Search Results (36)

The integration of emerging non-thermal technologies for the valorization of plant bioactive compounds is gaining increasing attention in food and natural product research. In this study, Atmospheric Room Temperature Plasma (ARTP) pretreatment combined with pulsed electric field (PEF) extraction was investigated as an innovative strategy to enhance the recovery of bioactive compounds from Cannabis sativa L. (cv. Futura 75) leaves. Response Surface Methodology (RSM) was employed to identify the best conditions within the investigated range for the ARTP process based on total phenolic content (TPC), DPPH^• radical scavenging activity, and ferric reducing antioxidant power (FRAP). Under the best conditions within the investigated range (16.6 mm electrode–sample distance, 1.6 mm sample thickness, 70% plasma intensity, 10 L min⁻¹ nitrogen flow, and 1 min treatment), ARTP pretreatment increased TPC, FRAP, and DPPH values by 23.0%, 68.6%, and 46.1%, respectively, compared with PEF extraction alone. HPLC–DAD analysis confirmed that ARTP pretreatment selectively enhanced the recovery of the identified phenolic compounds, particularly protocatechuic acid and luteolin-7-O-glucoside, supporting the observed increases in antioxidant capacity. The extract obtained under the best conditions within the investigated range was incorporated into a model vinegar system (6% v/v acetic acid), and its stability was evaluated over 28 days by investigating the effects of extract concentration, storage temperature, and light exposure on TPC and antioxidant capacity. Elevated temperature (40 °C) and light exposure accelerated antioxidant degradation, whereas the highest extract concentration (1.2 g L⁻¹), storage at ambient temperature (20 °C), and absence of light resulted in improved stability. These findings demonstrate that the combined ARTP–PEF approach enhances phenolic extraction from C. sativa leaves and yields extracts with improved stability, highlighting its potential for developing functional ingredients for acidified food systems. Full article

In recent years, consumers have been turning to natural products. Following this trend, the European Union has now recognized natural rosemary extract as a natural preservative (E392) in various foods. Considering this, the current study aimed to extract antioxidant compounds from rosemary leaves employing green techniques. More specifically, non-toxic solvents (water-ethanol) and green extraction techniques, such as Atmospheric Room Temperature Plasma (ARTP) for pretreatment of rosemary leaves and subsequently Pressurized Liquid Extraction (PLE) for the recovery of the antioxidant compounds, were used. Response Surface Methodology (RSM) was applied to optimize ARTP operating parameters, and the optimized ARTP + PLE extract was evaluated using spectrophotometric assays and HPLC-DAD. Under optimal conditions, the extract yielded a total polyphenol content (TPC) of ~72 mg gallic acid equivalents per g and an ascorbic-acid-associated reducing capacity (AACRC) of ~19 mg/g, while the extract exhibited relatively high antioxidant capacity as assessed by the FRAP (ferric-reducing antioxidant power) assay (~924 μmol ascorbic acid equivalents per g) and DPPH radical scavenging activity (~467 μmol ascorbic acid equivalents per g). Compared with the untreated PLE control, the ARTP-PLE extract showed increases of 3.1% in TPC, 2.03% in FRAP, 5.61% in DPPH and 25.86% in AACRC. Four commercial rosemary antioxidant preparations (E392) of different formulation types were also analyzed to contextualize the composition of the optimized extract. The commercial products, analyzed in their native form, displayed compositional patterns characteristic of oil-based formulations, whereas the ARTP + PLE extract reflected the profile of hydroethanolic rosemary extracts. These differences arise from extraction conditions and solvent systems. The comparison is descriptive and does not imply quality ranking or stability differences. Overall, the study provides insight into how plasma pretreatment can influence the composition of rosemary extracts within hydroethanolic systems. Full article

2,3-Butanediol (2,3-BD) is a versatile bulk chemical, serving as a high-boiling green solvent, a key monomer for polyesters and polyurethanes, and a crucial intermediate in the production of fine chemicals and cosmetics. Additionally, it is a key intermediate for pharmaceuticals and agrochemicals, supporting green chemistry. Microbial production of 2,3-BD offers a sustainable bioprocess but faces the challenge of high-concentration product inhibition. In an effort to improve 2,3-BD tolerance and production in Corynebacterium glutamicum, the engineered strain CGK4 was subjected to atmospheric and room temperature plasma (ARTP) mutagenesis, from which three superior mutant strains were isolated. Genomic and reverse engineering analyses revealed two mutations beneficial for tolerance and seven mutations beneficial for production. Among these, mutations atpG^L250P, cobT^V192V, and ctaE^L79P increased 2,3-BD titer by 18.0%, 14.5%, and 15.0%, respectively. Furthermore, we found that while certain mutations markedly enhanced stress tolerance, the highest-producing strains achieved a balanced metabolic state rather than maximal tolerance, indicating a non-linear relationship between the two traits. Finally, through mutation combination optimization, the optimal strain K4-cobT-ctaE was constructed. In shake-flask fed-batch fermentation, this strain produced 84.35 ± 3.04 g/L (2R,3R)-2,3-BD, which was 38.3% higher than that of the parental strain. Our findings provide a promising approach to addressing 2,3-BD tolerance challenges and promote the development of microbial production platforms for industrial application. Full article

Laurel (Laurus nobilis L.) is a characteristic species of the Mediterranean flora, valued for its medicinal, aromatic, and culinary uses. Many of these properties are attributed to its volatile constituents. In this study, the effect of Atmospheric Room Temperature Plasma (ARTP) pretreatment on laurel powder was evaluated, with emphasis on volatile recovery and food application. Volatile extraction was optimized using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography–mass spectrometry (GC-MS), investigating key parameters such as salt concentration, extraction temperature, equilibration time, extraction time, and fiber type. Subsequently, critical ARTP variables (nitrogen flow, treatment duration, treatment distance, substrate thickness, and plasma power) were optimized. Response Surface Methodology was applied in both optimization processes. The results demonstrated that fiber type was the most influential factor for volatile recovery, with extraction temperature also exerting a significant effect. A more nuanced pattern emerged during ARTP pretreatment, where moderate plasma intensities enhanced the recovery of several key volatiles. This trend was not uniform across compounds, indicating that plasma-induced microstructural changes interact with the physicochemical properties of individual analytes. To demonstrate food relevance, a ready-to-use broth model prepared with laurel powder confirmed improved headspace transfer of characteristic volatiles following ARTP treatment. Taken together, these findings suggest that ARTP can serve as a practical, non-thermal pretreatment for improving volatile release, supporting its potential use in the food, pharmaceutical, and cosmetic industries. Full article

Astaxanthin, one of the most commercially valuable carotenoids, is renowned for its potent antioxidant and anti-inflammatory properties and is experiencing growing demand across diverse industries. To enhance astaxanthin production in Paracoccus marcusii, compound mutagenesis was performed using ethyl methanesulfonate (EMS), ultraviolet (UV) radiation, and atmospheric room temperature plasma (ARTP) treatment. Subsequently, a high-throughput microbial microdroplet culture (MMC) system was employed to select fast-growing microdroplet, followed by screening for high astaxanthin-producing mutants on dual-inhibitor plates. The mutant M21 was isolated and exhibited a significant increase of 16.86% in astaxanthin content (1.53 mg/g) and a 19.81% increase in astaxanthin production (11.71 mg/L) compared with the wild type (WT) (p < 0.05). Moreover, the enhanced phenotype of M21 was genetically stable. Response surface methodology (RSM)-based optimization of fermentation conditions further increased astaxanthin content and production to 1.72 mg/g and 12.92 mg/L, respectively, corresponding to improvements of 16.44% and 23.02% over the WT, while simultaneously reducing culture time, total nitrogen requirements, and sodium lactate consumption, thereby lowering production costs. This study achieved significant enhancement of astaxanthin production through novel mutant breeding and fermentation optimization, underscoring the effectiveness of this integrated strategy for application in industrial biotechnology. Full article

Esters are predominant fragrance components in various traditional fermented foods. Hongqu rice wine, a beverage gaining popularity among young consumers in China, largely owes its aromatic profile to esterases derived from Monascus species. However, research on esterification characteristics of Monascus strains remains limited, constraining efforts to improve the quality and flavor of Hongqu rice wine. To better understand their esterification characteristics of commercial Monascus strains from different regions of China and further develop a high-quality esterifying Monascus strain for the liquor industry, we identified five Monascus isolates from red koji samples used in rice wine fermentation. Their esterification activity was evaluated by preparing red koji through solid-state fermentation of wheat bran under conditions simulating industrial production. Among the isolates, M. purpureus M21 exhibited the highest reported esterification activity to date, reaching 88.5 ± 8.6 U. Through atmospheric and room-temperature plasma (ARTP) mutagenesis breeding, the esterification activity of M. purpureus M21 was further enhanced by 41% to 124.8 U. In summary, this study not only figures out the properties of commercial esterifying Monascus from diverse regional sources but also significantly enhances the esterification performance of a potent esterifying Monascus strain without invoking GMO controversies. This high-performance esterifying Monascus strain presents a promising fermentation starter to enhance the flavor profile of Hongqu rice wine and diverse fermented beverages, thereby meeting evolving consumer preferences. Full article

Over the past few years, naturally sourced bioactive molecules have drawn increased attention for their antioxidant capacity and wide-ranging health effects. At the same time, interest in eco-friendly extraction approaches has risen sharply. Atmospheric Room Temperature Plasma (ARTP), a novel non-thermal pretreatment method, has emerged as a promising green technology due to its minimal environmental impact, cost-effectiveness, and superior extraction efficiency compared to conventional methods. In this study, ARTP pretreatment—optimized across variables such as treatment distance, substrate thickness, power, nitrogen flow, and duration—was combined with ultrasonic-assisted extraction to enhance the recovery of bioactive compounds from Moringa oleifera leaves. Both techniques were optimized using Response Surface Methodology (RSM). Under optimal conditions, the extract yielded a total polyphenol content of approximately 40 mg gallic acid equivalents per gram of dry weight. Antioxidant activity, assessed via ferric-reducing antioxidant power (FRAP) and DPPH radical scavenging assays, reached ~280 and ~113 μmol ascorbic acid equivalents per gram dry weight, respectively, and the ascorbic acid content was ~5.3 mg/g. These findings highlight the potential of ARTP as an effective and sustainable pretreatment method for producing high-value phytochemical extracts, with promising applications in the food, pharmaceutical, and cosmetic industries. Full article

Volvariella volvacea, a fungal species of Volvariella within the Pluteaceae family, is predominantly cultivated in southern China. Polysaccharides, the primary bioactive constituents of V. volvacea, exhibit diverse pharmacological activities. However, current cultivation practices face challenges due to the genetic heterogeneity of strains, leading to inconsistent content and compositional variability of polysaccharides and other functional components. ARTP, denoting atmospheric and room-temperature plasma, is a technology capable of generating plasma jets at ambient pressure with temperatures ranging from 25 to 40 °C. These jets feature high concentrations of highly reactive species, including but not limited to excited-state helium atoms, oxygen atoms, nitrogen atoms, and OH radicals. This study aims to develop high-yielding exopolysaccharide (EPS) strains through integrated ARTP mutagenesis and genome shuffling, thereby overcoming current cultivation bottlenecks. ARTP mutagenesis and genome shuffling significantly boosted EPS production in V. volvacea. ARTP generated nine stable mutants with >20% higher EPS yields. Subsequent genome shuffling (three rounds of protoplast fusion) produced the hybrid strain SL212, which achieved 46.85 g/L of EPS, an 111.67% increase over that of the parent strain under identical conditions. Metabolomics and transcriptomics analyses revealed that differential metabolites and genes were mainly enriched in galactose metabolism, ABC transporter pathways, and the tricarboxylic acid cycle. These pathways enhance monosaccharide biosynthesis and generate ATP, providing both precursors and energy for polysaccharide polymerization, thereby driving EPS overproduction. Preliminary mechanistic analysis identified the key contributing factors driving the elevated polysaccharide biosynthesis. Full article

The demand for L-tryptophan (L-Trp) has been rapidly increasing across various industries, including pharmaceuticals, food, and animal feed. However, traditional production methods have been unable to efficiently meet this growing demand. Hence, this study aimed to develop strategies for enhancing L-Trp production in Escherichia coli. Firstly, an L-Trp-producing strain was selected and subjected to atmospheric and room temperature plasma (ARTP) mutagenesis to generate a mutant library. This was followed by high-throughput screening using an L-Trp-specific riboswitch and a yellow fluorescent protein (YFP)-based biosensor in a flow cytometric cell sorting (FACS) system. Among the screened mutants, GT3938 exhibited a 1.94-fold increase in L-Trp production. Subsequently, rational metabolic engineering was applied to GT3938 by knocking out the L-Trp intracellular transporter gene (tnaB), enhancing the expression of the aromatic amino acid exporter (YddG) and optimizing precursor supply pathways. The resulting strain, zh08, achieved an L-Trp titer of 3.05 g/L in shake-flask fermentation, representing a 7.71-fold improvement over the original strain. This study demonstrated an effective strategy for industrial strain development by integrating biosensor-assisted, high-throughput screening with rational metabolic engineering. Full article

In this study, we established a mutagenesis and high-throughput screening system to select a high-yielding glutathione (GSH)-producing strain of Saccharomyces cerevisiae. The parent strain was mutated by atmospheric and room temperature plasma (ARTP) technology and cultivated using ethionine plate cultivation. Subsequently, high-throughput screening was performed using liquid deep microtiter plates (MTPs) for cultivation and a microplate reader for rapid GSH detection. The results demonstrated the successful selection of a stable mutant strain, S-272, which exhibited significantly enhanced GSH production. Fermentation validation in 5 L bioreactors revealed that S-272 achieved a 14.7% higher final GSH concentration and a 19.5% higher intracellular GSH content compared to the parent strain. The improved performance of S-272 was attributed to enhanced ethanol utilization, elevated activity of γ-glutamylcysteine synthetase (γ-GCS), and increased intracellular trehalose content. This study presents an effective strategy for developing high GSH-yield strains using ARTP complex mutagenesis technology combined with high-throughput screening. Full article

The valorization of agricultural residues, particularly corn stover, represents a sustainable approach for resource utilization and protein production in which high-performing microbial strains are essential. This study systematically evaluated fungal lignocellulolytic capabilities during corn stover solid-state fermentation and employed atmospheric and room-temperature plasma (ARTP) mutagenesis to enhance the degradative capacity of Trichoderma longibrachiatum. Comparative screening revealed that T. longibrachiatum exhibited superior comprehensive degradation of the major lignocellulosic components compared to other tested strains. ARTP mutagenesis yielded mutant strain TL-MU07, which displayed significantly enhanced enzymatic capabilities with improvements in FPase (22.1%), CMCase (10.1%), and xylanase (16.1%) activities, resulting in increased cellulose degradation (14.6%) and protein accumulation (14.7%). Proteomic analysis revealed 289 significantly differentially expressed proteins, with pathway enrichment demonstrating enhancement of glycosaminoglycan degradation, amino sugar metabolism, and membrane remodeling. Key mechanistic adaptations included downregulation of Zn(2)-C6 transcriptional repressors, upregulation of detoxification enzymes (ALDH-like proteins), and enhanced secretory pathway components. The ARTP-derived mutant strain TL-MU07 represents a valuable microbial resource for agricultural waste bioconversion, offering enhanced lignocellulolytic capabilities for industrial applications while elucidating specific proteomic changes associated with improved biomass degradation efficiency for sustainable protein production in the circular bioeconomy. Full article

Non-Saccharomyces yeasts have the potential to ameliorate wine ethanol levels, but such fit-for-purpose yeast strains are still lacking. Seventy-one indigenous non-Saccharomyces yeasts isolated from spontaneous fermentations of four wine regions in China (Ningxia, Xinjiang, Gansu, and Shaanxi) were screened for ethanol formation and were characterized for major metabolite profiles in synthetic grape juice fermentation to obtain non-Saccharomyces yeasts with low ethanol yields. Four Hanseniaspora strains with less volatile acidity production were primarily selected, and their ethanol yield was reduced by 22–32% compared to S. cerevisiae. These strains were further evaluated for oenological properties, namely ethanol and temperature tolerance, H₂S production, and killer activities against S. cerevisiae. Strain HuC-3-2 was then subjected to Atmospheric Room Temperature Plasma (ARTP) mutagenesis, and a mutant (HuC32-2-72) with rapid growth and optimized ethanol-reducing capability was obtained. The best-performing strains were further characterized in sequential fermentations with S. cerevisiae in Merlot juice, and resulted in a 1.4% v/v decrease in ethanol yield. Comprehensive analysis of yeast populations and the production of key metabolites highlighted important carbon sinks, as well as glycerol formation, partially accounting for the ethanol reduction. In addition to ethanol amelioration, the Hanseniaspora strains also led to alterations in many metabolites, including volatile compounds and some organic acids, which can further modulate wine aroma and flavor. Full article

Menaquinone-7 (MK-7) plays a crucial role in preventing fractures and certain cardiovascular diseases and is one of the essential vitamins in the human body. In this study, a strain of Bacillus subtilis that produces MK-7 was isolated from commercially available natto fermentation agents, with an MK-7 titer of 75 mg/L. It was named L-5. Firstly, by employing Atmospheric and Room Temperature Plasma (ARTP) mutagenesis technology and protoplast fusion techniques, mutants resistant to 1-hydroxy-2-naphthoic acid (HNA) and diphenylamine (DPA) were obtained, with the titer of MK-7 reaching 196 mg/L. It was named R-8. Based on whole-genome sequencing technology, four mutants involved in the MK-7 synthesis pathway of strain L-5 were identified: 2-succinyl-5-enol-pyruvate-6-hydroxy-3-cyclohexen-1-carboxylic acid, MenD (S249L); (1,4)-dihydroxy-2-naphthalic acid-heptaisoprenyltransferase, MenA (S196L); 1-deoxy-D-xylose-5-phosphate synthetase, Dxs (N60D, Q185H); and hydroxy acid reductive isomerase, Dxr (Q351K). The overexpression of these mutants led to increases in MK-7 production of 19 mg/L, 20 mg/L, 17 mg/L, and 16 mg/L, respectively, compared to the unmutated genes. These mutations have been shown to be effective. To further enhance the production of MK-7, the mutants menD (S249L), menA (S196L), Dxs (N60D, Q185H), and Dxr (Q351K) were co-expressed. The final titer of MK-7 reached 239 mg/L. This study provides theoretical support for the future genetic modification of key enzymes in the MK-7 biosynthetic pathway. Full article

Nattokinase (NK), a serine protease with high thrombolytic activity, has significant potential for application in foods intended for special health benefits. However, the NK production in wild-type Bacillus subtilis natto is relatively low. In this study, a high-yielding NK and genetically stable mutant strain (B. subtilis JNC002.001, 300.0 ± 4.7 FU/mL) was obtained through atmospheric and room temperature plasma (ARTP) mutagenesis. It increased NK activity by 1.84 times compared to the initial strain SD2, demonstrating significant prospects for NK production and food fermentation applications. Additionally, the B. subtilis JNC002.001 exhibited notable alterations in growth characteristics, glucose consumption, and sporulation. This study further elucidated the mechanism of enhanced NK production at the molecular level. Genome resequencing revealed that the mutant genes in JNC002.001 included 10 single nucleotide polymorphisms (SNPs) and one insertion, among which the kinA and gltA genes were associated with sporulation and NK synthesis, respectively. In terms of the transcriptional level, the NK-coding gene aprN was up-regulated 9.4 times relative to the wild-type strain. Most of the genes related to central carbon metabolism and the Sec secretion pathway were up-regulated. In addition, the expression of regulatory factors associated with the transcription of the aprN gene and the sporulation process provided evidence for high NK expression and sporulation deficiency in JNC002.001. These results could provide insights into the mechanism of NK production and facilitate the construction of engineered strains with high NK yield. Full article

Strong-flavor Baijiu, a widely popular distilled spirit in China, derives its characteristic aroma and quality largely from ethyl hexanoate, a key flavor compound. The concentration of ethyl hexanoate, influenced by its precursor hexanoic acid, is critical in defining the style and quality of this Baijiu variety. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis technology was applied to strains isolated from Strong-flavor Daqu to enhance their acid and ester production capabilities. A hexanoic acid-producing strain, identified as Bacillus velezensis WY4 through morphological, physiological, biochemical, and molecular analyses, was used as the starting strain. Following 90 s of ARTP exposure, a mutant strain, WY4-3, was successfully developed, achieving a balance between high mutation diversity and moderate lethality. WY4-3 exhibited robust growth across a pH range of 4.2 to 5.0 and demonstrated high ethanol tolerance. After five days of fermentation, WY4-3 produced 0.36 g/L of total acid and 0.528 g/L of total ester, surpassing the wild-type strain. Enzymatic activity assays revealed significant enhancements in amylase (9.13%), saccharifying enzyme (101.72%), and esterification (573.71%) activities in WY4-3. Validation in multiple artificial esterification systems further confirmed the superior ester production capacity of this mutant strain. These findings enrich the microbial germplasm resources for Baijiu brewing and provide a solid foundation for strain selection and genetic improvement in Baijiu production processes. This study highlights the potential of ARTP mutagenesis in optimizing brewing microorganisms and improving the quality and consistency of Strong-flavor Baijiu. Full article