Cellulase-assisted platelet-rich plasma release from nanofibrillated cellulose hydrogel enhances wound healing.

Platelet-rich plasma (PRP) is a source of growth factors, which are implicated in active tissue regeneration. However, after transplantation the efficacy of these bioactive compounds is often diminished due to rapid degradation and untargeted localization. For this reason, we evaluated the potential of nanofibrillated cellulose (NFC) hydrogel as a PRP carrier. NFC hydrogel is an animal-free biomaterial that, when doped with cellulase, can assist the release of PRP in a wound site. In this study, we examined the effects of 0.5% (m/v) NFC hydrogel formulations, including PRP and cellulase, on the migration and proliferation of skin cells via an in vitro scratch wound model. The suitability of the 0.8% NFC hydrogel formulations for accelerated wound healing and PRP carrying was studied in vitro in diffusion studies and in vivo in a full-thickness excisional wound model in SKH1 mice. None of the NFC hydrogel formulations with or without PRP and cellulase disturbed the normal cell behavior in vitro, and cellulase was successfully used to degrade NFC. NFC hydrogel slowed fibroblast migration rate in vitro. In vivo, NFC hydrogel treatment showed significantly enhanced re-epithelialization compared to control and supported collagen deposition. In addition, angiogenesis was significantly induced via PRP release after degrading NFC hydrogel with cellulase without abnormal host reaction. This study demonstrates the potential of NFC hydrogel with cellulase as a carrier for PRP with controlled release in future skin tissue engineering applications.

In vivo application of potent probiotics for enhancing potato growth and controlling Ralstonia solanacearum and Fusarium oxysporum infections.

Plant probiotics are live microbial cells or cultures that support plant growth and control plant pathogens through different mechanisms. They have various effects on plants, including plant growth promotion through the production of indole acetic acid (IAA), biological control activity (BCA), and production of cellulase enzymes, thus inducing systemic resistance and increasing the availability of mineral elements. The present work aimed to study the potential of Achromobacter marplatensis and Bacillus velezensis as plant probiotics for the field cultivation of potatoes. In vitro studies have demonstrated the ability of selected probiotics to produce IAA and cellulase, as well as antimicrobial activity against two plant pathogens that infect Solanum tuberosum as Fusarium oxysporum and Ralstonia solanacearum under different conditions at a broad range of different temperatures and pH values. In vivo study of the effects of the probiotics A. marplatensis and B. velezensis on S. tuberosum plants grown in sandy clay loamy soil was detected after cultivation for 90 days. Probiotic isolates A. marplatensis and B. velezensis were able to tolerate ultraviolet radiation (UV) exposure for up to two hours, the dose response curve exhibited that the D10 values of A. marplatensis and B. velezensis were 28 and 16 respectively. In the case of loading both probiotics with broth, the shoot dry weight was increased significantly from 28 in the control to 50 g, shoot length increased from 24 to 45.7 cm, branches numbers increased from 40 to 70 branch, leaves number increased from 99 to 130 leaf, root dry weight increased from 9.3 to 12.9 g, root length increased from 24 to 35.7 cm, tuber weight increased from 15 to 37.0 g and tubers number increased from 9 to 24.4 tuber, the rot percentage was reduced to 0%. The addition of both probiotic isolates, either broth or wheat grains load separately has enhanced all the growth parameters; however, better results and increased production were in favor of adding probiotics with broth more than wheat. On the other hand, both probiotics showed a remarkable protective effect against potato pathogens separately and reduced the negative impact of the infection using them together.

Growth performance, nutrient digestibility, fecal microbial diversity and volatile fatty acid, and blood biochemical indices of suckling donkeys fed diets supplemented with multienzymes.

BACKGROUND: As the foal grows, the amount of breast milk produced by the donkey decreases. In such cases, early supplemental feeding is particularly important to meet the growth needs of the foal. Foals have an incompletely developed gastrointestinal tract with a homogenous microbiota and produce insufficient amounts of digestive enzymes, which limit their ability to digest and utilize forage. Improving the utilization of early supplemental feeds, promoting gastrointestinal tract development, and enriching microbial diversity are the hotspots of rapid growth research in dairy foals. Plant-based feeds usually contain non-starch polysaccharides (NSPs), including cellulose, xylan, mannan, and glucan, which hinder nutrient digestion and absorption. In addition, proteins and starch (both biomolecules) form a composite system mainly through non-covalent interactions. The proteins wrap around the surface of starch granules and act as a physical obstacle, thereby inhibiting water absorption and expansion of starch and decreasing the enzyme's catalytic effect on starch. Glyanase, ß-mannanase, ß-glucanase, cellulase, protease, and amylase added to cereal diets can alleviate the adverse effects of NSPs. The current study determined the effects of adding multienzymes (glyanase, ß-mannanase, ß-glucanase, cellulase, protease, and amylase) to the diet of 2-month-old suckling donkeys on their growth performance, apparent nutrient digestibility, fecal volatile fatty acid (VFA) and pH, fecal bacterial composition, and blood biochemical indices. RESULTS: On day 120 of the trial, fecal samples were collected from the rectum of donkeys for determining bacterial diversity, VFA content, and pH. Moreover, fresh fecal samples were collected from each donkey on days 110 and 115 to determine apparent digestibility. The multienzymes supplementations did not affect growth performance and apparent nutrient digestibility in the donkeys; however, they tended to increase total height gain (P = 0.0544). At the end of the study, the multienzymes supplementations increased (P < 0.05) the Observed species, ACE, Chao1, and Shannon indices by 10.56%, 10.47%, 10.49%, and 5.01%, respectively. The multienzymes supplementations also increased (P < 0.05) the abundance of Firmicutes, Oscillospiraceae, Lachnospiraceae, Christensenellaceae, Christensenellaceae_R-7_group, and Streptococcus in feces, whereas decreased (P = 0.0086) the abundance of Proteobacteria. CONCLUSIONS: Multienzymes supplementations added to a basal diet for suckling donkeys can increase fecal microbial diversity and abundance.

Long noncoding RNA TRABA suppresses ß-glucosidase-encoding BGLU24 to promote salt tolerance in cotton.

Salt stress severely damages the growth and yield of crops. Recently, long noncoding RNAs (lncRNAs) were demonstrated to regulate various biological processes and responses to environmental stresses. However, the regulatory mechanisms of lncRNAs in cotton (Gossypium hirsutum) response to salt stress are still poorly understood. Here, we observed that a lncRNA, trans acting of BGLU24 by lncRNA (TRABA), was highly expressed while GhBGLU24-A was weakly expressed in a salt-tolerant cotton accession (DM37) compared to a salt-sensitive accession (TM-1). Using TRABA as an effector and proGhBGLU24-A-driven GUS as a reporter, we showed that TRABA suppressed GhBGLU24-A promoter activity in double transgenic Arabidopsis (Arabidopsis thaliana), which explained why GhBGLU24-A was weakly expressed in the salt-tolerant accession compared to the salt-sensitive accession. GhBGLU24-A encodes an endoplasmic reticulum (ER)-localized ß-glucosidase that responds to salt stress. Further investigation revealed that GhBGLU24-A interacted with RING-type E3 ubiquitin ligase (GhRUBL). Virus-induced gene silencing (VIGS) and transgenic Arabidopsis studies revealed that both GhBGLU24-A and GhRUBL diminish plant tolerance to salt stress and ER stress. Based on its substantial effect on ER-related degradation (ERAD)-associated gene expression, GhBGLU24-A mediates ER stress likely through the ERAD pathway. These findings provide insights into the regulatory role of the lncRNA TRABA in modulating salt and ER stresses in cotton and have potential implications for developing more resilient crops.

Effect of chemical pretreatment on quality attributes of the cashew apple.

Cashew apples, tropical pseudo fruit, are rich in bioactive compounds. It is still underutilized due to its high perishability and its astringent flavor. This study aims to extend its shelf life by chemical dip and dry method at the rural level. Inhibition of fruit-spoiling enzymes, such as polyphenol oxidase (PPO), peroxidase (POD), amylase, and cellulase, was a significant response in this method. Enzyme inhibition was carried out using chemicals: NaCl (1-10 mM), CaCl2 (1-10 mM), and ethylenediamine tetraacetic acid (0.1-1 mM). The effect of chemical concentration and dipping time was studied using a full factorial method at three levels (-1, 0, and 1). The dipping time ranged from 60 to 180 min, and chemical concentrations from 1 to 10 mM were studied. Optimal treatment conditions were obtained as follows: NaCl concentration of 9.45 mM, dipping time of 160 min, and CaCl2 concentration of 7.8 mM, dipping time of 160 min. NaCl pretreatment showed maximum inhibition of PPO (>80%) and POD (>80%), whereas CaCl2 pretreatment showed maximum inhibition of amylase (60.58%) and cellulase (80.23%). Hence, to avoid postharvest losses, pretreatment with NaCl and CaCl2 was adequate to preserve the texture and color of cashew apples. PRACTICAL APPLICATION: Chemical pretreatment can prevent the postharvest losses of cashew apples. Inhibition of PPO, POD, amylase, and cellulase is vital in the shelf-life extension of cashew apples. Sodium chloride dip is a cost-effective method for increasing the storability of cashew apples.

[Levofloxacin combined with cellulase can eradicate bacille Calmette-Guerin biofilm infection].

OBJECTIVE: To investigate the inhibitory effects of levofloxacin (LEV) combined with cellulase against bacille CalmetteGuerin (BCG) biofilms in vitro. METHODS: The mature growth cycle of BCG biofilms was determined using the XTT method and crystal violet staining. BCG planktonic bacteria and BCG biofilms were treated with different concentrations of LEV and cellulose alone or jointly, and the changes in biofilm biomass were quantified with crystal violet staining. The mature BCG biofilm was then treated with cellulase alone for 24 h, and after staining with SYTO 9 and Calcofluor White Stain, the number of viable bacteria and the change in cellulose content in the biofilm were observed with confocal laser scanning microscopy. The structural changes of the treated biofilm were observed under scanning electron microscopy. RESULTS: The MIC, MBC and MBEC values of LEV determined by broth microdilution method were 4 µg/mL, 8 µg/mL and 1024 µg/mL, respectively. The combined treatment with 1/4×MIC LEV and 2.56, 5.12 or 10.24 U/mL cellulase resulted in a significant reduction in biofilm biomass (P < 0.001). Cellulase treatments at the concentrations of 10.24, 5.12 and 2.56 U/mL all produced significant dispersion effects on mature BCG biofilms (P < 0.001). CONCLUSION: LEV combined with cellulose can effectively eradicate BCG biofilm infections, suggesting the potential of glycoside hydrolase therapy for improving the efficacy of antibiotics against biofilmassociated infections caused by Mycobacterium tuberculosis.

Evaluation of antioxidant capacity and digestive enzyme activities in Mytilus galloprovincialis exposed to nanoplastics under different patterns of hypoxia.

In the marine environment, plastic pollution may occur simultaneously with hypoxia. However, current ecological risk assessments of nanoplastics have rarely considered the impact of additional environmental factors, such as hypoxia. In this study, we investigated the effect of polystyrene nanospheres (PS-NPs) on the digestive performance (antioxidant system and digestive enzymes) of mussels Mytilus galloprovincialis under different patterns of hypoxia (normoxia, constant hypoxia, and fluctuating hypoxia). The result showed that PS-NPs caused oxidative damage in the digestive glands of mussels, while all patterns of hypoxia exacerbated this oxidative damage. Activities of four digestive enzymes (α-amylase, cellulase, trypsin, and lipase) were examined. Among these, the activity of the α-amylase was inhibited by PS-NPs, and the inhibition was aggravated by all the hypoxia patterns. The cellulase activity and trypsin activity was enhanced by PS-NPs, and the increase was further stimulated by hypoxia. Lipase activity was not affected by PS-NPs alone, but significant inhibition was detected after the coexposure to PS-NPs and hypoxia. Conclusively, the combined stress of hypoxia and nanoplastics can significantly affect the digestive performance of mussels and may alter the mussel nutrient uptake strategy. Our work has provided new insight into the ecological risk assessment of plastics under global climate change.

In Vitro Evaluation of Extracellular Enzyme Activity and Its Biocontrol Efficacy of Bacterial Isolates from Pepper Plants for the Management of Phytophthora capsici.

Phytophthora capsici is one of the most devastating fungal pathogens, causing severe diseases that lead to economic loss in the pepper industry. As a result of the infections, the chemical approach is becoming more popular. Biological control, on the other hand, is better suited to controlling fungal pathogens. The biological control approach significantly reduces the problems associated with chemical applications while restoring natural environmental balance. As a result, the overall findings indicate that certain bacterial isolates play a beneficial role in lytic enzyme production and biocontrol activities against P. capsici. Bacterial isolates obtained from the pepper plants were screened for lytic enzyme and anti-oomycete activity against Phytophthora capsici in Ethiopia. Sixty bacterial isolates were isolated and tested against Phytophthora capsici. From these bacterial isolates, different inhibition zones and hydrolytic enzyme production were detected. Biochemical tests using an automated machine (MALDI-TOF, VITEK 2 compact and 16S rRNA) revealed that three of them, AAUSR23, AAULE41, and AAULE51, showed a high inhibition zone and high production of hydrolytic enzymes and were identified as Enterobacter cloacae (AAUSR23), Pseudomonas fluorescens (AAULE41), and undetermined (AAULE51). The effects of diffusable metabolite isolate AAULE51 has a 66.7% inhibition zone against Phytophthora capsici, followed by AAULE41 and AAUSR23, which have 59.7% and 14.1% inhibition zones, respectively. These bacterial isolates showed high production of hydrolytic enzymes like protease, cellulase, chitinase, and lipase (5-34 diameter of inhibition zone). As a result, the overall findings show that selected bacterial isolates play a beneficial role in lytic enzyme production and for their biocontrol activities against P. capsici.

Multi-year and multi-site effects of recurrent glyphosate applications on the wheat rhizosphere microbiome.

Glyphosate (N-(phosphonomethyl)glycine) is broad-spectrum herbicide that is extensively used worldwide, but its effects on the soil microbiome are inconsistent. To provide a sound scientific basis for herbicide re-review and registration decisions, we conducted a four-year (2013-2016) study in which we consecutively applied glyphosate to a wheat (Triticum aestivum L.)-field pea (Pisum sativum L.)-canola (Brassica napus L.)-wheat crop rotation at five sites in the Canadian prairies. The glyphosate rates were 0, 1, 2, 4 and 8 kg ae ha-1, applied pre-seeding and post-harvest every year. The wheat rhizosphere was sampled in the final year of the study and analysed for microbial biomass C (MBC), the composition and diversity of the microbiome, and activities of ß-glucosidase, N-acetyl-ß-glucosiminidase, acid phosphomonoesterase and arylsulphatase. Glyphosate did not affect MBC, the composition and diversity of prokaryotes and fungi, and the activities of three of the four enzymes measured in the wheat rhizosphere. The one effect of glyphosate was a wave-like response of N-acetyl-ß-glucosaminidase activity with increasing application rates. The experimental sites had much greater effects, driven by soil pH and organic C, on the soil microbiome composition and enzyme activities than glyphosate. Soil pH was positively correlated with the relative abundance of Acidobacteriota but negatively correlated with that of Actinobacteriota and Basidiomycota. Soil organic C was positively correlated with the relative abundances of Proteobacteriota and Verrucomicrobiota, but negatively correlated with the relative abundance of Crenachaeota. The activity of acid phosphomonoesterase declined with increasing relative abundance of Acidobacteriota, but increased with that of Actinobacteriota and Basidiomycota. The activity of N-acetyl-ß-glucosaminidase also increased with increasing relative abundance of Actinobacteriota but decreased with that of Mortierellomycota. ß-glucosidase activity also decreased with increasing relative abundance of Mortierellomycota. The core fungal species observed in at least 90% of the samples were Humicola nigrescens, Gibberella tricincta and Giberella fujikuroi. Therefore, this multi-site study on the Canadian prairies revealed no significant effects of 4-year applications of glyphosate applied at different rates on most soil microbial properties despite differences in the properties among sites. However, it is important to keep evaluating glyphosate effects on the soil microbiome and its functioning because it is the most widely used herbicide worldwide.

Soybean isoflavones modulate gut microbiota to benefit the health weight and metabolism.

Soybean isoflavones (SIs) are widely found in food and herbal medicines. Although the pharmacological activities of SIs have been widely reported, their effects on the intestinal microecology of normal hosts have received little attention. Five-week-old Kunming (KM) mice were administered SIs (10 mg/kg/day) for 15 days. Food intake, body weight, and digestive enzyme activity were measured. Small intestine microbiota, including lumen-associated bacteria (LAB) and mucosa-associated bacteria (MAB), were analyzed using 16S ribosomal ribonucleic acid (16S rRNA) gene sequencing. Short-chain fatty acids (SCFAs) were analyzed using gas chromatography-mass spectrometry (GC-MS). The results showed that the mice that consuming SIs showed a higher food intake but a lower body weight gain rate than that of normal mice. Sucrase, cellulase, and amylase activities reduced, while protease activity increased after SIs intervention. Moreover, SIs increased the intestinal bacterial diversity in both LAB and MAB of normal mice. The composition of LAB was more sensitive to SIs than those of MAB. Lactobacillus, Adlercreutzia, Coprococcus, Ruminococcus, Butyricicoccus, and Desulfovibrio were the differential bacteria among the LAB of mice treated with SIs. In addition, acetic acid, valeric acid, isobutyric acid, isovaleric acid, and caproic acid decreased, while butyric acid and propionic acid increased in the mice treated with SIs. Taken together, SIs are beneficial for weight control, even in short-term interventions. The specific mechanism is related to regulating the gut microbiota, changing digestive enzyme activities, and further affecting carbohydrate absorption and metabolism.

ß-Glucosidases as dominant dose-dependent regulators of Oryza sativa L. in response to typical organic pollutant exposures.

Understanding the metabolic defense and compensation to maintain homeostasis is crucial for assessing the potential health risk of organic pollutants in crops. Currently, limited understanding is available regarding the targeted metabolic pathways and response mechanism under contaminant stress. This study showed that ciprofloxacin (CIP) at the environmental concentrations (1, 5, 25, 50 mg/L) did not significantly inhibit growth or cause severe oxidative damage to rice (Oryza sativa L.). Instead, the increment in CIP concentration induced a series of sequential metabolic disorders, which were characterized predominantly by primary and secondary metabolic disturbances, including phenylpropanoid biosynthesis, the carbohydrate, lipid and amino acid metabolism. After CIP in vivo exceeded a certain threshold level (>0.29 mg/g dry weight), ß-glucosidases (BGLUs) mediated the transition from the activation of the genes related to phenylpropanoid biosynthesis to the inhibition of the genes related to carbohydrate metabolism in rice. In particular, starch and sucrose metabolism showed the most profound perturbation stressed by environmental concentrations of CIP (5 mg/L) and other tested organic pollutants (10 µg/L of tricyclazole, thiamethoxam, polybrominated diphenyl ethers, and polychlorinated biphenyls). Besides, the key genes encoding endoglucanase and BGLU were significantly downregulated (|log2FC| > 3.0) under 100 µg/L of other tested organic pollutants, supporting the transition from the activation of secondary defense metabolism to the disruption of primary energy metabolism. Thus, in addition to bioaccumulation, changes in BGLU activity and starch and sucrose metabolism can reflect the potential adverse effects of pollutants on rice. This study explained the stepwise metabolic and transcriptional responses of rice to organic pollutants, which provided a new reference for the comprehensive evaluation of their environmental risks.

Effects of Resistant Dextrin from Potato Starch on the Growth Dynamics of Selected Co-Cultured Strains of Gastrointestinal Bacteria and the Activity of Fecal Enzymes.

Preparations of resistant dextrins have become an interesting topic of research due to their properties, which bear resemblance those of prebiotics, e.g., the improvement of metabolic parameters, increased efficiency of the immune system and induction of vitamin production. The aim of this study was to investigate the effects of the resistant dextrin produced from potato starch on the growth dynamics of typical gastrointestinal microbiota and the activity of fecal enzymes in order to assess a possible exhibition of prebiotic properties. In the study, in vitro cultivation of co-cultures of Lactobacillus, Bifidobacterium, E. coli, Enterococcus, Clostridium and Bacteroides spp. was conducted on media enriched with the resistant dextrin. The CFU/mL for each strain was measured in time periods of 24, 48, 72, 96 and 168 h. Furthermore, the activities of α-glucosidase, α-galactosidase, ß-glucosidase, ß-galactosidase and ß-glucuronidase were determined using spectrophotometric methods at a wavelength of 400 nm. The results show that the resistant dextrin can be utilized as a source of carbon for the growth of intestinal bacteria. Moreover, the results revealed that, after 168 h of cultivation, it enhances the viability of probiotic strains of Lactobacillus and Bifidobacterium spp. and decreases the growth of other intestinal strains (Clostridium, Escherichia coli, Enterococcus and Bacteroides), which is demonstrated by a high Prebiotic Index (p < 0.05). Furthermore, there was no significant change in the pH of the cultures; however, the pace of the pH decrease during the cultivation was slower in the case of culture with resistant dextrin. Furthermore, it was revealed that usage of the resistant dextrin as a medium additive noticeably lowered the activities of ß-glucosidase and ß-glucuronidase compared to the control (p < 0.05), whereas the activities of the other fecal enzymes were affected to a lesser degree. The resistant dextrins derived from potato starch are a suitable prebiotic candidate as they promote the growth of beneficial strains of gut bacteria and improve health markers, such as the activity of fecal enzymes. Nevertheless, additional in vivo research is necessary to further assess the suspected health-promoting properties.

Disease Inhibiting Effect of Strain Bacillus subtilis EG21 and Its Metabolites Against Potato Pathogens Phytophthora infestans and Rhizoctonia solani.

Potato production worldwide is plagued by several disease-causing pathogens that result in crop and economic losses estimated to billions of dollars each year. To this day, synthetic chemical applications remain the most widespread control strategy despite their negative effects on human and environmental health. Therefore, obtainment of superior biocontrol agents or their naturally produced metabolites to replace fungicides or to be integrated into practical pest management strategies has become one of the main targets in modern agriculture. Our main focus in the present study was to elucidate the antagonistic potential of a new strain identified as Bacillus subtilis EG21 against potato pathogens Phytophthora infestans and Rhizoctonia solani using several in vitro screening assays. Microscopic examination of the interaction between EG21 and R. solani showed extended damage in fungal mycelium, while EG21 metabolites displayed strong anti-oomycete and zoosporecidal effect on P. infestans. Mass spectrometry (MS) analysis revealed that EG21 produced antifungal and anti-oomycete cyclic lipopeptides surfactins (C12 to C19). Further characterization of EG21 confirmed its ability to produce siderophores and the extracellular lytic enzymes cellulase, pectinase and chitinase. The antifungal activity of EG21 cell-free culture filtrate (CF) was found to be stable at high-temperature/pressure treatment and extreme pH values and was not affected by proteinase K treatment. Disease-inhibiting effect of EG21 CF against P. infestans and R. solani infection was confirmed using potato leaves and tubers, respectively. Biotechnological applications of using microbial agents and their bioproducts for crop protection hold great promise to develop into effective, environment-friendly and sustainable biocontrol strategies. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Comparative analysis of physical and functional properties of cellulose nanofibers isolated from alkaline pre-treated wheat straw in optimized hydrochloric acid and enzymatic processes.

Two methods, HCl and enzymatic treatments, were evaluated for diversification of morphological and functional properties of cellulose nanofibers (CNF) from two- stage-alkaline pre-treated wheat straw (WS). The extraction conditions were optimized by a central composite designed experimental approach varying time (4-8 h) and temperature (80-120 °C) for the HCl-based treatment and time (4-8 h), and FiberCare dosage (50-100 endo-1,4-ß-glucanase unit/g) and Viscozyme (10-20 fungal ß-glucanase units/g) for the enzyme-based treatment. The CNF yields, morphological (polydispersity index (PdI), length and diameter), and functional (crystallinity and thermal degradation) properties were compared. The CNF produced by the HCl (HCN) and enzymatically (ECN) attained diameters ~17 nm had PdI, length, and crystallinity of 0.53, 514 nm & 70%, and 0.92, 1.0 µm & 48%, respectively. Thus, the HCN morphology suits homogenous nano-applications, whereas that of the ECN, would suit heterogenous nano-applications. The HCN and ECN yields were similar (~20%) with optimal production time of 7.41 and 4.64 h, respectively. Both the HCN & ECN can be classified as thermally stable nanocolloids with maximum thermal degradation temperatures of ~380 °C and Zeta potential ~-16 mV. The two CNF production methods have potential synergetic effects on CNF production, morphological, and functional properties.

'Biofilm Clippers'- enzyme formulation for bovine mastitic biofilm therapy.

Mastitis is one of the most important diseases that are threatening modern dairy farms. Biofilms of mastitic teat canal have serious clinical implications because of colonized pathogens having the ability to construct an extracellular polymeric substance (EPS) with increased tolerance to antimicrobials leads to difficulty in eradicating the infection. In this study, we investigated the synergistic biofilm disruptive effect of a combination of carbohydrate hydrolases targeting extracellular polysaccharides of biofilm matrix and we termed it as 'Biofilm Clippers (BC)'. Our findings demonstrate that the BC formulation exhibits intense biofilm-disrupting activity against Staphylococcus aureus biofilms. The results of the study showed that BC enables activity equivalent to physiologically achievable concentrations in disrupting biofilms of S. aureus in vitro. The synergistic anti-biofilm activities of BC on S. aureus biofilms demonstrated that the biofilm matrix is predominant of complex polysaccharides. Further, the confocal microscopic analysis demonstrates that the BC formulation is highly effective compared to the single treatment of either of the enzymes in disrupting the biofilm. To the best of our knowledge, this is the first report on the synergistic anti-biofilm activity of a class of enzyme formulation against mastitic biofilm mass. Even though a small study showed a promising effect on mastitic teat canal, further extensive investigation on a large number of bovines for mastitis therapeutic potential of this BC-derived product is now warranted.

Characterization of two extracellular ß-glucosidases produced from the cellulolytic fungus Aspergillus sp. YDJ216 and their potential applications for the hydrolysis of flavone glycosides.

A cellulolytic fungus YDJ216 was isolated from a compost and identified as an Aspergillus sp. strain. Two extracellular ß-glucosidases, BGL1 and BGL2, were purified using ultrafiltration, ammonium sulfate fractionation, and High-Q chromatography. Molecular masses of BGL1 and BGL2 were estimated to be 97 and 45 kDa, respectively, by SDS-PAGE. The two enzymes eluted as one peak at 87 kDa by Sephacryl S-200 chromatography, and located at similar positions in a zymogram after intact gel electrophoresis, suggesting BGL1 and BGL2 might be monomeric and dimeric, respectively. Both enzymes showed similar enzymatic properties; they were optimally active at pH 4.0-4.5 and 60 °C, and had similar half-lives at 70 °C. Two enzymes also preferred p-nitrophenyl glucose (pNPG) with the same Km and hardly hydrolyzed cellobiose, suggesting both enzymes are aryl ß-glucosidases. However, Vmax for pNPG of BGL1 (953.2 U/mg) was much higher than those of BGL2 (66.5U/mg) and other ß-glucosidases reported. When tilianin (a flavone glycoside of acacetin) was reacted with both enzymes, inhibitory activity for monoamine oxidase, relating to oxidation of neurotransmitter amines, was increased closely to the degree obtained by acacetin. These results suggest that BGL1 and BGL2 could be used to hydrolyze flavone glycosides to improve their inhibitory activities.

Potential Role of Exoglucanase Genes (WaEXG1 and WaEXG2) in the Biocontrol Activity of Wickerhamomyces anomalus.

The use of yeasts, including Wickerhamomyces anomalus, as biocontrol agents of fungi responsible for postharvest diseases of fruits and vegetables has been investigated for the past two decades. Among a variety of mechanisms, the production of glucanases coded by the "killer genes" WaEXG1 and WaEXG2 have been reported to play a role in the ability of yeast to inhibit other fungi. The objective of the present study was to determine the expression of these genes by RT-qPCR, utilizing gene-specific primers, when W. anomalus was grown on grape berries and oranges that were either non-inoculated or inoculated with Botrytis cinerea or Penicillium digitatum, or in minimal media supplemented with cell walls of various plant pathogens and different amounts of glucose. Results indicated that WaEXG2 was more responsive than WaEXG1 to the nutritional environment (including the addition of glucose to cell wall-amended media) in vitro and appeared to play a greater role in the cellular metabolism of W. anomalus. WaEXG2 expression also appeared to be more responsive to the presence of cell walls of P. digitatum and B. cinerea than other fungal species, whereas the same level of induction was not seen in vivo when the yeast was grown in wounded/pathogen-inoculated fruits.

Screening exogenous fibrolytic enzyme preparations for improved in vitro digestibility of bermudagrass haylage.

Our objectives were to evaluate the effects of 12 exogenous fibrolytic enzyme products (EFE) on ruminal in vitro neutral detergent fiber digestibility (NDFD) and preingestive hydrolysis of a 4-wk regrowth of bermudagrass haylage (BH), to examine the accuracy of predicting NDFD with EFE activity measures, and to examine the protein composition of the most and least effective EFE at increasing NDFD. In experiment 1, effects of 12 EFE on NDFD of BH were tested. Enzymes were applied in quadruplicate to culture tubes containing ground BH. The suspension was incubated for 24 h at 25 °C before addition of rumen fluid media and further incubation for 24 h at 39 °C. The experiment was repeated twice. In addition, regression relationships between EFE activity measures and NDFD were examined. Compared with the values for the control, 9 EFE-treated substrates had greater NDFD (37.8 to 40.4 vs. 35.6%), 6 had greater total VFA concentration (59.1 to 61.2 vs. 55.4 mM), and 4 had lower acetate-to-propionate ratios (3.03 to 3.16 vs. 3.24). In experiment 2, EFE effects on preingestive fiber hydrolysis were evaluated by incubating enzyme-treated and untreated bermudagrass suspensions in quadruplicate for 24 h at 25 °C and examining fiber hydrolysis measures. Compared with values for the control, 3 EFE reduced neutral detergent fiber concentration (62.8 to 63.7 vs. 67.3%), 10 increased release of water-soluble carbohydrates (26.8 to 58.5 vs. 22.8 mg/g), and 8 increased release of ferulic acid (210 to 391 vs. 198 µg/g). Regression analyses revealed that enzyme activities accurately [coefficient of determination (R(2)) = 0.98] predicted preingestive hydrolysis measures (water-soluble carbohydrates, ferulic acid), moderately (R(2) = 0.47) predicted neutral detergent fiber hydrolysis, but poorly (R(2) ≤ 0.1) predicted dry matter and NDFD. In experiment 3, proteomic tools were used to examine the protein composition of the most and least effective EFE at improving NDFD. Relative to the least effective, the most effective EFE at increasing NDFD contained 10 times more endoglucanase III, 17 times more acetylxylan esterase with a cellulose-binding domain 1, 33 times more xylanase III, 25 times more ß-xylosidase, and 7.7 times more polysaccharide monooxygenase with cellulose-binding domain 1 and 3 times more swollenin. The most effective EFE had a much greater quantity of fibrolytic enzymes and key proteins necessary for hemicellulose and lignocellulase deconstruction. This study identified several EFE that increased the NDFD and in vitro fermentation of 4-wk BH and revealed why some EFE are more effective than others.

Effect of Mediterranean saltbush (Atriplex halimus) ensilaging with two developed enzyme cocktails on feed intake, nutrient digestibility and ruminal fermentation in sheep.

The aim of this study was to assess the effects of feeding Atriplex halimus (AH) silage treated with two developed enzyme cocktails to sheep on feed intake, nutrient digestibility and ruminal fermentation. The AH silage was treated without or with 2 L of ZAD1(®) or ZAD2(®) /1000 kg with 5% molasses and ensiled for 30 days. Barley grain (300 g/head/day) was fed as an energy supplement once daily at 10.00 hours and AH silage with or without enzyme treatment was offered ad libitum to animals twice daily at 09.00 and 16.00 hours. Sheep were fed on four experimental forage diets comprised of AH silage and barley (D1), AH silage treated with ZAD1(®) and barley (D2), AH silage treated with ZAD2(®) and barley (D3) and AH silage treated with a combination of ZAD1(®) and ZAD2(®) (1:1) and barley (D4). Ensiling AH with enzymes reduced its contents of neutral detergent fiber and acid detergent fiber. The dry matter intake of AH of D2, D3 and D4 decreased (P < 0.001) as compared to D1. However, enzyme-treated diets had greater total digestible nutrients intake (P < 0.001) as compared to D1. The nutrients digestibility for D2, D3 and D4 were higher than those for D1 (P < 0.001), and were higher for D3 as compared to both D2 and D4. Sheep fed on D3 had highest (P < 0.001) ruminal total volatile fatty acids concentration, ammonia nitrogen concentration and microbial protein yield. It could be concluded that AH silage treated with ZAD1(®) or ZAD2(®) improved digestibility and rumen fermentation in sheep.

Purification and biochemical properties of a glucose-stimulated beta-D-glucosidase produced by Humicola grisea var. thermoidea grown on sugarcane bagasse.

The effect of several carbon sources on the production of mycelial-bound beta-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated beta-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The beta-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50 degrees C, respectively. The purified enzyme was thermostable up to 60 min in water at 55 degrees C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, o-nitrophenyl-beta-D-galactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-beta-D-fucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude beta-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea beta-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.