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thesis entitled \
A METHODOWI STUDY OF HETHIOHINB'
DETERMINATION BI MICROBIOHBICAL
ASSAY ‘

presented by
Mary Mills

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of the requirements for

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ajor professor

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A WHODOLOGY STUDY OF

METHIONINE DEIBRIVIDM ION BY MICROBIOLOGICAL ASSAY

BY
Nbry'yglls

A THESIS
Submitted to the School of Graduate Studies of‘Michigan
State College of.Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIEN

Department of Foods and Nutrition
School of.Home Economics

1951

"(g-€17.35”?

ACKNOWLEDGMENT

The writer wishes to express her sincere appreciation to
Dr. Margaret.A. Ohlson for her expert guidance and valuable
suggestions, and to Miss Ruth Ingalls for her interest,
constant encouragement, and willing instruction in the

laboratory without which this study could not have been

undertaken.

TABLE OF CONTENTS

Page
DHRODUCIION.................................. 1
REVIEWOFTFE LI‘I‘EMTURE.. 5
MEREJEI‘IPAL PROCEDURE...” ..... 13
RESUIES AND DISCUSSION........................ 19
SUMMRYAND CONCIUSIOI‘JS....................... 32
REFERENCES CITED.. 35

APPE\DHOOOO00.000.00.000...OOOOOOOOOOOOOOOOOO M

TABLES

Number Title Page
I. Double Strength Basal Medium for Streptococcus
Faecalis _R__ and Leuconostoc Mesenteroides
w”........................................ 16
II. A Comparison of Methionine Values Determined
by the Growth of Streptococcus Faecalis _R_
After Hydrolysis with 2N and 4N HCl........... . 20
III. A Comparison of Average Methinonine Values from
Four Analyses Determined by the Growth of
Streptococcus Faecalis 3 Following Hydrolysis
with 2N and 4N H01 Before and After Four
Weeks Storage of the Acid Hydrolysates. . . . . . . . 22
IV. Values Found in the Literature for Egg, Milk,
Pork and Cheese............................... 23
V. Recoveries of Methionine for Samples Measured
by the Growth of Streptococcus Faecalis 3.
After mtdrolysis with 2N and 4N HCl........... 25
VI. Comparison of Methionine Values in a Composite
of Foods Obtained by Analysis and by Calcu-
lat ion from Analyses of the Individual
Ingredients Determined by the Growth of
Streptococcus Faecalis fi...................... 26
VII. A Comparison of Methionine Values Obtained by
Analysis with Streptococcus Faecalis R and
Leuconostoc Mesenteroides P-GO. . . . . . . . . . . . . . . . 28

VIII. Methionine Consumed Per Day...................... 29

1‘

INI'RODUCI‘ ION

DTI‘RODUOI‘ ION

The critical nature of methionine as one of the essential amino acids
in both animal and human nutrition has been recognized for some time.
Recent work using the rat as the experimental animal has indicated the
possibility "that this amino acid does not act in the general maintenance
of body tissues, but in the synthesis of functional proteins and important
metabolites” (Brush, et al, 1947). Experimentation has demonstrated the
role of this amino acid in increasing the nitrogen retention in dogs and
rats (Allison, et al, 1947, Brush, et a1, 1947, and Cox, et al, 1947),
while similar work with man has indicated that the addition of’Dl-methionine
to a low-protein diet does not spare body protein (Johnson, et al, 1947,
'Cox, et al, 1947). An evaluation of more recent findings accumulated at
the University of Wisconsin indicated that the limiting amino acid in the
diets of four adult women, two of whom.exhibited a negative nitrogen
balance, was methionine, although other factors in addition to the low
methionine intake were considered in interpreting these data (Futrill, et
al, 1950).

As a result of the increasing interest in the role of this amino acid
in animal nutrition, the importance of a reliable method of analysis can
be realized. Chemical methods have been utilized to a large extent, but
the sensitivity of this type of analysis has been questioned due to the
possible interference of other sulfur-containing substances in the
reactions involved (Kuikea, et al, 1945, Stokes, et al, 1945, Block and
Bolling, 1951). In the past few years microbiological determinations have

been uddely utilized due to their highly specific and unusually sensitive

-2-

nature. The advantages and limitations of this method have been
adequately summarized by Snell (1945, 1946), and Dunn (1949). The use
of microbiological methods has made it unnecessary to remove extraneous
fat and carbohydrate before analysis as the samples can be hydrolyzed
without previous treatment and the amino acid content determined
(Schweigert, 1948). This use of the intact materials for analysis has
greatly facilitated the accumulation of data concerning the amino acid
content of food materials, and the knowledge of amino acid utilization
and excretion.

Data on the amino acid content of certain animal feeds have
indicated that it is possible to obtain reliable values by two methods
- (l) by an analysis of the complete feed, or (2) by analysis of the
individual ingredients and a summation of the results (Schweigert, 1947,
1948). The advantages in the application of the former method of
analysis to the determination of the amino acid content of a composite
of foods can readily be seen. The time saved in the number of necessary
analyses as well as the practicality of being able to analyze complex
food dishes without separation into individual constituents is easily
recognizable. However, certain data have been presented which suggest
that sugar-protein complexes which cannot be utilized by the micro-
organisms are formed when the acid hydrolysates of the food mixtures
are autoclaved at high temperatures, and, therefore, yield an inaccurate
picture of the amino acid content of the composite. Humin formation
and the products of the maillard browning reaction are two pathways which

have been suggested as producing amino acid loss.

-3-

The majority of the work done concerning the formation of this
sugar-protein complex has involved the lowered biological value of the
(protein food, due, perhaps, to the increased resistance of this complex
to enzyme attack (McCollum.and Davis, 1917, Stevens. and McGinnis, 1947,
Han, et al, 1948, Bank, at al, 1948, 1949, McInroy, et a1, 1949, Davis,
et a1, 1949, Evans and Butts, 1949, Friedman. and Kline, 1950, Lowry
and Thiessen, 1950, Griswold, 1951). Experimentation also has indicated
that the addition of reducing sugars (Lyman, et al, 1946a, Patton and
Hill, 1948) or starch (Lyman, et al, 1946a) prior to hydrolysis has
resulted in lowered methionine values as determined by microbiological
assay.

It has been the purpose of this study to determine the reliability
of methionine values obtained by analysis of a composite of foods by
comparing the values obtained by this method with those obtained by
calculation from the determination of the methionine content of the
individual foodstuffs. In addition, the effect of two strengths of acid
used in hydrolysis as well as the effect of storage on the methionine

content of the acid hydrolysates have been determined.

REVIEW OF THE LITERA'I'LME

REVIEW OF THE LITERATURE

The formation of the black, acid-insoluble humin of a protein
hydrolysate was shown by Gortner and his co-workers (Burr and Gortner,
1924, Gortner and Helm, 1917, 1920, Gortner and Blish, 1915, Holm.and
Gortner, 1920a, 1920b), to be due to the condensation of tryptophan
‘with an aldehyde. The soluble humin was found to be derived from
tyrosine. Due in part to the presence of this substance, the acid
hydrolysates of complex food materials which contain these amino acids
and carbohydrates with free aldehyde groups exhibit a darkened color
and the presence of a black precipitate. Kuiken, et a1, 1945, demon-
strated that the loss of tryptophan and tyrosine ,as a result of this
reaction was significant, and that there was also some loss of valine,
leucine and isoleucine as a result of humin formation although they did
not consider the loss great enough to affect seriously the usefulness of
determinations of these amino acids in protein or natural foodstuffs.

Lyman, et al, 1946a, suggested that one of the most important
problems related to the determination by microbiological assay of the
methionine content of foodstuffs containing relatively large amounts of
carbohydrate was to make certain that the hydrolysis of the material was
accomplished without undue loss of methionine as a result of reactions
such as humin formation. Their data showed that a small but measurable
loss of methionine did occur when casein was hydrolyzed in the presence
of sucrose, arabinose and starch. Hydrolysis with hydriodic acid was
shown to reduce the amount of humin formation, but was unsatisfactory for

microbiological work as demethylation resulted.

- 5 -

In 1912, Maillard pointed out that solutions containing amino acids
and reducing sugars turned brown upon heating, and in 1947, Hill and
Patton made use of the microbiological assay technique to gain further
insight into the maillard reaction. Their experimental results
indicated that good growth could be obtained by autoclaving a casein
hydrolysate in the presence of a reducing sugar, but that higher values
could be produced by autoclaving the two substances separately and then
combining aseptically, or by the use of sucrose in place of glucose in
the assay medium. They, therefore, suggested that optimal growth may
seldom be reached in microbiological assay due to the effects of the
Maillard reaction and pointed out that this would not be discovered
since both standard and unknown series would be autoclaved alike. It
was later suggested that partial inactivation of free amino acids and
amino vitamins resulting from glucose-heat treatment came apparently
from combination of the reactive groups with aldoses, their aldehyde
degradation products, or most prob bly, polymers of the latter (Patton,
et al, 1948a). In 1948, Hill and Patton demonstrated that both
L-lysine and L-methionine upon heating in the presence of glucose under-
went similar destruction as determined by microbiological assay. They
also indicated that the inactivation of nutrients could be minimized by
using sucrose instead of glucose in the medium. Their results suggested
that the browning reaction is promoted by alkalinity. As this work
involved the reaction of glucose with the free amino acid and it was of
interest to note the effect with the intact protein, these same workers
hydrolyzed casein with a free glucose solution and the amount of the nine

essential amino acids was determined in a treated and untreated sample.

- 7 -

It was found that only lysine, arginine and tryptOphan were inactivated
to an appreciable extent by the glucose-heat treatment (Patton, et al,
1948a). In a study to determine the effect of browning on the essential
amino acid content of soy bean globulin, Hill, Patton and Foreman,
1948b, demonstrated that, similar to the previous work with casein,

the amino acids which were attacked by browning an intact protein were
chiefly those containing functional nitrogen groups not involved in
peptide linkages. They found lysine to be the most susceptible to
attack followed by arginine, tryptophan and histidine. There was no
conclusive evidence that any of the other essential amino acids were
altered significantly. The work of Riesen, et al, 1947, indicated that
the amdno acids liberated by acid hydrolysis from.soy bean oil meal with
the exception of lysine, arginine and tryptophan were unaffected by heat
treatment. As soybeans contain aldoses, these results could quite
possibly be explained on the basis of the protein—glucose-heat reaction
observed by Patton, et al, 1948b.

By autoclaving a mixture of soybean protein and sucrose, Evans and
Butts, 1949, found that over 40 percent of the diamino acids, lysine and
arginine, were destroyed due apparently to the reaction of the free amino
groups with sucrose. Protein bound cystine was also partially destroyed.
They found no significant loss of methionine after autoclaving with
sucrose when the protein was determined microbiologically following
acid hydrolysis, but they did observe a significant loss when the
methionine was analyzed following enzymatic hydrolysis. Due to the
resistance of this complex to enzyme attack the decreased biological
value of these substances is not surprising. In 1950, Friedman and

Kline determined the reduced nutritive value of protein hydrolysate

(0

p3 leis dried with glucose for the growth of the rat as well as for h:
growth of the miereoigfli r. Both experiments were in agreement that

the MOSt significant changes occurred i1: the a no acids, bistidine
threonine, lysine, tryoto;han and Eh€1W§ldl ine, and that the other amino
acids were affected, but less appr r‘iab 1y. Ticy found an 18 percent loss
of metl ionine in the protein hydrolysate~gluccse dried senile when
determinations were made by microbiological assay. They pointed out

the relatively small alte' tio ns in the amino acid content of the sonrle
as measured by microbiological assay in c m-iris on with very in crtant
changes in the nutritive value for the rat.

Graham, {su and McGinnis,1949, sugg.sted the possible importance
of water in the prevention of amino acid destruction by browning when
determinations were made by microbiological assay. heir data indicated
that the destruction of methionine brought about by autoclaving in the
presence of glucose was significant in the absence of an excessive amount
of water, but that autoclaving methionine in an eight percent glucose
solution failed to bring about any marked destruction.

The preceeding presentation of data regarding the various results
wiich have been obtained due to the reaction of protein and carbohydrates
under stated conditions suggest possible difficulties which may be
encountered in the analysis of foods or combinations of foods which
contain both of these substances. Futrill, et al, 1951, reported data
in which the amino acid values of a composite of foods were obtained by
the microbiological analysis of the com; osite and by analyses of the
individua al foods comprising the composite. By the latter method, those
materials which were essentially protein or carbohydrate in na ture could

be analyzed separately and the comp sition of the composite computed from

- g _

these individual analyses. All of the foods were weighed for analysis
directly or for incorporation into the composite. The cereal mixture
was further prepared for analysis by drying. The meat, eggs and cheese
were dried and the fat was extracted. The milk sample, food mixture,
which consisted of fruits, vegetables and mixtures of foods, and the
composite were blended vithout further treatment and weighed for hydro-
lysis. Their results indicated that the determination of methionine

was subject to considerably higher losses in the presence of reducible
carbohydrate than the other essential amino acids with the exception of
lysine and tryptophan. They suggested the use of the factor 1.5 for the
correction of methionine values obtained from.analysis of a composite of
foods. The fact that the eggs, cheese and meat were fat extracted and
the composite was not must be taken into consideration in the evaluation
and use of this correction factor.

Various investigators have reported results concerning the relia-
bility of values obtained with different strengths of acid and varying
times of hydrolysis. In 1944, Schweigert, et al, found that autoclaving
at 15 pounds pressure for 5-10 hours with 2N H01 or refluxing for 24
hours with either 2N or 4N HCl or 5N'H2804 were satisfactory procedures
for the hydrolytic liberation of valine. In the followirg year,
Schweigert, et al, found that autoclaving 5-10 hours with either EN
32804 or 2N HCl was sufficient for the maximum liberation of leucine,
isoleucine and valine, and used a five hour autoclaving period with 2N
HCl for all subsequent assays. homahan and Snell, 1944, found that there

was little variation in the values for arginine and valine obtained from
casein after six hours hydrolysis whether two percent or 10 percent HCl was

used. Riesen, et al, 1946, found that there was no destruction of

-10..

mthionine vitl prolongel hydrolysis and they preferre alO hour period
with EN'HCl at 15 pounds pressure for tota net? ionine libe1ation.
Greenhut, et al, 1943, prepared hydrolySates by using 2N HCl for five hours
at 15 pounds pressure as they found no higher values for methionine, lysine
or threonine with an increase in either the strength of acid or the time
of hydrolysis. Stokes, et al, 1945, in their uniform assay for the
ten essential amino acids, hydrolyzed samples with 10 percent HCl for
5-30 hours and found hat five hours was sufficient to yield optimal
liberation of the amino acids present. Hydrolysis w th 2N HCl for five
hours at 15 pounds pressure has been the procedure used in this
laboratory (Ingalls, et al, 1950).

Data concerning the effect of storage upon the intact foodstuff
have been presented by investigators (Ingalls, et al, 1950, Evans, et al,
1949a, 1949b, 19490), but no information t:as fou nd in the literature
regarding the effect of storage upon the content of the acid hydrolysates.
As it is adv tejeous ur der certain circurs ta ces to repeat analyses, it
is of importance to know if the com; osition of the h ydrolysate remains

constant during the refr 5e rftion period. In order to obtain inferna-
tion of this kind for methionine, determinations were made following a
four week storage period of the acid hydrolysates.

An evaluation of the work that has been published concerning the
stability of methionine, as the free amino acid and as a breakdown
product of protein, to heat and to acid is necess ry in the a,3p1icati n
of the m101obiolo.:ical assay technique to the analysis of foods. Although
a variety of em per nents have been conducted which are relative to the
study presented here, data from analyses involving a uniform treatment

for a composite of foods and for the individual constituents have not

- 11 -

been published. As protein materials are readily subject to change and
alteration, any process other than those absolutely necessary might be
expected to affect the composition of the material analyzed. Therefore,
the samples in this study have been given the same minimum.amount of

treatment in preparation for analysis.

WEBMNFAL PROCEDURE

EXPERIMENTAL PROCEDURE

A day's diet was selected for analysis which followed the same
general pattern as that used for the metabolism study at Michigan
State College. (Table IX) In order to determine the reliability of
methionine values obtained from a composite of foods, the content of
this amino acid in the day's diet was obtained by two methods - by an
analysis of the complete food mixture, and by calculation from.the
analyses of the individual foods. The stability during storage of
the methionine in the acid hydrolysates was tested by a determination
of the samples before and after a four week refrigeration period. .A
comparison was also made of the methionine liberated by hydrolysis

with 2N’and 4N hydrochloric acid. Streptococcus faecalis §_was used as

 

the test organism. For the purpose of checking the growth response or
this organism in the presence of the products of the browning reaction,

one analysis was made using Leuconostoc mgsenteroides P-GO. It has been

 

demonstrated that this organism.needs the products of the browning
reaction for growth (Maatman, 1949).

The feeds were relegated into the following six categories for
analysis; (1) bread and cake; (2) fruits and vegetables; (3) eggs;
(4) milk; (5) meat, cheese and butter; (6) the composite of all the
foods in the day's diet. The samples were weighed, thoroughly blended
in a Waring Blender, made to volume with distilled water and placed in
glass bottles of adequate size for one analysis. These slurries were

preserved by freezing prior to hydrolysis.

.13 3-._....~ _. . o 3.123 1.. ..1 1 -., - ., 3.1,: .1 r. .. 1 qr “”1... n -d f’w
T-k) SUI“:L" L.1~5 U- lsJ‘v1~&b‘-’lo§J~vk‘~./‘AJ.U (”bibs - NIT bulk 114' .- ..‘,-U unit-- L31.

-- I . 1' '- L“« ' . v- I‘ 0- ‘1‘ a. v'-- '. Mr era-n1 -, :"
liberation of metaionine from tn: 100 s. DUZLLCutb sanpies mere

"sighed into Erlenmeyer flasks (Table X) and 100 mill il itsrs of the

of DL-u LfiCR’Aén slut' on co2M lhng 25 milligi‘ams of crystall ne
methiorine were added to th3 duplicate flash for each sanjle in order

that the percent recovery u ld be deterwinei. The flasks were plu33ed
pith rlass tool and autoclaved for five hours at 15 pounds pressure
(Stokes, et al, 1915, Schwei3ert, et al, 1944). then cool, the hydro-
lysates were filtered, made to volume with distilled water and stored
in stogpere -db ottles in th e refrigerator. One analysis was made
ivfiedifituly, and a SW} or nd dets"n1ra+i 1398 made one month later to
ascertain the effect of stars gs on the methionine content of the acid
hydrolysates.

The assay met ‘.od us ed mas essentially that of S. aozlich and Baumsnn,

191” The compo isition of the medium for Streptococcus faecali s R and

 

Leuconostoc mescnteroides P~€O was reported by Schweigert, et al, 1919,

 

in which the amino acids we‘e re 1eCed by FlonetrC1ted pe eptone, ch tine,
tyrosine and tryptOphan (Lyman, et al, 19460). Glucose was used as the
fermentable carbohydrate as previous. MC 1: in this la‘ar tory as well

as results reported by m_atwn1, 1919, had indi ed the t t}.e best

growth was not obtained with St1e33tococcus faecalis R when sucrose was

 

substituted in the medi m. Eastman (1949) also found that s cros could

not be substituted for glucose as the fermentibl e carlolydrs te for

CD

. Certain modifications in both the

Louccnastoc assenter031‘es P— 6

—-.-.

 

- 15 -

method and the medium were utilized as routinely follo1ed in this

laborat (Ingslls et al, 1950). In addition to t1 18 changes previously
reported, the concentrations of the following components of the basal
medium mere incroescd - H20 2-trea ted peptone, L-tryptophan, L—tyrosine,
L-oystine, folic acid and biotin. The composition of the medium as

used is recorded in Table I.

The organismsr 'e re maintain3d on stab cultures of yeast extract-
dsxtrose~agar. Transfers were made weekly, incubated at 57°C for 48
hours and held in the refrigerator. The inoculum for the assay tubes
was prepared by trans1er of the or3anisn desired from the stab culture
to five milliliters of the conplete double strength basal medium for that
Organism (Table I), diluted with an equal volume of distilled water.

The inoculum was incubated at 37°C for 21 hours, centrifuged, and the

} J.
(I)
D

of 0.9 percent sterile saline solution. One drop of th. sp: -nsion W

 

 

 

For analysis of the ar; wl .., the desired basal radius was prepared
omitting the nothionire. The pH was adju ted to 3.6~8.8 us; d 1'“ 7
TTIH as an srt3rnal indifie ““ f r c“";}tocwoevs fwonelis E; and to 8.9w
7.0 using b1 orthymol blue as tne external ind ioator for 11.; u oihstoc
mesontoroiios P~CC. One milliliter was added to each of the a‘"'"
tu1ns. T1? ‘1“:‘ 3 "are alluted to an "gproxc‘ite cozpentr1+ion, and

the pH 'djuste ed as indicated above. Duplica1e tlib9¢ at threg csnggn-

r 4" 3 ” I I -»- sh ~r~ 1 n.‘ . ‘ ' 3“ .‘ 1- ,, w : -‘-‘ "1 v‘ -
r313ors were prepared 1or oacn quwlc. Graded amwua1s of no 1‘9‘1:0

(9'

(l~‘ni“~“l) were adds? to a series of tubes in oiler to atttblisd

tatdird 01“?“ The 24 tutos used for tlfi is purpose consisted

[,3

1:1

of triplicate tubes for the first six dilutions and duplicate

supernatant liquid discarded. The otlls were resusperded in 20 milli Hit rs

- 15 -

Table I

DOUBLE SThENCTH BASAL MEDIUM

Glucose

Peptone, H202-treated
Adenine sulfate
Uracil ‘

Guanine hydrochloride

Xanthine

LJTryptophane 10
LATyrosine 20
L-Cystine 20
Riboflavin

Thiamin hydrochloride
Calc ium DL-pantothenat e
Nicotinic acid
Pyridoxine hydrochloride
Folic acid
Biotin
Para amino benzoic acid
Salts Cl

mgso417H20

NaCl

F6304. 7H20

111113040 711120

N

Sodium citrate
KZHPO4

For Streptococcus faccalis E

 

Sodium.acetate

Salts A2
HIE-P04
K2HPO4

For Leuconostoc mesenteroides P-60

($10938
0000

FOR

ONm-NNHHHOOOEONNEONIb
O O O.
OOOOOOOOOOOOOOOOO

001
00
010

4.0

0.5

 

Methionine 2
Water up to 10

iHenderson, L.M. and E.E. Snell, 194s.
“Snell, E.E. and F.M. Strong, 1939.

0.0
0.0

STREPTOCOCCUS FAECALIS R AND LEUCCNOSTOC MESEI‘H‘EROIDES P-60

grams
"

milligrams

03333333333

mi rograms

grams

0'

nulligrams
milliliters

tubes for the three hi3hest dilutions (‘3 chmei3 art, et al, lfiié). The
voluIne in each tube res made to two milliliters with distilled water,
the racks of tubes covered vith Several lagers of tovelinr and auto—
claved for 10 minut 3 st 15 *O“nds ores}; 1. ";;L 133 tflktn to
prevent srcesrive sterilization as it VcS four d that the excessive
nhirh resulted produced an erratic grow1h r::s:cnse of the
test C139nism. This otservrtion is in agreement vith the findings of
Feir, at al, 1915, and Rsbihowit? and Snell, 1947. A or cooling,

tea and incubot d in a water bath for 72 hours

The lactic acid produced during the vrc1uh period vas measured by
titration Viih 0.0?N s13ium Iyurcliie usin° th3tol blue as the internal

{3
U

indicator for Sirsgtccoccus fnwvelis and bromilyrol blue as the internal

 

ind’1t*cr for Ierconsstoc mescnterc1i1 A stream 0 air was introduced

 

simultaneous ly into each t be vith the alkali to llEU‘e uri orm color
formation.
The stands: d curve v9s onlstrroted by plotting the milliliters of

0.05N sodium hydroxide used in titration against the n.

O
H
O
C 1
LJ
611
O
"5

stonderd DL-methionizi9. The amount of 13thionine in tie s:«1*le3 9:;
determined by int ryolstion from this standfru curv:3 Tjgicz.1l

standard curVos for methionine using Streotocoocus faecslis and

_.

 

Leuconostoc nosentoroides are shown in Fig :'res I and II in the appendix.

 

* The 0.05N sodium.hy‘roxidc res ste.isid:33l using a standurd solution

of potassium acid phtholote.

RESULTS MID DISCUSSION

RESULTS AND DISCUSSION

.A comparison of methionine values determined by Streptococcus

 

faecalis 3 after hydrolysis with 2N and 4N hydrochloric acid before
and after four weeks storage of the acid hydrolysates is recorded

in Table II. Four successive analyses were made in the case of each
variable.

The greatest variation within these groups of four occurred in
samples 2, 3, 5, and 6. Sample 2 contained a mixture of fruits and
vegetables low in methionine content. As a result it was necessary to
use very concentrated aliquots of the hydrolysates for analysis and
proportionately large volumes of base were necessary for adjustment of
the pH. The resulting increase in salt concentration may have affected
the consistency of results (Dunn, et al, 1944). Sample 3 contained
only egg which was neither dried nor fat extnacted. Sample 5 contained
pork, bacon, cheese and butter. Sample 6 contained all the foods in
the day's diet. As the foods in all cases were analyzed as they were
prepared for consumption without further treatment, the variation might
be attributed to the difficulty in obtaining a uniform sample due to the
rather high fat concentration. The exceptionally low value obtained
with sample 5, analysis III, indicates the definite possibility of a
sampling error. There might also be the possibility of erratic growth
of the organism.in the presence of high fat concentration. It would be
of interest to analyze the same samples following fat extraction and
compare the results obtained.

The values derived from analyses following hydrolysis with 2N and

4N hydrochloric acid and from analyses made before and after a four

T able II

A COMPARISON OF MEFHIONINE VEIUES

DETERMINED BY THE GROWTH OF‘STREPTOCOCCUS FEECALIS R

AITER HYDROLYSIS WITH 2N and 4N'HCl

 

Before storage

 

 

 

 

 

 

 

 

 

Analytical
Sample 1 2N H01 4N HCl
12 II III Iv v v: VII VIII
No. msfem Ins/em msfem Ina/em Ins/em Ins/em me am msism
1. 1.07 1.08 1.09 1.11 1.07 1.06 1.08 1.13
2. .07 .05 .05 .06 .07 . .06 .05 .06
3. 3.08 2.93 3.12 3.34 3.13 3.09 3.36 3.38
4. .67 .67 .70 .68 .70 .63 .64 .65
5. 4.42 4.57 3.81 4.17 4.00 4.29 4.17 4.37
6. 1.13 1.10 1.13 1.10 1.05 1.05 1.09 1.07
After 4 weeks storage
1. 1.09 1.05 1.083 1.11 1.06 1.07 1.03 1.07
2. .06 .06 .06 .06 .06 .06 .06 .05
3. 3.01 2.94 3.20 3.37 3.04 3.22 3.25 3.11
4. .68 .63 .68 .67 .65 .64 .60 .66
5. 4.29 4.43 3.94 4.40 4.25 4.53 4.38 4.17
6. 1.10 1.03 1.13 1.15 1.12 1.08 1.04 1.08
1. The composition of the six samples is recorded infrableIX.
2. The Roman numerals indicate the successive analyses.
3. The third hydrolysate for analytical sample 1. was lost; therefore,

the average value obtained from analyses I, II, and IV after

storage was used for calculation.

- 91 -

week storage period of the acid hydrolysates exhibit no more variation
than was observed between determinations of identically treated
hydrolysates. It may be concluded, therefore, that the two strengths
of acid used in hydrolysis and the four week period of refrigeration
of the acid hydrolysates does not effect the methionine content of
the sample analyzed under the conditions of this experiment. The
average values obtained from the replicate analyses and recorded in
Table III clarify and emphasize these conclusions.
A compilation of methionine values published in the literature for

~~ milk, pork and cheese are recorded in Table IV. The values obtained
for milk in this experiment agree quite well with those of Hodson and
Krueger who used a similar method of analysis. The values found for egg
are lower than the majority of those quoted in the literature. This
could be attributed to a variety of factors. Csonka, et a1, 1947, 1950

think that the cystine and methionine content of e 5 cannot be considered

constant and may depend upon the hen's dietary protein. Their values

for the methi nine content of the entire egg varied from 2.8—3.8mg/gm
(1947) as determined by the colorimetric method of McCarthy and Sullivan
with corresponding variation in the quantity and type of protein in the
diet of the hen. Evans, et a1, 1949, found that the period of storage
affected the methionine content of the egg. as the eggs supplied by the
collate and used in this study were undoubtedly storage eggs the variation
could be attributed at least in part to this fact. Another factor which
must be considered in an evaluation of this kind is the type of treat-
ment imposed upon the food sample. All the values that were found in

the literature for eggs were obtained from the dried and fat extracted

material. Neither process was used in this erperiment l procedure.

A COMPARISON OF AVERAGE METHION

Table III

SE VaLUES FROM.FOUR ANALYSES
DETERIJ'IIED BY THE GROETH OF STREITOCOCCUS FAECALIS R

FOLLOWING HYDROLXSIS WITH 2N and 4N HCL

BEFORE AND.AFEER FOUR WEEKS STORAGE OF'THE ACID HYDROLYSATES

 

 

 

 

 

Analytical 2N HCl 4N'HCl
Sample 1
Before .After Before After
storage storage storage storage
No. Irefem nefem mefem Ins/en
1. 1.09 1.08 p 1.09 1.06
2. .06 .06 .06 .06
3. 3.12 3.13 3.24 3.16
4. .68 .67 .66 .64
5. 4.23 4.27 4.21, 4.33
6. 1.12 1.10 1.07 1.08
1

. The composition of the six samples is recorded in Table X.

VALUES FOUND IN THE LITERATURE FOR EGG, MILK, PCRK AND CHEESE

Table IV

 

 

Investigator Egg Milk Pork Cheese
msfem Ins/em Ike/em Ins/em
Lyman and Kniken, 1949 4.5* 1.0* 5.0* 6.3*
Evans, et al, 1949c 4.3:‘3.8* - - -
3.7#*
Csonka, et a1, 1947 2.8-3.8; - - -
Edwards, et al, 1946 4.8 - - -
Csonka and Danton, 1946 4.1, 4.3 - - -
Mitchell and Block, 1946 4.1 - - -
Dunn, 1947 4.0,* 3.8* - - -
Hess, 1949 6.2 - - -
Stokes, 1945 - 0.87*' - -
Hodson and Kruegar, 1946 - 0.60*
(0.5-0.8) - -
Lyman, et al, 1946b - - 5,o* -
Riesen, et a1, 1946 - - 5.6* -
Schweigert, et a1, 1949 - - 5.8* -
Bank, et a1, 1948 - - 3.8-3.9* -
Bank, et a1, 1949 - - 4.2* -
Horn, et a1, 1946 - 0.89*' 3.5* -
Baumgarten, et a1, 1946 - 0.93n - -
Block, 1946 — 1.12" - -
Block and Bolling, 1943 - 0.92 - -
Williamson, 1944 - 0.99 - -
Beach, 1943 - - 605;,6-9 _

 

‘EicrOBiological methods used.

lVariation in values correlated

entire egg, shell included, was analyzed.
#The eggs were stored nine months before this analysis was made.

'Values on the basis of ash-moisture free material.
"Values for dried skim milk.

with variation in the diet of the hen. The

- 24 -

It is impossible to make a direct comparison of the meat, cheese, butter
values found in this study with those reported in the literature as
methionine values for bacon and butter have not been published. It would
appear from an evaluation of the pork and cheese figures that the results
obtained in this experiment are within the expected range. 0

The percent recovery was determined for each analysis by the addition
of a known amount of the free amino acid and the results are recorded in
Table V. .All of the analyses for samples 1, 3, 4, 5, 6, with three
exceptions fall within a 10 percent range and all of the recoveries fall
within a 15 percent range. The recoveries for sample 2 which contains
the fruits and vegetable mixture are lower and less consistent. Futrill,
et a1, 1951, reported similar findings for a sample containing a come
parable mixture of foodstuffs. They also obtained 10w recoveries with
food samples containing high concentrations of carbohydrates. Similar
results were not found under the conditions of this experiment as may
be observed by the recoveries for samples 1 and 6.

A.comparison of the methionine values obtained by analysis of the
composite of foods and by the individual determination of samples 1-5
inclusive are recorded in.Table VI. The similarity between the results
obtained by both methods is apparent. The values for analysis III and
III' are the only ones which exhibit any appreciable variation, and in
these determinations the higher value was obtained from the analysis of
the composite. An explanation for this variation lies in the exception-
ally low value found for the meat, cheese, butter sample as previously
indicated. 4

During the course of this study a question was raised concerning

the possible stimulation of growth of Streptococcus faecalis by the

Table V

RECOVERIES OF METHIONHQ‘E FOR SW13

MEASURE) BY m GROWTH OF S'I‘REPI‘OCOCCUS FAECALIS R

AITER HYDROLYSIS WITH 2N AND 4N HCl

 

 

 

 

 

 

 

 

 

Analytical Before gorge
Sample 1
2N H01 4N H01
12 II III Iv v VI VII VIII
No. % 7% 95 7; 94: 95W ‘35 93
1. 104.0 92.0 102.2 99.5 108.8 97.4 95.7 102.1
2. 86.4 78.7 78.6 86.5 87.5 81.4 84.9 98.2
3. 104.4 94.0 97.9 98.2 85.1 100.8 99.2 109.5
4. 100.9 103.1 106.0 102.8 104.9 98.2 105.8 95.0
5. 94.3 92.3 103.0 99.9 99.5 97.4 98.6 108.5
6. 98.1 106.4 102.0 107.9 110.3 101.9 91.8 102.3
After 4 weeks storage
1. 112.0 102.3 3 94.3 106.6 94.9 114.0 92.7
2. 79.9 90.2 90.1 81.1 77.3 96.2 96.4 81.7
3. 107.6 100.2 105.0 104.2 96.1 103.2 101.8 93.8
4. 102.4 102.8 99.8 106.6 94.8 101.0 100.6 96.1
5. 97.7 93.3 91.4 93.5 99.5 104.0 96.3 - 101.0
6. 105.3 98.6 109.3 101.0 108.8 105.2 91.5 107.3

 

1‘ The composition of the six samples is recorded in Table X.
2° The Roman numerals indicate the successive analyses.

3.
The third hydrolysate for analytical sample 1 was lost; therefore,
an analysis could not be made after storage.

Table VI

COZTPARISON OF LCE‘I‘HIONDIE VALUES DJ A. COMJOSITE OF FOODS

OBTAINED BY ANALYS IS AND BY CALCUIA'I‘ I ON FROM IUL'LYSFS

OF THE DIDNDDUAL HIGl-‘JEDIEI‘ITS

Hydrolysis with 2N'HCl

DETERJ‘JIED BY THE GROWTH OF STREI’I‘OCOCCUS RECALIS R

 

 

 

 

 

 

 

 

 

 

 

 

Analytical
Samples 12 I' II II' III III' Iv IV'
Determined
No. refer: HIE/QR msfem mafam Ins/am mefam Ins/Em mefém
6. 1.13 1.10 1.10 1.03 1.13 1.13 1.10 1.15
10-50 1.09 1007 1010 1.07 1.01 1003 1.07 1010
inclusive
Hydrolysis with 4N HCl
V V” VI VI' VII VII' VII 'VIII'
'" -... Ins/5m me/e’m 312:7 am lug/ET? "YE/Em ref 51 I’d/75111 fifen
6. 1005 1.12 1.05 1.08 1009 1.04 1.07 1.08
10-5. 1.04 1.05 1.06 1.10 1.05 1.06 1.09 1.05
inclusive
1

' The composition of the six samples is recorded in.Table X.

‘ The Roman numerals indicate the successive analyses.

breahdoxn pr edit-+8 of the mteinwcarbohydr te reaction thich right
mask the otixr :ise lower res lts obtrired v.ith a mixture of Protein
and carbohydrrte flod'tiffs Therefore, one analysis was made in

rrhich the metiionine content was determined by the growth of IsIC onostcc

 

mosertero dos. Values were obtained from the same hydrolysates as were

 

used for Streptococcus faecalis 3, The results are recorded in Table VII.

 

The similarity between the values obtained with a single determination

would indi01te the relic ability of the results found with “l“fln+rnonnis

 

As it is of interest to determine the amount of variation which
truld be found if the methionine values were calculated as the grams of
methionine consumed per day, these values are recorded in Table VIII.
The variation is very slight with the exception of analyses III and III'
as would be expected from an evaluation of the results in Table VI. It
could be concluded, therefore, that under the conditions of this eszri-
ment the values obtained by analysis of a composite of foods contain
a day's list and by the calculation from the analyses of the individual
:edients composing the day's diet would yield comparable results.

The diffe erence in the conclusions derived from this experiment
and those reported by Futrill, et a1, 1951, cannot be fully exclained.
They found lower results from the analysis of the composite of foods
and s1 g3 sted the use of the factor 1. 5 in the calculation of th
methionine content of the day's diet if this method of anal,sis were
used. One possible explanation which must be considered is the variation
in the treatment of samples. Their cereal mixture was dried before
analysis. The eggs, cheese, and meat were dried and fat extracted whereas

the composite of foods was analyzed directly without further treatment.

Table VII
A COIPARISOIJ OF TEI‘HICNIIE VALUES
OB‘I‘ADTED BY Isl>fiiYS IS ".TITH
STREI’I‘OCOCCUS FIIECHIIS R AND

LEUCONOSTOC msnmaomrs 13--eol

 

 

Analytical S. faecalis R L. mesenteroides P-60
Sample
No. H‘s/Tom Ins/em
1. 1.11 1.07
2. .06 .04
3. 3.37 3.33
4. .67 .63
5. 4.40 4.70
6. 1.15 1.12

 

5;" Only one analysis was made with Leuconcostgg mesenteroides P-60.
‘ The composition of the six samples is recorded in Table X .

 

Table VIII

harmonma comm-ED PER 13.in

Samples hydrolyzed with 2N’HCl

 

 

 

 

 

 

 

 

 

 

 

 

 

Analytical Before storage
samples F
determined I° II III IV Average
No. 8111de emfday eflday emfday era/day
6. 1.41 1.38 1.41 1.38 1.40
l.-5. 1.36 1.3.7 1026 1034 1033
inclusive
After storage
6. 1.38 1.29 1.41 1.44 1.38
1.-5. 1.34 1.34 1.28 1.38 1054:
inclusive
Samples hydrolyzed with 4N'HC1
Analytical Before storage
samples
determined 'V 'VI VII 'VIII Average
No. emfday sin/day six/day sm/dw eta/day
6. 1.31 1.31 1.36 1.34 1.33
10-5. 1.31 1.32 1.31 .1037 1.33
inclusive
Eater storage
6. 1.40 1.35 1.30 1.35 1.35
10-5. 1.32 1.37 1.33 1.31 1033
inclusive

 

.MCA

1 Calculated from the values recorded in Table II.

3 The composition of the six samples is recorded in.Table X.

The Roman nunerals indicate the successive analyses.

"he samples in this study were all analyzed directly without fat

' 1
‘ d

gvtracticn or drjing of any of the fonds. A Lh;ugh a different

 

orS'nism - lea: hosts? ciircvcrum 8pil - has 1341 in the WiSCUPSin
study, it She‘s douttful that the conflictin; result: could be

attributed solely to this variation.

summary AND CONCLUS IOBB

Stir.» v :‘uTD SCALE-Ix, \o

The netlinnir.e CCZlT ent Cf a day's dist mas obtained by microbio-

y- ‘-,‘-z-- .‘w ‘-. .-./~. r-‘I: ‘ " -‘ "l 1.
lo"ica1 as 52‘ usinb Streptococcas f uc-iis a as thc test org nism. duo

 

procedures were used. Int1-.e fi_s , the entire day's diet rms eirtined

into 3 con csite and the c+1;cn'_re c ntent deterfiined by one anal sis.

In the secenl procedtre the day's dirt res divided into the follo'.inv
grec;s cf fctdsth’e: (1) brrad and cereal; (2) fruits and vegetables;

(3) egg; (4) milk; (5) meet, cheese and butter. The indiVidual groups

*«J
r\
('1
{.4
Q:
"3
Q;
(+-
.J
T)
F3
C
-+
b
f—o
f:
k 0
22’»
(U
C
n
:1
4-
C5
c+
O
’ ‘9
Ct
.54
a)
9‘
91
Q
04
DJ
('8
Cf

of foods were anv
calculatcd from these individual analyses. In both proced ass, analyses
were made after hydrolysis with EN end 4N hydrochlo oric acid in order to
datcrcire the effects of these t .o stren ngths of acid Itpon the liberatiun
of methionine frcw the sample during hydrolysis. In all cases, L na7.yses
were made before and after a four week storfge period of the acid
hydrolysates in order to dc+ermine the stability of methionine in acid
olution during a r frigeretien period.

No azprcciable v riation V88 obserVed either between the v ipes

+s

obtained from analyses site“ hydrolysis with the two strengths 0
hydroctleric acid or b tween the values obtained by analysts of the
acid tydr*7*‘eius before and after a four week refrigeration period.

Conlerable results were cbtaiz ed for tie meLhionine content of
the day's diet following either of the two procedures explaired above.
It may be concluded, therefore, that under the conditie-s of thi

Lie int? nt si.wi r sults may be obtained by analysis of a composite

(‘L

of foods or by calculeti n from the analyses of the individual

ingredients. Two distinct advantages may be seen in the use of the
former procedure. A.great deal of time can be saved due to the reduced
number of required analyses, and in addition, it is unnecessary to

separate complex food dishes into individual constituents before

analysis.

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REMJCES C ITED

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H‘
{"3

- 35 -

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- 37 -

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-38-

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-39..

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- 4o -

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- 41 -

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{L
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Snell, E. E. 1946 Microbiological methods in amino acid analysis.

Ann. N. Y. Acad. Sci. 42: 161.

Snell, E. E., and F. M. Strong 1939 A microbiological assay for
riboflavin. Ind. Eng. Chem., Anal. Ed., 11: 346.

Stevens, J. M., and J. McGinnis 1947 The effect of autoclaving lysine in
the presence of carbohydrate on its utilization by the chick. J. Biol.
Chem., 121: 431.

Stokes, J. L., and M. Gunness 1945 Microbiological methods for the

determination of amino acids I. Aspartic acid and serine. J. Biol.

Chem., 157: 651.

Stokes, J. L., h. Gunness, I. M. Dwyer and M. C. Casweil 1945
Microbiological methods for the determination of amino acids II.
A Uniform assay for the ten essential amino acids. J. Biol. Chem.,
_lgg: 35.

Williamson, M. B. 1944 Amino acid composition of human milk proteins.

J. Biol. Chem., 156: 47.

APPENDIX

v

. _ _ . A
.wJ‘I Aufis-U.‘

.g. Q-
'- r

g

:Killiliters of C

6.0

5.0

4.0

3.0

2.0

1.0

 

 

 

 

2 3 4 5 6 7 8 9 10

micrograms of DL-Methionine

Titration'Values Obtained from

Leuconostoc Mesenteroides P-60 for Known

 

Concentrations of DLPMethionine

Table IX

DAYS IZIIU FROM WHICH FOOD SJEIES WEE DERIVED

Breakfast

 

Grapefruit Juice _
Scrambled Eggs Bread, Butter
Milk

Lunch

Sandwich - Cheese, Bacon, Lettuce
Celery Bread, Butter
Baked Apple
Milk

Dinner

Roast Pork
Baked Potato Butter
Beets
Apricots on White Cake
Milk

Table X

DIVISION OF MENU I’I‘EIB

IITI‘O FOOD GROUPS FOR A-LQLYS IS

 

 

 

 

 

 

 

' Weight of
Analytical Food items Weight Total sample
sample included volume hydrolyzed
No . gm m1 gm
1. Bread 60 500 40
Cake
2. Grapefruit juice 40 500 35-40
Lettuce 6
Celery 10
Baked apple 40
Baked potato 4O
Beets 32
Apricots 32
3. Scrambled Egg 20 200 20
4. Milk 200 500 25
5. Bacon 3 500 25
Cheese 5
Roast Pork 15
Butter 12
6. Bread 10 1000 25
Cake 15
Grapefruit juice 20
Lettuce ' 3
Celery 5
Baked apple 20
Baked potato 20
Beets 16
Apricots 16
388 10
Milk 80
Bacon 3
Cheese 5
Pork 15

Butter 12

 

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