


































Energy and Earth Science 
Vol. 7, No. 2, 2024 

www.scholink.org/ojs/index.php/ees 

ISSN 2578-1359 (Print)   ISSN 2578-1367 (Online) 

 

27 

 

Original Paper 

A New Method for Producing Amino Acids and Preserving 

Tryptophan by Using Seafood and Waste 

Chen Junhao
1
 & Chen Guiqing

1
 

1
 Jinan University, Guangzhou 510630, China 

 

Received: November 5, 2024   Accepted: November 15, 2024    Online Published: January 7, 2025 

doi:10.22158/ees.v7n2p27                  URL: http://dx.doi.org/10.22158/ees.v7n2p27 

 

Abstract 

There are many problems in hydrochloric acid hydrolysis, and other acids are preferred. The main 

problem of hydrolysis is to use acid theory and calculation formula. Only the theory can be found from 

the textbook: the hydrolysis constant K=αNδ, which is problematic and not available. Had to establish 

a new theory and formula: “double Chen hydrolysis formula”. 

Tryptophan can be preserved by hydrolysis with citric acid. It does not cause racemization of amino 

acids. You get L-amino acids, you don’t get D-amino acids. It is neutralized with hydrated lime to form 

insoluble calcium citrate and filtered out without any impurities. It can also remove heavy metals such 

as arsenic, cadmium, mercury, lead and chromium. It does not pollute the environment, retains the 

advantages of hydrochloric acid hydrolysis and overcomes its disadvantages. Residual calcium citrate 

is a good calcium supplement for livestock and poultry—is environmentally friendly. 

Keywords 

amino acid, Hydrolysis, Citric acid, Calcium hydroxide, Triangular conical bottle, Reaction still 

 

1. Introduction 

Sea (fresh water) products and their leftovers are rich in 17 kinds of protein amino acids, which contain 

more amino acids such as glycine, alanine and glutamic acid, so the taste is particularly good. It also 

contains more non-protein amino acids-taurine, which is very important to the human body, is an 

essential substance for newborn brain development, and has the role of protecting myocardium, 

anti-arrhythmia, treatment of myocarditis, reducing blood pressure, blood lipids and blood sugar. In 

addition, it also contains essential elements and trace elements such as calcium, potassium, magnesium, 

zinc, iodine, selenium and phosphorus. 

 



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Our coastline stretches coastal areas are extremely rich in Marine resources! For example, Jiaodong 

area leaves about 2.5 tons of scallop skirt scraps for every 1 ton of wet scallop production. Scallop 

skirts abound throughout the country; A large number of shrimp heads after processing shrimp in 

various places; A large amount of mussel meat after freshwater pearl is produced in Jiang and Zhejiang 

regions. The sea red in some areas of Longhai and Jiaodong of Fujian Province, near the Wolfi Island 

of Zhejiang Province, is difficult to sell because of its large output. The cooking juice after processing 

scallops, Jiangyao, clams, sea red and oysters all over the coast is thrown away in large quantities, 

which can be fully utilized. 

Laver contains up to 32.5% to 39% amino acids, which is very rare and is very good for the production 

of amino acids and flavorings. Worth developing vigorously! 

Amino acids make proteins, without which there would be no life. Producing amino acids in large 

quantities and reducing their price is a necessary means for human health and longevity. 

The production of amino acids requires a balance of eight essential amino acids for the human body, in 

appropriate proportion to each other, in line with the FAO and the World Health Organization 

(FAO/WHO) model. Its production methods include hydrochloric acid hydrolysis method, enzyme 

hydrolysis method, chemical synthesis method and microbial fermentation method. Hydrochloric acid 

hydrolysis method has a long history, high production efficiency, cheap and easy to obtain raw 

materials, simple equipment, and can produce almost all kinds of amino acids at the same time, without 

causing racemization of amino acids. You get L-amino acids, you don’t get D-amino acids. But without 

it, all the tryptophan that humans cannot survive is destroyed. The residual acid can not be removed, 

the salt generated by neutralization is difficult to remove, the hydrolysis odor generated is difficult to 

eliminate, and the environment is polluted. Because these technical problems can not be solved for a 

long time, the great potential of hydrochloric acid hydrolysis can not be played out, has been on the 

edge of elimination, other production methods came into being, to be greatly developed, but they do 

not have the potential technical advantages of hydrochloric acid hydrolysis method! 

The biggest advantage of enzymatic hydrolysis is that the conditions are mild, and there is no need for 

high temperature, high pressure and acid-resistant equipment; The disadvantage is that the reaction 

time is long, and the hydrolysis is not easy to complete, the enzyme is very fragile, easy to inactivate, 

and the enzyme is generally more expensive and the efficiency is poor. Buffers must be added to 

stabilize the PH to ensure enzymatic hydrolysis. The inorganic salts produced after enzymatic 

hydrolysis cannot be removed, seriously affecting the purity of the product! The peptides produced 

have adverse allergic reactions in some human bodies. 

Tryptophan was obtained by alkaline hydrolysis. Some amino acids will racemize, from L-type amino 

acids to D-type amino acids that the human body can not absorb, and the loss of nutrients is large, and 

can not be used to produce food. 

 



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Traditional extraction method, chemical synthesis method and microbial fermentation method are 

difficult to achieve the purpose of industrial production because of the high cost and complex process 

of precursors. So we find other acids for hydrolysis, but the main problem of acid hydrolysis method is 

the theory and calculation of acid quantity. From the textbook catalytic theory: hydrolysis constant 

K=αNδ, α is the catalytic coefficient of acid, N is the molar concentration, δ and hydrolysis temperature 

T, time t in a certain range is positively correlated. The hydrolysis effect is proportional to N. 

The theory is flawed. 1) N increases with the decrease of water added. Due to the co-ionic effect, it 

inhibits each other, affecting the ionization of hydrogen atoms, but reducing the hydrolysis effect. 2) 

This theory fails to reflect the relationship between the amount of hydrolyzed acid and the different 

types of proteins and the different nitrogen content of hydrolysates. So this formula is not available. We 

had to build up new theories and formulas. 

 

2. Theoretical Analysis 

2.1 Hydrolysis Was Derived by the Acid Quantity Formula 

The hydrolysis capacity is only related to the H+ ion in the hydrolyzed acid molecule. For many (Y) 

elementary acids, an acid molecule with Y H atoms may ionize into H+ ions. Therefore, the hydrolysis 

effect is related to the equivalent number of hydrolyzed acid. The purpose of hydrolysis is to break the 

polypeptide chain of nitrogen molecules in the raw material into free amino acids. The more the 

number of nitrogen molecules in the raw material, the more the equivalent number of hydrolyzed acid 

is required. This is a proportional relationship, written as the equation: 

Equivalent number of hydrolyzed acid=number of raw nitrogen molecules ×CC...... (1), the 

proportional coefficient CC is called “double Chen hydrolysis coefficient”, referred to as “hydrolysis 

coefficient”. 

∵ Molecular number of hydrolyzed acid=weight of hydrolyzed acid/molecular weight of hydrolyzed 

acid; 

Equivalent number of hydrolyzed acid=number of molecules of hydrolyzed acid ×Y=(weight of 

hydrolyzed acid ×Y)/Molecular weight of hydrolyzed acid...... (2); 

Raw material nitrogen molecular number=raw material nitrogen content/molecular weight of nitrogen 

14.0067...... (3). 

Substitute (2) and (3) into (1): 

(Weight of hydrolyzed acid × Y)/Molecular weight of hydrolyzed acid=(nitrogen content of raw 

material /14.0067) ×CC. 

∴ hydrolytic acid weight=(molecular weight of hydrolytic acid / 14.0067) × nitrogen content of raw 

material ×CC ÷Y 

=(Molecular weight of hydrolyzed acid/14.0067) × raw protein content ×16%×CC÷Y...... ⑷ 

Double Chen hydrolysis formula. 



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General meat, poultry, eggs, fish, shellfish protein nitrogen content is 16%. 

2.2 Find Suitable CC and Hydrolysis Acid 

2.2.1 Double Chen Hydrolysis Coefficient CC 

Strong acid catalytic coefficient is high, CC can be smaller. Hard protein consists of bone, tendon, horn, 

hair, silk and other substances with large protein content, CC can be larger. CC is negatively correlated 

with hydrolysis pressure, temperature T, time t and air temperature. This is presented as Table 1 CC 

values for reference! 

2.2.2 Acid for Hydrolysis 

2.2.2.1 Inorganic Acid 

Iodate and bromate are dangerous or carcinogenic substances. Sulfuric acid is a binary strong acid, 

which is easy to injure and destroy things. Nitric acid is too aggressive. It explodes. Phosphoric acid 

has a burnt taste. Hydrochloric acid polluted the environment, destroyed tryptophan, and the salt 

neutralized by hydrolyzed residual acid was difficult to remove. They are not suitable for use! 

2.2.2.2 Organic Acids 

Because it is necessary to neutralize the residual acid with lime after hydrolysis, it is necessary to 

consider the saturated solubility of its calcifiers, which is too large to be easily removed, preferably 

below 0.1, and it is also necessary to consider the taste. Saturated solubility of the following organic 

acid calcifiers (g/100ml) 

Calcium formate 16.725. Calcium acetate 34. Calcium propionate 28.3, low threshold, bad smell. 

Calcium tartar 0.38, too exciting. Calcium lactate 4.54. Calcium gluconate 4.012. Apple calcium 0.8. 

Calcium oxalate 0.00067, taste heavy not available. Calcium citrate 0.0825, the best hydrolysis with 

citric acid, the catalytic coefficient α is acceptable, does not pollute the environment, the residual acid 

after hydrolysis is neutralized by hydrated lime to form calcium citrate precipitation, filtration removal, 

does not contain impurities, and preserves the very important tryptophan for the human body. The 

equipment is simple, the raw materials are cheap and easy to obtain, and the advantages of hydrochloric 

acid hydrolysis are retained and its disadvantages are overcome. 

The disadvantages of citric acid hydrolysis are low catalytic coefficient and large dosage, and reflux 

hydrolysis is only used for experimental purposes. The production must be hydrolyzed using a reactor. 

Alkaline hydrolysis is used to obtain tryptophan only. Most amino acids are racemic and do not 

produce food. 

Enzymatic hydrolysis is costly and inefficient. Neutralization produces inorganic salts that cannot be 

removed. 

Residual calcium citrate is the best calcium supplement for livestock, which is conducive to 

environmental protection! 

 

 



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3. Experimental Equipment and Method 

3.1 Hydrolysis Equipment and Conditions 

1) Reflux hydrolysis of 5L triangular cone bottle. With a 5L triangular cone bottle, plus a rubber plug, 

plug in the condensation tube, and connect the tap water to cool the hydrolyzed steam, so as not to burn 

the hydrolysate dry. It was heated in a temperature controlled electric furnace of 1500w and hydrolyzed 

at one atmosphere (1kg pressure) and 100
0
C for 12 hours. Take care to regulate the temperature to 

prevent excessive boiling and dry the hydrolysate liquid. 

2) Reactor hydrolysis. Use electric heating or oil heating 304 stainless steel reactor, volume according 

to need. With an anchor mixer. Run the blender at regular intervals for 10 minutes. Be careful not to fill 

the reactor too full, 70% can be. Hydrolysis at 2kg pressure, 112-118
0
C for 12 hours. Be careful not to 

heat too much to avoid a burnt taste. 

3.2 Citric Acid Hydrolysis. The Raw Material Is Hydrolyzed by Adding Citric Acid and Appropriate 

Amount of Water 

3.2.1 Calculation of Acid Quantity for Hydrolysis 

Citric acid C6H8O7 (192.125), ternary weak acid Y=3. The ionization constant Kα1=7.4×10
-4

, 

Kα2=1.7×10
-5

, Kα3=4.0×10
-7

. The citric acid required is calculated from Formula (4): 

M Hydrolytic acid=(molecular weight of hydrolytic acid/14.0067) × raw protein content ×16%×CC÷Y 

M Citric acid=(192.125÷14.0067)×(weight of raw material×protein content of the raw material) 

×16%×CC÷3 

=Weight of raw material×protein content of raw material ×CC×0.7315=weight of raw 

material×nitrogen content of raw material ×6.25×CC×0.7315...... 5. 

Add appropriate amount of water for hydrolysis. M stands for weight. Select CC refer to Table 1. 

3.2.2 Neutralization of Food Grade Hydrated Lime 

M calcium hydroxide=(222.284÷384.251)× M citric acid=0.5785×M citric acid... [6] 

In order to prevent residual calcium hydrogen citrate CaH(C6H5O7) and calcium hydrogen citrate 

CaH4(C6H5O7)2 from precipitating into calcium citrate Ca3(C6H5O7)2, and to completely remove heavy 

metals, so that the hydrolysate reaches PH12-13, more hydrated lime is added for this purpose. Since 

the saturated solubility of hydrated lime Ca(0H)2 at 20
0
C is 0.16(g/100g), PH12-13 is reached at this 

time. Excess lime can be filtered out, and the saturated solubility of lime in the solution of 0.16g/100g 

will not affect the subsequent treatment. 

3.2.3 After 3 Hours of Precipitation, Filter 

3.2.4 Post-Processing 

Under the strong alkaline PH12-13, the hydrolysate is neither delicious nor can amino acids be 

measured, and it is necessary to adjust it to weak acidity with acid. If neutralized with citric acid, small 

amounts of calcium hydrogen citrate CaH(C6H5O7) and calcium hydrogen citrate CaH4(C6H5O7)2 May 

be produced to precipitate. CaCl2 solution was formed by neutralization with food-grade hydrochloric 



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acid. The original Ca(0H)2 had a saturation solubility of 0.16(g/100g), and the resulting CaCl2 had a 

solubility of 0.23g/100g, which would not precipitate (the saturation solubility of CaCl2 at 20
0
C was 

74.5%). With food grade hydrochloric acid 0.157g/100g can be neutralized to PH7. It is best to add 

more hydrochloric acid to reach about PH5 and taste better. 

2HCl + Ca(OH)2——→ CaCl2 +2 H2O 

72.922  74.09468       110.99 

M CaCl2=(110.99÷74.09468)×M Ca(OH)2=1.4979×0.16g/100g=0.23g/100g 

MHCl=(72.922÷74.09468)×M Ca(OH)2=0.9842×0.16g/100g=0.157g/100g 

3.2.5 Expected Production V (volume) 

According to the definition of extraction rate γ=product nitrogen content÷raw material nitrogen content 

=[Product volume V(mL)×product specified amino nitrogen content (g/mL)]÷[raw material weight 

(g)×nitrogen content of the raw material %]. 

∴  Product volume V(mL)=[raw material weight (g)×nitrogen content of the raw material 

%×γ]÷Amino nitrogen content specified in the product (g/mL). 

∵ Nitrogen content of raw materials %=raw material protein content %×16%; 

Product volume V(mL)=raw material weight (g)×protein content of the raw material 

%×16%×γ÷Amino nitrogen of the product (g/mL)…⑺ 

Reactor hydrolysis γ=50%~70%, reflux hydrolysis temperature is greatly affected by temperature, 

especially in winter, γ=35%~50%. 

3.3 The Hydrolysis Capacity of Citric Acid Is Weaker Than That of Hydrochloric Acid, and It Is More 

Suitable For Hydrolyzing Seafood with Protein Content Below 30% 

 

4. Experimental Results and Analysis 

Scallop skirt was boiled and concentrated into scallop liquid (Table 2), which contained 4.82% total 

amino acids. Calculate the citric acid to be added from (5), add appropriate amount of water for reflux 

hydrolysis for 12 hours, hydrated lime neutralization, long-term precipitation, filtration and 

post-treatment. 

4.1 Experiment 1 

Scallop solution 2,120ml, select CC=5.1. Add 422g citric acid. After hydrolysis, hydrated lime 

neutralization, long-term precipitation, filtration and “post-treatment”. Scallop hydrolyzed juice 

1,300ml. As shown in Table 3, the total amino acid content is 2.81%. The extraction rate was γ=35.7%. 

4.2 Experiment 2 

Laver 180g, CC=7. Add 300g citric acid. After hydrolysis, hydrated lime neutralization, long-term 

precipitation, filtration and “post-treatment”. Seaweed seasoning liquid 1640ml. As shown in Table 5, 

the total amino acid content is 1.26%. Extraction rate: γ=35.3%. 

 



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The above two experimental testing companies could not detect tryptophan, so they had to change to 

Zhongke Company for testing, as shown in Table 6. 

4.3 Experiment 3 

Scallop solution 2100ml, CC=6.75. Add citric acid 138.2g. After hydrolysis, hydrated lime 

neutralization, long-term precipitation, filtration and “post-treatment”. 1,730ml of scallop juice. As 

shown in Table 7, the total amino acid content is 2.4% and tryptophan 0.09%. The extraction rate was 

γ=41%. 

4.4 Experiment 4 

Laver 83g (Table 4), select CC=8. Add 142g citric acid. After hydrolysis, hydrated lime neutralization, 

long-term precipitation, filtration and “post-treatment”. Get laver juice 260ml. As shown in Table 8, the 

total amino acid content is 5.1% and tryptophan is 0.15%. The extraction rate γ=49%. 

4.5 Experiment 5 

Pinctada martensii meat 750g, with citric acid 440g. After hydrolysis, hydrated lime neutralization, 

long-term precipitation, filtration and “post-treatment”. The total amino acid content was 3.27%. The 

extraction rate γ=(1300×3.27%) ÷(750×14.3%)=39.6%. 

4.6 Experiment 6 

1000L stainless steel reactor is adopted. Scallop skirt 270Kg, contains 8.8% protein, select CC=2, add 

citric acid 35kg. After hydrolysis, hydrated lime neutralization, long time precipitation, filtration and 

“post-treatment”. I have 500 liters of scallop juice. Contains 0.4% amino nitrogen. Extraction rate: 

γ=500×0.4%÷(270×8.8%×0.16%)=52.6%. Acceptance, smooth delivery. 

 

5. Conclusion 

1) The “double Chen hydrolysis formula” for calculating the amount of acid used for hydrolysis is 

derived, and the concept of “double aging hydrolysis coefficient CC” is introduced. 

2) Citric acid hydrolysis retains the advantages of hydrochloric acid hydrolysis and overcomes its 

disadvantages. After hydrolysis, it is neutralized with hydrated lime to produce calcium citrate 

precipitation, which is filtered to remove hydrolyzed residual acids, bases, salts and all heavy metals: 

As,Cd, Hg, Pb, Cr, etc. Does not pollute the environment. The equipment is simple, high efficiency, 

raw materials are cheap and easy to obtain, can produce amino acid, seasoning, compound condiments 

and so on. Suitable for hydrolysis of aquatic products, 

3) Seafood hydrolysis to taurine, very important to the human body. 

4) The head of prawn has many whiskers and shells which are difficult to handle. Amino acids can be 

extracted by hydrolysis with citric acid and flavoring can be produced. 

5) The hydrolysis of citric acid does not occur racemization to obtain L-amino acids, and does not 

produce D-amino acids. 

 



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6) The citric acid hydrolysis method removes all salts and impurities, preserving the most important 

tryptophan for the human body. Push enzymatic hydrolysis and alkaline hydrolysis off the technical 

stage, no need to use them! 

7) Residue is a good calcium supplement for livestock and poultry, which is conducive to 

environmental protection 

 

Attached test report 

 

Table 1. Hydrolysis Reinforcement Coefficient CC Reference Value 

 

 

Table 2. Experimental Scallop Liquid 

 



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Add too much water to increase CC. 

 

Table 3. Experiment 1 Results of Hydrolyzed Scallop Juice 

 

 

Table 4. Experimental Laver 

 



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Table 5. Experiment2 Hydrolyzed Laver Juice 

 

 

Table 6. Tryptophan in Hydrolyzed Scallop and Laver Juice 

 

 

 

 

 

 

 

 



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Table 7. Experiment 3 All Amino Acids in Scallop Liquid 

 

 

Table 8. Experiment 4 All Amino Acids in Laver 

 

 

 

 

 

 

 

 



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Table 9. Experiment 5 Test of Hydrolysate 

 

  

Table 10. Experiment 6 

  

 

The application of citric acid hydrolysis technology in the production of fresh scallop juice using a 

reactor in Yantai City, Shandong Province, China, won the “National Patent Technology Invention Gold 

Award”. 

Total amino acid氨基酸总量。Gly 甘氨酸。Ala 丙氨酸。Val 缬氨酸。Leu亮氨酸。Ile 异亮氨

酸。Phe 苯丙氨酸。Trp 色氨酸。Tyr 酪氨酸。Asp 天冬氨酸。His 组氨酸。Asn 天冬酰胺。



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Glu 谷氨酸。Lys 赖氨酸。Gln 谷氨酰胺 Met 甲硫氨酸。Arg 精氨酸。Ser 丝氨酸。Thr 苏氨

酸。Cys 半胱氨酸。Pro 脯氨酸。 

 

References 

Academic Proceedings on Amino Acids (pp. 1-192). (1983.). Amino acid Laboratory, Wuhan 

University. 

Amino acids and biological resources. (n.d.). Journal of Amino Acids and Biological Resources. School 

of Life Sciences, Wuhan University. 

Amino acids and human health. Biotechnology and application graduate thesis. (n.d.). Jining 

Vocational and Technical College. 

Chen Guiqing, & Chen Junhao. (2006). Methods for producing condiments by hydrolysis of organic 

acids. In Chinese Spiritual Civilization Treatise Volume III Comprehensive Treatise (pp. 341-343). 

Central Literary Publishing House. 

New trends in research, development, production and application of amino acid production by 

fermentation at home and abroad. Kou Guang will wait. (n.d.). School of Bioengineering, Tianjin 

University of Science and Technology. 

Production method of amino acid liquid of pearl oyster or ginseng of pearl oyster. (2017). Patent No. 

ZL 2015 1 057130.x. Authorization date November 21, 2017. 

Production status, development trend and existing problems of amino acid industry in China. (n.d.). 

School of Bioengineering, Jiangnan University. 

Research report on production technology of amino acid solution. (n.d.). Chemical market information 

network. 

 

 

 

 

 

 

 


