Alkaline Protease Activity Research Articles

Uncovering the physiological impacts of soybean meal replacement by Narbonne vetch (Vicia narbonensis) meal in rainbow trout (Oncorhynchus mykiss) diets: Towards the future and sustainable European aquaculture.

The identification and implementation of raw materials to replace fish meal (FM) or its major alternative, soybean meal (SBM), among other factors, is crucial for the sustainable growth of aquaculture. Narbonne vetch (Vicia narbonensis) meal (NVM) has previously been identified as a promising alternative raw material locally produced in Europe to replace SBM. Previous work has showed that 33% replacement of SBM by NVM treated with a commercially available phytase (Rovabio® PHY) does not compromise fish growth performance in a 63-day trial. Here, a deeper characterization of the potential use of NVM to replace SBM was performed to unveil any potential undesired physiological impact in rainbow trout (Oncorhynchus mykiss) juveniles with an initial weight of 38.04 ± 0.07 g. After a 63-day feeding trial, high (66%) SBM replacement by NVM (even when treated with Rovabio® PHY) had a negative impact on rainbow trout: decreasing the activity of alkaline proteases, trypsin, chymotrypsin, and α-amylase; inducing cell shrinkage in hepatocytes; and reducing plasma triglycerides and hepatic vitamin E (VE) levels. No biochemical alterations were observed in blood plasma glucose, cholesterol or redox enzymes, regardless of the % of SBM replacement and/or Rovabio® PHY treatment. In fish fed diets with 33% replacement of SBM by NVM (regardless of being treated with Rovabio® PHY or not), only a reduced content of plasma triglycerides, and a slight modification in the position of the nucleus in enterocytes (more intermediate) from the proximal intestine, were reported. Altered metabolism of both carbohydrates and VE was also confirmed at transcriptional level, possibly being related to the presence of non-starch polysaccharides (NSPs) in NVM. The present study identifies future strategies to improve the use of NVM as an alternative raw material to replace SBM, allowing greater sustainability, and a lower carbon footprint in European aquafeeds to be achieved.

Alkaline protease production by thermotolerant Bacillus sp. KU-K2, from non-rubber skim latex through the non-sterile system and its enzymatic characterization

The non-rubber skim latex was used as a substrate for alkaline protease production under a non-sterile system. Among 108 isolates of aerobic spore-forming bacteria, the isolate of KU-K2 was selected as a potent bacterial strain for alkaline protease production. The 16s rRNA gene sequence analysis showed that the KU-K2 strain was closely related to the B. licheniformis with 99.39% similarity. The alkaline protease production from non-rubber skim latex serum was optimized using the Plackett-Burman design (PB) and response surface methodology based on central composite design (CCD). The results showed that the optimized medium consisted of 9.82 g/L skim milk, 0.97 g/L glucose, 0.82 g/L K2HPO4, and 0.02 g/L CoCl2·6H2O gave the highest enzyme production at 6.14 ± 0.13 U/mL. The upscale production in the 5.0 L stirrer fermenter was achieved at 6.78 ± 0.32 U/mL when incubated at 37 °C, 250 rpm with 2.0 vvm of aeration rate for 48 h, which increased by 2.90 folds compared to the un-optimized medium. The optimal pH and temperature for alkaline protease activity were 10.0 and 60 °C, respectively. Zn2+, Fe3+, and Hg2+ metal ions strongly inhibited the crude alkaline protease. This study showed the potential of thermotolerant Bacillus sp. For alkaline protease production using non-rubber skim latex as a substrate which could be applied to reduce the industrial waste from latex industries.

PHYSICAL AND CHEMICAL CHARACTERIZATION OF ALKALINE PROTEASE FROM BACILLUS SUBTILIS VBC7 USING AGRO WASTE AS SUBSTRATE

Alkaline proteases are the most important group of industrial enzymes that hydrolyse the peptide bond of proteins into small peptides. The industrial demand of alkaline protease predominantly from the microbial origin has been recently increased and enhances the research for alkaline protease with high stability at extreme industrial conditions. Thus, this study is aimed to characterize the alkaline protease from bacterial isolate, Bacillus subtilis VBC7 screened from dairy waste dumped soil. Extracellular alkaline protease production was carried out in alkaline broth by submerged fermentation. The production medium with 10% ground nut extract at pH 10 and 40 °C enhanced the alkaline protease activity (712 U/mL) than other wastes such as coconut pulp extract and sesame seed extract. This optimized media increased the bacterial growth rate and activity of alkaline protease compared to the unsupplemented basal medium. Further, alkaline protease was partially purified and assessed their molecular weight (~30 k Da) in 12 % SDS -PAGE. The enzyme activity was observed at pH 10 and 40 °C and stable over a wide range of pH (5-12), and temperatures (10-70 °C). In addition, enzyme activity was stimulated in the presence of Mg2+, Ca2+ and Mn2+ and was unpretentious after treating with surfactants (SDS, Triton X-100, Tween 80), organic solvents (ethanol, methanol, chloroform, acetone and hexane) and protease inhibitors (EDTA and β-mercaptoethanol). These compatible features could lead the enzyme as a potential candidate among the industrial sectors.

Mango extract in tambaqui (Colossoma macropomum) diet: an in vitro and in vivo study

ABSTRACT Two experiments were carried out in order to evaluate the functional effect of mango extract in vitro and in vivo. The in vitro assay evaluated the growth of Lactobacillus spp; different treatments with mango extract (1% and 3%), cellulose (1% and 3%) and a control group were used. In the in vivo test, 100 juvenile tambaquis were used, with an initial average weight of 57.03 ± 1.05 g, distributed in twenty 1000 L experimental units, in a water recirculation system with a biofilter. Five diets were tested, with different concentrations of mango extract (0, 20, 40, 60 and 80 g kg−1 of diet). The in vitro assay showed that the addition of 3% mango extract presented functional-prebiotic potential against the Lactobacillus spp. strain. The recommended concentration of 40 g. kg−1 of mango extract in the diet for tambaqui improves performance. Some hematological variables showed significant variations but did not harm tambaqui. Mango extract improved muscle glycogen stores. Plasma albumin and nonspecific alkaline protease activity were responsive in fish fed mango extract. Mango extract increases the lactate concentration in the tambaqui intestine.

Effects of Different Vegetation Types on Soil Microbial Functional Genes and Enzyme Activities in Reclaimed Coal Mine

Long-term coal mining has seriously damaged soil structures and ecological environments. Reclaimed vegetation could effectively improve the ecological environment. There have been many studies on soil physical-chemical characteristics and microbial community structure, but there are few on soil functional microorganisms in mining areas. In this study, soil physical and chemical properties, soil enzyme activities, and the abundance of functional genes in carbon, nitrogen, phosphorus, and sulfur cycling were determined in five reclaimed lands of different vegetation types (Platycladus orientalis, Picea asperata, Pinus tabuliformis, Pinus sylvestris, and Sabina chinensis) in the Jinhuagong mining area, Datong City, Shanxi province. The results showed that the restoration types produced significant effects on soil physical and chemical properties, soil enzyme activities, and the abundance of 75 functional genes. The highest contents of total carbon, total nitrogen, and total sulfur were in the S. chinensis site. The activities of dehydrogenase and urease were the highest in P. asperata, and the lowest alkaline protease activity was in P. asperata. The alkaline protease activity was the highest in P. sylvestrist. The abundance of functional genes in the P. asperata forest was the highest, but the diversity index was significantly lower than that in other plots, which might be due to the fact that the total abundance of ureC, acsA, and mct in P. asperata accounted for more than 52%. Soil urease was significantly correlated with the abundance of six functional genes. The β diversity of the functional microbial community was significantly different among the different vegetation types, indicating that functional genes could better explain the effects of vegetation types on microbial function. The highest and lowest integrated fertility indexes were found in the P. sylvestris and P. orientalis plots, respectively. In conclusion, soil functional genes are sensitive indicators signifying the effects of different reclamation types on soil microorganisms, and P. asperata and P. sylvestris are more suitable for reclamation in this mining area.

Influence of the biofloc bacterial community on the digestive activity of Nile tilapia (Oreochromis niloticus)

High-throughput sequencing was performed to study the diversity of bacterial communities in the water of an aquaculture production system using Biofloc technology (BFT) and its effect on the activity of digestive enzymes in Nile tilapia, Oreochromis niloticus. Sex-reversed juveniles were reared in six circular indoor tanks (1 m3) for 12 weeks. The following treatments were performed: biofloc with 0% protein restriction in the diet (BFTH = high protein diet); biofloc with 11% dietary protein restriction (BFTL = low protein diet), and clear water control with 0% dietary protein restriction (CW). Biofloc groups showed a higher bacterial diversity and a completely different composition compared to CW, with significant increase of Actinobacteria and Planctomycetes and a decrease in Verrumicrobia and Fusobacteria. At the genus level, Flavobacterium, Thermomonas, Comamonas, Microbacterium, and Intestinibacter present in BFT significantly correlated with the activity of trypsin, aminopeptidase, and alkaline proteases. Twenty percent of the difference between the biofloc groups was due to the higher activity of acid proteases and aminopeptidases. The metagenome prediction revealed metabolic pathways differentially expressed in BFTH and BFTL. Most were similarly expressed in both treatments; except for the CoA degradation pathway I, which was expressed in BFTL, and the biosynthesis of isoprene II and mevalonate II in BFTH. The biofloc microbiota provided enzymes that, in synergy with the endogenous digestive enzymes of the fish, improved digestion and nutrient assimilation. The study provides knowledge on the exogenous enzymatic contribution and diversity of the bacterial groups present in the biofloc; the contribution of these microorganisms in digestive metabolism and fish performance is also highlighted.

Abscisic acid-polyacrylamide (ABA-PAM) treatment enhances forage grass growth and soil microbial diversity under drought stress.

Drought restricts the growth of alpine grassland vegetation. This study aimed to explore a new technical system to improve the drought resistance of forage grass. Qinghai cold-land Poa pratensis seedlings were used in the drought stress experiment. A combination of abscisic acid (ABA) and polyacrylamide (PAM) were used to affect the growth, leaf physiology, soil enzyme activity, and rhizosphere microbial diversity of P. pratensis. The fresh leaf weight and root surface area were significantly increased after ABA-PAM combined treatment, while root length was significantly reduced. Besides, the leaf catalase (CAT) and superoxide dismutase (SOD) enzyme activity, proline and chlorophyll content, increased after the treatment, while malondialdehyde (MDA) content decreased. The treatment also increased sucrase, urease, and alkaline protease activities in rhizosphere soil, while decreasing acid phosphatase and neutral phosphatase enzyme activities. ABA-PAM combined treatment enhanced the rhizosphere microbial community and forage drought resistance by altering the abundance of various dominant microorganisms in the rhizosphere soil. The relative abundances of Actinobacteria, Chloroflexi, and Acidobacteria decreased, while Proteobacteria, Firmicutes, and Ascomycota increased. Unlike the relative abundance of Gibberella that decreased significantly, Komagataeibacter, Lactobacillus, Pichia, and Dekkera were significantly increased. Single-factor collinearity network analysis revealed a close relationship between the different rhizosphere microbial communities of forage grass, after ABA-PAM treatment. This study implies that ABA-PAM combined treatment can improve the drought resistance of forages. Therefore, it provides a theoretical and practical basis for restoring drought-induced grassland degradation.

Genetic engineering for enhanced production of a novel alkaline protease BSP-1 in Bacillus amyloliquefaciens.

Alkaline protease has been widely applied in food, medicine, environmental protection and other industrial fields. However, the current activity and yield of alkaline protease cannot meet the demand. Therefore, it is important to identify new alkaline proteases with high activity. In this study, we cloned a potential alkaline protease gene bsp-1 from a Bacillus subtilis strain isolated in our laboratory. BSP-1 shows the highest sequence similarity to subtilisin NAT (S51909) from B. subtilis natto. Then, we expressed BSP-1 in Bacillus amyloliquefaciens BAX-9 and analyzed the protein expression level under a collection of promoters. The results show that the P43 promoter resulted in the highest transcription level, protein level and enzyme activity. Finally, we obtained a maximum activity of 524.12 U/mL using the P43 promoter after fermentation medium optimization. In conclusion, this study identified an alkaline protease gene bsp-1 from B. subtilis and provided a new method for high-efficiency alkaline protease expression in B. amyloliquefaciens.

Effects of Insect-Resistant Maize 2A-7 Expressing mCry1Ab and mCry2Ab on the Soil Ecosystem.

Transgenic maize 2A-7 expressing mCry1Ab and mCry2Ab has excellent resistance to lepidopteran pests. Previous studies have investigated the effects of several Bacillus thuringiensis (Bt) proteins on the soil. However, the effects of artificially modified Bt proteins on soil ecosystems are still unclear. To evaluate the effects of transgenic maize 2A-7 on soil, the physicochemical properties, enzyme activities and functional diversities of the microbial communities in rhizosphere soils from 2A-7 and its near-isogenic non-transgenic control Dongdan 6531 were analyzed at different developmental stages under field conditions. The alteration of six physicochemical properties (pH, total nitrogen, total phosphorus, organic matter, available phosphorus and alkali-hydrolyzed nitrogen) and six functional enzymes (catalase, alkaline phosphatase, sucrase, acid phosphatase, urease and alkaline protease) activities in the rhizosphere soils between the two maize cultivars were drastically correlated with plant growth stage, but not affected by the artificially modified Bt transgenes. An analysis of time-course Biolog data revealed that the functional diversity of microbial communities in the rhizosphere soil of 2A-7 and its control were similar at each developmental stage. The results suggest that transgenic maize 2A-7 has no significant impact on the soil ecosystem and provide valuable information on scientific safety assessments of 2A-7 and its commercial applications.

Macrobrachium rosenbergii fed with essential oil from Lippia alba in the diet in low and high stocking density

This study aimed to examine the zootechnical performance, whole-body composition, activity of digestive enzymes and histology of hepatopancreas of Macrobrachium rosenbergii fed diets incorporated with essential oil from Lippia alba (EOLA) in low (LSD - 50 prawns m−2) and high (HSD - 100 prawns m−2) stocking density. Three diets were formulated; each containing increasing levels of EOLA (0.00 [control], 0.5 and 1.0%) in triplicate for 70 days. Water quality, survival and growth did not differ between treatments. Crude protein was higher in prawns that received 0.5% EOLA in HSD than in LSD. The ethereal extract was higher in prawns in LSD than in HSD fed with EOLA. In addition, the ethereal extract was higher in animals fed diets with 1.0 than 0.0% EOLA in LSD. Prawns that received 0.0 and 1.0% EOLA, respectively, had significantly higher chymotrypsin and alkaline protease activity. In general, the activity of digestive enzymes increased in prawns fed with EOLA in HSD. Histological analyses of the hepatopancreas in all treatments proved to be healthy. In conclusion, the digestive enzymatic activity and crude protein were higher in HSD than in LSD, indicating that EOLA added to the diet is more critical for prawns reared in HSD. In addition, the intensive production of M. rosenbergii can be optimized in HSD (100 prawns m−2) with 1.0% EOLA in the diet.

Conjugational Transformation of Wild Type Bacillus halodurans CM1 by Methylated Recombinant Plasmid Harbouring a Gene Encoding for Alkaline Protease and the Protease Activity Assay of the Transformant

Protease contributes significantly to various industrial sectors, as shown by its increasing demand. An indigenous previously isolated bacteria, Bacillus halodurans CM1, is a wild-type bacterium whose ability to produce alkalotermophilic protease. In this study, the transformation of methylated pBBRE194 plasmid containing alkaline protease gene from this strain (PBBRE194-prot-CM1) into itself using a conjugation approach was conducted, and the measurement of the alkaline protease activity of the recombinant bacterium was carried out. The recombinant B. halodurans CM1 has been verified to carry the methylated pBBRE194 prot-CM1 by examining its ability to degrade protein in media containing skim milk and tetracycline, but also by amplifying tetracycline resistance gene sequence with 1,024 bp length of plasmid pBBRE194 prot-CM1 by PCR method from the recombinant bacterium. The results confirmed that recombinant B. halodurans CM1 was positively harboring plasmid pBBRE194 prot-CM1. Alkaline protease produced by recombinant CM1 reached higher activity than wild type between 18-36 h of cultivation. The alkalotermophilic wild type B. halodurans could accept the recombinant plasmid into their cells via conjugational transformation is firstly reported to our further knowledge.

Incorporation of Fructooligosaccharides in Diets Influence Growth Performance, Digestive Enzyme Activity, and Expression of Intestinal Barrier Function Genes in Tropical Gar (Atractosteus tropicus) Larvae

This study was conducted to investigate the effects of dietary fructooligosaccharides (FOS) on the growth, survival rate, digestive enzyms activity, and the expression of intestinal barrier function genes in tropical gar (Atractosteus tropicus) larvae. A total of 960 larvae (0.030 ± 0.006 g) were fed three diets supplemented with increasing FOS concentrations (2.5, 5, and 7.5 g kg−1) and a control diet for 15 days. Results revealed that a 7.5 g kg−1 FOS supplementation improved weight gain, specific growth rate, and survival rate (p < 0.05). Furthermore, 5 g kg−1 FOS supplementation increased alkaline protease and amylase activities and induced an upregulation of the claudin-17 gene expression (p < 0.05). Meanwhile, the inclusion of 7.5 g kg−1 FOS induced the upregulation of mucin 2 (muc-2), and the tight junction genes zo-2 and claudin-3 (p < 0.05). In addition, 2.5, 5, and 7.5 g kg−1 FOS promoted the downregulation of the claudin-15 gene expression (p < 0.05). At the same time, FOS inclusion did not increase the pro-inflammatory cytokine il-8 expression. We can conclude that 7.5 g kg−1 FOS supplementation improves growth performance, survival rate, and digestive capacity, and could contribute to the reinforcement of the intestinal barrier function of Tropical gar larvae.

Addition of essential oil from Lippia sidoides to the diet of tambaqui: An analysis of growth, metabolic and blood parameters, and intestinal enzymes

Plant essential oils can benefit fish growth and health when incorporated into fish diets. This study aimed to evaluate the effect of dietary supplementation of essential oil from Lippia sidoides (EOLS) on growth performance, hematological and biochemical variables, and digestive enzymes in tambaqui (Colossoma macropomum) juveniles. Five diets with different levels of EOLS—0.0 (control), 0.25, 0.50, 1.00, and 1.50 mL kg diet−1— were evaluated for 60 days. Carvacrol was the major chemical compound (44.50%) of EOLS. Supplementation of 0.50 mL EOLS kg diet−1 improved growth performance and increased hematocrit, hemoglobin rate, mean corpuscular hemoglobin concentration, plasma glucose, liver, and muscle glycogen values in juveniles. The tambaqui fed a diet supplemented with 1.00 or 1.50 mL EOLS kg−1 increased plasma triglycerides, albumin, lysozyme, and alanine aminotransferase levels, in addition to intestinal amylase and lipase activity. As the EOLS concentration in the diet increased, intestinal alkaline protease activity was reduced. In conclusion, the addition of 0.50 to 1.00 mL EOLS kg diet−1 is a promising alternative for tambaqui juveniles, as it has been shown to improve growth, metabolism, hematological parameters, and plasma lysozyme and intestinal amylase and lipase activity.

Efficient production of extracellular alkaline protease in Bacillus amyloliquefaciens by host strain construction

Bacillus amyloliquefaciens is an important industrial microorganism, which can be applied as a protein expression host. However, the extracellular protein expression ability of B. amyloliquefaciens is still low. Hence, it is necessary to develop a B. amyloliquefaciens host with a high extracellular protein expression capacity. In this study, B. amyloliquefaciens HZ-12 was the original strain, then seven important extracellular protease genes (epr, nprE, aprE-a, mpr, pbpF, vpr, ykct1), one important intracellular protease gene (aprX) and two redundant proteins genes (htrB, hag) were deleted in HZ-12, resulting in B. amyloliquefaciens BAX-10. In order to evaluate the extracellular protein expression ability of BAX-10, the alkaline protease gene aprE from Bacillus subtilis D7 was expressed in BAX-10. After fermentation, the alkaline protease activity of BAX-10/aprE reached 666.82 U/mL, which was 57% higher than that of the control strain HZ/aprE. Moreover, the deletion of the ten genes had no negative effect on the growth of B. amyloliquefaciens. In summary, B. amyloliquefaciens BAX-10 could be used as a potential platform host for expression of target extracellular proteins.

Dietary Use of the Microalga Chlorella fusca Improves Growth, Metabolism, and Digestive Functionality in Thick-Lipped Grey Mullet (Chelon labrosus, Risso 1827) Juveniles

In recent years, a clear emphasis has been placed on replacing fishmeal and fish oil in aquafeeds with other alternative ingredients, including algae, particularly in low trophic omnivorous fish species. This work aimed at evaluating the effects of moderate dietary supplementation with the green microalga Chlorella fusca on growth, metabolism, and digestive functionality in juvenile thick-lipped grey mullet (Chelon labrosus). Fish were fed a control diet (CT) or a diet containing 15% C. fusca (C-15) biomass during 90 days. C. labrosus fed with the C-15 diet showed higher growth performance (in terms of final weight and length, weight gain, and specific growth rate) than the control group. Somatic indices and muscle proximate composition were similar at the end of the feeding trial. Regarding fatty acids profile, C. fusca-fed fish showed a selective retention of docosahexaenoic acid (DHA) in the liver, and arachidonic acid (ARA), eicosapentaenoic acid (EPA), and DHA in the muscle. Dietary inclusion of this microalga significantly increased intestinal total alkaline protease, leucine aminopeptidase, and alkaline phosphatase activities in specimens fed with C-15 diet. Furthermore, intestine histological analysis revealed the absence of damage signs on gut morphology in fish fed the microalgae supplemented diet. Thick-lipped grey mullets fed the C-15 diet increased plasma glucose and decreased plasma lactate. Overall, the effects observed on liver (lipid metabolism, glycolysis and glycogenolysis) enzyme activities, together with adequate fatty acid profile, metabolic response, and gut morphology, and a significant increase in the intestinal mucosa’s digestive and absorptive capacity, could explain the positive effects on growth performance obtained in fish fed the microalgae-supplemented diet. In conclusion, the results obtained showed that C. fusca is suitable as dietary ingredient for feeding thick-lipped grey mullet juveniles.

Activities of Antioxidant and Proteolytic Systems and Biomarkers in the Fat Body and Hemolymph of Young Apis mellifera Females.

Simple SummaryThe proteolytic system consists of compounds that, similar to “scissors”, cut proteins found in bee cells (e.g., to activate these proteins) or released by pathogens. During these reactions, reactive oxygen species are created and then removed by antioxidants. The actions of the proteolytic and antioxidant systems are enhanced by biomarkers. These compounds are produced mainly in the fat body and then released into the hemolymph. We determined the activities of these compounds in various localizations/segments of the fat body and in the hemolymph in females with increased reproductive potential, i.e., queens and rebels, and in normal (sterile non-rebel) workers. Rebels are workers who resemble the queen in terms of anatomical, behavioural, and physiological features. It was revealed that the activities of these compounds in the rebels were between those of queens and normal workers. Normal workers had higher activities of the proteolytic and antioxidant systems in the fat body and hemolymph than the other females. These results are important for understanding the functioning of the fat body, the stress ecology, and the formation of the different castes of Apis mellifera females.The proteolytic and antioxidant systems are important components of humoral immunity, and these biomarkers indicate the immune status. These compounds are synthesized in the bees’ fat body and released into the hemolymph. Their functions maintain the organism’s homeostasis and protect it against adverse environmental factors (including pathogens). We determined the activities of acidic, neutral, and alkaline proteases and their inhibitors, as well as superoxide dismutase (SOD), catalase (CAT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and the level of total antioxidant potential (TAC). These compounds were investigated in the fat body and hemolymph in the females with increased reproductive potential, i.e., queens and rebels, and in normal (non-reproductive sterile non-rebel) workers. The phenoloxidase (PO) activities were determined in the hemolymph. The normal workers had higher activities of proteases and their inhibitors, SOD and CAT, in the fat body and hemolymph, compared to the queens and rebels. The protease inhibitors were not usually active in the queens. As we predicted, the rebels revealed values between those of the queens and normal workers. The highest activities of proteases and antioxidants were identified in the fat body from the third tergite in comparison with the sternite and the fifth tergite. These results are important for oxidative stress ecology and give a better understanding of the functioning of the fat body and the division of labor in social insects.

Effects of deleting peptidoglycan hydrolase genes on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease

In order to explore the effect of peptidoglycan hydrolase on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease, five peptidoglycan hydrolase genes (lytC, lytD, lytE, lytF and lytG) of B. amyloliquefaciens TCCC111018 were knocked out individually. The viable cell counts of the bacteria and their alkaline protease activities before and after gene deletion were determined. The viable cell counts of the knockout mutants BA ΔlytC and BA ΔlytE achieved 1.67×106 CFU/mL and 1.44×106 CFU/mL respectively after cultivation for 60 h, which were 32.5% and 14.3% higher than that of the control strain BA Δupp. Their alkaline protease activities reached 20 264 U/mL and 17 265 U/mL, respectively, which were 43.1% and 27.3% higher than that of the control strain. The results showed that deleting some of the peptidoglycan hydrolase genes effectively maintained the viable cell counts of bacteria and increased the activity of extracellular enzymes, which may provide a new idea for optimization of the microbial host for production of industrial enzymes.

Effect of different dietary proteins and fats on the digestive enzymes activities in the common carp fingerlings (Cyprinus carpio L.) reared in floating cages

The digestive enzyme activities of the common carp fed with different diets of protein sources were investigated during 150 days trial in order to obtain a theoretical basis for artificial diet preparation for the culture of carp fingerlings (Cyprinus carpio L.). The results showed that the fish at treatment 3 (protein 13.82%, fat 4.76% and carbohydrate 67.81%) had high levels of alkaline protease activity. Acid protease activity was high at the treatment 1 (protein 23.68%, fat 16.28% and carbohydrate 43.68%) (p<0.05). In general, the acid protease activity was higher than alkaline protease during the course of the study. Amylase activity reached a highest value in fish fed with none animal protein sources (treatment 3). Lipase activity showed a high level in fish fed with high lipid contents (treatment 1).

Selection and improvement of alternative raw materials for rainbow trout (Oncorhynchus mykiss) aquafeeds through a multiparametric screening tool

Aquaculture growth and sustainability mainly rely on the identification and implementation of alternative raw materials to replace fish meal (FM) and fish oil (FO) and/or its major substitute, the soybean meal (SBM). A five-step screening tool has been designed to identify and improve the use of promising alternative raw materials. To validate it, nine raw materials, including the standard reference (the SBM), were sequentially analyzed regarding (i) the total buffer capacity, alkaline protease activity inhibition and soluble protein content, (ii) soluble phosphorus and phenolic compound content, (iii) modification of nutrients bioavailability and presence of anti-nutritional factors after treatment with exogenous enzyme (Rovabio® Phy), (iv) release of nutrients after in vitro digestion, and (v) the palatability of the formulated diet. SBM partial replacement by selected raw material, the Narbonne vetch (Vicia narbonensis) meal (NVM), was evaluated in a 63-day nutritional trial using rainbow trout (Oncorhynchus mykiss) juveniles. One Control (no SBM replacement) and 4 experimental diets with SBM replacement in two levels (33% and 66%), treated or not with exogenous enzyme Rovabio® Phy, were compared. Fish growth performance and amino acid profile in fish fillet was not significantly affected when SBM was replaced by 33% of NVM treated with exogenous enzyme. Altogether, the present screening tool might be a wise strategy to identify promising alternative raw materials for European aquaculture sustainability, reducing the use of animals in experimentation, the SBM dependency from third countries, and its carbon footprint.

Dietary supplementation of Chlorella vulgaris improved growth performance, immunity, intestinal microbiota and stress resistance of juvenile narrow clawed crayfish, Pontastacus leptodactylus Eschscholtz, 1823

A 63- day feeding trial was conducted to study the effect of fishmeal substitution levels with Chlorella vulgaris meal (25, 50, 75 and 100%) on the growth performance, nutrient digestibility, immune responses and the stress resistance of juvenile narrow clawed crayfish, Pontastacus leptodactylus (6.19 ± 0.13 g). The highest values of final weight (25.52 g), specific growth rate (2.24% body weight day−1), protein efficiency ratio (3.24), the protein productive value (54.67%), in vivo ADCOM (88.40%) and in vivo ADCCP (93.50%) and the lowest feed conversion ratio (1.49) were observed in the juvenile crayfish fed the diet containing 75% fishmeal substituted level with chlorella meal (p < 0.05). With an increment in the dietary inclusion level of chlorella meal, the activities of alkaline protease (r2 = 0.97), lipase (r2 = 0.98), amylase (r2 = 0.97), phenoloxidase (r2 = 0.98), superoxide dismutase (r2 = 0.99), lysozyme (r2 = 0.96) and nitric oxide synthase (r2 = 0.98) were promoted as a third-order polynomial regression model (p < 0.05). There was an increasing trend in Lactobacillus count (r2 = 0.99) and a decreasing trend in Escherichia coli count (r2 = 0.97) with an increase in the fishmeal substitution level with chlorella meal. Based on the broken line regression model, the dietary fishmeal substitution level (%) with chlorella meal for maximum growth (SGR) and weight gain values and minimum FCR value of crayfish were estimated to be 85.49%, 78.14% and 78.13%, respectively.