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Abstract [Objectives] This study was conducted to optimize the determination conditions of amino acids from abalone. [Methods] The sample was treated by acid hydrolysis method and subjected to 2，4-2 nitro fluorobenzene column derivatization. The amino acid content in abalone was determined by HLPC， and the nutritional value of the amino acids was evaluated with egg protein model put forward by Institute of Nutrition and Food Hygiene， Chinese Academy of Preventive Medicine. [Results] Abalone contains full amino acids. According to the FAO/WHO ideal， it is a high-quality protein source and suitable for supplement of protein source for human body. [Conclusions] The experimental method has simple operation and could achieve a good effect with wide linear range and correlation coefficient over 0.999 8， and the obtained results are satisfactory.
Key words Abalone； Precolumn derivatization； HPLC； Amino acid； Evaluation
Abalone， originally known as Fu， belongs to Haliotis in Haliotidae family of Archaeogastropoda in Gastropo-da of Mollusca[1]. It is a famous and precious traditional food material， which tastes sweet and salty， and is neutral in nature， with the effects of nourishing the liver to improve visual acuity， treating yin deficiency by reinforcing body fluid and moistening dryness[2]. Abalone is mainly distributed in the surrounding waters of Hainan， Taiwan， Guangdong， Fujian and Liaoning in China[3].
There are various amino acid determination methods， mainly including high performance liquid chromatography （HPLC）， automatic amino acid analyzer with ninhydrin post-column derivatization， ion exchange chromatography and capillary tube method[4]， but they all have certain disadvantages. Automatic amino acid analyzer with ninhydrin post-column derivatization has no high sensitivity， and the instrument is very expensive； ion exchange chromatography has high requirements for reaction conditions and is much more complicated with high analysis cost and low work efficiency； and capillary tube method has poor reproducibility， and when the concentration is too high， its sensitivity decreases due to increase in electrostatic force and friction force caused by surrounding medium[5]. Precolumn derivatization-HPLC has no need for special reaction device and has the advantages of high instrument popularizing rate， flexible and diverse method， high sensitivity， good reproducibility and high resolution[6].
Materials and Methods
Materials and instruments Fresh living abalone was purchased from Sanya fishery quay （the abalone flesh was glossy， thick and solid， light brown， carnation or off-white， with inherent smell of abalone）； 18 amino acid standards （Solarbio）； 2，4-dinitrofluorobenzene （AR Xiya Reagent）； N，N-dimethylformamide （AR， Guangzhou Chemical Reagent Factory）； acetonitrile （HPLC， Shantou Xilong Chemistry Factory Co. Ltd.）； phenol （AR， Shantou Xilong Chemistry Factory Co. Ltd.）； acetone.
LC-2010A HT high performance liquid chromatograph （Shimadzu）； SHB-Ⅲ circulating water type multi-purpose vacuum pump （Zhengzhou Greatwall Scientific Industrial and Trade Co.， Ltd.）； DHG-9245 electro-thermostatic blast oven （Jintan Shenglan Instrument Manufacturing Co.， Ltd.）； JU-6224 ultrasonic generator （Shanghai JUMP Ultrasonic Equipment Co.， Ltd.）； HH digital thermostat water bath （Jintan Shenglan Instrument Manufacturing Co.， Ltd.）； RE-52 rotary evaporator （Shanghai Yarong Biochemical Instrument Factory）； DHS-3F pH meter （Shanghai Precision Instrument Co.， Ltd.）； 25 ml hydrolysis tube （pressure-resistant glass tube with screw cap）； ampoule.
Methods
Experimental principle
Acid hydrolysis used 6 mol/L hydrochloric acid as acidolysis agent， to hydrolyze proteins in abalone flesh at 110 ℃ to amino acids， and with 2，4-dinitrofluorobenzene as derivatization agent， and precolumn derivatization was performed to the 18 amino acids， to strengthen ultraviolet absorption. The sample was detected with Shimadzu LC-2010A HT high performance liquid chromatograph， at 360 nm. Qualitative analysis was performed from retention time， and quantitative analysis was performed by external standard method. As tryptophan was damaged by hydrochloric acid， the sample was also hydrolyzed with 5 mol/L sodium hydroxide solution， and tryptophan was determined by LC-2010A HT high performance liquid chromatograph （Shimadzu）.
Sample pretreatment
The shell of fresh abalone was removed， as well as viscera and edges of black mucous membrane. The edible part was flushed with distilled water， and the residual water was removed with filter paper. The edible part was boiled in distilled water for 15 min， and then taken out， followed by cooling and cutting. The pieces were placed in a watch glass and oven-dried in a blast oven at 60 ℃. After cooling， the material was pulverized， grinded and sieved with a 80 mesh sieve. The sample was then re-dried （105 ℃）， sealed and preserved in a dryer.
Acid hydrolysis of sample A certain amount the abalone sample （0.050 0 g） was accurately weighed and added into a hydrolysis tube. Then， 10 ml of 6 mol/L hydrochloric acid was added， and 3-4 drops of newly distilled phenol was added. After vortex oscillation， the sample was frozen in dry ice for 5 min， followed by evacuation to 7 Pa （≤0.05 mm mercury column） and introduction of highly-pure nitrogen gas， and the tube was screwed to seal it in nitrogen atmosphere. The hydrolysis tube was placed in a constant temperature drying oven at 105 ℃ for 22 h， and taken out. After cooling， mixing and opening， filtration was performed. The filtrate was transferred to a rotary evaporator and vacuumized at 60 ℃ to dryness， and if necessary， little distilled water could be added to repeat evaporation for 1-2 times. Little distilled water was added to dissolve the residue， and the solution was diluted to 10 ml. After mixing well， the solution was filtered with 0.45 μm filter membrane， obtaining filtrate which was stored in a refrigerator as acid-hydrolyzed stock solution for later derivatization.
Basic hydrolysis of tryptophan
A certain amount of the sieved abalone sample （0.050 0 g） was accurately weighed and added into a hydrolysis tube， and 10 ml of 5 mol/L sodium hydroxide solution was added. After vortex oscillation， the sample was frozen in dry ice for 5 min， followed by evacuation to 7 Pa （≤0.05 mm mercury column） and introduction of highly-pure nitrogen gas， and the tube was screwed to seal it in nitrogen atmosphere. The hydrolysis tube was placed in a constant temperature drying oven at 105 ℃ for 22 h， and taken out. After cooling， mixing and opening， filtration was performed. The filtrate was transferred to a rotary evaporator and vacuumized at 60 ℃ to dryness， and if necessary， little distilled water could be added to repeat evaporation for 1-2 times. Little distilled water was added to dissolve the residue， and the solution was diluted to 10 ml. After mixing well， the solution was filtered with 0.45 μm filter membrane， obtaining filtrate which was stored in a refrigerator as base-hydrolyzed stock solution for later derivatization.
Pre-column derivation
A certain amount of the acid-hydrolyzed stock solution （1 ml） was accurately weighed and added into an ampoule， and 0.5 ml of sodium carbonate solution （0.5 mol/L） was added. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， followed by mixing and sealing. The solution was heated in a water bath at 50 ℃ for 90 min in the shade. The solution was diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as sample solution. Chromatographic condition
Amino acid content was determined with Shimadzu LC-2010A HT high performance liquid chromatograph using Inertsil ODS-3 C18 column （4.6×150 mm， 5 μm）. The analysis cycle of one sample was 60 min. The HPLC was carried out with 0.05 mol/L sodium acetate-acetic acid buffer （containing 1% N，N-dimethylformamide， pH=6.4） as mobile phase A and acetonitrile-water （v/v=1∶1） as mobile phase B （70∶30） at a flow rate of 1 ml/min under the detection wavelength of 360 nm， the column temperature at 33℃ and a sample size of 10 μl. Under the separation conditions， the 18 amino acids were well separated.
Standard curve plotting and chromatogram analysis
Preparation of standard solution
Proper amounts of amino acid standards were weighed， dissolved with distilled water and diluted to 100 ml， 1 ml of which was accurately measured， placed in an ampoule bottle and added with 0.5 ml of sodium carbonate （0.5 mol/L）. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， followed by mixing and sealing. The solution was heated in a water bath at 50 ℃ for 90 min in the shade. The solution was diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required standard solution.
Chromatographic analysis
After hydrolysis of abalone， derivatization was performed with 2，4-dinitrofluorobenzene， and determination was performed with Shimadzu LC-2010A HT high performance liquid chromatograph， at 360 nm. The 18 amino acids were well separated under the separation conditions. The chromatograms of the standard solution， the acid-hydrolyzed sample solution， the acid-hydrolyzed blank solution， the base-hydrolyzed sample solution and base-hydrolyzed blank solution are shown in Fig. 1， Fig. 2， Fig. 3， Fig. 4 and Fig. 5， respectively.
Nutritional value modes of amino acid
According to the advises by FAO/WHO （Food and Agriculture Organization and the World Health Organization） and Institute of Nutrition and Food Hygiene， Chinese Academy of Preventive Medicine， the nutritional value of amino acids in abalone from Sanya was evaluated according to egg protein model.
Nitrogen amino acid score （AAS）， chemical score （CS） and essential amino acid score （EAAI） were calculated according to given formula[7-11].
Amino acid score=100×Essential amino acid content in each gram of to-be-evaluated protein （mg）/Essential amino acid content in each gram of protein in FAO/WHO model（mg） Chemical score=100×Essential amino acid content in each gram of to-be-evaluated protein （mg）/Essential amino acid content in each gram of egg protein （mg， each gram of egg as standard）
EAAI=ni=1aaiAAi
Wherein aa is the content of amino acid in sample （mg/g）； AA （FAO/WHO） is the content of the same amino acid in FAO/WHO evaluation mode （mg/g）； and AA （Egg） is the content of the same amino acid in whole egg protein （mg/g）. In the formula， aai is the percentage of certain amino acid contained in sample material to essential amino acids； and AAi is the essential amino acid contained in reference protein to essential amino acids. The evaluation criterions are as follows： EAAI>0.95 indicates high-quality protein source， 0.85 Agricultural Biotechnology2018
Results and Analysis
Plotting of standard curve
At first， 25， 50， 125， 250 and 500 mg/L mixed standard solutions of the 18 amino acids were prepared， and 1 ml of each solution was accurately added into an ampoule bottle and prepared into a testing solution according to the derivatization conditions. The obtained solutions were analyzed sequentially under set conditions. Standard curves were plotted with peak area as f（x） value and detection concentration （mg/L） as x value， and the obtained regression equations and correlation coefficients are shown in Table 1.
Above data and curve showed that the 18 amino acids had good linear relation in the linear range of 0-500 mg/L， and the correlation coefficients were all higher than 0.999 8.
Optimization of water hydrolysis conditions
Investigation of amount of hydrochloric acid
A certain amount of the sieved abalone sample （0.050 0 g） was accurately weighed， and added into four hydrolysis tubes， respectively. Into the four tubes， 5， 10， 15 and 20 ml of 6 mol/L hydrochloric acid were added， respectively， and hydrolysis was performed under acid hydrolysis conditions. Then， 1 ml of each of the solutions was accurately measured and added with 0.5 ml of sodium carbonate （0.5 mol/L）. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， followed by mixing and sealing. The solution was heated in a water bath at 60 ℃ for 90 min in the shade. The solution was diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different hydrochloric acid amounts on peak area was investigated， and the result is shown in Fig. 6. As shown in Fig. 6， in hydrochloric acid range of 10-20 ml， the peak area changed little， but if the amount of hydrochloric acid was too large， the chromatogram showed tailing phenomenon. Therefore， 10 ml of hydrochloric acid was selected for hydrolysis.
Investigation of hydrolysis time
A certain amount the sieved abalone sample （0.050 0 g） was accurately weighed， and added into four hydrolysis tubes， respectively， and hydrolysis was performed under acid hydrolysis conditions for 20， 22， 24 and 26 h， respectively. Then， 1 ml of each of the solutions was accurately measured and added with 0.5 ml of sodium carbonate （0.5 mol/L）. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， followed by mixing and sealing. The solution was heated in a water bath at 60 ℃ for 90 min in the shade. The solution was diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different hydrolysis time on peak area was investigated， and the result is shown in Fig. 7.
The results showed that during the hydrolysis， the best hydrolysis effect was achieved by performing hydrolysis using 10 ml of hydrochloric acid at 105 ℃ for 22 h.
Investigation of hydrolysis temperature
A certain amount of the sieved abalone sample （0.050 0 g） was accurately weighed and added into four hydrolysis tubes， respectively， and hydrolysis was performed under acid hydrolysis conditions at 100， 105， 110 and 115 ℃， respectively. Then， 1 ml of each of the solutions was accurately measured and added with 0.5 ml of sodium carbonate （0.5 mol/L）. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， followed by mixing and sealing. The solution was heated in a water bath at 60 ℃ for 90 min in the shade. The solution was diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different hydrolysis temperaturess on peak area was investigated， and the result is shown in Fig. 8.
The results showed that during the hydrolysis， the best hydrolysis effect was achieved by performing hydrolysis using 10 ml of hydrochloric acid at 105 ℃ for 22 h. Optimization of derivatization conditions
Investigation of amount of derivatization agent
At first， 1 ml of acid-hydrolyzed stock solution was accurately measured into four ampoule bottles， respectively， and into the four ampoule bottles， 0.5 ml of sodium carbonate （0.5 mol/L） was added， respectively. After oscillation， 0.5， 1， 1.5 and 2 ml of 1% DNFB acetonitrile solution were added， respectively， followed by mixing and sealing. The solutions were heated in a water bath at 60 ℃ for 90 min in the shade. The solutions were diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. The solutions were detected sequentially. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different derivatization agent amounts on peak area was investigated， and the result is shown in Fig. 9.
The results showed that under the condition of performing derivatization at 60 ℃ for 90 min， the peak area changed little with the addition of 1-2 ml of DNFB acetonitrile solution. Therefore， 1 ml of DNFB acetonitrile solution was selected.
Investigation of derivatization time
At first， 1 ml of acid-hydrolyzed stock solution was accurately measured into four ampoule bottles， respectively， and into the four ampoule bottles， 0.5 ml of sodium carbonate （0.5 mol/L） was added， respectively. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， respectively， followed by mixing and sealing. The solutions were heated in a water bath at 60 ℃ for 30， 60， 90 and 120 min， respectively， in the shade. The solutions were diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. The solutions were detected sequentially. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different derivatization agent amounts on peak area was investigated， and the result is shown in Fig. 10.
The results showed that under the condition of performing derivatization at 60 ℃ for 90 min with the addition of 1 ml of DNFB acetonitrile solution， the peak area was larger.
Investigation of derivatization temperature
At first， 1 ml of acid-hydrolyzed stock solution was accurately measured into four ampoule bottles， respectively， and into the four ampoule bottles， 0.5 ml of sodium carbonate （0.5 mol/L） was added， respectively. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， respectively， followed by mixing and sealing. The solutions were heated in a water bath at 40， 50， 60 and 70 ℃ for 90 min， respectively， in the shade. The solutions were diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. The solutions were detected sequentially. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different derivatization agent amounts on peak area was investigated， and the result is shown in Fig. 11. The results showed that under the condition of performing derivatization at 50 ℃ for 90 min with the addition of 1 ml of DNFB acetonitrile solution， the effect was the best.
Abalone sample determination results and analysis
As shown in Table 2， various amino acids， totally， 18 amino acids were detected， with a total content of 808.73 mg/g. Among them， glycine accounted for the most of the total nitrogen content， and had a content of 110.79 mg/g. Amino acid content in Sanya abalone was determined by precolumn derivatization-HPLC. Standard deviation （SD） was calculated with function STDEV in EXCEL， and relative standard deviation was calculated according to RSD=（SD/Mea）×100%. The calculation process could be completed in EXCEL.
The RSD values were all lower than 0.2%， so the experiment had good reproducibility.
Evaluation of nutritional value
Because EEAI reflects the fitting degree of the essential amino acid composition of abalone protein with the essential amino acid composition suitable for human[17-19]. Data in Table 3 showed that the proteins corresponding to abalone amino acids are almost high-quality proteins， and only tryptophan is a good protein source. Abalone had high amino acid contents， and essential amino acids accounted for 33.53% of total amino acids. Protein sources corresponding to amino acids in abalone are suitable for human as sources of protein supplements.
Non-essential amino acids include aspartic acid， glutamic acid， serine， glycine， proline， alanine， cystine and tyrosine； essential amino acids include threonine， valine， methionine， isoleucine， tryptophan， leucine， phenylalanine and lysine； semi-essential amino acids include histidine and arginine； flavor amino acids include aspartic acid， glutamic acid， glycine and alanine； sulfur-containing amino acids are methionine and cystine； and branched chain amino acids include valine， isoleucine and leucine.
Conclusions
In this study， amino acid content was determined by precolumn derivatization-HPLC， and the effects of hydrolysis conditions and derivatization conditions on peak area were investigated through optimization tests. The results showed that the optimal hydrolysis conditions were as follows： hydrochloric acid amount of 10 ml， hydrolysis time of 22 h， and hydrolysis temperature at 105 ℃. The optimal derivatization conditions： derivatization agent amount of 1 ml， derivatization time of 90 min and derivatization temperature at 50 ℃. It could be seen from the chromatogram of the sample that the 18 amino acids were well separated. The total content was 808.73 mg/g， the contents of essential amino acids， non-essential amino acids and flavor amino acids were 271.19， 435.44 and 329.52 mg/g， respectively， and essential amino acids accounted for 33.53% of the total amount of amino acids， 62.28% of non-essential amino acids and 40.75% of flavor amino acids. According to the evaluation criterions of Food and Agriculture Organization and World Health Organization （FAO/WHO）， protein sources corresponding to amino acids in abalone are suitable for supplement of protein sources for human body. Precision， detection limit and linear regression tests showed that the instrument had high precision with relative standard derivations lower than 0.21%； the detection limit was low， as low as 0.000 012 91 μg/L； the linear range was wider， and the standard solutions of various amino acids all had good linear relation in the range of 0-500 mg/L， and the correlation coefficients were all over 0.999 8. The method has the advantages of simple operation and satisfactory results. References
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Key words Abalone； Precolumn derivatization； HPLC； Amino acid； Evaluation
Abalone， originally known as Fu， belongs to Haliotis in Haliotidae family of Archaeogastropoda in Gastropo-da of Mollusca[1]. It is a famous and precious traditional food material， which tastes sweet and salty， and is neutral in nature， with the effects of nourishing the liver to improve visual acuity， treating yin deficiency by reinforcing body fluid and moistening dryness[2]. Abalone is mainly distributed in the surrounding waters of Hainan， Taiwan， Guangdong， Fujian and Liaoning in China[3].
There are various amino acid determination methods， mainly including high performance liquid chromatography （HPLC）， automatic amino acid analyzer with ninhydrin post-column derivatization， ion exchange chromatography and capillary tube method[4]， but they all have certain disadvantages. Automatic amino acid analyzer with ninhydrin post-column derivatization has no high sensitivity， and the instrument is very expensive； ion exchange chromatography has high requirements for reaction conditions and is much more complicated with high analysis cost and low work efficiency； and capillary tube method has poor reproducibility， and when the concentration is too high， its sensitivity decreases due to increase in electrostatic force and friction force caused by surrounding medium[5]. Precolumn derivatization-HPLC has no need for special reaction device and has the advantages of high instrument popularizing rate， flexible and diverse method， high sensitivity， good reproducibility and high resolution[6].
Materials and Methods
Materials and instruments Fresh living abalone was purchased from Sanya fishery quay （the abalone flesh was glossy， thick and solid， light brown， carnation or off-white， with inherent smell of abalone）； 18 amino acid standards （Solarbio）； 2，4-dinitrofluorobenzene （AR Xiya Reagent）； N，N-dimethylformamide （AR， Guangzhou Chemical Reagent Factory）； acetonitrile （HPLC， Shantou Xilong Chemistry Factory Co. Ltd.）； phenol （AR， Shantou Xilong Chemistry Factory Co. Ltd.）； acetone.
LC-2010A HT high performance liquid chromatograph （Shimadzu）； SHB-Ⅲ circulating water type multi-purpose vacuum pump （Zhengzhou Greatwall Scientific Industrial and Trade Co.， Ltd.）； DHG-9245 electro-thermostatic blast oven （Jintan Shenglan Instrument Manufacturing Co.， Ltd.）； JU-6224 ultrasonic generator （Shanghai JUMP Ultrasonic Equipment Co.， Ltd.）； HH digital thermostat water bath （Jintan Shenglan Instrument Manufacturing Co.， Ltd.）； RE-52 rotary evaporator （Shanghai Yarong Biochemical Instrument Factory）； DHS-3F pH meter （Shanghai Precision Instrument Co.， Ltd.）； 25 ml hydrolysis tube （pressure-resistant glass tube with screw cap）； ampoule.
Methods
Experimental principle
Acid hydrolysis used 6 mol/L hydrochloric acid as acidolysis agent， to hydrolyze proteins in abalone flesh at 110 ℃ to amino acids， and with 2，4-dinitrofluorobenzene as derivatization agent， and precolumn derivatization was performed to the 18 amino acids， to strengthen ultraviolet absorption. The sample was detected with Shimadzu LC-2010A HT high performance liquid chromatograph， at 360 nm. Qualitative analysis was performed from retention time， and quantitative analysis was performed by external standard method. As tryptophan was damaged by hydrochloric acid， the sample was also hydrolyzed with 5 mol/L sodium hydroxide solution， and tryptophan was determined by LC-2010A HT high performance liquid chromatograph （Shimadzu）.
Sample pretreatment
The shell of fresh abalone was removed， as well as viscera and edges of black mucous membrane. The edible part was flushed with distilled water， and the residual water was removed with filter paper. The edible part was boiled in distilled water for 15 min， and then taken out， followed by cooling and cutting. The pieces were placed in a watch glass and oven-dried in a blast oven at 60 ℃. After cooling， the material was pulverized， grinded and sieved with a 80 mesh sieve. The sample was then re-dried （105 ℃）， sealed and preserved in a dryer.
Acid hydrolysis of sample A certain amount the abalone sample （0.050 0 g） was accurately weighed and added into a hydrolysis tube. Then， 10 ml of 6 mol/L hydrochloric acid was added， and 3-4 drops of newly distilled phenol was added. After vortex oscillation， the sample was frozen in dry ice for 5 min， followed by evacuation to 7 Pa （≤0.05 mm mercury column） and introduction of highly-pure nitrogen gas， and the tube was screwed to seal it in nitrogen atmosphere. The hydrolysis tube was placed in a constant temperature drying oven at 105 ℃ for 22 h， and taken out. After cooling， mixing and opening， filtration was performed. The filtrate was transferred to a rotary evaporator and vacuumized at 60 ℃ to dryness， and if necessary， little distilled water could be added to repeat evaporation for 1-2 times. Little distilled water was added to dissolve the residue， and the solution was diluted to 10 ml. After mixing well， the solution was filtered with 0.45 μm filter membrane， obtaining filtrate which was stored in a refrigerator as acid-hydrolyzed stock solution for later derivatization.
Basic hydrolysis of tryptophan
A certain amount of the sieved abalone sample （0.050 0 g） was accurately weighed and added into a hydrolysis tube， and 10 ml of 5 mol/L sodium hydroxide solution was added. After vortex oscillation， the sample was frozen in dry ice for 5 min， followed by evacuation to 7 Pa （≤0.05 mm mercury column） and introduction of highly-pure nitrogen gas， and the tube was screwed to seal it in nitrogen atmosphere. The hydrolysis tube was placed in a constant temperature drying oven at 105 ℃ for 22 h， and taken out. After cooling， mixing and opening， filtration was performed. The filtrate was transferred to a rotary evaporator and vacuumized at 60 ℃ to dryness， and if necessary， little distilled water could be added to repeat evaporation for 1-2 times. Little distilled water was added to dissolve the residue， and the solution was diluted to 10 ml. After mixing well， the solution was filtered with 0.45 μm filter membrane， obtaining filtrate which was stored in a refrigerator as base-hydrolyzed stock solution for later derivatization.
Pre-column derivation
A certain amount of the acid-hydrolyzed stock solution （1 ml） was accurately weighed and added into an ampoule， and 0.5 ml of sodium carbonate solution （0.5 mol/L） was added. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， followed by mixing and sealing. The solution was heated in a water bath at 50 ℃ for 90 min in the shade. The solution was diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as sample solution. Chromatographic condition
Amino acid content was determined with Shimadzu LC-2010A HT high performance liquid chromatograph using Inertsil ODS-3 C18 column （4.6×150 mm， 5 μm）. The analysis cycle of one sample was 60 min. The HPLC was carried out with 0.05 mol/L sodium acetate-acetic acid buffer （containing 1% N，N-dimethylformamide， pH=6.4） as mobile phase A and acetonitrile-water （v/v=1∶1） as mobile phase B （70∶30） at a flow rate of 1 ml/min under the detection wavelength of 360 nm， the column temperature at 33℃ and a sample size of 10 μl. Under the separation conditions， the 18 amino acids were well separated.
Standard curve plotting and chromatogram analysis
Preparation of standard solution
Proper amounts of amino acid standards were weighed， dissolved with distilled water and diluted to 100 ml， 1 ml of which was accurately measured， placed in an ampoule bottle and added with 0.5 ml of sodium carbonate （0.5 mol/L）. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， followed by mixing and sealing. The solution was heated in a water bath at 50 ℃ for 90 min in the shade. The solution was diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required standard solution.
Chromatographic analysis
After hydrolysis of abalone， derivatization was performed with 2，4-dinitrofluorobenzene， and determination was performed with Shimadzu LC-2010A HT high performance liquid chromatograph， at 360 nm. The 18 amino acids were well separated under the separation conditions. The chromatograms of the standard solution， the acid-hydrolyzed sample solution， the acid-hydrolyzed blank solution， the base-hydrolyzed sample solution and base-hydrolyzed blank solution are shown in Fig. 1， Fig. 2， Fig. 3， Fig. 4 and Fig. 5， respectively.
Nutritional value modes of amino acid
According to the advises by FAO/WHO （Food and Agriculture Organization and the World Health Organization） and Institute of Nutrition and Food Hygiene， Chinese Academy of Preventive Medicine， the nutritional value of amino acids in abalone from Sanya was evaluated according to egg protein model.
Nitrogen amino acid score （AAS）， chemical score （CS） and essential amino acid score （EAAI） were calculated according to given formula[7-11].
Amino acid score=100×Essential amino acid content in each gram of to-be-evaluated protein （mg）/Essential amino acid content in each gram of protein in FAO/WHO model（mg） Chemical score=100×Essential amino acid content in each gram of to-be-evaluated protein （mg）/Essential amino acid content in each gram of egg protein （mg， each gram of egg as standard）
EAAI=ni=1aaiAAi
Wherein aa is the content of amino acid in sample （mg/g）； AA （FAO/WHO） is the content of the same amino acid in FAO/WHO evaluation mode （mg/g）； and AA （Egg） is the content of the same amino acid in whole egg protein （mg/g）. In the formula， aai is the percentage of certain amino acid contained in sample material to essential amino acids； and AAi is the essential amino acid contained in reference protein to essential amino acids. The evaluation criterions are as follows： EAAI>0.95 indicates high-quality protein source， 0.85
Results and Analysis
Plotting of standard curve
At first， 25， 50， 125， 250 and 500 mg/L mixed standard solutions of the 18 amino acids were prepared， and 1 ml of each solution was accurately added into an ampoule bottle and prepared into a testing solution according to the derivatization conditions. The obtained solutions were analyzed sequentially under set conditions. Standard curves were plotted with peak area as f（x） value and detection concentration （mg/L） as x value， and the obtained regression equations and correlation coefficients are shown in Table 1.
Above data and curve showed that the 18 amino acids had good linear relation in the linear range of 0-500 mg/L， and the correlation coefficients were all higher than 0.999 8.
Optimization of water hydrolysis conditions
Investigation of amount of hydrochloric acid
A certain amount of the sieved abalone sample （0.050 0 g） was accurately weighed， and added into four hydrolysis tubes， respectively. Into the four tubes， 5， 10， 15 and 20 ml of 6 mol/L hydrochloric acid were added， respectively， and hydrolysis was performed under acid hydrolysis conditions. Then， 1 ml of each of the solutions was accurately measured and added with 0.5 ml of sodium carbonate （0.5 mol/L）. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， followed by mixing and sealing. The solution was heated in a water bath at 60 ℃ for 90 min in the shade. The solution was diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different hydrochloric acid amounts on peak area was investigated， and the result is shown in Fig. 6. As shown in Fig. 6， in hydrochloric acid range of 10-20 ml， the peak area changed little， but if the amount of hydrochloric acid was too large， the chromatogram showed tailing phenomenon. Therefore， 10 ml of hydrochloric acid was selected for hydrolysis.
Investigation of hydrolysis time
A certain amount the sieved abalone sample （0.050 0 g） was accurately weighed， and added into four hydrolysis tubes， respectively， and hydrolysis was performed under acid hydrolysis conditions for 20， 22， 24 and 26 h， respectively. Then， 1 ml of each of the solutions was accurately measured and added with 0.5 ml of sodium carbonate （0.5 mol/L）. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， followed by mixing and sealing. The solution was heated in a water bath at 60 ℃ for 90 min in the shade. The solution was diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different hydrolysis time on peak area was investigated， and the result is shown in Fig. 7.
The results showed that during the hydrolysis， the best hydrolysis effect was achieved by performing hydrolysis using 10 ml of hydrochloric acid at 105 ℃ for 22 h.
Investigation of hydrolysis temperature
A certain amount of the sieved abalone sample （0.050 0 g） was accurately weighed and added into four hydrolysis tubes， respectively， and hydrolysis was performed under acid hydrolysis conditions at 100， 105， 110 and 115 ℃， respectively. Then， 1 ml of each of the solutions was accurately measured and added with 0.5 ml of sodium carbonate （0.5 mol/L）. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， followed by mixing and sealing. The solution was heated in a water bath at 60 ℃ for 90 min in the shade. The solution was diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different hydrolysis temperaturess on peak area was investigated， and the result is shown in Fig. 8.
The results showed that during the hydrolysis， the best hydrolysis effect was achieved by performing hydrolysis using 10 ml of hydrochloric acid at 105 ℃ for 22 h. Optimization of derivatization conditions
Investigation of amount of derivatization agent
At first， 1 ml of acid-hydrolyzed stock solution was accurately measured into four ampoule bottles， respectively， and into the four ampoule bottles， 0.5 ml of sodium carbonate （0.5 mol/L） was added， respectively. After oscillation， 0.5， 1， 1.5 and 2 ml of 1% DNFB acetonitrile solution were added， respectively， followed by mixing and sealing. The solutions were heated in a water bath at 60 ℃ for 90 min in the shade. The solutions were diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. The solutions were detected sequentially. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different derivatization agent amounts on peak area was investigated， and the result is shown in Fig. 9.
The results showed that under the condition of performing derivatization at 60 ℃ for 90 min， the peak area changed little with the addition of 1-2 ml of DNFB acetonitrile solution. Therefore， 1 ml of DNFB acetonitrile solution was selected.
Investigation of derivatization time
At first， 1 ml of acid-hydrolyzed stock solution was accurately measured into four ampoule bottles， respectively， and into the four ampoule bottles， 0.5 ml of sodium carbonate （0.5 mol/L） was added， respectively. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， respectively， followed by mixing and sealing. The solutions were heated in a water bath at 60 ℃ for 30， 60， 90 and 120 min， respectively， in the shade. The solutions were diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. The solutions were detected sequentially. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different derivatization agent amounts on peak area was investigated， and the result is shown in Fig. 10.
The results showed that under the condition of performing derivatization at 60 ℃ for 90 min with the addition of 1 ml of DNFB acetonitrile solution， the peak area was larger.
Investigation of derivatization temperature
At first， 1 ml of acid-hydrolyzed stock solution was accurately measured into four ampoule bottles， respectively， and into the four ampoule bottles， 0.5 ml of sodium carbonate （0.5 mol/L） was added， respectively. After oscillation， 1 ml of 1% DNFB acetonitrile solution was added， respectively， followed by mixing and sealing. The solutions were heated in a water bath at 40， 50， 60 and 70 ℃ for 90 min， respectively， in the shade. The solutions were diluted with 0.1 mol/L phosphate buffer （pH=7） to 10 ml， and filtered with 0.45 μm organic filter membrane， obtaining filtrate as the required sample solution. The solutions were detected sequentially. With the peak area of chromatographic peak subjected to deduction of blank as investigation index， the effect of different derivatization agent amounts on peak area was investigated， and the result is shown in Fig. 11. The results showed that under the condition of performing derivatization at 50 ℃ for 90 min with the addition of 1 ml of DNFB acetonitrile solution， the effect was the best.
Abalone sample determination results and analysis
As shown in Table 2， various amino acids， totally， 18 amino acids were detected， with a total content of 808.73 mg/g. Among them， glycine accounted for the most of the total nitrogen content， and had a content of 110.79 mg/g. Amino acid content in Sanya abalone was determined by precolumn derivatization-HPLC. Standard deviation （SD） was calculated with function STDEV in EXCEL， and relative standard deviation was calculated according to RSD=（SD/Mea）×100%. The calculation process could be completed in EXCEL.
The RSD values were all lower than 0.2%， so the experiment had good reproducibility.
Evaluation of nutritional value
Because EEAI reflects the fitting degree of the essential amino acid composition of abalone protein with the essential amino acid composition suitable for human[17-19]. Data in Table 3 showed that the proteins corresponding to abalone amino acids are almost high-quality proteins， and only tryptophan is a good protein source. Abalone had high amino acid contents， and essential amino acids accounted for 33.53% of total amino acids. Protein sources corresponding to amino acids in abalone are suitable for human as sources of protein supplements.
Non-essential amino acids include aspartic acid， glutamic acid， serine， glycine， proline， alanine， cystine and tyrosine； essential amino acids include threonine， valine， methionine， isoleucine， tryptophan， leucine， phenylalanine and lysine； semi-essential amino acids include histidine and arginine； flavor amino acids include aspartic acid， glutamic acid， glycine and alanine； sulfur-containing amino acids are methionine and cystine； and branched chain amino acids include valine， isoleucine and leucine.
Conclusions
In this study， amino acid content was determined by precolumn derivatization-HPLC， and the effects of hydrolysis conditions and derivatization conditions on peak area were investigated through optimization tests. The results showed that the optimal hydrolysis conditions were as follows： hydrochloric acid amount of 10 ml， hydrolysis time of 22 h， and hydrolysis temperature at 105 ℃. The optimal derivatization conditions： derivatization agent amount of 1 ml， derivatization time of 90 min and derivatization temperature at 50 ℃. It could be seen from the chromatogram of the sample that the 18 amino acids were well separated. The total content was 808.73 mg/g， the contents of essential amino acids， non-essential amino acids and flavor amino acids were 271.19， 435.44 and 329.52 mg/g， respectively， and essential amino acids accounted for 33.53% of the total amount of amino acids， 62.28% of non-essential amino acids and 40.75% of flavor amino acids. According to the evaluation criterions of Food and Agriculture Organization and World Health Organization （FAO/WHO）， protein sources corresponding to amino acids in abalone are suitable for supplement of protein sources for human body. Precision， detection limit and linear regression tests showed that the instrument had high precision with relative standard derivations lower than 0.21%； the detection limit was low， as low as 0.000 012 91 μg/L； the linear range was wider， and the standard solutions of various amino acids all had good linear relation in the range of 0-500 mg/L， and the correlation coefficients were all over 0.999 8. The method has the advantages of simple operation and satisfactory results. References
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