Factors Affecting the Hydrolysis Process by Protease Enzyme

partially destroyed. Glutamine and asparagine dissociate into glutamic acid, aspartic acid and NH4 + and most vitamins are destroyed (Nguyen Duc Luong, 2004) .

1.6.1.2 Alkaline hydrolysis

Amino acids can also be obtained by hydrolysis with NaOH, heated for many hours. The products obtained are mostly amino acids but are racemized, reducing nutritional value, creating lysineolanine, reducing lysine in the composition, and amino acids such as cysteine, serine and treonine are destroyed. Therefore, this method is rarely used in the food industry (Nguyen Duc Luong, 2004).

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1.6.1.3 Enzymatic hydrolysis

To obtain amino acid preparations, hydrolysis by protease enzymes is widely applied in many different scientific fields, especially more suitable for applications in food and pharmaceuticals because the use of enzymes has many advantages. The biggest advantage of this method is the control of the level of hydrolysis, the mild processing conditions, the obtained product has a high protein content without destroying the amino acid composition (Nguyen Duc Luong, 2004).

1.6.2 Factors affecting the hydrolysis process by protease enzyme

1.6.2.1 Effect of temperature

Due to the nature of enzymes being proteins, the rate of reaction increases with increasing temperature within a certain temperature range without affecting the structure of the enzyme. Each enzyme has a different optimum temperature. This difference depends on the origin of the enzymes, the conditions or the difference in sensitivity to temperature of the protein-enzyme molecule. The suitable temperature of many enzymes is about 40 - 50 o C, enzymes of plant and microbial origin have a higher suitable operating temperature (Pham Thi Tran Chau and Phan Tuan Nghia, 2006).

The optimal operating temperature of an enzyme is not fixed but varies depending on the substrate and hydrolysis time.

1.6.2.2 Effect of pH

Enzyme activity depends on the pH of the environment, because pH affects the ionization state of R groups in amino acids in the enzyme molecule and the substrate. The appropriate pH for enzyme activity is when the enzyme and substrate combine easily. Each enzyme is most active only at a certain pH range, called the optimal pH. The optimal pH of each enzyme is not fixed, it can change depending on the nature, concentration of the substrate and temperature (Pham Thi Tran Chau and Phan Tuan Nghia, 2006).

1.6.2.3 Effect of enzyme concentration and substrate concentration

Enzyme concentration greatly affects enzyme reaction. Under conditions of excess substrate, the reaction rate depends linearly on enzyme concentration.

When the substrate concentration is low, the level of contact between the enzyme and the substrate decreases, so the enzyme reaction decreases. The reaction rate reaches its maximum when all the enzyme is combined with the substrate (Pham Thi Tran Chau and Phan Tuan Nghia, 2006).

1.6.2.4 Effect of hydrolysis time

Hydrolysis time affects the efficiency of the hydrolysis process. The longer the hydrolysis time, the more conditions the protease has to hydrolyze the substrate more thoroughly. However, if the hydrolysis time is too long, it will lead to microorganisms working to produce more secondary products such as NH 3 , H 2 S, CO 2 , indole... On the contrary, if the hydrolysis time is shortened, the protein hydrolysis is not thorough, the hydrolysis efficiency is poor, causing waste of raw materials. Usually, in the beginning, the speed of the hydrolysis process occurs rapidly, later on, due to the decrease in substrate concentration while the concentration of the product increases, at the same time, due to the decrease in enzyme stability over time, the speed of the hydrolysis reaction gradually decreases (Tran Minh Tam, 1998).

1.6.2.5 Effect of contact area

In the process of hydrolysis, the important factor that promotes the hydrolysis process is the contact area. To create better conditions for enzyme hydrolysis is to increase

the ability to contact between enzyme and substrate, to do so, the substrate size must be reduced before hydrolysis (Nguyen Trong Can and Do Minh Phung, 1990).

In addition to the above factors, the hydrolysis process is also affected by other factors such as activators and inhibitors, metal anions, and the nature of the enzyme.

In summary, the hydrolysis process is affected by many factors, so depending on the hydrolyzed material, these factors must be optimized to achieve high hydrolysis efficiency.

1.6.3 Application of hydrolyzed protein

The product of protein hydrolysis is a hydrolyzed protein solution rich in low molecular weight peptides, especially di- and tri-peptides with few free amino acids, which is considered to have high nutritional value (Bhaskar et al., 2007).

Protease enzymes break down muscle protein into soluble and insoluble components. The insoluble components contain undesirable substances and fats that can be used in animal feed. The soluble components contain hydrolyzed proteins and low fat content. Hydrolyzed proteins can be used to enhance food flavor, supplement functional foods, or simply as nutritional additives to low-protein foods (Kurozawa et al., 2008). Fish protein hydrolysates (FPHs) have been successfully tested for incorporation into various food systems such as cereal products, fish and meat products, desserts and crackers, etc. (Kristinsson and Rasco, 2000).

Protein hydrolysates play an important role in animal nutrition, especially in enhancing immune resistance (Pasupuleti et al., 2010). FPHs have been used in aquaculture to enhance the growth and survival of fish (Kotzamanis, 2007). The results showed that peptides in protein hydrolysates affected the growth performance and immunity of seabass larvae. In another study, Nguyen Thi My Huong et al. (2012), conducted a feeding trial to evaluate the effects of tuna head hydrolysate supplementation on the survival and growth of shrimp ( Penaeus vannamei) and reported that tuna head hydrolysate significantly improved both growth and survival of shrimp.

FPHs can be used as a nitrogen source to sustain microbial growth. Ghorbel et al. (2005) used defatted protein hydrolysate from herring ( Sardinella aurita ) as a nitrogen source for extracellular lipase production by the filamentous fungus Rhizopus oryzae and reported higher lipase yields than when no protein hydrolysate was added.

Protein hydrolysates are also used in vaccine production and as plant growth regulators to increase commercial crop yields as well as for weed control in factories (Pasupuleti et al., 2010).

In recent years, there have been many studies on hydrolyzed animal proteins because of the outstanding properties that hydrolyzed proteins bring. Among them are studies on hydrolyzed pork, chicken and fish proteins (Soares et al., 2000; Vercruysse et al., 2005; Bhaskar et al., 2007; Kurozawa et al., 2008; Kurozawa et al., 2009; Rossi et al., 2009; Schmidt and Salas-Mellado, 2009; Silva et al., 2009; Zhang et al., 2010; Di Bernardini et al., 2011; Xijuan et al., 2012; Ha et al., 2013). Compared with beef, pork and chicken, crocodile meat contains less fat and more protein (Hoffman et al., 2000; Beilken et al., 2007). Therefore, crocodile meat is a more suitable raw material for hydrolysis to obtain protein hydrolysate.

Protein hydrolysis can generally be performed in much the same way as other meat proteins. The important thing to research is to find the right enzyme and reaction conditions as outlined above.

After hydrolysis, the liquid can be spray-dried into powder. This process will bring the advantages that the powder product is easy to store and has a longer shelf life, the powder product is easily added to various food products and is suitable for mixing with other ingredients. It can be said that spray-drying the liquid product into powder will increase the application potential of the hydrolyzed product.

1.7 Overview of protease enzymes

Enzyme preparations are produced more and more and are used in most fields such as: food processing, agriculture, animal husbandry, medicine...

1.7.1 General introduction to protease enzymes

Protease enzymes catalyze the hydrolysis of peptide bonds (-CO-NH) n in protein and polypeptide molecules to the final product of amino acids. In addition, many proteases are also capable of hydrolyzing ester bonds and transporting amino acids.

Protease is essential for living organisms, very diverse in function from the cellular, organ to body level, so it is widely distributed in many objects from microorganisms (bacteria, fungi and viruses) to plants (papaya, pineapple...) and animals (liver, calf stomach...). Compared with animal and plant proteases, microbial proteases have different characteristics. First of all, the microbial protease system is a very complex system consisting of many enzymes that are very similar in structure, mass and molecular shape, so it is very difficult to separate them in the form of homogeneous crystals.

Also because it is a complex of many different enzymes, microbial proteases often have broad specificity for thorough and diverse hydrolysis products.

1.7.2 Classification of proteases

Proteases (peptidases) belong to subclass 4 of class 3 (EC3.4). Proteases are divided into two types: endopeptidases and exopeptidases.

Based on the site of action on the polypeptide chain, exopeptidases are divided into two types:

+ Aminopeptidase: catalyzes the hydrolysis of peptide bonds at the free N-terminus of the polypeptide chain to release an amino acid, a dipeptide or a tripeptide.

+ Carboxypeptidase: catalyzes the hydrolysis of peptide bonds at the C-terminus of the polypeptide chain and releases an amino acid or a dipeptide.

Based on the kinetics of the catalytic mechanism, endopeptidases are divided into four groups:

+ Serine proteinase: are proteinases containing the –OH group of the serine radical in the active site and play a particularly important role in the catalytic activity of enzymes. This group includes two subgroups: chymotrypsin and subtilisin. The chymotrypsin group includes animal enzymes such as chymotrypsin, trypsin, elastase. The subtilisin group includes two types of bacterial enzymes such as Carlsberg subtilisin,

subtilisin BPN. Serine proteinases are typically highly active in the alkaline region and exhibit relatively broad substrate specificity.

+ Cysteine proteinase: proteinases containing a –SH group in the active site. Cysteine proteinases include plant proteinases such as papain, bromelin, some animal proteins and parasitic proteinases. Cysteine proteinases usually operate in the neutral pH range and have broad substrate specificity.

+ Aspartic proteinase: most aspartic proteinases belong to the pepsin group. The pepsin group includes digestive enzymes such as pepsin, chymosin, cathepsin, renin. Aspartic proteinases contain a carboxyl group in the active site and are often active at neutral pH.

+ Metallo proteinase: is a group of proteinases found in bacteria, molds as well as higher microorganisms. Metallo proteinases usually operate in the neutral pH region and their activity is greatly reduced under the effect of EDTA.

In addition, based on pH, protease activity is classified into three groups: Acid protease: pH 2-4; Neutral protease: pH 7-8; Alkaline protease: pH 9-11 (Nguyen Trong Can et al., 1998).

1.7.3 Catalytic mechanism of protease enzyme

Although the active sites of microbial proteases are different, they all catalyze peptide bond hydrolysis reactions according to the same general mechanism as follows:

E + S → ES → ES' + P 1 → E + P 2

In there:

E: Enzyme S: Substrate

ES: Enzyme-substrate complex

ES': Enzyme-Acylation Substrate Intermediate Complex (Acyl Enzyme)

P 1 : First product of the reaction chain (newly formed free amino group)

` P 2 : Second product of the reaction chain (newly formed free carboxyl group ) (According to Nguyen Van Mui, 2012).

1.7.4 Protease enzyme hydrolysis method

Protein

xt Polypeptide

H 2 O H 2 O

Peptides

H 2 O

Amino acid

Figure 1.5 : Intermediate products of protein hydrolysis

Protein hydrolysis is the process of breaking down protein chains at peptide bonds into intermediate products such as polypeptides, peptides and final products such as amino acid molecules (Bhaskar et al., 2007; McCarthy et al., 2013).

Currently, protein hydrolysis can be carried out by NaOH or by protease enzyme system. However, in the food industry, protein hydrolysis by protease enzyme system is often used due to high hydrolysis efficiency and better quality of hydrolysate compared to hydrolysis by NaOH. Alkaline hydrolysis is not used because it creates racemization phenomenon which reduces the nutritional value of amino acids.

1.8 Research status on protein hydrolysis using enzymes

1.8.1 In the world

In recent years, there have been many studies on hydrolyzed animal proteins because of the outstanding properties that hydrolyzed proteins bring. Including studies on hydrolyzed proteins of pork, chicken, fish, mussels, squid, by-products of seafood processing industry...

According to Zhuang et al. (2009), the optimization of antioxidant activity by response surface methodology in jellyfish collagen hydrolysate ( Rhopilema esculentum) was studied . To optimize the conditions for jellyfish collagen hydrolysis with the highest hydroxyl radical scavenging activity, collagen extracted from jellyfish was hydrolyzed with Trypsin enzyme. The optimal conditions obtained from the experiment were pH 7.75, temperature 48.77 o C and enzyme to substrate ratio 3.50%. Analysis of variance in response surface methodology showed that pH and enzyme to substrate ratio were important factors that significantly affected the process (p < 0.05 and p < 0.01, respectively). The hydrolysate of jellyfish collagen was separated by HPLC and three components (HF-1 > 3000 Da, 1000

HF-2 < 3000 Da and HF-3 < 1000 Da) were obtained. HF-2 had the highest hydroxyl radical scavenging activity with the highest yield compared to the other two components.

According to Fang et al. (2012), the optimization of antioxidant hydrolysate production from squid muscle protein was studied using response surface methodology. Squid muscle protein, extracted from squid products ( Ommastrephes bartrami ) was hydrolyzed by five protease enzymes (pepsin, trypsin, papain, alcalase and flavourzyme). DPPH free radical scavenging ability was used to evaluate the antioxidant activity of the hydrolysates. The results showed that the hydrolysates obtained with papain had the highest antioxidant activity. Response surface methodology was used to optimize the hydrolysis process conditions, including enzyme/substrate ratio (1 - 2%), reaction temperature (45 - 55 o C) and hydrolysis time (30 - 60 min). The optimum conditions obtained were as follows: enzyme/substrate ratio 1.74%, temperature 51 o C, time 46 min, accordingly, the DPPH free radical scavenging activity was 74.25%.

According to Kurozawa et al. (2008), they studied the optimization of protein hydrolysis from chicken meat using 2.4L alcalase enzyme and evaluated the effects of temperature (43 - 77 o C), enzyme / substrate ratio (0.8 - 4.2%) and pH (7.16 - 8.84) on the degree of hydrolysis and protein recovery. The enzymatic hydrolysis process was optimized for the maximum degree of protein hydrolysis and recovery. The results showed that the optimal conditions were determined as follows: temperature 52.5 o C, enzyme / substrate ratio 4.2% and pH 8. Under these conditions, the degree of hydrolysis obtained was 31% and the protein recovery was 91%. SDS-PAGE electrophoresis at 12% separating gel concentration and 4% collecting gel concentration showed that some protein bonds in the meat were cleaved after hydrolysis and the content of glutamic acid, aspartic acid, lysine and leucine was high.

Silva et al. (2009) studied the optimization of mussel meat protein hydrolysis using Protamex enzyme. The relationship of temperature (46 - 64 o C), enzyme / substrate ratio (0.48 - 5.52%) and pH (6.7 - 8.3) to the degree of hydrolysis was determined. Response surface methodology showed that the optimal conditions for enzymatic hydrolysis of mussel meat were pH 6.85; temperature 51 o C and enzyme / substrate ratio 4.5%. Under these conditions, the degree of hydrolysis obtained was 26.5% and the protein recovery rate was 65%.

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