Enzymatic Hydrolysis of Hairtail Surimi in an Ultra-High Pressure Bioreactor

Amino acids have been extracted from Hairtail surimi using enzymes in an 
ultra-high pressure bioreactor. The extraction efficiency of different enzymes 
including papain, trypsin, and proteases (acid, neutral, alkaline) also has 
been evaluated, and it has been discovered that neutral protease behaved the 
best. The amino acids were analyzed using automatic amino acid analyzer, and the enzymatic 
digestion conditions were optimized. For neutral protease, the optimal 
condition was 50℃, 250 MPa, pH 7.0. Material to 
liquid ratio of enzyme is 6%. More than 29 amino acids 
were detected after 24 hours of hydrolysis; the enzymatic hydrolysis rate can 
reach 83.29%. The results show that enzymatic digestion under 
ultra-high-pressure provides a very promising approach to extract amino acids 
from Hairtail surimi.


Introduction
Amino acids are used for a variety of applications in industry. The food industry is a major consumer of amino acids, in particular, glutamate, which is used as a flavor enhancer, and aspartame (aspartyl-phenylalanine-1-methyl ester) as a low-calorie artificial sweetener. Similar technology to that used for animal nutrition is employed in the human nutrition industry to alleviate symptoms of mineral deficiencies, such as anemia, by improving mineral absorption and reducing negative side effects from inorganic mineral supplementation. The earliest industrial production of amino acids in the world is the creation of the Japanese Ajinomoto company. First, "gluten" left by wheat flour to process starch is hydrolyzed with hydrochloric acid to obtain glutamate, and then neutralized by soda ash to obtain Sodium glutamate. Glutamate is the world's first industrially produced single amino acid product.
Since then, scientists have used protein hydrolysis to hydrolyze raw materials such as feathers, human hair, pig blood, meat chops and surimi into amino acids, but these amino acids are mostly "DL (Right-handed, Left-handed) mixed amino acids" and their resolution is very difficult until the specific catalysis of enzymes is used to obtain amino acids in a natural single configuration.
The industrial microbial fermentation process established in the 1960s brought the amino acid industry off. Since then, many common amino acid species (including glutamate, lysine, threonine, phenylalanine, etc.) can be produced by microbial fermentation, so that the yield is greatly increased and the cost is greatly reduced. But the fermentation process is a time-consuming and inefficient way. Percy Williams Bridgman received a Nobel Prize in 1946 for advancing high pressure process area of physics by several magnitudes of pressure (400 MPa to 40,000 MPa). In the last several decades, the field of biology has taken Bridgman's advice, and hydrostatic pressure has become a robust physicochemical tool for the study of biological macromolecules [1].
Chemical bonding is likely to change under high pressure, when the P*V (Pressure, Volume) term in the free energy becomes comparable to the energies of typical chemical bonds, i.e. at around 100 GPa.
High-Pressure Processing has been used in many fields of food industry, such as fruit products [2], seafood (Teixeira et al., 2013), vegetables [3], but it was often treated without exogenous enzyme, so the shape of fish needs a long time (1 or 2 months) to degrade into small fragments [4].
The aim of this work is to provide a specific and efficient treatment method for the exogenous enzyme hydrolysis of Hairtail surimi in an ultra-high pressure bioreactor.
The ultra-high pressure bioreactor utilizes biological tissue to hydrolysis by specific enzyme under ultra-high pressure conditions, producing favorable biological trait changes, and is regarded as a safe, green and effective biological treatment technology [5]. Using ultra-high pressure bioreactors, we extracted amino acids from meat emulsions under high pressure conditions for the first time to obtain higher extraction efficiency. We have found that ultra-high pressure conditions facilitate the enzymatic hydrolysis of meat emulsions, providing a new way to extract amino acids quickly and efficiently from meat emulsions.

Materials
All enzymes (papain, trypsin, and acid protease, neutral protease, alkaline protease) are bought from Nanning Pound Biotechnology Co., Ltd.
Fish meat of Hairtail is bought from local market.

Selection of enzymes
Weigh 10 g of processed Hairtail surimi, Water content (%) of which is 73.4%, and add it to 20 mL of distilled water. In the solution, different kinds of enzymes (papain, trypsin, and proteases) are added by 3% or 6% (W/W). The sample is placed in an ultra-high pressure biological reactor. In the reactor, the pressure is controlled to 250 MPa at 50˚C. Next, the reaction was carried out for 24 h. By detecting the protein in different samples, the efficiency is taken to determine the amount of the optimal enzyme preparation added [6].

Selection of Material to liquid ratio
In order to improve the efficiency of the use of ultra-high pressure bioreactors, the liquid ratio is an important factor. Weighed Hairtail surimi 10 g, under the optimum temperature, pH and enzyme amount of various enzymes, Regulate different ratios of solid to liquid (3%, 6%) at 250 MPa. Under the pressure, the reaction time was 4 hr. By detecting proteins in different samples Extraction efficiency, determine the best ratio of material to liquid [6].

Enzymatic assay
Optimization of enzymatic conditions such as optimum T and optimum pH is the same as in previous literature [6].

Water content determination
The moisture content of samples is determined by reference to the national standard method [7].

Total nitrogen determination
The nitrogen content of samples is determined by reference to the national standard method [8].

Amino acids Analysis
First: Pipette 2.0 mL of enzyme hydrolyzate into a 10 mL centrifuge tube, add 6.0mL of water ethanol to the centrifuge tube, shake well, centrifuge in a centrifuge for 10 min, remove the protein from the hydrolyzate. Enzymatic hydrolysis rate = total nitrogen content in the liquid obtained after enzymatic hydrolyzing/total nitrogen content in raw material.

Results
Water content of raw material Water content in Hairtail is shown in Table 1 Total Nitrogen content of raw material Nitrogen content in Hairtail is shown in Table 2 Comparison of enzymatic hydrolysis results of Hairtail under normal pressure and ultra-high pressure It can be seen from Figure 1 that after 24 hours at 50˚C, the fish meat under 100 MPa high pressure gradually digested into a paste, and no deterioration occurred, while the Hairtail under normal pressure still maintained the shape of the fish, but it was obviously spoiled.
The ultra-high pressure enzymatic hydrolysis process can completely eliminate the influence of spoilage bacteria, so that the enzymatic hydrolysis process can be controlled completely, In accordance with the direction of the experimental design, the solid fish protein can finally be hydrolyzed into water-soluble liquid amino acids. It can be seen from Figure 2 that the best enzymatic hydrolysis effect is that the enzymatic hydrolysis rate is 22.75% under the experimental conditions of enzymatic hydrolysis pressure of 250 MPa, enzymatic hydrolysis time of 2 hr and addition of 6% neutral protease.
It can be seen from Table 3

Selection of Material to Liquid Ratio
It can be seen from Figure 3 that the best enzymatic hydrolysis effect is that the enzymatic hydrolysis rate is 33.5% under the experimental conditions of

Selection of enzymatic hydrolysis time
The results from Figure 4 showed that the enzymatic hydrolysis rate continued to increase with the prolongation of the enzymatic hydrolysis time, and the enzymatic hydrolysis rate can reach 83% after the neutral protease was on the fish for 24 hours.

Amino acids Analysis
From Figure 3 and Table 4, we can find that the solid fish meat can be

Discussion
The results showed that the enzymatic hydrolysis under ultra-high pressure condition can obtain as high as 82.8% of enzymatic hydrolysis rate for hairtail meat. In this study, only a single sort of enzyme was used in the experiment. A A previous research analyzed amino acids contained in hairtail meat [9], only fourteen different kinds of amino acids were detected. In this study, up to twenty nine different kinds of amino acids were detected. This implicates that the enzymatic hydrolysis under ultra-high pressure condition can hydrolyze hairtail meat more sufficiently and the amino acids were destructed less during the enzymatic hydrolyzing process. Therefore, a lot more amino acids can be detected in the products from enzymatic hydrolysis under ultra-high pressure condition.

Conclusions
1) The ultra-high pressure enzymatic hydrolysis process can completely eliminate the influence of spoilage bacteria, so that the enzymatic hydrolysis process can be simply controlled, and finally the solid fish meat can be hydrolyzed into water-soluble liquid amino acids.
2) Using a single neutral protease, enzymatic hydrolysis rate of hairtail protein under ultra-high pressure conditions can reach up to 83%.
3) The enzymatic hydrolysis of the fish meat under the ultra-high pressure environment is very sufficient, and the various amino acids in the enzymatic hydrolysis process are not destroyed, and the enzymatic hydrolysate detects up to 29 kinds of free amino acids.