Development of an active biogenic silver nanoparticles composite film based on berry wax and chitosan for rabbit meat preservation.

This study aimed to develop an eco-friendly active biogenic nanocomposite film through the complexation of silver nanoparticles (AgNPs), berry wax (BYW), and chitosan (CT) for maintiaing rabbit functional meat freshness. AgNPs were synthesized using Chinese medicinal paeoniaceae petal extract, and they were loaded at various concentrations (0.5 %, 0.75 %, 1.0 %, 1.25 %, and 1.5 % based on CT w/w) into the CT/BYW complex. The AgNPs exhibited an average size of 55 nm and a zeta potential of -26.3 mV with a spherical shape. The particle size and zeta potential of the film dispersions were 370.5-529.5 nm and 40.17-49.345 mV, respectively. FTIR, SEM, and XRD results showed compatibility among AgNPs and CT/BYW structure. The film water vapor permeability and light transparency decreased from 6.5 to 3.5 and 10 to 0.78 %, respectively, while opacity increased from 1.76 to 9.96 % with increasing concentrations of AgNPs. Among them, the film composite CT/BYW/AgNPs1.5% had better antioxidant and antibacterial properties, which was then applied for rabbit meat preservation at 4 °C for 16 days of storage. CT/BYW/AgNPs1.25%-packed sample had lower values of TVB-N, TBARS, TVC, and pH with greater retention of color properties compared to the control sample, which describes its ability to maintain meat freshness.

Lactoferrin-chia seed mucilage complex coacervates for intestinal delivery of quercetin and fortification of set yogurt.

This study aimed to develop complex coacervates utilizing lactoferrin (LF) and chia seed mucilage (CSM) for promoting intestinal delivery of quercetin (Q) and fortification of set yogurt. Three cross-linkers, including calcium chloride (CC), transglutaminase (TG), and polyphenolic complex (HP), were used to further reinforce the coacervate network. Cross-linked coacervates had higher values of coacervate yield, encapsulation efficiency, and loading capacity. They efficiently preserved Q under gastric condition (⁓87%-99%), with CSM-TG-Q-LF being most effective for intestinal delivery of Q. Moreover, digested pellets of the cross-linked coacervates displayed better antioxidant activity than the uncross-linked coacervates with CSM-TG-Q-LF pellets showing maximum bioactivity. The Q-loaded coacervates demonstrated superior assembly in the yogurt matrix compared to the unencapsulated Q. Moreover, the coacervate systems, especially CSM-TG-Q-LF significantly improved the textural properties of yogurt and the stability of Q in it. Therefore, CSM-TG-LF is a promising carrier to promote intestinal delivery and food application of hydrophobic molecules.

Fabrication of biopolymer stabilized microcapsules for enhancing physicochemical stability, antioxidant and antimicrobial properties of cinnamon essential oil.

The current study entails the encapsulation validity to enclose naturally occurring food preservatives, such as cinnamon essential oil (CM), within various wall materials. This approach has demonstrated enhanced encapsulated compounds' stability, efficiency, and bioactivity. The base carrier system consisted of a solid lipid (Berry wax, RW) individually blended with whey protein (WYN), maltodextrin (MDN), and gum Arabic (GMC) as wall materials. The resulting formulations were freeze-dried: WYN/RW/CM, MDN/RW/CM, and GMC/RW/CM. The study comprehensively analyzed encapsulation efficiency, morphology, crystallinity, thermal, and physiochemical properties. When RW was combined with WYN, MDN, and GMC, the microcapsule WYN/RW/CM showed the highest efficiency at 93.4 %, while the GMC/RW/CM exhibited the highest relative crystallinity at 46.54 %. Furthermore, the investigation assessed storage stability, release of bioactive compounds, and oxidative stability during storage at 4 °C/ 25 % RH ± 5 % and 25 °C/40 % RH ± 5 % for 55 days, revealing optimal stability in the WYN/RW/CM microcapsule. Additionally, the antimicrobial activity was assessed at various concentrations of microcapsules, revealing their inhibitory effect against Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive) bacteria. The WYN/RW/CM microcapsule exhibited the highest inhibition activity in both strains, reaching 40 mm. This study demonstrates that combining WYN with RW as a wall material has greater efficiency in encapsulation and potential uses in various industrial sectors.

Berry wax improves the physico-mechanical, thermal, water barrier properties and biodegradable potential of chitosan food packaging film.

Chitosan (CT) is extensively applied in developing food packaging films due to its non-toxic, biodegradable, and good film-forming properties. But CT-based single polymer film has issues with poor physico-mechanical, thermal, and light barrier properties. Therefore, this study aimed to incorporate natural berry wax (BYW) at various concentrations (5 %, 10 %, 15 %, 20 %, and 25 %, wt%) into CT to improve the quality characteristics of CT film. The microstructure of the film matrix was effectively proven to be compatible with BYW through the utilization of SEM, XRD, and FTIR spectroscopy. The results demonstrated that the quality parameters of CT/BYW composite film were significantly affected by the increasing concentration of BYW. The integration of BYW with a concentration of 5 % to 20 % to CT substantially improved the film characteristics by reducing moisture content, swelling power, solubility, and water vapor permeability, increasing the film's opacity, thermal stability, and tensile strength as well as enhancing the biodegradable potential. Furthermore, CT/BYW films showed higher thermal stability and UV and visible light resistance compared to pure CT film. Taken together, the CT film with 20 % berry wax showed the best film characteristics and biodegradable potential, which could be promising for enhancing the shelf-life of various food products.

Red Wine High-Molecular-Weight Polyphenolic Complex Ameliorates High-Fat Diet-Induced Metabolic Dysregulation and Perturbation in Gut Microbiota in Mice.

Red wine polyphenolic complexes have attracted increasing attention as potential modulators of human metabolic disease risk. Our previous study discovered that red wine high-molecular-weight polymeric polyphenolic complexes (HPPCs) could inhibit key metabolic syndrome-associated enzymes and favorably modulate human gut microbiota (GM) in simulated colonic fermentation assay in vitro. In this work, the efficacy of HPPC supplementation (150 and 300 mg/kg/day, respectively) against high-fat diet (HFD)-induced metabolic disturbance in mice was investigated. HPPCs effectively attenuated HFD-induced obesity, insulin resistance, and lipid and glucose metabolic dysregulation and ameliorated inflammatory response and hepatic and colonic damage. It also improved the relative abundance of Bacteroidetes and Firmicutes, consistent with an anti-obesity phenotype. The favorable modulation of GM was further supported by improvement in the profile of fecal short-chain fatty acids. The higher dosage generally had a better performance in these effects than the low dosage. Moreover, serum metabolite profiling and pathway enrichment analysis revealed that HPPCs significantly modulated vitamin B metabolism-associated pathways and identified N-acetylneuraminic acid and 2-methylbutyroylcarnitine as potential biomarkers of the favorable effect on HFD-induced metabolic dysregulation. These findings highlight that dietary supplementation with red wine HPPCs is a promising strategy for the management of weight gain and metabolic dysregulation associated with HFD.

Seed mucilage-based advanced carrier systems for food and nutraceuticals: fabrication, formulation efficiency, recent advancement, challenges, and perspectives.

Seed mucilages are potential sources of natural polysaccharides. They are biodegradable, biocompatible, sustainable, renewable, and safe for human consumption. Due to the desirable physicochemical and functional properties (e.g. gelling, thickening, stabilizing, and emulsifying), seed mucilages have attracted extensive attention from researchers for utilization as a promising material for the development of advanced carrier systems. Seed mucilages have been utilized as natural polymers to improve the properties of various carrier systems (e.g. complex coacervates, beads, nanofibers, and gels) and for the delivery of diverse hydrophilic and lipophilic compounds (e.g. vitamins, essential oils, antioxidants, probiotics, and antimicrobial agents) to achieve enhanced stability, bioavailability, bioactivity of the encapsulated molecules, and improved quality attributes of food products. This review highlights the recent progress in seed mucilage-based carrier systems for food and nutraceutical applications. The main contents include (1) sources, extraction methods, and physicochemical and functional characteristics of seed mucilages, (2) application of seed mucilages for the development of advanced carrier systems, (3) major issues associated with carrier fabrication, and (4) mechanisms of carrier development, latest improvements in carrier formulation, carrier efficiency in the delivery of bioactive agents, and application in food and nutraceuticals. Furthermore, major challenges and future perspectives of seed mucilage-based carriers for a commercial application are discussed.

Pelargonidin-3-O-Glucoside Encapsulated Pectin-Chitosan-Nanoliposomes Recovers Palmitic Acid-Induced Hepatocytes Injury.

Pelargonidin-3-O-glucoside (Pg) is a well-known anthocyanin derivative possessing potential biological activity. Nonetheless, the bioactivity of Pg is limited due to instability in the physiological environment. Functionalized nanoliposomes using chitosan and/or pectin coating is an excellent carrier system for nanoencapsulation of food bioactive compounds such as Pg. Therefore, this study aimed to investigate the protective effect of Pg-loaded pectin-chitosan coated nanoliposomes against palmitic acid (PA)-induced hepatocytes injury in L02 cells. Firstly, Pg-loaded pectin-chitosan coated nanoliposomes were characterized using the DLS, HPLC, TEM, and cellular uptake study in L02 cells. Thereafter, we assayed the protective effect against PA-induced lipotoxicity, ROS and O2•- generation, mitochondrial dysfunction (MMP), and GSH depletion. Results showed that Pg-loaded nanoliposomes significantly reduced the PA-induced L02 cells toxicity via suppressing ROS production, O2•- generation, MMP collapse, and GSH reduction, whereas the free-Pg samples were not effective. On the contrary, the chitosan and/or pectin coated nanoliposomes showed higher results compared to coating-free nanoliposomes. Altogether, the results of our study ensured that Pg-loaded pectin-chitosan coated nanoliposomes was capable of reducing PA-induced hepatocytes injury. Thus, pectin-chitosan coated nanoliposomes can be useful for hepatocellular delivery of hydrophilic compounds with greater biological activity.

Green alternative methods for pretreatment of whole jujube before the drying process.

BACKGROUND: Jujube contains a waxy cuticle that acts as a barrier against fungal pathogens, prevents nutrition damage and leakage due to mechanical damage, and maintains water content. Chemical treatment before drying is the most commonly used method for whole jujube. Although chemical pretreatment can effectively enhance drying kinetics, it can lead to the loss of soluble nutrients and cause food safety issues due to chemical residues. Therefore, this study aimed to explore the effect of various pretreatments (cold plasma, cold plasma activated water, ultrasonics, thermosonication, and blanching) on the drying process and quality properties of whole jujube so as to find effective green alternatives to chemical pretreatment. RESULTS: The application of chemical, cold plasma, and thermosonication significantly altered the surface morphology of jujube by etching larger cracks and holes, which can facilitate the transfer of moisture, thereby improving the drying rate and the effective diffusivity. Chemical, cold plasma, and thermosonication pretreatment reduced drying time by 18%, 12%, and 7% respectively, thereby increasing the content of total phenolics by 13%, 12%, and 6% respectively, and enhancing antioxidant capacity (ferric reducing antioxidant power) by 13%, 11%, and 3% respectively. In addition, chemical and cold plasma pretreatment reduced the generation of 5-hydroxymethylfurfural by 25% and 15% respectively. CONCLUSION: Cold plasma is a promising green alternative method to chemical pretreatment for drying processes of whole jujube. © 2021 Society of Chemical Industry.

Advances in smart delivery of food bioactive compounds using stimuli-responsive carriers: Responsive mechanism, contemporary challenges, and prospects.

Many important food bioactive compounds are plant secondary metabolites that have traditional applications for health promotion and disease prevention. However, the chemical instability and poor bioavailability of these compounds represent major challenges to researchers. In the last decade, therefore, major impetus has been given for the research and development of advanced carrier systems for the delivery of natural bioactive molecules. Among them, stimuli-responsive carriers hold great promise for simultaneously improving stability, bioavailability, and more importantly delivery and on-demand release of intact bioactive phytochemicals to target sites in response to certain stimuli or combination of them (e.g., pH, temperature, oxidant, enzyme, and irradiation) that would eventually enhance therapeutic outcomes and reduce side effects. Hybrid formulations (e.g., inorganic-organic complexes) and multi-stimuli-responsive formulations have demonstrated great potential for future studies. Therefore, this review systematically compiles and assesses the recent advances on the smart delivery of food bioactive compounds, particularly quercetin, curcumin, and resveratrol through stimuli-responsive carriers, and critically reviews their functionality, underlying triggered-release mechanism, and therapeutic potential. Finally, major limitations, contemporary challenges, and possible solutions/future research directions are highlighted. Much more research is needed to optimize the processing parameters of existing formulations and to develop novel ones for lead food bioactive compounds to facilitate their food and nutraceutical applications.

Development and evaluation of a novel nanofibersolosome for enhancing the stability, in vitro bioaccessibility, and colonic delivery of cyanidin-3-O-glucoside.

The development of colon-specific carrier systems using polysaccharides for oral delivery of nutraceuticals is of great importance for the treatment and/or prevention of inflammatory bowel diseases. In this study, self-assembly with the assistance of vortexing and pulsed-ultrasonication was employed to develop a Fibersol®-2 (a digestion-resistant polysaccharide) and lipoid S75 based novel nanocarrier (denoted as nanofibersolosome) for the colonic delivery of cyanidin-3-O-glucoside (C3G). A series of nanofibersolosome formulations (CFS-0.5-4, 0.5-4 represent the ratios of Fibersol®-2:lipoid S75) were developed and their performance was compared with Fibersol®-2-free reference lipid formulation (CFS-0). The nanofibersolosomes (<150 nm) were spherical and unilamellar with high negative surface charge (-38 to -51 mV) and good encapsulation efficiency (EE > 90%). They performed much better than CFS-0 in retaining their physical properties during freeze drying, preventing particle aggregation, and retaining C3G during storage (4 and 25 ℃) and thermal treatments (40, 60, and 80 ℃). They also exhibited significantly higher stability during simulated gastrointestinal digestion than CFS-0. These desirable features of the nanofibersolosomes (especially CFS-0.5 and CFS-1) led to the efficient delivery of higher concentrations of C3G to the colon than CFS-0. Moreover, gastrointestinal-digested and colonic-fermented nanofibersolosome samples exhibited significantly higher DPPH radical scavenging activity and stronger promoting effect on short-chain fatty acid generation than CFS-0. These in vitro findings indicate that the novel nanofibersolosome possesses great potential for the colonic delivery of C3G and likely other hydrophilic labile phytochemicals that merits further evaluation in in vivo models.

Red Wine High-Molecular-Weight Polyphenolic Complex: An Emerging Modulator of Human Metabolic Disease Risk and Gut Microbiota.

Moderate red wine consumption has been linked to reduced chronic disease risk. Thus far, little has been known about the physicochemical properties and potential biological effects of high-molecular-weight polyphenolic complexes (HPPCs), a major fraction of red wine polyphenols. In this work, the stability and biochemical properties of HPPCs under simulated gastrointestinal conditions in vitro were studied. The results showed that HPPCs were resistant to simulated gastric digestion (SGD) and simulated intestinal digestion (SID). They exhibited significant inhibitory activity against key metabolic syndrome-associated digestive enzymes, achieving 17.1-90.9% inhibition of pancreatic α-amylase, lipase, and cholesterol esterase at 0.02-0.45 mg/mL. HPPCs were metabolized by gut microbiota (GM), leading to significantly enhanced antioxidant capacity when compared with the original, SGD, and SID samples. Furthermore, they favorably modulated GM profiles, which was accompanied by significantly increased short-chain fatty acid generation during the early colonic fermentation phase. These findings suggest that HPPCs are a promising modulator of human metabolic disease risk.

Effect of cold plasma pretreated hot-air drying on the physicochemical characteristics, nutritional values and antioxidant activity of shiitake mushroom.

BACKGROUND: Shiitake mushroom is one of the most popular delicious vegetables, although fresh shiitake mushroom has short shelf life as a result of biochemical degradation. Drying can prolong the shelf life of mushroom. Additionally, application of cold plasma pretreatments (CPT) before drying can preserve the product quality, processing costs and nutritional values. Therefore, we aimed to explore the effect of cold plasma pretreated hot-air drying at 50, 60 and 70 °C on the physicochemical characteristics, nutritional values and antioxidant activity of shiitake mushroom. RESULTS: Scanning electron microscopy micrographs showed that CPT induced the surface modification of fresh shiitake (such as cellular disarrangement, cellular shrinkages, disruption or break down of cell walls, and intracellular spaces and cavities) and facilitate the rapid drying than control samples. Furthermore, CPT improved the powder qualities (bulk density, water retention and swelling index) and preserved higher nutritional attributes (sugars, vitamins, phenolic acids contents and antioxidant activity) compared to the control groups. CONCLUSION: Conclusively, CPT could be a suitable alternative technique for improving drying characteristics and preserving nutritional attributes of agro-based products. © 2021 Society of Chemical Industry.

Suppression of palmitic acid-induced hepatic oxidative injury by neohesperidin-loaded pectin-chitosan decorated nanoliposomes.

The biological activity of neohesperidin (NH, a flavanone glycoside) is limited due to instability in the physiological environment. Thus, the current study aimed to explore the protective effect of NH-loaded pectin-chitosan decorated liposomes (P-CH-NH-NL) against palmitic acid (PA)-induced hepatic oxidative injury in L02 cells. The particles were characterized using DLS, TEM, HPLC, DSC, and cellular uptake study. Then, the protective effect of NH-loaded liposomal systems (NH-NLs) against PA-induced oxidative injury was evaluated in terms of cell viability study, intracellular ROS, superoxide ions (O2-), MMP, and cellular GSH determination. Our results exhibited that NH-NLs significantly lessened the PA-induced hepatic oxidative injury in L02 cells via decreasing ROS and O2- generation, reducing MMP collapse, and attenuating GSH reduction, whereas the free NH samples were ineffective. Furthermore, the coated NH-NLs were more effective than that of uncoated nanoliposome. Overall, our study confirmed that P-CH-NH-NL was capable of reducing PA-induced hepatic oxidative injury. Therefore, the pectin-chitosan decorated nanoliposome can be considered as an efficient delivery system for enhancing cellular uptake of lipophilic compound with controlled release and greater biological activity.

Cold plasma: An emerging pretreatment technology for the drying of jujube slices.

Jujube slices were pretreated by cold plasma for 15, 30, and 60 s on each side, followed by hot air drying at 50, 60, and 70 °C. Scanning electron microscopy investigation indicated that the application of cold plasma significantly changed the surface topography of jujube slice by etching larger cavities, which can facilitate moisture transfer and consequently enhance drying rate and effective diffusivity. Modified Henderson & Pabis model and Two-term model were the two most recommended models for describing the drying kinetics of jujube slices. Cold plasma pretreatment improved the contents of procyanidins, flavonoids, and phenolics by 53.81%, 33.89%, and 13.85% at most, respectively, and thereby enhanced antioxidant capacity by 36.85% at most. Besides, cold plasma pretreatment can reduce the production of 5-hydroxymethylfurfural by 52.19% at most. In summary, cold plasma can be used as a promising pretreatment tool for drying processes of jujube slices.

Improving the physicochemical stability and functionality of nanoliposome using green polymer for the delivery of pelargonidin-3-O-glucoside.

This study aimed to improve the physicochemical stability of nanoliposome (NL) with enhanced functionality for the delivery of Pelargonidin-3-O-glucoside (P3G) using biopolymers, i.e. chitosan (CH) and pectin (P). In this study, we successfully developed stabilized liposomal carriers, i.e. CH-conjugated NL (CH-NL) and P-conjugated CH-NL (P-CH-NL) using an optimum concentration of CH (0.6 wt%) and P (0.5 wt%). Results revealed that P-CH-NL had better physical stability to salt and pH with maximum P3G retention (>97%) under oxidative, thermal, and UV conditions. Nanoliposomes were more stable under refrigerated-storage and ensured high P3G retention (>96%). In vitro mucoadhesion study revealed that CH-NL had better mucin adsorption efficiency (59.72%) followed by P-CH-NL and NL. Furthermore, CH-NL and P-CH-NL alternatively had better stability to serum than NL. Taken together, the stabilization of nanoliposome using chitosan and pectin can be a promising approach for the delivery of hydrophilic compounds in association with enhanced stability and functionality.

Surface decoration of neohesperidin-loaded nanoliposome using chitosan and pectin for improving stability and controlled release.

The aim of this study was to improve the physicochemical stability of neohesperidin (NH) using nanoliposomal encapsulation in association with surface decoration strategy employing chitosan (CH) and pectin (P). Different nanoliposomal systems, i.e. NH-loaded nanoliposome (NH-NL), CH-coated NH-NL (CH-NH-NL), and P-coated CH-NH-NL (P-CH-NH-NL) were characterized through DLS, HPLC, TEM, and FTIR. The results confirmed good encapsulation efficiency (>90%) and successful layer formation with nano-sized and spherical carrier. Both CH-NL and P-CH-NL exhibited better physicochemical stability than NL under storage, thermal, pH, ionic, UV, oxidative, and serum conditions. In vitro mucin adsorption study revealed that CH-NL (60%) was more effective in mucoadhesion followed by P-CH-NL (46%) and NL (41%). Furthermore, P-CH-NL showed better performance in NH retention under different food simulants compared to CH-NH-NL and NH-NL, in which the release was mainly governed by the diffusion process. Thus, the P-CH conjugated nanoliposome could be a promising nano-carrier for neohesperidin.

Colonic delivery of pelargonidin-3-O-glucoside using pectin-chitosan-nanoliposome: Transport mechanism and bioactivity retention.

Colon-targeted delivery is an active area of research as it can improve drug stability, bioactivity, and lessen the systematic toxicity. In this study, the colon-specific delivery of pelargonidin-3-O-glucoside (P3G) was investigated using pectin (P)/chitosan (CH)-functionalized nanoliposome (NL). The food simulant stability, transport mechanism, and bioactivity retention potential of carrier systems were studied. Results showed that polymer-coated nanoliposomes (P-CH-NL and CH-NL) improved the thermal and food simulant stability as well as enhanced the P3G retention during the in vitro digestion. The maximum P3G retention after enzymatic and non-enzymatic digestion was observed by P-CH-NL and the values were 47.5% and 57.5%, respectively. However, all nanoliposomal carriers followed Fickian diffusion mechanism both in in vitro food simulants and in vitro digestion models. Digested functionalized nanoliposomes revealed higher antioxidant properties after gastric digestion. Following by simulated intestinal fluid digestion, ABTS antioxidant activity of P-CH-P3G-NL was 12.52% and 6.31% higher than that of P3G-NL and CH-P3G-NL, respectively, while DPPH scavenging capacity of P-CH-P3G-NL was 5.57% and 1.86% greater than that of P3G-NL and CH-P3G-NL, respectively. Therefore, the developed functionalized nanoliposome can be useful for colon-targeted delivery and applicable in functional foods and/or beverages.

In vitro study of bioaccessibility, antioxidant, and α-glucosidase inhibitory effect of pelargonidin-3-O-glucoside after interacting with beta-lactoglobulin and chitosan/pectin.

Polysaccharides and fruit extracts are applied in dairy products to enhance their nutritional property, but the effects of such formulations on the functions and biological activities are yet to be explored. Therefore, this study was aimed at evaluating the effect of interactions among milk protein (beta-lactoglobulin; BLG), polysaccharides (pectin, P; chitosan, CH), and anthocyanin (pelargonidin-3-O-glucoside; P3G) in improving the bioavailability and biological activity of P3G. After gastrointestinal digestion (GID), the content of free P3G in different model solutions were as follows: P3G-alone (73.59 µg/mL), P3G-P (66.59 µg/mL), P3G-CH (36.72 µg/mL), P3G-BLG (64.92 µg/mL), P3G-P-BLG (64.92 µg/mL), and P3G-CH-BLG (39.61 µg/mL). Less amount of free P3G in model solutions indicated increased complex formation of P3G with protein and/or polysaccharides during GID. These complexes resulted in protection and progressive release of P3G in the gastrointestinal tract. Chitosan exhibited more protection to P3G compared with P and BLG. In addition, α-glucosidase inhibitory activity and ROS scavenging activities of conjugated-P3G samples were potentially augmented after GID. However, the presence of polysaccharides and protein in the model solutions did not show any negative effect on the biological activity of P3G. Thus, pure P3G can be used as a nutritional ingredient in dairy industries.

Optimization of process parameters for improved production of biomass protein from Aspergillus niger using banana peel as a substrate.

This study was aimed to optimize the process variables for improved production of biomass protein using Aspergillus niger from banana fruit peel by the use of response surface methodology. A five-level-four factors central composite rotatable design was applied to elucidate the influence of process variables viz. temperature (20-40 °C), pH (4-8), substrate concentration (5-25%), and fermentation period (1-5 days) on biomass and protein content. The second-order polynomial models were established, which effectively explicated the variation in experimental data and significantly epitomized the appreciable correlation between independent variables and responses. After numerical optimization, the predicted optimum conditions (temperature of 31.02 °C, pH of 6.19, substrate concentration of 19.92%, and the fermentation period of 4 days) were obtained with biomass of 24.69 g/L and protein of 61.23%, which were verified through confirmatory experiments.