Separation, culture and freeze-drying method of parabacteroides dielsii

Abstract

The invention discloses a method for separating, culturing and freeze-drying parabacteroides dieldrii. The separation method of the parabacteroides dielsii comprises the following steps: S1, collecting a faeces sample of a healthy child to prepare a faeces suspension; s2, diluting the excrement suspension, coating the diluted excrement suspension in a culture medium containing antibiotics, and culturing under an anaerobic condition; s3, selecting single colonies with consistent forms, and performing pure culture to obtain single colonies; carrying out MALDI-TOF MS (Matrix-Assisted Laser Desorption / Ionization And then, through bidirectional Sanger sequencing, the strain is confirmed to be parabacteroides dieldrii. The invention provides full-process technical parameters related to low-oxygen exposure control, selective culture, separation and purification, genome identification, multiplication culture and cryopreservation or freeze-drying, and a parabacteroides dieldrii strain resource which has stable activity and phenotype after recovery and is suitable for culture amplification and preparation preservation can be obtained. The method can be used for basic research, large-scale culture and related product development of parabacteroides dieldrii.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for isolating, culturing and freeze-drying *Pseudomonas difficile*. Background Technology

[0002] *Pseudomonas distasonis* is an important commensal bacterium in the human gut, possessing polysaccharide utilization sites (PULs) and short-chain fatty acid metabolic potential. Recent studies have shown that *Pseudomonas distasonis* has potential probiotic functions in maintaining gut microbiota balance, regulating host metabolism, and related aspects of immunity and neurological function.

[0003] However, the isolation, purification, and preservation of *P. distasonis* remain challenging. During the isolation stage, *P. distasonis* often faces competition from other members of the same genus, such as *Parabacteroides*, as well as dominant anaerobic bacteria like *Bacteroides* and *Prevotella*, resulting in low isolation efficiency. After obtaining pure cultures, the strains are prone to phenotypic degeneration or decreased activity during multiple passages.

[0004] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for isolating, culturing and freeze-drying *Pseudomonas dilataniae*, in order to solve the problem of low isolation efficiency of *Pseudomonas dilataniae* in the prior art.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for isolating *Pseudomonas dignitaria*, comprising the following steps: S1. Collect fecal samples from healthy children and prepare a fecal suspension; S2. After dilution, the fecal suspension is spread on a culture medium containing antibiotics and cultured under anaerobic conditions. S3. Select single colonies with consistent morphology and obtain single colonies through pure culture; identify the single colonies by MALDI-TOFMS; and then confirm the strain as *Pseudomonas dignitaria* by bidirectional Sanger sequencing. The antibiotic-containing culture medium comprises: adding 0.1-1 g / L cysteine, 0.2-5 mg / L vitamin K1, 1-10 mg / L heme and 0.01-0.1% L-cysteine ​​to the basal culture medium.

[0007] Optionally, the antibiotic-containing culture medium comprises: adding 0.5 g / L cysteine, 1 mg / L vitamin K1, 5 mg / L heme and 0.05% L-cysteine ​​to the basal culture medium; the basal culture medium is mGAM.

[0008] Optionally, the culture medium containing antibiotics may further include: 1-3 μg / mL vancomycin and 10-100 μg / mL kanamycin.

[0009] Optionally, steps S1-S3 are all carried out under anaerobic conditions, wherein the anaerobic conditions are N2:CO2:H2=80:10:10.

[0010] Optionally, the healthy child is a 6-year-old healthy child who has not used antibiotics or other drugs and has no diseases in the 3 months prior to collection.

[0011] In a second aspect, the present invention provides a strain of *Pseudomonas dignitaria* obtained by the isolation method described above.

[0012] In a third aspect, the present invention provides a method for amplifying and culturing *Pseudomonas dignitaria*, using a culture medium containing 0.2-5 mg / L vitamin K1 and 1-10 mg / L heme, and culturing under anaerobic conditions; The *Pseudomonas dignitaria* was obtained using the isolation method described above.

[0013] Optionally, mGAM medium containing 1 mg / L vitamin K1 and 5 mg / L heme can be used.

[0014] In a fourth aspect, the present invention provides a method for cryopreserving *Pseudomonas difficile*, wherein the lyophilization protectant used in the cryopreservation method comprises 1-10% sucrose, 5-20% skim milk powder, and 0.01-0.1% L-cysteine ​​hydrochloride. The *Pseudomonas dignitaria* was obtained using the isolation method described above.

[0015] Optionally, the freeze-drying protectant used in the freezing method includes 5% sucrose, 10% skim milk powder, 0.05% calcium chloride, 0.05% magnesium chloride, and 0.01% L-cysteine ​​hydrochloride.

[0016] Beneficial Effects: This invention provides a method for the isolation, cultivation, and freeze-drying of *Pseudomonas diffusa*. Compared with existing technologies, the advantages of this invention are: Under anaerobic conditions, combined with a culture medium containing cysteine, L-cysteine, vitamin K1, and heme, and by using antibiotics to create selective pressure, a more suitable growth environment for *Pseudomonas diffusa* can be provided under mixed bacterial flora conditions. Based on this, *Pseudomonas diffusa* can be obtained by dilution plating, colony picking, purification and subculturing, and identification using MALDI-TOF and 16S rRNA sequencing. Furthermore, genomic analysis of the isolated *Pseudomonas diffusa* revealed that it carries multiple genes related to amino acid metabolism, neurotransmitter precursor synthesis, and vitamin synthesis, while no major drug resistance genes or virulence factor-related genes were detected. Attached Figure Description

[0017] Figure 1 The growth curves of *Pseudomonas dignitaria* under different culture conditions are shown.

[0018] Figure 2 The growth curves of *Pseudomonas dignitaria* strains before and after freeze-drying are shown. Detailed Implementation

[0019] This invention provides a method for isolating, culturing, and freeze-drying *Pseudomonas difficile*. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0020] An embodiment of the present invention provides a method for isolating *Pseudomonas dignitaria*, comprising the following steps: S1. Collect fecal samples from healthy children and prepare a fecal suspension; S2. After dilution, the fecal suspension is spread on a culture medium containing antibiotics and cultured under anaerobic conditions. S3. Select single colonies with consistent morphology and obtain single colonies through pure culture; identify the single colonies by MALDI-TOFMS; and then confirm the strain as *Pseudomonas dignitaria* by bidirectional Sanger sequencing. The antibiotic-containing culture medium comprises: adding 0.1-1 g / L cysteine, 0.2-5 mg / L vitamin K1, 1-10 mg / L heme and 0.01-0.1% L-cysteine ​​to the basal culture medium.

[0021] This invention provides a low redox environment and necessary metabolic cofactors for *Pseudomonas dignitaries* growth by adding cysteine, L-cysteine, vitamin K1, and heme to the culture medium. Combined with an antibiotic-containing medium, this creates selective pressure within the mixed bacterial community, helping to maintain optimal growth conditions for *Pseudomonas dignitaries* during the isolation phase. This reduces competition from other *Pseudomonas*, *Bacteroides*, or *Prevotella* species under mixed bacterial conditions. Furthermore, the combined use of morphological selection, MALDI-TOF mass spectrometry, and Sanger sequencing improves the accuracy of strain identification and ensures the successful identification of *Pseudomonas dignitaries*.

[0022] In some embodiments, the antibiotic-containing culture medium comprises: adding 0.5 g / L cysteine, 1 mg / L vitamin K1, 5 mg / L heme and 0.05% L-cysteine ​​to a basal culture medium; the basal culture medium is mGAM.

[0023] In this embodiment, based on the specific concentrations of each component of the culture medium, mGAM is used as the basal medium. This provides a more stable anaerobic nutrient environment for *Pseudomonas diglossi*, making the culture conditions more suitable for its growth requirements.

[0024] In some embodiments, the culture medium containing antibiotics further includes: 1-3 μg / mL vancomycin and 10-100 μg / mL kanamycin.

[0025] In this embodiment, the addition of vancomycin and kanamycin can inhibit some coexisting Gram-positive bacteria and Enterobacteriaceae strains, thereby reducing competitive interference from non-target bacteria.

[0026] In some embodiments, steps S1-S3 are all carried out under anaerobic conditions, wherein the anaerobic conditions are N2:CO2:H2=80:10:10.

[0027] In this embodiment, by maintaining an anaerobic environment of N2:CO2:H2 = 80:10:10 throughout steps S1-S3, the impact of oxygen exposure on the survival and growth of *Pseudomonas difficile* can be reduced, which helps to establish more suitable conditions for its separation and purification.

[0028] In some embodiments, the healthy child is a 6-year-old healthy child who has not used antibiotics or other drugs and has no diseases in the 3 months prior to collection.

[0029] This invention provides a strain of *Pseudomonas dilataniae* obtained using the isolation method described above.

[0030] This invention provides a method for amplifying and culturing *Pseudomonas dilataniae*, using a culture medium containing 0.2-5 mg / L vitamin K1 and 1-10 mg / L heme, and culturing under anaerobic conditions. The *Pseudomonas dignitaria* was obtained using the isolation method described above.

[0031] Because *P. distasonis* is a strict anaerobic bacterium, it is susceptible to oxidative damage and osmotic stress during freeze-drying and thawing, leading to cell membrane rupture, significantly reduced bacterial activity, and difficulty in ensuring thawing rate and freeze-drying stability. However, using a culture medium containing vitamin K1 and heme can provide *P. distasonis* with the cofactors required for its energy metabolism, enabling the strain to maintain good growth performance under anaerobic conditions. Figure 1 The growth curves shown indicate that the growth rate and final biomass of the strain in this type of culture medium are higher than those in the control conditions. This suggests that the culture medium containing vitamin K1 and heme can provide favorable conditions for the amplification and culture of *Pseudomonas dignitaria*.

[0032] In some embodiments, mGAM medium containing 1 mg / L vitamin K1 and 5 mg / L heme is used.

[0033] This invention provides a method for cryopreserving *Pseudomonas dilatatus*, wherein the lyophilization protectant used in the cryopreservation method comprises 1-10% sucrose, 5-20% skim milk powder, and 0.01-0.1% L-cysteine ​​hydrochloride. The *Pseudomonas dignitaria* was obtained using the isolation method described above.

[0034] A freeze-drying protectant composed primarily of sucrose, skim milk powder, and L-cysteine ​​hydrochloride provides osmotic pressure buffering and oxidative protection for the bacterial cells during freeze-drying and thawing. The number of colony-forming units changes little before and after freeze-drying; therefore, this protectant combination can reduce the loss of bacterial activity caused by freeze-drying.

[0035] In some embodiments, the freeze-drying protectant components used in the cryopreservation method include 5% sucrose, 10% skim milk powder, 0.05% calcium chloride, 0.05% magnesium chloride, and 0.01% L-cysteine ​​hydrochloride.

[0036] The present invention will be further described below through specific embodiments.

[0037] Example 1 This embodiment provides a method for isolating bacterial strains, as detailed below: 1. Sample collection and preprocessing In this embodiment, a healthy 6-year-old child was selected as the donor. The donor selection criteria met the "Chinese Expert Consensus on the Clinical Application and Management of Intestinal Microbiota Transplantation (2022 Edition)". The donor had not used antibiotics or other drugs in the three months prior to collection and had no diseases. Stool samples were collected from the child.

[0038] Samples were collected in an anaerobic preservative solution containing a reducing agent (0.1% cysteine, pH 7.0), and then transferred to an anaerobic chamber within 1 hour on an ice pack. Sterile PBS (containing 0.05% L-cysteine) was added to the anaerobic chamber, and large particles were removed by passing the sample through a 100 µm filter. This yielded a fecal suspension.

[0039] 2. Selective isolation and culture An anaerobic formulation of the basal medium (pH 7.2±0.1) was used: Under anaerobic conditions, the following components were added to the basal medium mGAM (Modified Gifu Anaerobic Medium): cysteine ​​0.5 g / L, vitamin K1 1 mg / L, heme 5 mg / L; antioxidant system: 0.05% L-cysteine. After thorough mixing, the mixture was dispensed and sterilized.

[0040] After the culture medium has been sterilized and cooled to 50°C, add antibiotics (low-dose vancomycin 2 μg / mL + kanamycin 50 μg / mL) (to inhibit Gram-positive and some Enterobacteriaceae, while retaining anaerobic Parabacterium; adjustments can be made based on initial screening results). Pour the culture medium onto plates under anaerobic conditions and allow it to solidify. The anaerobic environment is N2:CO2:H2 = 80:10:10. The above fecal suspension was diluted with 10 -4 -10 -7 Perform 10-fold serial dilutions. In an anaerobic chamber, take 100 μl of the diluted solution and spread it evenly on mGAM plates containing antibiotics. Incubate at 37°C for 72 hours; the anaerobic environment is N2:CO2:H2 = 80:10:10. After incubation, select colonies that meet the typical characteristics of *Parabacterium parabens*: grayish-white / beige, with neat edges and a slightly smooth, moist surface.

[0041] All of the above procedures were performed in an anaerobic chamber.

[0042] 3. Purification and preliminary identification Select single colonies with consistent morphology, purify and passage them on plates until they are morphologically consistent and free of contaminants.

[0043] After obtaining single colonies through pure culture, MALDI-TOF MS identification was performed. A small amount of bacterial cells was spotted onto a disposable stainless steel target plate, each spot approximately 1-2 mm in diameter. Formic acid / acetonitrile protein extraction was performed first (approximately 70% formic acid and acetonitrile in equal volumes, centrifuged and the supernatant collected) before spotting. At least two technical replicates were performed for each sample. After the spots were air-dried, 1 μL of saturated α-cyano-4-hydroxycinnamic acid (HCCA) matrix solution (prepared in 50% acetonitrile / 2.5% trifluoroacetic acid) was added, and the plates were dried at room temperature to crystallize. The instrument was used in standard positive ion linear mode, with an accelerating voltage of 20 kV and a scan range of 1-20 kDa to acquire spectra. External standard calibration and quality control were performed using E. coli ATCC 8739 extract or equivalent standards before acquisition. The original spectra were baseline-corrected, signal-smoothed, and peak-extracted using software to generate a master mass spectrum (MSP), which was then matched for similarity with a validated reference library. Scores were output based on the manufacturer's algorithm, and genus / species identification was determined according to thresholds (e.g., a score ≥2.0 was considered a reliable species, and 1.7–1.99 a genus). Negative / low-quality spectra (few peaks, S / N <3) were respotted or redeployed; suspicious junctions were confirmed by morphological and molecular sequencing (16S).

[0044] Genomic DNA was extracted from pure culture single colonies and used as a template for 16S rRNA gene amplification. PCR amplification was performed using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) (using a 25 μL system as an example: 1× high-fidelity buffer, 1.5 mM Mg). 2+ The following reagents were used: 200 μM dNTPs, 0.2 μM forward and reverse primers, 1 U high-fidelity DNA polymerase, and 10 ng template DNA. Cycling conditions were: 95°C pre-denaturation for 2 min; 30 cycles: 95°C for 30 s, 58°C annealing for 30 s, 72°C extension for 90 s; and 72°C final extension for 5 min. Electrophoresis confirmed an approximately 1.5 kb amplified fragment, which was then purified by gel electrophoresis and subjected to bidirectional Sanger sequencing. The forward and reverse sequences were subjected to quality shearing, error correction, and splicing to obtain a high-quality consensus sequence. Sequence similarity and coverage were calculated using the NCBI database. A sequence similarity of ≥99.0% and sufficient coverage (typically ≥95%) confirmed the gene as *P. distasonis*.

[0045] High-quality genomic DNA (OD260 / 280≈1.8-2.0) was extracted from pure culture single colonies after amplification and culture. Illumina short-read sequencing libraries (e.g., Nextera / TruSeq, 350-500 bp inserts) were constructed, and paired-end sequencing (PE150) was performed on the Illumina platform to obtain raw data with ≥100× genome coverage. Quality control and adapter removal were performed using fastp / Trimmomatic to filter out low-quality and host / contamination reads. De novo assembly was performed using SPAdes / MEGAHIT, and N50, total length, and GC content were assessed using QUAST. Integrity and contamination were assessed using CheckM or BUSCO, retaining assemblies with ≥95% integrity and ≤5% contamination. Gene annotation was performed using Prokka / PGAP; initial similarity screening was conducted using Mash / Sourmash, and then average nucleotide similarity (ANI) and genome coverage were calculated using pyani / fastANI and reference type strains (including P. distasonis and closely related species such as P. goldsteinii, P. merdae, P. johnsonii, etc.). Species determination was based on ANI ≥ 95% and sufficient coverage, with ANI ≥ 95% of the reference P. distasonis, while closely related species such as Parabacteroides goldsteinii were excluded.

[0046] Example 2 This embodiment performs virulence gene screening and functional gene mining on the strains isolated by the method provided in Example 1, as detailed below: Using the method shown in Example 1, 20 anaerobic bacteria were isolated from feces with high abundance of P. distasonis. The initial screening by MALDI-TOF pointed to 5 strains of P. distasonis. One strain was selected for whole genome sequencing, and the ANI of the type strain ATCC8503 (accession number GCF_000012845.1) reached 97.69%.

[0047] Whole-genome sequencing results showed no plasmids detected using PlasmidFinder (v2.1.6). PHASTEST did not identify any prophage regions. The genome is rich in genes related to aromatic amino acid transport and metabolism, as well as genes involved in the synthesis of vitamins B12 and B3. Results are shown in Table 1.

[0048] Table 1

[0049] Example 3 This embodiment involves the expansion culture of the strain isolated by the method provided in Example 1, as detailed below: The strain (P. distasonis) isolated by the method shown in Example 1 was selected and anaerobically cultured at 37°C in pre-medium until the logarithmic phase, with OD600≈0.6. Equal volumes were then aliquoted and inoculated into anaerobic liquid basal medium mGAM (GAM + Vitamin K1 1 mg / L + Heme 5 mg / L + Cysteine ​​0.5 g / L), with an initial inoculum size of OD600=0.05 (or 1×10⁻⁵). 6 CFU / mL), with n=3 replicates for each group. Cultured anaerobicly at 37℃ in an anaerobic workstation (N2:CO2:H2=80:10:10), with no shaking or gentle shaking at 100 rpm. OD600 was measured every 1 hour and recorded for 48 hours.

[0050] The results are as follows Figure 1 As shown in the figure, within the first 8 hours, the mGAM group experienced a rapid growth phase, with OD600 increasing from 0.05 to approximately 1.0-1.2; GAM and BHI increased to approximately 0.15 and 0.20, respectively, with significantly lower growth rates than mGAM. Between 24 and 36 hours, mGAM reached a plateau, with a peak OD600 of approximately 1.0-1.2. In contrast, BHI and GAM plateaued at approximately 0.30 and 0.25, respectively. This indicates that mGAM supplemented with vitamin K1, heme, and cysteine ​​significantly enhances the growth rate and terminal biomass of *P. distasonis*, making it a suitable and preferred base formulation for large-scale cultivation.

[0051] Example 4 This embodiment uses the strain isolated by the method provided in Example 1 as an example. The strain is freeze-dried, and the freeze-dried strain is then revived and its performance is tested, as follows: 1. Lyophilization treatment of the strain (1) Preparation of freeze-drying protectant Protection System A (General Protective Agent): 10% trehalose, 5% sucrose, physiological saline; Protective System B: 5% sucrose, 10% skim milk powder, 0.05% calcium chloride, 0.05% magnesium chloride, 0.01% L-cysteine ​​hydrochloride; (2) Bacterial resuscitation culture After reviving the strain (P. distasonis) isolated by the method provided in Example 1 in an anaerobic chamber, single colonies were picked and transferred to GAM liquid medium and anaerobically cultured at 37°C until the logarithmic growth phase (OD600 = 1.0-1.2). The bacterial suspension was sealed and transferred to a centrifuge, centrifuged at 8000 rpm for 5 minutes at 4°C. The supernatant was removed in the anaerobic chamber, and the precipitate was resuspended with a lyophilization protectant. The resuspended bacterial suspension (fresh bacterial suspension) was diluted and plated, and counted after anaerobic incubation for 48 hours.

[0052] (3) Freeze-drying treatment A single freeze-drying process was adopted, and the specific steps were as follows: the resuspended bacterial solution was sealed and frozen at -80℃. The next day, the frozen bacterial solution was freeze-dried for 24 hours to obtain freeze-dried powder, which was then dispensed into 5mL glass bottles and stored at 4℃.

[0053] (4) Counting of coating plates Take one bottle of lyophilized powder, add PBS to dissolve and dilute it in an anaerobic chamber, spread it on a plate, and count the samples after 48 hours of incubation.

[0054] (5) Results As shown in Tables 2 and 3, using protection system A, the bacterial count of *P. distasonis* decreased by 2-3 orders of magnitude after freeze-drying compared to before freeze-drying. Using protection system B, the bacterial count of *P. distasonis* decreased by 1-2 orders of magnitude after freeze-drying compared to before freeze-drying. This indicates that protection system B is more effective than protection system A.

[0055] Table 2 Experimental results of protection system A

[0056] Table 3 Experimental results of protection system B

[0057] 2. Recovery and Functional Backtesting The strains that had been freeze-dried using protection system B were revived, anaerobically cultured in mGAM liquid medium at 37°C, and their growth curves were measured. The results are as follows: Figure 2 As shown, the growth curve of the revived strain deviated by no more than 20% from that of the mother strain, indicating that freeze-drying treatment did not significantly affect the growth capacity of the strain. Single colonies of the revived strain and the mother strain were further collected, genomic DNA was extracted, and whole-genome SNP comparisons were performed. The SNP differences between the revived strain and the mother strain were less than 20, indicating that the freeze-drying and revival processes did not have a substantial impact on the genetic background of the strain.

[0058] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for isolating *Pseudomonas digiri*, characterized in that, Includes the following steps: S1. Collect fecal samples from healthy children and prepare a fecal suspension; S2. After dilution, the fecal suspension is spread on a culture medium containing antibiotics and cultured under anaerobic conditions. S3. Select single colonies with consistent morphology and obtain single colonies through pure culture; identify the single colonies by MALDI-TOF MS; and then confirm the strain as *Pseudomonas dignitaria* by bidirectional Sanger sequencing. The antibiotic-containing culture medium comprises: adding 0.1-1 g / L cysteine, 0.2-5 mg / L vitamin K1, 1-10 mg / L heme and 0.01-0.1% L-cysteine ​​to the basal culture medium.

2. The separation method according to claim 1, characterized in that, The antibiotic-containing culture medium comprises: adding 0.5 g / L cysteine, 1 mg / L vitamin K1, 5 mg / L heme and 0.05% L-cysteine ​​to the basal culture medium; the basal culture medium is mGAM.

3. The separation method according to claim 1 or 2, characterized in that, The culture medium containing antibiotics also includes: 1-3 μg / mL vancomycin and 10-100 μg / mL kanamycin.

4. The separation method according to claim 1, characterized in that, Steps S1-S3 are all carried out under anaerobic conditions, wherein the anaerobic conditions are N2:CO2:H2=80:10:

10.

5. The separation method according to claim 1, characterized in that, The healthy children were 6-year-old healthy children who had not used antibiotics or other drugs in the 3 months prior to collection and had no diseases.

6. A type of *Pseudomonas dignitaria*, characterized in that, Parabacterium digilei obtained by the isolation method described in claims 1-5.

7. A method for amplifying and culturing *Pseudomonas dignitaria*, characterized in that, Cultured under anaerobic conditions using a culture medium containing 0.2-5 mg / L vitamin K1 and 1-10 mg / L heme; The *Pseudomonas dilataniae* was obtained using the isolation method described in claims 1-5.

8. The amplification culture method according to claim 7, characterized in that, mGAM medium containing 1 mg / L vitamin K1 and 5 mg / L heme was used.

9. A method for cryopreserving *Pseudomonas difficile*, characterized in that, The freeze-drying protectant used in the cryopreservation method comprises 1-10% sucrose, 5-20% skim milk powder, and 0.01-0.1% L-cysteine ​​hydrochloride. The *Pseudomonas dilataniae* was obtained using the isolation method described in claims 1-5.

10. The cryopreservation method according to claim 9, characterized in that, The freeze-drying protectant used in the cryopreservation method comprises 5% sucrose, 10% skim milk powder, 0.05% calcium chloride, 0.05% magnesium chloride, and 0.01% L-cysteine ​​hydrochloride.

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