2024年4月7日发(作者：spss应用程序并行配置不正确)

1256

J.

Agric.

FoodChern.,

Vol. 27,

No.

6,

1979

Adler-Nissen

Block, R. J., Bolling,

D.,

to

the Borden

Co.,

U.S.

Patent 2 710858,

June 14, 1955.

Bray, W. E., “Clinical Laboratory Methods”, C. V. Mosby Co.,

St.

Louis, MO, 1951, p 239.

Cerbulis,

J., J.

Agric.

Food

Chem.

26, 806 (1978).

Chandler,

R.

L.,

OShaugnessy,

J.

C., Blanc,

F.

C.,

J. Water Pollut.

Control Fed.

48,

2971 (1976).

Claggett,

F. G.,

Wong,

J.,

Circular

No.

42, Salmon Wastewater

Clarification Part

11,

Fish Res. Bd.

of

Canada, Feb. 1969.

Hartman, G. H., Swanson,

A.

M.,

J. Dairy Sci.

49,

697 (1966).

Hentad,

O.,

Hvidsten, H.,

Acta

Agn’.

Scandinauica,

23,154 (1973).

Hidalgo,

J.,

Kurseman, J., Bohmen, H. V.,

J.

Dairy

Sci. 56,988

(1973).

Hopwood, A. P., Rosen,

G.

D.,

Proc. Biochem.

7,

15 (1972).

Jones,

S.

B., Kalan, E. B., Jones,

T. C.,

Hazel,

J.

F.,

J.

Agn’c.

Food

Chem.

20, 229 (1972).

Kramer,

S.

L., Waibel,

P.

E., Behrends, B.

R.,

El Kandelgy,

S.

M.,

J.

Agric.

Food

Chem.

26, 979 (1978).

Riehert,

S.

M.,

Diss. Abstr.

33,

3128-B

(1973).

Sanders, M. D.,

Znd. Eng. Chem.

6, 1151 (1948).

Spinelli, J., Koury, B.,

J.

Agric. Food Chem.

18,

284 (1970).

Van Steenberg, W., De Laval Co., Inc., Poughkeepsie,

NY,

personal

communication, 1979.

Waibel,

P.

E., Cuperlovic, M., Hurrell,

R,

F., Carpenter, K. J.,

J. Agric. Food Chem.

25, 171 (1977).

Received

for

review May

2,

1979. Accepted July 30, 1979.

Taken together, these data suggest that chemical treat-

ment of blood for reclamation of protein is feasible at the

industrial level. Industrial application of chemical coagu-

lation techniques could eliminate or diminish the need for

secondary treatment of blood wastewaters since chemical

coagulation is capable of quantitative removal of protein

in the primary step. We are currently studying other

chemical procedures of blood protein removal.

ACKNOWLEDGMENT

We are thankful to Delbert Doty for his insight and

suggestions concerning this research. The authors also

wish to thank Jack Barensfeld (A. W. Stadler, Inc.) and

Tom Dieter (Emge Packing Co.) for supplying industrial

whole blood samples. We are grateful to the Fats and

Proteins Research Foundation, Inc., Des Plaines, IL, for

bringing the blood processing problem

to our

attention and

for a grant which supported this research. This manuscript

was taken from data contained in the Master’s thesis of

A. Ratermann, submitted

to

the Department of Chemistry,

Murray State University, Aug. 1979.

LITERATURE CITED

American Public Health Association, “Standard Methods for the

Examination

of

Water and Wastewater”, 13th ed., Washington,

DC, 1971, p 244.

Determination of the Degree of Hydrolysis

of

Food Protein Hydrolysates by

Trinitrobenzenesulfonic Acid

Jens Adler-Nissen

An accurate, reproducible and generally applicable procedure for determining the degree of hydrolysis

of

food protein hydrolysates has been developed. The protein hydrolysate is dissolved/dispersed in

amino equivalents/L.

A

sample

hot

1%

sodium dodecyl sulfate to a concentration of 0.25-2.5

X

solution (0.250 mL) is mixed with 2.00 mL of 0.2125 M sodium phosphate buffer (pH 8.2) and 2.00 mL

of

0.10%

trinitrobenzenesulfonic acid, followed by incubation in the dark for 60 min at 50 “C. The

reaction is quenched by adding

4.00

mL of 0.100 N HCl, and the absorbance is read at

340

nm.

A

1.500

mM L-leucine solution

is

used

as

the standard. Transformation of the measured leucine amino equivalents

to degree of hydrolysis is carried out by means of a standard curve for each particular protein substrate.

Enzymatically hydrolyzed proteins possess functional

properties, such as low viscosity, increased whipping

ability, and high solubility, which make them advantageous

for use in many food products. Recent experiments have

indicated that

in order to obtain desirable organoleptic and

functional properties of soy protein hydrolysates, the hy-

drolysis must be carried out under strictly controlled con-

ditions to a specified (generally low) degree of hydrolysis

(DH) (Adler-Nissen, 1977; Adler-Nissen and Sejr Olsen,

1979). DH is defined as the percentage of peptide bonds

cleaved (Adler-Nissen, 1976). Therefore, a need exists for

a general method of determining DH of food protein hy-

drolysates, in particular for quality control. An obvious

method to consider for this purpose is the trinitro-

benzenesulfonic acid (TNBS) method, by which the con-

Novo Research Institute, Enzyme Applications Research

and Development, Novo Industri A/S, DK 2880 Bag-

svaord, Denmark.

0021-856 1/79/ 1427-1 256$01

.OO/O

centration of primary amino groups in the hydrolysate can

be determined.

Basically, this method is a spectrophotometric assay of

the chromophore formed by the reaction of TNBS with

primary amines (Figure

1).

The reaction takes place under

slightly alkaline conditions and is terminated by lowering

the pH. TNBS also reacts slowly with hydroxyl ions,

whereby the blank reading increases; this increase is stim-

ulated by light (Fields, 1971).

Since its introduction by Satake et al. (1960), the TNBS

method has enjoyed a widespread use for the determina-

tion of free amino groups of proteins and protein hydro-

lysates. However, the presence of insoluble proteinaceous

material in, e.g., the commercially used whipping agents

based on hydrolyzed soy protein necessitates certain mod-

ifications of the various existing procedures described in

the literature, as they seem to have been developed for

soluble materials only. Also, although

it is generally as-

sumed that a linear relationship between the color intensity

and the concentration of a-amino groups exists, we have

0

1979

American Chemical Society

Determination

of

Hydrolysis

of

Protein Hydrolysates

0-0421

NO2

'NO2

NO2

Figure

1.

Reaction

of

TNBS with

amino

groups.

observed in this study that there is a considerable differ-

ence between the different proteins with respect to the

actual value of the

(The intercept, which is mainly due to the €-amino

slope and intercept of this relationship.

is of considerable and varying magnitude.) Thus, some

groups,

way of standardizing the assay is needed. Finally, after

some initial experiments using either a version of the ori-

ginal TNBS procedure (Satake et

method described by Fields (1971),

al.,

1960)

o

a more thorough study was needed in order to obtain a

it

was concluded that

r

a more rapid

manual, accurate TNBS procedure for determining

of food protein hydrolysates in general. In particular, when

the DH

using the above-mentioned methods, a high spreading of

the results from repeated analysis on the same material

was observed. This was soon ascribed to difficulties in

dispersing the partially insoluble proteins and protein hy-

drolysates. With a view to these considerations it was

decided in advance that the following features should be

incorporated in the modified TNBS procedure.

sulfate (NaDodS04)

(1)

The sample should be dispersed in sodium dodecyl

tion of NaDodSOl in the TNBS method has been reported

rather than buffer alone. Incorpora-

by Habeeb (1966),

proteins in his samples. The use of mercaproethanol to

who used this agent for denaturing the

prevent protein aggregation is ruled out because this agent

reacts with TNBS (Kotaki et al., 1964).

thod is bicarbonate (pH 8.5). As we considered the forma-

(2)

The buffer used in most versions of the TNBS me-

tion of

decided to use another buffer system. Borate, which was

C02

during the acidification step a nuisance, it was

applied by, e.g., Fields (1971),

mendable if sugars are present

does not seem to be recom-

whereas phosphate is suitable (Burger,

(as

they may be in our case),

phate allows the choice of less alkaline pH values than

1974). Also, phos-

often used, which is advantageous

reaction increases considerably if pH is above 8.5 (Satake

in practice, as the blank

et al., 1960).

manual procedure, being neither

(3)

A

reaction time of

1

h was considered optimal for the

color intensity sensitive to a few minutes of deviation on

so

short as to make the

the reaction time, nor

the complete analysis in half a working day. Complete

so long

as

to make it difficult to run

reaction should be achieved within the reaction time to

ensure high reproducibility.

EXPERIMENTAL SECTION

(analytical grade) and NaDodSO, were obtained from

Materials.

Trinitrobenzenesulfonic acid dihydrate

Sigma. All the other analytical chemicals were from

Merck. The proteins (Kjeldahl nitrogen contents in par-

entheses) were soy protein isolate (Purina

Ralston Purina (14.1% N), casein according to Hammer-

500

E

from

sten (Merck) (14.0% N),

Bloom, alkaline extracted gelatin from Extraco, Sweden;

and gelatin (a commercial, low-

15.7% N). The enzymes used for the hydrolyses were

alcalase 6.0 FG and pancreatic trypsin Novo 6.0

from Novo Industri

S,

both

of

A/S.

The declared proteolytic activity

for

both enzyme preparations was 6 Anson units (AU)/g;

enzymes are approximately 25 AU/g for Alcalase (Novo

comparison the proteolytic activities of the crystalline

Industri 1978a) and approximately 20 AU/g for porcine

J.

Agric.

Food

Chem.,

Vol. 27,

No. 6,

1979

1257

trypsin (Novo Industri 1971).

were used in most of the experiments, were prepared ac-

The standard samples of soy protein hydrolysate, which

cording to the procedure described in the last subsection

of the experimental section.

M phosphate buffer [0.2125 M NaH2P04 is added

Reagents.

The following reagents were used: 0.2125

M Na2HP04

volumes is approximately 43:1000)],

until pH is 8.20

to

f

0.02 (the proportion of

0.2125

[TNBS

0.1

%

TNBS solution

(100 or

must be prepared immediately before use],

150 mL) covered with aluminium foil; the solution

is

dissolved

in

deionized water in a volumetric flask

1%

DodSO4.

NaDodS04, 1.500 mM leucine standard in

0.100

1%

N HCl,

Na-

reaction was carried out

TNBS

Reaction.

Unless otherwise stated, the TNBS

containing between 0.25

as

follows:

0.250

mL of a sample,

X

equiv/L,

buffer at pH 8.2. Two milliliters of

is

mixed

in

a test tube with 2.00 mL of phosphate

and 2.5

X

amino

is added and the

bath at

tubes and the water bath must be covered with aluminium

50

test

tube is shaken and placed in a water

0.10%

TNBS solution

f

1

"C for 60 min. During incubation the test

foil because the blank reaction is accelerated by exposure

to

to terminate the reaction, and the test tube is allowed to

light. After the 60 min 4.00 mL of 0.100 N HC1 is added

stand at room temperature (cooling below room tempera-

ture may cause turbidity because of the NaDodS04

ent) for

pres-

at 340 nm.

30

min before the absorbance is read against water

are carried out by replacing the sample with

The reactions on the blank and the standard solutions

SO4

spectively. The absorbances

and 1.500

1

%

NaDod-

X

M L-leucine in

1%

NaDodS04, re-

are determined as the averages of six individual determi-

of the blank and the standard

nations.

concentration

The conditions during the reaction are

=

0.10

M, TNBS concentration

as

follows: buffer

=

1.43

X

M, leucine concentration

perature

=

0.088

X

M,

pH 8.2, tem-

a pH below 3.5 will cause turbidity. The reaction can be

=

50

"C. pH after the HC1 addition is 3.7-3.9;

considered pseudo-first-order with respect to the amino

groups.

which are used more than once in the test are included:

Defiliitions and Symbols.

Only conceph and symbols

a,

units, a measure of proteolytic activity;

intercept on the

y

axis of a regression line; AU, Anson

gression line; DH, degree of hydrolysis, defined as the

b,

slope of a re-

percentage of peptide bonds cleaved, thus DH =

(h/h,)

X

substrate;

100%; E/S, enzyme-substrate ratio, based on protein

mated in kinetic experiments;

A,

absorbance;

A,,

maximum absorbance esti-

defined as the concentration in milliequivalents/

h,

hydrolysis equivalent,

tein of a-amino groups formed during hydrolysis;

g

of pro-

drolysis equivalent at complete hydrolysis to amino acids;

hbt,

hy-

hbt

vidual amino acids in

is calculated by summing up the contents of the indi-

constant;

g of protein;

A,

1

k,

first-order reaction

deviation within the groups in grouped regression analysis;

spectrophotometric wavelength;

so,

standard

sl

regression analysis;

,

standard deviation around regression line in grouped

determinations;

total standard deviation on

h

centration of Kjeldahl nitrogen multiplied by the appro-

S,

substrate concentration defined as con-

s(h),

priate factor. Sensitivity AA/Amequiv of NH2-

(change

equivalents.

in

X

A

for a given change in concentration of amino

L-'

either carried out by varying the reaction time in the

Kinetic Experiments.

The kinetic experiments were

TNBS reaction described above

or

by using the following,