Protein hydrolysates from silkworm (Bombyx mori) pupae protein treated with a novel neutral protease

Edible insects, regarded as a potential contributor to food security are currently given wide consideration due to their rich protein and other micronutrients contents. In this study, protease-assisted hydrolysis proposes an economically effective approach to hydrolyse proteins from silkworm (Bombyx mori) pupae to improve its functional properties. The proteolytic activity of a novel neutral protease (265.14 U/ml) with appreciable thermal activities, was identified using 16S rDNA as Stenotrophomonas maltophilia JW20 (SmNP20). The neutral protease with an apparent molecular weight of 28 kDa emerged active at pH 7 and maintained stability in pH range 6.0-8.0. The optimum temperature was 60 °C and stable at 55-60 °C, maintaining over 80% of its initial activity, with a half-life of 78.75, 89, 66.8 and 44 min at 50, 60, 70 and 80 °C. It was purified to 9.98-fold with a specific activity of 455.06 U/mg and 63.73% yield. The Km and Vmax values were 0.70 mg/ml and 9.48 μmol/min/mg, respectively. Enzymolysis with neutral protease enhanced the degree of hydrolysis (97.46±4.87%), increased water solubility over 50%, and a significant protein solubility of 63.44±0.65%. The Km and Vmax of the protein yield were 0.24 mg/ml and 165.63 μmol/min/mg respectively. A total of 17 amino acids have been detected in the hydrolysates obtained from the silkworm pupae protein. In comparison with neutrase and flavorzyme®, the enzyme possesses an elevated hydrolytic and catalytic efficiency. Emulsion activity and foam capacity ranged from 8-48 m2/g and 6-25% respectively. Hence, this study confirms the unique and efficient characteristics of an insect-enzyme correlation that is practically significant with potential improvement in nutritional composition and functional quality of insect proteins.

Production of an extracellular neutral protease by Bacillus aerius UB02 endophytic to carnivorous plant Utricularia stellaris

Aim: Endophytic bacteria indigenous to carnivorous plants have been explored for production of novel bioactive metabolites including extracellular enzymes. Bacillus aerius UB02, an extracellular neutral protease producing isolate endophytic to bladder of Utricularia stellaris L. f. was used in this study. Methodology: The bacterial isolate UB02 was identified following morpholological, physiological, biochemical and 16S rRNA gene sequence analyses. The media as well as the cultural conditions for production of protease were optimized. The extracellular protease was isolated and purified from the cell-free culture filtrate by ammonium sulphate precipitation, dialysis and DEAE Sephadex ion exchange column chromatography and the optimum conditions for its activity were determined. Results: The isolate Bacillus aerius UB02 (GenBank accession no. MK 696417, MCC accession no. 4132), produced significant amount of extracellular protease (38.29 U mg-1 protein) during growth in casein supplemented synthetic medium. However, peptone yeast extract glucose medium appeared to be the best for the synthesis of enzyme. Production of enzyme was enhanced by the inoculum density of 1.5% (v/v), culture volume: flask volume (CVF) ratio of 1:10, substrate concentration of 2.5% (w/v) with temperature and pH adjusted at 37°C and 7.4, respectively. Glucose (2.2%, w/v) and ammonium chloride (1.2 g/L) as carbon and nitrogen sources also favoured the enzyme production. The neutral protease with a molecular weight of approximately 35 kDa showed maximum activity at 40°C, pH 7.8 with 2% (w/v) casein. The enzyme exhibited Km and Vmax values of 6.81 mg ml-1 and 62.5 U mg-1 of protein, respectively, and was moderately thermostable. The protease activity was inhibited by Pb and Cd as well as 1,10-phenanthroline and β-mercaptoethanol. Interpretation: These findings will help not only in understanding the role of endophytic bacteria and the enzymes produced by them in the digestion of prey by carnivorous plant but could also be explored for application in the field of biotechnology.

Effects of Flaxseed and Multi-Carbohydrase Enzymes on the Cecal Microbiota and Liver Inflammation of Laying Hens

Background: The use of wheat and flaxseed to produce omega-3 (ω-3) enriched poultry meat and eggs is very popular in the world. However, wheat and flaxseed contain some anti-nutritional factors (ANFs), and enzymes are usually used to alleviate the deleterious influence of ANFs. Method: A 2 × 3 two factors design was used in the experiment. A total of 540 twenty-week-old Nongda-3 laying hens were randomly allocated to six dietary treatments, two diets (corn/flaxseed and wheat/flaxseed), and three enzymes (enzyme-a contains neutral protease 10,000, xylanase 35,000, β-mannanase 1500, β-glucanase 2000, cellulose 500, amylase 100, and pectinase 10,000 (U g−1); enzyme-b contains alkaline protease 40,000 and neutral protease 10,000 (U g−1); enzyme-c contains alkaline protease 40,000, neutral protease 10,000, and cellulase 4000 (U g−1). Results: There was an interaction between dietary treatment and supplemental enzymes for liver weight and liver inflammatory cytokines of broilers. A significant increase was observed in the fat weight of birds fed a corn diet as compared with a wheat diet. A corn diet and wheat diet with the addition of enzyme-a (p < 0.001) showed the highest level of liver fat followed by enzyme-c (p < 0.01) and enzyme-b. Moreover, a high level of secretory IL-1β, IL-6, and IL-10 and comparatively higher inflammatory changes in the liver tissue were found in birds fed a corn diet as compared with a wheat diet, and enzyme-b showed more beneficial effects as compared with enzyme-a and -c. The gut microbial composition of hens fed a corn diet was significantly different than that of birds fed a wheat diet. Bacteroides were significantly (p < 0.05) abundant in the corn-fed birds as compared with wheat-fed birds. However, Firmicutes were less abundant in the wheat-fed birds than the corn-fed birds (16.99 vs. 31.80%, respectively). The microbial community at the genus level differed significantly in the dietary groups and we observed that Bacteroides are the predominant cecal microbiota. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of co-factors, carbohydrates, vitamins, protein, and energy were expressed at slightly higher levels in the microbiota of the wheat-fed birds, whereas, metabolic pathways for nucleotides, lipids, and glycine were expressed at higher levels in the wheat-fed birds. Furthermore, expression of the growth and cellular processes pathway and endocrine system pathway levels were predicted to be higher for the wheat-fed group as compared with the corn-fed group. Conclusions: In conclusion, our findings suggest that inflammatory changes in laying birds were mediated by a corn diet with flaxseed and enzymes instead of a wheat diet. Additionally, in the wheat-fed group, enzyme-b and -c showed more encouraging results as compared to enzyme-a.

PSVI-5 Effects of exogenous glucoamylase enzymes or a combination with a neutral protease on total tract apparent digestibility and feces D-lactate in bulls fed a total mixed ration rich in rolled corn

The aim of this study was to evaluate the effect of 2 glucoamylases (GA) and the combination of one GA with a neutral protease on total tract apparent digestibility in bulls fed a total mixed ration (TMR) rich in rolled corn. Sixteen Angus beef bulls (266 ± 4.9 kg of initial BW, and 182 ± 1.7 d of age) were housed individually in pens and were distributed in 4 blocks of 4 animals balanced by BW. The experimental design was a 4 x 4 Latin square (4 blocks and 4 periods) with periods of 2 wks. Four treatments were tested; 1) control, a blank solution of 0.2% potassium sorbate and 0.6% sodium benzoate corresponding to the preservatives used in the enzyme preparation, 2) a GA preparation from Trichoderma reesei; 3) a GA preparation from Aspergillus fumigatus; 4) and a GA from Aspergillus fumigatus and a Bacillus amyloliquefaciens neutral protease preparation. These enzyme solutions were daily mixed was mixed in a dry TMR with 70% of rolled corn at a rate of 10 mL for 40 kg TMR. From day 8 to 14 chromium oxide (1 mg/kg) was mixed with the diet and from day 12 to 14 feces were collected to estimate total tract apparent digestibility and D-lactate concentration. Intake was recorded daily. Enzyme supplementation, regardless of enzyme type, increased (P &lt; 0.05) DM (from 66.7% to 73.1% ± 2.01), OM (from 66.7% to 72.9% ± 2.03), and starch (from 74.7% to 81.8% ± 2.25) total tract apparent digestibility, and did not affect feces D-lactate concentration (1.26 vs 1.00 ± 0.200 mM for control and enzyme supplemented, respectively). So, any of the enzyme preparations tested (glucoamylases from Trichoderma reesei or Aspergillus fumigatus, or Aspergillus fumigatus glucoamylase n combination with the Bacillus amyloliquefaciens protease) increased by 9.5% starch digestibility.

Effects of three kinds of protease on growth performance, apparent digestibility of nutrients and caecal microbial counts in weanling pigs

This experiment investigated the effects of three single kinds of protease on performance, serum parameters, apparent digestibility of nutrients and caecal microorganisms in weanling pigs. One hundred and ninety-two White × Landrace × Duroc hybrid pigs (7.51 ± 0.81 kg) were allotted to 1 of 4 treatments for 28 days. No protease was added to the control diet and the three experimental diets were supplemented with the same proportion of 10 IU/kg of alkaline, acidic and neutral protease, respectively. The supplementation of alkaline protease increased average daily gain and reduced serum concentrations of cholesterol and triglyceride compared with the control group (P &lt; 0.05). The three single kinds of protease supplementation decreased the levels of serum urea nitrogen and increased the serum concentration of aspartate aminotransferase (P &lt; 0.05). The apparent digestibility of crude protein was increased by the dietary supplementation of alkaline or acidic protease compared with that of pigs in the control group (P &lt; 0.05). Results indicate that the alkaline protease supplementation improved the performance and apparent digestibility of crude protein in weanling pigs.

Effects of Exogenous Glucoamylase Enzymes Alone or in Combination with a Neutral Protease on Apparent Total Tract Digestibility and Feces D-Lactate in Crossbred Angus Bulls Fed a Ration Rich in Rolled Corn

The aim of this study was to evaluate the effect of two glucoamylases (GA) and the combination of one GA with a neutral protease on apparent total tract digestibility in beef bulls fed a total mixed ration (TMR) rich in rolled corn. Sixteen Angus beef bulls (266 ± 4.9 kg of initial BW, and 182 ± 1.7 d of age) were distributed in 4 blocks, each block consisted of 4 animals balanced by BW. The experimental design was a 4 × 4 Latin square (4 blocks and 4 periods, 2 w per period). Four treatments were tested; (1) control, (2) GA preparation from Trichoderma reesei (TrGA); (3) GA from Aspergillus fumigatus (AfuGA); (4) AfuGA in combination with a neutral protease from Bacillus amyloliquefaciens (BamPro). Apparent total tract digestibility and fecal D-lactate concentration were analyzed. Enzyme supplementation, regardless of enzyme type, increased apparent total tract digestibility of dry matter (from 66.7% to 73.1% ± 2.01), and starch (from 74.7% to 81.8% ± 2.25), without affecting feces D-lactate concentration. Irrespective of glucoamylase type, glucoamylase supplementation improved apparent digestibility of dry matter and starch, and the addition of a protease did not have additional benefits on nutrient digestibility.