Selection of a proteolytic enzyme to solubilize lean beef tissue

Selection of a proteolytic enzyme to solubilize lean beef tissue G. M. O'Meara and P. A. Munro* Department o f Chemical and Materials Engineering, University o f Auckland, Auckland, N e w Zealand

(Received 5 July 1983; revised 12 October 1983) Many proteases are available for the hydrolysis o f various protein substrates. The qualitative effect o f most experimental variables on reaction progress is known, so it is possible to devise a rational procedure for selecting the best enzyme. Reaction time and enzyme concentration shouM be chosen in the region where they have little effect on reaction progress. Substrate concentration shouM be low to avoid possible product inhibition. Each enzyme shouM be tested at its optimum pH, and at a range o f temperatures around {mainly below) the reported temperature optimum. Enzyme cost and other relevant factors shouM also be considered in the enzyme selection. Using this selection procedure Alcalase was chosen as the most appropriate enzyme for solubilizing lean beef tissue. Keywords: Proteases; meat hydrolysis; Alcalase; enzyme selection

Introduction Lean meat wastes, such as the meat left on bones and heads, may be upgraded to edible products by solubilizing them with proteolytic enzymes. 1'2 Many are now available in quantities sufficient for commercial production of protein hydrolysates, and it was necessary to select the most suitable protease or proteases. A review of the literature indicated that different workers had used widely different enzyme selection procedures. Hale a determined the enzyme concentration required to achieve 60% solubilization of fish protein in 24 h. Reaction temperature and pH for each enzyme were chosen near the optimum values suggested by the manufacturer. Cheftel et al. 4 studied solubilization of fish protein concentrate by measuring nitrogen released into the supernatant. Reaction temperature was maintained at 37°C and pH was varied around the optimum for each enzyme. Reaction time was usually 24 h and several enzyme-substrate ratios were examined. Arzu et al. s studied the release of soluble protein into the supernatant after a 5h reaction time with cotton-seed protein. Reaction temperature was 45°C for each enzyme and pH was varied within the optimum ranges suggested by the respective enzyme manufacturers. Connelly et al. 6 and Criswell et al. 7 measured the release of soluble solids to determine the extent of hydrolysis of meat protein. Reactions were conducted for 4 h at 50°C using several enzyme-substrate ratios. In the five studies mentioned the only reaction variables systematically altered were pH and enzyme-substrate ratio. Reaction time and substrate concentration were arbitrarily chosen. In four studies temperature was arbitrarily f'Lxed, and in the fifth a published optimum temperature was used for each enzyme. In this paper the development *To whom correspondence should be addressed. 0141 --0229/84/040181-05 $03.00 © 1984 Butterworth & Co. (Publishers)Ltd

of a rational enzyme selection procedure is discussed and the application of this procedure to the selection of a protease for solubilizing lean beef tissue is described. A rational e n z y m e selection procedure

The qualitative effect of the experimental variables on enzymic protein hydrolysis reactions is well known. Reaction rate is initially rapid and gradually decreases to become slow late in the reaction, l,a For comparison of enzymes the reaction time should be based on a suitable time for an industrial reaction process, and should be in the region where reaction rate is slow. A plot of reaction progress versus enzyme concentration for a fixed reaction time is similar in shape to the reaction progress versus time curve.2 For comparion of enzymes it is therefore desirable to use an enzyme concentration in the region where it has little effect on reaction progress. Substrate concentration may be chosen largely for experimental convenience, provided it is low enough for product inhibition to be insignificant. Curves of reaction progress versus pH or temperature both exhibit optima, so it is important to compare enzymes somewhere near their respective optima. Most commercial protease preparations have a broad pH range of 1.5-2.5 pH units over which they are reasonably active. The optimum pH varies between different protein substrates but is usually within a range of only 0.5-1.0 pH units. Provided the pH optima suggested by the manufacturer or in the literature for different substrates are within a narrow range, it is satis-. factory to test each enzyme at a single pH chosen from reported optima. The temperature optimum depends on reaction time in a complex manner, with longer reaction times corresponding to lower optimum temperatures. Increasing reaction temperature increases the rates of both enzyme reaction (activation energy typically 100 kJ mo1-1)

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Papers and enzyme inactivation by denaturation (activation energy typically 400kJmol-1), and may also alter the susceptibility of the substrate to proteolysis, e.g. collagen denaturation (G. M. O'Meara, unpublished work). Testing enzymes only at an optimum temperature suggested by the manufacturer or in the literature is, therefore, not satisfactory unless the accompanying reaction time and the effect of reaction time on the temperature optimum are both known. Enzyme manufacturers often quote optimum temperatues for relatively short reaction times, e.g. 10min. 9 These temperatures Will therefore be too high for reaction times of several hours, and the enzymes should also be tested at lower temperatures. Based on the above qualitative effects the following enzyme selection procedure was developed: (a) obtain a range of suitable proteases; (b) assemble data on their pH and temperature optima, with associated reaction times, from both manufacturers data and published literature; (c) choose a suitable hydrolysis parameter for experimental comparison of the proteases, e.g. solubilization of insoluble solids, or release of soluble peptides; (d) choose a reaction time based on what would be suitable industrially for the particular substrate; (e) perform several preliminary reactions with the substrate to determine the shape of the reaction progress curve, and to select enzyme and substrate concentrations which produce reasonable reaction progress; (f) using the chosen reaction time, enzyme concentration and substrate concentration, test each enzyme at its optimum pH and at several temperatures around the suggested temperature optimum; (g) select for further consideration those enzymes which produce reasonable solubilization, or extent of reaction; (h) compare these enzymes on the basis of price and any other relevant criteria.

E n z y m e selection Buffer (85ml) was placed in a 250ml conical flask, heated to the reaction temperature, and then lOg thawed meat was added and the pH adjusted. Enzyme (10mg) in 5 ml of buffer was added and the flask was placed in a shaking water bath at the required temperature. After a reaction time of 3 or 5 h, insoluble tissue was separated from the reaction liquid by vacuum filtration on Whatman No. 4 filter paper. The insoluble tissue was then washed with distilled water at 50°C and dried overnight in a forced draught oven at 105°C. Solubilization was then calculated from the relationship: DT - IT solubilization- - x 100% DT where DT is the dry weight of initial substrate solids and IT the dry weight of final insoluble substrate solids.

Results and discussion

Table 1 lists the proteases tested, together with published optimum reaction conditions. From this table an optimum pH was chosen for each protease (two pH values were used for Protease XIII because of limited and conflicting published data), and three or four temperatures were chosen around the suggested temperature optimum. Since our interest was in solubilizing meat tissue for removal from bones and heads, solubilization was chosen as a suitable parameter for comparison of the proteases. A hydrolysis time of 3 to 5 h is likely to be the longest acceptable industrially. This would allow one batch per working day. A reaction time of 3 h was therefore chosen for experiments. Preliminary experiments indicated that 1.0 mg protease/g wet meat tissue gave a reasonable solubilization in 3 h, and reaction progress curves (Figure 1) indicated that the reaction was rapid for 2 h and was slow between 3 and 5h. A low substrate concentration was

Materials and methods

I00,

Materials Thirteen protease preparations were obtained. Alcalase 1.5 M (from Bacillus licheniformis, 1.70 Anson units/g) and Neutrase 0.5 L (from Bacillus subtilis, 0.553 Anson units/g), Novo Industri A/S, Bagsvaerd, Denmark, were gifts from Chemby Marketing Ltd, Auckland. Rhozyme 41 concentrate (from Aspergillus oryzae, activity factor 5.98) and Rhozyme P53 concentrate (from Bacillus subtilis, activity factor 32.92) were gifts from Rohm and Haas N.Z. Ltd, Auckland. Bromelain (B-2252, EC 3.4.22.4), chymotrypsin (C-4129, EC3.4.21.1), flcin (F-8629, EC3.4.22.3), pancreatin (P-1750, from porcine pancreas), papain (P-3375, EC3.4.22.2), pepsin A (P-7000, EC3.4.23.1), protease V (Pronase AS, P-5005, from Streptomyces griseus EC 3.4.24.4), protease XIII (Molsin, P-2143, EC 3.4.23.6) and trypsin (T-8128, EC3.4.21.4) were purchased from Sigma Chemical Co., St Louis, USA. Beef trimmings were obtained from a local slaughterhouse. All visible fat was removed, and the lean beef was minced through 4 mm holes and stored at - 2 0 ° C . Buffers used to control pH during enzyme selection experiments were prepared according to Long 1° as follows: pH 2.5, 0.2 M HC1-KC1; pH 5.0, 0.1 M citric acid-sodium citrate; pH 7.0 and 7.5, 0.1 M KH2PO4-NaOH; pH 8.0 and 8.5, 0.1 M Tris-HC1.

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Microb. Technol., 1984, vol. 6, April

O

8C O

60

o

(,9

40

20

o' 0

I

I

I

I

I

I

2

$

4

5

Time (h)

Figure 1 Reaction progress curve for the solubilization of meat b y Alcalase 1.5 M. Reaction conditions were 50°C, pH 8.5 and 1.0 mg Alcalase 1.5 M/g wet meat tissue

Selection o f an enzyme to solubilize beef tissue: G. M. O'Meara and P. A. Munro Table 1 Suggested reaction pH and temperature f o r proteases

Enzyme

Temperature (°C)

pH

Reaction time c (h)

Alcalase

60 a 50--55 b 55 b 30--60 a (50 b) 60 a

8--10 (8.5) a 8.0 b 8.5 b 4--9 a (6.0 b) 8.0 a

0.16 1.5-2 5 (24) 24

Bromelain

6.~ a

0b --30--50 a (40 b)

Chymotrypsin Ficin

Neutrase Pancreatin

Papain

--

7.0 a

--

6.5-7.5

56 a 40 a 45 b 40 b 6 0 - - 7 0 a (65b) 6 0 - - 7 0 a (65 b) 50 b

6a 7--9 a 8.5 b 7.5 b 5--6 a (6.0 b) 6--8 a (7.0 b) 6a

--

7a

50 a 50 a (50 b) 70 a 40--55 a

5a 7.5 b 2 a (2 b) 1--4 a 1.6 a 7--8 a (7.5 b) 8.0 a 7.8--8.5 a (8 b)

--

3.5 a

60 a 50 a 50 b 60 a 50 b 40 a (40 b)

2.8 a 6--9 a 7a 5--8.5 a 6.5 b 7--9 a (7.5 b) 7--9 a

40 b 40--50 a (50 b)

Pepsin A

-

Protease V (Pronase) Protease X III

(Molsin)

Rhozyme 41 Rhozyme P53 Trypsin

0b a Optimum reaction bConditions c

13

--

Casein

16

--

Casein,

egg albumin and haemoglobin (denatured and undenatured) Denatured haemoglobin

16

-

11

Fish protein Fish protein (Fish protein) (Fish protein) Meat scrap C a s e i n , egg albumin Gelatin Denatured haemoglobin (Fish protein) Several proteins, pH 1.8 Soy proteinate (Fish protein) Soy proteinate Fish protein Trypsinogen, chymotrypsinogen Soy proteinate -Meat scrap -Soy protein (Fish protein) Proteins Denatured haemoglobin

11 11 11 11 7 13

75~

---(24) 24

0.16 -

5 24 (24) (24) 4 --

--(24) 24 (24) 24 ---

24 -4 --(24) ---

9

8 11 11 12 14 13 15 11 16

9

13

14 11 13 12 11 12 17 A

18

12 19 7

19 20 11 13 14

reference quoted reactions but not specified as optimum in experiments

c o n d i t i o n s s p e c i f i e d in t h e

used for

Reaction times

a

Reference

Denatured haemoglobin Soy protein Fish protein (Fish protein) Soy proteinate Casein, haemoglobin Denatured haemoglobin -N-AcetyI-L-tryptophanamide (Fish protein) Gelatin

-

7. 7--9 a 8a 5--8 a (6.0 b) 5.0 a

Substrate

used

chosen to reduce possible product inhibition. This also ensured that agitation during reaction, and filtration to determine the solubilization, were straightforward. Experiments were also performed at a reaction time of 5 h, to check that the chosen enzyme concentration was high enough for most of the reaction to be completed within 3 h. Table 2 presents the results of the enzyme selection experiments. Generally, most of the reaction was completed in 3 h and solubilization at 5 h was only slightly higher than that at 3 h. Similarly, the use of a slightly higher enzyme concentration would have had only a small effect on solubilization after 3 h. No results are given for pepsin A since the procedure for determining solubilization was unsatisfactory with this enzyme. Filtration rates were very low and no reproducible values for solubilization were obtained. The optimum temperatures suggested by manufacturers and published work are generally higher than those indicated in Table 2. Novo 9 using a reaction time of 0.16h (lOmin), suggested optimum temperatures of 60°C for Alcalase 1.5 M and 56°C for Neutrase 0.5 L (Table 1). Using reaction times of 3 or 5 h with meat, the optimum temperatures were 50-60°C for Alcalase 1.5 M and about 40°C for Neutrase 0.5 L (Table 2). Rohm and Haas Co. 19 do not specify a reaction time, but suggest optimum tempera-

tures of 50°C for Rhozyme 41 and 60°C for Rhozyme P53. Corresponding optimum temperatures for 3 or 5 h meat hydrolysis reactions were about 40°C for Rhozyme 41 and 50°C for Rhozyme P53 (Table 2). Presumably Rohm and Haas Co. have also used a short reaction time to determine their optimum temperatures. In general, enzyme manufacturers specify high temperature optima corresponding to short reaction times, and significantly lower temperatures should be used for reaction times of several hours. Inspection of published experimental temperatures (Table 1) also indicates that many workers have used much higher temperatures than those found optimal in this work. Fujimaki et al. !2 reacted bromelain with soy proteinate for 2 4 h at 60°C, whereas Table 2 suggests a temperature optimum (for 3 h) of 40°C. Enzyme inactivation was well advanced after 3 h at 60°C (Table 2). Hale u reacted papain with fish protein for 24h at 65°C, whereas our results suggest a temperature optimum between 40 and 50°C (for 3 - 5 h). For protease V our experiments suggest an optimum temperature just above 40°C (for 3 - 5 h), whereas Hale 11 used 50°C for 24h against fish protein, and Fujimaki et al. 12 used 70°C for 24 h against soy proteinate. Table 2 shows that seven of the enzymes tested gave good meat solubilization (greater than ~75%) at their optimum conditions (i.e. Alcalase 1.5 M, bromelain, chymo-

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Papers Table 2 Enzyme selection results

% Solubilization Enzyme

Initial reaction pH

Reaction time (h)

30°C

40°C

50°C

60°C

AIcalase 1 . 5 M

8.5

Bromelain

7

3 5 3 5

--48 62

71 77 76 78

Chymotrypsin

8

3

71

83

5 3 5 3 5 3 5 3 5 3 5 3 5 3 5

77 65 68 63 62 ----------64 59 38 34 52 61 -----

86 64 69 65 72 62 62 67 74 70 76 51 55 29 37 64 66 -51 76 80 20 21 ---

79 81 73 76 73 78 59 68 63 66 71 77 71 74 66 76 60 54 38 49 54 59 48 54 77 79 22 20 32 35

74 80 60 63 ----34 37 36 43 59 63 39 39 43 46 43 45 27 26 35 33 -23 22 32 28

Ficin

7

Neutrase 0 . 5 L

7

Pancreati n

7.5

Papain

7

Protease V

7.5

Protease X l l l

2.5 3.5

Rhozyme 41 Rhozyme

P53

7 7.5

Trypsin

7.5

No enzyme

7 8.5

3 5 3 5 3 5 3 5 3 5

Enzyme cost c (NZ $/kg) 10a 278b 8650 b 290b 7a 124b 213 b 687o b 1060 b

198gd 111 ad

1330b

a Prices f o r e n z y m e s c o m m e r c i a l l y available in bulk in New Zealand (September 1983) b Catalogue prices for research enzymes (Sigma Chemical Co., February 1983) c NZ dollars/kg. Approximate exchange rate, 1.0 £ = 2.3 SNZ dprices f o r preparations having activity factor used in this work (quoted prices were on a unit activity factor basis)

trypsin, pancreatin, papain, protease V and trypsin). These were selected for further consideration. The costs of these enzyme preparations, obtained from various sources, are listed in Table 2. Chymotrypsin, protease V and trypsin are expensive research enzymes whose high solubilization results may be partly attributed to the relatively high purity of the preparations used. They were unlikely to be obtained from commercial sources at a price comparable to that of Alcalase 1.5 M for example, and so were rejected. Pancreatin is likely to interest the meat industry since the protease can be produced on site from pancreas glands. However, the lipase activity of pancreatin would be unacceptable in meat solubilization processes, since the free fatty acids produced would lower the quality of the accompanying fat. Isolation of a purified protease from the pancreas would therefore be required. Bromelain and papain deserve further Consideration, since in most countries they are available in bulk from commercial sources at much lower prices than those quoted in Table 2. Alcalase 1.5 M is also worth further consideration, since it is available cheaply in bulk quantities. Alcalase 1.5M was finally chosen for further experimental work 1'2'21 because it had produced a higher solubilization than bromelain or papain, and because its optimum reaction conditions of 50-60°C and pH 8.5 were much less conducive to microbial growth during enzyme reaction than those of bromelain (40-50°C, pH 7.0) or papain ( 4 0 50°C, pH 7.0). Conceptual improvements can readily be made to this enzyme selection procedure, e.g. varying pH around the optimum, or varying enzyme concentration and determining

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the amount of each enzyme needed to produce a given solubilization (80% for example). However, most of such alterations to the above procedure would make it far more time consuming. Similarly, some short cuts could easily be made to the experimental selection procedure, e.g. omit the 5 h reaction time since it served only as a check. This enzyme selection procedure is presented as a conceptual framework to enable a rational choice of one enzyme when a number of alternatives are available to attack a given substrate.

Acknowledgements This research was partially supported by a grant from the Meat Industry Research Institute of New Zealand, Hamilton, which is gratefully acknowledged. Drs C. E. Devine and J. E. Swann (MIRINZ) provided useful comments during the preparation of this manuscript.

References 1 O'Meara, G. M., Munro, P. A. and Spedding, P. L. Proc. 9th Australasian Conf. Chem. Eng. Christchurch, New Zealand, 1981, pp. 511-518 2 O'Meara,G. M. and Munro, P. A. Meat ScL in press 3 Hale, M.B. FoodTechnol. 1969,23,107-110 4 Cheftel, C., Ahem, M., Wang, D. I. C. and Tannenbaum, S. R.J. Agric. FoodChem. 1971, 19, 155-161 5 Arzu, A., Mayorga, H., Gonzalez, J. and Rolz, C. J. Agric. Food Chem. 1972, 20, 805-809 6 ConneUy, J. J., Vely, V. G., Mink, W. H., Sachsel, G. F. and Litchfield, J. H. Food Technol. 1966, 20, 829-834

Selection o f an enzyme to solubilize beef tissue: G. 114.O'Meara and P. A. Munro 7 8 9 10 11 12 13

Chriswell, L. G., Litchfield, J. H., Vely, V. G. and Saehsel, G. F. Food Teehnol. 1964, 18, 1493-1497 Adler-Nissen,J., Poulsen, G. and Andersen, P. E. Ann. Nutr. Aliment. 1978, 32,205-216 Novo Industri A/S, Information Bulletin number 163-GB, Novo Industri A/S, Bagsvaerd, Denmark, 1978 Long, C. Biochemists' Handbook E. & F. N. Spon Ltd, 1961, pp. 27-43 Hale, M. B. Making t~sh Protein Concentrates by Enzymatic Hydrolysis Report NMFS SSRF-657, National Marine Fisheries Service, Seattle, USA, 1972 Fujimaki, M., Kato, H., Arai, S. and Tamaki, E. Food Technol. 1968, 22 (7), 77-81 Yamarnoto, A. in Enzymes in Food Processing 2nd ed. (Reed,

14 15 16 17 18 19 20 21

G., ed.) Academic Press, New York, 1975, pp. 124-174 Miyada,D. S. and Tappel, A. L. Food Res. 1956, 21,217-225 Hess, G. P. in The Enzymes 3rd edition (Boyer, P. D., ed.) Academic Press, New York, 1971, vol. 3, pp. 213-249 Whitaker, J. R. Food Res. 1957, 22,483-493 Cheftel, C. Ann. Technol. Agric. 1972, 21,423-433 Sodek, J. and Hoffman, T. Methods Enzymol. 1970, 19, 372-397 Rohm and Haas Co. Publication AG-374a, Rohm and Haas Co., Philadelphia, 1977 Roozen, J. P. and Pilnik, W. Process Biochem. 1973, 8 (7), 24-25 O'Meara,G. M. PhD Thesis University of Auckland, 1983

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