Advances in the Study on Nutrient Requirements of Grouper (Epinephelus sp. ): a Review

The paper reviews the recent advances in studying grouper nutrition requirement for the development of cost-effective and environmentally friendly artificial diets. It consists of seven parts: protein and amino acid, lipid and essential fatty acid, carbohydrate, vitamin, mineral, alternative protein source, broodstock and larval nutrition. The review provides some basic information for further investigation of nutrient requirements of groupers.     

Recovery of Chitin and Protein from Shrimp Head Waste by Endogenous Enzyme Autolysis and Fermentation

The industrial processing of shrimp produces massive quantities of solid waste that is a notable source of animal protein, chitin, carotenoids, and other bioactive compounds that are not appropriately utilized. In the present study, chitin and protein extraction from shrimp head with autolysis and fermentation using Bacillus licheniformis were investigated. The results showed that when shrimp heads were autolyzed with a natural pH at 50℃ for 4 h, the total amino acid nitrogen in the supernatant was 5.01 mg mL^-1. Then, when a 50%(v/m) inoculum of the hydrolysate was incubated at 60℃ for 10 h, a deproteinization rate of 88.3% could be obtained. The fermented supernatant was processed into a dry protein powder, while the residues were demineralized by 10% citric acid for chitin. The recovered protein powder contained 5.5% moisture, 11.5% ash, and 66.7% protein, while the chitin contained 3.5% moisture, 2.1% ash, and 3.1% protein. In addition, amino acids, minerals, heavy metals, the degree of acetylation, microstructure, and Fourier-transform infrared(FT-IR) spectroscopy results were analyzed. Furthermore, the statistics of the large scale trial after treatment with 20 kg of shrimp heads were analyzed. Thus, this work made the shrimp waste utilization environmentally sound and valuable.     

Cloning and sequence analysis of a full-length cDNA of <Emphasis Type="Italic">SmPP1cb</Emphasis> encoding turbot protein phosphatase 1 beta catalytic subunit

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 （PP1） is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase l（PPlcb）, was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPPlcb, by the rapid amplification of cDNA ends （RACE） technique. The cDNA sequence of SmPPlcb we obtained contains a 984 bp open reading frame （ORF）, flanked by a complete 39 bp 5＇ untranslated region and 462 bp 3＇ untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B （PP2B）. And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPPlcb is extremely conserved in both amino acid and nucleotide acid levels compared with the PPlcb of other vertebrates and invertebrates, and its Kozak motif contained in the 5＇UTR around ATG start codon is GXXAXXGXXATGG, which is different from mammalian in two positions A6 and G3, indicating the possibility of different initiation of translation in turbot, and also the 3＇UTR of SmPPlcb is highly diverse in the sequence similarity and length compared with other animals, especially zebraf＇lsh. The cloning and sequencing of SmPPlcb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.     

Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXXATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.     

Effects of nutrients and light intensity on the growth and biochemical composition of a marine microalga Odontella aurita

Algal biotechnology has advanced greatly in the past three decades. Many microalgae are now cultivated to produce bioactive substances. Odontella aurita is a marine diatom industrially cultured in outdoor open ponds and used for human nutrition. For the first time, we have systematically investigated the effects of culture conditions in cylindrical glass columns and fiat-plate photobioreactors, including nutrients （nitrogen, phosphorus, silicon, and sulfur）, light intensity and light path, on O. aurita cell growth and biochemical composition （protein, carbohydrate, β-1,3-glucan, lipids, and ash）. The optimal medium for photoautotrophic cultivation of O. aurita contained 17.65 mmol/L nitrogen, 1.09 mmol/L phosphorus, 0.42 mmol/L silicon, and 24.51 mmol/L sulfur, yielding a maximum biomass production of 6.1-6.8 g/L and 6.7-7.8 g/L under low and high light, respectively. Scale-up experiments were conducted with fiat-plate photobioreactors using different light-paths, indicating that a short light path was more suitable for biomass production of O. aurita. Analyses of biochemical composition showed that protein content decreased while carbohydrate （mainly composed of 15-1,3-glucan） increased remarkably to about 50% of dry weight during the entire culture period. The highest lipid content （19.7% of dry weight） was obtained under 0.11 mmol/L silicon and high light conditions at harvest time. Fatty acid profiles revealed that 80% were Cx4, C~6, and C20, while arachidonic acid and eicosapentaenoic acid （EPA） accounted for 1.6%-5.6% and 9%-20% of total fatty acids, respectively. High biomass production and characteristic biochemical composition profiles make O. aurita a promising microalga for the production ofbioactive components, such as EPA and D-1,3-glucan.     

Fish protein recovered using ph shifting method and its physicochemical properties

The solubility of meat protein of croaker and jack mackerel was significantly affected by pH shifting. The protein yield of alkali-aided processing is higher than those of conventional and acid-aided processing. The addition of sarcoplasmic protein increases the breaking force and deformation value. The breaking force of recovered protein gel from alkali-aided processing is decreased by the addition of NaCI. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） revealed that fish protein of alkali is similar to that at pH7.0. Alkali-aided processing for recovering fish protein is a valuable method for increasing the utilization of frozen and pelagic fishes and for making kamaboko products.     

Effects of Calanoid Copepod Schmackeriapoplesia as a Live Food on the Growth,Survival and Fatty Acid Composition of Larvae and Juveniles of Japanese Flounder,Paralichthys olivaceus

Zooplankton constitutes a major part of the diet for fish larvae in the marine food web, and it is generally believed that copepods can meet the nutritional requirements of fish larvae. In this study, calanoid copepod Schmackeria poplesia, rotifer Brachionus plicatilis and anostraca crustacean Artemia sp. were analyzed for fatty acid contents, and were used as live food for cul- turing larval Japanese flounder, Paralichthys olivaceus. The total content of three types of HUFAs （DHA, EPA and ARA） in S. po- plesia was significantly higher than that in the other two live foods （P〈O.O1）. Three live organisms were used for raising larvae and juveniles of Paralichthys olivaceus respectively for 15 and 10 d. Then the growth, survival and fatty acid composition of the larvae and juveniles were investigated. The results showed that the larvae and juveniles fed with copepods （S. poplesia） had significantly higher growth rate than those fed with the other two organisms （P〈0.01）. The survival of the flounder larvae fed with copepods was significantly higher than that of the others （P〈0.01）, and the survival of the juvenile fish fed with copepods was higher than that fed with Artemia （P〈0.05）. The contents of three types of HUFAs （DHA, EPA and ARA） and the ratio of DHA/EPA in larval and juve- nile flounder P. olivaceus were analyzed. The results showed that the contents of DHA, EPA and ARA in the larvae and juveniles fed with S. poplesia were higher than those fed with a mixed diet orArtemia only, and the ratio of EPA/ARA in larvae and juveniles of P. olivaceus fed with S. poplesia was lower than that in the case of feeding with a mixed diet or Artemia only. The present data showed that copepod is the best choice for feeding the larvae and juveniles of fish considering its effects on the survival, growth and nutrition composition of the fish.     

Study on Application of Polyenzyme Method to Offal of Harengula zunasi

The new polyenzyme method for making gravy from Harengula zunasi offal involves protein enzymolysis with flavorase after proper alkaline and neutral protease levels were established by orthogonal trials to select the best hydrolytic conditions for processing offal with alkaline and neutral protease. The conditions for the polyenzyme method were pH of 7.0, temperature of 50℃ , alkaline and neutral protease concentration of 1.5% respectively, hydrolysis time of 120 min, andflavorase concentration of 2.0% , for 60 min. The new gravy-making technology yields a nutritious and delicious gravy containing 40.3 % of total essential amino acids, with delicious amino acids Glu, Asp, Gly, Ala, Pro and Ser comprising 49.5% , total and amino nitrogen being respectively1.9 and 1.1 g/100 g (amino acid nitrogen being 61.0% of total nitrogen), The polyenzyme method was used to make 14.8% protein gravy from Harengula zunasi offal. In addition, in inorganic elements, the phosphorus content is the highest.     

Molecular Cloning,Expression and Characterization of Peroxisome Proliferators-Activated Receptors Gamma in the Sea Urchin(Strongylocentrotus intermedius)

Peroxisome proliferators-activated receptor gamma(PPARγ) plays important regulatory roles in adipocyte differentiation. In this study, we cloned the full-length sequence of the PPARγ gene and analyzed its expression profile in different developmental stages and tissues of Strongylocentrotus intermedius. The full-length cDNA of PPARγ contains 2286 base pairs(bp) with a putative open reading frame of 1755 bp, and the gene encodes encoding a polypeptide of 584 amino acid residues. The predicted molecular mass of the protein is 67.27 kDa, and its theoretical isoelectric point(pI) is 10.07. The protein contains conserved motifs, including an RRM(RNA recognition motif) domain. PPARγ expression with the highest level was observed in the gonad, and the lowest was observed in the tube feet of S. intermedius. Time-course expression measurements at different developmental stages showed that the highest expression level of PPARγ is in the eggs and its weakest expression level is in the 32-cells stage. Knock-down of PPARγ by specific siRNA revealed that UCP2 expression is significantly decreased in the gonads and intestines 48 h post-transfection, indicating that the UCP2 is a downstream target gene of PPARγ.This finding suggests that PPARγ and UCP2 have positive regulatory effects in regulating adipocyte differentiation. Changes in fatty acid levels in the gonads before and after PPARγ interference were assessed, and decreased C18:2(trans, n-6) and C20:3(n-6) levels were observed 48 h after siRNA transfection. The results showed the function of PPARγ in fatty acid anabolism, The data are helpful to improve the current understanding of the fatty acid synthesis pathways and regulatory mechanisms in S. intermedius. They also provide an experimental basis for improving fatty acid synthesis in sea urchins, which is important for cultivating sea urchins with high nutritional value.     

Comparative Study on the AUergenicity of DifferentLitopenaeus vannamei Extract Solutions

Allergen extracts are widely used for allergy diagnosis and treatment. The application of shrimp extract is hampered dueto the low protein concentration and the inconsistent allergenicity. Extracting solutions are considered to be the primary limitingfactor of protein extraction from crustaceans. This study aimed to select an optimal solution for shrimp protein extraction by com-paring the allergenicity of different shrimp extracts. The effect of 7 existing or modified extracting solutions were evaluated, includ-ing the glycerol-NaC1 solution, the glycerol Cocaine＇s solution, the buffered saline solution, the Cocaine＇s solution, the Glucoseleaching solution, 1 mol L-1 KC1 solution, and 0.01 mol L-1 phosphate buffered saline solution with and without dithiothreitolor （DTT）.The quantitative （protein concentration） and qualitative parameters （SDS-PAGE protein patterns and immuno-reactivity） were deter-mined using the sodium dodecyl sulfate polyacrylamide gel electrophoresis, enzyme linked immunosorbent assay and immunoblottingassay. Results showed that the 1 molL-1 KC1 solution with DTT was optimal for shrimp protein extraction, which yielded high con-centrafion and allergenicity in the protein extract, including major and minor allergens. The 1 tool L-l KC1 solution with DDT is pro-posed for preparation of shrimp extract and associated allergy diagnosis, as well as potential applications for other crustaceans.     

Effects of Dietary Protein Level on Growth and Utilization of Protein and Energy by Juvenile Mangrove Red Snapper （ Lutjanus argentimaculatus ）

A feeding trial was conducted in a recirculating water system to investigate the effects of dietary protein levels on growth, feed utilization, hepatosomatic index and liver lipid deposition of juvenile red snapper, Lutjanus argentimaculatus (average initial wet weight 8.0 ± 0.39 g and total length 3.14 ± 0.3 cm). In the experiment, six fishmeal-based diets were formulated to contain various protein levels (20% to 45% in 5% increments), with dietary energy ranging from 2210.7kJ lOOg to 2250.2kJlOOg dry matter. The protein to energy ratios of diets ranged from 8.58 mg protein kJ⁻¹ to 20.03 mg protein kJ⁻¹. Diets were fed for 90d to triplicate groups of fish stocked in 0.128m³ seawater tanks, 25 individuals each. The daily ration of 2% wet body weight was offered to the fish thrice a day. The fish at the end of the study had more than ten-fold (77.0g) increase in weight compared to the initial (8.0g). Fish fed diets of 40% and 45% protein produced significantly (P<0.05) higher weight gain of 77.2g and 76.5g, and specific growth rate (SGR) of 2.65% and 2.62% than those of 67.0 g and 68.3g, and 2.49% and 2.51% of the other diets. The broken-line regression of SGR against dietary protein level yielded an optimum dietary protein requirement of 42.6% (Y=−1.6295 + 0.1114 X ²,P<0.05). Survival remained 100% among groups. Feed conversion ratio decreased from 0.45 for fish fed 20% dietary protein to 0.35 for fish fed 45% dietary protein. Nitrogen intake increased with an increase in dietary protein, which in turn resulted in an increase in nitrogen gain of fish whole body. Fish fed 40% and 45% protein diets showed higher (P<0.05) nitrogen gain (0.27g and 0.26g) than those (0.23g and 025g) fed all other diets. Gross energy intake (GEI) in fish fed 45% protein was lower (600.67kJ) than that (607.97 kJ) of 40% protein diet, though the differences were not statistically significant (P>0.05); GEI ranging from 677.31 kJ to 663.20 kJ at remaining four diets (20% to 35% protein) did not appear to differ significantly (P>0.05). The highest energy gain of 518.33 kJ was obtained with fish fed 40% protein, resulting in the highest energy retention efficiency of 85.26%. The hepatosomatic index of fish fed diets of 20%, 25%, 30% and 35% protein were significantly (P<0.05) higher (2.09% to 2.57%) than those (1.44% and 1.41%) of fish fed diets containing 40% and 45% protein. Liver lipid contents decreased from 8.72% to 7.0% in fish fed dietary protein of 20% to 45% in 5% increments. Results suggest that the diet containing 40% to 42.6% protein with a P/E ratio of 17.6 mg protein kJ⁻¹ is required for good growth of L. argentimaculatus weighing between 8.0 g and 85.2 g under the culture conditions of the present study.     

Fatty acid composition analyses of the DCMU resistant mutants of <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> (eustigmatophyceae)

Ultraviolet mutagenesis was applied to Nannochloropsis oculata and three mutants resistant to 3-(3, 4-dichlorophenyl)-1,1-dimethylurea (DCMU) were isolated. The cellular chlorophyll a and total lipid content of the wild are higher in the medium supplemented with DCMU than in the control without DCMU. Without DCMU, the growth rates and chlorophyll a contents of the mutants are similar to those of the wild. Significant changes of fatty acid content and composition have occurred in DCMU-resistant mutants growing in the medium supplemented with DCMU. The total lipid, palmitic acid (16:0), palmitoleic acid (16:1ω9) and oleic (18:1ω9) contents decrease significantly, while the vaccenic acid (18:1ω11) increases significantly and the EPA content of dried powder increases slightly in the mutants. The study may provide a basis to improve EPA content in Nannochloropsis oculata in the future.     

Structure and function of the digestive system of <Emphasis Type="Italic">solen grandis</Emphasis> dunker

Structure and function of the digestive system of a bivalve mollusc, Solen grandis, were studied using light microscopy and histochemical methods. The wall of digestive tube consists of four layers: the mucosal epithelium, connective tissue, muscular and fibrosa or serosa (only in the portion of rectum) from the inner to the outer. The ciliated columnar epithelial cells, dispersed by cup-shaped mucous cells, rest on a thin base membrane. There are abundant blood spaces in connective tissue layer. The digestive diverticula are composed of multi-branched duct and digestive tubules. The digestive tubules are lined with digestive and basophilic secretory cells, and surrounded by a layer of smooth muscle fibers and connective tissues. Activities of acid and alkaline phosphatases, esterase and lipase are detected in the digestive cells, and the epithelia of stomach and intestine, suggesting that these cells are capable of intracellular digesting of food materials and absorbing. Besides, acid phosphatase and esterase activities are present in the posterior portion of esophagus. Phagocytes are abundant in blood spaces and the lumens of stomach and intestine, containing brown granules derived from the engulfed food materials. The present work indicates that phagocytes play important roles in ingestion and digestion of food materials, which is supported as well by the activities of acid phosphatase, esterase and lipase detected in blood spaces.     

Expression and localization of a novel phosducin-like protein from amphioxus <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>

A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri. Supported by the Pilot Projects of Knowledge Innovation Project of Chinese Academy of Sciences (No. KZCX2-YW-211-03 and MGE2008KG06)     

Dietary lipid and gross energy affect protein utilization in the rare minnow <Emphasis Type="Italic">Gobiocypris rarus</Emphasis>

An 8-week feeding trial was conducted to detect the optimal dietary protein and energy, as well as the effects of protein to energy ratio on growth, for the rare minnow (Gobiocypris rarus), which are critical to nutrition standardization for model fish. Twenty-four diets were formulated to contain three gross energy (10, 12.5, 15 kJ/g), four protein (20%, 25%, 30%, 35%), and two lipid levels (3%, 6%). The results showed that optimal dietary E/P was 41.7–50 kJ/g for maximum growth in juvenile rare minnows at 6% dietary crude lipid. At 3% dietary lipid, specific growth rate (SGR) increased markedly when E/P decreased from 62.5 kJ/g to 35.7 kJ/g and gross energy was 12.5 kJ/g, and from 75 kJ/g to 42.9 kJ/g when gross energy was 15.0 kJ/g. The optimal gross energy was estimated at 12.5 kJ/g and excess energy decreased food intake and growth. Dietary lipid exhibited an apparent protein-sparing effect. Optimal protein decreased from 35% to 25%–30% with an increase in dietary lipid from 3% to 6% without adversely effecting growth. Dietary lipid level affects the optimal dietary E/P ratio. In conclusion, recommended dietary protein and energy for rare minnow are 20%–35% and 10–12.5 kJ/g, respectively.     

EFFECTS OF FISH OIL,DHA OIL AND LECITIHIN IN MICROPARTICULATE DIETS ON STRESS TOLERANCE OF LARVAL GILTHEAD SEABREAM（SPARUS AURATA）

The effects of natural fish oil,DHA oil and soybean lecithin in microparticulate diets on stress tolerance of larval gilthead seabream(Sparus aurata)were investigated after 15 days feeding trials.The tolerance of larval gilthead seabream to various stress factors such as exposure to air(lack of dissolved oxygen),changes in water temperature(low)and salinity(high) were determined.This study showed that microparticulate diet with natural fish oil and soybean lecithin was the most effective for in-creasing the tolerance of larval gilthead seabream to various stresses,and that microparticulate diet with natural fish oil and palmitic acid(16:0)was more effective than microparticulate diet with DHA oil and soybean lecithin.     

Effects of sodium nitrate and sodium acetate concentrations on the growth and fatty acid composition of <Emphasis Type="Italic">Brachiomonas submarina</Emphasis>

One isolate of Brachiomonas submarina was tested for its ability to grow heterotrophicly on 5 different organic compounds. Sodium acetate and glucose were found to be effective in supporting the growth. Sodium acetate was chosen as the organic nutrient to test the combined effects of organic and inorganic solutions on the growth and fatty acid composition of Brachiomonas submarina. The best growth rates were achieved at 3 mmol L⁻¹ CH₃COONa and 0.88 mmol L⁻¹ NaNO₃ in heterotrophic condition, and 4 mmol L⁻¹ CH₃COONa and 3.52 mmol L⁻¹ NaNO₃ in mixotrophic condition. The differences between fatty acid contents were significant. The total polyunsaturated fatty acids (T. P. U. F. As) varied from 55.79% to 67.72% in heteritrophic growth and from 52.39% to 65.55% in mixotrophic growth. It is concluded that CH₃COONa and NaNO₃ at 3 mmol L⁻¹ and 3.52 mmol L⁻¹ should respectively be used in order to achieve the highest growth rate and fatty acid content.     

龙虎斑鱼肌肉营养成分分析

对龙虎斑鱼的肌肉进行营养成分分析和肉质评价,结果表明,龙虎斑鱼新鲜肌肉中粗脂肪质量分数4.18%,粗蛋白质量分数17.05%,粗灰分质量分数1.24%,水分质量分数80.19%.肌肉蛋白质中共检测出氨基酸种类有17 种,占新鲜肌肉样品的质量分数为17.88%;7 种必需氨基酸（EAA）占新鲜肌肉样品的质量分数为7.79%,占氨基酸总量的43.57%;4 种鲜味氨基酸（DAA）占新鲜肌肉样品的质量分数为6.17%,占氨基酸总量的34.51%;必需氨基酸指数（EAAI）为101.35,其氨基酸组成比例符合FAO/WHO 标准.新鲜肌肉中共检测出脂肪酸种类有14 种.其中7 种饱和脂肪酸（SFA）占总脂肪酸的质量分数为47.29%;4 种单不饱和脂肪酸（MUFA）占总脂肪酸的质量分数为18.86%;3 种多不饱和脂肪酸（PUFA）占总脂肪酸的质量分数为33.86%,DHA 质量分数占脂肪酸总量的30.24%.龙虎斑鱼营养价值较高,海鲜味浓郁.     

Study on application of polyenzyme method to offal of Harengula zunasi

The new polyenzyme method for making gravy fromHarengula zunasi offal involves protein enzymolysis with flavorase after proper alkaline and neutral protease levels were established by orthogonal trials to select the best hydrolytic conditions for processing offal with alkaline and neutral protease. The conditions for the polyenzyme method were pH of 7.0, temperature of 50°C, alkaline and neutral protease concentration of 1.5% respectively, hydrolysis time of 120 min, and flavorase concentration of 2.0%, for 60 min. The new gravy-making technology yields a nutritious and delicious gravy containing 40.3% of total essential amino acids, with delicious amino acids Glu, Asp, Gly, Ala, Pro and Ser comprising 49.5%, total and amino nitrogen being respectively 1.9 and 1.1 g/100 g (amino acid nitrogen being 61.0% of total nitrogen), The polyenzyme method was used to make 14.8% protein gravy fromHarengula zunasi offal. In addition, inorganic elements, the phosphorus content is the highest. This project was aided financially by the Guangdong Science and Technology Office (No. 2KM06002S).