes, but lower than with 8% and 10% inoculum sizes. At pH 9.0, with increasing inoculum size, the highest OD of the fermentation broth... 600 The value gradually increases.

[0097] Table 7. Effects of culture medium pH and inoculum size on the growth of *Paracoccus vulgaris*.

[0098] .

[0099] 4. Antibiotic susceptibility of variable paracocci

[0100] Antibiotic susceptibility testing can evaluate the biosafety of bacterial strains, determine whether they carry drug-resistant genes, and avoid the risk of drug-resistant transmission. In this invention, the susceptibility of *Paragonimus westermani* KY-T1 to common antibiotics was tested using the disk susceptibility testing method. The results are shown in Table 8. The strain of this invention is sensitive to all 23 antibiotics tested, and there is no risk of drug-resistant transmission, demonstrating extremely high safety for use.

[0101] Table 8 Antibiotic susceptibility of Paracoccus vulgaris KY-T1

[0102] .

[0103] Example 3:

[0104] The degradation effect of nitrite by Paracoccus variableis

[0105] Preparation of fermentation broth: The cultured Paracoccus versutus KY-T1 seed culture was inoculated at a ratio of 2% into 100 ml of liquid fermentation medium and cultured at 37℃ and 200 rpm for 24 hours. The viable cell count of the fermentation broth was ≥2×10⁻⁶. 10 CFU / ml, the fermentation broth for the following strains was prepared according to this method. The cultured fermentation broth was stored at 4°C for later use.

[0106] The liquid fermentation medium consisted of 20.0 g / L corn starch, 20.0 g / L glucose, 5.0 g / L yeast extract, 10.0 g / L fish peptone, 1.5 g / L calcium chloride, 3.0 g / L magnesium sulfate, 2.0 g / L sodium chloride, 2.0 g / L dipotassium hydrogen phosphate, 0.3 g / L manganese sulfate, 0.01 g / L ferrous sulfate, 2.0 ml / L defoamer, and a pH of 7.2 ± 0.1.

[0107] The effect of Paracoccus versutus KY-T1 on nitrite removal from nitrite-containing culture media:

[0108] The bacterial count of the prepared Paracoccus versutus KY-T1 fermentation broth was adjusted to 2 × 10⁻⁶. 9 CFU / ml was added to nitrite medium at a ratio of 1%-1‰, and cultured at 30℃ and 200rpm for 24h. The degradation efficiency of nitrite by Paracoccus KY-T1 was then determined.

[0109] The degradation efficiency of nitrite nitrogen by *Paragonimus westermani* in laboratory simulated nitrite-containing culture medium is shown in Table 9. At a 1% inoculum, the degradation efficiency reached 97.43% in 4 hours and 100% in 16 hours; at a 1‰ inoculum, the degradation efficiency was 98.97% in 24 hours.

[0110] Table 9. Degradation efficiency of nitrite nitrogen in culture medium by *Paracococcus variegata*

[0111] .

[0112] Example 4:

[0113] Effect of culture medium salinity on the efficiency of nitrite degradation by Paracoccus versutus KY-T1

[0114] In aquaculture waters, especially in marine aquaculture, the sodium chloride content has a significant impact on the efficiency of denitrifying bacteria in degrading nitrite. In this example, different concentrations of sodium chloride were added to the nitrite culture medium used in Example 3 to test the degradation efficiency of nitrite by Paracoccus KY-T1 under different salinity conditions.

[0115] The prepared Paracoccus versutus KY-T1 fermentation broth was added at a ratio of 1‰ to nitrite culture medium containing different concentrations of sodium chloride (15‰, 20‰, 25‰, 30‰). The culture was carried out at 30℃ and 200rpm for 48h, and the degradation efficiency of nitrite by Paracoccus versutus KY-T1 was determined.

[0116] Table 10 shows the degradation efficiency of Paraacoccus versutus for nitrite nitrogen under different salinity conditions. In the control medium without additional sodium chloride, the degradation efficiency was 98.60% after 24 hours. In the media with additional sodium chloride at 15‰, 20‰, 25‰, and 30‰, the degradation efficiencies of nitrite nitrogen after 24 hours were 99.79%, 94.59%, 90.77%, and 84.20%, respectively. The degradation efficiency of all groups was 100% after 48 hours. The Paraacoccus versutus KY-T1 strain of this invention still has a high efficiency in degrading nitrite nitrogen under high salt concentrations.

[0117] Table 10 Effect of salinity on the efficiency of *Paracococcus variableis* in degrading nitrite nitrogen.

[0118] .

[0119] Example 5:

[0120] Effect of dissolved oxygen content on the efficiency of nitrite degradation by Paracoccus versutus KY-T1

[0121] In aquaculture, high dissolved oxygen levels are typically required to ensure the health of farmed aquatic animals, especially under high-density farming conditions. Low dissolved oxygen can lead to decreased feed intake, slow growth rates, oxygen deficiency causing fish to surface for air, and stress. However, under high-density farming conditions, ammonia nitrogen and nitrite levels in the water are more likely to exceed safe limits, causing ammonia nitrogen and nitrite poisoning in fish. Most denitrifying bacteria require anaerobic or hypoxic conditions to complete denitrification and degrade nitrite in the water. High dissolved oxygen levels will reduce the degradation rate of nitrite nitrogen. This example measured the degradation efficiency of nitrite nitrogen by *Paragonimus westermani* KY-T1 under dissolved oxygen conditions at different rotation speeds.

[0122] The prepared Paracoccus versutus KY-T1 fermentation broth was added to nitrite medium at a ratio of 1‰ and cultured at 30℃ for 48 h at 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, and 240 rpm. The degradation efficiency of nitrite by Paracoccus versutus KY-T1 was then determined.

[0123] Table 11 shows the degradation efficiency of Paracoccus versutus for nitrite nitrogen under different shaking speeds and dissolved oxygen conditions. At lower shaking speeds, the degradation efficiency of Paracoccus versutus for nitrite nitrogen increases with increasing shaking speed and dissolved oxygen. When the shaking speed reaches 160 rpm to 240 rpm, the degradation efficiency can reach 100%. High dissolved oxygen has no effect on the degradation efficiency of Paracoccus versutus for nitrite nitrogen. The Paracoccus versutus KY-T1 of this invention still has a high degradation efficiency for nitrite nitrogen under high shaking speed conditions.

[0124] Table 11 Effect of dissolved oxygen content on the nitrite degradation efficiency of Paracoccus versutus KY-T1

[0125] .

[0126] Example 6:

[0127] The degradation effect of ammonia nitrogen by Paracoccus KY-T1

[0128] The effect of Paracoccus versutus KY-T1 on the removal of ammonia nitrogen in culture medium

[0129] The ammonia nitrogen culture medium is as follows:

[0130] Add 10.0 g / L glucose, 0.6 g (NH4)2SO4, 0.01 g MnSO4, 0.03 g MgSO4, 0.25 g Na2HPO4, 0.75 g KH2PO4, 0.2 g CaCl2, and 0.03 g FeSO4 to water to a final volume of 1 L and adjust the pH to 7.2.

[0131] The prepared Paracoccus versutus KY-T1 fermentation broth was added to ammonia nitrogen-containing culture medium at a ratio of 1‰ (V / V), and cultured at 30℃ and 200rpm for 6 days. The degradation efficiency of ammonia nitrogen by Paracoccus versutus KY-T1 was then determined.

[0132] The results are shown in Table 12. The *Paragonimus westermani* provided by this invention can not only efficiently degrade nitrite nitrogen in water, but also, compared with the blank control group, degrade ammonia nitrogen in water to a certain extent.

[0133] Table 12. Effect of Paracoccus versutus KY-T1 on ammonia nitrogen removal in culture medium.

[0134] .

[0135] Example 7:

[0136] A comparison of the efficacy of different formulations of Paracoccus versutus KY-T1 and commercially available nitrite-lowering products.

[0137] Formulation 1: *Paragonimus westermani* KY-T1 fermentation broth, pH 6.5-7.5, effective bacterial concentration 2×10⁻⁶. 9 CFU / ml;

[0138] Formulation 2: KY-T1 acidified liquid formulation of *Paragonimus westermani*, pH 5.0-5.5. This formulation is based on Formulation 1, with citric acid added to adjust the pH of the fermentation broth to 5.0-5.5. The effective bacterial concentration is 2 × 10⁻⁶. 9 CFU / ml;

[0139] Formulation 3: *Paragonimus westermani* KY-T1 powder; The fermentation broth of *Paragonimus westermani* was freeze-dried at low temperature to prepare a lyophilized powder. Maltodextrin was then added to the lyophilized powder to prepare a powder formulation with an effective bacterial concentration of 2 × 10⁻⁶. 9 CFU / g;

[0140] Competitor 1: Commercially available nitrite-reducing powder product 1;

[0141] Competitor 2: Commercially available nitrite-reducing powder products;

[0142] Competitor 3: Commercially available liquid products for reducing nitrite 1;

[0143] Competitor 4: Commercially available liquid nitrite-reducing products 2;

[0144] Different formulations of the above-mentioned *Paragonimus westermani* and competing formulations were added to nitrite culture medium I and nitrite culture medium II at a ratio of 1‰ (V / V for liquid products and W / V for solid powder products). The mixtures were incubated at 30℃ and 200rpm for 24h, and their nitrite degradation efficiency was determined.

[0145] The nitrite culture medium is as follows:

[0146] Culture medium 1: 15g glucose, 0.49g sodium nitrite, 1.5g dipotassium hydrogen phosphate, 0.7g potassium dihydrogen phosphate, 0.5g magnesium sulfate, pH 7.2±0.1, C / N ratio 60.35.

[0147] Culture medium 2: glucose 15g, sodium nitrite 0.49g, dipotassium hydrogen phosphate 1.5g, potassium dihydrogen phosphate 0.7g, magnesium sulfate 0.5g, peptone 10.0g, beef meal 3.0g, sodium chloride 5.0g, pH 7.2±0.1, C / N ratio 3.18.

[0148] Compared with competing products with the same function on the market, the *Paragonimus westermani* and its preparation provided by this invention have a better degradation effect on nitrite nitrogen in different component culture media, and the effect is more stable. Specific data are shown in Table 13. It can be seen that when the C / N ratio in the culture medium is reduced to 3.18, it still has a good degradation effect on nitrite nitrogen.

[0149] Table 13 Comparison of the effects of different formulations of Paracoccus versutus KY-T1 and commercially available nitrite-lowering products.

[0150] .

[0151] Example 8:

[0152] The effect of Paracoccus versutus KY-T1 on nitrite degradation in shrimp pond water samples

[0153] Water samples collected from shrimp ponds in Beihai City were used as the original water samples. Paracoccus dysporum KY-T1 fermentation broth was added at a ratio of 1‰ (V / V), and the samples were cultured at 30℃ and 200rpm for 120h. The degradation efficiency of KY-T1 on nitrite in shrimp pond water samples was then determined.

[0154] Table 14 shows that Paracoccus variegata has a high degradation efficiency in shrimp farming water samples at a dosage of 1‰, with a degradation rate of 92.59% at 24h and 96.30% at 48h.

[0155] Table 14. Degradation effect of *Paragonimus westermani* on nitrite nitrogen in aquaculture water samples.

[0156] .

[0157] Example 9:

[0158] Evaluation of the efficacy of Paracoccus versutus KY-T1 formulation in Datang

[0159] The fermentation broth of *Paragonimus westermani* KY-T1 (with a viable bacterial count ≥2×10⁻⁶) was transferred to a fermentation tank. 10 The product was prepared by adding 3% sodium acetate to CFU / ml of the product. Its effect on the degradation of nitrite nitrogen in aquaculture water was evaluated in aquaculture sheds. The specific procedure was as follows: 1L of the preparation containing *Paragonimus westermani* KY-T1 was mixed with 0.5-1kg of brown sugar or 1.5-3kg of molasses and 15-20L of water. After activation for 3-4 hours, the mixture was applied to the entire pond. 1L of activated product was applied to 3-4 mu (approximately 0.2-0.2 hectares) of water surface at a water depth of 1 meter. Water samples were taken before application, 3 days after application, and 5 days after application to test the concentration of nitrite nitrogen in the aquaculture water. The results are shown in Tables 15 and 16.

[0160] Paracoccus versutus KY-T1 exhibits a stable degradation effect on nitrite nitrogen in the water even under complex environmental conditions in the pond.

[0161] Table 15. Degradation effect of *Paragonimus westermani* on nitrite nitrogen in pond culture.

[0162] .

[0163] Table 16. Degradation effect of *Paragonimus westermani* on nitrite nitrogen under different water quality conditions in pond culture.

[0164] .

[0165] Example 10:

[0166] Selection of activated carbon source for the preparation of *Paragonimus westermani* in Datang

[0167] Activating microbial preparations before use in ponds aims to restore the physiological activity and reproductive capacity of bacteria from a dormant state, promote their proliferation, allow them to adapt to the aquatic environment in advance, and enhance their functions in decomposing organic matter, purifying water, and converting nitrogen. This allows them to quickly form a dominant bacterial community after being introduced into the water, improving efficacy and preparation utilization. This example clarifies the impact of different carbon source types on the activation efficiency of *Paragonimus westermani* preparations, further promoting the effectiveness of *Paragonimus westermani* and its products in practical applications.

[0168] Activation conditions: temperature 28℃; activation water: tap water aerated for 24-48 hours; activation time: samples were taken at 4 hours and 8 hours for testing.

[0169] Paracoccus variegata can be rapidly activated and grown in different carbon sources such as brown sugar, molasses, glucose, sodium acetate, and sodium citrate. The specific results are shown in Table 17.

[0170] Table 17 Activation effect of *Paragonimus westermani* on different carbon sources

[0171] .

[0172] Example 11:

[0173] Screening for the optimal fermentation medium for Paracoccus KY-T1

[0174] The culture media to be screened are as follows:

[0175] Culture medium 1 (NB medium): peptone 10.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, pH 7.2±0.1, carbon-to-nitrogen ratio (C / N) = 3.85:1

[0176] Culture medium 2 (patent CN120624203A discloses the culture medium): sucrose 20.0 g / L, peptone 10.0 g / L, yeast extract 10.0 g / L, sodium nitrate 2.0 g / L, magnesium sulfate 0.1 g / L, calcium carbonate 10.0 g / L, pH 7.2±0.1, carbon-nitrogen ratio C / N=3.41:1.

[0177] Culture medium 3: Corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 2.0 ml / L, pH 7.2 ± 0.1, carbon-nitrogen ratio C / N = 14.77:1.

[0178] Culture medium 4: Corn starch 20.0 g / L, soybean meal 20.0 g / L, glucose 5.0 g / L, peptone 3.0 g / L, yeast extract 5.0 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, manganese sulfate 0.3 g / L, potassium dihydrogen phosphate 2.0 g / L, calcium carbonate 3.0 g / L, defoamer 1.0 ml / L, pH 7.2±0.1, C / N ratio of the culture medium = 9.40:1.

[0179] Culture medium 5: maltodextrin 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, defoamer 2.0 ml / L, pH 7.2 ± 0.1, C / N ratio of the culture medium C / N = 14.77:1.

[0180] Culture medium 6: glucose 40.0 g / L, yeast extract 10.0 g / L, fish peptone 10.0 g / L, sodium acetate 5.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 2.0 ml / L, pH 7.2 ± 0.1, carbon-nitrogen ratio C / N = 12.11:1.

[0181] Culture medium 7: Corn starch 10.0 g / L, glucose 30.0 g / L, yeast extract 20.0 g / L, fish peptone 6.0 g / L, sodium acetate 5.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 2.0 ml / L, pH 7.2 ± 0.1, C / N ratio of the culture medium C / N = 11.65:1.

[0182] Culture medium 8: Corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, hydroxypropyl methylcellulose 25.0 g / L, defoamer 2.0 ml / L, pH 7.2 ± 0.1, C / N ratio of the culture medium C / N = 14.77:1.

[0183] Culture medium 9: Corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.015 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 2.0 ml / L, pH 7.2 ± 0.1, C / N ratio of the culture medium C / N = 14.77:1.

[0184] Culture medium 10: Corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.015 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, hydroxypropyl methylcellulose 42.0 g / L, defoamer 2.0 ml / L, pH 7.2 ± 0.1, C / N ratio of the culture medium C / N = 14.77:1.

[0185] Culture medium 11: maltodextrin 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.015 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 2.0 ml / L, pH 7.2 ± 0.1, C / N ratio of the culture medium C / N = 14.77:1.

[0186] Culture medium 12: maltodextrin 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.015 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, hydroxypropyl methylcellulose 42.0 g / L, defoamer 2.0 ml / L, pH 7.2 ± 0.1, C / N ratio of the culture medium C / N = 14.77:1.

[0187] Culture medium 13: maltodextrin 35.0 g / L, glucose 5.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.015 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, defoamer 2.0 ml / L, pH 7.2 ± 0.1, C / N ratio of the culture medium C / N = 14.77:1.

[0188] Culture medium 14: glucose 40.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 2.0 ml / L, pH 7.2 ± 0.1, carbon-nitrogen ratio C / N = 14.77:1.

[0189] Culture medium 15: Corn starch 10.0 g / L, glucose 30.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 2.0 ml / L, pH 7.2 ± 0.1, C / N ratio of the culture medium C / N = 14.77:1.

[0190] Culture medium 16: glucose 40.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, sodium acetate 5.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 2.0 ml / L, pH 7.2 ± 0.1, C / N ratio of the culture medium C / N = 15.72:1.

[0191] Culture medium 17: Corn starch 10.0 g / L, glucose 30.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, sodium acetate 5.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 2.0 ml / L, pH 7.2 ± 0.1, C / N ratio of the culture medium C / N = 15.72:1.

[0192] Fermentation was conducted using different culture media to investigate the effect on the viable count of *Paracoccus versutus* culture. *Paracoccus versutus* KY-T1 culture stored at -80℃ was rapidly warmed to 37.0±1.0℃ for 2–3 min and inoculated into 100 ml of basal medium (NB) at a 1:50 ratio for rejuvenation. The culture was incubated at 37.0±1.0℃ for 12–16 hours. The seed culture maturation condition was an OD600 value of 2–3 (with pure water as a blank control). The cultured seed culture was then inoculated at a 1:50 ratio into different culture media formulations and ratios and incubated at 37.0±1.0℃ at a rotation speed of 180–220 rpm for 24 hours. Fermentation cultures were diluted, spread, and counted to determine the viable count in each culture medium. The results are shown in Table 16.

[0193] Table 18 Screening results of shake flask culture media

[0194] .

[0195] The results showed that culture medium composition, carbon-nitrogen ratio, and carbon source structure all affected fermentation levels. Specific data revealed that in high-density fermentation of *Paragonimus westermani*, carbon source structure was the primary factor influencing viable cell count. The combined slow-release carbon sources, corn starch and maltodextrin, had a greater effect on improving fermentation levels than single readily available carbon sources such as glucose. The viable cell count of the readily available carbon source group, pure glucose, was significantly lower than that of the group with the added slow-release carbon source. The fermentation levels of the pure glucose carbon source groups in media 14 and 16 were 15.86 billion CFU / ml and 14.86 billion CFU / ml, respectively. Under the same carbon-nitrogen ratio and other consistent conditions, the viable cell count in the combined carbon source group was higher than that in the single glucose group. Under the same carbon-nitrogen ratio and other consistent conditions, corn starch had a better effect on improving the viable cell count of *Paragonimus westermani* than maltodextrin. The highest viable cell count in the starch group was 37.5 billion CFU / ml (culture medium 9), while the highest in the maltodextrin group was 34.5 billion CFU / ml (culture medium 13).

[0196] In the high-density batch fermentation of *Paragonimus westermani* in shake flasks, the inorganic salt composition of the culture medium determined the upper limit of the fermentation level. High phosphorus and high calcium content reduced the fermentation level, resulting in lower viable cell counts compared to the low phosphorus and low calcium group. The fermentation levels of the high phosphorus and high calcium groups in media 3 and 5 were 24.7 billion / ml and 21.56 billion / ml, respectively, while the fermentation levels of the low phosphorus and low calcium groups in media 9, 11, and 13 were 37.5 billion / ml, 26.67 billion / ml, and 34.5 billion / ml, respectively. Regarding the influence of carbon source structure and carbon-nitrogen ratio on the fermentation level, in the batch fermentation of shake flasks, the culture medium with a carbon-nitrogen ratio of 14.77:1 had a higher viable cell count than the culture medium with a carbon-nitrogen ratio of 12.11:1 (culture medium 6) and a carbon-nitrogen ratio of 11.65:1 (culture medium 7). In culture medium 7, reducing fish peptone and increasing yeast extract further increased the viable cell count, indicating that the nitrogen source composition of yeast extract was more suitable for the growth of *Paragonimus westermani* in shake flask culture.

[0197] Based on the applicant's previous research, sodium acetate showed a higher activation rate than carbon sources such as brown sugar, molasses, and glucose when activating *Paragonimus westermani* products in large-scale fermentation (Table 15). Considering whether it could improve the fermentation level of *Paragonimus westermani* in high-density fermentation, 5‰ sodium acetate was added to media 16 and 17. The fermentation culture counting results showed that sodium acetate, as a carbon source, could slightly improve the fermentation level of *Paragonimus westermani* in shake-flask high-density fermentation. The viable cell counts in media 14 and 15 without sodium acetate were 14.86 billion / ml and 16.57 billion / ml, respectively, while the viable cell counts in fermentation broths of media 16 and 17 with sodium acetate were 15.36 billion / ml and 17.5 billion / ml, respectively.

[0198] According to existing reports, adding some stability protectants during microbial fermentation can extend the shelf life of fermentation products. Considering the future promotion and use of *Paragonimus westermani*, a certain proportion of hydroxymethylpropyl cellulose (HPMC) was added during culture medium screening to test its effect on fermentation level. In culture medium 8, 2.5% HPMC was added to culture medium 3, and the fermentation level decreased from 24.7 billion / ml to 14.6 billion / ml. In culture media 10 and 12, 4.2% HPMC was added to culture media 9 and 11, respectively, and the fermentation levels decreased from 29.33 billion / ml and 26.67 billion / ml to 2.37 billion / ml and 7.97 billion / ml, respectively.

[0199] Example 11:

[0200] Example: Application of culture medium: Fermentation experiment in a fermenter

[0201] A 30L fermenter was used for the scale-up fermentation of *Paragonimus westermani*. Experiments were conducted with different inoculum ratios and fermentation parameters. The viable cell count after fermentation was determined using the plate dilution method. Specific fermentation conditions are as follows:

[0202] 1. Primary seed preparation: Take the glycerol tubes of *Paragonimus westermani* KY-T1 working batch from the ultra-low temperature freezer, rapidly warm them to 37.0±1.0℃ for 2-3 minutes, and inoculate 1 ml of the inoculum into a sterile Erlenmeyer flask containing 50 mL of culture medium near an alcohol lamp flame in a clean bench. Incubate at 37.0±1.0℃ for 12-16 hours. Seed culture maturation conditions: OD600 value 2-3 (OD value with pure water as a blank control).

[0203] 2. Secondary seed preparation: Under aseptic conditions, inoculate 50 mL of the qualified seed solution into a sterilized 300 mL culture medium at an inoculation rate of 5% (1:20), and incubate at 37.0±1.0℃ for 6-8 hours. Maturation conditions for 300 mL seed solution: OD600 value 2-3.

[0204] 3. Fermentation test in a fermenter

[0205] Fermentation Experiment 1: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at a natural pH during the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.5, OD value remained unchanged for 3 consecutive hours, fermentation time: 20 hours. The fermentation medium consisted of: corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, sodium acetate 1.0 g / L, and defoamer 2.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 16.71:1.

[0206] Fermentation Experiment 2: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, with natural pH throughout the process; stirring: initial 150, increasing or decreasing by 50 each time during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, increasing or decreasing by 0.5 each time during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 21 hours. The fermentation medium consisted of: corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, and antifoaming agent 2.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 14.77:1.

[0207] Fermentation Experiment 3: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at natural pH during the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0. Fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 22 hours. The fermentation medium consisted of: corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, and antifoaming agent 2.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 14.77:1.

[0208] Fermentation Experiment 4: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at natural pH during the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0. Fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 23 hours. The fermentation medium consisted of: corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, and antifoaming agent 2.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 14.77:1.

[0209] Fermentation Experiment 5: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowing for natural pH during the process; stirring: initial 150, increasing or decreasing by 50 each time during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, increasing or decreasing by 0.5 each time during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 22 hours. The fermentation medium consisted of: corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.015 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, and defoamer 2.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 14.77:1.

[0210] Fermentation Experiment 6: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at a natural pH during the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 17 hours. The fermentation medium consisted of: corn starch 35.0 g / L, glucose 5.0 g / L, yeast extract 5.0 g / L, fish peptone 8.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, sodium acetate 1.0 g / L, and defoamer 2.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 16.71:1.

[0211] Fermentation Experiment 7: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at natural pH during the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 22 hours. The fermentation medium consisted of: corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.015 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 0.1 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 1.5 ml / L, pH 7.2 ± 0.1, and a carbon-to-nitrogen ratio (C / N) of 14.77:1.

[0212] Fermentation Experiment 8: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at a natural pH during the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 22 hours. The fermentation medium consisted of: maltodextrin 35.0 g / L, glucose 5.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.015 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 0.1 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, and defoamer 2.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 14.77:1.

[0213] Fermentation Experiment 9: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at a natural pH throughout the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0. Fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 25 hours. The fermentation medium consisted of: maltodextrin 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.015 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 0.1 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, and defoamer 2.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 14.77:1.

[0214] Fermentation Experiment 10: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at a natural pH during the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 25 hours. The fermentation medium consisted of: maltodextrin 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.015 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 0.1 g / L, manganese sulfate 0.3 g / L, ferrous sulfate 0.01 g / L, HPMC 25.0 g / L, and antifoaming agent 2.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 14.77:1.

[0215] Fermentation Experiment 11: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at a natural pH during the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 25 hours. The fermentation medium consisted of: corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 1.5 g / L, magnesium sulfate 3.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, and antifoaming agent 1.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 14.77:1.

[0216] Fermentation Experiment Twelve: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at natural pH during the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 25 hours. The fermentation medium consisted of: corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.15 g / L, magnesium sulfate 0.3 g / L, dipotassium hydrogen phosphate 0.2 g / L, and antifoaming agent 2.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 14.77:1.

[0217] Fermentation Experiment Thirteen: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowing for natural pH during the process; stirring: initial 150, increasing or decreasing by 50 each time during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, increasing or decreasing by 0.5 each time during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 24 hours. The fermentation medium consisted of: corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, magnesium sulfate 0.3 g / L, dipotassium hydrogen phosphate 2.0 g / L, ferrous sulfate 0.01 g / L, and antifoaming agent 2.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 14.77:1.

[0218] Fermentation Experiment Fourteen: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowing for natural pH during the process; stirring: initial 150, increasing or decreasing by 50 each time during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, increasing or decreasing by 0.5 each time during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 22 hours. The fermentation medium consisted of: corn starch 20.0 g / L, glucose 20.0 g / L, yeast extract 5.0 g / L, fish peptone 10.0 g / L, calcium chloride 0.02 g / L, magnesium sulfate 0.75 g / L, dipotassium hydrogen phosphate 3.0 g / L, potassium dihydrogen phosphate 1.5 g / L, manganese sulfate 0.01 g / L, ferrous sulfate 0.01 g / L, and defoamer 1.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 14.77:1.

[0219] Fermentation Experiment 15: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at natural pH during the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 24 hours. The fermentation medium consisted of: corn starch 10.0 g / L, glucose 25.0 g / L, yeast extract 8.0 g / L, fish peptone 15.0 g / L, calcium chloride 0.02 g / L, magnesium sulfate 0.75 g / L, dipotassium hydrogen phosphate 3.0 g / L, potassium dihydrogen phosphate 1.5 g / L, manganese sulfate 0.01 g / L, ferrous sulfate 0.01 g / L, and defoamer 1.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 10.36:1.

[0220] Fermentation Experiment 16: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 3.0. OD value growth slowed or stopped for 3 consecutive hours, fermentation time: 30 hours. The fermentation medium consisted of: corn starch 10.0 g / L, glucose 10.0 g / L, yeast extract 10.0 g / L, fish peptone 20.0 g / L, calcium chloride 0.02 g / L, magnesium sulfate 1.0 g / L, dipotassium hydrogen phosphate 4.0 g / L, potassium dihydrogen phosphate 2.0 g / L, manganese sulfate 0.01 g / L, ferrous sulfate 0.01 g / L, defoamer 2.0 ml / L, and pH 7.2 ± 0.1.

[0221] Fed-feed fermentation: Prepare 1L of 500g / L glucose solution as solution A, and 1L of peptone (100g / L), yeast extract (100g / L), and magnesium sulfate (5g / L) as solution B. Sterilize each solution at 121.0℃ for 15 minutes, allow to cool naturally, and then mix thoroughly to use as the fed-feed solution.

[0222] Timing for refeeding: When dissolved oxygen first rises above 60 or residual sugar is <2g / L.

[0223] Feeding strategy: By increasing the rotation speed (up to 400 rpm) and ventilation rate (up to 3.0 rpm), using exponential flow feeding, dissolved oxygen is controlled at 20%-40%.

[0224] During the feeding period, the pH should be controlled at 7.0-7.2 using a 50% phosphoric acid and 40% sodium hydroxide solution;

[0225] 50% Phosphoric Acid Solution: Prepare a 50% phosphoric acid solution, sterilize at 121.0℃ for 15 minutes, and allow to cool naturally before use.

[0226] 40% sodium hydroxide solution: Weigh an appropriate amount of NaOH and slowly pour it into a beaker containing an appropriate amount of purified water. Dissolve it completely to prepare a 40% NaOH solution for later use.

[0227] Stop feeding when fermentation is complete.

[0228] Fermentation Experiment 17: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 3.0. OD value growth slowed or stopped for 3 consecutive hours, fermentation time: 28 hours. The fermentation medium consisted of: corn starch 10.0 g / L, glucose 5.0 g / L, yeast extract 10.0 g / L, fish peptone 20.0 g / L, calcium chloride 0.02 g / L, magnesium sulfate 1.0 g / L, dipotassium hydrogen phosphate 4.0 g / L, potassium dihydrogen phosphate 2.0 g / L, manganese sulfate 0.01 g / L, ferrous sulfate 0.01 g / L, defoamer 2.0 ml / L, and pH 7.2 ± 0.1.

[0229] Fed-feed fermentation: Prepare 1L of 500g / L glucose solution as solution A, and 1L of peptone (100g / L), yeast extract (100g / L), and magnesium sulfate (5g / L) as solution B. Sterilize each solution at 121.0℃ for 15 minutes, allow to cool naturally, and then mix thoroughly to use as the fed-feed solution.

[0230] Timing for refeeding: When dissolved oxygen first rises above 60 or residual sugar is <2g / L.

[0231] Feeding strategy: By increasing the rotation speed (up to 400 rpm) and ventilation rate (up to 3.0 rpm), using exponential flow feeding, dissolved oxygen is controlled at 20%-40%.

[0232] During the feeding period, the pH should be controlled at 7.0-7.2 using a 50% phosphoric acid and 40% sodium hydroxide solution;

[0233] 50% Phosphoric Acid Solution: Prepare a 50% phosphoric acid solution, sterilize at 121.0℃ for 15 minutes, and allow to cool naturally before use.

[0234] 40% sodium hydroxide solution: Weigh an appropriate amount of NaOH and slowly pour it into a beaker containing an appropriate amount of purified water. Dissolve it completely to prepare a 40% NaOH solution for later use.

[0235] Stop feeding when fermentation is complete.

[0236] Fermentation Experiment 18: Secondary seed culture was inoculated into a 30L fermenter at a bacterial culture to culture medium ratio of 1:50, filling the tank to 50% capacity. Tank temperature: 35±0.5℃; tank pressure: 0.05Mpa; pH: adjusted to 7.2 before sterilization, allowed to remain at a natural pH during the process; stirring: initial 150, gradually increasing or decreasing by 50 during the process and aeration, with a maximum stirring speed of 400. Initial aeration: 0.5, gradually increasing or decreasing by 0.5 during the process and stirring, with a maximum aeration of 2.0; fermentation endpoint: dissolved oxygen recovered, pH stabilized between 6.5 and 8.0, OD value remained unchanged for 3 consecutive hours, fermentation time: 24 hours. The fermentation medium consisted of: corn starch 20.0 g / L, soybean meal 20.0 g / L, glucose 5.0 g / L, peptone 3.0 g / L, yeast extract 5.0 g / L, magnesium sulfate 3.0 g / L, sodium chloride 2.0 g / L, manganese sulfate 0.3 g / L, potassium dihydrogen phosphate 2.0 g / L, calcium carbonate 3.0 g / L, and defoamer 1.0 ml / L. The pH was 7.2 ± 0.1, and the carbon-to-nitrogen ratio (C / N) was 9.40:1.

[0237] Table 19 Fermentation Levels in Fermentation Tanks

[0238] .

[0239] Similar to the results of shake-flask fermentation, under batch-to-batch non-feeding conditions in fermenters, slow-release carbon sources such as maltodextrin and corn starch are more conducive to the high-density fermentation of *Paragonimus westermani* than fast-acting carbon sources such as glucose. Under the condition of consistent trace element content, the fermentation level of the composite carbon source containing corn starch was higher than that containing maltodextrin. Fermentation experiment 5 used corn starch and the viable cell count was 42 billion CFU / ml. Fermentation experiments 8 and 9 used different concentrations of maltodextrin and the viable cell counts were 36 billion CFU / ml and 34.33 billion CFU / ml, respectively. Adding HPMC to the fermenter also significantly reduced the viable cell count. Fermentation experiment 10 added 2.5% HPMC to the culture medium of fermentation experiment 9, and the viable cell count decreased to 24.16 billion CFU / ml. Contrary to the results of shake-flask fermentation, in fermenters, under the same conditions of other components and contents, the viable cell count in the high-concentration calcium chloride fermentation broth was higher than that in the low-concentration calcium chloride medium. In fermentation experiments two, three, and four, with calcium chloride at 1.5 g / L, the viable cell counts in the fermentation broth were 44.67 billion CFU / ml, 51.5 billion CFU / ml, and 45.33 billion CFU / ml, respectively. In fermentation experiment five, with calcium chloride at 0.015 g / L, the viable cell count was 42 billion CFU / ml. In the high-density fermentation process in fermenters, adding sodium acetate to the medium can increase the viable cell count in the fermentation broth. In fermentation experiment one, with 1 g / L of sodium acetate added, the viable cell count was 57 billion CFU / ml, a higher fermentation level than in fermentation experiments two, three, and four without sodium acetate. In fermentation experiment six, further increasing the corn starch content and decreasing the fish peptone content increased the viable cell count in the fermentation broth to 62.3 billion CFU / ml.

[0240] The above results show that in high-density fermentation in fermenters, the viable bacteria content of fed-feed fermentation is significantly higher than that of non-fed fermentation. In this invention, the highest viable bacteria content in non-fed fermentation is 62.3 billion CFU / ml (Fermentation Experiment 6), while the highest viable bacteria content in fed fermentation can reach 122.3 billion CFU / ml (Fermentation Experiment 17).

[0241] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An isolated strain of Paracoccus versutus KY-T1, the preservation number of Paracoccus versutus KY-T1 being CCTCC NO: M2026770.

2. The *Paragonimella hyopneumoniae* KY-T1 according to claim 1, characterized in that, The described paracoccus KY-T1 contains the specific sequence shown in SEQ ID NO.

2.

3. A compound comprising Paracoccus KY-T1 as described in claim 1.

4. The application of the *Paragonimus westermani* KY-T1 as described in claim 1 or the compound as described in claim 2 in the preparation of water quality conditioning and improving agents.

5. The application of the *Paragonimus westermani* KY-T1 as described in claim 1 or the compound as described in claim 2 in water quality conditioning.

6. The application according to claim 4 or 5, wherein the water quality adjustment is to reduce the content of ammonia nitrogen and / or nitrite in the water.

7. In the application according to claim 4 or 5, the Paracoccus KY-T1 or its compound is pre-activated using a carbon source during application, wherein the carbon source is brown sugar, molasses, glucose, sodium acetate and / or sodium citrate.

8. A high-density large-scale fermentation method applicable to *Paragonimococcus KY-T1* as described in claim 1, comprising inoculating *Paragonimococcus KY-T1* into a fermentation medium at an inoculum size of 1% to 10%, wherein the fermentation medium has the following formulation: corn starch 10.0 g / L, glucose 5-10.0 g / L, yeast extract 10.0 g / L, fish peptone 20.0 g / L, calcium chloride 0.02 g / L, magnesium sulfate 1.0 g / L, dipotassium hydrogen phosphate 4.0 g / L, potassium dihydrogen phosphate 2.0 g / L, manganese sulfate 0.01 g / L, ferrous sulfate 0.01 g / L, antifoaming agent 2.0 ml / L, pH 7.2 ± 0.1, with the remainder being water, wherein the preservation number of *Paragonimococcus KY-T1* is: CCTCC NO: M2026770.

9. The method according to claim 8, wherein the large-scale fermentation is fed-batch fermentation, and feeding is carried out when the dissolved oxygen first rises to above 60 or the residual sugar is <2g / L. During the feeding process, the dissolved oxygen is controlled at 20%-40% and the pH is 7.0-7.

2. The feeding is prepared by mixing 1L of solution A and 1L of solution B. Solution A is a 500g / L glucose solution, and solution B is a mixture of 100g / L peptone, 100g / L yeast extract, and 5g / L magnesium sulfate.

10. The application of the fermentation method according to claim 8 in the fermentation production of Paracoccus KY-T1.

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