ABSTRACT

RECOVERY AND BIOLOGICAL VALUE OF PROTEIN F R O M WA S T E
EFFLUENT OF POTATO CHIP PROCESSING METHODS
OF REC O V E RY AND BIOLOGICAL VALUE AS A F FECTED BY VARIETY AND TREATMEN T
By
Erhard Meister-Clemons

Simple physical-chemical methods for the recovery of p r otein from
w a ste effluent of potato processing were investigated.

The nutrition a l

value of precipitated protein of simulated wa s t e effluent and from w h o l e
tubers of three varieties from which it was made was biologically and
microbio l o gically assessed using weanling voles

(Microtus p e n n s y l v a n i c u s )

and Streptococcus z y n o g e m e s .
Heat application

(98°) at pH 4 to 4.5 or adjustment of pH w i t h

F e C l 3 to pH 4 yielded highest amounts of recoverable protein.
alone

(pH 3 to 3.5) or addition of lime

the pH to 9 with either H 3 P O 4 or F e C ^

(to pH 11.5)

A c i d treatment

followed by lowering

were almost as effective.

Sedimentation of protein was accomplished by settling for one h o u r
or b y centrifugation.

There appeared little difference in the amount of

protein removed by centrifugational forces up to 10,000 x G with only
minimal gains from 10 to 40,000 x G.
Protein efficiency indices

( PEI), determined in the vole assay,

approached those of casein and were equal or better than PEl's of the whole

Erhard M e i ster - C l em o n s

tubers.

The microbiological assay gave similar results, w i t h "biological

values" ranging from

66

to 75.

In both assays heat and/or acid treatment

was least damaging to protein quality.
It was estimated that a third of the crude protein in the w a s t e
effluent or approximately 130 kg of dried protein/day could be easily
recovered in an average potato chip plant.
was e x c e l l e n t .

The quality of the pro t e i n

RECOVERY AND BIOLOGICAL VALUE OF PROTEIN
F ROM W A S T E EFFLUENT OF POTATO CHIP PROCESSING
METHODS OF RECOVERY AND BIOLOGICAL VALUE
AS AFFECTED BY VARIETY AND TREATMENT

By
Erhard Meister-Clemons

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

MA S T E R OF SCIENCE

Department of Crop and Soil Sciences
1975

ProQ uest Number: 10008721

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uest
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ACKNOWLEDGMENT

The author wishes to express his sincere appreciation to his m aj o r
professor, Dr. N.R.

Thompson,

for his guidance throughout this study.

The experience and opportunities received through involvement in many
aspects of potato processing w e r e greatly appreciated.

My gratitude

goes to Drs. E.C. Elliott, D. Penner,

0. Mickelson,

and S. Wellso for their time, advice,

laboratory facilities and help in

the revision of the draft.

C. Cress,

C.M. Har r i s o n

I would like to thank Mr. R. Kitchen for his

technical assistance during the conduct of this work.

TABLE OF CONTENTS
P age
LIST OF TABLES.

.

.

.

.

.

.

.

.

.

v

LIST OF FIGURES

.

.

.

.

.

.

.

.

.

vi

INTRODUCTION
C HAPTER 1.
I.

R E VIEW OF LITERATURE

.

.

Potatoes: General Considerations
A.

II.

.

.

.
.

.
.

.

Historical Background of Potato Processing
Pollution Problems of the Potato Industry .

A.
B.

III.

F.
G.

C.

.

3

.

.

.

3

.

.

.

.

.

History of Pollution in the Potato Industry.
.
Characterization of Processing Wastes from Chip
Manufactoring
.
.
.
.
.
.
.
.
.
.
.

3
.

4
.

.

Nitrogen Containing Substances of the Potato
.
Nutritional Value of the Nitrogenous Substances
of the Potato
.
.
.
.
.
.
.
.
.
.
Protein Evaluation Methods .
.
.
.
.
.
.

.

5
6

.

Biological Treatment of Wastes .
.
.
.
.
.
Potato W aste for Fuel Production
.
.
.
.
.
Agricultural Use of Waste
.
.
.
.
.
.
.
W aste Effluent as Culture M edia for Micro-organisms.
Primary Treatment of Effluentts for the Recovery
of Solid Waste .
.
.
.
.
.
.
.
.
.
Recovery of Starch .
.
.
.
.
.
.
.
.
Physical-Chemical Treatment for the Recovery of
Suspended and Dissolved Solids .
.
.
.
.
.

LITERATURE CITED

1

.

Composition and Quality of Crude Potato Protein
A.
B.

.

.

Disposal and Utilization of Potato Wastes
A.
B.
C.
D.
E.

IV.

..........................................

.

7
7
8
8
8

9
9
10
12
12
14
15

16

iii

Page
C HAPTER 2.

PHYSICAL-CHEMICAL METHODS FOR THE RECOVERY OF
PROTEIN FROM W ASTE EFFLUENT OF POTATO CHIP
P R O C E S S I N G ..........................................

20

A B S T R A C T ........................................................

20

INTRODUCTION

21

.

.

.

M A T ERIALS AND METHODS
I.

.

.

..........................................

Preparation of Protein Water

II.
III.
IV.

Analytical Procedures

.

.

.

23

.

23

Separation of Precipitates

.

.

.

Treatments for Protein Precipitation

.
.

RESULTS AND DISCUSSION.
L ITERATURE CITED

.

.

23

.

.

.

2

4

.

25

.

.

26

.

.

40

'
jr

CHAP T E R 3.

ABSTRACT

PROTEIN QUALITY OF PRECIPITATE FROM EFFLUENT OF
POTATO CHIP PROCESSING MEASURED BY BIOLOGICAL METHODS
.

.

.

.

.

.

42

I N T R O D U C T I O N ..................

.

MATERIALS AND METHODS

.

.

.

RESULTS AND DISCUSSION.
L ITERATURE CITED

A PPENDICES

.

.

.

42

.

.

.
.

.

.

43
44
48

.

56

.

58

iv

LIST OF TABLES

Page

CHAPTER 2
1.

2.

3.

4.

5.

Chemical analysis of water taken from Michigan
State University Campus wells .
.
.
.
.
A verage composition of potato effluent of two
local potato chip plants
.
.
.
.
.
.

27

.

.

27

Residual crude protein (ppm) in solution after gravity
settling of protein from waste w ater of low and high
c oncentration at pH 4, with and without heat treatment.

.

Composition of crude protein of Russet Burbank potatoes:
w h o l e tubers, distilled and tap water extracts.
. . .
Residual crude protein (ppm) in solution after treatment
of protein water with ferric chlorid and HC1 respectively

30

36

.

38

C H APTER 3
1.

Diet composition

2.

Comparison of litter, variety and treatment effects
of vole feeding trial .
.
.
.
.
.
.
.
.

3.

4.

(%)

.

.

.

.

.

.

.

.

49

.

51

Food intake, weight gain and protein efficiency index (PEI)
of eleven diets tested in vole feeding trial.
Average values of six voles per diet
.
.
.
.
.
.

53

"Biological value" of crude protein of three potato varieties
subjected to six treatments, determined by S . zymogenes
.

55

v

LIST OF FIGURES

Page
C HAPTER 1
1.

4

Potatoes used for processed food items

C H APTER 2
1.

2.

3.

The effect of pH and heat treatment on the sedimentation
of protein from tap water extract
.
.
.
.
.
.

32

The effect of pH and heat treatment on the
of protein from a distilled w ater extract

32

sedimentation
.

Residual protein in solution after raising the pH w i t h
CaOH, followed by lowering it with either H^PO^ or FeCl^.

vi

38

INTRODUCTION

World wide population growth;
resources such as land, water,

rising affluence;

energy,

and fertilizer;

shortage of
and symptoms

of ecological overstress caused an international scarcity of major
agricultural commodities.

Besides social and economic improvements,

the

alleviation of food shortages and/or protein malnutrition by means of
increasing crop yields and quality,

developing n ew food sources, and

reducing wastes is part of an integrated system to meet food demands.
The traditional approach to increasing production has only limited
scope in view of limiting resources.
important.

Controlling waste is vitally

Land is lost through erosion; water through mismanagement;

crops by degradation of disease,
poor handling,

insects, and rodents;

storage, processing,

food through

and retailing.

The importance of potatoes in medieval societies of South America
and Europe is history.
in the world,

Today,

the potato ranks fourth as a food crop

and second in the U. S.

In this country, processed

products account for 50% of the potatoes consumed.

They help diversify

one's diet and reduce preparation time.

Unfortunately,
processing.

approximately 10-20% of the potato is lost during

This contributes to the pollution of public waters.

in the interest of the public,

as well as the potato industry,

It is

to recover

as much of the valuable nutrients as possible and reduce waste discharge.

1

2

To see the problem of waste production,
in the right perspective
(1)

reclamation,

and disposal

one has to appreciate the following p o i n t s :

It was the introduction of snack and convenience foods by the

p rocessing industry that brought the decline in potato consumption to
a halt in the 1 9 5 0 ’s.

(2)

Potato processing plants are in business

to produce an edible product,
less waste,

and are more concerned w ith creating

than recovering it.

(3)

Economic exploitation of the

waste stream may be possible by large plants,

but perhaps only

collectively by the smaller chip factories in municipal areas.

(4)

Types of waste vary within and between plants.
Equipment

for the recovery of starch from potato processing wat e r

is available, but used only by one plant in Michigan.

Potato processors

and representatives of the potato starch industry met to discuss methods
and their feasibility for collection of this by-product.
followed,

A survey

indicating that some plants could recover up to 1.3 million

pounds of starch per year.
This study was undertaken to investigate methods for the recovery
of proteins from the waste stream of potato chip processing,
evaluate the nutritional value of the recovered product.

and to

CHAPTER 1

REVIEW OF LITERATURE

REVIEW OF LITERATURE

I.

Potatoes:

General Considerations

Veg e t a ble foods such as potatoes have b een culturally associated w i t h
diet patterns of less affluent times and less affluent people.

Per capita

consumption is inversely related to the rising standard of living because of
a shift to more expensive and less available animal food.
As a part of the anti-affluence image, potatoes are primarily thought
of as "stomach fillers," being fattening and of low nutritional value.
Although,
protein,

in its fresh state,

the potato contains approximately 2-3% crude

or 10— 17% of the dry weight,

major cereals.

Kofranyi and Jekart

it is equivalent or better than the

(1967) reported that in human subjects

potato protein is almost equivalent to whole egg protein and better than
beef,

tuna, whole milk, wheat flour,

protein.

corn,

rice,

soybean,

and kidney bean

Potatoes contain low amounts of fat, and enrich our diet w i t h

other essential nutrients such as minerals and vitamins.

A.

Historical Background of Potato Processing

Potatoes in their original form are inconvenient and time consuming
to prepare.

Increasing involvement of w omen in activities outside of the

household was partly responsible for the decline in potato consumption.
The availability of processed snack and convenience foods by the m id fifties
halted the decline.

Consumption of fresh produce continued to decrease.

Potato processing dates back to at least 200 A.D. w h e n the natives
of Peru dehydrated potatoes by allowing them to freeze at night and thaw

3

4

during daylight hours, yielding a product called "chuno".

In the U.S.,

potato processing has risen from practically nothing in 1940 to about
30% of all potatoes consumed in 1959 and 52% in 1971.
(Figure 1).

Initially potato chips accounted for most of the

p r o c essing while in 1971 frozen french fries represented the largest
share w i t h 40%,
with

20

followed by chips with 26%,

%, starch manufacturing w i t h

products accounting for the rest,

6

(50% in 1959),

dehydration

%, and other frozen or canned

(American Potato Yearbook,

1974).

Figure 1

POTATOES USED FOR PROCESSED FOOD ITEMS
MIL. C W T . * ~

% D EHY DRAT E D
S HOESTRI NGS

^xCHIPS/Sf:-:

M M
1962

1964

frozen

1966

;:*

1968

1970

1972A 1974

CROP YEAR
* FRCSH

U .S. D E P A R T M E N T O F A G R I C U L T U R E

II.

ir,H T F tlU IV A l EN T

A PRC L I M i N A

NEG

RY

A M S 3 3 4 7 3 19)

A G R IC U L T U R A L M A R K E T IN G

S E R V IC E

Pol l u tion Problems of the Potato Industry
Eutrophication caused by discharge of human,

agricultural and indus­

trial was t e s into rivers is a serious problem, both from an aesthetic and
hea l t h point of view.

Public pressure and concern by the government r e s ulted

in the Clean Water Restoration Act of 1972, which calls for elimination

5

of discharge into navigable waters by 1985

(Federal Register,

The n u t ritive requirement for growth of algae are,
CO 2 , N, P, K, Mg,

Ca,

S, Fe,

in descending order:

to men t i o n the major ones.

biolog i c a l ly oxidized by bacteria.

1973).

Carbon can be

Nitrogen is generally removed w i t h

a ctiviated sludge and/or anaerobic denitrification.

Breaking the food

chain b y N-reduction is not particularly favored because of the ability
of some algae,

especially the objectionable blue-greens,

atmospheric nitrogen.
remove P.

A favored method to break the food chain is to

Low cost acid salts of aluminum and iron are capable of reducing

P to a cceptable levels.
as c alcium hydroxyapatite

A.

to fix

Lime can be used to precipitate orthophosphates
(Wilcox,

1974).

History of Pollution in the Potato Industry

The potato processing industry is one sector of the food industry
wh ere serious waste problems are caused by potential foodstuffs.
For m any years,

the potato industry grew at a very healthy rate and

w a s t e waters were dumped in nearby rivers.

There was little concern about

po l l ution since most of the larger plants producing frozen french fries
or d ehydrated products were located in sparsely populated areas.
until the early sixties did problems become apparent in Maine.

Not
M u nic i p a l

stabilization lagoons have been overloaded in the Red River Valley.

Over

25,000 fish w ere killed in the Snake River of Idaho, and the water supply
of the city of Twin Falls was jeopardized

(Willard,

1962).

These incidents

brought the interested parties together for research in this area.
Because of the magnitude,
from potato starch factories.

the main effort was directed towards wastes
In starch manufacturing all solubles,

27%

of the dry weight of the potatoes, were dumped into the nearest stream.
Acc o r d i n g to Xander and Hoover

(1974)

there are many plants processin g a

6

m i l l i o n pounds a day, w i t h a 5-day biochemical oxygen demand
equivalent to that of a city of 300,000 people.

(BOD)

Because of the smaller size

of most potato chip factories and the lower pollution potential,
p r o b l e m seems to be minor,

the

but finding a solution for the large number of

plants m a y be m o r e difficult.

B.

Characterization of Processing Wastes from Chip Manufactoring

A composite of w aste from a processing plant can be classified into
four general categories:
solids.

screenable,

settleable,

Its composition is largely determined by the process by w h i c h it

is produced.

From a waste treatment point of v i e w potato processing should

be broken down in the following areas:
(2)

colloidal and soluble

peeling and trimming,

frying.

Therefore,

silt,

(3) slicing accompanied w ith washing and

the types of waste vary between;

cooked and non-cooked;
Dirt,

(1 ) w ashing of unpeeled potatoes,

dissolved,

stones,

(4)

edible and non-edible;

fine pulped and particulate matter.

and other foreign matter that result from

w a s h i n g of raw potatoes are readily removed by sedimentation in settling
ponds
fill.

(Dostal and Boydston,

1969).

The collected solids can be used for land­

These wastes shall not be discussed further.
It has been estimated that up to 80% of the organic matter in the

factory comes from the peeling process

(Graham eb a_l. , 1969).

Losses are

min i m i z e d by choosing the best raw material and selecting appropriate peeling
processes.
industry,

Abrasive peeling,

the method most widely used by the chip

results in effluent consisting of cell debris,

and soluble cell contents.

New,

large,

round potatoes w i t h smooth skins

and s hallow eyes produce the least waste
h igh as 50% have b een reported, but Smith
as normal.

granular starch,

(Greig,
(1968)

1957).

P e eling losses as

considers losses of 10%

Steam peeling results in lower losses, but the material is

7

cooked,
means

starch is gelatinized and can no longer be removed by physical

(Dickinson,

1964).

Lye peeling results in even lower losses and the

w a s t e c o mposition is similar to steam peeling,

except for the high con c e n ­

tration of sodium and the high pH

(Harrington,

1957).

reduced peeling losses by up to

% and the recovered by-product had a

solid content of about 18-20%

95

Dry caustic peeling

(Graham et a l . , 1969).

Was h i n g slices yields a waste stream containing starch granules,
nitrogenous constituents

(see IV A), minerals,

sugars and other solubles.

Washing losses are roughly proportional to the n ew surface exposed by cutting,
and are a pproximately 12% for potato chips compared w ith 3% for half-i n c h
french fries

III.

(Hautela and Weaver,

1971).

Disposal and Utilization of Potato Wastes
Laws,

bad experiences such as in the Snake River,

environmental

considerations to preserve these waters for food, water supply, and re c r e a ­
tion no longer permit discharge of industrial and municipal wastes into
streams,

A.

lakes,

or tidal waters.

Biological Treatment of Wastes

Organic matter can be removed from a solution by micro-organisms
through biological oxidation.

Local factories m ay buy the right to dispose

of their w a ste into municipal facilities

(Dickinson,

added financial burden on the potato chip industry.

1964), but this is an
Activated sludge and

aerated lagoons could be very effective for BOD removal
et a l . , (1964).

Difficulties arose from BOD overload,

(up to 95%) Buzell
especially during

the w i n t e r months, w h i c h resulted in bad odors and the development of the
purple sulphur bacteria,
areas,

Chromaticium

(Olson et^ a l . , 1964).

In m any u r ban

space is not available for large ponds to provide sufficient ret e n t i o n

8

times.

Trickling filters or biotowers seem to offer an alternative

1964).

The disadvantage of these conventional methods is that they are

destructive,

B.

(Sproul,

costly and return nothing.

Potato W aste for Fuel Production

Haeusler and M a lcher

(1972) report the successful application of

m e t h a n e fermentation, utilizing gasses given off by methanogenic organisms
during anaerobic fermentation as fuel.

Wet combustion of waste was proposed

as a possible alternative by Hurwitz and Dunday

C.

(1960).

Agricultural Use of Waste

D i c k inson

(1964) reported that composting of potato solids w i t h far m ­

yard m a n u r e is practical,

but the scale is obviously limited.

Where

sufficient agricultural areas w ith suitable soils are available,
disposal is feasible

(de Haan et a l ., 1971).

spray

Unswollen starch w h i c h does

not readily decompose m ay interfere with consumption and digestion by
grazing animals

(Dickinson,

1964).

Sodium content of caustic w hich can

destroy colloidal structure of soils and pollute surface run-off and
ground water are other concerns.

D.

Waste Effluent as Culture Media for Micro-organisms

Selected strains of fungii can reduce nitrogen and phosphorous
compounds in waste streams to low levels and produce a useful by-product.
W a s t e effluent was used as substrate for:
C andida utilis

(Reiser,

biosynthesis of protein w i t h

1964) and Torula cremonis

(Janicki et al.,

1964);

simultaneous production of protein and antibiotics with yeast and the
m old A c t inomyces
acetone,

(Wieczer,

1963);

butanol and ethanol

p roduc t i o n

(Borud,

1971).

synthesis of vitamin B 1 2

(Malcher,

1971);

(Dietrich 1962),

and for alcohol and yeast

9

E.

Primary Treatment of Effluents for the Recovery of Solid Waste

S c r e e n i n g : Removal of rejected potatoes,

trimmed potato fragments,

eyes and sprouts is often an essential step not only from a wa s t e treat­
ment point of view,

but it also protects pumps and other equipment.

effectiveness of screening is determined by the m e s h size.
lower limit for m e s h size is 6-7 for fixed screens,
disc and 40 for v i brating screens

(Ballance,

The practical

120 for drum,

1964).

The

20 for

Screening will remove

a p p r o ximately 50% of the suspended and 90% of the settleable matter
(US-Public Health Service,
Sedimentation:

1955).

Settling of 95% of the suspension was achieved during

a hol d i n g period of seven hours

(US-Public He a l t h Service,

1955).

It is

a v e r y effective m e thod of removing solids but has two major disadvantages:
the sludge w i t h d r a w n has a dry matter content of only 2-4% and sedimenta­
tion tanks are bulky

(Ballance,

1964).

The sediment could easily be

dewatered in a centrifuge to an easily handled product and was an excellent
cattle feed

(Kueneman,

1964) .

According to Olson elt a^L.

(1964) 5-day

BOD is reduced by nearly 75% through primary treatment.

F.

Recovery of Starch

Starch granules are insoluble in cold water, but may be suspended and
form a slurry.
M aywald 1956),

Heating beyond a critical temperature
causes the granules to swell.

(56-67°C,

Schoch and

This process is referred to

as gelatinization, w h ereby the solubility increases markedly.
Starch granules can be removed by settling through either gravity
or centrifigal forces.

Commercial hydrocyclones designed for the size

of potato chip plants are available, but mechanical separation no longer
applies after starch has been gelatinized.

10

G.

P hysical-Chemical Treatment for the Recovery of Suspended and
Dissolved Solids

Preci pitation and recovery of proteins:

Proteins,

like amino acids,

are a mpholytes and their solubility is dependant on their functional
groups,

temperature,

pH,

ionic strength and the presence of organic

solvents and is lowest at the isoelectric point.

The solubility of

g lobulin is m a rkedly increased by low concentrations of neutral salts,
a p h e n o m e n on called salting-in, but proteins are precipitated from aqueous
solution by high concentrations.
than mono-valent,
point.

Poly-valent ions are more effective

and salting-out is more effective at the isoelectric

Isoelectric precipitation of potato protein has b een used by

N euberger and Sanger

(1942), and Xander and Hoover

(1959).

Many ions form insoluble salts w ith proteins and are used as
p r e c i pitating agents.
picric,

perchloric,

Acids such as p h o t o t u n g s t i c , trichloroacetic,

etc.

form insoluble salts w ith proteins w h e n the

latter are in cation form, and have been used for deproteinizing protein
w a ter

(Neuberger and Sanger,

1942).

The standard m ethod for determining

the amount of protein nitrogen is calculated from N precipitated by 10%
TCA

(AACC, 1967).

Heavy m e t a l ions are used for precipitating proteins

on the alkaline side of their isoelectric point, but these compounds are
of little use for recovery of proteins from a waste stream.

Denatura t i o n

of proteins wh i c h is caused by a loss of its native conformation is
generally accompanied w i t h reduced solubility.
by heating,

Most proteins are denatured

freezing-thawing, high concentration of urea,

w a t e r m i s c ible solvents.

Of these methods heat denaturation for recovery

of potato protein has been used by Neuberger and Sanger
et al.
Borud

radiation and

(1942).

Heisler

(1959) precipitated protein by heating the "fruit juice" to 80°C.
(1971) achieved immediate flocculation of proteins by heat treatment

of potato juice to 120°C.

11

Distillation:

This process is based on vaporizing the liquid waste,

leaving the dissolved solids in an enriched solution,
the w a t e r vapor.

and then condensing

This process is more of theoretical interest since high

temperature treatment of the waste is undesirable and evaporation under
reduced pressure tends to be expensive,
Free ze C r y s t a l l i z a t i o n :

e.g.

freeze-drying.

This is another m ethod involving phase

change and has b een under development in the desalination industry,
but has not yet b e e n applied commercially

(Probstein,

1972).

The use

of freezing as a concentration process is based on the fact that w he n
ice is crystallized from an aqueous solution,

the ice crystal is pure

water, w i th all of the impurities left in the original solution.

Thus,

wit h suitable means to separate the ice from the mother liquid,pure
water and high concentration w aste could be recovered.
be:

Advantages would

low energy consumption in comparison with distillation and less

d e struction of proteins and other compounds.
R everse O s m o s i s :

This process separates salt and other solids

from water under the action of a hydrostatic pressure applied across
a semipermeable membrane.

Energy costs are quite low, but the

economic limitation is related to the high cost of membranes and their
maintenance.

Application of reverse osmosis to wastes from potato starch

manufa c t u ring was investigated by Porter e_t a l . (1970) .

Great diff­

iculties were encountered in a pilot plant study from potato chip
effluents by Seyfert
Ion E x c h a n g e :

(1974; pers.

comm.).

This is based on absorption and removal of charged

m o l e c u l e s by a resin from w hich they are eluted in a regeneration step.
Rec o v e r y of free amino compounds from starch waste by ion exchange was
investigated by Heisler e_t aJL. (1962) , amino acids and p o t a s s i u m by

12

Heisler et a l . (1972).

Even though the total purification process

reduced the chemical oxygen demand by 78% and total solids by 84%,
Stabile et a l . (1971) found that protein recovery followed by removal
of other constituents using ion exchange is economically not feasible.

IV.

C o m p osition and Quality of Crude Potato Protein

A.

N itrogen Containing Substances of the Potato

In potato tubers crude protein

(N x 6.25)

is found in three

m o r p h ological and physiological forms:
Structural proteins or bound proteins are part of the living
cytoplasm and its organelles.

They include nucleo- and l i p o - p r o t e i n s ,

proteins of mitochondria and amylo-plasts,

etc.

This p ortion is often

referred to as insoluble protein or scleroprotein and it accounts for
at least 7-10% of the total nitrogen

(Neuberger and Sanger,

1942).

Crystalline proteins were found by some authors in the less starchy
subperidermal tissue and in the pith of the tuber.
of p r o t e i n crystals,

These are deposits

formerly thought to be associated w ith virus disease.

V a r i e t a l differences have been described by Hoelzl and Blancher
Cell sap proteins consist of soluble proteins,

(1959).

classified according

to their solubility, and a non-protein fraction composed of short peptides,
free amino acids,
acids,

etc.

This extractable nitrogen fraction,

90% of the N,
accounts
8-12%

amides and minor constituents such as vitamins,

(Levitt,

accounting for about

is the portion most extensively investigated.

for 25-50%

(Neuberger and Sanger,

nucleic

P rotein

1942), nucleic acids for

1954), most of the remainder can be attributed to amides

and amino acids.

13

Extractable p r o t e i n .

Osborne and Campbell

(1896)

the p r o t e i n present in potatoes was a single substance,
w h i c h they called tuberin.

solubility by Lindner e_t_ _al.

in addition,

Further separation according

to

(1960) yielded six protein fractions,

tuberin accounting for more than three-fourths.
(1972)

a globulin,

Groot et a l . (1947) described,

an alb u m i n n ow termed tuberinin.

Lue s c h e r

suggested that

In m ore recent work,

found similar relative amounts.

The albumin particles are corpuscular and the globulin molecules
are longitudinal,
higher viscosity.

having a greater dissymetry,

a lower solubility,

and

The albumin is denatured w h e n heated at 5 0 - 6 0 ° C .

The v i s c o sity thereby increases more than 100-fold, because of the loss
of its globular structure

(Jirgenson,

1946).

He also suggests that

tuberin and tuberinin are interconvertible by changing the pH.
Lindner ej; al.

treatment yielded m o r e tuberin.

(1960)

Acid

found a very

similar composition of the two proteins, but Luescher's data suggests
that they differ.
Separation of proteins using paper electrophoresis yielded six
c omplex bands

(Zwartz,

1966).

Band heights w ere typical for varieties.

Environmental components showed little or no effect, but virus infection
d r a s t i c a l ly changed the pattern.

Refined separation by acrylamide

electrophoresis resulted in the separation of up to 25 proteins wh i c h
could be used for v a riety identification

(Loeschke and Stegemann,

No n - protein nitrogen is mainly composed of: short peptides;
amino acids w i t h glutamic,
m ost abundant*

amides,

1966) .
free

aspartic, ^ - a minobutyric and alanine being

the principle ones being asparagine and glutamine.

These free amino acids and amides have a key role in the nitrogen
m e t a b o l i s m of plants as well as animals.

Their relative amounts are

14

d e t e rmined by the physiological stage of the tuber and are subject to
changes by environmental conditions, namely nutrient availability
(Mulder and Bakema,

B.

1954).

Nutritional Value of the Nitrogenous Substances of the Potato

The nutritional value of crude potato protein compares favorably
w i t h other plant proteins.
value ranged from 61— 89.

For 258 samples examined,

the biological

L o w values w ere caused by either deficiency

or surplus of n itrogen fertilization (Schupan,

1959).

N i trogen balance

studies w ith human adults showed that potato protein was superior to
most major plant protein and approached the value of whole egg
(Kofranyi and Jekart,

1967).

Removal of the skin,

that accounted for 7.6% of the dry w eight and

for about 10% of the total N,
and better N utilization

led to higher weight gains of growing rats

(Chick and Slack,

1949).

This difference could

partly be explained through the lower digestibility of the insoluble
ni trogen present in the skin.
growth,

but gave,

The non-protein fraction did not support

in combination with tuberin,

the latter by itself.

a better growth than

This supplementary effect could not be explained

in terms of their contents of essential amino acids.
The sulfur containing amino acids are the first to limit growth of
animals in potato protein

(Schupan,

1958).

Supplementing potato p r o t e i n

w i t h methionine increased both digestibility and weight gain of growing
voles

(Rios et. al.

1972) .

Kies and Metzfox

(1972)

improved nitrogen

b a lance in human subjects w ho consumed dehydrated potato flakes by adding
methionine.

Using Streptococcus z y m o g e n e s , Luescher

(1972)

found in

a seedling population that crude protein was negatively correlated w i t h

15

both methionine

(r = -.45) and the biological value

(r = -.55).

Most

of the v a riability in the methionine was in the non-protein fraction.
Hoff et al.
increased,

C.

(1971)

found that as the total nitrogen content of a va r i e t y

non-protein N went up but methionine decreased.

Protein Evaluation Methods

D e t e r m ination of protein content in foods is based on measurement
of the nitrogen content.

There are several methods available, but

the Kjeldahl method is most widely used and is standardized
1970).

(AOAC,

Crude protein is estimated by multiplying the amount of n i t r o ­

gen by 6.25.
The nutritive value is determined by the amount and the relative
av a i l a b i l ity of the amino acids.

Speedy and accurate methods are

available for measuring amino acid composition
but they do not indicate the availability.
used by Luescher
(1973),

(Speckmann et a T . , 1956),

Microorganisms have bee n

(1972) , voles by Rios e_t a_l. , (1972) , rats

and human subjects by Kofranyi et al.,

q ua l i t y of potatoes.

(1967)

by Peare

to assess protein

LITERATURE CITED

A m e r i c a n Association of Cereal Chemists.
1962.
The association.
St. Paul, Minn.
1962,
A m e r i c a n Potato Yearbook.
Vol. XXII. 1974.
Plains, New Jersey, 07076, USA.

AACC approved methods.
p. 45.

S.E. Walker

e d .,

Scotch

A m e r i c a n Public Health Association.
1955.
Standard Methods for the
Examination of Water, Sewage and Industrial Wastes.
Publication
Office, 1790 Broadway New York, N.Y.
A s s o c i a t i on of Official Agricultural Chemists.
1970.
l l ^ ed.
Was h ington DC. Association of Official Agricultural Chemists,
p. 801.
Ballance, R.C.
1964.
A review of primary treatment processes.
Int.
Symp. Util. & Disposal of Potato Waste, Proceedings, pp. 200-211.
Borud, 0. J.
1971.
Verwertung von Abfaellen der kartoffelverarbeitenden Industrie.
Die Staerke 23
(5): 172-176.
Buzzel, J.C., Caron,
A - L . , Ryckman, S. J. and Sproul, 0. J.1964.
Biological treatment of protein water from manufacture of potato
starch.
Water and Sewage Works III, p. 327.
Chick, H. and Slack, E. B.
1949.
Distribution and nutritive value
of the nitrogenous substances in the potato.
Biochem. J.
45:
211-221.
Dickinson, D.
1964.
Int. Symp. Util.
pp. 246-257.

Treatment of effluent from potato processing.
& Disposal of Potato Waste, Proceedings,

Dietrich, K.
1962.
Synthesis of V i t . B ^
Die Staerke, 13 (1): 11-15.
Dostal, K. A. and Boydston, J.R.
w a ste treatment.
Proc. 1 8 ^
USDA.
pp. 7-14.

from potato starch effluent.

1969.
The state of art of potato
Natl. Potato Util. Conf. ARS 74-49,

Federal Register 38, 13523 and 13528, May 22, 1973.
Federal Water
Pollution Control Act as amended by Public Law 92-500 of 1972.
Ford, J. E. 1962.
A microbiological method for assessing the
nutritional value of proteins.
Br. J. Nutr. 16:409-425.
Graham, R. P., Huxsoll, C. C. Hart, M. R . , Weaver, M. L. and Morgan,
A. I.
1969.
"Dry" caustic peeling of potatoes.
18 t “ Natl.
Potato Util. Conf.
ARS 74-49, USDA. 14-21.
Greig, W. S. 1957. Trends and inplant costs in the potato peeling i n d u s ­
try.
Proc. 8 *^ Annual Potato Util. Conf., Berkely, Calif., Aug.
21-23. pp. 77-82.
16

17
G r o o t , E.H. , Janssen, L. W., Kentrie, A., Oosterhuis, H. K. and Trap,
H.J.L.
1947.
A n e w protein in potatoes.
Biochemica et Biophysica
A cta 1: 410-414.
H aan d e , F. A. M . , Hoogeveen, G.J. and Vis, F. R.
1973.
agricultural use of potato starch waste water. Neth.
21: 85-94.
Harrington, W.
U.S. Pat.

Aspect of
J. Agr. Sci.

. 1957.
Removing cooked tissue from peeled potatoes.
2,797,165.

0

H a e u s l e r , J. and Malcher, J. 1972.
Unschaedlichmachung der Abwaesser
durch bessere Ausnuetzung des Rohstoffes bei der Kar t o f f e l s t a e r k e —
erzeugung.
Staerke 24 (7): 229-235.
Hautala, E. and Weaver, M. L. 1971.
Reuse of water from reclamation
of nutrients from potato cutting wastes.
Proc. 20 t ^1 Natl.
Potato Util. Conf.
July 29-31.
1970. Riverside Calif. ARS 74-55,
USDA. pp. 77-79.
Heisler, E. G. , Siciliano, J. and Krulick, S.
1972.
Potato starch
factory w aste effluents.
II Development of a process for recovery
of amino acids, proteins and potassium.
J. Sci. Food and A g r i c u l ­
ture 23:
745-762.
Heisler, E. G . , Siciliano, J . , Treadway, R. H. and Woodward, C. F. 1959.
Recovery of free amino compounds from potato starch processing
w a t e r by use of ion exchange.
Am. Potato J. 36:
1-11.
Heisler, E. G. , Siciliano, J . , Treadway, R.H. and Woodward, C.F.
1962.
Rec o very of free amino compounds from potato processing water by
use of ion exchange.
II. Large-scale laboratory experimentation.
Am. Potato J.
39. 78-82.
Hoelzl, J. and Blancher E.
1959.
Ueber das Vorkommen und die
Sortenabhaengigkeit der Eiwisskristalle in der Kartoffelknolle
(Solanum tuberosum) Qual. Plant. Mater. Veg. 8 : 1-24.
Hoff,

J. E . , Jones, C. M . , Wilcox, G. E. and Castro, D.
1971.
The
effect of nitrogen fertilization on the composition of the free
amino acid pool of potato tubers.
Am. Potato J. 48: 390-394.

Hurwitz, E. and Dundas, W. A. 1960.
Wet oxydation of sewage sludge.
W a t e r Pollution Control Federation 32:
9.

J.

Janicki, J . , Szebiotko, K. and Stawicki, S.
1964.
Potatoes and their
industrial wastes as media for yeast production.
Intl. Sympl Util.
& Disposal of Potato wastes, Proceedings.
pp.
135-142.
Jirgenson, B.
1946.
1 *
484-494.
Kies,

Investigations of potato proteins.

J. Polym.

Sci.

C. and Metzfox, H.
1972.
Effect of amino acid supplementation
of dehydrated potato flakes on protein nutritive value for human
adults.
J. Food.
Sci. 37:
378-380.

18

Kofranyi, E. and Jekart, F.
1967.
Zur Bestimmung der biologischen
Wer t i gkeit v o n Nahrungsproteinen, XII.
Die Mischung v o n Ei mit
Reis, Mais, Soja, Algen.
Hoppe S e y l e r 1s Z. Physiol.
Chem.
348:
84-88.
Kueneman, R.W.
1964.
Design of biological oxidation systems for
pu r i f ication of organic wastes, particularly from potato processing.
Intl.
Symp. Util. & Disposal of Potato Waste, Proceedings,
pp. 236-245.
Levitt, J.
1954.
The cytoplasmic particulates and proteins of potato
tubers.
II Nitrogen, phosphate and carbohydrate content.
Physiol.
P l a n e t a r i u m 7:
117-123.
Lindner, K. , Jaschnik, S. and Korpaczy, I.
1960.
Aminosaeurezusamm e n s e t z u n g und biologischer Wert der Kartoffeleiweissfraktionen.
Qual.
Plant.
Mater.
Veget. 7:289-94.
Luescher, R.
1972.
Genetic variability of "available" methionine,
total protein, specific gravity and other traits in tetraploid
potatoes.
Ph.D.
Thesis, Michigan State University, East
Lansing, Michigan.
Loeschke, V. and Stegemann, H.
1966.
Proteine der Kartoffel in
Ab h a e ngigkeit v o n Sorte und Virosen.
(Polyacrylamid E l e k t r o p h o r e s i s ) . Phytochemistry 5:
985-991.
Malcher, J.
1971, Geringe Abwasserbelastung durch vollstaendige Nutzung der Kartoffeltrockensubstanz bei der S t a e r k e g e w i n n u n g . Die
Staerke 3:
95-103.
Mulder, E.G. and Bakema, K.
1956.
Effect of the nitrogen, potassium,
m a g n e s i u m nutrition of potato plants on the content of free amino
acids and on the amino acid composition of the protein of tubers.
Plant, and Soil.
7:
135-166.
Neuberger, A. and Sanger, F.
J.
36, 662-671.

1942.

The nitrogen of the potato.

Biochem.

Olson, O. 0., van Heuvelen, W. and Vennes, J. W.
1964.
Experimental
treatment of potato wastes in North Dakota, USA.
Intl.
Symp.
Util. & Disposal of Potato Wastes, Proceedings.
pp.
315-344.
Peare, R. M.
1973.
Potato protein as affected by:
A. variety and
environment and B. air separation of the dried flour.
Master's
Thesis.
Mich i g a n State University, East Lansing, Michigan.
Porter, W. L., Siciliano, J., Krulick, S. and Heisler, E. G.
1970.
Res v erse Osmosis:
Application to potato starch w a s t e effluents.
M e m b rane Science and Technology (Plenum Press, 1970).
pp.
220-230.
Probstein,

R. F.

1972.

Desalination.

American Scientist 61:

Reiser, C. 0.
1954.
Torula yeast from potato starch wastes.
& F ood Chem.
2:70.

280-293.
Agr.

19

Rios Iriate, B. J., Thompson, N.R. and Bedford, C. L.
1972.
potato flakes.
Evaluating by the m eadow vole (Microtus
p e n n s i l v a n i c u s ) . Am. Potato J. 49:
255-260.

Protein in

Schoch, T. J. and Maywald, E. C.
1956.
Microscopic examination of
m o d i fied starches.
Anal.
Chem.
28 (3):
382-7.
Schupan, W.
1958.
Proteins et amino acids indispensable des vegeta u x
a limentaires et de leur diverses organes.
Q u a l . Plant. Mater.
Veg.
3-4:
19-33.
Schupan, W.
1959.
The influence of increasing nitrogen fertilizers
on the content of essential amino acids and the biological
albumine evaluation of potatoes.
Z.
Pflanzenernaehrung.
Duengung.
Bodenk.
8 6 :
1-14.
Smith, 0.
1968.
Potatoes:
Production, Storing, Processing.
The Avi
P u blishing Company, Inc.
Wesport, Connecticut.
pp.
262-393.
Speckmann, D. H., Stein, W. H. and Moore, S.
1956.
Automatic
r ecording apparatus for use in chromatography of amino acids.
Federation Proceedings 15: 358.
Stabile, R. L.,
evaluation
effluent.
Colorado.

Turkot, V. A. and Aceto, N. C.
1971.
Economic
of processes for the recovery of potato starch waste
Proc. Nat. Symp. Fd. Process.
Wastes, Denver,
p. 185.

Vlasblom, M. and Peters, H.
1958.
Dutch patent 87,150.
1957.
German
patent 1,021,243.
Recovery of proteins from potato w ash water.
Wilcox- R. L.
1974.
Removing in excess of 99% phosphorous at ELY,
Minnesota.
In Water-1973.
G. F. Bennet e d . AIChE-Syposium series
136, vol. 70.
pp.
358-366.
1"li

Willard, M.
1962.
Introduction panel on waste disposal.
12
Natl.
Potato Conference, May 1962.
Bakersfield, Calif.
pp.
37-38.
Xander, P. A. and Hoover, E. F.
1959.
Method of producing protein
hydrolysate from potatoes and potato waste.
US-Pat.
2,879,264
(March 24, 1959).
Zwartz, J. A.
1966.
Potato varieties and tuber protein electropherog ram c h a r a c t e r i c t i c s . Europ.
Potato J. 9. 111-128.

CHAPTER 2

PHYSICAL-CHEMICAL METHODS F OR THE RECOVERY OF PROTEIN
F ROM WASTE EFFLUENT OF POTATO CHIP PROCESSING

20

PHYSICAL-CHEMICAL METHODS F OR THE RECOVERY OF PROTEIN
F R O M W ASTE EFFLUENT OF POTATO CHIP PROCES S I N G 1
Erhard Meister and Norman R. Thompson

ABSTRACT
Simple physical-chemical methods for the recovery of protein
from potato chip processing were evaluated.

It was estimated that

an average potato chip plant, processing 18.4 metric tons of potatoes
per week,

could recover approximately 170 kg of dried potato protein

(550 kg of food containing 30% of p r o t e i n ) .
in settling tanks for 60 minutes yielded,

Sedimentation of protein

after appropriate treatments,

the same amounts of recoverable protein as centrifugation at speeds
b e l o w 10,000 x G.

Drum drying of precipitate appears to be the most

suitable m ethod to produce a concentrated feed.

Improved recovery

can be expected from higher protein concentrations,

thus use of a

hy d r o c y c lone to remove starch granules and recycling of water would
b e advantageous.

Application of heat at pH 4-4.5 was generally more

efficient for p rotein recovery, but lowering the pH to 3-3.5 with
no heat gave similar results.

Protein yields were improved if the

w a ste was kept in m otion during floe formation.
r ed u c t i o n was highest if no heat was applied.

Total dry matter
FeCl^

(at pH 4) was

a slightly more effective precipitating agent than either HC1 or H 3 P O 4 .

1Received for publication
A rticle No.

Michigan Agr.

Exp.

Sta. Journal

^Grad. Asst, and Professor, respectively, Department of Crop and Soil
Sciences, Michigan State University, East Lansing, Michigan 48824.

21

F r o m an environmental point of v i e w the latter is not very desirable.
P r o t e i n recovery was similar when pH was raised to 11.5 and then
l owered w i t h either H^PO^ or F e C ^ t o pH 9, but large amounts of chemi­
cals w ere required.

The composition of the nitrogenous compounds

e x t r a c t e d is dependent on the type of water used.
the efficiency of protein recovery.

This also determines

Approximately 30-40% of the

crude p rotein or 80— 90% of the coagulable protein, presently wasted,
could easily be recovered by any one of the above procedures.
INTRODUCTION
The potato processing industry is one sector of the food industry
w h e r e serious waste problems are caused by potential food stuffs.
M i c higans potato chip manufacturers are searching for economical
solutions to minimize losses and to meet local and federal standards
for the effluent discharged.

The Clean Water Restoration Act of

1972 calls for elimination of waste discharged into navigable water
by 1985

(Federal Register,

1973).

M o s t of the potato chip plants are located in municipal areas
w h e r e space for conventional treatment or agricultural use of the
effluent is not available.

Peel,

potato fragments and other particu­

late solids can be readily removed by screening or settling
1964).

(Ballance,

Recovery of dissolved and suspended solids in the waste

effluent, primarily from washing of tubers and potato slices,
still inadequate.

is

The waste effluent contains approximately 50%

starch and 30% crude p r o t e i n 0

22

Most of the w ork on potato w aste has been done on effluent from
starch m a n u f a cturing where the problem is magnified because of the
i
larger size of the factories and only starch is retained and the
other solids wasted.
has been patented

In Europe, a process for the recovery of prot e i n

(Vlasblom and Peters,

1958).

A starch factory

e l i m inated its waste by a process that called for an investment
of 17 m i l l i o n German Marks, of which the Dutch government paid 11
million.

The U . S 0 D 0A. investigated the application of ion exchange

for the recovery of amino acids, proteins and potassium (Heisler
et a l . (1972) and organic acids and phosphate

(Schwartz et a l . (1972).

But Stabile ^t a l . (1971) found that protein recovery followed by
removal of other constituents using ion exchange is not economically
feasible.

Reverse osmosis treatment of waste was investigated by

Porter et al.

(1970), but great difficulties were encountered in a

pilot plant study with potato chip effluents
comm.).

(Seyfert,

1974, person,

In the small potato chip plants simple methods for by-product

recovery are required.

Both commercial hydrocyclones for the recovery

of starch and a m a rket for the product are available

(Pettay,

1975).

It was the purpose of this study to examine optimal conditions for
recovery of proteins
to heat treatments,

by simple means.

Special attention is given

since waste heat generated by the cooker could

be r e c o vered in a heat e x c h a n g e r 0

23

MATERIALS A ND METHODS
I.

Pr e p aration of Protein Water
Pr e liminary w ork was carried out on processing water received

fro m a Detroit potato chip factory.

Because of the inconvenience

of transporting a dilute solution a long distance and possible com­
p ositional changes, it was decided to simulate processing water in
the laboratory by preparation of a dilute potato extract.

Potatoes

w e r e w a s h e d thoroughly, peeled and ground in a Wa r i n g Blender.
slurry was diluted w i t h tap water
times its volume.

Initially,

at 2000 x G for five minutes,

(composition,

The

see Table 1) to 10

the resulting juice was centrifuged
the supernatant filtered through paper

napkins to remove suspended cell debris and kept at 10°

.

It was

later found that filtering the juice through several layers of chee s e ­
cloth followed by settling for 30 minutes produced protein water of
a composition similar to the samples obtained from processing plant
A

(see Table 2).
The potatoes used were Russet Burbanks grown on the M o ntcalm

Experiment Station during the 1974 season.

Tubers were kept in cold

storage until utilized.
II.

A nalytical Procedures
The dry m atter of the samples was determined by the official

A O A C v a c u u m oven method

(A0AC, 1970).

The v acuum oven was operated

for 12 hours at 70° under partial vacuum.

24

The n itrogen content of the samples was m easured by the official
A OAC m i c ro-Kj e l d a h l method

(AOAC,

1970).

The terms total, coagulable and precipitated protein refer to
crude protein
(1 0 % w/v)

(N x 6.25), protein coagulable by trichloracetic acid

and protein precipitated by the treatments as specified

below. P r ecipitated protein was expressed in percent of coagulable
protein. All values w ere adjusted for dilution caused by treatments.
The composition of crude protein of whole Russet Burbank tubers
a n d d i s tilled and tap water extracts was determined according to
L i n d n e r et a l . , (1960), outlined by Luescher,

(1972).

Flour of freeze

dried tubers and of freeze dried protein water were each blended with
the extracting solutions for four minutes at room temperature. ~ T h e
isolates were dialyzed in cellophane bags against
v olumes of distilled water for 48 hours at 4°.

100

times their

During dialysis the

w a t e r was changed 4 times.
III.

Separation of precipitates
Initial attempts to filter the slurries with several size filters

w e r e fruitless since starch and protein floe immediately plugged the
cloth.
Gra v i t y s e t t l i n g .

The influence of concentration on settling times

of samples treated with or without heat was investigated.

Protein

w a t e r w a s prepared as described above with the exception that slurries
w e r e d iluted either five or ten times their volume to give different
concentrations.

The pH of the protein water was adjusted with 2N HC1 x

25

to p H 4 and one half was heated to boiling
cooling in ice water,

(98°) followed by immediate

the other half was stirred during the time the

former was heated and cooled.

One liter of each treatment combination

was poured into a settling cylinder.

Samples w e r e withd r a w n from

the center at ten minute intervals and analyzed for nitrogen and dry
matter. This was repeated twice.
Centrifugation.

Separation of protein by 7 different centrifugational

forces for 15 minutes was compared with gravity settling for 1 hour.
Two different extracts of protein water were heated to 98° at pH 4.
D u p licate samples were centrifuged in a laboratory centrifuge
Superspeed RC-2) at speeds of 2, 4,

6

(Sorvall

, 10, 20, 30, and 40,000 x G

and residual nitrogen in the supernatant was determined.
IV.

Treatments for protein p r e c i p i t a t i o n .
Gravity settling for 1 hour was chosen for the evaluation of

each treatment combination.
drawn with a syringe after

Aliquots of the supernatant were w i t h ­
1

hour of sedimentation, placed in screwcap

b ottles and stored in a refrigerator for analysis.
Hea t versus pH t r e a t m e n t .

The effect of p H on protein precipitation

was tested in the range of p H 1 to 7.

The pH of 500 ml samples of

p r o t e i n water was adjusted with 2N HC1 or 2N H 3 PO 4

to

p H levels w hile stirring w i t h a magnetic stirrer.

Four 100 ml aliquots

8

different

of protein w ater of the different pH levels were placed in Erlenmeyer
flasks.

They w e r e assigned to water baths of 23°,

and heated under a low shaking motion.

60°, 80° and 98°,

They were immediatly cooled

26

in ice w ater after reaching the desired temperature and allowed to
settle.
The same procedure was followed w i t h protein water obtained f r o m
a di s t i l led w a t e r potato extract, but only temperatures of 23° and
98° w ere compared.
FeCl^ versus HC1 as c o a g u l a n t .

The p H ’s of 100 ml samples of protein

w a t e r was adjusted w ith either 1M F eCl 3 or 2N HC1 over the same pH
range.

Samples w ere agitated for 20 minutes in a shaker at 23° before

they w e r e subjected to sedimentation.
Lime, H^P0/t and FeClq trea t m e n t .

A newly prepared slurry of CaO and

dis t i l l e d w a t e r was used to gradually raise the pH of 3 liters of
/

p ro t e i n w ater to pH 12.

This was followed by lowering the pH with

either 2N H 3 P O 4 or 1M FeCl^.

100 ml aliquots were withdrawn at inter­

m e d i a t e p H ’s and subjected to settling.
RESULTS A N D DISCUSSION
T wo local potato chip plants co-operated in the conduct of this
res e a r c h providing several samples of typical effluents from their
plants.

Table 1 gives the average dry matter and crude protein content

of the samples.

Values for the total dry matter content for plant

B are of the same magnitude as reported in the literature
et al., 1962).

(Willard

The higher values in plant A are attributable to the

use of a hydrocyclone w hich removes the bulk of the starch and allows
p artial recycling of effluent.

In the light of reduced waste discharge

the latter s ystem would be more desirable.

Effluent of plant A w a s

27

T a b l e 1.

pH

Chemical analysis of the water taken from Mich i g a n U n iversity
campus wells*

Cl
mg/L

7.5

Alkalinity
m g /L CaCO^

4.9

* Data:

304

D fItri

Table 2.

processing
plant

A

B

Total Hardness
mg/L CaC 0 3

Sulfate
mg/L SO 4

318

Nitrate
mg/L N

Mn
Tot.Kjeldahl
Fe
mg/L N
mg/ L m g / L

.46

.03

21

(1973)

Average composition of potato effluent of two local potato
chip plants.

sample*

dry matter
mg/liter

t

crude protein
mg/liter

1

11200

4307

2

8400

2960

3

9400

3524

4

10600

3360

5

3310

1578

6

1725

771

7

1550

547

8

2100

895

* Samples 1 to 4 give average composition of effluent leaving the
hyd r o c y c l o n e on five different days in plant A.
Samples 5 to 8 were
collected on the same day at 4 different locations in factory B.

.73

.01

28

simulated for the study of protein recovery.
Smith (c.f. 1968) reports that an average potato plant processes
appro x i m a tely 18.4 m e tric tons of potatoes per week and uses 460,000
liters of wa t e r per day.

Assuming that the w aste composition of plant

B is typical, one can calculate the daily loss of protein per day
w h i c h amounts to about 385 kg of crude protein, of which approximately
a third to one half can be easily precipitated.

This gives an esti­

mate for easily recoverable protein per day of 170 kg for an average
potato chip plant.
Se p a r a t i o n of protein from heat treated waste w a t e r .
Samples of potato waste water with an initial crude protein
c o n c e n t r ation of 5832 ppm. on the average were acidified, heated to
98° and immediately cooled in ice water.

Of 3582 ppm. protein pre­

cipitated b y 10% TCA, approximately 84% was removed at speeds of 2
to 10,000 x G, 87%,

94%, and 99% settled at 20, 30, and 40,000 x G

respectively. The lower centrifugational forces did not yield any
apprec i a ble amounts beyond the 82% which was achieved by gravity
se ttling for 60 minutes.

The latter yielded a white slurry that could

e a s i l y b e drained off and contained from 6-8.5% dry matter, whereas
a pasty cake

(15-20% DM) was recovered by centrifugation.

Drying studies with similar wastes from potato starch m a n u f a c ­
turing

(Strolle et al., 1973) gave good results w i t h double drum drying,

w h e r e freeze drying would be too expensive and air drying in a conven­
tional tray dryer gave a black, hard, hornlike product.

The drum

29

drier r e quired m a terial containing 12-15% solids.

The cakes produced

b y c e n t r ifugation at 500,000 rpm contained 25-35% solids and had to
be diluted.

It appears that low centrifugational forces would yield

a product suitable for drum drying.

The slurry from settling w ould

r equire additional concentration but its volume is about twenty times
smaller than the original volume of the waste.
D ep ending on the individual case, settling or centrifugation
m i g h t be preferred.

In this study separation of protein by settling

w a s chosen because of the large number of samples that had to be
treated simultaneously.
Effect of concentration and heat treatment on s e t t l i n g .
Res idual crude protein measured over a time span of 80 minutes
is g i ven in Table 3.

Sedimentation was faster in heat treated samples,

b u t differences w ere not significant beyond 60 minutes.
cant interaction,

time x treatment,

The signifi­

indicates that initial sedimenta­

tion was faster at low protein concentrations but that the percentage
of p r o t e i n settled during the entire period was lower.
al.

Strolle et_

(1973) came to the same conclusion using waste from a starch plant.

These data suggest that the efficiency of protein recovery could be
increased if higher w aste strength could be obtained.

W a t e r usage

s ho u l d be reduced if possible and the waste effluent recycled.

This

w a s a c h ieved in plant A by using a hydrocyclone for starch recovery
but p rotein was not recovered.

30

Table 3.

Residual crude protein (ppm) in solution after gravity settling of
p rotein from waste water of low and high protein concentration at
p H 4, w ith and without heat treatment.*

settling time
minutes

high concentration
23°C
98°C

low concentration
23°C
98°C

0

8333 a,A

8337 a,A

4232 a,A

4232 a,A

10

7310 b ,A

7107 b ,A

3672 b ,A

3607 b,A

20

6594 c,A

6054 c ,A

3297 c ,A

3111 c,A

30

6169 d ,A

5631 d ,B

3001 be,A

2829 c d ,A

40

5783 e,A

5092 e,B

2801 c ,A

2623 de,A

50

5497 f ,A

4807 e,B

2742 c ,A

2503 d e ,A

60

5088 f g ,A

4838 e ,A

2642 c,A

2379 e ,A

70

5049 g ,A

4827 e,A

2496 d ,A

2355 e,A

80

4949 g,A

4875 e,A

2475 d ,A

2367 e,A

TCA standard

1874

3898

* average value of two replications

1

Studentized range test:
- time comparisons within treatments
Values with the same letter

(within c o l u m n s ) :

(a,b . . .) are not significantly different.

- temperature means within time and concentration (between columns):
Values with the same letter

(A) are not significantly different.

31

Heat versus pH t r e a t m e n t .
The solubility of proteins is markedly influenced by the pH and
is m i n i m u m at the iso— electric point.

Since a potato extract represents

a m i x t u r e of different proteins and other constituents w e cannot expect
a n a r r o w zone of low solubility.
s ummarized in Figures 1 and 2.

The data of this experiment are
No heat-pH combination was as effec­

tive as T C A in precipitating protein.

Neuberger and Sanger

(1942)

c ompared T C A and heat with NaCl at 80° and found that T C A was slightly
m o r e effective.

Efficiency was improved at higher NaCl concentration.

P relim i n ary w o r k with neutral salts

(NaCl,

(NH^^SO^,

N a 2 S 0 ^) was

not ver y successful at low salt concentrations and high concentrations
are not justifiable in recovery of proteins from a waste stream.
The curves in Figure 1 indicate an optimal pH range for protein p r e ­
ci p i t a t i on f rom pH 3.5-4.5.

The highest temperature was most effective

at almost all pH*s except the very low ones.

The temperature effect

w a s m o s t pronounced at neutral pH and was negligible around pH 4.
A t h i g h e r acidities,

room temperature yielded better results. Eighty

degrees was included because Heisler et al.,

(1959) reported that

the simplest method of carrying out precipitation was by heating to
80° or acidification to pH 3.0 or slightly below.
that n e i ther treatment gave optimal responses.

These data suggest

Strolle et_ a l ., (1973)

who used steam injection found that heat alone was not v ery effective
but that lowering the pH improved the efficiency of the treatment.
His data did not show the optimal pH range observed here.

32

Figure 1:

The effect of pH and heat treatment on the s e d i m e n t a t i o n
of protein from tap w a t e r extract.

Figure 2: The effect of pH and heat treatment on the s e d i m e n t a t i o n
of p rotein from a distilled w a t e r extract.

33

100

80
d
•H
a)
o

u

80

Oh

C

a>
rd

ca
^

rH

a»
d

AO

20

*H
cn
a)

u

1

2

3

A

5

6

7

pH

34

A l t hough heat coagulation has been proposed by many authors
(Vlasblom and Peters,

1958; Borud, 1971;

data indicated that at low pH ( ^ 3.5)
at low temperatures.

Strolle et al.,

1973) our

good results could be achieved

Aggregation was much improved through the slight

shaking m otion during floe formation and resulted in faster and better
settling.
Using distilled water in the initial simulation of potato waste
effluent,

a rather surprising observation was made.

was rather poor, especially w h e n no heat was applied

Sedimentation
(Figure 2).

These results did not agree at all with the observations made on
actual waste water.

This suggested the investigation of possible

differences in the composition of the proteins extracted

(see below)

but it also points out the difficulties with comparisons of results
of different sources.
If HC1 was substituted for H 3 P O 4 results were almost identical
and are not given.

From a cost point of view as well as considerations

of a d d i n g H3PO4 to public water H C 1 should be preferred.
If one looks only at the protein recovered, heating of the pro ­
tein water after acidification with HC1 appears to be the best pro­
cedure.

But the decision becomes even more difficult if one also

looks at the total dry weight recovered.

The original solution c on ­

tained a total of 14,700 ppm solids of which 4,705 ppm was crude
protein.
tate,

After heating to 98° at pH 4 and separation of the pre c i p i ­

2,744 ppm crude protein and 7,894 ppm solids remained in the

35

solution. A cid treatment at pH 3 reduced the residual protein to 2,905
p p m but the solids w ere decreased to 6,853 ppm.

It is assumed that

this difference in reduction of total solids is primarily due to
starch. Potato starch gelatinizes above 50-67°

(Schoch and Maywald,

1956) , thereby yielding a dispersion of granule fragments,

starch

aggregates and molecules, which do not settle as readily but contribute
to the pollution problem.

If recovered, starch could be a valuable

feed constituent.
T he protein content of the recovered food ranged from 27 to 35%.
It w a s lower for acid precipitate since more starch settled with the
proteins.
Composition of protein of water extracts and whole tu b e r s .
The m ajor difference between the nitrogenous components in dis­
tilled and tap wa t e r extracts versus those in the original tubers
is the increased non-protein-N in the extracts
tein fractions,

(Table 4). Of the pr o ­

the water soluble tuberinin and the unknown nitrogen

compounds were increased most*

Tuberin, the less soluble but major

prot e i n fraction in potato tubers and the residue was markedly lower
in b oth extracts.

The distilled water extract contained much less

of the tuberin fraction but more tuberinin and non-protein-N.

It

appears that the different response of the two extracts to protein
p r e c i pitation can b e explained partly by the different compositions
of the extracts.

The distilled water extract contained m ore of the

h i g h l y soluble fractions.

36

Table 4.

Composition of crude protein of Russet Burbank potatoes:
tubers, distilled and tap water extracts.

whole tuber
%

Crude pro tein

100

a

distilled
water
%

100

b

w h ole

tap
water
%

100

c

Protein fractions
39.2

13.7

27.3

Globulin II

0.5

1.1

0.8

Tuberinin

1.5

6.9

6.2

Pro l amin

0.8

1.2

0.6

Glutelin

0.1

0.1

0.1

u n known nitrogen
compounds

0.9

4. 9

4.3

Residue

7.8

4.1

4.2

Nonprotein N

49.2

67.8

56.4

Non p r o t e i n N
(12% TCA)

-O

00

Tuberin

71.3

58.

a Sample size

60gm,

116 mg crude protein/gm

b Sample size

15gm,

469 m g crude protein/gm

c Sample size

20gm,

321 mg crude protein/gm

2

37

C o m p a r i s o n of ferric chloride and H C 1 .
F e r ric chloride is one of the principal coagulants used in sewage
work.

It is cheap, has acid properties and the trivalent iron ion

is a good nucleating site for large floe formation

(Daniels,

Table 5 shows that it compares favorably w i t h HC1,

its pH optimum

for pro t ein precipitation is higher (pH 4.0)

than for HC1

1973).

(pH 3.0).

The advantage of ferric chloride is that the water does not have to
b e heated.

The iron recovered w i t h the protein could add to the n u t r i ­

tional significance of a recovered feed.

Amine e_t al.

(1972) found

in c h icken assays that reduced iron ranked second only to ferrous
sulfate in efficiency as an iron supplement.
Lime, H ^ P O a and FeCl^ treat m e n t .
In the sugar beet industry,

the Steffen process, using a c ombi n a ­

tion of lime and H 3 P O 4 for the recovery of protein has been widely
used
ment.

(Schneider, 1968).

Figure 3 summarizes the data of this experi­

P rotein yields are continuously increased as the pH is raised

to pH 12 and then lowered by H 3 PO 4 or FeCl^.

The data indicates

that lowering the pH to 9.0 w ith either F e C l 3 or H 3 P O 4 yielded the
h i g h e s t quantities of recoverable protein.

Protein recovery was good,

but b e c a use of the high amounts of Ca in the product its usefulness
as pro t e in feed is questionable.

It w ould better qualify as a Ca

supplement•
A serious disadvantage of lime and H 3 P O 4 treatment is that water
has to be neutralized before being discharged.

Also, phosphorus can

38

Table 5.

Residual crude protein (ppm) in solution after treatment of protein
w a t e r w i t h ferric chloride and HC1 respectively.

pH

HC1

FeCl3

6.0

3101

2659

5.0

2707

2264

4.0

2501

2167

3.0

2382

2228

2.0

2584

2652

1.0

3024

3207

3456

3468

1982

2012

original
concentration
TCA standard

Figure 3:

Residual protein in solution after raising the pH with
CaOH, followed by lowering it with either H^PO^ or FeCl^.
e

•H
<0

O 100

CaO

cx
80

FeCl

40
•H

20
0

8

9

lo

11

12

pH

39

be r e adily precipitated with lime above pH 11.8 but its solubility
is m u c h increased at pH 9 (Wilcox,

1974),

thus leaving high amounts

of residual phosphorous in the effluent stream after the above treat­
ment .

40

LITERATURE CITED
Amine,

E . K . , Neff, K. and Hegsted, D.M. , 1972.
of available iron using chicks or rats.
Vol. 20, No. 2: 246-251.

Biological estimation
J. Agr. Food Chem.

A s s o c i a t i on of Official Agricultural Chemists.
1970.
11th ed.
W a shington DC.
Association of Official Agricultural Chemists,
p. 801.
Ballance, R.C.
1964.
A Review of primary treatment processes.
Int.
Symp. Util. & Disposal of Potato Waste, Proceedings.
p p . 200-211.
Borud, O.J.
1971.
Verwertung von Abfaellen der kartoffelverarbeitenden
Industrie.
Die Staerke 23(5): 172-176.
Daniels, S.L.
1974. A survey of flocculating agents-process description
and design considerations.
In "Water-1973,"
G.F. Bennet ed.,
A l ChE - Symposium series 136, vol. 70.
p p . 266-281.
Federal Register 38, 13523 and 13528, May 22, 1973.
Federal Water
P ollution Control Act as amended by Public Law 92-500 of 1972.
Heisler, E.G., Siciliano, J. and Krulick, S., 1972.
Potato starch
factory waste effluents.
II Development of a process for
recovery of amino acids, proteins and potassium.
J. Sci. Food
and Agriculture 23: 745-762.
Heisler, E.G., Silciliano, J . , Treadway, R.H. and Woodward, C.F., 1959.
R e covery of free amino compounds from potato starch processing
water by use of ion exchange.
Am. Potato J.
36: 1-11.
Itri d 1, F.M.
1973. Water quality management, Technical Report No. 31.2.
August 15, 1973.
Institute of Water Research, Michigan State
University, E. Lansing, Michigan
48824.
Lindner, K . , Jaschnik, S. and Korpaczy, I.
1960.
Aminosaeurezusammensetzung und biologischer Wert der Kartoffeleiweissfraktionen. Qual. Plant. Mater. Veget. 7: 289-94.
Luescher, R.1972.
Genetic variability of "available" methionine,
total protein, specific gravity and other traits in tetraploid
potatoes, Ph.D. Thesis, Michigan State University, East Lansing,
Michigan
48824
Neuberger, A. and Sanger, F.
1942.
Biochem. J.
36, 662— 671.

The nitrogen of the potato.

41

Pettay,

B.
1975.
Y ou can make money in pollution control.
January 1975, p. 50-52.

Chipper,

Porter, W.L., Siciliano, J . , Krulick, S. and Heisler, E.G., 1970.
R everse Osmosis: Application to potato starch waste effluents.
Memb r a n e Science and Technology (Plenum Press, 1970). p p . 220-230.
Schoch, T.J. and M a y w a l d , E.C., 1956.
Microscopic examination of
modified starches.
Anal. Chem. 28 (3): 382-7.
Schwartz, J.H., Krulick, S. and Porter, W.L., 1972.
Potato starch
factory waste effluents.
III.
Recovery of organic acids and
phosphates.
J. Sci. Food and Agriculture
23: 977-985.
Smith, 0., 1968.
Potatoes: Production, Storing, Processing.
The Avi
Publishing Company, Inc. Westport, Connecticut, pp. 262-393.
S t a b i l e , R.L. , Turkot, V . A . , and Aceto, N . C . , 1971.
Economic evaluation
of processes for the recovery of potato starch waste effluent.
Proc. nat. Symp. F d . Process. Wastes, Denver, Colorado, p . 185.
Strolle, E . O . , Cording, J. and Aceto, N . C . , 1973.
Recovering potato
proteins coagulated by steam injection heating.
J. Agr. Food
Chem. 21 (6 ): 974-977.
Vlasblom, M. and Peters, H . , 1958.
Dutch patent 87,150.
1957.
German
patent 1,021,243.
Recovery of proteins from potato w ash water.
Wilcox,

R.L., 1974.
Removing in excess of 99% phosphorous at ELY,
Minnesota.
In Water-1973.
G.F. Bennet ed. AIChE-Symposium
series 136, vol. 70. pp. 358-366.

Willard, M . , 1962.
Introduction panel on waste disposal.
12th Natl.
Potato Conference, May 1962.
Bakersfield, California, p p . 37-38.

CHAPTER 3

PROTEIN

QUALITY OF PRECIPITATE FROM WASTE EFFLUENT OF POTATO CHIP
PROCESSING MEASURED BY BIOLOGICAL METHODS

42

PRO T E I N QUALITY OF PRECIPITATE FROM W ASTE EFFLUENT OF POTATO CHIP
PROCESSING MEASURED BY BIOLOGICAL M E T H O D S 1
Erhard Meister and Norman R. Thompson^

ABSTRACT
The nutritional value of precipitated protein of simulated waste
effluent and of crude protein of whole tubers was biologically and
m i c r o - b i ologically assessed using weanling voles
cus) and Streptococcus z y m o g e n e s .

(Microtus pennsylvani-

The nutritional value of the protein

fraction from Sebago was superior to Russet Burbank and an unidentified
v ariety received from a potato chip plant.
index

(PEI)

The protein efficiency

for Sebago was not significantly different from casein.

P E I ’s for precipitated protein were higher than for whole tubers for
the latter two varieties but not for Sebago.

No differences in P E I fs

w e r e found between samples heated to 98° and those treated at room
temperature.

The "biological values" determined by S_, zymogenes

followed the same pattern for both varietal and treatment differences,
giving highest values for Sebago protein.
b e t w e e n heat and acid

No differences were found

(HC1 or FeCl 3 > treatment but values for whole

tubers and samples treated with lime and H 3 P O 4 or F e C l 3 were all lower.
This paper demonstrates that a potato chip plant could reduce water
consum p t ion and discharged waste by relatively simple means and obtain

^Received for publication
A rticle No.

Michigan A g r . Exp.

Sta. Journal

^Grad. Asst, and Professor, respectively, Department of Crop and Soil
Sciences, Michigan State University, East Lansing, MI
48824.

43

a high quality feed containing approximately 30% protein with a high
b i o l o g i c a l value.
INTRODUCTION
Mi c higans potato chip industry is searching for methods to reduce
p r o c e s s i n g losses and to economically exploit the waste effluent.
After removal of primary wastes by screening,
a p p r o x i m ately 50% of what remains.
removed w i t h hydrocyclones.

A sizable portion can easily be

The product can be m arketed in either

d ry or w et form (Pettay, 1975).
of proteins, accounting for

starch accounts for

1/3

Simple procedures for the recovery
to

1/2

of the nitrogenous substances

in the effluent, were reported by Meister and Thompson

(1975).

Several

treatment combinations reduced the protein in the effluent by 85 to
95%. The recovered material contained approximately 30% protein.
P roteins are subject to alterations by both physical and chemical
treatments.

These and the source of the protein may affect their

n u t r i t i o n a l value.

It is important to select a procedure for w a s t e

r e c l a m a t ion that is least damaging to the protein to obtain a high
quality product.
N it rogen balance studies with human adults showed that potato
pro t e i n was superior to most m ajor plant proteins and it approached
the value of whole egg

(Kofranyi and Jekart,

1967).

The sulfur con­

taining amino acids are first limiting in potato protein

(Schupan,

1958). W eight gains of growing voles w e r e dependent on the potato
v a r i e t y fed and were improved w i t h methionine supplementation

(Rios

44

e t a l * 1972) .

"Available methi o n i n e 11 and a "biological" value of

potato p rotein was evaluated w i t h Streptococcus zymogenous
1972).

The same organism was used by Ford

(1962)

(Luescher,

to study heat damage

to different proteins.
The purpose of this study was to examine the effect of cultivar
differences and methods for protein precipitation on the biological
va lue of the recovered potato protein.
MATERIALS AND METHODS
Two cultivars, Russet Burbank and Sebago grown on the Mont c a l m
Experiment Station during the 1974 season and an unidentified variety
from a Detroit chip plant were selected for this study.

Whole tuber,

heat and acid precipitate of each variety were tested and compared
to casein as a standard.

Abrasive peel from the unknown variety

was included as an additional protein source.
Pr e paration of samples for vole d i e t .
For whole tuber diets, average size tubers of each variety were
selected, washed, autoclaved at 15 p.s.i.
sliced and freeze-dried.

for 15 minutes,

peeled,

To simulate waste water, Russet Burbank

and Sebago tubers w e r e washed,

ground for 10 minutes in a Waring blender,

the m ash diluted w i t h water to five times its volume,

filtered through

several layers of cheese cloth and left for 30 minutes to settle the
starch.

Half of each diluted extract and half of a w ater sample

received from Detroit were acidified w i t h HC1 to pH 3.0,

samples w er e

stirred for 15 minutes then subjected to 90 minutes of sedimentation.

45

The other half of each sample was heated to 98° after adjustment of
the pH to 4.5,

this was followed by immediate cooling in ice water

and settling.

The peel and

w a t e r b a t h for 15 minutes at

the acid precipitates

were heated in a

60° to gelatinize the starch.

All

samples

w e r e freeze— dried.
Pro t e i n evaluation by animal a s s a y .
In this study meadow voles

(Microtus pennsylvanicus) were selected

as experimental animals because of their rapid growth and small food
r e q u i r e m e n t s .^
b y Elliott

Diets were made up according to methods described

(1963) and (Shenk and Elliott, 1969).

Composition of con­

trol and experimental diets

is given in Table 1.

Freeze dried

w e r e ground in a Wiley Mill

to pass a 40 mesh sieve,

and assay

samples
diets

m a d e up to contain seven percent protein.
In the feeding experiments, weanling voles 12-14 days old and
w e i g h i n g from 12.5-16.0 grams were used.

For two days they received

a starter diet followed by the experimental diets over a six day period.
Food and water were available ad l i b i t u m .

The animals were closely

observed during the feeding trial and weights taken initially and
every two days thereafter.

The voles were housed individually in

plastic-bottomed cages with corncob bedding and non-absorbent cotton
for nesting.

^Litters of weanling voles were received from Dr. Elliott, Department
of Crop and Soil Sciences, Michigan State University, East Lansing,
M i c h i g a n 48824.

46

Diets w e r e mixed with the amount of water necessary to m ake a
dough-like consistency,
v o l e feeder.

then molded into wafers that would fit the

They w ere dried at 105°F for 48 hours, wrapped in a l u m i ­

n u m foil and stored at — 18° until needed.

They were thawed at room

temperature for 16-18 hours before weighing and feeding.

The moisture

content of an aliquot was determined using the vacuum oven method
(AOAC, 1970).

The food consumption was determined by the loss in

we i g h t of the entire feeder.
The experimental design was a randomized block design.

The most

uni f o r m voles of two litters of the same harem were randomly assigned
to the eleven diets forming one block and this was repeated
At the end of the feeding period, protein efficiency indices

6

times.
(PEI)

w e r e computed from gain and food intake.
Protein evaluation by Streptococcus zymog e n e s .
The "biological" value of protein was assessed microbiologically.
Ford's procedure, adapted for potato protein by Luescher
Peare

(1973), was followed.

(1971) and

Casein was used as a standard.

Samples containing the equivalent of 50 mg crude protein were
placed into

4

-ounce screw-cap bottles,

20

ml citrate cyanide buffer

w a s added, and the pH adjusted to 7.2 with 1 N KOH.

The samples were

h e a t e d in a w ater bath to 56° for 3 hours w ith intermittent shaking
and adjustment of the pH to 7.2, they were diluted to 100 ml with
distilled water.

47

Tri p licate portions of 4 ml of the digest were pipetted into
16 x 150 m l test tubes.

Two ml of basal m edium (see Luescher,

1971)

was added and the volume brought to 10 ml with distilled water.
capping each tube,
12

minutes,

After

samples were sterilized with flowing steam for

and after cooling one drop of inoculum culture

,

diluted

1:10 w i t h sterilized .85% saline solution, was added and tubes were
incubated for 48 hours at 37°.
After incubation,
minutes,

tubes were heated in flowing steam for 10

stoppered and shaken vigorously, and set aside for 30 seconds.

The optical

densities of the cultures were then measured with a

Hitachi Perkin-Ulmer 139 UV-VIS spectrophotometer at 580 nyw.

The

"biological value" of the sample was expressed in percent of growth
compared to a tube containing the same amount of casein protein.
The above procedure was carried out on two consecutive days and
average values for absorption on each day were used to calculate the
"biological value."
Preparation of samples for microbiological a s s a y s .
Five tubers of each of the three varieties were chosen at random,
four longitudinal slices were cut from the middle of each tuber, quickly
frozen, combined and freeze— dried.

^The o r g anism used for these tests was Streptococcus zymogenes NCDO
592, obtained from the National Collection of Dairy Organism,
Institute for Research in Dairying, Shinefield, Reading (UK).

48

D il ute potato extracts were made as described. Aliquot samples
w e r e treated as described below:
(a) Acidification w ith HC1 to p H 3.0.
(b) Acidification with HC1 to pH 4.5, heated to 98°.
(c) Acidification with F e C l 3 to pH 4.0.
(d) Raising pH to 11.5 with lime followed by adjustment to
pH 9 w ith H 3 P O 4 .
(e) Raising pH to 11.5 with lime followed by adjustment to
p H 9 w ith FeClg.
A ll sediments w e r e dialized in cellophane bags-* for 48 hours to avoid
interferences with the assay.

The samples were freeze dried, ground

in a Wiley mill through a sixty mesh screen and the nitrogen content
w a s determined by the micro-Kjeldahl method

(AOAC, 1970).

RESULTS A ND DISCUSSION
The protein quality of whole tubers, heat and acid precipitates
of three varieties, and a sample of abrasive peel were compared with
casein in a feeding trial with weanling voles.

A description and

the exact composition of the diets is given in Table 1.

The analysis

of v a riance for just the three treatments of the three cultivars indi­
cated that treatment effects were less pronounced than varietal differ
ences

(Table 2).

indices

Food intakes, weight gains and protein efficiency

(PEI’s) are summarized in Table 2.

Food consumption was the

same for all diets, although differences were found between litters.
E x a mination of the raw data showed that two litters w i t h high initial

Number 27/100 was obtained from Union Carbide.

49

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Diet

Composition*

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Cleveland,

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epotato starch:
cooked, oven-dried and ground.

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Ohio.

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5o

b o d y w e i ghts

(14— 15— 5 g) ate more than the others, but the weight gains

w e r e not significantly different.
varied considerably.
effici e n cy ratios
b y Peare
et al.

Consequently, PEl's of litters

PET values are in the same range as protein

(PER) found for potato protein in rat feeding trials

(1972). They are much higher than reported for voles by Rios

(1972) who fed diets containing 5.28% crude protein, considered

to be below the level for minimal growth.

PEI values were highest

for casein but protein of Sebago compared favorably with it.

Values

for the other two varieties were lower, especially of whole tubers.
W i t h the exception of the heat precipitate of Sebago

(SH), PEl's of

pro t e i n recovered by either method of precipitation were equal or
superior to crude protein of whole tubers.
and Slack

(1948)

Chick and Cutting,

(1943)

found that nonprotein nitrogen alone did not support

growth of weanling rats and that tuberin prepared from the sap by
h e a t i n g at 80° at pH 4 was not superior to that of the mixture of
p r o t e i n and non-protein in the whole potato.
are in accordance with these results.

Only the data from Sebago

These findings suggest that

the n u tritional value of the protein fraction of Sebago is superior
to Russet Burbank and the unidentified variety.

The presumably better

balanced protein of Sebago could compensate for the lower nutritive
v a l u e of the non-protein fraction.

Evidence for such an interpretation

is given by the higher PEl's of Sebago protein to protein of the other
precipitates, and the fact that PEl's for recovered protein were equal
to crude protein of whole tubers for Sebago but not for the other

51
Table 2.

Comparison of litter, variety and treatment effects of vole
feeding trial.

Analysis of v ariance for PEI

source

df

litter

5

2.330

.466

variety

2

4.066

2.032

(a)

10

1.319

.132

treatment

2

1.547

.774

7.075*

var. x tmt.

4

1.905

.476

4.355**

30

3.277

.109

53

14.445

error

error

(b)

total

***

significant at P = .05,

SS

.01,

MS

.001 respectively.

F

3.531*
15.403**

52

varieties.

The relative amount of n on—protein N was the same for

all varieties

(approximately 54-60%).

The abrasive peel from the unidentified variety did not support
growth

(Table 3).

Chick and Slack (1949) have given evidence that

removal of the skin and outer cortex, which are the parts richest
in "insoluble protein" increased the nutritive value of the remainder
in a rat feeding trial.

In these trials with field voles,

salvage

an d processing of the skin into a feed is not justifiable because
of its low nutritional value.
It was anticipated that the different procedures employed to
precipitate the proteins might reduce the nutritional value of the
protein.

Two m ajor sources of spoilage were expected to occur:

non-enzymatic browning

(Maillard reaction,

(1)

1912) and (2) structural

changes such as denaturation and aggregation.

The former involves

condensation between aldehyde groups of reducing sugars and free amino
groups, primarily the €-amino groups of lysine, a reaction that in­
creases rapidly at higher temperatures.

The data from the vole feeding

trial did not reveal any appreciable differences between heat and
no heat treatment.

The high moisture content and low pH during heating

could have slowed dwn the reaction.

Also differences m a y not have

appeared because the second sample was heated

(60°) to gelatinize

the starch.
Streptoccus zymogenes was considered an ideal organism to test
the different varietal and treatment differences.

Besides reduced

53

Table 3.

Food intake, weight gain and protein efficiency index (PEI) of
eleven diets tested in vole feeding trial.
Average values of
six voles per diet.

Diets

Food intake
g / 6 days

Weight gain
g / 6 days

PEI

Casein

30.0

6.25 a

3.02 a

Burbank tubers

35.8

4.75 cd

1.89 e

Burbank heat prec.

35.5

5.31 be

2.19 c

Burbank acid prec.

28.4

4.13 de

2.07 c

Sebago tubers

35.3

5.98 ab

2.51 b

Sebago heat prec.

40.3

6.26 a

2.21

Sebago acid prec.

28.2

5.82 ab

2.96 ab

Unknown tubers

32.2

3.93 e

1.81 e

Unknown heat prec.

33.8

4.93 c

2.09 c

U nknown acid prec.

35.4

5.41 be

2.18 c

U nknown peel

34.7

.06 f

.02

c

d

Studentized range test:
Values with the same letter are not significantly different at the
5% level.

54

availa b i l ity of lysine. Ford

(1962) demonstrated significant reduction

of available arginine, histidine, methionine, valine,

leucine,

isoleu­

cine, and tryptophan of heated m i l k and fish protein using the above
bacteria.

Luescher

(1972) successfully used the same organism to

discri m i nate between available methionine of different potato cultivars.
The var i ety Sebago gave a higher biological value for almost all
treatments

(Table 4).

Highest biological values were observed by

precipitates of treatment with HC1 alone, HC1 plus heat and F e C ^ .
Treatment w i t h H 3 P O 4 or F e C l 3 subsequent to coagulation w ith lime
gave lowest values, but not different from whole tubers.

55
Table 4.

"Biological value" of crude protein of three potato varieties
subjected to six treatments, determined by S. z y m o g e n e s .

Treatment

Russet Burbank

Sebago

Unknown

whole tubers

67 D

70 ED

68 CD

aheat precipitate

70 CD

75 AB

69 CD

bacid precipitate

72 BC

77 A

69 CD

cF e C l 3 precipitate

72 BC

74 AB

69 CD

dCaO and H^PO^ prec.

66 D

68 CD

67 D

eCaO and T e C l 3 prec.

68 C

68 CD

67 D

Studentized range test:
Values with the same letter

(A, B,

. . .) are not significantly different

at the 5% level.

aheated to 98° after acidifying with 2N

HC1 to pH 4.5

^sedimentation at

23° after acidifying with 2N HC1 to pH 3.0

csedimentation at

23° after acidifying with 2M F e C l 3 to pH 4.5

^sedimentation at
23° after raising pH to 11.5 with CaO,
it to p H 9.0 with H^PO^
e sedimentation at 23° after raising pH to 11.5 with CaO,
to pH 9.0 with H^PO^

followed

by lowering

followed by lowering

56

LITERATURE CITED
As s o c i a t i o n of Official Agricultural Chemists.
1970. 1 1 th ed.
W a s h i n g t o n DC.
Association of Official Agricultural Chemists,
p. 801.
Chick, H. and Cutting, M.E.M., 1943.
Nutritive value of the nitro­
genous substances in the potato.
Lancet 245: 677-669.
Chick, H. and Slack, E.B. 1949.
Distribution and nutritive value of
the nitrogenous substances in the potato.
Biochem. J. 45:211-221.
Elliott, F.C., 1963.
The m e adow vole (Microtus Pennsylvanicus) as a
b io a s say test organism for individual forage plants.
Michigan
State A g r . Exp. Station, Quarterly Bulletin, Vol. 46, No. 1: pp 58— 72.
Ford,

J.E., 1962.
A microbiological method for assessing the nutritional
v a l u e of proteins.
Br. J. Nutr.
16:409-425.

Kofranyi, E. and Jekart, F . , 1967.
Zur Bestimmung der biologischen
W ertigkeit von Nahrungsproteinen, XII. Die Mischung von Ei mit
Reis, Mais, Soja, Algen.
Hoppe Seyler's Z. Physiol. Chem.
348:84-88.
Luescher, R. , 1971.
Evaluation of methods to determine the sulfur
c ontaining amino acids in potatoes.
M a s t e r ’s Thesis, Michigan
State University, East Lansing, Michigan 48824.
Luescher, R. , 1972.
Genetic variability of "available" methionine,
total protein, specific gravity and other traits in tetraploid
potatoes.
Ph.D. Thesis, Michigan State University, East Lansing,
M i c h i gan 48824.
Maillard, L . C . , 1912.
Action des acides amines sur les sucres.
Formation de melanoidines par voie m e t h o d i q u e . C.R. Acad.
154:66.
Pettay, B . , 1975.
You can make money in pollution control.
January 1975:50-52.

Sci.

Chipper,

Peare, R . M . , 1973.
Potato protein as affected by: A. variety and
environment and B. air separation of the dried flour.
M a s t e r ’s
Thesis.
Michigan State University, East Lansing, M i chigan 48824.
Rios Iriate, B.J., Thompson, N . R . , and Bedford, C.L., 1972.
Protein
in potato flakes.
Evaluating by the meadow vole (Microtus
p e n n s i l v a n i c u s ). Am. Potato J. 49:255-260.

57

Schupan, W. 1958.
Proteins et amino acids indispensable des vegetaux
alimentaires et de leur diverses o r g a n e s . Qual. Plant. Mater.
Veg. 3-4:19-33.
Shenk, S.J. and Elliott, F.C., 1969.
Technical notes.
A diet feeder
for wea n l i n g m eadow voles (Microtus pe n n s y l v a n i c u s ) . Am. Assoc.
Lab. Animal Sci- 1 9 ( 4 ) :522.
Slack, E.B., 1948.
161:211-212.

Nitrogen constituents of the potato.

Nature

A P^P E N D I C E S

Appendix 1
S edimentation of protein from tap water extract of Russet Burbank
tubers after heating to 98°C at pH 4.5 by gravity settling and 7
different centrifugational forces.
replications,

rep

Randomized block design w i t h 2

8 treatments with duplicate observations per treatment.

gravity
2

centrifugational forces in 1000 x G
4
6
10
20

30

40

1

3075

2985

3000

2977

3030

2917

2602

2422

2

2705

2650

2590

2575

2553

2482

2311

2150

mean

2890

2817

2795

2776

2791

2699

2456

2286

Studentized range test:
U nderlined means are not significantly different at the 5% l e v e l ,
using the studentized multiple range test.
A n a lysis of variance

source

df

SS

MS

F

replication

1120504

1

1120504

223.178***

treatment

1210124

7

172874

34.432***

rep x tmt

35144

7

5021

4168

16

2369941

31

sample
total

*** significant at P =

.001

58

APPENDIX 2

Sedimentation of protein from tap water extracts of Russet Burbank
tubers at p H 4 as a function of protein concentration,
temperature and settling time.
2 concentrations,

2

coagulation

Split plot design with 2 replications,

temperatures and 8 time intervals

(10 min.).

Analysis of variance

source

df

r e plication

1

2551

2551

concentration

1

152989866

152989866

error

1

51769

51769

temperature

1

913923

913923

temp x cone

1

307426

307426

error

2

12432

6211

time

8

51636030

6454504

time x temp

8

321570

40196

time x cone

8

5763635

720454

time x temp x cone

8

419240

52405

32

592389

18512

71

213010523

error
total

n.s.

(a)

(b)

(c)

SS

MS

non-significant at P = .05

*,*** significant at P = .05 and P = .001 respectively

59

F

.04 n.s.
2955.23***

147.14***
49.49*

348.66***
2.17 n.s.
38.84***
2.83*

APPENDIX 3

P r e c ipitaion of protein from a distilled water extract of Russet
Burbank tubers as influenced by 9 pH and 2 temperature levels.

Split

plot design with 2 replications,

heating

9 pH levels,

room temperature,

to 98°C and 2 observations per treatment.

Analysis of variance

source

SS

replication

MS

df

F

261623

1

261624

66.41***

8080439

8

1010055

240.94***

33536

8

4192

temperature

2606239

1

2606239

1085.07***

pH * temperature

3374821

8

421852

175.63***

21617

9

2402

15429

36

428

14393704

71

pH
error

error
sample
total

(a)

(b)

***significant at P = .001

60

61

Re s idual crude protein

(ppm) in solution after pH and heat

t r e a t m e n t .*

PH

r e p lication I
23°C
98 C

replication II
23°C
98°C

mean

23° C

98°C

6.3

4144

4152

4303

4285

4224 a,A

4218 a,A

6.0

4145

4142

4261

4248

4204 a,A

4195 a,A

5.5

4100

4032

4216

4128

4158 a b ,A

3157 a,A

5.0

4026

3099

4143

3216

4084 abc,A

3157 c,B

4.5

3842

2866

3899

2868

3870 be,A

2867 d,B

4.0

3867

2922

3998

2971

3933 c ,A

2947 c d ,B

3.5

4107

3665

4227

3904

4164 ab,A

3785 b,B

3.0

4187

4068

4252

4269

4219 a,A

4169 a,A

2.5

4137

4166

4325

4620

4231 a,A

4243 a,A

original
sol.

4151

4295

4223

TCA

2523

2451

2481

^average values of duplicate sample

(ppm)

Studentized range test:
- Temperature means for a given pH:
Values w i t h the same letter
different at the 5% level.

(A,B,..) are not significantly

- pH means for a given temperature:
Values with the same letter
different at the 5% level.

(a,b,..) are not significantly

APPENDIX 4

Precipitation of protein from a tap water extract of Russet Burbanks
tubers by 9 pH and 4 temperature levels.
replications , pH levels,

Split plot design with 2

4 temperatures and duplicate observations.

Analysis of variance

SS

source

df

MS

F

replication

3418918

1

3418918

77.374***

pH

5858462

7

5658462

128.051***

44189

7

44189

heat

310339

3

310338

8.891***

p H * heat

500989

21

500989

14.353***

83729

24

34904

238

64

5564227

127

error

error
sample
total

(a)

(b)

***significant at P = .001

62

63

Residual crude protein

(ppm) in solution after pH and heat

00

o
0
n

t r e a t m e n t .*

23°C

60°C

6.8

4702 a,A

4344 a b ,AB

3917 b ,BC

3527 b, C

6.0

4682 a,A

3836 bc,B

3403 be,BC

3142 b e ,C

5.0

3360 b ,A

3177 d ,AB

2908 c ,AB

2786 c,B

4.5

2957 be,A

2956 d,A

2854 c ,A

2745 c, A

4.0

2905 be,A

2984 d ,A

2906 c ,A

2744 c ,A

3.0

2865 c ,B

3537 cd,A

3140 c ,AB

3498 b ,A

2.0

3339 b ,B

3902 be,A

3992 b ,A

4167 b ,A

1.0

4646 a,A

4612 a,A

3656 a,A

4706 a ,A

PH

98°C

original
sol.

4705

TCA

2661

^average values of duplicate samples and

2

replications

Studentized range test:
- Temperature means for a given pH:
Values w i t h the same letter
different at the 5% level.

(A,B,..) are not significantly

- pH means for a given temperature:
Values w ith the same letter
different at the 5% level.

(a,b,..) are not significantly

APPENDIX 5

C omparison of ferric chloride versus hydrochloric acid as
p recipitating agent for protein from a dilute potato extract.
plot des i gn w i t h 2 replications,

2 treatments at

6

Split

different pH levels.

Analysis of variance

source

df

SS

MS

F

replication

1

1097600

1097600

17.77 n.s.

treatment

1

356482

356482

5.77 n.s.

error

1

61778

61778

pH

6

8703489

1450581

82.58***

tmt x p H

6

932467

155411

8.84***

40

702606

17565

55

11854423

error
total

n.s.
***

(a)

(b)

non-significant at P = .05
significant at P = .001

64

APPENDIX 6

R e s idual crude protein in solution after raising the pH with CaO
to pH 12 followed by lowering it with H_PO, and Fed,,.
3 4
3
design w i t h 2 replication,

PH

Randomized block

20 treatments and duplicate observations.

CaO

h

3P 0 4

FeCl3

7.0

3444

2622

2317

8.0

3432

2577

2320

2541

2717

2375

2387

2412

2455

2459

2502

2596

2562

8.5

—

9.0

3185

9.5

—

10.0

2840

11.0

—

12.0

2657

original
solution

3448

TCA

2125

Analysis of variance

df

SS

MS

1

233820

233820

9 6 . 22***

treatment

19

9183968

483366

198.92***

error

59

77593

1315

total

79

9495381

source

replication

*** significant at P = .001

65

F

APPENDIX 7
Vole Feeding Trial

Eleven diets were tested in a randomized block design,

food intake

and weight gain was measured and the protein efficiency index calculated.
Replications were made up of voles of two litters originating from the
same harem.
Analysis of variance for food consumed
source

df

litter

5

1285.54

257.11

diets

10

795.32

79.53

error

50

3919.03

78.38

total

65

5999.89

SS

MS

F
3.280*
1.015 n„

Analysis of variance for weight gained
source

df

litter

5

SS

MS

F

9.92

1.98

1.198 n.
11.502***

diets

10

190.41

19.04

error

50

82.77

1.65

total

65

283.11

Analysis of variance for PEI
MS

SS

F

source

df

litter

5

2.26

.452

4.318**

diets

10

39.56

3.956

37.79***

error

50

5.23

total

65

47.07

n.s.

non-significant at P =
significant at P = .05,

.1046

.05
.01,
66

.001 respectively