Phytochemical analysis, in vitro and in silico effects from Alstonia boonei De Wild stem bark on selected digestive enzymes and adipogenesis in 3T3-L1 preadipocytes.

BACKGROUND: Obesity is a global health issue arising from the unhealthy accumulation of fat. Medicinal plants such as Alstonia boonei stem bark has been reported to possess body weight reducing effect in obese rats. Thus, this study sought to investigate the in vitro and in silico effects of fractions from Alstonia boonei stem bark on selected obesity-related digestive enzymes and adipogenesis in 3T3-L1 preadipocytes. METHOD: Two fractions were prepared from A. boonei: crude alkaloid fraction (CAF) and crude saponin fraction (CSF), and their phytochemical compounds were profiled using Liquid chromatography with tandem mass spectrometry (LCMS/MS). The fractions were assayed for inhibitory activity against lipase, α-amylase and α-glucosidase, likewise their antiadipogenic effect in 3T3-L1 adipocytes. The binding properties with the 3 enzymes were also assessed using in silico tools. RESULTS: Eleven alkaloids and six saponin phytochemical compounds were identified in the CAF and CSF using LCMS/MS. The CAF and CSF revealed good inhibitory activity against pancreatic lipase enzyme, but weak and good activity against amylase respectively while only CSF had inhibitory activity against α-glucosidase. Both fractions showed antiadipogenic effect in the clearance of adipocytes and reduction of lipid content in 3T3-L1 adipocytes. The LCMS/MS identified compounds (41) from both fractions demonstrated good binding properties with the 3 enzymes, with at least the top ten compounds having higher binding energies than the reference inhibitors (acarbose and orlistat). The best two docked compounds to the three enzymes were firmly anchored in the substrate binding pockets of the enzymes. In a similar binding pattern as the reference acarbose, Estradiol-17-phenylpropionate (-11.0 kcal/mol) and 3α-O-trans-Feruloyl-2 α -hydroxy-12-ursen-28-oic acid (-10.0 kcal/mol) interacted with Asp197 a catalytic nucleophile of pancreatic amylase. Estradiol-17-phenylpropionate (-10.8 kcal/mol) and 10-Hydroxyyohimbine (-10.4 kcal/mol) interacted with the catalytic triad (Ser152-Asp176-His263) of pancreatic lipase while Estradiol-17-phenylpropionate (-10.1 kcal/mol) and 10-Hydroxyyohimbine (-9.9 kcal/mol) interacted with Asp616 and Asp518 the acid/base and nucleophilic residues of modelled α-glucosidase. CONCLUSION: The antiobesity effect of A. boonei was displayed by both the alkaloid and saponin fractions of the plant via inhibition of pancreatic lipase and adipogenesis.

Sleep-promoting activity of amylase-treated Ashwagandha (Withania somnifera L. Dunal) root extract via GABA receptors.

Ashwagandha (Withania somnifera L. Dunal), an Indian medicinal plant that has been used for centuries to treat insomnia, exhibits a variety of biological activities, such as improving cognitive function, immunity and anxiety. In this study, the effect of enzyme-treated Ashwagandha root extract (EA) and on sleep was evaluated using rodent models. Starch contained in the Ashwagandha root extract was removed by amylase treatment to prepare EA. To evaluate the sleep-promoting activity of EA, a pentobarbital-induced sleep test and electroencephalogram analysis were performed. In addition, the sleep-promoting mechanism of EA was elucidated by analyzing the expression of sleep-related receptors. In the pentobarbital-induced sleep test, EA dose-dependently increased sleep duration. Additionally, electroencephalogram analysis revealed that EA significantly increased δ-wave and non-rapid eye movement sleep times, which are involved in deep sleep, thereby improving sleep quality and quantity. EA also effectively relieved caffeine-induced insomnia symptoms. Furthermore, the γ-aminobutyric acid (GABA) content in the brain and mRNA and protein expression of GABAA, GABAB1, and serotonin receptors were significantly increased by EA compared to the normal group. In particular, EA showed sleep-promoting activity by binding to various GABAA receptor sites. Collectively, EA exhibited sleep-promoting activity through the GABAergic system and may be used as a functional material to improve sleep deprivation.

The effect of amylase supplementation on individual variation, growth performance, and starch digestibility in broiler chickens.

The objective of this study was to evaluate the variance of starch digestibility in broilers individually fed diets without or with supplemental exogenous amylase. A total of 120 d-of-hatch male chicks were individually reared from 5 to 42 d in metallic cages and fed maize-based basal diets or diets containing 80 kilo-novo-α-amylase units/kg (60 birds or replicates per treatment). Beginning on d 7, feed intake, body weight gain, and feed conversion ratio were recorded; partial excreta collection was conducted every Monday, Wednesday, and Friday until 42 d, when all birds were sacrificed for individual collection of duodenal and ileal digesta. Lower feed intake (4,675 vs. 4,815 g) and feed conversion ratio (1.470 vs. 1.508) were observed in amylase-fed broilers during the overall period (7-43 d; P < 0.01), whereas body weight gain was not affected. Amylase supplementation improved total tract starch (TTS) digestibility (P < 0.05) on each day of excreta collection (except for d 28, where no difference was found), averaging 0.982 vs. 0.973 compared to basal-fed broilers from d 7 to 42. Both apparent ileal starch (AIS) digestibility and apparent metabolizable energy (AMEN) were increased (P <0.05) from 0.968 to 0.976 and from 3,119 to 3,198 kcal/kg, respectively, with enzyme supplementation. Activity of amylase in the duodenum was higher (18.6 vs. 50.1 IU/g of digesta) in supplemented birds. Amylase supplementation led to a reduced coefficient of variation for both TTS (averaged 2.41 vs. 0.92% from 7 to 42 d) and AIS digestibilities (1.96 vs. 1.03%), as well as AMEN (0.49 vs. 0.35%), when compared to the nonsupplemented group, indicating lower individual heterogenity. An age effect was detected for TTS digestibility, as both groups saw an increase during the first weeks (slightly more pronounced in the supplemented group); older birds (d 30 onwards) presented a lower TTS digestibility compared to ages between 7 and 25 d. In conclusion, amylase supplementation in maize diets for broilers can attenuate individual bird variation for starch and energy utilization by increasing amylase activity and enhancing starch digestibility.

Characterizing the Effect of Amylase Inhibitors on Maltodextrin Metabolism by Gut Bacteria Using Fluorescent Glycan Labeling.

Diet-derived polysaccharides are an important carbon source for gut bacteria and shape the human gut microbiome. Acarbose, a compound used clinically to treat type 2 diabetes, is known to inhibit the growth of some bacteria on starches based on its activity as an inhibitor of α-glucosidases and α-amylases. In contrast to acarbose, montbretin A, a new drug candidate for the treatment of type 2 diabetes, has been reported to be more specific for the inhibition of α-amylase, notably human pancreatic α-amylase. However, the effects of both molecules on glycan metabolism across a larger diversity of human gut bacteria remain to be characterized. Here, we used ex vivo metabolic labeling of a human microbiota sample with fluorescent maltodextrin to identify gut bacteria affected by amylase inhibitors. Metabolic labeling was performed in the presence and absence of amylase inhibitors, and the fluorescently labeled bacteria were identified by fluorescence-activated cell sorting coupled with 16S rDNA amplicon sequencing. We validated the labeling results in cultured isolates and identified four gut bacteria species whose metabolism of maltodextrin is inhibited by acarbose. In contrast, montbretin A slowed the growth of only one species, supporting the fact that it is more selective. Metabolic labeling is a valuable tool to characterize glycan metabolism in microbiota samples and could help understand the untargeted impact of drugs on the human gut microbiota.

Supplementation of amylase or amylase + xylanase improves performance and metabolism of broilers fed with diets containing newly harvested maize.

How supplementation with amylase or amylase + xylanase in newly harvested maize-based diets affects broiler nutrient metabolism and performance is unclear. Thus, this study evaluated whether the supplementation of amylase (CN) or amylase + xylanase (CAX) improves performance and metabolism of broilers fed with newly harvested maize-based diets during a 6-week production. The results showed that the body weight gain of broilers fed with CA or CAX diet was higher than that with the control (CN) diet at 1-21 d of age; however, an opposite trend was observed for feed/gain (p < 0.05). Furthermore, 150, 64 and 35 different metabolites were found between CA/CN, CAX/CN and CAX/CA, respectively. Overall, amylase supplementation improved broiler growth performance at 1-21 d of age, and the positive effects of amylase on nutrient utilization were mostly related to nicotinate, retinol and glutathione metabolism improvement. Moreover, CAX diet increased apparent metabolizable energy and growth performance of broilers at 22-42 d of age, and the difference might be related to sphingolipid, porphyrin and chlorophyll metabolism regulation. The findings prove amylase + xylanase supplementation is an effective method to improve the nutritional value of newly harvested maize for broilers.

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.

Genistein attenuated oxidative stress, inflammation, and apoptosis in L-arginine induced acute pancreatitis in mice.

AIM: Acute pancreatitis is a common and potentially serious condition. However, a specific treatment for this condition is still lacking. Genistein, with its anti-oxidant and anti-inflammatory effects, could possibly be used to tackle the underlying pathophysiology of acute pancreatitis. Therefore, the aim of this study was to investigate the effects of genistein on oxidative stress, inflammation, and apoptosis in acute pancreatitis induced by L-arginine in mice. METHODS: Twenty-four male ICR mice were equally divided into 4 groups: Control (Con); Acute pancreatitis (AP) group: Two doses of i.p. 350 mg/100 g body weight (BW) of L-arginine were administered 1 h apart; AP and low-dose genistein (LG) group: mice were given i.p. injection of 10 mg/kg genistein 2 h prior to L-arginine injection followed by once-daily dosing for 3 days; and AP and high-dose genistein (HG) group: mice were given 100 mg/kg genistein with the similar protocol as the LG group. Pancreatic tissue was evaluated for histopathological changes and acinar cell apoptosis, malondialdehyde (MDA) levels, immunohistochemical staining for myeloperoxidase (MPO), nuclear factor-kappa beta (NF-kB), and 4-hydroxynonenal (4-HNE). Serum levels of amylase (AMY), c-reactive protein (CRP), and interleukin (IL)-6 were measured. RESULTS: Significant increases in the degree of acinar cell apoptosis, pancreatic MDA, serum IL-6 and amylase, MPO, NF-kB and 4-HNE positivity were observed in the AP group. All these parameters declined after low- and high-dose genistein treatment. Severe pancreatic inflammation, edema, and acinar cell necrosis were observed in the AP group. Significant improvement of histopathological changes was seen in both low- and high-dose genistein groups. There were no significant differences in any parameters between low and high doses of genistein. CONCLUSION: Genistein could attenuate the severity of histopathological changes in acute pancreatitis through its anti-oxidant, anti-inflammatory, and anti-apoptotic properties.

Enzymes in the time of COVID-19: An overview about the effects in the human body, enzyme market, and perspectives for new drugs.

The rising pandemic caused by a coronavirus, resulted in a scientific quest to discover some effective treatments against its etiologic agent, the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). This research represented a significant scientific landmark and resulted in many medical advances. However, efforts to understand the viral mechanism of action and how the human body machinery is subverted during the infection are still ongoing. Herein, we contributed to this field with this compilation of the roles of both viral and human enzymes in the context of SARS-CoV-2 infection. In this sense, this overview reports that proteases are vital for the infection to take place: from SARS-CoV-2 perspective, the main protease (Mpro ) and papain-like protease (PLpro ) are highlighted; from the human body, angiotensin-converting enzyme-2, transmembrane serine protease-2, and cathepsins (CatB/L) are pointed out. In addition, the influence of the virus on other enzymes is reported as the JAK/STAT pathway and the levels of lipase, enzymes from the cholesterol metabolism pathway, amylase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and glyceraldehyde 3-phosphate dehydrogenase are also be disturbed in SARS-CoV-2 infection. Finally, this paper discusses the importance of detailed enzymatic studies for future treatments against SARS-CoV-2, and how some issues related to the syndrome treatment can create opportunities in the biotechnological market of enzymes and the development of new drugs.

Paeoniflorin Can Improve Acute Lung Injury Caused by Severe Acute Pancreatitis through Nrf2/ARE Pathway.

Objective: To evaluate the potential therapeutic effect of paeoniflorin on acute lung injury induced by severe acute pancreatitis (SAP) and to initially explore the possible protective mechanisms of paeoniflorin. Method: The SAP lung injury rat model was established by retrograde injection of 5% sodium taurocholate to the cholangiopancreatic duct. H&E staining was used to detect pathological changes in rat lung tissue. W/D ratio method, serum amylase (AMY), and lipase activity were used to assess the degree of lung injury in rats. Oxidation indicators such as LDH, MDA, and SOD in lung tissue were measured. Levels of inflammatory factors TNF-α, IL-6, and IL-10 were measured in bronchoalveolar lavage fluid (BALF). At the same time, Western blot was used to detect the expression of related proteins in the Nrf2/ARE signaling pathway. Results: In SAP rats, paeoniflorin treatment could significantly alleviate lung injury conditions such as pulmonary edema and inflammatory cell infiltration in lung tissue and reduce serum amylase and lipase activities. Paeoniflorin can reduce the content of LDH and MDA in lung tissue and increase the content of SOD. In addition, ELISA results showed that paeoniflorin could inhibit the levels of TNF-α and IL-6 in BALF and upregulate the levels of IL-10. Paeoniflorin could upregulate the expression of Nrf2/ARE signaling pathway proteins Cyt-Nrf2, HO-1, and NQO1 in lung tissue of SAP rats. Conclusion: Paeoniflorin may improve acute lung injury in rats with severe pancreatitis by inhibiting inflammation and oxidative stress response. These effects may be related to activating the Nrf2/ARE signaling pathway.

Acute toxic effects of thiamethoxam on Chinese mitten crab Eriocheir sinensis.

The information about toxic effects of thiamethoxam on non-target aquatic organisms is still incomplete. The semi-static toxicity test method was used to investigate the acute toxic effects of thiamethoxam on Eriocheir sinensis. The results showed that the median lethal concentration (LC50) of thiamethoxam to E. sinensis at 96 h was 510 µg/L, and the safety concentration (SC) was 51 µg/L. After 96 h exposure to thiamethoxam, the survival rates of crabs at concentrations of 0, 151.11, 226.67, 340, and 510 µg/L were 100%, 76.19%, 64.29%, 61.91%, and 46.43%, respectively. A significant (P < 0.05) decrease of the number of hemocytes was observed in thiamethoxam groups. With the increase of thiamethoxam concentration, the phagocytic activity of hemocytes, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and the activities of protease, amylase, and lipase of crabs increased firstly and then decreased, and the above indexes reached the maximum in 151.11 µg/L thiamethoxam group. Collectively, a high concentration of thiamethoxam (510 µg/L) had a great effect on the gene expression of immune metabolism-related factors in hepatopancreas and gill of crabs. These findings indicated that thiamethoxam exposure had the ability to impair immune and metabolic systems and resulted in the reduction of survival rate of crabs.

Corn drying temperature, particle size, and amylase supplementation influence growth performance, digestive tract development, and nutrient utilization of broilers.

Broiler live performance may be influenced by postharvest corn drying temperature, and results could depend on particle size after grinding. The supplementation with an exogenous amylase may improve performance parameters, but responses to enzymes are also affected by particle size. Two parallel experiments were conducted to evaluate the effects of hard-kernel corn dried at 2 temperatures (35°C and 120°C), ground at 2 particle sizes (coarse or fine), and 3 supplementation levels (0, 133, and 266 g ton-1) of an exogenous amylase on live performance, gastrointestinal organ development, energy utilization, and nutrient digestibility. Twelve dietary treatments resulting from a 2 × 2 × 3 factorial arrangement of drying temperature, particle size, and amylase supplementation were evaluated in both experiments. A total of 1,920 day-old male chicks were randomly allocated to 96 floor pens, while 480 chicks were distributed among 4 battery brooder units. Ileal and fecal samples were collected to determine energy utilization and nutrient digestibility using titanium dioxide as inert marker. At 42 D, organs were collected, and relative weight or length was determined. Data were analyzed using a three-way ANOVA in a randomized complete block design. Feeding fine corn-based diets showed improvements on live performance for both studies. At 40 D, supplementing 266 g ton-1 of amylase improved feed conversion ratio (P < 0.05) by 1 point compared to chickens that consumed nonsupplemented diets and feed with amylase at 133 g ton-1. Broilers fed coarse corn-based diets had heavier gizzard (P < 0.001) and liver (P < 0.05) than chickens that consumed fine corn-based diets. In addition, starch digestibility was improved by amylase (P < 0.05) at 133 g ton-1 and by feeding coarse corn-based diets (P = 0.06). For chicks raised in cages (16 D), AMEn was increased (P < 0.01) by amylase supplementation regardless of its inclusion level. In conclusion, drying temperature and particle size interactions influenced broiler live performance, gastrointestinal organ development, nutrient digestibility, and energy utilization, and these parameters were improved by supplementing amylase.

Effects of high-amylase corn on performance and carcass quality of finishing beef heifers.

Developed initially for use in fuel ethanol production, Enogen Feed Corn (EFC; Syngenta Crop Protection) is genetically modified to express high concentrations of α-amylase in the corn kernel. Experiments were conducted to evaluate processing characteristics of EFC, in vitro digestion, and effects on feedlot performance, carcass characteristics, and liver abscess incidence. Experiment 1 used a randomized complete block design (3 × 3 × 5 factorial) to evaluate starch availability, in situ dry matter disappearance (ISDMD), in vitro gas production (IVGP), and volatile fatty acid (VFA) profiles of in vitro cultures. Grains (EFC or mill-run control [CON]) were flaked to a density of 360 g/L, and mixtures with 0%, 25%, 50%, 75%, or 100% EFC were prepared. Grains were tempered with added moisture (0%, 3%, or 6%) prior to steam conditioning for 15, 30, or 45 min. No two- or three-way interactions were observed. Adding moisture improved starch availability (linear; P < 0.01), and tended to improve ISDMD (linear, P = 0.06). Steam conditioning for 30 min improved starch availability, IVGP, and production of acetate, propionate, butyrate, valerate, and total VFA (P < 0.01) compared with conditioning for 15 or 45 min. Starch availability, ISDMD, IVGP, acetate, propionate, valerate, and total VFA production increased with an increasing proportion of EFC (linear, P < 0.01). Experiment 2 used 700 beef heifers (394 ± 8.5 kg initial body weight [BW]) fed finishing diets with steam-flaked corn as CON or EFC for 136 d. Targeting similar starch availabilities, grains were processed to 360 g/L (CON) and 390 g/L for CON and EFC, respectively. Heifers were blocked by BW, stratified, and then randomly assigned to 28 dirt-surfaced pens (25 animals per pen). Dry matter intakes were similar between treatments (P = 0.78), but cattle fed EFC had greater average daily gain (P < 0.01), improving feed efficiency by 5% (P < 0.01). Hot carcass weight was 6 kg greater for EFC cattle (P <0.01) than CON. No differences were observed for longissimus muscle area (P = 0.89), 12th-rib fat thickness (P = 0.21), or USDA yield grade (P = 0.13). Cattle fed CON had greater marbling scores than EFC (P = 0.04), but this did not affect the USDA quality grade (P > 0.33). Cattle fed EFC had 23% fewer abscessed livers than CON (P = 0.03). High-amylase corn may be used to improve microbial digestion, mill-throughput, and cattle performance, and it may mitigate liver abscesses.

'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.

Development of an enzymatic assay for the quantitative determination of trypsin inhibitory activity in wheat.

Wheat is one of the world's most widely consumed staple food. However, the number of people suffering from wheat-related disorders has increased drastically. Amylase-trypsin inhibitors (ATIs) have recently been identified as one of the main triggers of non-celiac wheat sensitivity (NCWS). In this study, an enzymatic assay for the determination of trypsin inhibition activity in hexaploid wheat was developed. This method was optimized with respect to several parameters, such as extraction and incubation procedures, and was validated according to international standards, concerning accuracy, precision and robustness of the method. Results revealed that linear inhibition and thus accuracy occurred only in a narrow concentration range. However, after optimization of settings the novel method was found to be satisfactory for accurate determination of trypsin inhibition in wheat. Purification of the wheat extract with immobilized trypsin beads led to the identification of CM inhibitors (chloroform/methanol soluble proteins) as main contributors of trypsin inhibition.

Amylase increases energy and nutrient density of Super Cereal Plus porridge as prepared and accepted by Rwandan caregivers.

Adding amylase to Super Cereal Plus (SC+A) improves energy and nutrient intake as porridge energy density reaches 1.0 kcal/g, meeting the recommended ≥0.8 kcal/g for prepared foods for young children. Caregiver response to SC+A in terms of adjusting porridge preparation using printed pictogram instructions was not yet investigated. The study assessed (a) porridge preparation by caregivers; (b) porridge energy density; (c) sensory porridge acceptability; and (d) understanding of preparation instructions. An 8-day follow-up intervention study was conducted amongst caregivers of children aged 6-23 months (n = 238) in Rwanda. Caregivers prepared porridge using SC+A whilst referring to printed pictogram instructions at the study site on Days 1 and 8 and received flour for preparation at home on Days 2-7. At the site, data were collected on porridge preparation procedures, energy density, consistency, acceptability, and interviews (n = 12), and focus group discussions (n = 6) were conducted. Mean porridge dry matter (DM) increased from 21.3 ± 4.4% (Day 1) to 25.1 ± 4.8% (Day 8; p < 0.0005). Flour and water were mixed before cooking by 95% of the participants, as per printed instructions. Sensory porridge acceptability was high, and the printed instructions enabled caregivers to prepare an accepted and energy dense porridge. The preferred water/flour volume ratio was 2.5 instead of 3. In conclusion, Rwandan caregivers prepared well-accepted SC+A porridges with a preferred consistency and mean DM content of 25.1% (1.0 kcal/g), after 1 week practicing at home. This supports introducing SC+A with the tested instructions at scale.

Nutrient digestibility, ruminal fermentation, and milk yield in dairy cows fed a blend of essential oils and amylase.

Two experiments were carried out to evaluate a blend of essential oils (EO) combined with amylase as an alternative to ionophores and its potential for reducing the use of antibiotics in the dairy industry. In experiment 1, 8 rumen-cannulated Holstein cows (576 ± 100 kg of body weight, 146 ± 35 d in milk, and 35.1 ± 4.0 kg/d of milk yield at the start of the experiment) were assigned to a 4 × 4 Latin square experiment with 21-d periods to determine the influence of feed additives on total apparent digestibility of nutrients, ruminal fermentation, N utilization, microbial protein synthesis, blood glucose and urea concentrations, and milk yield and composition in dairy cows. Treatment sequences assigned to cows in each block included no feed additives (control; CON); monensin (MON) added at 13 mg/kg of diet dry matter (DM); a blend of EO supplemented at 44 mg/kg of diet DM; and EO treatment combined with α-amylase at 330 kilo novo units/kg of diet DM (EOA). Differences among treatments were studied using orthogonal contrasts as follows: CON versus feed additives (MON, EO, and EOA), MON versus EO and EOA, and EO versus EOA. No differences were detected in nutrient intake and digestibility in cows. In general, feed additives decreased ruminal NH3-N concentration of cows, notably when diet was supplemented with MON. Furthermore, feed additives increased ruminal concentrations of acetate, butyrate, and branched-chain fatty acids. Cows fed treatments containing EO and EOA exhibited lower pH, higher NH3-N, and a trend to greater total volatile fatty acid concentration in the ruminal fluid compared with cows fed MON. Treatments containing EO increased ruminal butyrate concentration compared with MON. No treatment × time interaction effect was observed on ruminal fermentation measurements. Cows fed diets supplemented with feed additives had greater efficiency of N transfer into milk (milk N:N intake), whereas cows fed EOA exhibited greater N transfer into milk than those fed EO. Treatments had no effect on milk yield and composition, but feed additives increased the milk yield efficiency (milk yield divided by dry matter intake), whereas treatments containing EO had similar milk yield efficiency compared with MON. For experiment 2, 30 multiparous Holstein cows (574 ± 68 kg of body weight, 152 ± 54 d in milk, and 30.9 ± 4.1 kg/d of milk yield at the start of the experiment) were enrolled to a randomized complete block design experiment. The MON, EO, and EOA treatments were randomly assigned to cows within blocks (n = 10), and feed additives were provided throughout a 9-wk period. No differences were found in nutrient intake and digestibility, but cows fed EOA tended to exhibit greater dry matter intake than those fed EO. Blood metabolites and milk production were not affected by treatments. However, cows fed MON or EOA had greater milk protein content than those cows fed treatments containing EO. Feeding EO with or without amylase had similar response to feeding MON in terms of feed intake and milk yield, with a small negative effect on milk protein yield when feeding EO alone. Feed additives increased the concentrations of acetate, butyrate, and branched-fatty acids in ruminal fluid, whereas treatments containing EO had greater ruminal butyrate and NH3-N concentrations. Therefore, either EO or EOA can replace MON in diets of dairy cows while maintaining performance.

Effect of exogenous amylase on lactation performance of dairy cows fed a high-starch diet.

Exogenous amylase supplementation can increase starch and fiber digestibility in lactating dairy cows. We evaluated the effect of exogenous amylase supplementation on diets with high starch concentration (32% of dry matter). Twenty-eight Holstein cows (171 ± 80 d in milk, 4 primiparous) received a standard diet for 14 d and then a treatment for 63 d, in a covariate-adjusted randomized block design with repeated measures over time. Treatments were amylase [0.5 g of Ronozyme RumiStar (DSM Nutritional Products, Basel, Switzerland) per kg of total mixed ration dry matter] or control. The diets contained (% of dry matter): 39.4% corn silage, 11.2% rehydrated and ensiled mature corn grain, and 11.7% finely ground mature corn. Amylase increased milk yield (32.3 vs. 33.0 kg/d) and reduced dry matter intake (20.7 vs. 19.7 kg/d), increasing feed efficiency (1.52 vs. 1.63). Amylase also increased milk lactose synthesis (1.49 vs. 1.56 kg/d) and plasma glucose concentration (59.3 vs. 68.6 mg/dL). Secretions of milk fat and protein did not differ. Although milk urea N did not differ, amylase reduced the concentration of urea N in blood, suggesting an increase in ruminal starch degradation. However, the total-tract apparent digestibility of starch (96.3% of intake) and neutral detergent fiber (44.4% of intake), ruminal fermentation profile, and microbial yield estimated by urinary allantoin excretion did not differ. Cows fed amylase sorted in favor of long feed particles and against short particles, had shorter chewing activity (780 vs. 699 min/d), and had fewer meals per day (11.5 vs. 9.7). Amylase improved the feed efficiency of lactating cows fed a high-starch diet; the enzyme increased milk yield and reduced intake.

Nitration of Wheat Amylase Trypsin Inhibitors Increases Their Innate and Adaptive Immunostimulatory Potential in vitro.

Amylase trypsin inhibitors (ATI) can be found in all gluten containing cereals and are, therefore, ingredient of basic foods like bread or pasta. In the gut ATI can mediate innate immunity via activation of the Toll-like receptor 4 (TLR4) on immune cells residing in the lamina propria, promoting intestinal, as well as extra-intestinal, inflammation. Inflammatory conditions can induce formation of peroxynitrite (ONOO-) and, thereby, endogenous protein nitration in the body. Moreover, air pollutants like ozone (O3) and nitrogen dioxide (NO2) can cause exogenous protein nitration in the environment. Both reaction pathways may lead to the nitration of ATI. To investigate if and how nitration modulates the immunostimulatory properties of ATI, they were chemically modified by three different methods simulating endogenous and exogenous protein nitration and tested in vitro. Here we show that ATI nitration was achieved by all three methods and lead to increased immune reactions. We found that ATI nitrated by tetranitromethane (TNM) or ONOO- lead to a significantly enhanced TLR4 activation. Furthermore, in human primary immune cells, TNM nitrated ATI induced a significantly higher T cell proliferation and release of Th1 and Th2 cytokines compared to unmodified ATI. Our findings implicate a causative chain between nitration, enhanced TLR4 stimulation, and adaptive immune responses, providing major implications for public health, as nitrated ATI may strongly promote inhalative wheat allergies (baker's asthma), non-celiac wheat sensitivity (NCWS), other allergies, and autoimmune diseases. This underlines the importance of future work analyzing the relationship between endo- and exogenous protein nitration, and the rise in incidence of ATI-related and other food hypersensitivities.

The impact of direct-fed microbials and enzymes on the health and performance of dairy cows with emphasis on colostrum quality and serum immunoglobulin concentrations in calves.

Thirty-six cows were blocked by calving date and randomly assigned to one of three treatments. Cows were on treatments 3 weeks prepartum through 8 weeks post-partum. Treatments were as follows: (i) no direct-fed microbial (DFM) or cellulase and amylase enzymes (C), (ii) 45.4 g/day of DFM (D) or (iii) 45.4 g/day of DFM and 18.2 g/day of enzyme (DE). Total mixed ration fed and refused were measured daily to determine dry matter intake (DMI). Blood samples were taken three times weekly and analysed for ß-hydroxybutyrate, glucose and non-esterified fatty acids. Body weight (BW) was measured weekly. Colostrum was weighed and analysed for IgA and IgG concentration. Calves were fed 4 L of colostrum within 2 hr of birth. Calf blood samples were taken at 0 and 24 hr for analysis of IgA and IgG concentrations and apparent efficiency of absorption. Milk yield was measured daily and samples collected weekly. Initial BW was different among treatments with D being lesser than C or DE treatments. Body weight, weight gain, efficiency of gain, DMI and blood parameters were unaffected. Treatment did not affect colostrum yield. Ash percentage of colostrum tended to increase with D and DE, while IgA and total solids yield decreased with D. Colostrum fat yield was decreased in D and DE. Treatments did not impact BW, serum IgA and IgG concentrations or apparent efficiency of absorption of calves. Post-partum BW, DMI, blood parameters, milk production and composition were unaffected by treatment. However, cows on D gained more BW and tended to have greater efficiency of gain compared to those on DE, but were similar to C. Somatic cell scores were greatest for D. Results indicate that DFM and enzyme supplementation did not improve health and performance of dairy cattle during the pre- and post-partum periods under conditions of this study.

Clinical applications of amylase: Novel perspectives.

BACKGROUND: Amylase was the first enzyme to be characterized, and for the previous 200 years, its clinical role has been restricted to a diagnostic aid. Recent interface research has led to a substantial expansion of its role into novel, viable diagnostic, and therapeutic applications to cancer, infection, and wound healing. This review provides a concise "state-of-the-art" overview of the genetics, structure, distribution, and localization of amylase in humans. METHOD: A first-generation literature search was performed with the MeSH search string "Amylase AND (diagnost∗ OR therapeut$)" on OVIDSP and PUBMED platforms. A second-generation search was then performed by forward and backward referencing on Web of Knowledge™ and manual indexing, limited to the English Language. RESULTS: "State of the Art" in amylase genetics, structure, function distribution, localisation and detection of amylase in humans is provided. To the 4 classic patterns of hyperamylasemia (pancreatic, salivary, macroamylasemia, and combinations) a fifth, the localized targeting of amylase to specific foci of infection, is proposed. CONCLUSIONS: The implications are directed at novel therapeutic and diagnostic clinical applications of amylase such as the novel therapeutic drug classes capable of targeted delivery and "smart release" in areas of clinical need. Future directions of research in areas of high clinical benefit are reported.