Characterization of AI-2/LuxS quorum sensing system in biofilm formation, pathogenesis of Streptococcus equi subsp. zooepidemicus.

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of both humans and animals. Quorum sensing (QS) plays an important role in the regulation of bacterial group behaviors. The aim of this study was to characterize the LuxS in SEZ and evaluate its impact on biofilm formation, pathogenesis and gene expression. The wild-type SEZ and its LuxS mutant (ΔluxS) were examined for growth, biofilm formation, virulence factors, and transcriptomic profiles. Our results showed that LuxS deficiency did not affect SEZ hemolytic activity, adhesion or capsule production. For biofilm assay demonstrated that mutation in the luxS gene significantly enhances biofilm formation, produced a denser biofilm and attached to a glass surface. RAW264.7 cell infection indicated that ΔluxS promoted macrophage apoptosis and pro-inflammatory responses. In mice infection, there was no significant difference in mortality between SEZ and ΔluxS. However, the bacterial load in the spleen of mice infected with ΔluxS was significantly higher than in those infected with SEZ. And the pathological analysis further indicated that spleen damage was more severe in the ΔluxS group. Moreover, transcriptomics analysis revealed significant alterations in carbon metabolism, RNA binding and stress response genes in ΔluxS. In summary, this study provides the first evidence of AI-2/LuxS QS system in SEZ and reveals its regulatory effects on biofilm formation, pathogenicity and gene expression.

A distinct variant of the SzM protein of Streptococcus equi subsp. zooepidemicus recruits C1q independent of IgG binding and inhibits activation of the classical complement pathway.

Streptococcus equi subsp. zooepidemicus (SEZ) is a major equine pathogen that causes pneumonia, abortion, and polyarthritis. It can also cause invasive infections in humans. SEZ expresses the M-like protein SzM, which recruits host proteins such as fibrinogen to the bacterial surface. Equine SEZ strain C2, which binds only comparably low amounts of human fibrinogen in comparison to human SEZ strain C33, was previously shown to proliferate in equine and human blood. As the expression of SzM_C2 was necessary for survival in blood, this study investigated the working hypothesis that SzM_C2 inhibits complement activation through a mechanism other than fibrinogen and non-immune immunoglobulin binding. Loss-of-function experiments showed that SEZ C2, but not C33, binds C1q via SzM in IgG-free human plasma. Furthermore, SzM C2 expression is necessary for recruiting purified human or equine C1q to the bacterial surface. Flow cytometry analysis demonstrated that SzM expression in SEZ C2 is crucial for the significant reduction of C3b labelling in human plasma. Addition of human plasma to immobilized rSzM_C2 and immobilized aggregated IgG led to binding of C1q, but only the latter activated the complement system, as shown by the detection of C4 deposition. Complement activation induced by aggregated IgG was significantly reduced if human plasma was pre-incubated with rSzM_C2. Furthermore, rSzM_C2, but not rSzM_C33, inhibited the activation of the classical complement pathway in human plasma, as determined in an erythrocyte lysis experiment. In conclusion, the immunoglobulin-independent binding of C1q to SzM_C2 is associated with complement inhibition.

Metabolic labeling of hyaluronan: Biosynthesis and quantitative analysis of 13C,15N-enriched hyaluronan by NMR and MS-based methods.

Hyaluronan (HA), a member of the GAG family of glycans, has many diverse biological functions that vary a lot depending on the length of the HA chain and its concentration. A better understanding of the structure of different-sized HA at the atomic level is therefore crucial to decipher these biological functions. NMR is a method of choice for conformational studies of biomolecules, but there are limitations due to the low natural abundance of the NMR active nuclei 13C and 15N. We describe here the metabolic labeling of HA using the bacterium Streptococcus equi subsp. Zooepidemicus and the subsequent analysis by NMR and mass spectrometry. The level of 13C and 15N isotope enrichment at each position was determined quantitatively by NMR spectroscopy and was further confirmed by high-resolution mass spectrometry analysis. This study provides a valid methodological approach that can be applied to the quantitative assessment of isotopically labeled glycans and will help improve detection capabilities and facilitate future structure-function relationship analysis of complex glycans.

Genetic variation reveals the enhanced microbial hyaluronan biosynthesis via atmospheric and room temperature plasma.

This study reveals the genetic and biochemical changes underlying the enhanced hyaluronan (HA) biosynthesis in Streptococcus zooepidemicus. After multiple rounds of atmospheric and room temperature plasma (ARTP) mutagenesis combined with novel bovine serum albumin/cetyltrimethylammonium bromide coupled high-throughput screening assay, the HA yield of the mutant was increased by 42.9% and reached 0.813 g L-1 with a molecular weight of 0.54 × 106 Da within 18 h by shaking flask culture. HA production was increased to 4.56 g L-1 by batch culture in 5-L fermenter. Transcriptome sequencing exhibits that distinct mutants have similar genetic changes. Regulation in direction of metabolic flow into the HA biosynthesis, by enhancing genes responsible for the biosynthesis of HA including hasB, glmU and glmM, weaking downstream gene (nagA and nagB) of UDP-GlcNAc and significantly down-regulating transcription of wall-synthesizing genes, resulting in the accumulation of precursors (UDP-GlcA and UDP-GlcNAc) increased by 39.74% and 119.22%, respectively. These associated regulatory genes may provide control point for engineering of the efficient HA-producing cell factory.

Tapping on the Potential of Hyaluronic Acid: from Production to Application.

The manufacture, purification, and applications of hyaluronic acid (HA) are discussed in this article. Concerning the growing need for affordable, high-quality HA, it is essential to consider diverse production techniques using renewable resources that pose little risk of cross-contamination. Many microorganisms can now be used to produce HA without limiting the availability of raw materials and in an environmentally friendly manner. The production of HA has been associated with Streptococci A and C, explicitly S. zooepidemicus and S. equi. Different fermentation techniques, including the continuous, batch, fed-batch, and repeated batch culture, have been explored to increase the formation of HA, particularly from S. zooepidemicus. The topic of current interest also involves a complex broth rich in metabolites and residual substrates, intensifying downstream processes to achieve high recovery rates and purity. Although there are already established methods for commercial HA production, the anticipated growth in trade and the diversification of application opportunities necessitate the development of new procedures to produce HA with escalated productivity, specified molecular weights, and purity. In this report, we have enacted the advancement of HA technical research by analyzing bacterial biomanufacturing elements, upstream and downstream methodologies, and commercial-scale HA scenarios.

Gasdermin D protects against Streptococcus equi subsp. zooepidemicus infection through macrophage pyroptosis.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus, SEZ) is an essential zoonotic bacterial pathogen that can cause various inflammation, such as meningitis, endocarditis, and pneumonia. Gasdermin D (GSDMD) is involved in cytokine release and cell death, indicating an important role in controlling the microbial infection. This study investigated the protective role of GSDMD in mice infected with SEZ and examined the role of GSDMD in peritoneal macrophages in the infection. GSDMD-deficient mice were more susceptible to intraperitoneal infection with SEZ, and the white pulp structure of the spleen was seriously damaged in GSDMD-deficient mice. Although the increased proportion of macrophages did not depend on GSDMD in both spleen and peritoneal lavage fluid (PLF), deficiency of GSDMD caused the minor release of the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) during the infection in vivo. In vitro, SEZ infection induced more release of IL-1ß, IL-18, and lactate dehydrogenase (LDH) in wild-type macrophages than in GSDMD-deficient macrophages. Finally, we demonstrated that pore formation and pyroptosis of macrophages depended on GSDMD. Our findings highlight the host defense mechanisms of GSDMD against SEZ infection, providing a potential therapeutic target in SEZ infection.

miR-223-3p contributes to suppressing NLRP3 inflammasome activation in Streptococcus equi ssp. zooepidemicus infection.

Streptococcus equi subsp. zooepidemicus (SEZ) is an essential pathogen in a range of species, causing a worldwide variety of diseases, such as meningitis, endocarditis, and septicaemia. Studies have shown that microRNAs (miRNAs), which regulate target genes at the post-transcriptional level, play an important regulatory role in the organism. In this study, the infection of J774A.1 murine macrophages with SEZ up-regulated NLRP3 inflammasome and downstream pathways accompanied by miR-223-3p down-regulation. Through computational prediction and experimental confirmation, we have shown that miR-223-3p directly targets the NLRP3 mRNA. Consequently, overexpression of miR-223-3p suppressed NLRP3 inflammasome activation and downstream pathways in response to SEZ infection. The miR-223-3p inhibitor exhibited the opposite effect, causing hyperactivation of NLRP3 inflammation activation and downstream pathways. Additionally, we further demonstrated that miRNA-223-3p inhibited the secretion of IL-1ß and IL-18 by regulating the NLRP3/caspase-1 pathway. Furthermore, intravenous administration of miR-223-3p significantly decreased inflammation in mice in response to SEZ. In conclusion, our results demonstrated that miR-223-3p contributes to suppressing the NLRP3 inflammasome activation in SEZ infection, contributing novel evidence to identify a therapeutic target for treating SEZ.

A honeycomb-like hydrogel in-situ constructed by Streptococcus zooepidemicus and TOCN for the proliferation of bacteria.

Cellulose, as a significant natural carbohydrate polymer, can be transformed into polysaccharide nanofiber. A novel in-situ hydrogel was constructed using TEMPO-oxidized cellulose nanofiber (TOCN) and metabolites of Streptococcus zooepidemicus, such as hyaluronic acid (HA) and lactic acid (LA). In the presence of LA, HA underwent a gelation process, and S. zooepidemicus was embedded in the skeleton of the hydrogel network. This in-situ hydrogel accommodated the bacteria by providing a suitable environment for their growth and protecting them from external stress. The elastic modulus of the in-situ hydrogel was maintained at 4000 G' in Pa, thus ensuring that it was protected from damage even after being immersed in the medium. The hydrogel continued to show normal fermentation activities upon being transferred into the seed or fermentation medium. These findings indicated that the in-situ hydrogel could act as a wellspring that spills out the scattered bacteria.

Physicochemical characterization and in vitro antioxidant activity of hyaluronic acid produced by Streptococcus zooepidemicus CCT 7546.

Hyaluronic acid (HA) is a biopolymer with applications in different areas such as medicine and cosmetics. HA is currently either isolated from animal sources or produced by microbial fermentation. Animal HA presents some disadvantages such as high cost and risk of viral cross-species or another infectious agent. In the present study, we evaluated the physicochemical characteristics and in vitro antioxidant capacity of HA produced by Streptococcus zooepidemicus CCT 7546. In addition, commercial sodium hyaluronate (SH) from an animal source was used as control. The microbial HA yield after purification was 69.8 mg/L. According to Fourier transform infrared spectroscopy, it was seen that bacterial and animal HA spectra are overlapped. The thermogravimetric analysis revealed that microbial HA was more stable than its equivalent from the animal source. However, scanning electron microscopy indicates that the purification method used in the animal product was more effective. Microbial HA showed activity in total antioxidant capacity (14.02 ± 0.38%), reducing power (18.18 ± 6.43%), DPPH radical-scavenging (5.57 ± 0.23 kmol TE/g), and hydroxyl radical-scavenging (28.39 ± 2.40%) tests. Therefore, in vitro antioxidant tests demonstrated that the antioxidant action mechanism occurs through scavenging reactive oxygen species (ROS) and donating electrons/hydrogen atoms.

The dilution effect of media culture on mixing time, Kla O2, and hyaluronic acid production in S. zooepidemicus fed-batch culture.

OBJECTIVES: Microbial production of biopolymers is typically associated with high viscosity and suitable mixing plays an important role in their production. Due to the nature of Streptococcus strains in high production of lactic acid and consequently high consumption of NaOH, which is associated with increased viscosity and reduced mixing caused by hyaluronic acid production, the injected NaOH accumulates and causes cells loss, and decreases in quantity and quality of the produced hyaluronic acid. RESULTS: In this study, the effect of increasing dilution of media culture of Streptococcus zooepidemicus fed-batch culture during pH control by NaOH on mixing time, volumetric oxygen transfer coefficient, and increasing hyaluronic acid production in a 2-L fermenter were studied. The results showed that significant increasing dilution causes reduction mixing time, remarkable improvement volumetric oxygen transfer coefficient, hyaluronic acid production enhancement from 6.6 to 8.4 g/L, and diminution the consumption of NaOH. CONCLUSION: Dilution of media culture of S. zooepidemicus fed-batch culture by the pH controlling agent achieved one of the highest amounts of hyaluronic acid that was reported recently. This method does not require any automatic control and can be used at a low cost to produce other soluble extracellular biopolymers.

Deciphering the role of dissolved oxygen and N-acetyl glucosamine in governing higher molecular weight hyaluronic acid synthesis in Streptococcus zooepidemicus cell factory.

The present study is focused on systematic process and kinetic investigation of hyaluronic acid (HA) production strategy unraveling the role of dissolved oxygen (DO) and N-acetyl glucosamine (GlcNAc) towards the enhancement of HA titer and its molecular weight. Maintaining excess DO levels (10-40% DO) through DO-stat control and the substitution of GlcNAc at a range (5-20 g/L) with glucose (Glc) critically influenced HA production. DO-stat control strategy yielded a promising HA titer (2.4 g/L) at 40% DO concentration. Controlling DO level at 20% (DO-stat) was observed to be optimum resulting in a significant HA production (2.1 g/L) and its molecular weight ranging 0.98-1.45 MDa with a consistent polydispersity index (PDI) (1.57-1.69). Substitution of GlcNAc with Glc at different proportions explicitly addressed the metabolic trade-off between HA titer and its molecular weight. GlcNAc substitution positively influenced the molecular weight of HA. The highest HA molecular weight (2.53 MDa) of two-fold increase compared with glucose as sole carbon substrate and narrower PDI (1.35 ± 0.18) was achieved for the 10:20 (Glc:GlcNAc) proportion. A novice attempt on modeling the uptake of dual substrates (Glc and GlcNAc) by Streptococcus zooepidemicus for HA production was successfully accomplished using double Andrew's growth model and the kinetic parameters were estimated reliably.

Films Based on Blends of Polyvinyl Alcohol and Microbial Hyaluronic Acid

Abstract The aims of this work were to produce hyaluronic acid (HA) by Streptococcus zooepidemicus ATCC 39920 in a low cost sugarcane molasses fermentation medium and to employ the produced HA to obtain films blends based on polyvinyl alcohol (PVA). The films were produced using solution casting method and they were characterized according to their microstructure, mechanical and barrier properties. HA was added in different concentrations (0, 5, 10 and 15% (w/w)), and glycerol was used as a plasticizer (25 g/100 g solids). All formulations resulted in easily manipulated films with good appearance. The addition of HA on PVA films increased their thermal stability, solubility, swelling index, water vapor permeability and elongation. Microbial HA sample combined with PVA showed to be a promising material to biomedical application, and an addition between 5 and 10% (w/w) was sufficient to improve PVA films properties.

Prominent Binding of Human and Equine Fibrinogen to Streptococcus equi subsp. zooepidemicus Is Mediated by Specific SzM Types and Is a Distinct Phenotype of Zoonotic Isolates.

Streptococcus equi subsp. zooepidemicus is an important pathogen in horses that causes severe diseases such as pneumonia and abortion. Furthermore, it is a zoonotic agent, and contact with horses is a known risk factor. In this study, we investigated the working hypothesis that the zoonotic potential varies among S. equi subsp. zooepidemicus strains in association with differences in M-like protein-mediated binding of host plasma proteins. We demonstrate via in-frame deletion mutagenesis of two different S. equi subsp. zooepidemicus strains that the M-like protein SzM is crucial for the binding of fibrinogen to the bacterial surface and for survival in equine and human blood. S. equi subsp. zooepidemicus isolates of equine and human origins were compared with regard to SzM sequences and binding of equine and human fibrinogens. The N-terminal 216 amino acids of the mature SzM were found to exhibit a high degree of diversity, but the majority of human isolates grouped in three distinct SzM clusters. Plasma protein absorption assays and flow cytometry analysis revealed that pronounced binding of human fibrinogen is a common phenotype of human S. equi subsp. zooepidemicus isolates but much less so in equine S. equi subsp. zooepidemicus isolates. Furthermore, binding of human fibrinogen is associated with specific SzM types. These results suggest that SzM-mediated binding of human fibrinogen is an important virulence mechanism of zoonotic S. equi subsp. zooepidemicus isolates.

Biosynthesis of Hyaluronic acid polymer: Dissecting the role of sub structural elements of hyaluronan synthase.

Hyaluronic acid (HA) based biomaterials have several biomedical applications. HA biosynthesis is catalysed by hyaluronan synthase (HAS). The unavailability of 3-D structure of HAS and gaps in molecular understanding of HA biosynthesis process pose challenges in rational engineering of HAS to control HA molecular weight and titer. Using in-silico approaches integrated with mutation studies, we define a dictionary of sub-structural elements (SSE) of the Class I Streptococcal HAS (SeHAS) to guide rational engineering. Our study identifies 9 SSE in HAS and elucidates their role in substrate and polymer binding and polymer biosynthesis. Molecular modelling and docking assessment indicate a single binding site for two UDP-substrates implying conformationally-driven alternating substrate specificities for this class of enzymes. This is the first report hypothesizing the involvement of sites from SSE5 in polymer binding. Mutation at these sites influence HA production, indicating a tight coupling of polymer binding and synthase functions. Mutation studies show dispensable role of Lys-139 in substrate binding and a key role of Gln-248 and Thr-283 in HA biosynthesis. Based on the functional architecture in SeHAS, we propose a plausible three-step polymer extension model from its reducing end. Together, these results open new avenues for rational engineering of Class I HAS to study and regulate its functional properties and enhanced understanding of glycosyltransferases and processive enzymes.

[Construction of engineered Streptococcus zooepidemicus for the production of hyaluronic acid ligosaccharide].

Hyaluronic acid (HA) is widely used in many fields, such as medicine, cosmetics and food. The bioactivity of HA depends on its molecular weight (Mw). Owing to the important physiological activities and special physiological functions, HA oligosaccharides have important application prospects in medicine fields. Streptococcus zooepidemicus has wide applications in commercial production of HA, due to its short fermentation cycle and strong production intensity. In order to efficiently synthesize HA oligosaccharides and solve the dissolved oxygen in the fermentation process, in this study, we overexpressed HA synthase (HasA) and introduced and optimized the leech hyaluronidase LHAase in Streptococcus zooepidemicus WSH-24. As a result, HA oligosaccharides were efficiently produced with improved dissolved oxygen. After 24 h, HA oligosaccharides production intensity reached to 294.2 mg/(L·h), and the concentration accumulated to 0.97 g/L in flask cultures, which was 1.82 times of the wild strain. Impressively, HA oligosaccharides were increased to 7.06 g/L in 3 L fermentor. The constructed Streptococcus zooepidemicus strain for producing HA oligosaccharides would have broad application prospects.

A hyaluronan-based polysaccharide peptide generated by a genetically modified Streptococcus zooepidemicus.

Polysaccharide peptides (or protein-bound polysaccharides, PSPs) are commonly found in mushrooms and plants and possess important nutritional properties and health benefits. The pathogenic bacterium Streptococcus zooepidemicus does not inherently produce PSPs but secretes the capsular polysaccharide hyaluronan. However, in a previous investigation of the catalytic mechanism of UDP-glucose dehydrogenase (UGDH), a PSP of peptide-bound hyaluronan was found to be produced by S. zooepidemicus through the in vivo expression of a mutant of the gene encoding UGDH. In the present study, this hyaluronan-derived PSP was structurally characterized by FT-IR, NMR, and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the data confirmed that the polysaccharide backbone, hyaluronan, is covalently bound to the side-chain peptides via an amide linkage. More importantly, the bacterial production of a PSP via this genetic modification method should inspire further research on the in vitro enzymatic synthesis of PSPs or even naturally occurring polysaccharide derivatives and may provide a theoretical foundation for investigating the in vivo synthetic mechanism of PSPs.

Identification of a Quorum Sensing System Regulating Capsule Polysaccharide Production and Biofilm Formation in Streptococcus zooepidemicus.

Streptococcus zooepidemicus is an important opportunistic pathogen of several species including humans. This organism is also well-known as the main producing strain in industrial production of hyaluronic acid (HA), which is the component of its capsule polysaccharide. How its virulence and capsule polysaccharide production is regulated remains poorly understood. Intercellular chemical signaling among bacteria provides communities of microbes the opportunity to coordinate gene expression to facilitate group behavior, such as pathogenicity, capsule polysaccharide production, etc. Yet no conserved cell-to-cell signaling system has been elucidated in S. zooepidemicus. Encoded within the genome of S. zooepidemicus is one Rgg regulator encoding gene (rgg) with low similarity to both rgg2 and rgg3 from Streptococcus pyogenes. A small ORF (named as shp) encoding a novel short hydrophobic peptide (SHP) was found in the vicinity of rgg. We found that the active form of pheromone is short and hydrophobic (LLLLKLA), corresponding to the C terminal 7 amino acids of the pre-peptide Shp, which shows divergent sequence to all peptide pheromones reported in streptococci. In response to active SHP, Rgg functions as a transcriptional activator to induce the expression of shp, forming a positive feedback circuit. Bacteria social behaviors, such as capsule polysaccharide production and biofilm formation, were significantly affected when the rgg-shp pathway was inactivated. These data provide the first demonstration that Rgg/Shp signaling pathway comprises an active quorum sensing system in S. zooepidemicus.

UDP-glucose Dehydrogenase: The First-step Oxidation Is an NAD+-dependent Bimolecular Nucleophilic Substitution Reaction (SN2).

UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid by NAD+-dependent two-fold oxidation. Despite extensive investigation into the catalytic mechanism of UGDH, the previously proposed mechanisms regarding the first-step oxidation are somewhat controversial and inconsistent with some biochemical evidence, which instead supports a mechanism involving an NAD+-dependent bimolecular nucleophilic substitution (SN2) reaction. To verify this speculation, the essential Cys residue of Streptococcus zooepidemicus UGDH (SzUGDH) was changed to an Ala residue, and the resulting Cys260Ala mutant and SzUGDH were then co-expressed in vivo via a single-crossover homologous recombination method. Contrary to the previously proposed mechanisms, which predict the formation of the capsular polysaccharide hyaluronan, the resulting strain instead produced an amide derivative of hyaluronan, as validated via proteinase K digestion, ninhydrin reaction, FT-IR and NMR. This result is compatible with the NAD+-dependent SN2 mechanism.

CD44 facilitates adherence of Streptococcus equi subsp. zooepidemicus to LA-4 cells.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) causes a wide variety of infections in many species. CD44 is a transmembrane adhesion molecule, expressed by various cell types, which has been implicated in several infection processes. The aim of this study was to examine the role of CD44 in S. zooepidemicus adherence to LA-4 cells (mouse lung adenoma). Dose-dependent adhesion with LA-4 may be effectively studied by flow cytometry. Adherence of S. zooepidemicus is reduced after treatment of cells with anti-CD44 antibody. Treatment of S. zooepidemicus with recombinant CD44 significantly reduced bacteria adherence. In addition, CD44 can directly bind to wild-type S. zooepidemicus, while the binding was decreased in the capsule deletion isogenic mutant. These data suggest that CD44 facilitates adherence of S. zooepidemicus to LA-4 cells.

Recovery and Purity of High Molar Mass Bio-hyaluronic Acid Via Precipitation Strategies Modulated by pH and Sodium Chloride.

The effects of ethanol/broth proportions and the number of steps at varying pH in the presence or absence of sodium chloride (NaCl) were studied as precipitation strategies for the recovery and purification of high molar mass bio-hyaluronic acid (Bio-HA). Bio-HA was synthesized by Streptococcus zooepidemicus in a culture medium containing glucose and soy peptones. A single-step precipitation was more attractive than multistep precipitation in terms of recovery and purity as well as decreased use of ethanol. The best conditions in the absence and presence of salt were 2:1 ethanol/broth (v/v) at pH 4 (55.0 ± 0.2% purity and 85.0 ± 0.7% recovery) and 2:1 ethanol/broth (v/v) at pH 7 + 2 mol L-1 NaCl (59.0 ± 0.9% purity and 82.0 ± 4.3% recovery). Dynamic light scattering (DLS) spectra showed different particle sizes as a consequence of the changes in the molecular structure of HA, mainly with changes in pH. Although slight changes in distribution were observed, the average HA molar mass was not affected by the precipitation strategy, remaining on the order of 105 Da. Therefore, pH and NaCl modulated the precipitation performance of HA. These findings are relevant to further optimizing the precipitation step, thus minimizing costs in the later stages of HA purification.