Chemical and Antioxidant Properties of Solvent and Enzyme-Assisted Extracts of Fucus vesiculosus and Porphyra dioica.

Extraction strategies impact the efficiency and nature of extracted compounds. This work assessed the chemical composition and antioxidant capacity of ethanolic, hydroethanolic, and aqueous versus enzyme-assisted extracts (isolated or with the sequential use of alcalase®, cellulase®, and viscozyme®) of the macroalgae Fucus vesiculosus (brown, Phaeophyceae) and Porphyra dioica (red, Rhodophyta. For both macroalgae, enzyme-assisted extraction (EAE) was the most efficient process compared to solvent-assisted extraction (SAE), independent of solvent. Fucus vesiculosus extraction yields were higher for EAE than for SAE (27.4% to 32.2% and 8.2% to 30.0%, respectively). Total phenolics content (TPC) was at least 10-fold higher in EAE extracts (229.2 to 311.3 GAE/gextract) than in SAE (4.34 to 19.6 GAE/gextract) counterparts and correlated well with antioxidant capacity (ABTS and ORAC methods), with EAE achieving values up to 8- and 2.6-fold higher than those achieved by SAE, respectively. Porphyra dioica followed F. vesiculosus's trend for extraction yields (37.5% to 51.6% for EAE and 5.7% to 35.1% for SAE), TPC, although of a lower magnitude, (0.77 to 8.95 GAE/gextract for SE and 9.37 to 14.73 GAE/gextract for EAE), and antioxidant capacity. Aqueous extracts registered the highest DPPH values for both macroalgae, with 2.3 µmol TE/gextract and 13.3 µmol TE/gextract for F. vesiculosus and P. dioica, respectively. EAE was a more efficient process in the extraction of soluble protein and reducing sugars in comparison to SAE. Furthermore, an improved effect of enzyme-assisted combinations was observed for almost all analyzed parameters. This study shows the promising application of enzyme-assisted extraction for the extraction of valuable compounds from F. vesiculosus and P.dioica, making them excellent functional ingredients for a wide range of health and food industrial applications.

Dietary adaptation to high starch involves increased relative abundance of sucrase-isomaltase and its mRNA in nestling house sparrows.

Dietary flexibility in digestive enzyme activity is widespread in vertebrates but mechanisms are poorly understood. When laboratory rats are switched to a higher carbohydrate diet, the activities of the apical intestinal α-glucosidases (AGs) increase within 6-12 h, mainly by rapid increase in enzyme transcription, followed by rapid translation and translocation to the intestine's apical, brush-border membrane (BBM). We performed the first unified study of the overall process in birds, relying on activity, proteomic, and transcriptomic data from the same animals. Our avian model was nestling house sparrows (Passer domesticus), which switch naturally from a low-starch insect diet to a higher starch seed diet and in whom the protein sucrase-isomaltase (SI) is responsible for all maltase and sucrase intestinal activities. Twenty-four hours after the switch to a high-starch diet, SI activity was increased but not at 12 h post diet switch. SI was the only hydrolase increased in the BBM, and its relative abundance and activity were positively correlated. Twenty-four hours after a reverse switch back to the lower starch diet, SI activity was decreased but not at 12 h post diet switch. Parallel changes in SI mRNA relative abundance were associated with the changes in SI activity in both diet-switch experiments, but our data also revealed an apparent diurnal rhythm in SI mRNA. This is the first demonstration that birds may rely on rapid increase in abundance of SI and its mRNA when adjusting to high-starch diet. Although the mechanisms underlying dietary induction of intestinal enzymes seem similar in nestling house sparrows and laboratory rodents, the time course for modulation in nestlings seemed half as fast compared with laboratory rodents. Before undertaking modulation, an opportunistic forager facing limited resources might rely on more extensive or prolonged environmental sampling, because the redesign of the intestine's hydrolytic capacity shortly after just one or a few meals of a new substrate might be a costly mistake.

A systematic review of in vitro studies evaluating the inhibitory effects of polyphenol-rich fruit extracts on carbohydrate digestive enzymes activity: a focus on culinary fruits consumed in Europe.

Evidence shows that polyphenols can attenuate postprandial blood glucose responses to meals containing digestible carbohydrate. Polyphenol-rich plant extracts are emerging as potential ingredients in functional foods and/or beverages despite limited understanding of their physiological effects. Many studies have investigated the mechanisms of polyphenol-rich fruit extracts on inhibition of digestive enzymes. However, the evidence available has yet to be critically evaluated systematically. This report reviews the in vitro literature to quantify the effect of fruit polyphenol extracts on the activities of digestive carbohydrases. A systematic literature search was conducted using six science databases. Included studies, totaling 34 in number, were in vitro digestion models which quantified gut digestive enzyme(s) activity on starch digestion in the presence of fruit polyphenol extracts. Most studies assessed the effects of fruit extracts on either α-amylase (n = 30) or α-glucosidase (n = 30) activity. Studies were consistent overall in showing stronger inhibition of α-amylase compared to α-glucosidase by proanthocyanidin- and/or ellagitannin-rich fruit extracts. Recommendations are proposed for future reporting of this type of research to enable meaningful synthesis of the literature as a whole. Such knowledge could allow effective choices to be made for development of novel functional foods and beverages.

Genomic diversification of giant enteric symbionts reflects host dietary lifestyles.

Herbivorous surgeonfishes are an ecologically successful group of reef fish that rely on marine algae as their principal food source. Here, we elucidated the significance of giant enteric symbionts colonizing these fishes regarding their roles in the digestive processes of hosts feeding predominantly on polysiphonous red algae and brown Turbinaria algae, which contain different polysaccharide constituents. Using metagenomics, single-cell genomics, and metatranscriptomic analyses, we provide evidence of metabolic diversification of enteric microbiota involved in the degradation of algal biomass in these fishes. The enteric microbiota is also phylogenetically and functionally simple relative to the complex lignocellulose-degrading microbiota of terrestrial herbivores. Over 90% of the enzymes for deconstructing algal polysaccharides emanate from members of a single bacterial lineage, "Candidatus Epulopiscium" and related giant bacteria. These symbionts lack cellulases but encode a distinctive and lineage-specific array of mostly intracellular carbohydrases concurrent with the unique and tractable dietary resources of their hosts. Importantly, enzymes initiating the breakdown of the abundant and complex algal polysaccharides also originate from these symbionts. These are also highly transcribed and peak according to the diel lifestyle of their host, further supporting their importance and host-symbiont cospeciation. Because of their distinctive genomic blueprint, we propose the classification of these giant bacteria into three candidate genera. Collectively, our findings show that the acquisition of metabolically distinct "Epulopiscium" symbionts in hosts feeding on compositionally varied algal diets is a key niche-partitioning driver in the nutritional ecology of herbivorous surgeonfishes.

The effect of carbohydrases or prebiotic oligosaccharides on growth performance, nutrient utilisation and development of small intestine and immune organs in broilers fed nutrient-adequate diets based on either wheat or barley.

BACKGROUND: Non-starch polysaccharides are large complex molecules and are found in cereal grains. This study was conducted to investigate the effect of carbohydrase enzymes or prebiotic oligosaccharides on growth performance, nutrient utilisation and weight of organs associated with the immune system in broilers fed wheat- or barley-based diets. RESULTS: In wheat-based diets, feed intake was lower (P < 0.05) following xylo-oligosaccharide supplementation, whereas in barley-based diets feed intake was greater (P < 0.05) following ß-glucanase supplementation. Gross energy digestibility was improved (P < 0.01) when either level of xylanase was added to wheat diets. Ileal digestible energy was greater (P < 0.01) in wheat diets including an additive compared with the control diet. In wheat diets, bursa weight was lower (P < 0.05) following xylo-oligosaccharide supplementation compared with the control treatment. CONCLUSIONS: The current study showed that supplemented carbohydrases or prebiotic oligosaccharides could alter the development of immune organs or small intestine without any significant effect on growth performance in broilers receiving nutrient-adequate diets. © 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effect of different salinities on gene expression and activity of digestive enzymes in the thick-lipped grey mullet (Chelon labrosus).

The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquaculture.

Carbohydrases and Phytase in Poultry and Pig Nutrition: A Review beyond the Nutrients and Energy Matrix.

This review aimed to clarify the mechanisms through which exogenous enzymes (carbohydrases and phytase) influence intestinal health, as well as their effects on the nutrients and energy matrix in diets fed to poultry and pigs reared under sanitary challenging conditions. Enzyme supplementation can positively affect intestinal microbiota, immune system, and enhance antioxidant status. Although enzymes have been shown to save energy and nutrients, their responses under sanitary challenging conditions are poorly documented. Immune system activation alters nutrient partitioning, which can affect the matrix values for exogenous enzymes on commercial farms. Notably, the carbohydrases and phytase supplementation under sanitary challenging conditions align with energy and nutritional valorization matrices. Studies conducted under commercial conditions have shown that matrices containing carbohydrases and phytase can maintain growth performance and health in poultry and pigs. However, these studies have predominantly focused on assessing a single level of reduction in energy and/or available phosphorus and total calcium, limiting our ability to quantify potential energy and nutrient savings in the diet. Future research should delve deeper into determining the extent of energy and nutrient savings and understanding the effects of alone or blended enzymes supplementation to achieve more specific insights.

Effect of emulsifier and multicarbohydrase enzyme supplementation on performance and nutrient digestibility in broiler diets containing rapeseed meal.

The aim of this study was to determine the effect of emulsifier and multicarbohydrase enzyme supplementation on performance, nutrient utilization, and apparent metabolizable energy-nitrogen (AMEN) value of broiler diets containing rapeseed meal (RSM) as well as their influence on the gut morphological structures, excretion of total and free sialic acid, and cecum concentration of short-chain fatty acids (SCFAs) in broiler chickens. A total of 384 male broiler chicks were assigned to four dietary treatments. The diet of the control treatment (CON) consisted of soybean, maize, and RSM (5% in starter, 7% in grower, 15% in finisher) with soybean and palm oils. The diets used for the experimental treatments were the control diet supplemented with an emulsifier (EMU), enzyme (ENZ), or both (EMU + ENZ). The duodenum (n = 10/treatment) and ileum (n = 10/treatment) digesta samples were assessed to determine nutrient digestibility: crude protein (CP), ether extract (EE), starch, Ca. Throughout the experimental period, EMU + ENZ treatment indicated the lowest total average feed intake and feed conversion ratio, with the highest average weight gain among the studied treatments (P < 0.05). The EMU + ENZ treatment also resulted in higher (P < 0.05): apparent prececal digestibility (APD) of CP, total tract neutral detergent fibre (NDF) degradation, apparent total tract digestibility (ATTD) of EE, villus height to crypt depth ratio (P < 0.1). The highest APD of EE was noted in the EMU treatment (P < 0.05). No significant differences were found in the AMEN values of the diets. A greater jejunum villi surface area was found in groups supplemented by enzyme compared to CON (P < 0.05). The EMU + ENZ treatment presented lower sialic acid excretion in the ileum and concentration of cecum SCFAs compared to the CON treatment (P < 0.05). The obtained results indicate that simultaneous usage of additives had beneficial effect on production parameters, nutrient digestibility, NDF degradation, as well as gut mucosa morphology. Based on the SCFAs concentration results, separate or simultaneous addition of emulsifier or/and enzyme did not provoke excessive fermentation activity of cecal bacteria.

Enrichment of a Plant Feedstuff Mixture's Nutritional Value through Solid-State Fermentation.

Plant feedstuffs are the main ingredients of animal feed. Owing to food-feed competition, increasing the utilization efficiency of these feedstuffs is important for animal nutrition. This can be achieved via solid-state fermentation (SSF). SSF of a plant feedstuff mixture (PFM) (25% rapeseed meal, soybean meal, rice bran, and sunflower meal) by three fungi (Aspergillus ibericus MUM 03.29, Aspergillus niger CECT 2088, and Aspergillus niger CECT 2915) resulted in an increase in protein content by 5%, irrespective of fungi, a reduction in cellulose content by 9 to 11%, and of hemicellulose content by 21 to 34%, relative to unfermented PFM. Enzyme production was measured: the highest cellulase (123.7 U/g), xylanase (431.8 U/g), and beta-glucosidase (117.9 U/g) activity were achieved with A. niger CECT 2088. Principal component analysis showed a positive correlation between all fermented PFMs and enzyme production, protein content, digestibility, and fiber reduction. Bioprocessing of the PFM by SSF increased its nutritional value and digestibility, making it more appealing for animal feeds.

Influence of fiber type and carbohydrase supplementation on nutrient digestibility, energy and nitrogen balance, and physiology of sows at mid and late gestation.

Carbohydrase supplementation in grow-finish pig diets improves energy, nutrient digestibility, and gastrointestinal function, but their efficacy in gestation diets is understudied. The experimental objective was to evaluate the efficacy of a multicarbohydrase to improve digestion, energetics, and various physiological functions in gestating sows fed soluble and insoluble fiber diets. On day 28 of gestation, 36 sows (186 ±â€…4.6 kg body weight), blocked by parity, were randomly assigned to a 2 × 2 factorial arrangement of dietary treatments (n = 9). Factors included fiber type of insoluble (IF; 20% dried distiller grains with solubles) or soluble fiber (SF; 20% sugar beet pulp) and with (+) or without (-) enzyme (0.05%, Rovabio Advance P10; Adisseo, Antony, France). Diets were fed from days 28 to 109 of gestation at a feeding level of 2.1 kg (SID-Lys 11 g/d and 4.5 net energy-Mcal/d). Two separate 9-d metabolism periods were conducted on days 50 to 59 (mid) and 99 to 108 (late) of gestation. During each period, days 1 to 3 served as an adaptation period, days 4 to 7 total urine and feces were collected (96-h) and followed by a 48-h lactulose-mannitol study. Serum and plasma were collected on days 50 and 99. Data were analyzed as repeated records using a linear mixed model with block as a random effect and fiber type, enzyme, and period and their interactions as fixed effects. Sows fed SF+ had increased serum IL-1ra (Fiber × Enzyme P = 0.035), and IL-2 (Fiber × Enzyme P = 0.042). In the presence of IF, multicarbohydrases increased serum lipopolysaccharide-binding protein, but not when supplemented with SF (Fiber × Enzyme P = 0.028). Circulating IL-8 and TNF-α were decreased in sows fed multicarbohydrases (P < 0.05). Multicarbohydrase supplementation increased the apparent total tract digestibility (ATTD) of gross energy (GE), dry matter, and neutral detergent fiber by 2.8%, 3.4%, and 8.3%, respectively (P < 0.05). Compared to IF-, the ATTD of hemicellulose was 5.3% greater in sows fed IF+ but did not differ from SF- and SF+ (Fiber × Enzyme P = 0.037). Sows fed IF+ had the greatest ATTD of insoluble dietary fiber (Fiber × Enzyme P = 0.011). Sows fed multicarbohydrases excreted less energy in their urine (519 vs. 469 GE kcal/d; Enzyme P = 0.033) and in their feces (985 vs. 900 GE kcal/d; Enzyme P = 0.003). This resulted in an improvement in both digestible energy (Enzyme P < 0.01) and metabolizable energy (Enzyme P = 0.041), irrespective of fiber type. In conclusion, multicarbohydrase supplementation increased the digestibility and energetic contribution of fiber, irrespective of adaptation time or fiber type, but modulation of inflammatory responses was unique to dietary fiber type.

This study evaluated the effects and mechanisms of supplementing multicarbohydrases to gestating sow diets containing either corn-dried distiller grains or sugar beet pulp fibrous co-products. Findings revealed significant energy, dry matter, and fiber digestibility enhancements with carbohydrase supplementation, irrespective of adaptation time. However, the interaction between enzyme supplementation and fiber type led to distinct immune marker responses. Specifically, granulocyte-macrophage colony-stimulating factor, interleukin-1 receptor antagonist, and interleukin-2 responses differed based on the presence of insoluble or soluble fiber. Sows in late gestation have greater nutrient and energy digestibility and altered nitrogen balance relative to day 50 of gestation. These findings highlight the potential benefits of incorporating carbohydrases into gestating sow diets to improve nutrient utilization and metabolic efficiency. Moreover, the study underscores the intricate interplay between enzyme supplementation, adaptation time, dietary fiber characteristics, and immune responses, offering valuable insights into the underlying mechanisms governing sow physiology during gestation and the influence of enzyme supplementation.

Effects of Dietary Carbohydrases on Fecal Microbiome Composition of Lactating Sows and Their Piglets.

Corn-soybean meal diets are commonly used in the pork industry as a primary source of energy and protein. However, such a diet generally contains non-starch polysaccharides (NSPs) which present a challenge in finding ways to improve their availability and digestibility. Dietary multi-carbohydrases (MCs) have been proposed as an efficient approach to utilize NSPs, and can result in improved growth performance and host intestinal fitness. In this study, we evaluated the effects of MC in lactation diets on gut microbiota composition of lactating sows and their litters. The experimental design contained two dietary treatments, a diet based on corn-soybean meal (CON), and CON supplemented with 0.01% multigrain carbohydrases (MCs). Sow and piglet fecal samples were collected on days 7 and 28 after farrowing. Based on the results from 16S rRNA gene amplicon sequencing, MC led to changes in species diversity and altered the microbial compositions in lactating sows and their piglets. Specifically, the MC treatment induced an increase in the proportions of Lactobacillus in piglets. Clostridium and Spirochaetaceae showed a significantly reduced proportion in MC-treated sows at day 28. Our results support the beneficial effects of dietary carbohydrases and their link with improved production due to better host fitness outcomes and gut microbiota composition.

A proteomic approach to identify digestive enzymes, their exocytic and microapocrine secretory routes and their compartmentalization in the midgut of Spodoptera frugiperda.

A proteomic approach was used to identify the digestive enzymes secreted by exocytosis and by microapocrine vesicles and enzyme midgut compartmentalization in Spodoptera frugiperda larvae. For this, proteomic analyses were performed in isolated midgut enterocyte microvillar membrane, in a fraction enriched in microapocrine vesicles (separated in soluble and membrane fractions), in the washings of the peritrophic membrane to isolate its loosely- and tightly-bound proteins, and in the peritrophic membrane contents. PM washings correspond to proteins extracted from the mucus layer surrounding PM. Serine endopeptidases (trypsins, chymotrypsins and serine endopeptidase homologs that have substitutions in the catalytic residues) and lipases are mainly secreted by exocytosis. Aminopeptidases are mainly microvillar enzymes and some are secreted membrane-bound to microapocrine vesicles, whereas carboxypeptidase isoforms follow different secretory routes. The results also showed that most polymer hydrolases (such as amylase and endopeptidases) are not retained in the ectoperitrophic fluid (found in PM washings but absent from PM contents). On the contrary, most enzymes involved in intermediate digestion (exemplified by carboxypeptidase and aminopeptidase) do not pass through the peritrophic membrane. Finally, the data revealed that the protein composition of PM includes peritrophins classified as peritrophic membrane proteins, PMP, and chitin deacetylase.

Valorization of Brewer's Spent Grain Using Biological Treatments and its Application in Feeds for European Seabass (Dicentrarchus labrax).

Brewer's spent grain (BSG) is the main brewery industry by-product, with potential applications in the feed and food industries due to its carbohydrate composition. In addition, the lignocellulosic nature of BSG makes it an adequate substrate for carbohydrases production. In this work, solid-state fermentation (SSF) of BSG was performed with Aspergillus ibericus, a non-mycotoxin producer fungus with a high capacity to hydrolyze the lignocellulosic matrix of the agro-industrial by-products. SSF was performed at different scales to produce a crude extract rich in cellulase and xylanase. The potential of the crude extract was tested in two different applications: -(1) - the enzymatic hydrolysis of the fermented BSG and (2) - as a supplement in aquafeeds. SSF of BSG increased the protein content from 25% to 29% (w/w), while the fiber content was reduced to 43%, and cellulose and hemicellulose contents were markedly reduced to around 15%. The scale-up of SSF from 10 g of dry BSG in flasks to 50 g or 400 g in tray-type bioreactors increased 55% and 25% production of cellulase and xylanase, up to 323 and 1073 U g-1 BSG, respectively. The optimum temperature and pH of maximal activities were found to be 55°C and pH 4.4 for xylanase and 50°C and pH 3.9 for cellulase, cellulase being more thermostable than xylanase when exposed at temperatures from 45°C to 60°C. A Box-Behnken factorial design was applied to optimize the hydrolysis of the fermented BSG by crude extract. The crude extract load was a significant factor in sugars release, highlighting the role of hydrolytic enzymes, while the load of fermented BSG, and addition of a commercial ß-glucosidase were responsible for the highest phenolic compounds and antioxidant activity release. The lyophilized crude extract (12,400 and 1050 U g-1 lyophilized extract of xylanase and cellulase, respectively) was also tested as an enzyme supplement in aquafeed for European seabass (Dicentrarchus labrax) juveniles. The dietary supplementation with the crude extract significantly improved feed and protein utilization. The processing of BSG using biological treatments, such as SSF with A. ibericus, led to the production of a nutritionally enriched BSG and a crude extract with highly efficient carbohydrases capable of hydrolyzing lignocellulosic substrates, such as BSG, and with the potential to be used as feed enzymes with remarkable results in improving feed utilization of an important aquaculture fish species.

Potential of Red, Green and Brown Seaweeds as Substrates for Solid State Fermentation to Increase Their Nutritional Value and to Produce Enzymes.

Seaweeds are valuable feedstocks with the potential to be used as ingredients in aquafeeds. However, their use are still limited, given their recalcitrant polysaccharide structure. To break this structure, a biotechnological approach such as solid-state fermentation (SSF) by filamentous fungi can be used, which simultaneously increases the nutritional value of the biomass. However, SSF has hardly been studied in seaweeds; thus, in this study, five different seaweeds (Gracilaria sp., Porphyra dioica, Codium tomentosum, Ulva rigida, and Alaria esculenta) were used as substrates in SSF with Aspergillus ibericus MUM 03.49 and A. niger CECT 2915. Firstly, the seaweeds were fully characterized, and, then, changes in the crude protein and carbohydrate contents were assessed in the fermented biomass, as well as any carbohydrases production. The SSF of U. rigida with both fungi resulted in the maximum xylanase and ß-glucosidase activities. The maximum cellulase activity was achieved using Gracilaria sp. and U. rigida in the SSF with A. niger. The protein content increased in C. tomentosum after SSF with A. ibericus and in U. rigida after SSF with both fungi. Moreover, U. rigida's carbohydrate content decreased by 54% and 62% after SSF with A. ibericus and A. niger, respectively. Seaweed bioprocessing using SSF is a sustainable and cost-effective strategy that simultaneously produces high-value enzymes and nutritionally enhanced seaweeds to be included in aquafeeds.

Effect of a carbohydrase admixture in growing pigs fed wheat-based diets in thermoneutral and heat stress conditions.

The efficacy of exogenous carbohydrases in pig diets has been suggested to depend on enzyme activity and dietary fiber composition, but recent evidence suggests other factors such as ambient temperature might be important as well. Therefore, we investigated the effect of heat stress (HS) on the efficacy of a multienzyme carbohydrase blend in growing pigs. Ninety-six (barrows: gilts; 1:1) growing pigs with initial body weight (BW) of 20.15 ± 0.18 kg were randomly assigned to six treatments, with eight replicates of two pigs per pen in a 3 × 2 factorial arrangement: three levels of carbohydrase (0, 1X, or 2X) at two environmental temperatures (20 °C or cyclical 28 °C nighttime and 35 °C day time). The 1X dose (50 g/tonne) provided 1,250 viscosimetry unit (visco-units) endo-ß-1,4-xylanase, 4,600 units α-l-arabinofuranosidase and 860 visco-units endo-1,3(4)-ß-glucanase per kilogram of feed. Pigs were fed ad libitum for 28 d and 1 pig per pen was sacrificed on day 28. There was no enzyme × temperature interaction on any response criteria; thus, only main effects are reported. Enzyme treatment quadratically increased (P < 0.05) BW on day 28, average daily gain (ADG) (P < 0.05), and average daily feed intake (ADFI) (P < 0.05) with the 1X level being highest. HS reduced the BW at day 14 (P < 0.01) and day 28 (P < 0.01), ADG (P < 0.01), and ADFI (P<0.001). There was a trend of increased feed efficiency (G:F) (P < 0.1) in the HS pigs. HS increased apparent jejunal digestibility of energy (P < 0.05) and apparent ileal digestibility of calcium (P < 0.01). At day 1, HS reduced serum glucose (P < 0.001) but increased nonesterified fatty acid (P < 0.01). In the jejunum, there was a trend of increased villi height by carbohydrases (P < 0.1), whereas HS reduced villi height (P < 0.05). HS increased the jejunal mRNA abundance of IL1ß in the jejunum (P < 0.001). There was a trend for a reduction in ileal MUC2 (P < 0.1) and occludin (P < 0.1) by HS, and a trend for increased PEPT1 (P < 0.1). There was no effect of HS on alpha diversity and beta diversity of the fecal microbiome, but there was an increase in the abundance of pathogenic bacteria in the HS group. In conclusion, HS did not alter the efficacy of carbohydrases. This suggests that carbohydrases and HS modulate pig performance independently.

Cocktail of carbohydrases from Aspergillus niger: an economical and eco-friendly option for biofilm clearance from biopolymer surfaces.

Biofilm formation on both biotic and abiotic surfaces accounts for a major factor in spread of antimicrobial resistance. Due to their ubiquitous nature, biofilms are of great concern for environment as well as human health. In the present study, an integrated process for the co-production of a cocktail of carbohydrases from a natural variant of Aspergillus niger was designed. The enzyme cocktail was found to have a noteworthy potential to eradicate/disperse the biofilms of selected pathogens. For application of enzymes as an antibiofilm agent, the enzyme productivities were enhanced by statistical modelling using response surface methodology (RSM). The antibiofilm potential of the enzyme cocktail was studied in terms of (i) in vitro cell dispersal assay (ii) release of reducing sugars from the biofilm polysaccharides (iii) the effect of enzyme treatment on biofilm cells and architecture by confocal laser scanning microscopy (CLSM). Potential of the enzyme cocktail to disrupt/disperse the biofilm of selected pathogens from biopolymer surfaces was also assessed by field emission scanning electron microscopy (FESEM) analysis. Further, their usage in conjunction with antibiotics was assessed and it was inferred from the results that the use of enzyme cocktail augmented the efficacy of the antibiotics. The study thus provides promising insights into the prospect of using multiple carbohydrases for management of heterogeneous biofilms formed in natural and clinical settings.

A novel multi-enzyme preparation produced from Aspergillus niger using biodegradable waste: a possible option to combat heterogeneous biofilms.

Extracellular polymeric substance (EPS) produced by the microorganisms provides protection and stability to them when they are encased within biofilms. Heterogeneous polysaccharides form a major constituent of the EPS and are crucial for the formation and integrity of the biofilms/slime. Thus, breakdown of polysaccharides might help in dispersion of biofilms from abiotic surfaces. In the present study we isolated a fungus, Aspergillus niger APS, capable of concurrently producing a cocktail of carbohydrases and optimized the conditions for higher yields of all the enzymes by one variable at a time (OVAT) approach. The optimization studies resulted in 1.5 to 12 fold augmentation in the enzyme yields using biodegradable waste. Further, keeping in view the heterogeneous nature of polysaccharides in biofilm matrix, the in-house produced enzyme cocktail was used for the dispersal of biofilms formed by Salmonella enterica serovar Typhi, Escherichia coli and Staphylococcus aureus. Treatment with enzyme preparation caused 90.23 ± 4.0, 82.64 ± 5.0 and 76.32 ± 5.0% reduction of the biofilms formed by these organisms respectively which was also evidenced by Field emission scanning electron microscopy (FESEM) revealing the loss of biofilm architecture. Interestingly, the enzyme cocktail could also remove viscous slime formed under natural conditions in the kitchen drainage pipe (KDP). To the best of our knowledge, this is the first report on biotreatment of abiotic surfaces for removal of biofilms/slime formed under natural conditions. The study thus indicates the prospects of using multiple carbohydrases as an anti-biofouling agent on abiotic surfaces like equipments as well as implants/prostheses and pipelines.

Evolution of World and Brazilian Markets for Enzymes Produced by Solid-state Fermentation: A Patent Analysis.

BACKGROUND: The use of enzymes in various industrial processes has become increasingly frequent. When added to productive processes, it can accelerate reactions and generate a number of new products. The solid state fermentation (SSF), among other applications, has been employed also to obtain enzymes. OBJECTIVE: The purpose of this prospection was to map registered patent documents about enzymes production by this type of fermentation in the world, identify the most obtained enzymes with patent documents and compilate information about the world and Brazilian enzyme markets. METHODS: The experimental design was carried out by the keyword-driven scope through the advanced search in the Espacenet database European Patent Office (EPO). The keywords selected were solid-state fermentation and the International Patent Classification code, C12N9 (enzymes; proenzymes), for prospecting of interest. RESULTS: In 2012, there was the higher number of registered patents (12). China holds 84% of deposited patents. Among the types of depositors, 54% of the selected patent documents were deposited by universities and institutes, and 44% by companies. 76.5% of the evaluated patents used fungi as enzyme producer. Analyzing the enzymes obtained in the registered patents, it is verified that the majority belongs to the group of carbohydrases with 43%, followed by proteases (25%), which are also the two classes of enzymes most commercialized in the market. CONCLUSION: China holds the majority of the registered patents but North America gets the largest global enzyme market revenue followed by Europe and Pacific Asia. Carbohydrases were the most commercialized enzymes and with the highest number of patents registered. Among the carbohydrases, cellulases, xylanases and amylases are the most frequent in patent registration while being fungi produced.

Dietary Soluble and Insoluble Fiber With or Without Enzymes Altered the Intestinal Microbiota in Weaned Pigs Challenged With Enterotoxigenic E. coli F18.

Post-weaning diarrhea caused by enterotoxigenic E. coli (ETEC) causes significant economic losses for pig producers. This study was to test the hypotheses that an ETEC challenge disrupts intestinal microbial homeostasis and the inclusion of dietary soluble (10% sugar beet pulp) or insoluble fiber (15% corn distillers dried grains with solubles) with or without exogenous carbohydrases will protect or restore the gut microbial homeostasis in weaned pigs. Sixty crossbred piglets (6.9 ± 0.1 kg) were blocked by body weight and randomly assigned to one of six treatments (n = 10), including a non-challenged control (NC), ETEC F18-challenged positive control (PC), ETEC-challenged soluble fiber without (SF-) or with carbohydrases (SF+), and ETEC-challenged insoluble fiber without (IF-) or with carbohydrases (IF+). Pigs were housed individually and orally received either ETEC inoculum or PBS-sham inoculum on day 7 post-weaning. Intestinal contents were collected on day 14 or 15. The V4 region of the bacterial 16S rRNA was amplified and sequenced. High-quality reads (total 6,671,739) were selected and clustered into 3,330 OTUs. No differences were observed in α-diversity among treatments. The ileal microbiota in NC and PC had modest separation in the weighted PCoA plot; the microbial structures were slightly altered by SF+ and IF- compared with PC. The PC increased ileal Escherichia-Shigella (P < 0.01) and numerically decreased Lactobacillus compared to NC. Predicted functional pathways enriched in the ileal microbiota of PC pigs indicated enhanced activity of Gram-negative bacteria, in agreement with increased Escherichia-Shigella. The SF+ tended to decrease (P < 0.10) ileal Escherichia-Shigella compared to PC. Greater abundance of ileal Streptococcus, Turicibacter, and Roseburia and colonic Prevotella were observed in SF- and SF+ than PC (P < 0.05). Pigs fed IF + had greater Lactobacillus and Roseburia than PC pigs (P < 0.05). The ETEC challenge reduced total volatile fatty acid (VFA) compared with NC (P < 0.05). The SF+ tended to increase (P < 0.10) and SF- significantly increased (P < 0.05) colonic total VFA compared with PC. Collectively, ETEC challenge disrupted gut microbial homeostasis and impaired microbial fermentation capacity. Soluble fiber improved VFA production. Dietary fiber and carbohydrases altered microbiota composition to maintain or restore microbial homeostasis.

Green processing: minimising harmful substances in leather making.

Conventional leather processing poses serious threat to the environment due to its numerous chemical treatments which include hazardous chemicals such as sodium sulphide and lime. To minimise the pollutants and harmful substances during leather processing, an enzymatic rehydration-dehairing-fibre-opening process has been achieved in shortest possible time compared to conventional process. The physicochemical characteristics of experimental leathers were found to be comparable with those of conventionally processed leathers. The releases of sugar and proteoglycans were found to be in congruence with the scanning electron micrographs and histology. TGA and DSC results ascertained the stability of enzymatically processed leathers. Pollution load in terms of TOC, BOD, COD, and TDS was reduced up to 80% compared to that of the conventional process. The present work provides immense potential for a new approach in leather making with environmental safeguards. Graphical abstract.