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‘ 13* “d “—42% k. g '63 4-51
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ENZYME DEGRADATION DURING SHEARING AND FOAM FORMATION

By

Katherine Alice Linz

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Chemical Engineering

1983

ABSTRACT

ENZYME DEGRADATION DURING SHEARING AND FOAM FORMATION

By

Katherine Alice Linz

In order to examine the mechanism of enzyme degradation
in a shear field, rennet solutions were subjected to turbu-
lent shear in a stirred tank. The results suggest that
shear caused little or no loss of activity and that the
formation of foam in the stirring process was the more
likely cause of activity loss. Addition of an antifoaming
agent (polypropylene glycol) to the tank during stirring
resulted in no detectable loss of activity. Conversely,
foaming caused by sparging (without stirring) with air or
nitrogen resulted in as much as 95% loss. It is suggested
that activity loss occurred during the process of bubble

formation.

ACKNOWLEDGMENTS

I would like to thank Dr. Donald Anderson for all

his support and guidance during my research.

ii

TABLE OF CONTENTS

List of Tables

List of Figures
Table of Nomenclature
Introduction
Experimental Methods
Apparatus

Methods and Results
Discussion
Recommendations

Appendix A: Data for
Milk-Rennet System

Appendix B: Discussion on
Couette Viscometer

List of References

iii

vi

oomc'mp

18

19

23
2n

Table

A1

A2

A3

A4

A5

A6

A7

A8

A9

A10

LIST OF TABLES

Title

 

Sparging of Rennet Solution with Air
Sparging of Rennet Solution with Air

Sparging of Rennet Solution with
Nitrogen

Sparging of Rennet Solution with
Nitrogen for 5 Minutes

Coagulation.Time of Milk as a Function
of Reciprocal Concentration of Rennet

Rennet Activity Loss During Shear in a
Stirred Tank (c=0.66%. 2500 RPM)

Rennet Activity Loss During Shear in a
Stirred Tank (c=0.66%, 2750 RPM)

Rennet Activity Loss During Shear in a
Stirred Tank (c=0.66%. 3000 RPM)

Rennet Activity Loss During Shear in a
Stirred Tank (c=o.55%. 2500 RPM)

Rennet Activity Loss During Shear in a
Stirred Tank (c=0.55%, 2750 RPM)

Rennet Activity Loss During Shear in a
Stirred Tank (c=0.55%, 3000 RPM)

Rennet Activity Loss During_Shear in a
Stirred Tank with 1.11x10 5% Poly-
propylene Glycol

Rennet Activity Loss During_Shear in a
Stirred Tank with 1.39x10 5% Poly-
propylene Glycol

Rennet Activity Loss During Shear in a

Stirred Tank with 3.56x10‘5% Poly-
propylene Glycol

iv

17

17

19

19

19

20

20

20

21

21

21

22

Figure

LIST OF FIGURES

Title

 

Viscosity of Milk-Rennet Solution as
a Function of Time During Assay
Procedure

Coagulation Time of Milk as a Function
of Reciprocal Concentration of Rennet

Schematic of Stirred Tank Used for
Shear Damage Studies

Rennet Activity Loss During Shear in
a Stirred Tank (c=0.66%)

Rennet Activity Loss During Shear in
a Stirred Tank (c=0.55%)

Rennet Activity Loss During Shear in
a Stirred Tank with Polypropylene
Glycol (.5.56x10 5% -Polypropylene
Glycol. I1.39x10:5% - Polypropylene
Glycol,.A1.11x10 5% - Polypropylene
Glycol. 9N0 Polypropylene Glycol)

10

11

12

13

1L»

15

Symbol

TABLE OF NOMENCLATURE

vi

Definition
Concentration

Initial concentration
Constant

Molar concentration

Exposure time in stirred
tank

Coagulation time

Delay time

INTRODUCTION

In 1970. Charm and Wong3 showed that the enzymes
catalase, carboxypeptidase, and rennet lost activity when
sheared in a coaxial cylinder viscometer. They attributed
the loss of activity to the shearing alone. They also found
that when shearing was stopped, rennet regained much of its
activity over a period of 100 minutes. Tirrell and Middleman8
found that urease lost activity when sheared during the
hydrolysis of urea and that some of the damage was reversible.
However. in 1979. Thomas and Dunhill7 studied catalase and
urease in a sealed couette viscometer using the same shear
rates as Charm and Wong and found no significant loss of
activity of the enzymes over extended periods of time. When
they repeated the same experiments in an open viscometer, the
enzymes lost activity not only when sheared, but also under
zero shear. In neither case was the activity loss as great
as reported by Charm and Wong. Thomas and Dunhill suggested
that enzyme degradation thought to be caused by shearing may
in fact, be a result of other factors such as:7 1) pressure,
2) heat denaturation. 3) gas-liquid surface denaturation
caused by oxidation, A) cavitation. and 5) metal contamina-
tion. In an open system, denaturation by oxidation and foam-

ing are especially important to take into consideration.

EXPERIMENTAL METHODS

The coagulation of milk by rennet is a two step process.
Milk contains micelles which are stabilized by k-casein. In
the first step the k-casein is hydrolysed. leaving the
micelles unstable. The casein micelles aggregate in the
second step to form a coagulum.6 Several studies have shown
that coagulation time varies with pH, temperature, and the

type of milk used.2’u

5

Kopelman and Cogan used the sudden increase in vis-
cosity at the onset of coagulation as an indicator of coagu-
lation time.’ This time is correlated with enzyme concentra-
tion and used as an assay. Viscosity was followed continu-
ously, using a Brookfield viscometer, by Kopelman and Cogan
and in this work. A small sample adaptor was used here.
Rennet solutions. contained 4% of a 1 M acetate buffer
(pH 5.7) to keep coagulation times in a reasonable range
and were maintained at 14°C. Reconstituted milk was made
with 16% skim milk powder in 0.01 M CaCl2 solution. The
milk was gently shaken for 15 minutes and heated to #000
in a water bath for 10 minutes prior to each assay. The
reaction was started by adding 0.3 ml of enzyme solution to
16 ml of heated milk in a test tube. The milk-enzyme solu-
tion was gently mixed and quickly added to the viscom-t-r
which was maintained at 40°C in a water bath. Th- output

from the viscometer was continuously recorded on a strip

i
Rennet was obtained from the Sigma Chemical Cczplny.

3
chart recorder. At the onset of coagulation, a sharp in-
crease in viscosity is observed. Coagulation time, tc. was
found by extending the tangent to the viscosity curve back
to the base line (see Figure 1). The intersection of the

base line and the tangent is defined as to.

5

Kopelman and Cogan found that coagulation time de-

creased with increasing rennet concentration and coagulation

times were correlated by the relationship:

tc = t0 + Kl/c

to: coagulation time

to: delay time characteristic of
the system
1: constant
: percent enzyme concentration in
enzyme solution

K
c

In this work. concentrations from 0.05% to 0.7% were assayed
and a calibration plot of data for known concentrations is

Shown in Figure 2.

APPARATUS

Stirred Tank
The stirred tank used to shear rennet solutions is
shown in Figure 3. The tank was designed following Standard

1 often used in industry: a) fluid depth

Tank Configurations
equal to the tank diameter, b) impeller diameter equal to
one third of the tank diameter. c) impeller distance from
the bottom equal to one third of the tank diameter. d) im-
peller blade width equal to one fifth of the impeller diam-
eter. The impeller, as shown in Figure 3 has six blades.
each 0.56 centimeters in length. The temperature of the
enzyme solution was maintained by circulating water at 14°C

through the tank jacket. A cover was used to prevent spil-

lage from the tank.

METHODS AND RESULTS

Shearing in Stirred‘Tank

The stirred tank was filled with 450 ml of distilled
water containing 4% of 1 M acetate buffer. Two enzyme concen-
trations were used for this test: a) 0.55% and b) 0.67%.
The enzyme solution was cooled to IAOC before stirring. For
each concentration separate tests were run at impeller
Speeds of 2500, 2750, and 3000 RPM. Samples were taken
intermittently over a 100 minute period. For each sample,
Figure 2 was used to find the apparent concentration from the
recorded coagulation time. This concentration was compared
with the initial concentration to find the percent loss of
activity for the solution. The results for the two concen-
trations are shown in Figures 4 and 5. In all cases coagu-
lation time increased with the length of time in the stirred
tank. As the impeller speed was increased. more activity
was lost at corresponding times in the tank. For example,
from Figure A it can be seen that the solution had 67% of
the initial activity left after 50 minutes in the tank at
2500 RPM. while at 3000 RPM only 32% of the activity was
left. It is interesting to note that the lower concentration
lost more activity at corresponding times in the tank and at
all impeller speeds. than the larger concentration. In all
experiments a stable foam developed almost immediately after
shearing began. There was noticeably more foam at higher

Speeds and with the larger enzyme concentration.

6

Shearingin Stirredgignkwith Antifggm

Polypropylene glycol. an antifoaming agent, was added
to a 0.55% rennet solution and sheared at 3000 RPM in the
stirred tank. Four concentrations of antifoam were tested,
taking samples over a 100 minute period. Figure 6 shows
that with an antifoam concentration of 5.56 x 10'5%. no
significant activity was lost after 70 minutes of shearing.
Very little foam developed at this antifoam concentration
and the foam collapsed within a few seconds when the impeller
was stopped to take samples. As the polypropylene glycol
concentration was decreased, more foam developed while

shearing and more activity was lost.

Foaming of Rennet Solutions Without Shea;

A sparger was used to foam rennet solutions to find the
effect of foaming alone on the activity. A 50 ml solution
with 0.55% rennet was sparged in a graduated cylinder until
21 ml of the liquid was foamed. While sparging. samples
were taken of the foam and the liquid. After sparging was
stopped. another sample was taken of the liquid including
the collapsed foam. These samples were assayed and compared
to an initial sample taken before sparging. The results.
given in Table 1. show that 50% of the initial activity was
gone from the sample taken after sparging. The samples
taken during the foaming process indicate that the foam had
a significantly higher activity than the liquid. A small
amount of brown precipitate was observed in the foam.

A second test was performed in which an enzyme solution

(c=0.55%) was sparged for a period of 15.8 minutes. Three

7
foam samples were taken while sparging. as well as a liquid
sample after sparging was stopped. As Table 2 shows. the
final solution had only 5.1% of the original activity.

The sparging experiments were repeated using nitrogen
rather than air. In the first experiment 50 ml of a 0.55%
rennet solution was sparged until 26 ml of the liquid was
foamed. Samples were taken of the liquid before sparging,
the foam and liquid while sparging, and the liquid after
sparging. Table 3 shows that. as before, the rennet was
more concentrated in the foam. The liquid after foaming
lost 40% of the initial activity. Table A shows the results
when a rennet solution was sparged for 5 minutes. The

liquid after sparging lost 70% of the initial activity.

DISCUSSION

Activity loss was found in the shearing and foaming
experiments. In both cases, the loss was related to the
amount of foam formed as well as the duration of the foam-
ing process. As found by Thomas and Dunhill7, the enzyme
proved to be more shear resistant at the higher enzyme con-
centration. When antifoam was added to the stirred tank.
the enzyme damage was reduced or eliminated.

That activity was lost in the sparging experiments
without shear. suggests that factors other than shear may
be responsible for the enzyme damage in these experiments
and in previous Work. The experimental results in Table 1
indicate that the rennet concentration in the foam was much
higher than in the liquid being sparged. This was also
shown in the stirred tank by stirring a 0.387% rennet solu-
tion at 2500 RPM for 50 minutes. Samples were taken of the
foam and liquid. ‘The rennet concentration in the foam was
0.20% while the concentration in the liquid was 0.097%.

The brown precipitate found in the foam in both the stirred
tank and the sparger experiments is further evidence that the
rennet concentrated at the surface of the liquid where foam-
ing occurred.

When an antifoaming agent, polypropylene glycol was
added to the stirred tank before shearing. there was a
noticeable decrease in foam development. There was a corre-
lation between activity loss and the amount of foam that

developed from stirring. Whether the antifoam protected the

9
enzyme by preventing the formation of foam or by some other
means is not known.

In order to check whether enzyme damage occurred at
the time of foam development or with time in the foam phase,
a 0.55% rennet solution was sheared in the stirred tank at
3000 RPM for 30 minutes. After stirring, two samples were
taken of the foam. 0ne sample was collapsed immediately
with polypropylene glycol and assayed. The other sample
was allowed to sit for two hours in an open container before
it was collapsed and assayed. The apparent rennet concen-
trations for the two samples were essentially the same
suggesting that enzyme damage occurred during the formation
of the foam. This is consistent with previous studies of
denaturation of plasma-lipoproteins in bubble or film
oxygenation of dog blood plasma.9

The sparging experiments with nitrogen were done to
determine if enzyme denaturation was caused by oxidation or
the foaming process itself. As shown in Table 4, a rennet
solution lost 70% of the initial activity when sparged with
nitrogen for 5 minutes suggesting that the foaming process
is responsible for enzyme damage.

In conclusion, this study suggests that previous reports
of damage to enzymes when subjected to shear may be incorrect
and that much. if not all. of the damage likely occurred at
forming gas-liquid interfaces independent of the presence of

shear.

VISCOSITY, CENTIPOISES

25"'

201'-

153-

ICIh-

 

 

 

 

o J
100 150 200

TIME, SECONDS

Figure 1. Viscosity of Milk-Rennet Solution as a
Function of Time During Assay Procedure

 

lO

COAGULATION TIME, MINUTES

 

 

. 1 1 1 L J
o 5 ‘ 1o 15 20 25

1/c(%‘1)

 

Figure 2. Coagulation Time of Milk as a Function of
Reciprocal Concentration of Rennet

11

 

 

 

 

 

 

 

 

    

 

F
fi 0 cooling
8.41 cm :0 water
A_A.1 -- AI,- outlet
- 8.41 cm 7cm
Tel 2.8cm |-
Impeller
coollng 2-80'“ (bottom View)
water a: /

 

 

 

inlet cooling water jacket ‘

Figure 3. Schematic of Stirred Tank Used for
Shear Damage Studies

12

0'

REMAINING ACTIVITY clc

.0 .° .° 7‘
'Q (D (O (3
I

II

.0
a:
I

2500 RPM

.0
01
I

9
1:.
I

2750 RPM

.0
co
I

3000 RPM

.0
'9
I

1.
(L1 -

0.0.111111'1 11
102030405060708090100

TIME, MINUTES

Figure A. Rennet Activity Loss During Shear in a
Stirred Tank (c=0.66%)

 

13

0

REMAINING ACTIVITY GIc

0.00.0:
CD \l a: “I C)
III

I.
1.

P
m.
I

2500 RPM

.0
a.
I

2750 RPM

9
a)
I

.1
3000 RPM

.0
n
I

P
.115
I

 

0° llllLlllLJ
' 1o 20 so 40 so so 70 so eo1oo

TIME, MINUTES

Figure 5. Rennet Activity Loss During Shear in
a Stirred Tank (c=0.55%)

 

1h

O

REMAINING ACTIVITY c/c

1.0

0.9 N

0.8 P"

OJBW-

 

0.0

Figure 6.

 

 

'1

I 11111! 1 l l
102030405060708090100

TIME, MINUTES

Rennet Activity Loss During Shear in a
Stirred Tank with Polypropylene Glycol
( .5.56x10'5% - Polypropylene Glycol,
I1.39x10 5% - Polypropylene Glycol.
1A1411x10-5% — Polypropylene Glycol,
.No Polypropylene Glycol)

15

 

Table 1

Sparging of rennet solution with air*

 

 

 

 

 

Sample % Activity
Initial llquld (c=0.55%) 100.0
Foam - while sparging 66.6
Liquid - while sparging 41 .2
Liquid - after sparging 50.0

 

 

*Initial 50 ml sample was sparged until 21 mi
became foam

 

 

 

Table 2
Sparging of rennett solution with air
Sample % Activity

Initial liquid. 100.0
Foam - 3 minutes 81 .4
Foam - 8 minutes 18.0
Foam - 15.8 minutes 5.8
Liquid - after sparging 5.1

 

 

 

 

16

 

Table 3
Sparging of rennet solution with nitrogen*

 

 

 

 

Sample % Activity
Initial Liquid (c = 0.55%) 1 00.0
Foam - while sparging 146.0
Liquid - while sparging 65.0
Liquid - after sparging 60.0

 

 

*lnitial 50 ml sample was sparged until 26 ml
became foam

 

Table 4

Sparging of rennet solution with nitrogen
for 5 minutes

 

 

 

Sample % Activity
Initial Liquid 1 00.0
Liquid - after sparging 31 .4
5 minutes

 

 

17

 

 

RECOMMENDATIONS

Further study is recommended in the following areas.
The stirred tank experiments should be repeated using
several rennet concentrations to verify the pattern found
in earlier experiments in which lower enzyme concentrations
lost more activity at corresponding times and impeller
speeds. The second recommendation is to repeat all experi-
ments using other enzymes such as urease, catalase, and
carboxypeptidase. Charm and Wong3 and Tirrel8 reported
that these enzymes lost activity in a shear field. It is

important to discover if the reported activity loss was due

to bubble formation as shown with rennet.

18

APPENDI CES

APPENDIX A

DATA FOR MILK-RENNET SYSTEM

TABLE A1
Coagulation Time of Milk as a Function of
Reciprocal Concentration of Rennet

 

c gggio ml) 1 0 0‘1 tc,minutes
007 1.44 203
.06 1.68 2.4
.05 2.01 2.8
.04 2.51 3.6
.03 3-3Li 3-9
.02 5.01 5.9
.01 10.01 9.4
.005 20.01 17.8
TABLE A2

Rennet Activity Loss During Shear in a
Stirred Tank (c=0.66%, 2500 RPM)

 

Time,mins tc,mins c 1%) % Activity
0.00 2.2 .87 100
5.00 2.3 .80 92

19.15 2.4 .74 85

33.40 2.6 .67 77

47.95 2.7 .59 68

62.70 2.8 .57 66

77-33 3-0 .49 56

92.20 3.1 .47 53
TABLE A3

Rennet Activity Loss During Shear in a
Stirred Tank (c=0.66%, 2750 RPM)

 

 

Time,min§ tc,mins c 1%) % Activity
0.00 2.3 _80 100
5.00 2.5 .71 89

19.62 2.5 .63 78
39.03 3.1 .41 55
49.15 3.6 .36 45
64.80 4.0 .30 38
80.92 5.1 .22 27
98.20 8.1 .12 15

19

TABLE A4
Rennet Activity Loss During Shear in a
Stirred Tank (c=0.66%, 3000 RPM)

 

Time,mins tc,mins c 1%) % Activity
0.00 2.3 .87 100
5.00 2.6 .63 72

19.50 3.0 .50 57

34.50 3.6 .36 41

50.20 4.2 .28 32

66.62 5.3 .20 23

84030 609 015 17
TABLE A5

Rennet Activity Loss During Shear in a
Stirred Tank (c=0.55%. 2500 RPM)

 

 

Timeymins tc,mins c 1%) % Activity
0.00 2.6 .63 100
5.00 2.7 .59 94

19.58 3'0 OLI'9 78

34.50 3.1 .47 74

49.60 3.5 .38 60

65.00 4.1 .29 47

81057 L708 023 37

98.30 5.7 .18 29
TABLE A6

Rennet Activity Loss During Shear in a
Stirred Tank (c=0.55%o 2750 RPM)

 

 

Time,mins tc,mins c 1%) % Activity
0.00 2.4 .74 100
5.00 2.5 .71 96

19.32 207 '57 77
33092 3eLI' 039 53
49.40 4.2 .29 39
66.00 5.0 .22 30
83.85 6.0 .18 24

20

TABLE A7
Rennet Activity Loss During Shear in a
Stirred Tank (0:0. 55%, 3000 RPM)

 

 

Time,mins tc,mins c 1%) % Activity
0.00 2.7 .59 100
5.00 2.2 .44 7A

19.98 .3 .27 47

36.20 6.9 .15 25

56.70 8.9 .11 18
TABLE A8

Rennet Activity Loss _During Shear in a Stirred
Tank with 1. 11x10 5% Polypropylene Glycol

 

 

Time,mins tg,mins c 1%) % Activity
0.00 3.0 .48 100
5'00 303 .40 BL}

20.80 3.9 .32 68

36.20 4.5 .25 53

53.00 5.1 .22 45

70.50 5.9 .18 38

88.92 6.9 .15 31
TABLE A9

Rennet Activity Loss _During Shear in a Stirred
Tank with 1. 39x10 5% Polypropylene Glycol

 

 

Time,mins talmins c 1%) % Activity
0.00 2.7 .57 100
5.00 2.8 .56 97

19.88 3.2 .44 76
35.12 3.6 .36 63
50.80 3.9 .31 55
67.10 4.2 .29 50
83.60 4.7 .24 43

21

TABLE A10
Rennet Activity Loss_During Shear in a Stirred
Tank with 5.56x10 5% Polypropylene Glycol

 

 

Time,mins tc,mins c 1%) % Activity
0.00 3.38 .35 100
5.00 3.38 .35 100

20.80 3.28 .36 101
36.30 3.45 .34 98
52.00 3.48 .32 96
68.30 3.45 .34 98

22

APPENDIX B

APPENDIX B

DISCUSSION ON COUETTE VISCOMETER

In a separate set of experiments a couette viscometer
was used to shear rennet solutions. Under laminar conditions.

shear stress in a couette viscometer can be defined as

follows.1
5 = u/b
s= shear stress (sec-1)
u= linear velocity of fluid
b= gap width of viscometer
Where:

u= RN

R= outer radius of viscometer
N: revolutions per second

By shearing rennet solutions at different speeds in the
couette viscometer and measuring the activity of the rennet
over time it was hoped to find a correlation between loss

of activity in the viscometer and in the stirred tank. If
such a correlation was found, the shear field in the stirred
tank could be modeled.

A rennet concentration of 0.55% was used in the couette
experiments. The solutions were sheared at 233 RPM and 417
RPM. In both cases the activity loss was minimal. No corre-
lation could be made between the shear field in the couette
viscometer and the stirred tank. When bubble formation
rather than shear was found to be the cause of activity loss
it was decided to exclude the couette viscometer data from

the main body of this report.
23

LIST OF REFERENCES

LIST OF REFERENCES

Beck, Carl Robert, Prediction of Shear Induced Enzyme
Activity Loss in Flow System, PhD Dissertation,
Michigan State University, 1979.

Castle, A.V. and Wheelock, J.V., Journal of Dairy
Research, 32, 15(1972).

Charm, S.E. and Wong, B.L., Biotechnol. Bioeng., 12,
1103(1970).

Fox, P.F., Journal of Dairy Science, 52, 8(1969).

Kopelman,I .J. and Cogan, U., Journal of Dairy Science,

2). 196(1976)-

Miyoshi, Masamitsu, Yoon, Chang-Hoon, Ibuki, Fumio, and
Kanamori, Masao, Journal Nurt. Sci. Vitaminol., 21.
309(1975)

Thomas, C. R. and Dunnill, P., Biotechnol. Bioeng., 21,
2279(1979)

Tirrel, Matthew and Middleman, Stanley, Biotechnol.
Bioeng.. _Z; 299(1975)

Zapol, Warren.M., Levy, Robert I., Kolobow, Theodor,

Spragg, Roger, and Bowman, Robert L., Current Topics in
Surgical Research, 1, 449(1969).

24