Impact of β-Mannanase from indigenous fungal isolates on growth, metabolism and immunity of omnivore fish

Abstract

Nonstarch polysaccharides (NSP), in plant-based animal feed, are one of the major anti-nutritional factors which interfere with the digestion and absorption of the nutrients. Among NSP mannan occurs in the form of glucomannan and galactomannan and is widely present in plant-based feed sources like soybean meal, sesame meal, copra meal, palm kernel meal, guar gum meal, corn meal, canola meal and Corn distillers dried grain with solubles (DDGS) Thus mannan is a part of most feed formulations. As monogastric animals including fish lack the digestive enzymes needed to degrade mannan, this results in decreased nutrient digestibility and poor growth performance. Studies on poultry and swine have shown that β-mannanase can hydrolyze mannans in plant-based feed, thus increasing mineral bioavailability and nutrient digestibility while also providing health beneficial prebiotics in the form of mannan oligosaccharides (MOS). Most of the commercial β-mannanase enzyme preparations are well suited to function in gut of poultry and swine. But the gastrointestinal tract environment of omnivore fish is different from other monogastric animals like pigs and poultry. This creates a need for a suitable enzyme candidate that can used as a fish feed additive and can function well in the unique gastrointestinal tract environment of fish. In order to produce β-mannanase with the properties suitable for feed applications in aquaculture, new enzymes with desired characteristics need to be isolated from novel strains. Without supplementation of plant-based feed with suitable enzyme that degrade hemicellulose fraction in feed, the replacement of fishmeal with plant-based protein sources would not be feasible in aquaculture. Therefore, the present study's goal was to identify and characterize a mannan-degrading microbial enzyme from a novel fungal strain that may be employed as a feed supplement in plant-based feeds in aquaculture. The study was conducted in three phases. In the first phase, isolation of various fungal strains from soil samples obtained from different parts of Punjab was carried out. Among many different strains tested for mannanase production, one fungal strain turned out to be an efficient producer of β-mannanase enzyme. Molecular and phenotypic identification of this strain revealed it to be Aspergillus niger. The DNA sequence was submitted in NCBI with accession number MN239884 and the strain was designated as Aspergillus niger AD-01. Different parameters for maximum production of β mannanase from Aspergillus niger AD-01 were optimized. Optimum production of mannanase occurred at 30◦C, pH=5, substrate concentration=1% locust bean gum, nitrogen xiv source=yeast extract, days of incubation=5 days. When produced under optimized conditions, the specific activity of β-mannanase reached 12.49U/mg. After optimization of mannanase production its purification was carried out. Mannanase was purified in two steps. In the first step precipitation with ammonium sulphate was done and the enzyme was precipitated out. The precipitated enzyme was dialyzed against sodium phosphate buffer (PH=6) for overnight and then run through size exclusion Column containing sephadex gel G-75. The fractions containing highest activity of the enzyme were collected. The specific activity of purified enzyme reached 78.07 U/mg and it was purified by 6-fold with a percentage yield of 24%. Characterization of the enzyme revealed it to be a thermostable, low temperature active, weakly acidic and protease resistant enzyme. It’s optimum temperature and pH were 40◦C and 6, respectively. The enzyme exhibited considerable stability when incubated in buffers with pH ranging from 4-8. Mannanase isolated from Aspergillus niger AD-01showed good thermostability. The enzyme was very stable at 37ºC and it retained half of its activity at 40◦C after 30 min but at 50ºC after 10 min incubation, its activity was reduced to mere 20%. The enzyme showed good resistance towards proteases and NaCl (1M and 2M) at 37◦C and pH=6.Mannanase enzyme did not lose its activity in presence of different metal except Ba2+ and Hg2+ where drastic reduction in activity was observed. Inhibition by Hg2+ suggests the presence of a crucial sulfhydryl group in the enzyme. This weakly acidic, protease resistant, low temperature active profile showed the potential of β-mannanase for use as a feed additive for agastric or omnivorous fish. A 90-day feeding experiment was conducted in the second phase to examine the potential positive effects of β-mannanase supplementation in the plant-based feed on intestinal and biochemical enzymes activity, growth performance, digestibility of nutrients, immunological response and hematological indices of common carp. The experiment was carried out in triplicate, and 225 carp fingerlings (weighing a total of 13.17 ±0.12 g) were placed in 15 fiberglass tanks (15 fish in each tank). Five groups of fish were created with 45 fish in each group. The control group was provided with 35% basal crude protein diet and had 0 Ukg-1 diet of β-mannanase while Group A1 and A2 were supplemented with (500 and 1000 Ukg-1 diet of) commercial mannanase enzyme and group B1 and B2 with (500 and 1000 Ukg-1 diet of) isolated β-mannanase enzyme. Sampling was done at the end of the 90 day long feeding trial and it was observed that groups of fish that were supplemented with β mannanase enzyme in the feed had enhanced growth performance, survival rate, immunological indices (respiratory burst activity, lysozyme activity, immunoglobulin activity xv and phagocytic index) and hematological indices (Hb, MCHC, white blood cells and red blood cells). The activity of biochemical indices including AST and ALT decreased in the experimental groups but a reverse trend was observed with CHO and TG. The digestibility of different nutrients like fats, protein and carbohydrates also increased. Additionally, MyoD expression in muscles and TNF-α gene expression in muscles, intestine and liver was enhanced. The effect of mannanase supplementation on digestive enzymes was also investigated. The activity of all the digestive enzymes tested. i.e., cellulase, protease and amylase was increased significantly (P < 0.05) in treatment groups. Both sources of enzyme supplemented at two dosage levels had similar effect on different parameters of fish (P> 0.05). In the third phase of the study the effect of β-mannanase supplementation on the intestinal health of common carp was investigated. For this the gene expression of crucial digestive and immune enzyme related genes and fish gut microbial diversity was studied. Again a 90-day feeding trial was run. The fish were divided into three groups: the control had 35% basal crude protein diet with 0 Ukg-1 diet of mannanase and the experimental groups (A1 and A2) were provided with 500 and 1000 Ukg-1 diet of isolated β-mannanase. Sampling was done at the end of the 90-day long feeding trial, and fish gut microbial diversity and the expression level of the selected immune and digestive enzyme related genes was studied. For investigation of the effects of β-mannanase supplementation on gut microbiota, the V4 region of bacterial 16S rRNA and ITS region of fungal 18sRNA was amplified and sequenced. Shannon and Simpson index showed that the experimental groups of fish had greater microbial diversity in the gut than control group. Enhanced microbial diversity in the experimental groups is an indication of the health of the fish and positive effects of β mannanase supplementation in the diet. In the experimental groups Lactococcus, Lactobacillus, Lachnospira and Geotrichum increased and disease-causing opportunistic pathogens like Vibrio and Aeromonas were absent. Further evidence of beneficial effect of β mannanase supplementation was provided by enhanced gene expression of important digestive enzyme (Amy, Lip, Trp, FAS, FASB) related and immune related (SOD, Lys, IL-β, Def, Nk-lys) genes. Overall results indicated that the present study identified, isolated and characterized a stable, salt-tolerant, cold active extracellular endo-β-mannanase produced by Aspergillus Niger-AD-01, a new mannanase-producer. β-mannanase displayed good activity against xvi locust bean gum, guar gum, konjac powder and soybean meal. Furthermore, β-mannanase supplementation irrespective of microbial origin showed a beneficial effect on apparent digestibility coefficient of nutrients, the growth, body composition, intestinal enzyme activity, hemato-immunological indices, metabolic enzymes, and expression of genes related to growth and immunity in C. carpio. In addition, the supplementation of β-mannanase modulated the gut microbiota of C.carpio, while certain beneficial microorganisms were plentiful, opportunistic pathogens were absent. Mannanase also upregulated the expression of key digestive and immune-related genes in the intestine. Based on these findings, supplementation of β-mannanase may be recommended as a feed additive for low-cost production of common carp.