| Introduction:In recent years, the utilization of Housefly (Musca domestica) has attracted the attention of domestic and foreign researchers. In addition to rich in nutrients such as protein, fat, polysaccharides and minerals, housefly larvae maggots also contain chitin, antimicrobial peptides, agglutination prime, lysozyme and other biologically active substances. Housefly larvae maggots have a wide range of applications in the bio-pharmaceutical, food and feed development, et al. In Livestock and aquatic animal breeding research, because of the higher protein content in Housefly larvae meal, it is generally believed that Housefly larvae meal is a highly quality substitute for fish meal, and can be used as protein source in the animal feed. Some Studies suggest that the high-quality fats, minerals or chitin, antimicrobial peptides and other biologically active substances contained in the maggot can promote animal growth and enhance animal immunity.Compare to several other trace active ingredient, the chitin in housefly larvae maggots shell in a higher proportion of up to20-30%. Chitin is a long-chain polymer of a N-acetyl glucosamine. Chitin has a variety of biological activity and can be used as the growth-promoting agent, immunopotentiator and broad-spectrum antimicrobial agent in animals. In maggot, chitin and protein conjugate to a proteoglycans, and associated with the calcium carbonate. It is not dissolve in water, when added directly to the feed, is difficult to be digested. In some studies, maggot chitin is consider to be a kind of anti-nutritional factors affect the animal’s digestion and absorption functions, and is no role of nutrition. As a new type of feed enzymes, chitinase can break down glycosidic bonds in chitin and eliminate its anti-nutritional factors role, and change it to a water soluble oligosaccharide. The water soluble chitin oligosaccharide has a biological activity similar or higher than chitin. Chitin oligosaccharides can be used as a immunoenhancer and a nutritional polysaccharide. In order to improve animal absorption and utilization of other nutrients, the chitinase can be used as a feed enzyme preparation in animal feed which contains the maggot meal to eliminate the anti-nutritional factors with chitin.By gene recombination technology, some specific enzyme gene has been transfer to a suitable host stain and the fermentation has become an effective way to product feed enzymes. As a novel protein expression technology, yeast surface display system can be used to express a variety of biological activity enzymes and fix it to the cell surface result a yeast whole cell catalyst. Using of the whole cell enzyme as feed additives can avoid the purification and solid support procedures of the extracellular expression. In order to eliminate the anti-nutritional factors role of housefly maggot chitin, Saccharomyces cerevisiae, a generally recognized as safe organism, was used as an expression host to produce a heat stable chitinase on its surface. The whole yeast cell enzyme was used as a enzyme preparation to feed Carassius auratus. The feeding trials was used to evaluating the effect of whole yeast cell enzyme as a feed enzyme additives to eliminate anti-nutritional factor role by growth performance and non-specific immunity of Carassius auratus. The aim of this project is to develop a feed enzymes preparation which can eliminate the anti-nutritional factor of housefly maggot chitin and can promote animal growth and enhance fish immunity, further improve the utilization value of the housefly maggot meal as a feed additive.This paper consists of five parts:(1) Introduction (background, purpose and scheme of the project);(2) Conventional nutrients analyze of the house fly maggot and the preparation of chitin from maggot.(3) The effects of the house fly maggot meal on the growth performance and the nonspecific immunity of Carassius auratus;(4) Development a heat stable whole-cell enzyme with chitin degradation activity by yeast surface display engineering;(5) The effects of whole yeast cell enzyme and housefly maggot meal in feed on the growth performance and the nonspecific immunity of Carassius auratus.MethodsOn the basis of nutrients content determination (moisture, protein and fat) of the house fly maggot, house fly meal is used as a substitute for fish meal in the fish feed. And a12-week feeding trial on the Carassius auratus was done to evaluation The effects of the house fly maggot meal on the growth performance and the nonspecific immunity of Carassius auratus. On a basis of a comprehensive analysis of the experimental results, a heat stable whole-cell enzyme with chitin degradation activity is development by yeast surface display engineering to eliminate anti-nutritional factor role of maggot chitin. The whole yeast cell was added in the fish diet containing Housefly maggot and a12-week feeding trial on the Carassius auratus was done to evaluation the effects of the house fly maggot meal on the growth performance and the nonspecific immunity of Carassius auratus.1. The moisture, protein, fat content of house fly maggot meal and fish meal were determined by direct drying, spectrophotometry and acid hydrolysis, respectively. The maggot cuticle chitin was extracted by acid-base method and microwave heating. 2. The house fly maggot meal was replace for fish meal in different proportions and a12-week feeding trial on the Carassius auratus was done to evaluation The effects of the house fly maggot meal on the growth performance and the nonspecific immunity of Carassius auratus.(1) Calculate the growth parameter (weight gain, relative growth rate, specific growth rate and feed conversion ratio) and survival rate.(2) Count the number of red blood cells and white blood cells of fish by dilution method and the leukocyte count by Wright-Giemsa composite staining.(3) Application of nitro blue tetrazolium reduction assay to detec phagocytosis of blood neutrophils, and the zymosan stimulation and nitro tetrazolium blue reduction method to detect respiratory burst activity of leukocyte.(4) Using the turbidimetric method, xanthine oxidase and iron reduction capacity assay for determine lysozyme activity, anti dismutase activity and total antioxidant capacity in serum and liver pancreas.(5) The iodine-starch colorimetry and arginine ethyl ester method were used to detect amylase and trypsin activity of intestine and hepatopancreas.(6) The meat ratio of Carassius auratus was determined. The moisture, protein, fat content of fish muscle was determined by direct drying, spectrophotometry and acid hydrolysis, respectively.3. Development of a heat stable whole cell enzyme with chitinolytic activity by Yeast surface display engineering.(1) The ChiC gene was amplified from Serratia marcescens AS1.1652strain chromosomal DNA by PCR using two primers constructed based on ChiC gene of the Serratia marcescens GEI srain(GenBank accession number GQ855219).A yeast surface display vector pYD1-SMChiC was constructed, and electrotransfered into the Saccharomyces cerevisiae EBY100. SMChiC was anchored on yeast cell wall by a-agglutinin receptor system. The expression product was identified by Western blot. The presence of SMChiC-Aga2p on the cell surface of yeast was verified by indirect immunofluorescence.(2) The expression conditions of purified enzyme or whole yeast cell was optimized. The chitinolytic activity of purified enzyme or whole yeast cell was detected by SDS-PAGE zymography, agar plate method and DNS method.(3) The effect of temperature and pH on chitinolytic activity of the yeast cells or purified product was determined.4. The Saccharomyces cerevisiae cell with chitinolytic activity (yeast enzyme) was added to the fish feed which containing the house fly maggot meal in different proportions and a12-week feeding trial on the Carassius auratus was done to evaluation The effects of the house fly maggot meal on the growth performance and the nonspecific immunity of Carassius auratus.(1) Calculate the growth parameter (weight gain, relative growth rate, specific growth rate and feed conversion ratio) and survival rate.(2) Count the number of red blood cells and white blood cells of fish by dilution method and the leukocyte count by Wright-Giemsa composite staining.(3) Application of nitro blue tetrazolium reduction assay to detec phagocytosis of blood neutrophils,and the zymosan stimulation and nitro tetrazolium blue reduction method to detect respiratory burst activity of leukocyte.(4) Using the turbidimetric method, xanthine oxidase and iron reduction capacity assay for determine lysozyme activity, anti dismutase activity and total antioxidant capacity in serum and liver pancreas.(5) The iodine-starch colorimetry, arginine ethyl ester method was used to detect amylase and trypsin activity of intestine and hepatopancreas.(6) The meat ratio of Carassius auratus was determined. The moisture, protein, fat content of fish muscle were determined by direct drying, spectrophotometry and acid hydrolysis, respectively.(7) The hepatopancreas, spleen, kidney and intestine sections stained with hematoxylin and eosin were use to evaluate the effect of yeast enzyme on the organizational structure of Carassius auratus.Result1. The conventional nutrient content measured of house fly maggot meal and fish meal:moisture were6.95±0.11%and8.74±0.13%, respectively; protein content(dry weight%) were61.51±1.08%and60.32±1.10%, respectively, and there was no statistically significant between maggot meal and fish meal; fat content(dry weight%) were13.71±0.13%and7.32±0.11%. The maggot cuticle chitin was accounted for27.62±0.26%of the dry shell weight.2. The house fly maggot meal was replace for fish meal in different proportions and a12-week feeding trial on the Carassius auratus was done, the results showed that, compared to the control group:(1) The house fly maggot meal could enhance the weight gain(F=7.735, P=0.009), relative growth rate(F=7.143,P=0.012) and specific growth rate(F=7.141, P=0.012), and could reduce the feed conversion ratio(F=7.208, P=0.012). The10%maggot meat group had the most significant effect (P-values were0.009,0.012,0.012and0.012, respectively),5%(P-values were0.261,0.349,0.353and0.184, respectively) and15%(P-values were0.069,0.099,0.094and0.063, respectively) maggot meal group had no significant difference with the control group.(2) The phagocytosis(F=17.817, P=0.001)and respiratory burst activity(F=53.138, P <0.001)of leukocyte, SOD activity(F=10.593, P=0.004) and total antioxidant capacity (T-AOC)(F=9.042, P=0.006)of serum, SOD activity(F=4.972, P=0.031) and total antioxidant capacity(F=9.343, P=0.005)of hepatopancreas were enhanced in different degrees, and the10%maggot meal group had the most significant effect (P-values were0.001,0.001,0.004,0.006,0.046and0.015, respectively). The serum lysozyme activity was higher than the control group, but the difference was not significant (F=0.118, P=0.947).(3)The neutrophil proportion(F=0.612, P=0.626), basophils proportion(F=0.225, P=0.876), leukomonocyte proportion(F=0.387, P=0.766), histoleucocyte proportion(F=0.409, P=0.751), erythrocyte content(F=0.354, P=0.788)and leukocyte count(F=0.248, P=0.861)had no significant change。(4) The amylase activity(F=0.273, P=0.844), trypsin activity(F=1.815, P=0.222)of fish intestinal and amylase activity(F=1.698, P=0.244), trypsin activity(F=3.301, P=0.079)of fish hepatopancreas had no significant difference.(5) Adding house fly maggot meal to the fish feed had no significant affect on the meat ratio(F=3.272, P=0.080)and moisture(F=0.284, P=0.836), protein content(dry weight%)(F=1.714, P=0.241), fat content(dry weight%)(F=0.026, P=0.994) offish muscle.3. The ChiC gene was amplified from Serratia marcescens AS1.1652strain chromosomal DNA. One open reading frame (ORF) of1,440base pair encoding a protein of480amino acids was identified in the sequenced DNA region. The degrees of identity to the other strains (stain2170, strain141, strain xd1, stain BJL200)were96%,96%,96%and98%at the DNA level, and99%,98%,98%and99%at the protein level, respectively.4. The presence of SMChiC-Aga2p on the cell surface of Saccharomyces cerivisiae was verified by indirect immunofluorescence.The molecular mass of Aga2-SMChiC fusion protein of AGA2was about67kDa identified by Western Blot. The chitinase expressed in yeast was confirmed by agar plate method and SDS-PAGE zymography, and had a degrading activity on both α-and β-chitin. 5. The whole yeast cell had chitinolytic activity between20℃-80℃.It was still able to detect9%of the initial activity at80℃. The optimum reaction temperature was52℃. The thermal stability tests show that the yeast cell retained88.6%of its initial activity after70℃treatment for4h, even retained some activity after boiled it for1h, the zymogram test results also confirmed that purified enzyme had a certain heat stability at high temperature. The whole yeast cell had chitinolytic activity between pH2.2-8, the optimum pH was5.0, and whole cell enzyme could maintain a certain stability under acidic conditions.6. The Saccharomyces cerevisiae cell with chitinolytic activity(yeast enzyme) was added to the fish feed which containing the house fly maggot meal in different proportions and a12-week feeding trial on the Carassius auratus was done, the results showed that, compared to the control group:(1) The house fly maggot meal could significant enhance the weight gain(F=37.289, P<0.001), relative growth rate(F=33.053, P<0.001) and specific growth rate (F=31.435, P<0.001), and could significant reduce the feed conversion ratio (F=47.163, P<0.001).0.5%yeast enzyme group (P-values were0.004,0.006,0.008,0.001, respectively),1.0%yeast enzyme group (P-values were0.001,<0.001,<0.001,<0.001, respectively) and1.5%yeast enzyme group (P-values were0.001,0.003,0.003,<0.001, respectively) had the higher weight gain, relative growth rate, specific growth rate and the lower feed conversion ratio compared to the control group.(2) The phagocytosis(F=36.935, P<0.001)and respiratory burst activity(F=96.737, P <0.001)of leukocyte were enhanced.0.5%yeast enzyme group (P-values were0.014,<0.001, respectively),1.0%yeast enzyme group (P-values were<0.001,<0.001, respectively) and1.5%yeast enzyme group (P-values were0.037,<0.001, respectively) had the higher values compared to the control group. (3) The serum lysozyme activity(F=34.057, P<0.001), SOD activity(F=33.490, P<0.001), total antioxidant capacity(F=20.579, P<0.001)and the hepatopancreas lysozyme activity(F=35.497, P<0.001), SOD activity(F=16.673, P=0.001), total antioxidant capacity(F=24.374,P<0.001)were enhanced in different degrees.0.5%yeast enzyme group (P-values were0.001,0.001,0.002,<0.001, respectively),1.0%yeast enzyme group (P-values were0.001, P<0.001,0.001,<0.001,respectively) and1.5%yeast enzyme group (P-values were0.001,0.001,0.001,<0.001, respectively) had the higher serum lysozyme activity, SOD activity, total antioxidant capacity and hepatopancreas lysozyme activity compared to the control group.1.0%yeast enzyme group (P=0.001) had higher SOD activity compared to the control group.1.0%yeast enzyme group (P<0.001) and1.5%yeast enzyme group (P=0.004) had the higher hepatopancreas total antioxidant capacity compared to the control group.(4) The neutrophil proportion(F=1.590, P=0.266), basophils proportion(F=0.277, P=0.875), leukomonocyte proportion(F=0.877, P=0.492), histoleucocyte proportion(F=1.923, P=0.204), erythrocyte content(F=0.344, P=0.795)å'Œleukocyte count(F=0.061, P=0.979)had no significant change。(5) The amylase activity(F=0.240,P=0.866), trypsin activity(F=1.110, P=0.400)of fish intestinal and amylase activity(F=1.285, P=0.344), trypsin activity(F=1.110, P=0.400)of fish hepatopancreas had no significant difference. The amylase and trypsin activity of the fish intestinal and hepatopancreas had no significant difference (P>0.05).(6) Adding yeast enzyme to the fish feed had no significant affect on the meat ratio(F=0.478, P=0.706)and moisture(F=1.706, P=0.243), protein content(dry weight%)(F=0.016, P=0.997), fat content(dry weight%)(F=0.076, P=0.971) of fish muscle. (7) Pathological microscopic examination results showed that the major organs organ of Carassius auratus fed with the yeast cell had no abnormal changes.The experiment had no adverse impact to the organ of Carassius auratus.ConclusionThe protein content of the house fly maggot meal was similar to that of fishmeal, but the chitin containing in maggots shell is insoluble in water, and may act as anti-nutritional factors. When fish meal were replaced partially with maggot meal, the higher growth, lower feed coefficient, higher antioxidant capacity,higher leukocyte phagocytosis and respiratory burst of Carassius auratus were observed, and the efficiency of feed utilization were improved. Thereby, house fly maggot meal can enhance the non-specific immune function of the fish, and without adversely affecting the fish meal quality. The results show that the house fly maggot meal can partially replace for fish meal as a high-quality feed protein source.The Serratia marcescens ChiC was displayed on the yeast cell wall surface. The whole yeast cell enzyme had chitin degradation activity and heat stable characteristics, moreover, it still maintained a certain stability in low pH conditions. The whole yeast cell enzyme which had chitin degradation activity can eliminate the anti-nutritional factors of maggot chitin, boost the fish growth, improve the efficiency of feed utilization, improve leukocyte phagocytosis and respiratory burst capacity, improve fish lysozyme activity, superoxide dismutase and total antioxidant capacity, and enhance fish non-specific immune function. Added the yeast cells in fish feed hand no adversely affecting on blood environment, the main organ tissue structure and the meal quality of fish. The results show that the yeast cell which displayed the Serratia marcescens ChiC on its surface can add into fish feed and act as a feed enzymes preparation which can degradation anti-nutritional factors. |
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