REHYDRATION OF FREEZE-DRIED PORK AS RELATED
1‘0 pH AND PROTEiN DENATURATEON

Thesis For Ht. 909ch of M. S.
MiCHIGAN STATE UNIVERSITY
Joan Ruth Sudan
1963

This is to certify that the

thesis entitled
REHYDRATION 0F FREEZE-DRIED PORK AS RELATED TO

pH AND PROTEIN DENATURATION

presented by

Joan Ruth Suden

has been accepted towards fulfillment
of the requirements for

M.S. degree in Food Science

4 ~74 sci/W2

Major professor

Date May 13L 1963

 

0-169

   
   
 

  

LIBRAR Y
Michigan State

‘1‘ LindVUfSicy

ABSTRACT
REHYDRATION OF FREEZE-DRIED PORK AS RELATED TO

pH AND PROTEIN DENATURATION
by Joan Ruth Suden

‘With the advent of WOrld war II, extensive investigations occurred
in the preparation of dehydrated foods for the armed forces. Of all the
dehydration methods studied, freeze-drying produced dehydrated foods of
the highest quality.

Despite all the advantages of freeze-drying, freeze-dried meat re-
hydrates to only 80-90% of the original water content, is tougher, and
has a drier texture than that of the control. The objectives of this
study were: (1) To investigate the effect of pH on the percentage rehy-
dration of freezeadried pork; and (2) To determine the degree of protein
denaturation and its relationship to rehydration.

Results indicated that there was no significant correlation between
percentage rehydration and either pH of the rehydrating solution or pH
of the rehydrated meat. Freeze-dried pork showed no Optimum pH for re-
hydration.

Freeze-dried pork was found to rehydrate to a much lower level than
beef. The percentage rehydration of freeze-dried pork ranged from 48.54%
to 92.41% with a mean percentage rehydration of 73.75% i 9.26. Fat con-
tent did not influence rehydration.

An increase in the pH of freeze-dried pork occurred when the fillets
were rehydrated in deionized water. A loss of acidic volatiles during
dehydration was indicated. If the volatiles were trapped and utilized in

the reconstitution of the dried meat solids, the original pH of the fresh

meat slurry was regained.

Joan Ruth Suden

Results showed that no significant correlations existed between per-
centage rehydration and sarc0plasmic protein nitrogen, 0.53 p.(KCl-
bicarbonate) extractable protein nitrogen, soluble fibrillar protein
nitrogen or non-protein nitrogen content. However, as the percentage
of rehydration increased, there was a marked increase in the protein
content of the rehydrating solution.

The sarc0plasmic protein nitrogen content of freeze-dried and rehy-
drated pork decreased from that of the fresh control. Thus denaturation
of the sarc0plasmic proteins of pork occurred during the freeze-dehydra-
tion process.

Rehydrating solutions of similar ionic strength had identical, qual-
itative amino acid compositions. The qualitative amino acid composition
of the rehydrating solutions was not influenced by pH. However, a change
in ionic strength varied the qualitative amino acid composition of the

rehydrating solution.

REHYDRATION OF FREEZE-DRIED PORK.AS RELATED TO

pH AND PROTEIN DENATURATTON

By

Joan Ruth Suden

A THESIS

S ubmit ted to
‘Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER.OF SCIENCE
Department of Food Science

1963

ACKNOWLEDGMENTS

The author wishes to express her sincere gratitude and appreciation
to Dr. A. M. Pearson for his continuous guidance and encouragement during
the course of this study and preparation of this manuscript.

Sincere thanks are extended to Dr. Leroy R. Dugan for his guidance
and assistance in the use of the freeze-drying facilities in his labora-
tory, to Mrs. Dora Spooner for her aid in the statistical analysis and
to Mrs. Beatrice Eichelberger for typing this manuscript.

Most of all, the author wishes to acknowledge her husband, Edward,

for his patience, encouragement and unending faith.

ii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . .

LITEMTURE REVIEW 0 O O O O O O O O O 0

Physical prOperties of freeze-dried

Factors influencing rehydration

Protein denaturation . .

EXPERIMENTAL METHODS . . . . .

Fat and moisture analysis . . .

Nitrogen analysis . . . . . . . . .

Non-protein nitrogen determination

Measurement of pH . . . .
Centrifugation . . . . .
Reagents . . . . . . . .
Statistical analysis . .
Experimental meat . . . .
Sample preparation . . .
Fresh meat . . . . . . .
Freeze-dehydration . . .
Rehydration of fillets .
Protein fractionation . .
Amino acid composition of

Volatile loss detection .

rehydrating solution

iii

Page

11
ll
11
ll
11
12
12
12
12
12
12
13
13
l4
18

20

RESULTS AND DISCUSSION . . . . . .
Influence of pH on percentage

Protein denaturation . . . .

SUMMARY AND CONCLUSIONS . . . . .

LITERATURE CITED . . . . . . . . .

APPENDIX 0 O O O O O O O O O O O O

rehydration'

iv

Page
22
22

3O

42

45

48

Table

10

11

12

13

LIST OF TABLES
Page

Means and standard deviations for pH of rehydrating
solution, pH of rehydrated meat and percentage rehydration 22

Correlation coefficients between pH of rehydrating solution,
pH of rehydrated meat and percentage of rehydration . . . 23

Correlation coefficients between the pH of rehydrating solu-
tion and pH of rehydrated pork . . . . . . . . . . . . . 24

Analysis of variance of percentage rehydration and pigs . 25

Mean percentage rehydration and fat content of fresh
longissimus dorsi . . . . . . . . . . . . . . . . . . . . 27

Influence of freeze-drying on the pH of rehydrated pork . 28

Influence of freeze-drying on the loss of volatiles from
pork 0 O O O O O O O O O O O O O O . O O O O O O O O O I I 29

MEans and standard deviations for the nitrogen content of
the protein fractions extracted from freeze-dried loins
expressed as mg. N/g. solids . . . . . . . . . . . . . . 31

Analysis of variance for non-protein nitrogen content
among three freeze-dried loins . . . . . . . . . . . . . 33

The nitrogen content of the protein fractions extracted
from the fresh longissimus dorsi controls expressed as
mg. N/g. SOIidS O O O O O O O I O O O O O O O O O I O O O 33

Correlations between percentage rehydration and the nitro-
gen content of the extracted protein solutions expressed
as mg. N/g. solids . . . . . . . . . . . . . . . . . . . 34

A comparison of the nitrogen content (mg. N/g. solids) of
the protein fractions extracted from fresh and freeze-

dried loins O O O O O O O O C O O I O O O I O O O O O O O 36
Correlations between the sarc0plasmic protein nitrogen
content and pH . . . . . . . . . . . . . . . . . . . . . 38

Figure

LIST OF FIGURES
Page

Scheme of analysis for the quantitative determination
of sarcoplasmic protein nitrogen, non-protein nitrogen
and total fibrillar protein nitrogen . . . . . . . . . . 15

Scheme of analysis for the quantitative determination of
fibrillar protein nitrogen solubility . . . . . . . . . . 16

Scheme of analysis for the qualitative determination of
amino acid composition of the rehydrating solution . . . l9

Histogram of percentage rehydration for freeze-dried
Pork fillets O O O O O O O O I O O I O O O O O O O O O O 25

Chromatograms of the qualitative amino acid composition
of total amino acid content (Hydrolyzate I) and total
non-protein nitrogen amino acid content (Hydrolyzate II) 39

Chromatograms of the qualitative amino acid composition

of free amino acid content (Solution F) and known amino
ac ids (Kn-MS) O O O O O O O O I O O O I O O O O O O O O 40

vi

Appendix

LIST OF APPENDIX TABLES

Composition of rehydrating solutions . . . . . . . .

Composition of rehydrating solutions (continued) . .

Colors given with the ninhydrin-cupric nitrate spray
of Moffat and Lytle (1959) . . . . . . . . . . . . .

Formulas used in calculations . . . . . . . . . .

Formulas used in calculations (continued) . . . .

Formulas used in calculations (continued) . . . .

Formulas used in calculations (continued) . . . .

Correlations
Correlations
Correlations

Correlations
pooled data

between all investigated factors of loin 1
between all investigated factors of loin 2

between all investigated factors of loin 3

between all investigated factors of

Complete calculated data for the longissimus dorsi

freeze-dried

101:)» 1 O O O O O O O O 0 O O O O O 0

Complete calculated data for the longissimus dorsi

freeze-dried

loin 1 (continued) . . . . . . . . .

Complete calculated data for the longissimus dorsi

freeze-dried

loin 1 (continued) . . . . . . . . .

Complete calculated data for the longissimus dorsi

freeze-dried

10111 2 O O O O O O I O O I O O O O 0

Complete calculated data for the longissimus dorsi

freeze-dried

loin 2 (continued) . . . . . . . . .

Complete calculated data for the longissimus dorsi

freeze-dried

of

loin 2 (continued) . . . . . . . . . . .

vii

Page
48

49

50
51
52
53
54
55
56

57

58

59

60

61

62

63

64

Appendix

Page

Complete calculated data for the longissimus dorsi of
freeze-dried loin 3 . . . . . . . . . . . . . . . . . 65

Complete calculated data for the longissimus dorsi of
freeze-dried loin 3 (continued) . . . . . . . . . . . 66

Complete calculated data for the longissimus dorsi of
freeze-dried loin 3 (continued) . . . . . . . . . . . 67

viii

INTRODUCTION

The ideal dehydrated food was defined by Cooding and Rolfe (1955)
as one having the appearance, palatability, and nutritional quality of
the freshly prepared food. The dehydrated food would reconstitute rapid-
ly when water was added. It would also have a long storage life under a
range of conditions, a high packaging density, low processing losses and
economy in manufacture.

With the advent of WOrld War II, extensive investigations occurred
in the preparation of dehydrated foods for the armed forces. Of all the
dehydration methods studied, freeze-drying produced dehydrated foods of
the highest quality.

The advantages of freeze drying have been outlined by Flosdorf
(1949). The low temperatures of Operation avoided chemical changes in
labile components and the loss of volatile constituents was minimal. No
bubbling, foaming, or shrinkage of the material occurred. The tendency
for coagulation was at a minimum and no case hardening was apparent.
Under the frozen conditions of drying, neither bacterial growth nor enzy-
matic changes occurred.

DeSpite all of the above factors, freeze-dried meats only rehydrate
to 80-90% of the original water content. The reconstituted freeze-dried
meat is tougher and has a drier texture than that of the control. The

water-holding capacity of the meat has been altered.

The objectives of this study are twofold:

(1) To investigate the effect of pH on the percentage rehydration of
freeze-dried pork.

(2) To determine the degree of protein denaturation and its relation-

ship to rehydration.

LITERATURE REVIEW

Physical properties of freeze-dried meat

The physical characteristics of freeze-dried beef (biceps femoris)
were outlined by Tappel 23 a1. (1955). The composition was detenmined
as 80-85% protein and 13-17% lipid. These workers found a 3% moisture
content in l-inch thick pieces, which had been dried for 24 hours. The
biceps femoris retained a structure similar to balsa wood with no volume
change due to the freeze-drying process. Its density (0.33 g/cc.),
porosity (80%) and thermal conductivity (0.02 B. Th. IL/h./ft./°F) were
detenmined after dehydration. The low thermal conductivity was accounted
for by the wood-like physical structure of the meat. The muscle was pink
in color but changed to tan on storage. Harper and Tappel (1957) have
explained the color change as being due to the low oxygen tensions in
the freeze-dryer. They suggested that oxymyoglobin is deoxygenated to
form myoglobin which is labile to oxidation on storage.

According to Harper and Tappel (1957), freeze-dried pork was very
similar to freeze-dried beef in structure and texture. They reported
that the color of pork was initially a light pink, which soon changed to
light tan. The color remained unchanged until active browning deteriora-
tion developed.

Hankins.g§.§l. (1946) reported that dehydrated pork actually con-
tained less fat and more protein than was calculated from the composition

of the raw meat. They also showed that the chemical composition of the

-3-

-4-

dehydrated product was influenced by the composition of the raw material,
extent of the drying, and any losses in constituents in addition to mois-
ture, that occurred during processing. The investigations of Doty 2E 11.
(1953) indicated that the histological changes found in freeze-dried
beef were very slight and the gross chemical composition of meat on a

moisture-free basis was apparently little changed by dehydration.

Factors influencing rehydration

'Wang 2; 31. (1953) studied the effect of various freezing methods
on the reconstitution of freeze-dried beef. Reconstitution of the freeze-
dried samples was compared by measuring the increase in moisture content
and of muscle fiber diameter. Meat pre-frozen at -17°C, -80°C and ~150°C
was reconstituted. Samples prefrozen at -l7°C had the largest amount of
interfibral Space, the greatest degree of recovery of muscle fiber dia-
meter, and the fastest initial water penetration.

Investigations by wang gt 31. (1954) have shown that the rate of
freezing had a marked effect on the size of the ice crystals formed and
their location in the foodstuff. 'With rapid freezing, the ice crystals
were extremely small and mostly inside the cells. As freezing rate de-
creased, the size of ice crystals increased and the frequency of loci
decreased. The freezing changed from an intracellular to an intercellu-
1ar pattern, and eventually quite severe mechanical damage occurred
within the cell structure. As the ice was sublimed, pores of large dia-

meter remained in the dry tissue. The material that had been frozen

slowly offered a reduced resistance to the escape Of the water vapor from
the ice surface as compared with rapidly frozen material.

Luyet (1962) compared the effects Of various freezing rates on the
structure Of freeze-dried muscle. The experimental results Of Luyet
corraborated those of NangIg£_§l, (1954).

According to Greaves (1954), freeze-dried serum went into solution
at a much faster rate, if prepared from rapidly frozen serum. Experimen-
tal results Of Gooding and Rolfe (1957) indicated that rapidly frozen
meat failed tO give a product which could be easily reconstituted. The
TMinistry Of Agriculture, Fisheries and Food (1961) recommended the use
of an intermediate rate of freezing, such as that obtained by blast freez-
ing, in order to gain an Optimum crystal size. Harper and Tappel (1957)
suggested that meat be frozen by pre-freezing in external freezing equip-
ment, since case-hardening occurred during evaporative_freezing within
the drying cabinet.

The rehydration of freeze-dried meat has been investigated by Auer-
bach gt 31. (1954). These investigators reported that meat cut across
the grain reconstituted more rapidly and more completely than that cut
longitudinal to the grain. The thickness Of the sample was found to
influence both the rate and level of rehydration. A l-inch section re-
hydrated for 3 hours did not reach the same level Of rehydration that a
1/2 inch section achieved in 3 minutes.

Auerbach et a1. (1954) also reported that reconstitution was un-

affected by water in the temperature range 22-55°C. The Optimum pH of

rehydration was reported as 7.00. These workers stated that reconsti-
tution was more rapid and more complete in a vacuum. .Although the per-
centage rehydration varied from 80-90%, after cooking the texture
usually was drier than that of the controls.

According to Turner (1956) increased hydration Of raw dehydrated
meat occurred when salts were added to the rehydration water. He reported
that the addition Of 1.5% to 2% NaCl with 0.1% to 0.15% tetrasodium py-
rophosphate (Na4P207 . 10 H20) improved the texture and overall accepta-

bility of the final product.

Protein denaturation

Taylor (1953) reported that many proteins have been freeze-dried with-
out Obvious denaturation and appear to be stable indefinitely in the dry
state. At least two food proteins, egg albumen, according to Bull (1944),
and rabbit myosin, according to Bailey (1956), cannot be freeze-dried
without denaturation.

The changes caused by freeze-drying in the histological micro-structure
and in the molecular structure of the fiber substance were studied by
Connell (1957). He reported that the fibers lost their close contact
and it was possible to separate them easily. There were also aggregations
of fibers, and areas of fused fibers separated by large Spaces. The in-
vestigator suggested that the change in molecular structure Of the fibers
was due to denaturation Of actomyosin during dehydration and was accom-

panied by a loss Of the gel-forming ability Of protein and water.

Connell (1958) studied the effect of drying on fish muscle proteins.
He reported that the protein-gel system of the dried fish was more dis-
organized than that of fresh fish, even though the microsc0pica1 appear-
ance Of the muscle cell was unchanged. The true water-binding capacity
of the proteins was greatly reduced. The solubility of the proteins of
freshly prepared dehydrated fish in 0.5 M KCl (pH 7.00) was found tO be
very much less than that of the proteins of fresh fish.

Hunt and Matheson (1958) reported a decrease in the actomyosin ATP-
ase activity Of cod and beef muscle on dehydration. The results Of Connell
(1958) and Of Hunt and Matheson (1958) indicated that the main structural
protein complex Of muscle, actomyosin, had been denatured on drying.

Brooks (1958) attributed the dry, tough texture Of freeze-dried meat
to the loss of water-holding capacity by the muscle proteins. He sug-
gested that protein denaturation during drying may be responsible.

The results Of Hamdy £5 a1, (1959) confinmed those Of Brooks (1958).
They added various solutes to the water used for rehydration in order to
investigate the water-holding prOperties of freeze-dried beef. These in-
vestigators showed that the rehydration of freeze-dried meat was not
greatly improved as a result Of changing the ionic atmOSphere in meat.

Hamdy g£_§l, (1959) reported that freeze dehydration Of beef at a
43°C plate temperature and 1500 [AHg resulted in a considerable decrease
in the concentration Of the water soluble nitrogen. Upon heating the
freeze-dried samples, the amount Of juice released was much more than
that Of the reapective controls prior to freeze-drying. At a plate temp-

erature of 22-30°C, 300-400’p Hg, these workers detected no effect on

the pH of meat. Similarly, there was no measurable effect on the water
soluble proteins or the 4% TCA soluble proteins of both the control and
those samples to which salt was added. They also detected no changes in
pH as a result of frozen storage or freeze-dehydration of the meat samples.
The evidence indicated that denaturation of the proteins had resulted in
changes in permeability of the fibers.

According to Deatherage and Hamm.(1960), quick freezing of muscle
tissue did not decrease the hydration of muscle nor cause protein denatur-
ation. 0n the other hand, slow freezing caused a small but significant
decrease in the water-holding capacity of meat. It was postulated that
some alteration of protein structure was caused by the formation of the
large ice crystals between the cells. With slow freezing, the liklihood
of denaturation increased as the proteins were in the presence of a con-
centrated salt solution for a longer period of time.

The denaturation of proteins during freeze-drying was studied by
Hamm.and Deatherage (1960) using water-holding capacity and buffering
capacity at different pH values, as well as measurement of the dye bind-
ing ability of the free acidic and basic groups of muscle proteins. In
general fresh meat exhibited a greater water-holding capacity than freeze-
dried at a pH of 5.6 (the natural pH of meat). Over a range of pH, freeze-
dried meat was found to exhibit greater water-binding capacities than
fresh meat at a pH higher than 6.5 but less at a pH lower than 6.5. The
minimum water binding capacity for both products was at pH 5.0, where

differences were greatest between the fresh and freeze-dried meat.

Hamm and Deatherage (1960) also found that differences existed between
fresh and freeze-dried meat in their buffering capacities. The difference
was greatest between pH 6 and 7, where for both the water extract and
structural proteins, freeze-dried meat exhibited a higher buffering capa-
city. Results obtained from dye binding by free acidic and basic groups
supported the assumption that more free acidic groups were liberated on
the basic side of the isoelectric point.

Deatherage and Hamm (1960) also concluded that the removal of water
gives rise to a decreased number of protein groups available to bind
water after reconstitution. They further suggested that drying resulted
in a more closed protein structure due to the salt and/or hydrogen bridge
type of bonds, which can be reversed at high or low pH.

Hamm and Deatherage (1960) also reported a drop of muscle hydration
with an increase of temperature. However, the rehydrated samples had the
same moisture content. They also studied the influence of the shape of
meat on the water-holding capacity. They showed that the water-holding
capacity of all freeze-dried beef samples was less than that of fresh
meat. The hydration of the powdered ground meat was greater than that
of the rehydrated and ground cubed meat. This in turn was somewhat
higher than hydration of the ground dehydrated meat, which was rehydrated
without powdering. These results indicated that the grinding of meat
before drying was disadvantageous.

According to Hamdy gt 31. (1959) the electrophoretic patterns of
the myosin extract of beef muscle appeared to be greatly affected during

freeze dehydration.

-10-

Cole and Smithies (1960) investigated the electrophoretic patterns
of a 0°15,P extract of freeze-dried beef. Their results indicated that
few changes had been introduced through freeze drying. In addition,
actomyosin extracts from freeze-dried beef sedimented at a faster rate
than actomyosin from frozen beef. There was no marked decrease in the
level of Specific ATP-ase activity.

A more intensive and extensive investigation of the ATP-ase acti-
vities of a beef actomyosin extract was performed by Cole (1962). It
was found that stimulation of ATP-ase by 2,4-dinitrOphenol was less for
both freeze-dried and frozen tissues than it was for fresh meat extract.
No significant difference between frozen and freeze-dried extracts was

reported.

EXPERIMENTAL METHODS

Fat and moisture analysis

MOisture was determined by drying 5 g. samples of ground meat in a
disposable aluminum dish for 24 hours in a 105°C oven. The dried samples
were used for fat extraction with the Goldfish apparatus according to

the procedure described by Hall (1953).

Nitrogen analysis

All nitrogen analyses were performed, in duplicate, by the micro-
Kjeldahl method as outlined by the American Instrument Company (1961).
Nitrogen contents were reported as mg. of protein nitrogen or non-protein

nitrogen per m1. of solution, or per g. of solids.

Non-protein nitrogen determination

Non-protein nitrogen was determined after precipitating the proteins
by adding 5 ml. of 10% trichloroacetic acid to 15 ml. of all extracted
protein solutions. After 15 minutes the material was filtered through
Whatman No. 1 filter paper and the filtrate was analyzed for nitrogen
content. The value was multiplied by 1.33 in order to obtain the non-

protein nitrogen content per m1. of the original solution.

Measurement ofng

All pH measurements were made with a Beckman Model G pH meter. The
electrodes were placed directly into the ground meat sample or protein
solution, and the Observed .values were recorded to the nearest hundredth

unit.

-11-

-12-

Centrifugation
Centrifuging was performed at 2500 rpm (1400 x gravity) in a model

PR-2 refrigerated International Centrifuge at 4°C.

Reagents

American Chemical Society reagent grade chemicals and deionized

distilled water were used throughout the experiment.

Statistical analysis
Simple correlation coefficients, means, and standard deviations

were calculated as described by Snedecor (1956).

Experimental meat

 

All meat samples were obtained from carcasses of hogs slaughtered in
the Michigan State University abattoir. No attempt was made to relate
treatment effects to the previous history of the animal, since meat from

the same animal served as the untreated control in all studies.

Sample preparation
The longissimus dorsi muscle was used in all studies. All separable
fat and visible connective tissue were removed from the excised muscle

samples.

Fresh‘meat
One hundred g. samples were removed from the posterior, middle and
anterior portions of the muscle. The composite sample was ground twice

through a 1 cm. plate and twice through a 2 mm.plate of a Hobart grinder.

-13-

The grinder head and.plates were pre-chilled to 4°C in all cases to
prevent heat denaturation of the sample. The pH of the fresh meat was
recorded. A portion was frozen and analyzed later for total nitrogen,

fat and moisture content.

Freeze-dehydration

The remainder of the longissimus dorsi muscle was sliced into 26-
28 fillets about 1/4 inch in thickness, weighed to the nearest tenth
of a green and prefrozen in aluminun foil at -28.9°C blast for 3 hours.
The frozen samples were freeze-dried for 20-24 hours in a Stokes Freeze-
Drier, Laboratory Model 2003 F-2 using a vacuun of 150 P Hg., with a
plate temperature ranging from 28-30°C. Upon removal from the Stokes
apparatus, the samples were immediately reweighed. The fillets were
wrapped individually in aluminum foil and stored under nitrogen in a
desiccator at room temperature. Length of Storage never exceeded four

weeks.

Rehydration of fillets
Fillets of known weight were immersed in 150 ml. of rehydrating
solution in covered casserole dishes. Detailed composition of all rehy-
drating solutions is contained in appendix A. In order to submerge the
samples, glass weights were utilized. The fillets were rehydrated 4 1/2
hours at 4°C. All rehydration procedures were carried out in duplicate.
The rehydrated pork was blotted with'Whatman No. 1 filter paper for

l/2 minute on each side to remove excess moisture. Samples were weighed

-14-

and the percentage rehydration was calculated according to the formula,
-g§=x 100, where WR_- g. moisture regained in rehydration, W1 - g. mois-
ture lost in freeze-dehydration. The pH of the rehydrated fillet was
recorded. The protein nitrogen and non-protein nitrogen of the rehy-

drating solution were measured.

Protein fractionation

The protein fractionation procedure was adapted from that of Hegarty
(1963), with modifications according to the methods of Cole and Smithies
(1960) and of Seagran (1958), and is described below.

All fractionation procedures were carried out in duplicate at 4°C.
The scheme of analysis is shown in figure 1. It was used for the quanti-
tative determination of sarCOplasmic protein nitrogen, non-protein nitro-
gen, and total fibrillar protein nitrogen. The scheme shown in figure
2 was used for the determination of fibrillar protein solubility.

Five g. of fresh or rehydrated pork were weighed into a 250 ml.
centrifuge tube. Eighty m1. of a phOSphate buffer, pH 7.6,,1 - 0.05,
(0.156 M K2HP04; 0.0035 M KH2P04) was used to transfer the sample to a
micro blender container. Protein denaturation due to excessive foaming
was avoided by comminuting with the waring blendor, in which the Speed
was adjusted by means of a Powerstat transformer setting of 40. The
samples were blendorized for a 10 second burst followed by a 3 minute
rest period. This process was repeated three times. After blendorizing,
the suspension was transferred back to its original tube. The contain-

er was rinsed with 20 m1. of extracting solution. After one hour the

-15-

Blendorized a 5 g. sample in
80 m1. of P04 buffer +'a 20
‘ml. rinse with same buffer

25

 

residue

added 100 m1. P04

 

residue (Z) supernat

residue

alkali insoluble

 

 

added 200 ml. 0.1 M
NaOH, after 12 hours
filtered through gauze

 

mixed, after one hour
centrifuged 25 minutes

after one hour,
centrifuged for

‘minutes

supernatant

buffer,

ant

nitrogen solution
(B), extracted at
low ionic strength

 

 

filtrate

solution (A), containing

material, connective total fibrillar protein

tissue (discarded)

15 m1. aliquot of
B treated with TCA
‘for non-protein
nitrogen determin-

 

 

nitrogen ation
residue filtrate
discarded solution (C)’contain-
precipitate ing non-protein

nitrogen

Figure 1. Scheme of analysis for the quantitative determination of sarco-
plasmic protein nitrogen, non-protein nitrogen and total fibrillar
protein nitrogen.

-l6-

Blendorized a 5 g. sample in
80 m1.KC1 buffer-+ a 20 ml.
rinse with same solution

after one hour,
centrifuged for 25
'minutes

residue supernatant

 

 

added 100 ml. KCl
buffer, mixed, after
one hour centrifuged
for 25 minutes

 

 

residue supernatant
discarded solution (D),

nitrogen extracted
at high ionic
strength

 

 

 

Figure 2. Scheme of analysis for the quantitive determination of fibrillar
protein nitrogen solubility.

-17-

material was centrifuged for 25 minutes and the supernatant was decanted.
One hundred m1. of extracting solution was added to each tube. Complete
dispersion of the precipitate was achieved by Stirring with a glass rod.

After one hour the material was centrifuged and the supernatant de-
canted as before. The two decanted solutions were combined and designated
as B, or the protein solution extracted at low ionic strength. Two ali-
quots of 15 ml. were taken for nitrogen and non-protein nitrogen analyses.
The filtrate resulting from the TCA precipitation was designated as C.
The residue (Z) resulting from extraction with phOSphate buffer was ex-
tracted with 200 m1. of 0.1 M NaOH for 12 hours at room temperature. The
volume of the tube contents was measured after filtration through gauze.
A very small amount of residue (alkali insoluble material, i.e., collagen
and elastin) was removed by filtration. An aliquot of the filtrate (A)
was taken for nitrogen analysis. The procedure in figure 2 is exactly the
same as the first two steps outlined in figure 1, except that the extract-
ing solution was a KClecarbonate buffer, pH 8.25, P" 0.53, (0.5 M KCl;
0.03 M NaHCO3). The solution extracted by this scheme was designated D.

Solutions A, B, C5 and D were analyzed for nitrogen and results desig-
nated as A9, Bn, etc. which represent the following fractions:

A? - total fibrillar protein nitrogen

Bn - nitrogen extractable at low ionic strength

Cn - non-protein nitrogen

Dn - nitrogen extractable at high ionic strength
Bn - Cn - sarcoplasmic protein nitrogen

Dn - Bn a soluble fibrillar protein nitrogen

n n
D - (C -+ HI) = connective tissue protein nitrogen.

-13-

The above values were averages of duplicate analyses recorded to the
second decimal place. Variation between duplicates for B“, On, Dn was
normally 0 - .02 mg. per m1. Variation in the second decimal place in
the case of A? was found to be 0 - .04 mg. with an extreme range in one

or two cases of .09 mg. per ml.

Amino acid composition of rehydrating solution

The scheme of analysis outlined in figure 3 was utilized for the
qualitative detenmination of the amino acid composition of the rehydrating
solutions.

Forty ml. of rehydrating solution were hydrolyzed with 40 ml.12 N
HCl for 24 hours and then filtered. The resultant hydrolyzate (I) repre-
sented the total amino acid content of the rehydrating solution. Forty
ml. of rehydrating solution were mixed with 160 ml. of 100% ethyl alcohol
to precipitate the proteins. After 30 minutes, the material was filtered
through Whatman No. 1 filter paper. The residue was discarded. The fil-
trate was concentrated to 16 ml. with a Rinco rotary evaporator at 40°C.
One ml. of the concentrated solution (F) was removed. This solution re-
presented the free amino acid content of the original rehydrating solution.
Fifteen ml. of 12 N HCl were added to the remaining 15 m1. of concentrate.
The solution was hydrolyzed for 24 hours and then filtered. The hydroly-
zate (II) contained the total non-protein nitrogen amino acids.

The amino acids were separated on a one-dimensional descending paper
chromatograph. A l-n-butanol-acetic acid-water (upper layer) of a 4:1:5

by volume solvent system was used. The ninhydrin-nupric nitrate Spray of

-19-

Rehydrating solution

 

40 m1. rehydrating solution +' 40 ml. rehydrating solution + 160
40 ml. 12 N HCl are hydrolyzed ml. 100% E t OH
for 24 hours
filter through after 30 minutes
Whatman No. 1 filter through
filter paper ‘Whatman No. 1
\/ filter paper

 

Hydrolyzate (1)
total amino acid content

 

 

 

 

residue supernatant
discard concentrate
to 16 ml.
15 ml. condgntrate +-15 m1. SolutionfitF), 1 ml.
12 N HCl are hydrolyzed for concentrate contain-
24 hours ing free amino acids

filter through
Whatman No. 1
(filter paper

 

\

Hydrolyzate (II)
Total non-protein nitrogen
amino acid content

Figure 3. Scheme of analysis for the qualitative determination of amino
acid composition of the rehydrating solution.

-20..

Moffat and Lytle (1959) permitted complete resolution of the incompletely
separated amino acid spots. The three solutions (Hydrolyzate I, II, and
solution F) were placed on Whatman No. 1 paper in 100 microliter aliquots.
The chromatogram was developed until the solvent front had advanced 32 to
35 cm.beyond the point at which the sample was applied. The paper was re-
'moved from the apparatus and dried in a 105°C oven for 5 minutes. The
chromatogram was Sprayed with the ninhydrin-cupric nitrate indicator and
dried in a 105°C oven for 2 minutes. The colors given with this reagent

are listed in appendix B.

Volatile loss detection

A 20 g. portion of the ground fresh meat was blendorized with 80 m1.
of deionized water for one minute in a waring blender adjusted with a
Powerstat transformer setting of 60. The pH of the slurry was taken. The
meat slurry was transferred to a 1000 ml. round bottom flask, which was
then slowly rotated in an ethanol-dry ice bath. This caused the meat
slurry to be frozen as a thin Shell on the flask's surface. The flask
was attached to a vacuum distillation apparatus, which consisted of one
ethanol-dry ice trap and two liquid nitrogen traps. A welch Duo-Seal
vacuun pump created a vacuum ranging from 170 [.1 to 50/; Hg. Complete de-
hydration occurred within 6-9 hours. The ethanol-dry ice trap contained
all of the water removed from the meat slurry. The water was thawed and
its pH was recorded.

The volatiles lost in the dehydration process were trapped in the
two liquid nitrogen traps. The volatiles were distilled into the thawed
water using nitrogen to flush out the containers. After each trap had

been distilled, the pH of the solution was taken.

-21-

The solution consisting of all volatile constituents removed during
the dehydration of the slurry was added to the dehydrated meat solids.

The pH of the reconstituted meat slurry was then measured.

RESULTS AND DISCUSSION

Influence ofng on percentage rehydration

A total of 70 fillets taken from the longissimus dorsi of three hogs
was used in this study. Duplicate samples were rehydrated in either 10
or 11 different buffers of 0.051P.covering a pH range of 3.62 to 9.05.
Four samples were rehydrated in a buffer of 0'1.P’ pH 3.05 and 2 samples
were rehydrated in a buffer of 0.1 p, pH 2.55.

The means and standard deviations for pH and percentage rehydration
are presented in table 1. There were no significant differences between
the means for the three loins in either the pH of the rehydrating solution
or the pH of the rehydrated meat.

Table 1. Means and standard deviations for pH of rehydrating solution,
_pH of rehydrated meat and percentage rehydration

Means and standard deViations

 

 

Pooled
Item Loin 1 Loin 2 Loin 3 data
pH of rehydrating
solution 6.40 i 1.81 6.35 i 1.74 6.38 i 1.85 6.38 i 1.80

pH of rehydrated

meat 5.55 i .51 5.47 i .44 6.41 i .48 5.47 i .48
Percentage
rehydration 67.41*i 8.86 80.61**i7.38 72.71*i 6.18 73.75 i 9.25

 

 

**Significantly different at the 1% level from the underlined observations.
* Significantly different at the 5% level.

Table 2 summarizes the correlation coefficients of pH versus percent-

age rehydration. The association between percentage rehydration and either

-22...

-23-

pH of the rehydrating solution or pH of the rehydrated meat was not sta-
tistically significant. Thus, freeze-dried pork showed no optimum pH for
rehydration. This is in cOntrast to the work of Auerbach gt a1. (1954),
who reported that the highest level of rehydration for freeze-dried beef
occurred at pH 7.00. This indicates that a difference between freeze-
dried pork and beef exists, which, perhaps, can be attributed to differ-
ent physiological characteristics of the two Species.
Table 2. Correlation coefficients between pH of rehydrating solution,

pH of rehydrated meat andgpercentage of rehydration

Correlation coefficients

 

 

pH rehydrating solution pH rehydrated pork
Loin vs vs
percentage rehydration percentage rehydration
1 -.217 -.064
2 -.081 -.027
3 0.091 ’ -.067
Pooled
data -.O71 -.O78

 

A direct correlation between pH of the rehydrating solution and the
pH attained by the rehydrated pork was observed for all three loins in
this study. The correlation coefficients are shown in table 3. This
straight line relationship was not surprising as the pH of meat would be
expected to change on the addition of an acidic or basic solution. The
amount of change in pH would be dependent on the buffering capacity of

the proteins. Sherman (1961) reported that the pH of fresh pork is effected

-24-

Table 3. Correlation coefficients between the pH of rehydrating solution
and pH of rehydrated pork

 

Loin Correlation coefficients
1 +3916**
2 +.721**
3 -+.846**
Combined data +,824**

 

**Significant at 1% level.

by the addition of neutral salts and polyphOSphates. In the present
study, no attempt was made to determine the effect of neutral salts and
polyphOSphates on the pH of freeze-dried pork.

The analysis of variance between percentage rehydration for differ-
ent loins is summarized in table 4. The F ratio of 17.27 was highly
significant at the 1% level. The Studentized range test indicated that
there was a Significant difference between the means for percentage rehy-
dration of the loins from the three different hogs utilized in this study.
The occurrence of a significant deviation between loins may have been
caused by breed differences or possibly by an individual reaction to the
dehydration process.

The experimental data indicates that freeze-dried pork rehydrates
to a much lower level than beef. Percentage rehydration of freeze-dried
pork ranged from 48.54% to 92.41%. The mean percentage rehydration of
70 samples is 73.75% and the standard deviation is 9.26. The distribu-

tion of percentage rehydration is summarized in the histogram shown in

figure 4.

-25-

Table 4. Analysis of variance of percentage rehydration and pigs

 

 

 

 

 

 

 

 

 

 

 

Sum of Mean
Source d.f. squares square F
Percentage rehydration 69 5996.43
Loins 2 2039.96 1019.98 l7.27**
Individuals 67 3956.47 59.05
**Highly significant at 1% level.
it
254» 24
20
20..
18
U)
.2
@150
S
U)
“3 10 s.
H
.8
g 5 4 3
l
0 r———__ a?
40’ 6 8 9 1 0

 

 

 

 

 

Percentage Rehydration

Figure 4. Histogram of percentage rehydration for freeze-dried pork
fillets.

According to Tappel gthgl. (1955) l-inch pieces of the biceps femoris

 

of beef attained an 80-90% level of rehydration. Harper and Tappel (1957)

have stated that freshly prepared freeze-dried beef rehydrates to a

maximum level of 80-100% of its original water content. Although freeze-

-26..

dried pork is very Similar to freeze-dried beef in structure and texture,
there is a noticeable difference in their rehydration characteristics.

The conditions of this investigation varied from those of Tappel,
who utilized a -17.8°C freezing temperature, a 0.1 to 0.2 mm.Hg.pressure
and a plate temperature of 45°C. A freezing temperature of -28.9°C, a
0.15 mm. Hg. pressure and a plate temperature of 25-30°C were used in
this study. A rapid freezing rate tends to increase product quality,
while a lower plate temperature during the freeze-drying process tends
to reduce protein denaturation. Thus, the experimental conditions of
this investigation should have caused an equivalent or greater percentage
rehydration than that reported for freeze-dried beef by Harper and Tappel
(1957). However, the results of this study indicated that freeze-dried
pork will usually rehydrate in the range of 64-83% which is considerably
lower than the 80-100% reported by Harper and Tappel (1957) for freeze-
dried beef.

Results outlined in table 5 Show that loin l, which had a low fat
content, did not rehydrate to a higher level than loin 3, which had a
high fat content. These results are in direct Opposition to those of
Harper and Tappel (1957), who stated that the high fat content of pork
was responsible for a decreased percentage of rehydration. According to
Orme 35 31. (1958), the fat content from beef longissimus dorsi ranged
from 1.90 to 11.21%. The mean percent fat averaged 4.25 and 8.59% for
good and prime steers, reapectively. Harrington and Pearson (1962)
reported that the intramuscular fat content of pork longissimus dorsi

muscle differing greatly in marbling averaged 3.45% with a range from

-27-

Table 5. Mean percentage rehydration and percent fat content of fresh
longissimus dorsi

 

Mean percentage

 

Loin % fat ' rehydration
1 2.40 67.41 i 8.86
2 2.94 80.61 i 7.38
3 4.26 72.71 i 6.18

 

1.1 to 7.4%. Pearson 25 El. (1962) using another group of pigs found

the percentage fat of pork longissimus dorsi ranged from 2.14 to 8.14%.
Thus, the percentage intramuscular fat in beef and pork lgpgissimus dorsi
does not differ greatly. Therefore, if the fat content influenced rehy-
dration, no marked difference in rehydration would be expected between
beef and pork. It is also interesting to note that loin 2, which had

an intermediate fat content, rehydrated more completely than either of
the others.

The percentage fat reported in this study was obtained from a com-
posite sample taken from the whole loin. It is known that the fat content
of the longissimus dorsi varies with sampling position. Since the fat
content of the individual rehydrated fillets of loins l, 2, and 3, had
been calculated from the fat content of the fresh control, a fourth loin
was freeze-dried and rehydrated. Results showed that the correlation
(r - .168) between fat content and percentage rehydration was not statis-
tically significant. Therefore, fat content does not greatly influence

percentage rehydration.

-28-

The differences in percentage rehydration between loins from differ-
ent pigs may be caused by a combination of factors including pre-slaughter
treatment, electrolyte content of muscle and Over-all muscle composition.

One of the outstanding features of this series of experiments was
the increase in the pH of freeze-dried pork when rehydrated in deionized
water. The influence of freeze-drying on the pH of rehydrated pork is
shown in table 6. Seven out of eight rehydrated fillets achieved a higher

pH than that of the controls.

Table 6. Influence of freeze-drying on theng of rehydrated pork

 

 

pH
Loin Fresh ' Rehydrated duplicates1
l 5.32 5.69 5.65
2 5.35 5.25 5.45
3 5.51 5.75 ' 5.75
4 5.29 5.45 5.45

 

ISamples rehydrated in deionized water at 4°C.

In order to determine whether the change in pH was caused by protein
denaturation or a loss of volatile constituents, the volatiles were col-
lected and added back during rehydration. Results indicate that the
volatile losses are responsible for some of the changes in pH, and if
trapped and utilized in reconstitution of the dried meat solids, the ori-
ginal pH of the fresh meat slurry could be regained. Table 7 presents a

summarization of the results of the volatile-loss determination.

-29-

Table 7. Influence of freeze-drying on the loss of volatiles from pork.

 

 

 

- Trial - _ ApH I
Conditions (1)- (2)- pH1 - sz
Pressure 170-70 )1 Hg. 50 )1 Hg.
Dehydration time 9 hours 6 hours
J£i_______
Meat slurry, fresh 5.23 5.15
Volatile Fractions
Trap 1: Solution 1
(thawed water) 6.45 5.62 +0.83
Trap 2: Solution 2
(Solution 1 + volatiles
of lst liquid N2 trap) 5.68 5.30 +0.38
Trap 3: Solution 3
(Solution 2 + volatiles
of 2nd liquid N2 trap) 4.49 5.62 -1.13
Meat slurry, reconstituted
(dehydrated solids + solution 3) 5.28 5.18

 

The differences observed between trials in the pH of solutions 1, 2,
and 3 (table 7) may be exPlained by the varying experimental conditions.
A much higher evacuation of the flask in trial 2 was achieved. Thus, the
meat slurry in trial 2 could be completely dehydrated in a shorter period
of time. A complete equilibration of the volatiles between the traps in
trial 2 could not be accomplished due to the shorter dehydration time.
The total change in pH between trials was calculated as +.08 units, which

was within the experimental error of i .15. Although the volatiles were

-30-

not found in exactly the same traps in the two trials, the total change
in pH was equivalent.

The change in pH indicated that volatiles which are acidic in nature
are removed during the freeze-drying process. The removal of acidic vola-
tiles during the freeze-drying process, accounts for the rise in pH found
to occur on rehydration of freeze-dried pork. The nature of the volatiles
was not studied in this investigation, however, it is likely that C02,
H23, and short-chained fatty acids would be among the volatile constitu-
ents.

Flosdorff (1949) stated that the loss of volatile constituents during
freeze-drying was minimal. Hamm and Deatherage (1960) refer to an occa-
sional but not significant shift of pH in freeze-dried beef on rehydration.
The significant pH change of freeze-dried pork on rehydration again indi-
cates that freeze-dried pork.is dissimilmrto freeze-dried beef in its
rehydration characteristics. This may be due to the inherent differences

in the structure of the two species.

Protein denaturation

Seventy rehydrated fillets taken from the loggissimus dorsi muscle
of three pigs were used to investigate the degree of protein denaturation
caused by the freeze-drying process. Duplicate samples of each rehydrated
fillet and the fresh control were fractionated according to the schemes
outlined in figures 1 and 2. The protein content, expressed as mg. nitro-
gen per g. of solids, was determined for the following fractions: (1) sar-

c0plasmic protein nitrogen, (2) 0.53,p (KCl-bicarbonate) extractable protein

-31-

nitrogen, (3) soluble fibrillar protein nitrogen, and (4) non-protein

nitrogen. The protein and non-protein nitrogen contents of the various

rehydrating solutions were also determined.‘

The means and standard deviations for the nitrogen content of the

various protein fractions extracted from the freeze-dried loins are pre-

sented in table 8. There were no significant differences between the

means for the three loins in the sarCOplasmic protein nitrogen and 0.53

}1(KCl-bicarbonate) soluble protein nitrogen.

The differences between

the means for the three loins in the protein and non-protein nitrogen

contents of the rehydrating solution were not statistically significant.

Table 8. Means and standard deviations for the nitrogen content of the
protein fractions extracted from freeze-dried loins expressed

as mg. N/g. solids.

Means and standard deviations

 

 

Item Loin 1;, Loin 2 Loin 3 Pooled data

Sarcoplasmic protein

nitrogen 26.93 i 4.01 27.23 i 4.79 28.04 i 7.24 27.41 i 5.58
0. 53 p soluble

protein nitrogen 35.07 i 6.15 33.48 i 5.03 35.99 i 7.89 34.84 i 6.56
Soluble fibrillar

protein nitrogen 8.29 i 6.21 6.57 i 4.62 8.08 i 4.97 7.63 i 5.34
Non-protein

nitrogen 12.88 i 1.82 10.72*i 1.69 13.91 i 3.05 12.63 i 2.53
Rehydrating solution

protein nitrogen 2.25 i .654 2.95 i .981 2.63 i .615 2.62 i .821
Rehydrating solution

non-protein

nitrogen 4.49 i .556 5.34 i .686 5.62 i .740 5.17 i .820

 

*Significantly different at 1% level from the underlined observations. All

other values were not significantly different.

-32-

A large standard deviation for the soluble fibrillar protein content
was obtained for all three loins. It is impossible to arrive at direct
conclusions on the basis of these results due to the wide range in the
data. The analysis of variance for the soluble fibrillar protein content
among the three loins indicated that there was no statistical difference
between loins. The range in data obtained from freeze-dried loins could
have resulted from incomplete extraction of the fibrillar proteins.

Bailey (1954) stated that extractability of proteins was not solely
determined by solubility. He concluded that extractability of the intra-
cellular protein fraction appeared to be determined by pH, ionic strength
of the extracting solution, type of extractant, and by adequacy of grind-
ing. Dyer.ggnal. (1950) also concluded that the most important point in
the extraction of protein was a sufficiently fine subdivision of the mus-
cle fibrils. In the present study, the-freeze-dried fillets were hand-
minced, thus, the size of the mince varied. In further studies, the
experimental error could probably be reduced by increasing the size of
the fillets in order to permit mechanical grinding, and thereby, obtain
a more uniform particle size.

The analysis of variance for non-protein nitrogen content among three
freeze-dried loins is summarized in table 9. The F value of 9.11 was
highly significant at the 1% level. The Studentized range test indicated
that the non-protein nitrogen content of loin 2 was significantly different
from that of loins l and 3. The non-protein nitrogen contents of loins
l and 3 did not differ significantly. The variation in non-protein nitro-

gen content of the three loins could possibly arise from different amounts

of decomposition products of metabolism in the three animals.

-33-

Table 9. Analysis of variance for non-protein nitrogen content among
three freeze-dried loins.

 

Sums of Mean F
Source d.f. squares square value
Non-protein nitrogen
content 69 443.28
Loins 2 94.71 47.36 9.11**
Individuals 67 348.57 5.20

 

**P'< .01

The nitrogen content of the protein fractions extracted from the
fresh longissimus dorsi controls is shown in table 10. The sarcoplasmic
protein nitrogen, the 0.53 y.(KCl-bicarbonate) extractable protein nitro-
gen and the soluble fibrillar protein nitrogen fractions of loin 2 con-
tained more nitrogen than either loin l or 3. Loins l and 3 did not
differ significantly in the nitrogen content of the various protein frac-
tions. The differences observed in the non-protein nitrogen content may
be due to individual animal differences.

Table 10. The nitrogen content of the protein fractions extracted from
the fresh longissimus dorsi controls expressed as mg. N/g.

 

 

 

 

solids
Nitrogen content
Item. Loin 1 Loin 2 Loin 3
SarOOplaemic protein nitrogen 30.45 47.51 35.04
0.53 P.extractable protein nitrogen 36.98 57.74 39.11
Soluble fibrillar protein nitrogen 6.53 10.23 4.07
Non-protein nitrogen 14.31 13.95 16.47

 

The correlations between percentage rehydration and the nitrogen con-
tents of the extracted protein solutions are presented in table 11. There

was no significant correlation between percentage rehydration and sarco~

-34-

plasmic protein nitrogen, 0.53 p.(KCl-bicarbonate) extractable protein
nitrogen, soluble fibrillar protein nitrogen or non-protein nitrogen of
the three freeze-dried loins. Direct correlations of 0.541 for loin 2
(P'< .01) and 0.413 for loin 3 (P'< .05) were Obtained between percent-
age rehydration and the protein content of the rehydrating solution.

The correlation between percentage rehydration and the protein content
of the rehydrating solution for loin l was positive, but was not signi-
ficant. The pooled data, however, had a correlation of 0.461, which was
significant at the 1 percent level.

Table 11. Correlations between percentage rehydration and the nitrogen

content of the extracted protein solutions expressed as mg.
N/g. solids
Correlation coefficients

 

 

Pooled
Protein fractions Loin 1 Loin 2 Loin 3 data
SarCOplasmic protein
nitrogen +.224 -.07l '+.072 +.059
0.53‘p.extractable
protein nitrogen -.001 -.096 +.095 -.06l
Soluble fibrillar protein
nitrogen -.152 +.024 +.055 -.1l6
Rehydrating solution
protein nitrogen +.O62 +.541** +.413* +.461**
Non-protein nitrogen +.19 +.14 +.21 -.083

 

*Significant at 5% level
**Significant at 1% level

The trend established by loins 2 and 3 indicated that the greater

the percentage rehydration, the more proteins would be leached from the

-35-

freeze-dried fillets. The larger the amount of water reabsorbed by the
meat, the more proteins that could be extracted and transported into the
rehydrating solution. No statistically significant correlations were
found between the pH and the protein content of the rehydrating solution
for the three loins. However, when the data were pooled for analysis,

a correlation of-+.274, (P'< .05) was obtained. It may be concluded that
an interaction between percentage rehydration and pH of the rehydrating
solution may effect the protein nitrogen content of the rehydrating solu-
tions.

A comparison of the nitrogen content of the protein fractions ex-
tracted from fresh and freeze-dried loins is shown in table 12. The
sarCOplasmic protein fraction for all three loins noticeably decreased
in nitrogen content on freeze dehydration. As shown in table 3, a cer-
tain amount of protein nitrogen was leached out by the rehydrating solu-
tions. If one assumes the nitrogen content of the rehydrating solution
to be composed entirely of sarcoplasmic protein nitrogen, a decrease in
sarc0plasmic protein content on freeze dehydration is still evident.

The non-protein nitrogen content of all three loins decreased after
freeze-dehydration and rehydration had occurred. However, when the non-
protein nitrogen content of the rehydrating solution (table 3) is consi-
dered, the total non-protein nitrogen content of all three freeze-dried
loins was greater than that of the fresh controls.

Hamdy er al. (1959) reported a decrease in the water soluble nitro-
gen content of reconstituted beef, which had been freeze-dried at a plate

temperature of 43°C and a pressure of 1500 P.Hg. Freeze-drying at a

-35-

Table 12. A comparison of the nitrogen content (mg. N/g. solids) of
the protein fractions extracted from fresh and freeze-dried

 

 

 

 

 

loins
Nitrogen content 6mg. N/g. solids)
Loin 1 -Loin 2 Loin 3
Freeze- Freeze- Freeze-

Protein fractions Fresh dried Fresh dried Fresh dried

SarcOplasmic protein
nitrogen 30.45 26.93 47.51 27.23 35.04 28.04

0. 53 )1 extractable
protein nitrogen 36.98 35.07 57.74 33.48 39.11 35.99

Soluble fibrillar

protein nitrogen 6.53 8.29 10.23 6.57 4.07 8.08
Non-protein
nitrogen 14.31 12.88 13.95 10.72 16.47 13.91

 

’1Mean value of nitrogen content for freeze-dried loins is recorded.

plate temperature of 22-30°C and 300-400 p.Hg. chamber pressure resulted
in no detectable effect on the water soluble nitrogen content. Kronman
and Winterbottom (1960) stated that freezing of beef resulted in a de-
creased extractability of water soluble proteins, as well as in a loss
of specific electrOphoretic and ultracentrifugal components.

Results of the present study indicated that the sarCOplasmic protein
nitrogen content of pork decreased, when the fillets were freeze-dehydrated
at a 28-30°C plate temperature and a pressure of 150 p.Hg. Denaturation
of the sarcOplasmic proteins of pork appear to result from freeze-drying
as evidenced by a decrease in the concentration of the nitrogen content
of the sarOOplasmic protein fraction. The freeze—dehydration process
may in some way effect the bonds that are due to electrostatic interaction

between polar groups and to 'van der Waals forces between non-polar groups

-37-

of the meat proteins. Irreversible structural changes may occur which
influence percentage rehydration and the water holding prOperties of
pork.

The amount of 0.53 p (KCl-bicarbonate) extractable protein nitrogen
of reconstituted pork also decrdased. This is a composite protein frac-
tion consisting of both sarCOplasmic and soluble fibrillar proteins.
Since the sarc0plasmic protein fraction decreased, the decrease in the
0.53 p.extractable protein nitrogen was expected.

Due to the large standard deviation of the values obtained for the
soluble fibrillar protein nitrogen content of all three freeze-dried
loins, conclusions concerning the possible influences of freeze-drying
on the fibrillar proteins can not be resolved.

Correlations between the sarcoplasmic protein nitrogen content and
pH of the rehydrating solution or pH of the meat are expressed in table
13. A highly significant direct correlation between the pH of the rehy-
drating solutions and the nitrogen content of the sarOOplasmic protein
fraction was obtained for loins l, 2 and for the pooled data. Although
possitive correlations were found for loin 3, they were not significant.
The trend established for the pooled data, however, is not surprising
as the effect of pH on protein extractability is well-known.

Investigation of the influence of pH on the amino acid composition
of the rehydrating solutions was perfOrmed on loin 4. The qualitative
amino acid composition of the rehydrating solutions at pH 2.55. 5.85,
9.05 and deionized water was determined. The amino acids in the various

rehydrating solutions were fractionated (figure 3) into total amino acid

-33-

content (Hydralyzate 1), total non-protein nitrogen amino acid content

(Hydrolyzate II), and free amino acids (solution F).

Table 13. Correlations between the sarOOplasmic protein nitrogen con-
tent andng

Correlation coefficients

 

 

Loins
Pooled
1511 l 2 3 data
Rehydrating .
solution +.574** .646** +.370 +.483**
Meat '+.719** +.517** -+.030 '+.3l6**

 

**Significant at 1% level

A qualitative separation of the amino acids was achieved by utiliz-
ing one-dimensional descending paper chromatography. Photographs of the
chromatograms were taken and the results of this study are shown in fig-
ure35 and 6. The chromatograms indicated that .the rehydrating solutions
of deionized water (a), pH 5.15 (c) and pH 9.05 (d) were similar in their
qualitative amino acid composition. Thus, pH did not influence the
qualitative amino acid composition of rehydrating solutions. No attempt
was made in the present study to determine the quantitative amino acid
composition.

At pH 2.55 the rehydrating solution (b) contained a distinctly dif-
ferent qualitative amino acid composition than the other rehydrating
solutions. The ionic strength of the pH 2.55 rehydrating solution was
0.1, while all other rehydrating solutions investigated had an ionic
strength of 0.05. Therefore, a change in ionic strength of the rehydrat-

ing solution greatly influenced the fingerprinting of amino acids obtained

-39-

 

ring- ,_ 7 , -7 ___,_ , _ _7-_ 7 , _

 

 

Hydrolyzate I

Q...
" vow

 

L' .‘— ' ' ,fl—i '__‘—‘.,—".‘-.‘—'—- 7 "

 

Hydrolyzate 11

Figure 5. Chromatograms of the qualitative amino acid composition of
total amino acid content (Hydrolyzate I) and total non-pro-
tein nitrogen amino acid content (Hydrolyzate II},(a, b, c,
and d represent the rehydrating solutions: deionized water,
pH 2.55, pH 5.85, and pH 9.05, reSpectively.)

-40-

—
O
'L

 

,, _—__-—___———_-———- _ A

Solution F

1-

 

 

‘ -
V

 

. 1*
~-

 

 

Knowns

Figure 6. Chromatograms of the qualitative amino acid composition of
free amino acid content (Solution F) and known amino acids

(Knowns) (a, b, c, and d°represent the rehydrating solutions:
deionized water, pH 2.55, pH 5.85, and pH 9.05, respectively.)

-41-

for the total amino acid content (Hydrolyzate I), the total non-protein
nitrogen.amino acid content (Hydrolyzate II), and free amino acids
(Solution F). This is an expected result as ionic strength influences
protein solubility and thus, also influences the qualitative amino acid

composition.

SUMMARY AND CONCLUSIONS

Seventy freeze-dried fillets taken from the longissimus dorsi mus-
cle of three hogs were utilized in this investigation. The fillets
were rehydrated in buffers varying in pH in order to determine the in-
fluence of pH on percentage rehydration. The degree of protein denatur-
ation caused by freeze-dehydration was studied by comparing the nitrogen
content of the extracted protein fractions of the freeze-dried, rehy-
drated fillets with the respective protein fractions of the fresh
controls.

The percentage rehydration of freeze-dried pork ranged from 48.54%
to 92.41% with a mean percentage of 73.75% i 9.26. Freeze-dried pork
was found to rehydrate to a much lower level than beef. There was a
significant difference between the means for percentage rehydration of
the loins obtained from the three different hogs used in this study.

A direct correlation between pH of the rehydrating solution and
the pH attained by the rehydrated pork was observed for all three loins
in this investigation. However, percentage rehydration was not signifi-
cantly influenced by either pH of the rehydrating solution or pH of the
rehydrated meat. Thus, freeze-dried pork showed no Optimun pH for rehy-
dration.

An increase in the pH of freeze-dried pork was noted when the fillets
were rehydrated in deionized water. Acidic volatile losses during the

freeze-dehydration process were investigated and found to be reSponsible

-42-

-43-

for most of the changes in pH. If these volatiles were trapped and
utilized in reconstitution of the dried meat solids, the original pH
of the fresh meat slurry could be regained. The significant pH change
of freeze-dried pork on rehydration again indicated that freeze-dried
pork is dissimilar to freeze-dried beef in its rehydration characteris-
tics.

A correlation of only +.l68 between fat content and percentage
rehydration indicated that fat content had little effect on percentage
rehydration in pork.

Results indicated that there was no significant correlation between
percentage rehydration and sarc0plasmic protein nitrogen, 0.53 p.(KCI-
bicarbonate) extractable protein nitrogen, soluble fibrillar protein
nitrogen or non-protein nitrogen content. A positive correlation existed
between percentage rehydration and the protein content of the rehydrating
solution. Thus, the greater the percentage rehydration, the larger the
amount of proteins that are leached into the rehydrating solution.

The non-protein nitrogen content of freeze-dried loin 2 was signi-
ficantly different from that of freeze-dried loins l and 3. The non-
protein nitrogen content of the fresh controls also varied. On freeze-
dehydration, the total non-protein nitrogen content increased.

The sarCOplasmic protein fraction noticeably decreased in nitrogen
content on freeze dehydration and rehydration. Thus, apparent denatura-
tion of the sarOOplasmic proteins of pork resulted on freeze-drying.

Due to the large standard deviation for the soluble fibrillar protein

-44-

fraction, no conclusions concerning the possible influences of freeze-
drying on the denaturation of fibrillar proteins could be fonmulated.
The qualitative amino acid composition on reconstitution with
deionized water, pH 5.85, or pH 9.05 rehydrating solutions was similar.
A dissimilar composition was indicated for the pH 2.55 rehydrating
solution. The pH 2.55 buffer had an ionic strength of 0.1, while all
others studied had an ionic strength of 0.05. Therefore, pH did not
influence the qualitative amino acid composition of the rehydrating
solutions. A change in ionic strength, however, greatly influenced

the qualitative amino acid composition of rehydrating solutions.

LITERATURE CITED

American Instrument Co. 1961. The determination of nitrogen by the
Kjeldahl procedure including digestion, distillation and titration.
American Instrument Co., Reprint No. 104.

Auerbach, E., wang, H., Maynard, N., Doty, D. M., and Kraybill, H. R.
1954. A histological and histochemical study of beef dehydration.
V. Some factors influencing the rehydration level of frozen-dried
muscle tissue. Food Res. '12, 557.

Bailey, K. 1954. Structure proteins. 11. Muscle. The Proteins. Vol.
II B, 854. H. Neurath and K. Bailey, eds., Academic Press, New
York.

Bailey, K. 1956. .Muscle Proteins. Brit. Med. Bull.‘1g, 183.

Biochemists' Handbook. 1961. Cyril Long, editor. D. Van Nostrand
Company, Inc., New York.

Brooks, J. 1958. The structure of the animal tissues and dehydration.
Fundamental Aspects of the Dehydration of Foodstuffs. Society of
Chemical Industry, London, p. 8.

Bull, H. B. 1944. Adsorption of water vapor by proteins. J. Amer.
Chem. Soc. 66, 1499.

Cole, L. J. N. 1962. A comparison of the effects of freezing and dry-
ing on the rehydratability of freeze-dried beef. p. 217. Fisher,
F. R., ed., Freeze-Drying of Foods, National Academy of Sciences -
National Research Council. 'Washington 4, D. C.

Cole, L. J. N. and Smithies, W. R. 1960. Methods of evaluating freeze-
dried beef. Food Res. 22, 363.

Connell, J. J. 1957. Some aspects of the texture of dehydrated fish.
J. Sci. Food and Agric..§, 526.

Connell, J. J. 1958. The effect of drying and storage in the dried state
on some prOperties of the proteins of food. Fundamental Aspects of
the Dehydration of Foodstuffs. Society of Chemical Industry, London,
p. 167.

Deatherage, F. E. and Hamm, R. 1960. Influence of freezing and thawing
on hydration and charges of the muscle proteins. Food Res. 25, 623.

-45-

-46-

Doty, D. N., Weng, H., and Auerbach, E. 1953. Dehydrated foods: Chem-
ical and histological prOperties of dehydrated meat. J. Agr. and
Food Chem. 1, 664.

Dyer, W. J., French, H. V. and Snow, J. M. 1950. Proteins in fish muscle.
1. Extraction of protein fractions in fresh fish. J. Fish. Res. Bd.,
Can. _7_, 585. ‘

Flosdorf, E. W. 1949. Freeze-drying (Drying by Sublimation) Reinhold
Publishing Co., New York.

Gooding, E. G. B. and Rolfe, E. J. 1955. The vacuum contact-plate
dehydration of foodstuffs. I. A first appraisal. J. Sci. Food
and Agric. .6, 427.

Gooding, E. G. B. and Rolfe, E. J. 1957. Some recent work on dehydration
in the United Kingdom. Food Tech. 11, 302.

Greaves, R. I. N. 1954. Theoretical aspects of drying by vacuum Subli-
mation. p. 87. Harris, R. J. 0., Bd., Biological Applications of
Freezipg and Drying, Academic Press, New York.

Hall, J. L. 1953. Methods of estimating degree of fatness in carcasses
and cuts. Ether extraction.method of estimating degree of fatness
in carcasses and cuts. Proc. Recip. Meat Conf. .1, 122.

Hamdy, M. K., Cahill, V. R., and Deatherage, F. E. 1959. Some observa-
tions on the modification of freeze dehydrated meat. Food Res.
24, 79.

Hamm, R. and Deatherage, F. E. 1960. Changes in hydration and changes
of muscle proteins, during freeze-dehydration of meat. Food Res.
.25, 573.

Hankins, O. G., Ernst, A. J., Kauffman, W. R. 1946. Chemical composition
of raw and dehydrated meat. Food Res. 1}, 501.

Harper, J. C. and Tappel, A. L. 1957. Freeze drying of food products.
.Academic Press, New York. Advances in Food Res. _1, 172.

Harrington, G. and Pearson, A. M. 1962. Chew count as a measure of
tenderness of pork loins with various degrees of marbling. J. Food
Sci. 21, 106.

Hegarty, G. R. 1963. Solubility and emulsifying characteristics of
intracellular beef muscle proteins. Unpublished Ph.D. thesis,
iMichigan State University.

Hunt, S. M. V. and.Matheson, N. A. 1958. The effects of dehydration on
actomyosin in fish and beef muscle. Food Tech.qlg, 410.

-47-

Kronman, M. J. and Winterbottom, R. J. 1960. Post mortem changes in
the water-soluble proteins of bovine skeletal muscle during aging
and freezing. J. Agric. Food Chem. 8, 67.

Luyet, B. J. 1962. Effect of freezing rates on the structure of freeze-
dried materials and on the mechanism of rehydration. p. 194. Fisher,
F. R., ed., Freeze-Drying of Foods, National Academy of Sciences -
National Research Council. washington 4, D. C.

Ministry of Agriculture, Fisheries and Food. 1961. The Accelerated
Freeze-Drying (AFD) Method of Food Preservation. London.

Moffat, E. D. and Lytle, R. I. 1959. Polychromatic technique for the
identification of amino acids on paper chromatograms. Anal. Chem.
31, 926.

Orme, L. E., Pearson, A. M., Bratzler, L. J., and Magee, W; T. 1958.
Specific gravity as an objective measure of marbling. J. of Animal
Sci. E, 693.

Pearson, A. M., Harrington, 6., west, R. G. and Spooner, M. E. 1962.
The browning produced by heating fresh pork. 1. The relation of
browning intensity to chemical constituents and pH. J. Food Sci.
Egg 177.

Seagran, H. L. 1958. Contribution to the chemistry of the king crab.
Comm. Fisheries Rev. 11, 15.

Sherman, P. 1961. The water binding capacity of fresh pork. 1. The
influence of sodium chloride, pyroPhosphate, and polyphosphate on
water absorption. Food Tech. 15, 79.

Snedecor, G. W. 1956. Statistical Methods. 5th ed. The Iowa State
College Press, Ames, Iowa.

Tappel, A. L., Conroy, A., Emerson, M. R., Regier, L. W., and Stewart,
G. F. 1955. Freeze-dried meat. 1. Preparation and pr0perties.
Food Tech. 2, 401.

Taylor, J. F. 1953. The isolation of proteins. The Proteins IA, p. 29.
H. Neurath and K. Bailey, eds., Academic Press, New York.

 

Turner, E. W. 1956. The future of dehydrated meat as a convenience
food item. Proc. of the 8th Res. Conf., American Meat Institute,
Chicago, p. 37. .

wang, H., Andrews, F., Rasch, E., Doty, D. M., and Kraybill, H. R. 1953.
A histological and histochemical study of beef dehydration. 1. Rate

of dehydration and structural changes in raw and cooked meat. Food
Res. 18, 351.

Whng, H., Auerbach, E., Bates, V., Doty, D. M., and Kraybill, H. RE 1954.
A histological and histochemical study of beef dehydration. IV.
Characteristics of muscle tissues dehydrated by freeze-drying tech-
niques. Food Res. 12, 543.

APPENDIX

-43-

Appendix A. Composition of rehydrating solutions.

Reference: Biochemists Handbook 1961

l. Hydrochloric acid egpotassium chloride

25°C, I a 0.1 A ml. 0.2 MI- HCl +’C ml. 0.2 M - KCl, diluted to l 1.

_al. A_ _c__
2.20 42 458
2.41 25 475
2.80 10 490
3.11 5 495

2. Acetic acid - sodium acetate

25°C, I - 0.05. A ml. M - acetic acid-+ 50 m1. M - NaOH diluted to

1 liter.
in. A
3.6 650
3.8 428
4.0 288
4.2 200
4.4 145
4.6 110
4.8 87.7
5.0 73.8
5.2 65.0
5.4 59.5
5.6 56.0

5.8 53.8

-49-

Appendix A. Composition of rehydrating solutions. (continued)

3. Potassium dihydrggen phOSphate - disodium hydrogen phosphate .

25°C, I = 0.05. A ml. 0.5 M - KH2P04'+ B ml. 0.5 M - NazHPO4 diluted

 

 

to 1 1.

pH;_ A B
6.0 74.2 8.58
6.2 64.6 11.8
6.4 53.4 15.5
6.6 42.0 19.3
6.8 31.4 22.8
7.0 22.4 25.8
7.2 15.4 28.2
7.4 10.3 30.0
7.6 6.74 31.0
7.8 4.36 31.8
8.0 2.80 32.4

4. Sodium bicarbonate - sodium carbonate

25°C, I = 0.05. A ml. M.- NaHCO3 +'B m1. M - Na2C03 diluted to 1 1.

LE- _A_ .11.
9. 39.8 3.41

9.2 35.5 4.83

-50-

Appendix B. Colors given with the ninhydrin-cupric nitrate Spray of
MOffat and Lytle (1959). (The order in which the amino acids
are listed is also the order in which they appear on descend-
ing chromatograms.)

Cystine Gray

Lysine Reddish brown, pink ring forms on standing

Histidine Light brown with dark brown ring inside a yellow ring
Asparagine Golden

Arginine Dark purple

Serine Greenish brown, red ring forms on standing

ASpartic acid

Glycine
Threonine

Glutamic acid

Light blue (if removed from the oven too soon, the
asPartic acid Spot will be bright green.)

Orange brown with bright orange ring
Greenish brown, changes to purplish brown on standing

Purple, fades slightly on standing

Alanine Dark purple

Proline Light green with yellow ring

Cysteine Gray

Tyrosine Light brown

Valine Purple

Methionine Grayish purple with yellow ring

TryptoPhan Brown with bright blue ring, ring fades rapidly
Isoleucine Light blue

Phenylalanine Greenish yellow

Leucine Light purple with yellow ring

-51-

Appendix C. Formulas used in calculations.

Percentage moisture in rehydrated Samples: ‘Mr

% moisture (Mr) - (Mf x Wf) ' ("f " Wd) + (Wr ' Wd) x 100
Wr

1'

where: 'Mf percentage moisture in fresh loin
Wf = weight of fillet prior to freezing (g)

- weight of fillet after dehydration (g)

of
l

weight of fillet after rehydration (g)

2
ll

Percentage fat in rehydrated samples: Fr

 

‘7. fat (Fr) = fliiaflfl x 100
r

where: Ff percentage fat in original loin
Wf = weight of fillet prior to freezing (g)

Wr = weight of fillet after rehydration (g)

Weight of solids in rehydrated or fresh sample: WS

 

Weight of solids (WS) = S - Ser)- S(Fr)
(rehydrated) = S (1 -‘Mr - Fr)

sample weight (g)

where: S

M1. = percentage moisture in rehydrated sample

Fr percentage fat in rehydrated sample.
Weight of solids (fresh) is obtained by substituting Mf and Ff in the above
formula where

IMf = percentage moisture in fresh sample

Ff = percentage fat in fresh sample

-52-

Appendix C. Formulas used in calculations. (continued)

Percentage rehydration of freeze-dried meat.

% rehydration = Wr ' Wd X 100
wf-wd

where: Wr weight of fillet after rehydration (g)
Wd = weight of fillet after dehydration (g)

Wf = weight of fillet prior to freezing (g)

Total fibrillar protein nitrogen content of solution A.

Total fibrillar protein nitrogen a AP =

 

Xf mg N2 xV m1 x S g. a Xf V mg N2
ml 3 g. WS g W3 g solids

where: Xf fibrillar protein nitrogen content of solution A (mg N2/ml.)

V volume of 0.1 N NaOH (m1.)

S = sample weight (g)

WS = weight of solids in rehydrated (or fresh) sample (g)

Total water soluble protein nitrogen content of solution B.

Total water soluble protein nitrogen = Bn =

Xw mg N2 X 200 m1 extracting solution X S g = EH (2002 (mg N2 )
ml S g Ws g Ws g solids

where: Xw - water soluble protein nitrogen content of solution B
(we Nz/ml)

S = sample weight (g)

Ws a weight of solids in rehydrated (or fresh) sample (g)

-53-

Appendix C. Formulas used in calculations. (continued)

Non-protein nitrogen content of solution C.

Non-protein nitrogen = Cn =

Xc mg N2 X 1.33 X 200 ml extracting solution X S g

= x. (266.67) (mg N2
WS g solids

 

where: Xc = non-protein nitrogen content of solution C (mg Nz/ml)
S = sample weight (g)

ws

weight of solids in rehydrated (or fresh) sample (g)

Total salt soluble protein nitrogen content of solution D.

Total salt soluble protein nitrogen = Dn =

 

XS 98 N2 X 200 ml extracting solution X S g
ml

3 8 Ws g
=x 200 (mg N2
WS g solids

where: XS = salt soluble protein content of solution B (mg N2/ml)

S - sample weight (g)

Ws = weight of solids in rehydrated (or fresh) sample (g)

Total protein content of the rehydrating solution (RP)

Total protein content of the rehydrating solution = R9 =

m1 Wr g W3 g Wr W3

(mg N2 3
g solids

-54-

Appendix C. Formulas used in calculations. (continued)

where: Xr = rehydrating solution protein content (mg N2/m1)
Sr = sample weight of rehydrated meat (g)
Wr = weight of fillet after rehydration (g)
Wd 8 weight of fillet after dehydration (g)

WS = weight of solids in rehydrated sample (g)

Non-protein nitrogen content of the rehydrating solution: REP

Non-protein nitrogen content of the rehydrating solution: RPP =

an mg N2 x 1.33 [150 - (Wt - Wdfl m1 x Sr -
ml Wr 8 WS 8

 

1.33 (Km, Sr)(150 - w. + wd) (mg N2 )
‘Wr WS g solids

where: an rehydrating solution non-protein content (mg Nz/ml)

Sr = sample weight of rehydrated meat (g)

2:
H.
I

- weight of fillet after rehydration (g)
Wd = weight of fillet after dehydration (g)

WS = weight of solids in rehydrated sample (g)

Actual protein content of rehydrating solution.

Actual protein content = (Rn - REP) mg N2 )
g solids

Where RR = total protein nitrogen content of the rehydrating solution
(mg N2/g solid)

REP = non-protein nitrogen content of the rehydrating solution
(mg N2/g solids)

Hmm.+ wHH.+ aw¢¢.+

3o. Vv m .3.

Amo. Vv m a.

noa.t mom.+ *Rmmm.+ Noo.+ ohm.+

.... *Rwow.+ oom.+ 50H.1
0... NOHO+

mom.+ com
nouuwu oHououmaooo
dowunaom wawumnvhnom
woo.+ mm~.+ oom.+ mqa.u «mm.n
NmH.+ «no.+. qwo.w o~a.+ nom.u
eamqm.u omH.u

dwououn HOOOH

am¢¢.t caboose
HwHHHunfim HouOH

samno.+ Noo.+ amm¢.+

-55..

mmd.+ nowouuwa
owououm noau
unaom wowumuousom
.... tsmom.+ Nom.- NmH.- moN.- ma~.- nowononn anooono
HOHHHHnHm OHAOHom
.... wm~.+ Hoo.- me.+ moo.+ nowonono onooono

manouomuuxo A mm.o

.... ¢~N.+. seman.+ seamm.+

nowouuwo damn

tone owammamooumm
awouoaa

«co.- nam.a
demuoum Gowouuwa nowouuwa
HouOH amaaaunwm awououm

Houoa

doaumnuhnou N
oHououe
coaunaom

nowouufia

toam.+ note an
comouuwo oowuouu umoa Summon
ufiouone oHououe uhfimm we we
Hmaaaunfim OH wuowuuxo oaamman N
mowuonphnmm mannaom . nn.o tooumm

.H SHOH mo muouomm pouowaumo>aa Ham consume moowuoHouuoo

.Q Nemaonm<

-57-

30. Vv m in

 

 

Go. vv m 1.
n8 .- 89+ 33 .+ n: .+ m8 .. 8m .- omo.+ mmo.+ H8 .. non-83o
oaououmuooo SOHO

uSHOm wowuonwmnom
o o oo **©Hmo+ NNNOI wo~.+ *mN‘NOIT N¢N 0+ NmH 0+ mmNol mONoI HHHHQUOH“ HmUOH.

.... o-.: mmm.+ aoo.+ mo~.u mNH.+ oam.u amoq.u awououe
HOHHHHAHM HMOOH

.... oma.u Hma.u Nmo.u emH¢.+ mmN.+ Ho¢.+ cowouuao naououa
ooaunHOm wowuouvhsmm

. o o o yo®©¢ 0+ wON ol mmOo-T me ol HNM ol HHGWOHUHH— d—Hmu—OHQ

Hmaaaunwm oaeoaom

. . . . .33.: .+ 39+ to..- a: .+ none-Sn anon-8o
magnuoouuxo & mm.o

.. .. N8 .+ omo.+ in .+ sows-on.- anon-on.-

OHEmOHmooumw
.... noo.1 Hmo.+. coauonmhnou N
o o o o toqw 0+ ““08 mg

aqueoum nfiououn dowoaufio nwonuHa comouuwa dowouuwc dowumuo uoofi Mommas
HOOOH Hmaaaunam awououa owououe aaouoad uwouona theom mm mm
Hence coauoaon umaaaunam oanmuomuuxo OHEmmHa x
moaumuvknmm manoaow 1 mm.o nooumm

 

.m GHOH mo mHOuOmm wouowwumo>aw Ham consume moowumaouuoo

om anoond<

-59-

AmvfiHOm .m\Nz .wfivt

 

nn.nn 4n.4H an.na nn.4a nn.~n 4a.na nn.na nn.na inononono ononono-ooz .o

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aoHunHom wawumupmfium .w
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nn.an nn.nn 4o.non na.no 4n.non nn.4n on.4on an.nnn toononono
. .nnononn nnannnnnn annoy .n

na.~ an.n NH.~ oo.n nn.n nn.~ oo.a 4H.H toonononn onooono
doaunHOm wowuonpmnum .m

nn.n -- an.n 4n.a nn.an nn.a an.o~ an.n «nononono anooono
Hosannnnn nanoaon .4

an.nn no.a~ n~.on n~.on nn.o4 no.4n 4n.~4 on.nn tan.nn.oO nononona
oHououa mannaom uHmm .m

nH.on oo.n~ «n.0n an.n~ no.n~ a4.n~ mm.HN nn.~n noononoao
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na.no na.nn 4n.oa nn.ao nn.nn on.na on.nn na.na nononnnnnon a .H
nn.n nn.n no.n no.n nn.4 nn.4 nn.4 nn.4 none no
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u n ma AH Hm m N a Honanz magnum

 

.H GHOH wowuwtouooum mo «whom mnawmmflwmoa age you dump wouoanoamo ouoanaoo

 

.m xwuaoam<

-60-

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nwououd mannaom uaom .m

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.m newcome<

-61-

AanHOm .w\Nz .maoa

 

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o4.n on.4 oo.n oS.4 n~.4 a~.4 s nononono onooonn-ooo
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nonaononu ononaon .4

oo.~n oo.o4 o4.4n nn.44 4a.on no.o4 tag no.oo nononono
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owououm oaemmamooumm .N
mm.Ho 4o.n4 nn.na H4.no oo.oo no.no nonoonnnoon e .H
oo.n oo.o on.o .no.o oo.o Ho.o onoa_no
one nonnnonon nH.o na.n «5.5 na.a nonnno an
an an NH n on on nooano onoaon

 

AmonafiuaOOV

 

.H caoH vowuvnonooum mo Hmuon mnEHmmwwdoa win you dump moumHnOHoo Ouonaoo

.n snoooooa

-62-

Son-8 .knz .ne.

 

 

 

H~.0H m4.4H NN.NH m~.~H an.oa 4m.~a 4H.NH oo.oH soowouuao oaououauooz .m

oh.o Hm.4 NN.m Nn.4 m4.¢ ow.4 Nm.o mN.n soowouuao nwouountnon
aoaunHOm mowumuvhsom .w
n¢.m~H n4.mma no.4ma oo.mmH no.moH wo.m0H mm.~ma oa.nma .snowonuau owououa Houoa .n

oH.4m oo.om 4m.om mo.om 44.Hn n~.oo ow.mm m~.m~ oowouuao
tonooono noaannonu Hoooa .o

nn.n am.m ma.m ma.~ an.~ on.~ mo.a 4o.H soononuae cannona
coauSAOm wnfiuoawhnum .m

mo.mH nu Hm.oH 44.H mH.o NN.H mm.m mw.m soowouuwo oaouonn
nonnnnnnn onooaon .4

4o.n4 aa.on na.nn on.on oa.on no.4n on.o~ o~.4~ SA: no.oo nononono
owououd mannaom uaom .m

oo.on oo.nn Nn.nn on.nn Ho.ou ao.~n ao.nn n4.on «nononono
caboose owanoanoouom .N
4m.Hm H4.~n mm.nn a4.~n no.Hn 4n.4n oo.on a4.oo aoaonnoneon a .H
mn.m m~.m om.m 04.m mm.4 no.4 o~.4 nn.¢ unoa_mo
on.n on.o on.4 no.4 na.n oa.n oo.n oo.n nounno no
on H n n o 4 on nH nooenn onoaon
.N aHOH pownwtouooum mo HwHOO mnafimmwwdoa onu you dump tonnanoamo ouoameoo .H Ravaomgd

-63-

Anowaom .wNNz .weva

 

 

 

4H.HH no.on an.na 44.HH n4.HH on.n on.HH oo.HH noononono onooonn-ooz .o

4n.o on.o 44.4 o~.o no.n on.o no.n oo.n noononono noooonn-ooo
uoaunHOm unqumuwhsum .w
no.nNH 4S.H~H oo.onn oo.nnn no.onn oo.nnn 4H.Hna n4.nnn «nononono onooonn annoy .a

oh.na n4.4a nn.na n~.on 4a.no an.4a Ho.4n no.oo tnononono
. nnooono nonaonoon Hoooa .o

no.4 oa.n on.~ on.n nn.~ on.~ o4.~ no.n snononono onooonn
coauSHOm wnaumupmfimm .m

oo.o no.n -- oo.n n~.oa Ho.o no.nn oo.o soononnno nnooonn
nonannonn ononnon .4

no.4n an.an 4o.on an.nn an.nn nn.nn nn.o4 an.on tfimno.oo nononoao
owouoad oHebHon uHmm .m

4o.nn 4n.on an.on n4.4n o4.nn ao.nn oo.nn Ho.o~ «nononono
owououm owemoamooaom .N
on.nn oa.4n n4.ou mm.Hn nn.oa oo.na no.na ~a.4n nononnonnon S .H
nn.n on.n no.n oo.o nn.n on.o on.o on.o noon no
on.a on.a on.a oH.S a.o a.o OH.o oH.o nonnno no
on Na on an nu a 4H on nooann oaoann
AnuncwuaooV .N QHOH mowuutonooum mo “anon mosqmmwmnoa OSu you wont woundsoamo ouoanaoo .H Nawdoem¢

-64-

Amunaom .w\Nz .wEvs

 

 

 

4n.on nn.o an.4n no.nn on.n on.o 4n.HH no.o .ooononono onooonn-ooz .o

no.4 on.o an.n no.4 nH.o nn.n o4.n nn.n .ooononono onooonn-ooo
nonusaom wcaumuvmeom .w
oo.nnn ao.onn oH.n4H no.onn an.o~H Ho.nna no.onn 4n.ao n-oononono onooonn noooa .a

oo.oa no.oo nn.nn on.nn no.4o oo.oo n4.oo no.on n-oononnno
. onooono nonnnnonn Hoooa .o

oa.~ H~.n nn.n o~.~ oo.n Ho.n on.H 4n.n -.oonononn noooono
nonunHOm onuOHO550m .m

~4.nn on.nn -- an.n 04.0 on.n no.a on.o n-oononono onooono
nonnnnonn ononnon .4

on .on an .4n no .8 4n§n nn .nn an .on on .04 4n.on s1 no.8 nononon-
Someone OHeoHon uamm .m

o4.nn 4o.nn nn.on on.on no.o~ no.on on.nn oo.on n-oononono
onououd owammHaoouom .N
o~.nn nn.~n no.nn oo.no no.nn on.~o no.no no.oo nonnonoonon a .H
n4.n nN.n on.n o~.o on.n nn.n on.n nn.n none no
one nonnoonoo nn.o nn.o no.n no.o no.o no.o nonnno no
no on 4n an o o an n nooano onoann
AponoHOGOOv .N oHoH poaupuouoouw mo «whom moefinnnwooH man you some noumflnoaoo ouoaeaoo .H Noncomd<

-65-

notoa0m .wxmz .wava

 

no.4n o4.nn nn.nn 4n.nn nn.an no.nn o~.na no.nn o nononono onooono-noz .o

Hm.m n~.4 4n.4 ma.» ~4.m na.m nH.m ~4.o a nowouunu unonoua-aoo
GOHOSHOm woauouphnmm .w
on.Hnn an.nnn 4o.4nn on.onn on.nnn no.onH no.44n no.nnH noononono onooonn Hooon .o

Ho.n4 Ho.no ~n.4n 4n.on on.on nn.nn n~.no 4o.no snonononn
. onooonn nonnnnonm Hoooa .o

na.n an.n nn.n n4.n on.~ nn.n on.H nn.n noononono nnooono
nonunaon wanuoupheom .m

-- no.nn on.o nn.n nn.o nn.on 44.nn o~.oH a nononono onooono
noannnonn ononnon .4

H4.4n oo.n4 on.nn no.nn no.nn nn.on no.an nn.on no; nn.no nononono
Someone manaaom uHmm .m

4o.nn on.nn nn.on oo.nn 4o.nn on.nn no.4n an.nn tnononooo
nnmuoun unamoanoonom .N
nn.oo nn.no o4.4o NH.nn no.no o4.oo o4.nn nn.nn nononnoonon e .n
nn.n na.n no.4 no.4 oo.4 no.4 no.4 oa.4 none no
nn.n nn.n no.4 no.4 no.n no.n no.n on.n nonnno no
on o no on on n n on noose: oaoann

 

.m SHOH nonsptouooum mo Hmnou manammwwooa one you memo OOOOHSOHOO ouoamaoo

 

.h Moonomm<

-66-

Anpnaom .w\~z .wavs

 

nn.on 4o.on no.4n n4.on oo.o oo.nn oo.o nn.on inononnno onooono-ooz .o

n4.o nn.o nn.o nn.4 nn.o oo.o on.n on.o n-nononono anooono-ooo
nonunHOn monumHO%£mm .w
on.nnn nn.onn on.onn no.nnn nn.nnn nn.nnH no.onn nn.oo neononooo onooonn Hooon .o

nn.no nn.no 4n.4o nn.an Ho.nn nn.oo 4n.no 4o.n4 inononono
. nnouonn nonnnnonn annoy .o

n4.4 n4.~ no.o no.o nn.~ no.o nn.n no.o noonononn onooono
nonunHOm wonuouo55Om .m

44.n no.o no.4 4n.n nn.nn ne.n Ho.nn n4.o n.oononono onooono
nonnnnonu onooaon .4

Ho.nn 4n.nn nn.on nn.on oo.Hn nn.on oo.n4 oo.n4 no; nn.nv nononnno
someone OHADHOm uaom .m

no.4n an.nn nn.on no.4n 4n.n~ an.nn nn.on no.nn noononono
awououd OHEmOHdooumm .N
4o.~n oa.no nn.no a4.4o no.4o oo.oo Ho.na Ho.no nonoonoonon a .H
no.o no.o oo.o nn.o nn.o nn.o nn.o nn.o oooa no
no.“ nn.o nn.o nn.o no.o no.o no.o no.o nonnoo no
4H n 4n no on an on a nooann onoaon

 

 

AmOOoHOGOOV .m onoH Ocean-onooum mo Hmuov mneHmmeooa ago How ammo OOOMHSOHMO ouoHanu

.n noononna

-67-

Anonnon .n\~z .neot

 

Ho.nn no.4n anoann on.4n nn.nn on.nn nn.nn nn.nn noononono noooono-ooz .o

nn.o nn.o no.4 no.o no.o o4.n nn.o 4n.4 toononooo cannonn-oon
nonusaom woauoupnnom .w
nn.nnn 4n.oHH no.4nn on.nnn n4.o4n nn.nnn 4n.nnn 4o.onn noonononn onooono Hooon .o

nn.nn 4n.na oa.4n nn.oo on.nn on.na on.no n4.no noonononn
. nnooonn nonannonm Hoooa .o

an.n nn.o nn.o no.o o4.n no.o no.o na.n noononnno noooono
GOHuSHOn wanuouomaom .m

on.aH oo.o nn.o o4.nn no.4 no.nn no.4 nn.n «nononono noooonn
nonnnnonn onoonon .4

on.nn 4o.4n nn.nn on.n4 no.on oo.oo no.nn nn.nn nan nn.nn nonononn
uaouonm OHQSHOm uHmm .m

54.nn no.nn 4n.on no.on nn.nn nn.nn on.nn nn.nn «nononono
compose OHEmdeooumm .N
nn.oa no.no 4n.no 4o.4o 4n.no nn.nn on.no o4.oo nonoonoonon S .H
no.o no.o nn.o on.o n4.o nn.o no.o no.o none no
one nonnoonon nn.o nn.o on.“ nn.o no.o no.5 nonnoo no
nn n on on on on on nn noosno onoaon

 

 

AwmsoquOOV .m GHOH nonstaonooum mo “whom mnafimmawnoa man you upon nonnanoamo ouoH@Eoo

.h wwwnomm<

I I‘ll '1 .1 '1 .ll 1 I 1'

 

 

 

 

 

 

 

 

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MICHIGAN STATE UNIVERSITY LIBRARIES

I III III Illlllllll

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