The branching ratio of enzymatically synthesized α-glucans impacts microbiome and metabolic outcomes of in vitro fecal fermentation.

The aim of this study was to evaluate the impacts of enzymatically synthesized α-glucans possessing α-1,4- and α-1,6-glucose linkages, and varying in branching ratio, on colonic microbiota composition and metabolic function. Four different α-glucans varying in branching ratio were synthesized by amylosucrase from Neisseria polysaccharea and glycogen branching enzyme from Rhodothermus obamensis. The branching ratios were found to range from 0 % to 2.8 % using GC/MS. In vitro fecal fermentation analyses (n = 8) revealed that the branching ratio dictates the short-chain fatty acid (SCFA) generation by fecal microbiota. Specifically, slightly branched (0.49 %) α-glucan resulted in generation of significantly (P < 0.05) higher amounts of propionate, compared to more-branched counterparts. In addition, the amount of butyrate generated from this α-glucan was statistically (P > 0.05) indistinguishable than those observed in resistant starches. 16S rRNA sequencing revealed that enzymatically synthesized α-glucans stimulated Lachnospiraceae and Ruminococcus related OTUs. Overall, the results demonstrated metabolic function of colonic microbiota can be manipulated by altering the branching ratio of enzymatically synthesized α-glucans, providing insights into specific structure-function relationships between dietary fibers and the colonic microbiome. Furthermore, the slightly branched α-glucans could be used as functional carbohydrates to stimulate the beneficial microbiota and SCFAs in the colon.

A structure-functionality insight into the bioactivity of microbial polysaccharides toward biomedical applications: A review.

Microbial polysaccharides (MPs) are biopolymers secreted by microorganisms such as bacteria and fungi during their metabolic processes. Compared to polysaccharides derived from plants and animals, MPs have advantages such as wide sources, high production efficiency, and less susceptibility to natural environmental influences. The most attractive feature of MPs lies in their diverse biological activities, such as antioxidative, anti-tumor, antibacterial, and immunomodulatory activities, which have demonstrated immense potential for applications in functional foods, cosmetics, and biomedicine. These bioactivities are precisely regulated by their sophisticated molecular structure. However, the mechanisms underlying this precise regulation are not yet fully understood and continue to evolve. This article presents a comprehensive review of the most representative species of MPs, including their fermentation and purification processes and their biomedical applications in recent years. In particular, this work presents an in-depth analysis into the structure-activity relationships of MPs across multiple molecular levels. Additionally, this review discusses the challenges and prospects of investigating the structure-activity relationships, providing valuable insights into the broad and high-value utilization of MPs.

New insights into starch, lipid, and protein interactions - Colon microbiota fermentation.

Starch, lipids, and proteins are essential biological macromolecules that play a crucial role in providing energy and nutrition to our bodies. Interactions between these macromolecules have been shown to impact starch digestibility. Understanding and controlling starch digestibility is a key area of research. Investigating the mechanisms behind the interactions of these three components and their influence on starch digestibility is of significant practical importance. Moreover, these interactions can result in the formation of resistant starch, which can be fermented by gut microbiota in the colon, leading to various health benefits. While current research has predominantly focused on the digestive properties of starch in the small intestine, there is a notable gap in understanding the colonic microbial fermentation phase of resistant starch. The benefits of fermentation of resistant starch in the colon may outweigh its glucose-lowering effect in the small intestine. Thus, it is crucial to study the fermentation behavior of resistant starch in the colon. This paper investigates the impact of interactions among starch, lipids, and proteins on starch digestion, with a specific focus on the fermentation phase of indigestible carbohydrates in the colon. Furthermore, valuable insights are offered for guiding future research endeavors.

Microbial Fermentation Enhances the Effect of Black Tea on Hyperlipidemia by Mediating Bile Acid Metabolism and Remodeling Intestinal Microbes.

Black tea (BT), the most consumed tea worldwide, can alleviate hyperlipidemia which is a serious threat to human health. However, the quality of summer BT is poor. It was improved by microbial fermentation in a previous study, but whether it affects hypolipidemic activity is unknown. Therefore, we compared the hypolipidemic activity of BT and microbially fermented black tea (EFT). The results demonstrated that BT inhibited weight gain and improved lipid and total bile acid (TBA) levels, and microbial fermentation reinforced this activity. Mechanistically, both BT and EFT mediate bile acid circulation to relieve hyperlipidemia. In addition, BT and EFT improve dyslipidemia by modifying the gut microbiota. Specifically, the increase in Lactobacillus johnsonii by BT, and the increase in Mucispirillum and Colidextribacter by EFT may also be potential causes for alleviation of hyperlipidemia. In summary, we demonstrated that microbial fermentation strengthened the hypolipidemic activity of BT and increased the added value of BT.

The effects of fermentation time on sourdough bread: An analysis of texture profile, starch digestion rate, and protein hydrolysis rate.

To ensure the best quality bread, it is important to consider the speed of digestion of starch and proteins, as well as how time fermentation and storage time influence the rate of starch digestion and the texture of the bread. This study compared the effect of fermentation time and days of storage on the texture, physicochemical, protein and starch digestibility of sourdough bread. Texture profile analysis showed that the fermentation time in recently baked sourdough bread affects hardness, chewiness, and springiness. The electrophoretic profile showed a decrease in band thickness with increase in fermentation time, consistent with a higher percentage of protein digestion. While fermentation time did not significantly affect rapidly digestible starch (RDS) and slowly digestible starch (SDS), storage time resulted in a decrease in RDS and an increase in SDS. Sourdough breads had higher levels of resistant starch (RS). The digestibility characteristics of protein and starch, as well as texture properties, are significantly influenced by fermentation and storage time. The evidence suggests that sourdough bread has the potential to improve the digestion of protein and to effectively regulate the glycemic response, which is due to its higher levels of SDS and RS.

Differential responses of rumen and fecal fermentation and microbiota of Liaoning cashmere goats after 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester supplementation.

The 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester (HMBi), a rumen protective methionine, has been extensively studied in dairy cows and beef cattle and has been shown to regulate gastrointestinal microbiota and improve production performance. However, knowledge of the application of HMBi on cashmere goats and the simultaneous study of rumen and hindgut microbiota is still limited. In this study, HMBi supplementation increased the concentration of total serum protein, the production of microbial protein in the rumen and feces, as well as butyrate production in the feces. The results of PCoA and PERMANOVA showed no significant difference between the rumen microbiota, but there was a dramatic difference between the fecal microbiota of the two groups of Cashmere goats after the HMBi supplementation. Specifically, in the rumen, HMBi significantly increased the relative abundance of some fiber-degrading bacteria (such as Fibrobacter) compared with the CON group. In the feces, as well as a similar effect as in the rumen (increasing the relative abundance of some fiber-degrading bacteria, such as Lachnospiraceae FCS020 group and ASV32), HMBi diets also increased the proliferation of butyrate-producing bacteria (including Oscillospiraceae UCG-005 and Christensenellaceae R-7 group). Overall, these results demonstrated that HMBi could regulate the rumen and fecal microbial composition of Liaoning cashmere goats and benefit the host.

From the grapevine to the glass: A wine metabolomics tale by FT-ICR-MS.

Wine is one of the most consumed beverages around the world. Its unique characteristics arise from numerous processes, from the selection of grapevine varieties and grapes, the effect of the terroir and geographical origin, through the biochemical process of fermentation by microorganisms, until its aging. All molecules found in wine define its chemical fingerprint and can be used to tell the story of its origin, production, authenticity and quality. Wine's chemical composition can be characterized using an untargeted metabolomics approach based on extreme resolution mass spectrometry. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is currently the most powerful analytical technique to analyse such complex sample, providing the most comprehensive analysis of the chemical fingerprint of wine.

Strategies for the efficient biosynthesis of ß-carotene through microbial fermentation.

ß-Carotene is an orange fat-soluble compound, which has been widely used in fields such as food, medicine and cosmetics owing to its anticancer, antioxidant and cardiovascular disease prevention properties. Currently, natural ß-carotene is mainly extracted from plants and algae, which cannot meet the growing market demand, while chemical synthesis of ß-carotene cannot satisfy the pursuit for natural products of consumers. The ß-carotene production through microbial fermentation has become a promising alternative owing to its high efficiency and environmental friendliness. With the rapid development of synthetic biology and in-depth study on the synthesis pathway of ß-carotene, microbial fermentation has shown promising applications in the ß-carotene synthesis. Accordingly, this review aims to summarize the research progress and strategies of natural carotenoid producing strain and metabolic engineering strategies in the heterologous synthesis of ß-carotene by engineered microorganisms. Moreover, it also summarizes the adoption of inexpensive carbon sources to synthesize ß-carotene as well as proposes new strategies that can further improve the ß-carotene production.

Strategies for the synthesis of the osmolyte glucosylglycerate and its precursor glycerate.

Glycosidic osmolytes are widespread natural compounds that protect microorganisms and their macromolecules from the deleterious effects of various environmental stresses. Their protective properties have attracted considerable interest for industrial applications, especially as active ingredients in cosmetics and healthcare products. In that regard, the osmolyte glucosylglycerate is somewhat overlooked. Glucosylglycerate is typically accumulated by certain organisms when they are exposed to high salinity and nitrogen starvation, and its potent stabilizing effects have been demonstrated in vitro. However, the applications of this osmolyte have not been thoroughly explored due to the lack of a cost-efficient production process. Here, we present an overview of the progress that has been made in developing promising strategies for the synthesis of glucosylglycerate and its precursor glycerate, and discuss the remaining challenges. KEY POINTS: • Bacterial milking could be explored for fermentative production of glucosylglycerate • Glycoside phosphorylases of GH13_18 represent attractive alternatives for biocatalytic production • Conversion of glycerol with alditol oxidase is a promising strategy for generating the precursor glycerate.

Levels of lipid-derived gut microbial metabolites differ among plant matrices in an in vitro model of colon fermentation.

This study explored differences in microbial lipid metabolites among sunflower seeds, soybeans, and walnuts. The matrices were subjected to in vitro digestion and colonic fermentation. Defatted digested materials and fiber/phenolics extracted therefrom were added to sunflower oil (SO) and also fermented. Targeted and untargeted lipidomics were employed to monitor and tentatively identify linoleic acid (LA) metabolites. Walnut fermentation produced the highest free fatty acids (FFAs), LA, and conjugated LAs (CLAs). Defatted digested walnuts added to SO boosted FFAs and CLAs production; the addition of fibre boosted CLAs, whereas the addition of phenolics only increased 9e,11z-CLA and 10e,12z-CLA. Several di-/tri-hydroxy-C18-FAs, reported as microbial LA metabolites for the first time, were annotated. Permutational multivariate analysis of variance indicated significant impacts of food matrix presence and type on lipidomics and C18-FAs. Our findings highlight how the food matrices affect CLA production from dietary lipids, emphasizing the role of food context in microbial lipid metabolism.

Correlation between monosaccharide, oligosaccharide, and microbial community profile changes in traditional soybean brick (meju) fermentation.

Meju is essential for making diverse traditional fermented soybean foods in Korea. To understand the changes in carbohydrates during fermentation, we aimed to identify autochthonous microorganisms from spontaneously fermented meju and compare the alterations in monosaccharides and oligosaccharides throughout the fermentation process. Microbial diversity was determined using a metabarcoding approach, and monosaccharide and oligosaccharide profiles were obtained by HPLC-Q-TOF MS and HPLC-MS/MS analyses, respectively. The dominant bacterial genera were Weissella, Lactobacillus, and Leuconostoc, while Mucor was highly abundant in the fungal community. The total monosaccharide content increased from Day 0 to Day 50, with the highest amount being 4.37 mg/g. Oligosaccharide profiling revealed the degradation of soybean dietary fiber during fermentation, and novel oligosaccharide structures were also discovered. Correlation analysis revealed that the fungus Mucor was positively related to pentose-containing oligosaccharides, galactose, and galacturonic acid, indicating that Mucor may degrade soybean dietary fibers such as xylogalacturonan, arabinogalactan, and rhamnogalacturonan. The negative relationships between the abundances of Weissella and oligo- and monosaccharides suggested that the bacteria may utilize saccharides for fermentation. These findings provide insights into the mechanisms underlying carbohydrate degradation and utilization; the key components involved in saccharide transformation that contribute to the characteristics of traditional meju were subsequently identified.

The bacteriome-coupled phage communities continuously contract and shift to orchestrate the traditional rice vinegar fermentation.

Amounts of microbiome studies have uncovered the microbial communities of traditional food fermentations, while in which the phageome development with time is poorly understood. Here, we conducted a study to decipher both phageome and bacteriome of the traditional rice vinegar fermentation. The vinegar phageomes showed significant differences in the alpha diversity, network density and clustering coefficient over time. Peduoviridae had the highest relative abundance. Moreover, the phageome negatively correlated to the cognate bacteriome in alpha diversity, and undergone constantly contracting and shifting across the temporal scale. Nevertheless, 257 core virial clusters (VCs) persistently occurred with time whatever the significant impacts imposed by the varied physiochemical properties. Glycoside hydrolase (GH) and glycosyltransferase (GT) families genes displayed the higher abundances across all samples. Intriguingly, diversely structuring of toxin-antitoxin systems (TAs) and CRISPR-Cas arrays were frequently harbored by phage genomes. Their divergent organization and encoding attributes underlie the multiple biological roles in modulation of network and/or contest of phage community as well as bacterial host community. This phageome-wide mapping will fuel the current insights of phage community ecology in other traditional fermented ecosystems that are challenging to decipher.

Metabolomics and HS-SPME-GC-MS-based analysis of quality succession patterns and flavor characteristics changes during the fermentation of Lycium barbarum and Polygonatum cyrtonema compound wine.

This work set out to investigate how the physicochemical markers, volatiles, and metabolomic characteristics of mixed fermented the fermentation of Lycium barbarum and Polygonatum cyrtonema compound wine (LPCW) from S. cerevisine RW and D. hansenii AS2.45 changed over the course of fermentation. HS-SPME-GC-MS combined with non-targeted metabolomics was used to follow up and monitor the fermentation process of LPCW. In total, 43 volatile chemical substances, mostly alcohols, esters, acids, carbonyl compounds, etc., were discovered in LPCW. After 30 days of fermentation, phenylethyl alcohol had increased to 3045.83 g/mL, giving off a rose-like fresh scent. The biosynthesis of valine, leucine, and isoleucine as well as the metabolism of alanine, aspartic acid, and glutamic acid were the major routes that led to the identification of 1385 non-volatile components in total. This study offers a theoretical foundation for industrial development and advances our knowledge of the fundamental mechanism underlying flavor generation during LPCW fermentation.

Starter molds and multi-enzyme catalysis in koji fermentation of soy sauce brewing: A review.

Soy sauce is a traditional fermented food produced from soybean and wheat under the action of microorganisms. The soy sauce brewing process mainly involves two steps, namely koji fermentation and moromi fermentation. In the koji fermentation process, enzymes from starter molds, such as protease, aminopeptidase, carboxypeptidase, l-glutaminase, amylase, and cellulase, hydrolyze the protein and starch in the raw ingredients to produce short-chain substances. However, the enzymatic reactions may be diminished after being subjected to moromi fermentation due to its high NaCl concentration. These enzymatically hydrolyzed products are further metabolized by lactic acid bacteria and yeasts during the moromi fermentation process into organic acids and aromatic compounds, giving soy sauce a unique flavor. Thus, the starter molds, such as Aspergillus oryzae, Aspergillus sojae, and Aspergillus niger, and their secreted enzymes play crucial roles in soy sauce brewing. This review comprehensively covers the characteristics of the starter molds mainly used in soy sauce brewing, the enzymes produced by starter molds, and the roles of enzymes in the degradation of raw material. We also enumerate current problems in the production of soy sauce, aiming to offer some directions for the improvement of soy sauce taste.

A modular and synthetic biosynthesis platform for de novo production of diverse halogenated tryptophan-derived molecules.

Halogen-containing molecules are ubiquitous in modern society and present unique chemical possibilities. As a whole, de novo fermentation and synthetic pathway construction for these molecules remain relatively underexplored and could unlock molecules with exciting new applications in industries ranging from textiles to agrochemicals to pharmaceuticals. Here, we report a mix-and-match co-culture platform to de novo generate a large array of halogenated tryptophan derivatives in Escherichia coli from glucose. First, we engineer E. coli to produce between 300 and 700 mg/L of six different halogenated tryptophan precursors. Second, we harness the native promiscuity of multiple downstream enzymes to access unexplored regions of metabolism. Finally, through modular co-culture fermentations, we demonstrate a plug-and-play bioproduction platform, culminating in the generation of 26 distinct halogenated molecules produced de novo including precursors to prodrugs 4-chloro- and 4-bromo-kynurenine and new-to-nature halogenated beta carbolines.

Reducing transmission of high-risk antibiotic resistance genes in whole-crop corn silage through lactic acid bacteria inoculation and increasing ensiling temperature.

The microbial hosts of antibiotic resistance genes (ARGs) found epiphytically on plant materials could grow and flourish during silage fermentation. This study employed metagenomic analysis and elucidated the occurrence and transmission mechanisms of ARGs and their microbial hosts in whole-crop corn silage inoculated with homofermentative strain Lactiplantibacillus plantarum or heterofermentative strain Lentilactobacillus buchneri ensiled under different temperature (20 and 30 °C). The results revealed that the corn silage was dominated by Lactobacillus, Leuconostoc, Lentilactobacillus, and Latilactobacillus. Both the ensiling temperature and inoculation had greatly modified the silage microbiota. However, regardless of the ensiling temperature, L. buchneri had significantly higher ARGs, while it only exhibited significantly higher mobile genetic elements (MGEs) in low temperature treatments. The microbial community of the corn silage hosted highly diverse form of ARGs, which were primarily MacB, RanA, bcrA, msbA, TetA (58), and TetT and mainly corresponded to macrolides and tetracyclines drug classes. Plasmids were identified as the most abundant MGEs with significant correlation with some high-risk ARGs (tetM, TolC, mdtH, and NorA), and their abundances have been reduced by ensiling process. Furthermore, higher temperature and L. buchneri reduced abundances of high-risk ARGs by modifying their hosts and reduced their transmission in the silage. Therefore, ensiling, L. buchneri inoculation and higher storage temperature could improve the biosafety of corn silage.

Effects of dietary forage neutral detergent fiber and rumen degradable starch ratios on chewing activity, ruminal fermentation, ruminal microbes and nutrient digestibility of Hu sheep fed a pelleted total mixed ration.

Pelleted total mixed ration (P-TMR) feeding, which has become a common practice in providing nutrition for fattening sheep, requires careful consideration of the balance between forage neutral detergent fiber (FNDF) and rumen degradable starch (RDS) to maintain proper rumen functions. The present study aimed to investigate the effects of the dietary FNDF/RDS ratio (FRR) on chewing activity, ruminal fermentation, ruminal microbes, and nutrient digestibility in Hu sheep fed a P-TMR diet. This study utilized eight ruminally cannulated male Hu sheep, following a 4 × 4 Latin square design with 31 d each period. Diets consisted of four FRR levels: 1.0 (high FNDF/RDS ratio, HFRR), 0.8 (middle high FNDF/RDS ratio, MHFRR), 0.6 (middle low FNDF/RDS ratio, MLFRR), and 0.4 (low FNDF/RDS ratio, LFRR). Reducing the dietary FRR levels resulted in a linear decrease in ruminal minimum pH and mean pH, while linearly increasing the duration and area of pH below 5.8 and 5.6, as well as the acidosis index. Sheep in the HFRR and MHFRR groups did not experience subacute ruminal acidosis (SARA), whereas sheep in another two groups did. The concentration of total volatile fatty acid and the molar ratios of propionate and valerate, as well as the concentrate of lactate in the rumen linearly increased with reducing dietary FRR, while the molar ratio of acetate and acetate to propionate ratio linearly decreased. The degradability of NDF and ADF for alfalfa hay has a quadratic response with reducing the dietary FRR. The apparent digestibility of dry matter, organic matter, neutral detergent fiber, and acid detergent fiber linearly decreased when the dietary FRR was reduced. In addition, reducing the dietary FRR caused a linear decrease in OTUs, Chao1, and Ace index of ruminal microflora. Reducing FRR in the diet increased the percentage of reads assigned as Firmicutes, but it decreased the percentage of reads assigned as Bacteroidetes in the rumen. At genus level, the percentage of reads assigned as Prevotella, Ruminococcus, Succinivibrio, and Butyrivibrio linearly decreased when the dietary FRR was reduced. The results of this study demonstrate that the dietary FRR of 0.8 is crucial in preventing the onset of SARA and promotes an enhanced richness of ruminal microbes and also improves fiber digestibility, which is a recommended dietary FRR reference when formulating P-TMR diets for sheep.

Forage neutral detergent fiber (FNDF) and rumen degradable starch (RDS) are key components of carbohydrates in the diet for ruminants, which would reflect saliva secretion and the acid production potential of feed. However, appropriate FNDF to RDS ratios (FRR) applicable to ruminants under the condition of pelleted total mixed ration (P-TMR) feeding have not been reported. In this study, we investigated the effects of the dietary FRR on chewing activity, ruminal fermentation, ruminal microbial communities, and nutrient digestibility of Hu sheep under P-TMR feeding. The results indicate that reducing dietary FRR levels would induce acidosis in sheep, which negatively affected fiber utilization and ruminal bacterial communities. The FRR of 0.8 was a recommended dietary FRR when formulating a P-TMR diet for fattening sheep, as indicated by decreased ruminal acidosis risk and increased richness of ruminal microbes in the rumen as well as nutrient digestibility.

Metabolic Pathway Coupled with Fermentation Process Optimization for High-Level Production of Retinol in Yarrowia lipolytica.

Retinol is a lipid-soluble form of vitamin A that is crucial for human visual and immune functions. The production of retinol through microbial fermentation has been the focus of recent exploration. However, the obtained titer remains limited and the product is often a mixture of retinal, retinol, and retinoic acid, necessitating purification. To achieve efficient biosynthesis of retinol in Yarrowia lipolytica, we improved the metabolic flux of ß-carotene to provide sufficient precursors for retinol in this study. Coupled with the optimization of the expression level of ß-carotene 15,15'-dioxygenase, de novo production of retinol was achieved. Furthermore, Tween 80 was used as an extractant and butylated hydroxytoluene as an antioxidant to extract intracellular retinol and prevent retinol oxidation, respectively. This strategy significantly increased the level of retinol production. By optimizing the enzymes converting retinal to retinol, the proportion of extracellular retinol in the produced retinoids reached 100%, totaling 1042.3 mg/L. Finally, total retinol production reached 5.4 g/L through fed-batch fermentation in a 5 L bioreactor, comprising 4.2 g/L extracellular retinol and 1.2 g/L intracellular retinol. This achievement represents the highest reported titer so far and advances the industrial production of retinol.

Fungal-Bacterial Mutualism: Species and Strain-Dependent Simultaneous Modulation of Branched-Chain Esters and Indole Derivatives in Fermented Sausages through Metabolite Cross-Feeding.

The precise impact of species and strain diversity on fungal-bacterial interactions and the overall community functioning has remained unclear. First, our study revealed how Debaryomyces hansenii influences diverse bacteria to accumulate key metabolites in a simulated fermented food system. For flavor, D. hansenii promoted the accumulation of branched-chain esters in Staphylococcus xylosus by promoting growth and facilitating the precursor branched-chain acids transformations but hindered the accumulation of Staphylococcus equorum. Furthermore, fungal-bacterial interactions displayed diversity among S. equorum strains. For bioactive compounds, species and strain diversity of lactic acid bacteria (LAB) also influences the production of indole derivatives. Then, we investigated specific metabolic exchanges under reciprocal interaction. Amino acids, rather than vitamins, were identified as the primary drivers of the bacterial growth promotion. Moreover, precursor transformations by D. hansenii played a significant role in branched-chain esters production. Finally, a synthetic community capable of producing high concentrations of branched-chain esters and indole derivatives was successfully constructed. These results provide valuable insights into understanding and designing synthetic communities for fermented sausages.

Screening of cellulose-degrading bacteria and optimization of cellulase production from Bacillus cereus A49 through response surface methodology.

Cellulose-degrading microorganisms hold immense significance in utilizing cellulose resources efficiently. The screening of natural cellulase bacteria and the optimization of fermentation conditions are the hot spots of research. This study meticulously screened cellulose-degrading bacteria from mixed soil samples adopting a multi-step approach, encompassing preliminary culture medium screening, Congo red medium-based re-screening, and quantification of cellulase activity across various strains. Particularly, three robust cellulase-producing strains were identified: A24 (MT740356.1 Brevibacillus borstelensis), A49 (MT740358.1 Bacillus cereus), and A61 (MT740357.1 Paenibacillus sp.). For subsequent cultivation experiments, the growth curves of the three obtained isolates were monitored diligently. Additionally, optimal CMCase production conditions were determined, keeping CMCase activity as a key metric, through a series of single-factor experiments: agitation speed, cultivation temperature, unit medium concentration, and inoculum volume. Maximum CMCase production was observed at 150 rpm/37 °C, doubling the unit medium addition, and a 5 mL inoculation volume. Further optimization was conducted using the selected isolate A49 employing response surface methodology. The software model recommended a 2.21fold unit medium addition, 36.11 °C temperature, and 4.91 mL inoculant volume for optimal CMCase production. Consequently, three parallel experiments were conducted based on predicted conditions consistently yielding an average CMCase production activity of 15.63 U/mL, closely aligning with the predicted value of 16.41 U/mL. These findings validated the reliability of the model and demonstrated the effectiveness of optimized CMCase production conditions for isolate A49.