Expansin SlExp1 and endoglucanase SlCel2 synergistically promote fruit softening and cell wall disassembly in tomato.

Fruit softening, an irreversible process that occurs during fruit ripening, can lead to losses and waste during postharvest transportation and storage. Cell wall disassembly is the main factor leading to loss of fruit firmness, and several ripening-associated cell wall genes have been targeted for genetic modification, particularly pectin modifiers. However, individual knockdown of most cell wall-related genes has had minimal influence on cell wall integrity and fruit firmness, with the notable exception of pectate lyase. Compared to pectin disassembly, studies of the cell wall matrix, the xyloglucan-cellulose framework, and underlying mechanisms during fruit softening are limited. Here, a tomato (Solanum lycopersicum) fruit ripening-associated α-expansin (SlExpansin1/SlExp1) and an endoglucanase (SlCellulase2/SlCel2), which function in the cell wall matrix, were knocked out individually and together using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9-mediated genome editing. Simultaneous knockout of SlExp1 and SlCel2 enhanced fruit firmness, reduced depolymerization of homogalacturonan-type pectin and xyloglucan, and increased cell adhesion. In contrast, single knockouts of either SlExp1 or SlCel2 did not substantially change fruit firmness, while simultaneous overexpression of SlExp1 and SlCel2 promoted early fruit softening. Collectively, our results demonstrate that SlExp1 and SlCel2 synergistically regulate cell wall disassembly and fruit softening in tomato.

Nitrogen sources alter ligninase and cellulase activities of thermophilic fungi isolated from compost and vermicompost.

Fungi harboring lignocellulolytic activity accelerate the composting process of agricultural wastes; however, using thermophilic fungal isolates for this process has been paid little attention. Moreover, exogenous nitrogen sources may differently affect fungal lignocellulolytic activity. A total of 250 thermophilic fungi were isolated from local compost and vermicompost samples. First, the isolates were qualitative assayed for ligninase and cellulase activities using Congo red (CR) and carboxymethyl cellulose (CMC) as substrates, respectively. Then, twenty superior isolates harboring higher ligninase and cellulase activities were selected and quantitatively assayed for both enzymes in basic mineral (BM) liquid medium supplemented with the relevant substrates and nitrogen sources including (NH4)2SO4 (AS), NH4NO3 (AN), urea (U), AS + U (1:1), or AN + U (1:1) with final nitrogen concentration of 0.3 g/L. The highest ligninase activities of 99.94, 89.82, 95.42, 96.25, and 98.34% of CR decolorization were recorded in isolates VC85, VC94, VC85, C145, and VC85 in the presence of AS, U, AS + U, AN, and AN + U, respectively. Mean ligninase activity of 63.75% in superior isolates was achieved in the presence of AS and ranked the highest among other N compounds. The isolates C200 and C184 exhibited the highest cellulolytic activity in the presence of AS and AN + U by 8.8 and 6.5 U/ml, respectively. Mean cellulase activity of 3.90 U/mL was achieved in AN + U and ranked the highest among other N compounds. Molecular identification of twenty superior isolates confirmed that all of them are belonging to Aspergillus fumigatus group. Focusing on the highest ligninase activity of the isolate VC85 in the presence of AS, the combination can be recommended as a potential bio-accelerator for compost production.

Agroindustrial and food processing residues valorization for solid-state fermentation processes: A case for optimizing the co-production of hydrolytic enzymes.

In the pursuit of sustainability, managing agro-industrial and food processing residues (AFR) efficiently is crucial. This study proposes a systematic approach to convert AFR into valuable products via solid-state fermentation (SSF). Using fungal enzyme production as a case study, this adaptable methodology suits any SSF bioprocess. Initially, AFR's physicochemical properties were evaluated to assess their feasible use as carbon sources and solid matrices for SSF. Then, five strains were screened for their capability to produce enzymes (Xylanase, X; pectinase, P; cellulase, C). Apple pomace (AP) and brewery spent grain (BSG) with Aspergillus sp. (strain G5) were selected. Subsequent steps involved a two-phase statistical approach, identifying critical factors and optimizing them. Process conditions were screened using a Plackett-Burman design, narrowing critical variables to three (BSG/AP, pH, humidity). Response Surface Methodology (Central Composite Design) further optimized these factors for co-synthesis of X, P, and C. The humidity had the most significant effect on the three responses. The optimum conditions depended on each enzyme and were further validated to maximize either X, P or C. The obtained extracts were used for pectin extraction from orange peels. The extract containing primarily xylanase (X = 582.39, P = 22.86, C = 26.10 U mL-1) showed major pectin yield recovery (12.33 ± 0.53%) and it was obtained using the optimal settings of BSG/AP (81/19), humidity (50.40%), and pH (4.58). The findings will enable adjusting process conditions to obtain enzymatic cocktails with a tailored composition for specific applications.

High-solids enzymatic saccharification of starch-rich raw herbal biomass residues for producing high titers of glucose.

The bioresource utilization of herbal biomass residues (HBRs) has been receiving more attention. Herein, three different HBRs from Isatidis Radix (IR) and Sophorae Flavescentis Radix (SFR) and Ginseng Radix (GR) were subjected to batch and fed-batch enzymatic hydrolysis to produce high-concentration glucose. Compositional analysis showed the three HBRs had substantial starch content (26.36-63.29%) and relatively low cellulose contents (7.85-21.02%). Due to their high starch content, the combined action of cellulolytic and amylolytic enzymes resulted in greater release of glucose from the raw HBRs compared to using the individual enzyme alone. Batch enzymatic hydrolysis of 10% (w/v) raw HBRs with low loadings of cellulase (≤ 10 FPU/g substrate) and amylolytic enzymes (≤ 5.0 mg/g substrate) led to a high glucan conversion of ≥ 70%. The addition of PEG 6000 and Tween 20 did not contribute to glucose production. Furthermore, to achieve higher glucose concentrations, fed-batch enzymatic hydrolysis was conducted using a total solid loading of 30% (w/v). After 48-h of hydrolysis, glucose concentrations of 125 g/L and 92 g/L were obtained for IR and SFR residues, respectively. GR residue yielded an 83 g/L glucose concentration after 96 h of digestion. The high glucose concentrations produced from these raw HBRs indicate their potential as ideal substrate for a profitable biorefinery. Notably, the obvious advantage of using these HBRs is the elimination of the pretreatment step, which is typically required for agricultural and woody biomass in similar studies.

Biosurfactant promoted enzymatic saccharification of alkali­pretreated reed straw.

The decrease of cellulase activity and unproductive adsorption of lignin are important obstructive factors for inefficient enzymatic hydrolysis. This paper applied five different kinds of biosurfactants including rhamnolipid, sophorolipid, chitin, tea saponin, and sodium lignosulfonate in the enzymatic hydrolysis process of alkali-pretreated reed straw (RS) to enhance the saccharification efficiency. When 8 g/L sophorolipid is added, the efficiency of enzymatic hydrolysis is 91.68 %, which is 30.65 % higher than that without using any biosurfactant. The efficiency of enzymatic hydrolysis can be further increased to 99.56 % when 7.5 g/L sophorolipid and 1.5 g/L tea saponin are added together. This is because the sophorolipid, rhamnolipid, and chitin can synergistically hamper the enzymatic inactivation during enzymatic hydrolysis, while tea saponin and sodium lignosulfonate can inhibit the non-productive adsorption of lignin. This work proposed a very effective method to improve the efficiency of enzymatic hydrolysis and reduce the dosage of the enzyme by adding biosurfactants.

Comparison of Alkaline Sulfite Pretreatment and Acid Sulfite Pretreatment with Low Chemical Loading in Saccharification of Poplar.

Sulfite pretreatment is a productive process for lignin dissolution in lignocelluloses and to reduce the hydrophobicity of lignin by sulfonation, thus promoting the hydrolyzability of the substrate. Previously, sulfite pretreatment needs high dosages of chemicals and thus results in the high cost of the pretreatment and the great pressure of environmental pollution. To overcome these problems, it was crucial to research whether alkaline sulfite pretreatment (ALS) and acid sulfite pretreatment (ACS) with low chemical loading could enhance the saccharification of poplar. In this work, the results indicated that with low loading of chemicals in sulfite pretreatment, ALS pretreatment (1.6% Na2SO3 and 0.5% NaOH) at 180 °C removed more lignin, resulted in lower hydrophobicity and higher cellulase adsorption capacity of poplar than ACS pretreatment (1.6% Na2SO3 and 0.5% H2SO4) at 180 °C. A satisfying glucose yield of 84.9% and a xylose yield of 76.0% were obtained from poplar after ALS pretreatment with 1.6% Na2SO3 and 0.5% NaOH at 180 °C for 1 h using 10 FPU cellulase/g dry matter, saving sodium sulfite by 60.0% compared to the loading of sulfite in traditional sulfite pretreatment. The strategy developed in this work reduced chemical loading and cellulase loading in alkali sulfite pretreatment for the saccharification of poplar.

Plant regeneration from leaf mesophyll derived protoplasts of cassava (Manihot esculenta Crantz).

A high yield of isolated protoplast and reliable regeneration system are prerequisite for successful somatic hybridization and genome editing research. However, reproducible plant regeneration from protoplasts remains a bottleneck for many crops, including cassava. We evaluated several factors that influence isolation of viable protoplasts form leaf mesophyll, induction of embryogenic calli, and regeneration of plants in three cassava cultivars; Muchericheri, TMS60444 and Karibuni. A relatively higher protoplast yield was obtained with enzyme mixture containing 5 g/L Macerozyme and 10 g/L cellulase. Muchericheri recorded relatively higher protoplast yield of 20.50±0.50×106 whereas TMS60444 (10.25±0.25×106) had the least protoplast yield in 10 g/L cellulase and 4 g/L cellulase. Freshly isolated protoplast cells were plated on callus induction medium (CIM) solid medium containing MS basal salt, 60 g/L D-glucose, 30 g/L sucrose, B5 vitamins, 100 mg/L myo-inositol, 0.5 mg/L copper sulphate, 100 mg/L casein hydrolysate, 4.55 g/L mannitol, 0.1 g/L MES, 10 mg/L picloram and 3 g/L gelrite to induce protoplast growth and development. The three cultivars reached colony formation but no further development was observed in this culture method. Protoplast growth and development was further evaluated in suspension culture using varying cell densities (1, 2 and 3× 105 p/mL). Development with highest number of minicalli was observed in cell density of 3× 105 p/mL. Minicalli obtained were cultured on CIM supplemented with 10mg/L picloram. Callus induction was observed in all cell densities with the cultivars. Highest somatic embryogenesis was observed in 2× 105 p/ml while no somatic embryogenesis was observed in cell density of 1×105 p/mL. Somatic embryos were matured in EMM medium supplemented with 1 mg/L BAP, 0.02 mg/L NAA and 1.5 mg/L GA3 then germinated in hormone free medium for plant regeneration. This protocol which used simple mixture of commercial enzymes is highly reproducible and can be applied in biotechnology research on cassava.

Customized optimization of lignocellulolytic enzyme cocktails for efficient conversion of pectin-rich biomass residues.

Pectin is a major component in many agricultural feedstocks. Despite the wide use in industrial production of cellulases and hemicellulases, the fungus Trichoderma reesei lacks a complete enzyme set for pectin degradation. In this study, three representative pectinolytic enzymes were expressed and screened for their abilities to improve the efficiency of T. reesei enzymes on the conversion of different agricultural residues. By replacing 5 % of the T. reesei proteins, endopolygalacturonase and pectin lyase remarkably increased the release of sugars from inferior tobacco leaves. In contrast, pectin methylesterase showed the strongest improving effect (by 31.1 %) on the hydrolysis of beetroot residue. The pectin in beetroot residue was only mildly degraded with the supplementation of pectin methylesterase, which allowed the extraction of pectin keeping the original emulsifying activity with a 51.1 % higher yield. The results provide a basis for precise optimization of lignocellulolytic enzyme systems for targeted valorization of pectin-rich agricultural residues.

Enhancing the Activity of Carboxymethyl Cellulase Enzyme Using Highly Stable Selenium Nanoparticles Biosynthesized by Bacillus paralicheniformis Y4.

The inorganic selenium is absorbed and utilized inefficiently, and the range between toxicity and demand is narrow, so the application is strictly limited. Selenium nanoparticles have higher bioactivity and biosafety properties, including increased antioxidant and anticancer properties. Thus, producing and applying eco-friendly, non-toxic selenium nanoparticles in feed additives is crucial. Bacillus paralicheniformis Y4 was investigated for its potential ability to produce selenium nanoparticles and the activity of carboxymethyl cellulases. The selenium nanoparticles were characterized using zeta potential analyses, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). Additionally, evaluations of the anti-α-glucosidase activity and the antioxidant activity of the selenium nanoparticles and the ethyl acetate extracts of Y4 were conducted. B. paralicheniformis Y4 exhibited high selenite tolerance of 400 mM and the selenium nanoparticles had an average particle size of 80 nm with a zeta potential value of -35.8 mV at a pH of 7.0, suggesting that the particles are relatively stable against aggregation. After 72 h of incubation with 5 mM selenite, B. paralicheniformis Y4 was able to reduce it by 76.4%, yielding red spherical bio-derived selenium nanoparticles and increasing the carboxymethyl cellulase activity by 1.49 times to 8.96 U/mL. For the first time, this study reports that the carboxymethyl cellulase activity of Bacillus paralicheniforis was greatly enhanced by selenite. The results also indicated that B. paralicheniformis Y4 could be capable of ecologically removing selenite from contaminated sites and has great potential for producing selenium nanoparticles as feed additives to enhance the added value of agricultural products.

A review on pectinase properties, application in juice clarification, and membranes as immobilization support.

Pectic substances cause haziness and high viscosity of fruit juices. Pectinase enzymes are biological compounds that degrade pectic compounds. Nontoxicity and ecofriendly nature make pectinases excellent biocatalysts for juice clarification. However, the poor stability and nonreusability of pectinases trim down the effectiveness of the operation. The immobilization techniques have gained the attention of researchers as it augments the properties of the enzymes. Literature has reported the stability improvement of enzymes like lipase, laccase, hydrogen peroxidase, and cellulase upon immobilization on the membrane. However, only a few research articles divulge pectinase immobilization using a membrane. The catalysis-separation synergy of membrane-reactor has put indelible imprints in industrial applications. Immobilization of pectinase on the membrane can enhance its performance in juice processing. This review delineates the importance of physicochemical and kinematic properties of pectinases relating to the juice processing parameters. It also includes the influence of metal-ion cofactors on enzymes' activity. Considering the support and catalytic-separation facets of the membrane, the prediction of the membrane as support for pectinase immobilization has also been carried out.

Non-targeted metabolomics reveals the stress response of a cellulase-containing penicillium to uranium.

Human industrial activities have caused environmental uranium (U) pollution, resulting in uranium(VI) had radiotoxicity and chemical toxicity. Here, a cellulase-producing Penicillium fungus was screened and characterized by X-ray fluorescence (XRF), and Fourier transform infrared reflection (FT-IR), as well as by GC/MS metabolomics analysis, to study the response to uranium(VI) stress. The biomass of Penicillium decreased after exposure to 100 mg/L U. Uranium combined with carboxyl groups, amino groups, and phosphate groups to form uranium mineralized deposits on the surface of this fungal strain. The α-activity concentration of uranium in the strain was 2.57×106 Bq/kg, and the ß-activity concentration was 2.27×105 Bq/kg. Metabolomics analysis identified 118 different metabolites, as well as metabolic disruption of organic acids and derivatives. Further analysis showed that uranium significantly affected the metabolism of 9 amino acids in Penicillium. These amino acids were related to the TCA cycle and ABC transporter. At the same time, uranium exhibited nucleotide metabolism toxicity to Penicillium. This study provides an in-depth understanding of the uranium tolerance mechanism of Penicillium and provides a theoretical basis for Penicillium to degrade hyper-enriched plants.

Extraction of Radix trichosanthis Polysaccharides for Potential Antihyperlipidemic Application.

This study focused on the optimization of ultrasound-assisted compound enzyme extraction for polysaccharides (RTPs) from Radix trichosanthis by orthogonal experiment and response surface methodology, and then its extraction kinetics model and antihyperlipidemic activities were studied. The optimum extraction process was as follows: cellulase-1.0%, papain-1.0%, pectase-0.5%, pH-5, extraction temperature-50°C, and liquid-to-solid ratio-30 mL/g; prediction value of RTPs was 7.54%; the experimental yield of RTPs was 7.22%, while 50 minutes was optimized in Weibull kinetics model. Then high-dose groups of RTP extract could reduce the TC, TG, and LDL-C levels and increase the level of HDL-C in high-fat mice, with the ability to lower the MDA content and enhance SOD level.

Endophytic fungi of Brunfelsia uniflora: isolation, cryopreservation, and determination of enzymatic and antioxidant activity.

Brunfelsia uniflora (Pohl.) D. Don (Solanaceae), commonly known as manacá-de-cheiro, is widely distributed in Brazil and used by local indigenous peoples as an antirheumatic, antisyphilitic, depurative, emetic, vermifuge, and purgative agent. Several studies have examined the biological activities and phytochemical profile of Brunfelsia; however, few have focused on the diversity of endophytic microorganisms that colonize members of the genus. This study aimed to isolate and cryopreserve endophytic fungi from B. uniflora and determine their cellulase, laccase, and antioxidant activities. Endophytic fungi were isolated from B. uniflora stems, cultured on wheat grains, immersed in a 150 g L-1 aqueous sucrose solution, and cryopreserved at - 80 °C for 1 and 6 months. Cellulase activity was determined by a qualitative test using carboxymethylcellulose medium and laccase activity by a quantitative test based on the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate). Prior to antioxidant activity assays, fungi were grown in malt extract broth for production of mycelial biomass. A methanolic extract was prepared for evaluation of DPPH· scavenging activity, FRAP activity, and total phenolic content. A total of 46 endophytic fungal isolates were obtained from B. uniflora stems and classified into 24 groups according to morphological similarities. B. uniflora was shown to harbor different genera of ascomycete fungi as endophytic organisms. Mycelial viability was observed after 1 and 6 months of cryopreservation at - 80 °C. Fungi exhibited cellulase and laccase activities. Isolate CE23 had the highest laccase activity after 7 days of cultivation. Twelve isolates were found to have low total phenolic contents and DPPH· and FRAP activities.

Allelopathic inhibitory effect on the growth of microcystis aeruginosa by improved ultrasonic-cellulase extract of Vallisneria.

The unique and efficient characteristics of allelopathy in submerged plants make it an environmentally friendly method to control harmful algal blooms. Increased research focus has been placed on the improved allelochemical extraction methods of submerged plants because of their cost-utility relationships. In this study, the growth inhibition effect of Vallisneria extract on Microcystis aeruginosa (M. aeruginosa) cells through the combination of enzyme and ultrasonic-assisted extraction method was analyzed. By establishing a co-cultivation experiment, the growth indicators, photosynthetic system, and oxidative stress system of M. aeruginosa were determined. The reactive oxygen species (ROS) and superoxide dismutase (SOD) activity, as well as the catalase (CAT) and Malondialdehyde (MDA) levels of algal cells were found to have increased significantly after co-cultivation, which indicated that the Vallisneria ultrasonic-cellulase extract could induce oxidative stress in Microcystis aeruginosa cells. The Vallisneria extract could promote at low concentrations and inhibit at high concentrations on the growth of Microcystis aeruginosa. The effective suppression of growth of algae cells with the extract was observed at 5 g/L (fresh weight). The results showed that the Vallisneria ultrasonic-cellulase extract had a significant inhibitory effect on M. aeruginosa, making the effective ingredients a useful reference for algae inhibitors.

Isolation and characterization of a mesophilic cellulolytic endophyte Preussia africana from Juniperus oxycedrus.

The medicinal plant Juniperus oxycedrus is less recognized for the diversity of its fungal endophytes and their potential to produce extracellular enzymes. The present study is the first report on the isolation and identification of a mesophilic endophytic strain JO-A, Preussia africana, from fresh stems of the J. oxycedrus endemic tree in the Ifrane region-Morocco, and the evaluation of its ability to produce cellulases. A one-time multi-parameter one-factor screening was optimized to select factors that enhance cellulase production in P. africana. The maximum production of both CMCase and FPase activities were 1.913 IU.mL-1 and 0.885 IU.mL-1, respectively, when the medium was supplemented with 2% w/v glucose. These remarkable titers were tenfold greater than those obtained under the initial non-optimized conditions. This mesophilic P. africana JO-A strain grows and actively produces cellulases at 37 °C demonstrating its great potential for various biotechnology applications. The cellulolytic extract showed the highest enzymatic activities at pH 5.0 and 50 °C with a half-life of 24 h at 50 °C.

Effect of Extraction Procedures with Ultrasound and Cellulolytic Enzymes on the Structural and Functional Properties of Citrus grandis Osbeck Seed Mucilage.

The structural and functional properties of Citrus grandis Osbeck (CGO) seed mucilage by different extraction practices, including conventional citrate buffer, ultrasonic-assisted (UAE), enzymatic-assisted extraction (EAE) with cellulase or Celluclast® 1.5 L and various ultrasonic-assisted enzymatic extraction (UAEE) procedures were investigated. It was found that CGO seed from agricultural and processing byproducts is an excellent new source of high methoxyl pectin with quite high intrinsic viscosity (about 108.64 dL/g) and molecular weight (about 1.9 × 106) as compared with other pectin sources. UAEE with Celluclast® 1.5 L enhanced the extraction yield most pronouncedly (about 2.3 times). Moreover, the monosaccharide composition of CGO seed mucilage is least affected by EAE with Celluclast® 1.5 L. In contrast, EAE with cellulase dramatically reduces the galacturonic acid (GalA) content to less than 60 molar%, and increases the glucose (Glc) content pronouncedly (to about 40 molar%), which may be considered as an adverse effect in terms of pectin purity. Though extraction procedures involved with ultrasound and cellulolytic enzymes generally show a decrease in GalA contents, weight average molar mass and intrinsic viscosity, EAE with Celluclast® 1.5 L is least affected, followed by UAE and UAEE with Celluclast® 1.5 L. These features can be leveraged in favor of diversified applications.

Different Effects of Lard and Vegetable Blend Oil on Intestinal Microorganisms, Enzyme Activity and Blood Routine in Mice.

The intake of moderate oils and fats is necessary to maintain the body's energy balance, and the fatty acid composition of different oils and fats varies in their nutrition and function. The study aimed to investigate the effects of lard and vegetable blend oil on gut microbiota, intestinal enzyme activities, and blood routine. Kunming mice were assigned to the three groups: (1) Control group (CK) was gavage administration with distilled water, (2) Plant oil group (ZWY) was gavage administration with edible vegetable blend oil, (3) Lard group (DWY) was gavage administration with lard. After 42 days, microbiological, digestive enzymes, and blood routine were performed. Compared with the CK group, Escherichia coli, Lactobacilli, and Bifidobacteria were significantly decreased (p < 0.05), the activities of protease, cellulase, amylase, and xylanase were markedly reduced (p < 0.05), the hemoglobin was significantly increased (p < 0.05) in the ZWY group and DWY groups, and the hematocrit was increased in the ZWY group (p < 0.05), while other routine blood indices were increased (p > 0.05). Compared to the ZWY group, the activity of cellulase and amylase were significantly increased (p < 0.05), the intestinal microorganism and the routine blood indexes had no significant difference in the DWY group. Lard and vegetable blend oil diet affected the composition of the intestinal microorganisms, and the functions of digestive enzymes. Meanwhile, the levels of digestive enzymes may be correlated with the intestinal microbiota.

Medium supplementation and thorough optimization to induce carboxymethyl cellulase production by Trichoderma reesei under solid state fermentation of nettle biomass.

In the present study, the production of cellulase by Trichoderma reesei under solid-state fermentation of nettle biomass was promoted through supplementation of the culture media using carbonaceous additives and comprehensive optimization of the cultivation via the Taguchi method. CMCase activities about 5.5-6.1 U/gds were obtained by fermentation of the autoclave-pretreated biomass, among various chemical and physical pretreatments. Then, several additives including Tween 80, betaine, carboxymethyl cellulose, and lactose were individually or in combination added to the culture media to induce the enzyme production. The results proved that such additives could act as either inducers or inhibitors. Furthermore, CMCase activity surprisingly increased to 14.0 U/gds by supplementing the fermentation medium with the optimal mixture of additives including 0.08 mg/gds Tween 80, 0.4 mg/gds betaine, and 0.2 mg/gds carboxymethyl cellulose. Factor screening according to Plackett-Burman design confirmed that the levels of Urea and MgSO4 among basal medium constituents as well as pH of the medium were significantly affected CMCase production. By optimizing the levels of these factors, CMCase activity of 18.8 U/gds was obtained, which was noticeably higher than that of fermentation of the raw nettle. The applied procedure can be promisingly used to convert the nettle biomass into valuable products.

α-Amylase and cellulase production by novel halotolerant Bacillus sp.PM06 isolated from sugarcane pressmud.

A novel Bacillus sp.PM06 isolated from sugarcane waste pressmud was tested for extracellular α-amylase and cellulase enzyme production. The effect of different substrates, nitrogen sources, pH, and temperature on growth and extracellular enzyme production was examined. Bacillus sp.PM06 was able to grow with starch and carboxymethyl cellulose (CMC) as a sole source of carbon and ammonium chloride was found to be the best nitrogen source. Maximum enzyme production was obtained at 48 H for both α-amylase and cellulase. The optimal condition for measuring enzyme activity was found to be pH 5.5 at 50 °C for α-amylase and pH 6.4 at 60 °C for cellulase respectively. It was found that Bacillus sp.PM06 exhibited halotolerance up to 2 M Sodium chloride (NaCl) and Potassium chloride (KCl). The isolate could produce α-amylase in the presence of 2 M NaCl and 1 M KCl. However, the strain produced cellulase even in the presence of 2 M NaCl and KCl. Concomitant production of both enzymes was observed when the medium was supplemented with starch and CMC. A maximum of 31 ± 1.15 U/mL of amylase and 15 ± 1.5 U/mL of cellulase was produced in 48 H. The enzyme was partially purified by Ammonium sulphate (NH4 )2 SO4 precipitation with 2.2 and 2.3-fold purification.

Impact of feed supplement with alfa-amylase and beta-glucanase on ingestive-related biomarkers registered with real-time sensors.

The aim of this study was to investigate the impact of feed supplements with alfa-amylase and beta-glucanase (Optipartum C+ 200) on ingestive-related behaviour biomarkers registered with real-time sensors: rumination behaviours and reticulorumen parameters (pH and temperature). Cows (n=20) in the treatment group (TG) were fed with Optipartum C+ 200 (Enzymes feed supplement: Alfa-Amylase 57 Units; Beta-Glucanase 107 Units) from 21 days before calving until 30 days after calving with a feeding rate of 200 g/cow/day. Cows (n=22) in the control group (CG) were fed a feed ration without feed supplement. Measurements started from 6 days before calving and continued until 21 days after calving. The following indicators were registered: with the RumiWatch System: Rumination time; Eating time; Drinking time; Rumination chews; Eating chews; Drinking gulps; Bolus; Chews per minute; Chews per bolus. With the SmaXtec system: the temperature, pH of the contents of the cows' reticulorumens, and cows' walking activity. According to our results, feed supplementation with alfa-amylase and beta-glucanase (Optipartum C+ 200) in the TG group resulted in increases in the following parameters: 9% rumination time and eating time, 19% drinking time, 11% rumination chews, 16% eating chews, 13% number of boluses per rumination, 5% chews per minute and 16% chews per bolus. The rumination time showed a strong, positive relation with rumination chews and bolus indicators in both groups (TG and CG) (p⟨0.001); while the rumination time in both groups of cows showed an opposite direction and was negatively related to eating time and eating chews (p⟨0.05). We found a 1.28 % lower reticulorumen pH and a 0.64 % lower reticulorumen temperature in cows fed with the supplement compared with cows in the control group. Cows in TG were 8.80% more active than those in the CG group. For improvement of ingestive-related behaviour we suggest adding a feed supplement with alfa-amylase and beta-glucanase (Optipartum C+ 200).