Objective To study the single and combined-enzymic hydrolysis conditions of Oreochromis niloticus skin collagen.Methods Based on the inhibitory activities of six kinds of single enzymic hydrolysates to angiotensin converting enzyme(ACE),bromelain and alcalase(2.4L) were selected as combined-enzyme.The optimal hydrolytic conditions of the skin collagen with the two enzymes were determined by orthogonal test L_(16)(4~(5)).Results and Conclusion The results showed that the optimal hydrolytic conditions of bromelain and alcalase 2.4L were temperature 45℃,pH4.5,E/S 4000U?g~(-1),time 4h,concentration of substrate 6%;and temperature 55℃,pH7.5,E/S 6000U?g~(-1),time 2h,concentration of substrate 4%,respectively.Furthermore,hydrolytic effect would be better when using bromelain in advance,followed by alcalase 2.4L.The hydrolytic degree of the hydrolysates prepared under the provided technology was 30.43%,the ACE inhibitory rate would be as high as(68.65%) in vitro.

Objective To research the optimum hydrolysis conditions of oyster protein so as to raise the protein recovery and degree of hydrolysis.Methods Considering protein recovery,degree of hydrolysis,bitterness and clarity,the best enzyme was selected;Orthogonal test was designed to determine the best enzyme hydrolysis conditions;Based on hydrolysis and protein recovery,the effect of heat treatment,ultrasound and microwave processing and handling on enzymatic hydrolysis of oyster were investigated.Results and Conclusion Bromelain protease was suitable for the proteolysis of oyster,and the optimal conditions were:pH 6,temperature 55℃,substrate concentration 1∶3,E/S=800U/g,4 hours.In the optimal condition,the recovery of protein and degree of hydrolysis were 67.55%and 29.86% respectively in the hydrolysate.Heating,ultrasound technology and microwave treatments before hydro-lysis were harmful to enzymatic hydrolysis of oyster.

In this paper, the origins, characteristics and poisoning mechanisms of shellfish poisons including the paralytic shellfish poison(PSP),the diarrheic shellfish poison(DSP), the neurotoxic shellfish poison(NSP) and the amnestic shellfish poison(ASP) were introduced briefly. Methods of assay and detoxification of shellfish poisons were also discussed in detail.

Bioactive peptides in the marine organisms such as sponges,ascidian,fishes,shellfish,etc. and their bioactivities of antineoplastic, antimicrobial, antihypertension and antioxidation were reviewed in this paper.

BACKGROUND: Agiotensin Ⅰ -converting enzyme (ACE) inhibitory peptides which are separated from red tilapia skin collagen should be researched further.OBJECTIVE: To obtain a peptide of high ACE inhibitory activity through enzymic hydrolysates of red tilapia skin collagen.DESIGN: Enzymic hydrolysates were done with orthogonal experimental method; decompression peptide was separated with ultrafiltration, gel chromatography and hypertensive liquid chromatography.SETTING: Laboratory of Aquatic Products, Chinese Marine University.MATERIALS: The red tilapia weighing (500+50)g was donated by Branch Factory of Jimo Redian Factory. ACE was isolated from hog lung.METHODS: The experiment was carried out in the Laboratory of Aquatic Products, Chinese Marine University from May Bergman.①The collagen from red tilapia skin was extracted using the method described by Grossman and Bergman.The collagen extraction was hydrolyzed with compound enzymes in the order of bromelain and Alcalase 2.4 L.The red tilapia collagen hydrolysates (RTCH) were subsequently boiled to inactivate the enzyme. The resultant RTCH was fractionated through three different UF membrane bioreactor system having a range of molecular weight cut-offs (MWCO) of Mr 6 000, 4 000 and 1 000. Three portions were obtained: RTCH-I (M, 6000-4000), RTCH- Ⅱ (Mr 4000-1000)and RTCH-Ⅲ(Mr＜1000).②The ACE inhibitory 50%of ACE activity was defined as the IC50 value.③RTCH-Ⅲ(1.5 Ml) with the highest activity among RTCHs was loaded onto a Sephadex G-25 column (1.6×100 cm), and the absorbance of theeluent was monitored at 220 nm. Four samples of primary hydrolysates, RTCH- Ⅰ, RTCH- Ⅱ and RTCHⅢ were separated to collect active fractions which were pooled and lyophilized, immediately. The lyophilized fraction was separated with HPLC ODS-C18 analysis column to obtain component of high activity. Meanwhile, the same method was used to measure ACE inhibitory rate.④Each sample was hydrolyzed with 6 mol/L hydrochloric acid containing 1g/L thioglycolic acid at 110 ℃ for 24 hours in vacuum. The amino acid compositions of the resultant hydrolysates were determined on an amino acid auto analyzer, and molecular weight was measured with HPLC technique.MAIN OUTCOME MEASURES：①ACE inhibitory activity of fractionated RTCHS;②Purification of ACE inhibitory peptide;③Amino acid analysis of ACE inhibitory peptides.RESULTS:①ACE inhibitory activityof fractionated RTCHs:IC50values of RTCH-Ⅰ,RTCH-Ⅱand RTCH-Ⅲ were 3.30,2.23 and 0.31 g/L,and inhibitory of RTCH-Ⅲ was the highest.②Purification of ACE inhibitory peptide: The IC50 value of the four peak were 3.5, 2.12, 1.56 and 0.65 g/L, respectively. Results in Figures 2, 3, 4 and 5 showed that the high active fractions were: 6K4, 4K2, 1K2 and ACF2, and the inhibitory ratio were 11.1%, 89.9%, 65.0% and 49.7%, respectively.Among of these fractions,the 4K2 exhibited the highest inhibitory rate.③Amino acid analysis of ACE inhibitory peptides: Separated peptide products had more aromatic and hydrophobic amino acids.CONCLUSTON: Enzymic hydrolysates of red tilapia skin collagen have high ACE inhibitory peptide which is separated with ultrafiltration, gel chromatography and hypertensive liquid chromatography.

Objective To establish a rapid and accurate analysis method for food-derived ACE inhibitory peptides activity in vitro.Methods Reaction time of ACE and substrate was by measuring the hippuric acid liberated in the ACE reaction mixture at regular intervals;An optimal RP-HPLC method to measure food-derived ACE inhibitory peptides activity in vitro was set up.The hippuric acid from ACE reaction mixture(sea cucumber peptides were regarded as ACE inhibitor) was estimated by Zorbax SB-C_(18) analytical column with acetonitrile and ultrapure water as mobile phase.Results The reaction time of ACE with substrate was determined at sixty minutes;The elution was carried out with the ratio of acetonitrile to ultrapure water was 1:1(0.1%TFA) at a flow rate of 0.4 mL?min~(-1).The ahsorbance of the eluent was monitored at 228 nm,and column temperature was 25℃.The relationship between hippuric acid concentration and peak area exhibited a good linearity in the concentration ranges of 0～200?g?mL~(-1) and 200～800?g?mL~(-1).The RP-HPLC method was further validated by captopril,the oyster hydrolysate and the anchovy hydrolysate.Conclusion The method has been proved to be convenient,accurate and suitable for the analysis of foodderived ACE inhibitory peptides activity in vitro.

The patient was a 21 days-old baby girl,admitted to Guangdong Women and Children Hospital because of "poor intake,seldom crying and no activity in 1 day".The major clinical manifestations included hypotonia,aggravation of the conscious disturbance,pancytopenia,intractable acidosis and hyperammonemia,so,inherited metabolic disorders should be considered.Screening of inherited metabolic diseases with blood and urine samples,genetic test and active treatments were carried out.After targeted next-generation sequencing,a novel homozygotic frame shift mutation in PCCB gene:c.838_839insC (L280Pfs * 11) was identified,which was validated by Sanger sequencing.This mutation had not been reported in the mutation database,and bioinformatic analysis of this mutation indicated disease-causing.So,the diagnosis of propionic acidemia was identified.The baby was in a critical condition,and despite active treatment,her conscious disturbance was aggravated,and the spontaneous breathing disappeared.Subsequently,the baby died of pneumonia.Propionic acidemia is a relatively common genetic metabolic disease in newborns.The severity and the clinical phenotypes of propionic acidemia varied,which often made the diagnosis difficult.When the baby is presented with developmental delay,hypotonia,recurrent convulsion and vomiting,etc,which can't be explained by common diseases of children,propionic acidemia may be considered.Next generation sequencing analysis of the complicated cases can easily to pinpoint a disease-causing gene,which lays a solid foundation for accurate diagnosis and treatment of the patients.