Search Results (25)

Agar can be degraded into agar-oligosaccharides by physical, chemical, and biological methods, but the further industrial application of agar-oligosaccharides has been limited by the environmental pollution of traditional agar-oligosaccharides preparation methods and the lack of novel agarase. In this study, we reported the screening of 12 strains with agar-degrading activity from sea cucumber intestine and mucus using a combination of Gram’s iodine staining and 3,5-dinitrosalicylic acid (DNS) method, during which five fungal strains exhibited high agarase activity. Their production of different agarases and agar-oligosaccharides could be visualized by zymogram assay and thin-layer chromatography. A strain ACD-11-B with the highest agarase activity showed 99.79% similarity to Aspergillus sydowii CBS593.65 for ITS rDNA sequence. Strain ACD-11-B produced five possible agarases with predicted molecular weights of 180, 95, 43, 33, and 20 kDa, approximately. The optimal temperature and pH of the crude enzyme production by strain ACD-11-B were 40 °C and 6.0. The crude enzyme was stable at 30 °C, and Ca²⁺, K⁺, and Na⁺ could increase the activity of the crude enzyme. Its agarases demonstrated remarkable salt tolerance and substrate specificity, with neoagarobiose (NA2) identified as the main degradation product. These results indicate that the fungal strain ACD-11-B can secrete agarases with potential in industrial applications, making it a new producer strain for agarase production. Full article

This study aimed to evaluate the applicational possibilities of enzymatically modified apple pomace (AP) in conjunction with probiotics as value-added ingredients for the production of jelly candies. AP was enzymatically modified with Pectinex^® Ultra Tropical, Viscozyme^® L, and Celluclast^® 1.5 L (Novozyme A/S, Bagsværd, Denmark), and the soluble and insoluble dietary fibre content was determined using the Megazyme kit (Megazyme International Ireland Ltd., Wicklow, Ireland), reducing sugar content using the 3,5-dinitrosalicylic acid assay. The technological properties of the modified AP, such as its swelling capacity, water-retention capacity, oil-retention capacity, bulk density, and static and thermal emulsion stability, were evaluated. Enzymatically modified AP hydrolysed with Celluclast^® 1.5 L was used for the production of jelly candies supplemented with Bifidobacterium animalis DSM 20105. The survival of probiotics in the jelly candies during in vitro digestion, the viability of probiotics during candy storage, and candy quality characteristics were analysed. Enzymatically modified AP had different carbohydrate compositions and technological properties, depending on the enzyme preparation used. Although the viability of probiotics in the jelly candies decreased during storage, a significantly higher viability of B. animalis was determined in jelly candies supplemented with hydrolysed AP compared with control candies made without AP after digestion in the saline, gastric, and intestine phases. This study shows that Celluclast^® 1.5 L can be used for increasing the soluble dietary fibre in AP (18.4%), which can be further applied, in conjunction with B. animalis, for added-value jelly candy production. Full article

Brown-rot fungi are large fungi that can decompose the cell walls of wood; they are notable for their secretion of diverse and complex enzymes that synergistically hydrolyze natural wood cellulose molecules. Fomitopsis pinicola (F. pinicola) is a brown-rot fungus of interest for its ability to break down the cellulose in wood efficiently. In this study, through a combination of rDNA-ITS analysis and morphological observation, the wood decay pathogen infecting Korean pine (Pinus koraiensis Siebold and Zucc.) was identified. Endoglucanase (CMCase) and β-glucosidase were quantified using the DNS (3,5-Dinitrosalicylic acid) method, and the cellulase activity was optimized using a single-factor method and orthogonal test. The results revealed that the wood-decaying fungus NE1 identified was Fomitopsis pinicola with the ITS accession number OQ880566.1. The highest cellulase activity of the strain reached 116.94 U/mL under the condition of an initial pH of 6.0, lactose 15 g·L⁻¹, KH₂PO₄ 0.5 g·L⁻¹, NH₄NO₃ 15 g·L⁻¹, MgSO₄ 0.5 g·L⁻¹, VB₁ 0.4 g·L⁻¹, inoculated two 5 mm fungal cakes in 80 mL medium volume cultured 28 °C for 5 days. This laid a foundation for improving the degradation rate of cellulose and biotransformation research, as well as exploring the degradation of cellulose by brown rot fungi. Full article

An abundance of refractory cellulose is the key limiting factor restricting the resource utilization efficiency of silkworm (Bombyx mori) excrement via composting. Screening for cellulose-degrading bacteria is likely to provide high-quality strains for the safe and rapid decomposition of silkworm excrement. In this study, bacteria capable of degrading cellulose with a high efficiency were isolated from silkworm excrement and the conditions for cellulase production were optimized. The strains were preliminarily screened via sodium carboxymethyl cellulose culture and staining with Congo red, rescreened via a filter paper enzyme activity test, and identified via morphological observation, physiological and biochemical tests, and phylogenetic analysis of the 16S rDNA sequence. Enzyme activity assay was performed using the 3,5-dinitrosalicylic acid method. DC-11, a highly cellulolytic strain, was identified as Bacillus subtilis. The optimum temperature and pH of this strain were 55 °C and 6, respectively, and the filter paper enzyme activity (FPase), endoglucanase activity (CMCase), and exoglucanase activity (CXase) reached 15.40 U/mL, 11.91 U/mL, and 20.61 U/mL. In addition, the cellulose degradation rate of the treatment group treated with DC-11 was 39.57% in the bioaugmentation test, which was significantly higher than that of the control group without DC-11 (10.01%). Strain DC-11 was shown to be an acid-resistant and heat-resistant cellulose-degrading strain, with high cellulase activity. This strain can exert a bioaugmentation effect on cellulose degradation and has the potential for use in preparing microbial inocula that can be applied for the safe and rapid composting of silkworm excrement. Full article

Interactions between food components have a positive impact in the field of food science. In this study, the effects of tea polyphenol on the structural and physicochemical properties of Chinese yam starch using autoclave-assisted pullulanase treatment were investigated. X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, rapid visco analysis, differential scanning calorimetry, and the 3,5-dinitrosalicylic acid method were applied in this study. The results showed that the Chinese yam starch–tea polyphenol complex formed a structural domain with higher thermal stability along with lower pasting viscosities than native starch. The in vitro digestibility of Chinese yam starch decreased with the addition of the tea polyphenol, and the amount of resistant starch content in the complex was 56.25 ± 1.37%, significantly higher than that of native starch (p < 0.05). In addition, the complex showed a B+V-type crystalline structure, which confirmed that the interaction modes between the starch and tea polyphenol include hydrogen bonding and hydrophobic interactions. Moreover, the appearance of an irregular sponge network structure of the complex further supported the interactions between the starch and tea polyphenol. This study provides a theoretical basis for the development of functional foods using Chinese yam starch. Full article

Maize is a crucial cereal crop in China, serving both as a staple food and an essential industrial resource. Northern corn leaf blight (NCLB) is a disease of corn caused by a fungus, Exserohilum turcicum (sexual stage Setosphaeria turcica). This study aimed to assess the biocontrol potential of various Trichoderma strains against Exserohilum turcicum 101 in Jilin, China. Through dual culture tests, the Trichoderma strains were categorized into four groups based on their antagonistic abilities. Eleven Trichoderma strains exhibited strong antagonistic behavior, with comparable or faster growth rates than E. turcicum 101. Microscopic observations confirmed that T. asperellum 576 hyphae effectively encircled E. turcicum 101 hyphae, reinforcing their antagonistic behavior. The production of non-volatile and volatile substances by the Trichoderma strains was evaluated, with T. asperellum 576 showing the highest potency in producing non-volatile and volatile substances, leading to an impressive 80.81% and 65.86% inhibition of E. turcicum 101 growth. Remarkably, co-culture suspensions of T. asperellum 576 + E. turcicum 101 and T. atroviride 393 + E. turcicum 101 exhibited strong antifungal activity. Furthermore, the activities of chitinase, β-1.3-glucanase, and cellulase were evaluated using the 3, 5-dinitrosalicylic acid (DNS) method. T. asperellum 576 + E. turcicum 101 displayed stronger cell wall degradation enzyme activity compared to T. atroviride 393 + E. turcicum 101, with values of 8.34 U/mL, 3.42 U/mL, and 7.75 U/mL, respectively. In greenhouse conditions, the application of a 10⁷ spores/mL conidia suspension of T. asperellum 576 significantly enhanced maize seed germination and plant growth while effectively suppressing E. turcicum 101 infection. Maize seedlings inoculated/treated with both E. turcicum 101 and T. asperellum 576 demonstrated substantial improvements compared to those inoculated solely with E. turcicum 101. The T. asperellum 576 treatment involved a 10⁷ spores/mL conidia suspension applied through a combination of foliar spray and soil drench. These findings highlight T. asperellum 576 as a promising biocontrol candidate against northern leaf blight in maize. Its antagonistic behavior, production of inhibitory compounds, and promotion of plant growth all contribute to its potential as an effective biocontrol agent for disease management. Full article

The inhibition of carbohydrate digestion by plant bioactive compounds is a potential dietary strategy to counteract type 2 diabetes. Indeed, inhibition of α-amylase, a key enzyme that carries out the bulk of starch digestion, has been demonstrated for a range of bioactive compounds including anthocyanins; however, sample pigmentation often interferes with measurements, affecting colorimetric assay outcomes. Therefore, the present study compared the performance of a direct chromogenic assay, using 2-chloro-4 nitrophenyl α-D-maltotrioside (CNPG3) as a substrate, with the commonly used 3,5-dinitrosalicylic acid (DNS) assay. The direct chromogenic assay demonstrated a 5–10-fold higher sensitivity to determine α-amylase inhibition in various samples, including acarbose as a reference, pure anthocyanins, and anthocyanin-rich samples. The IC₅₀ values of acarbose presented as 37.6 μg/mL and 3.72 μg/mL for the DNS assay and the direct chromogenic assay, respectively, whereas purified anthocyanins from blackcurrant showed IC₅₀ values of 227.4 µg/mL and 35.0 µg/mL. The direct chromogenic assay is easy to perform, fast, reproducible, and suitable for high-throughput screening of pigmented α-amylase inhibitors. Full article

Studies on the degradation of plant cell wall polysaccharides by fungal extracellular enzymes have attracted recent attention from researchers. Xylan, abundant in hemicellulose, that play great role in connection between cellulose and lignin, has seen interest in its hydrolytic enzymatic complex. In this study, dozens of fungus species spanning genera were isolated from rotting leaves based on their ability to decompose xylan. Among these isolates, a strain with strong xylanase-producing ability was selected for further investigation by genome sequencing. Based on phylogenetic analysis of ITS (rDNA internal transcribed spacer) and LSU (Large subunit 28S rDNA) regions, the isolate was identified as Penicillium oxalicum. Morphological analysis also supported this finding. Xylanase activity of this isolated P. oxalicum 5-18 strain was recorded to be 30.83 U/mL using the 3,5-dinitro-salicylic acid (DNS) method. Further genome sequencing reveals that sequenced reads were assembled into a 30.78 Mb genome containing 10,074 predicted protein-encoding genes. In total, 439 carbohydrate-active enzymes (CAZymes) encoding genes were predicted, many of which were associated with cellulose, hemicellulose, pectin, chitin and starch degradation. Further analysis and comparison showed that the isolate P. oxalicum 5-18 contains a diverse set of CAZyme genes involved in degradation of plant cell wall components, particularly cellulose and hemicellulose. These findings provide us with valuable genetic information about the plant biomass-degrading enzyme system of P. oxalicum, facilitating a further exploration of the repertoire of industrially relevant lignocellulolytic enzymes of P. oxalicum 5-18. Full article

β-Chitin is an important carbon fixation product of diatoms, and is the most abundant nitrogen-containing polysaccharide in the ocean. It has potential for widespread application, but the characterization of chitin-related enzymes from β-chitin producers has rarely been reported. In this study, a chitin deacetylase (TwCDA) was retrieved from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) database and was heterologously expressed in vitro for functional analysis. The results showed that both the full-length sequence (TwCDA) and the N-terminal truncated sequence (TwCDA-S) had chitin deacetylase and chitinolytic activities after expression in Escherichia coli. High-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC-MS) indicated that TwCDA and TwCDA-S could catalyze the deacetylation of oligosaccharide (GlcNAc)₅. TwCDA had higher deacetylase activity, and also catalyzed the deacetylation of the β-chitin polymer. A dinitrosalicylic acid (DNS) assay showed that TwCDA-S had high chitinolytic activity for (GlcNAc)₅, and the optimal reaction temperature was 35 °C. Liquid chromatography combined with time-of-flight mass spectrometry (LC-coTOF-MS) detected the formation of a N-acetylglucosamine monomer (C₈H₁₅NO₆) in the reaction mixture. Altogether, we isolated a chitin deacetylase from a marine diatom, which can catalyze the deacetylation and degradation of chitin and chitin oligosaccharides. The relevant results lay a foundation for the internal regulation mechanism of chitin metabolism in diatoms and provide a candidate enzyme for the green industrial preparation of chitosan and chitin oligosaccharides. Full article

Wenling Medic (Medicago polymorpha) is common in southern China and has long been utilized as a vegetable in eastern China, as well as a significant raw ingredient for livestock and pickled meals. As a legume, there is still a research vacuum, and the first problem Wenling Medic faces in production is the problem of seed germination. The germination percentage of Wenling Medic was low, according to production practice and laboratory tests performed in this work. Furthermore, after more than two years of storage, the germination percentage of Wenling Medic dropped sharply, and it lacked a long enough seed life. An attempt was made to restore the viability of the seeds using the polyethylene glycol (PEG) osmoregulation-mediated priming method, after the physiological and biochemical characteristics of the seeds were assessed using the anthrone method, Bradford assay, 3,5-dinitrosalicylic acid (DNS) method, and thiobarbituric acid (TBA) method. The findings showed that it has a relatively short storage age and, with a useable life of only two years under normal aging circumstances, is not deactivated soon after harvesting. In addition, whereas protein content and α-amylase concentration did not significantly correlate (p > 0.05) with storage life, the conductivity of exudate, malondialdehyde content, and carbohydrate content did (p < 0.05). The seed viability was not considerably increased by the saturation initiation mediated by osmoregulation, utilizing PEG. In conclusion, the decrease in Wenling Medic seeds’ germination ability was substantially connected with higher levels of lipid peroxidation and decreased carbohydrate levels, but not with protein concentrations or α-amylase activity. The timing of dehydration may need to be carefully controlled when using PEG osmoregulation to prime Wenling Medic seeds. Full article

The evolution of green technology for the simple and ecological formation of silver nanoparticles (AgNPs) inspired the present work for simple and efficient detection of reducing sugars (RS) in foods. The proposed method relies on gelatin as the capping and stabilizing agent and the analyte (RS) as the reducing agent. This work may attract significant attention, especially in the industry, for testing the sugar content using gelatin-capped silver nanoparticles as it not only detects the sugar in food, but also determines the content (%), which could be an alternative technique to the conventionally used DNS colorimetric method. For this purpose, a certain amount of maltose was mixed with a gelatin-silver nitrate. Different conditions that may affect the color changes at 434 nm owing to the in situ formed AgNPs, such as gelatin-silver nitrate ratio, PH, time, and temperature, have been investigated. The 1:3 mg/mg ratio of gelatin-silver nitrate dissolved in 10 mL distilled water was most effective in color formation. The development of AgNPs color increases within 8–10 min at PH 8.5 as the selected optimum value and at the optimum temperature of 90 °C for the evolution of the gelatin-silver reagent’s redox reaction. The gelatin-silver reagent showed a fast response (less than 10 min) with a detection limit for maltose at 46.67 µM. In addition, the selectivity of maltose was checked in the presence of starch and after its hydrolysis with α-amylase. Compared with the conventionally used dinitrosalicylic acid (DNS) colorimetric method, the proposed method could be applied to commercial fresh apple juice, watermelon, and honey to prove its viability for detecting RS in fruits; the total reducing sugar content was 287, 165, and 751 mg/g, respectively. Full article

Nepenthes mirabilis pitcher fluid is known to hydrolyse lignocellulosic mixed agro-waste (MAW) into fermentable sugars through a cocktail of oxidative and hydrolytic enzymes. However, the influence of factors such as pH, pore size, temperature and trace elements on its functionality is not adequately understood. This study aims to explore the potential of Nepenthes mirabilis pitcher fluid for the pre-treatment of MAW (>106 µm) by assessing the influence of the factors mentioned above on the yield of total reducible sugars (TRSs). The association between the trace element solution, pH, and temperature was evaluated using standard methods: Dinitrosalicylic acid (DNS) assay for the concentration of TRSs, and BET assay for the surface area and pore properties of the samples. The results showed that the highest concentration of TRSs (407.50 g/L) was at pH 2, albeit below ambient temperature, while pores (>106 µm) of agro-waste can accommodate <10 kDa enzymes, i.e., the enzymes could be adequately embedded within the pores of the milled agro-waste used. In conclusion, supplementing the pitcher fluids with a trace element solution did not improve the yield of TRS, but a low pH at below ambient temperature was more effective. Full article

Many studies have demonstrated the herbicidal effects of several essential oils and their possible use as substitutes for chemical herbicides. Several enzymes play a very significant role in seed germination: among these, α-amylase could be involved in essential oil phytotoxic processes. The aims of this study were to compare the chemical composition of the essential oils of two ecotypes of O. heracleoticum growing in Cilento (Southern Italy) and to study their possible use as natural herbicide using Raphanus sativus, Sinapis arvensis and Lolium multiflorum seeds. Moreover, a possible inhibitory activity on the α-amylase enzyme extracted from germinating seeds was evaluated as a possible mechanism of action. Both oils, characterized by GC-MS, belonged to a carvacrol chemotype. The alpha-amylase activity was determined using DNSA (dinitrosalicylic acid) assay quantifying the reducing sugar produced. Furthermore, the essential oils demonstrated phytotoxicity at the highest dose tested, and an inhibitory effect on α-amylase, probably correlated with the phytotoxic effects, was registered. The oils showed interesting phytotoxic and alpha-amylase inhibitory activities, which deserve to be further investigated. Full article

The effects of hydrolysis methods (hydrothermal, acid, alkali, hydrothermal-enzyme, acid-enzyme, and alkali-enzyme) on hydrolysate characteristics and photo fermentative hydrogen production (PFHP) of corn straw (CS) and sorghum straw (SS) were investigated. The optimum production of reducing the sugar of straw in different solvent environments was studied by one-step hydrolysis and co-enzymatic hydrolysis pretreatment through a 3,5-dinitrosalicylic acid method. The hydrogen production process by photolytic fermentation of hydrolysates of Rhodobacter sphaeroides HY01 was further analyzed through a gas chromatograph, including the differences in accumulated PFHP yield, chemical oxygen consumption (COD), and volatile fatty acid (VFA) composition. The results showed that the highest reducing sugar yield was obtained by the acid method among one-step hydrolysis. In contrast, acid-enzyme hydrolysis can further increase the reducing sugar yield, which reached 0.42 g·g⁻¹-straw of both straws. Both CS and SS had the highest hydrogen yield from acid-enzyme hydrolysate, 122.72 ± 3.34 mL·g⁻¹-total solid of straw (TS) and 170.04 ± 4.12 mL·g⁻¹-TS, respectively, compared with their acid hydrolysates with 40.46% and 10.53% higher hydrogen yields, respectively. The use of enzymatic hydrolysis showed a significantly higher hydrogen yield for CS compared to SS, indicating that acid hydrolysis was more suitable for SS and acid-enzyme hydrolysis was more suitable for CS. Full article

In this study, the potential for a collection of wild-type L. lactis strains to metabolize the breakdown products of lignocellulose was investigated. The strains, isolated from a variety of environmental sources including grass and vegetables, were analyzed for their ability to ferment pentose sugars and their cellulolytic ability. In total, 21 environment-derived L. lactis strains were evaluated. Eleven of the 21 L. lactis isolates were found to have the potential to ferment pentose sugars commonly produced by lignocellulose breakdown. A 3,5-dinitrosalicylic acid (DNS)-based cellulase assay was performed, and 10 of the 21 L. lactis isolates showed cellulolytic activity. Six strains were able to both metabolize pentose sugars and showed cellulolytic activity: these included green pea isolates DPC 6754, DPC 6755, DPC 6756, and DPC 6758, the grass isolate DPC 6760, and the mung bean sprouts isolate KF147. For the first time, certain wild-type non-engineered L. lactis were found to possess cellulolytic activity. Moreover, these two abilities do not appear to be correlated. These findings highlight that environment-derived L. lactis, a species with a history of safe use in food production, has the potential for second-generation bioconversion processes, and the potential to re-utilize plant biomass found in waste streams. Full article