By-Products Technology

Module 2. Skim milk and its by-products

Lesson 15
ENZYMATIC PRODUCTION OF CASEIN HYDROLYSATES

15.1 Introduction

Proteolytic enzymes are used to produce casein hydroysates. These enzymes have ability to hydrolyse proteins to peptides and amino acids. The chain length of peptides formed is dependent upon the extent of hydrolysis, condition of hydrolysis, type, concentration and activity of enzyme, and type of protein to be hydrolysed. Proteolytic enzymes could be obtained from plant (Papain, Ficin, Bromelain), animal (Pepsin, Trypsin, Rennin) microorganisms (Neutrase, Alcalase, Esperase, Pronase etc.).

15.2 Classification of Enzymes

On the basis of their specificity of action, enzymes can be classified into:

15.2.1 Endopeptidases

These enzymes have preference for certain side chains on amino acids adjoining peptide bond and are usually divided into three groups:

i. Pepsin type of protease is characterized by a preference for amino acids with free carboxyl groups.

ii. Trypsin types are characterized by a preference for amino acids with basic group.

iii. Chymotrypsin types are characterized by a preference for amino acids with aromatic or bulky chains.

Papainase type enzymes (Papain, chymopapain, ficin, bromelain) are endopeptidases, but are difficult to classify under this scheme.

15.2.2 Exopeptidases

These are mostly microbial enzymes that split terminal amino acids from one end of the chain by hydrolysis of peptide bond. They can be further divided in two groups:

i. Carboxy (exo) peptidases that act on the terminus of chain carrying free carboxyl group .

ii. Amino (exo) peptidases that start from the other end i.e. the terminus of chain carrying free amino group.

Choice of the protease for protein hydrolysis depends mainly on its specificity and also on its pH optimum, heat stability and the presence of activators or inhibitors. pH optima of pepsin, papain and pancreatin are 3.0, 7.0 and 8.0, respectively. Individual endopeptidases do not split all or even a majority of the peptide bonds in a protein system leading to the formation of bulky, hydrophobic acid chains, which give bitter taste. Exopeptidases are reported to hydrolyse carboxyl and amino terminal amino acids of such peptides, thus eliminating the bitter taste.

15.3 Enzymatic Production of Casein Hydrolysates

Hydrolysis of casein has been mostly carried out as a single stage process (where enzyme is added once during the hydrolysis period) and a two-stage process (where two or more enzymes are added at subsequent intervals of hydrolysis). The general steps in the manufacture of protein hydrolysates include suspension of protein in appropriate amount of water, incubation with enzyme at appropriate pH and temperature in the presence of preservative like chloroform, pasteurize to inactivate enzymes, removal of insoluble material by centrifugation, concentration, drying and packing in moisture proof container.

15.3.1 Single stage hydrolysis

In single-stage hydrolysis process, the common enzymes used have been pepsin, papain, trypsin, pancreatin and microbial proteinases, though other proteolytic enzymes: Corolase PS, Corolase L10, Maxatase LS 400,000, Novozym 257 etc. can also be used. In one method, three enzymes - pancreatin, proteinase of Aspergillus oryzae and Lactobacillus helveticus were added to 10% casein solution and incubated at 50°C for 24 h. Inactivation of proteolytic enzymes was done by heating at 85°C for 15 min. The solution was cooled to 5°C and centrifuged at 1,200 x g for 20 min to remove the precipitate. About 91% of the total casein nitrogen remained in the supernatant.

15.3.2 Two stage hydrolysis

In a two stage process, combination of different enzymes have been used. Some of important two stage casein hydrolysis processes suggested are as follow:

• Clegg et al. (1974) described a process for the production of enzymatic hydrolysate of casein. Twelve Kg of casein was suspended in 220 l water at pH 6.2-6.3 and digested with papain at 40°C for 8 h, and then with a pig kidney homogenate (serving as a source of exopeptidases) at pH 7.8-8.0 for 24 h. The hydrolysate was then passed through a Russel separator to remove insoluble material followed by pasteurizatin at 83-85°C for 3-5 min (Fig. 15.1). After concentration up to 20-23% total solids in a Wiegand evaporator and holding at 60^oC, the product was spray dried. The process was completed in 60 h. However, this was not an efficient process for production of debittered hydrolysate, as it is costly, time consuming and results in production of significant amount of free amino acids. First endopeptidase papain (4%) and then an exopeptidase leucine aminopeptidase (0.015%) are used to obtain casein hydrolysates with reduced bitterness and high (46%) free amino acids.

• Cogan et al. (1981) suggested use of papain, pepsin and Rhozyme enzymes.
  

• Khanna and Gupta (1996) reported enzymatic production of casein hydrolysates in a short hydrolysis period of 8 h. Sodium caseinate solution (10% TS) at pH 7.0 was observed to be optimum for hydrolysis with papain. Four percent papain in the first stage and 0.4% pancreatin in the second stage, each stage with 4 h of hydrolysis, were found to give optimum degree of hydrolysis (Fig. 15.2). However, the casein hydrolysate so produced was definitely bitter. The bitterness was removed using activated carbon treatment. Minimum 15% activated carbon treatment was necessary for debittering the casein hydrolysate, though this treatment resulted in 40.90% N loss through adsorption on activated carbon. The yield and recovery of liquid casein hydrolysate were 47.98% and 46.23%, respectively. The liquid product had 10.25% TS, 1.93% nitrogen, 1.19% ash and a low viscosity of 1.99 cP at 20°C.

• Peptic pre-digestion of protein followed by hydrolysis with pancreatin or trypsin, papain followed by leucine aminopeptidase and pancreatin followed by pepsin have also been used.

15.4 Continuous Hydrolysis of Proteins in Membrane Reactor

The development of membrane technology has led to a new concept: continuous reaction and simultaneous separation of the product from the reaction mixture (Mannheim and Cheryan, 1990). A membrane module with the appropriate pore size and physicochemical properties is incorporated into the reactor, which contains the enzyme. Feed is continuously pumped into the reaction mixture while product is continuously withdrawn as the permeate. The molecular size of the product can be controlled by proper selection of the pore size of the membrane. The enzyme is recycled and reused, thus improving enzyme utilization and overall

productivity. The main system components include a vessel coupled in a semi-closed-loop configuration to a membrane module via a recirculation pump. Provisions are made for control of temperature, pH and agitation in the reaction vessel. The casein suspension is preadjusted to the required pH and temperature (in the food vessel) and pumped into the reaction vessel at a flow rate equal to the permeate flux. The reaction mixture is continuously recycled with a pump inserted in-line between the reaction vessel and the membrane module inlet. The membrane modules may be polysulfone hollow fibers of 5,000 and 10,000 molecular weight cut-off.

Chiang et al. (1995) worked on producing casein hydrolysate continuously by hydrolysis of bovine casein with protease type XXIII (from Aspergillus oryzae) in a pilot scale formed-in-place membrane reactor. A high percentage (> 99%) of TCA-soluble nitrogen in the hydrolysate (product) was achieved after 45 min at 37°C and pH 7. The product was completely soluble over pH range 2-9. Water sorption increased 4-6.5 times at water activity of 0.35-0.95 as compared with intact casein. The immunologically active casein and immunologically active whey proteins in the product were reduced 99 and 97%, respectively.

Selected references

Thakar, P.N., Joshi, N.S., Patel, J.R 1991. Protein hydrolysates- A Review. Indian J. Dairy Sci., 44: 9.
Cogan, U., Moshe, M. and Mokady, S. 1981. Debittering and nutritional upgrading of enzymic casein hydrolysates. J. Sci. Food Agric., 32: 459-564.
Chiang, Wen Dee., Cordle, C.T. and Thomas, R.L. 1995. Casein hydrolysate produced using a formed-in-place membrane reactor. J. Food Sci., 60 (6): 349.
Clegg, K.M., Smith, G. and Walker, A.L. 1974. Production of an enzymic hydrolysate of casein on a kilogram scale. J. Food Technol., 9: 425-430.
Helbig, N.B., HO, L., Christy, G.E. and Nakai, S. 1980. Setittering of skim milk hydrolysates by adsorption for incorporation into acid beverages. J. Food Sci., 45: 331-337.
Jenny Ann John and Ghosh, B.C. 2008. Bitterness and its reduction in milk protein hydrolyzates. Indian Dairyman, 60 (1): 36-45.
Khanna, R.S. and Gupta, V.K. 1996. Process optimization for the production of buffalo milk casein hydrolysate. Indian J. Dairy Sci., 49: 386.
Kodjev, A., Ratchev, R. and Panova, V. 1974. Methods for obtaining the protein hydrolysates with improved flavour qualities. IV Int. Congr. Food Sci. Technol., Id: 3, Cited: Dairy Sci. Abstr., 37: 5411.
Mannheim, A. and Cheryan, M. 1990. Continuous hydrolysis of milk protein in a membrane reactor. J. Food Sci., 55 (2): 381-386.
Vegarud, G.E. and Langsrud, T. 1989. The level of bitterness and solubility of hydrolysates produced by controlled proteolysis of caseins. J. Dairy Res., 56: 375-379.