EPA-600/3-76-074
August 1976
Ecological Research Series
GROWTH RESPONSES OF CHICKS FED MICROBIAL
PROTEIN PRODUCED FROM ORGANIC WASTES
Municipal Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related .fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical1
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-074
August 1976
GROWTH RESPONSES OF CHICKS FED MICROBIAL
PROTEIN PRODUCED FROM ORGANIC WASTES
by
Ozie L. Adams
Erly J. Thornton
Tennessee State University
Nashville, Tennessee 37203
Grant No. R802679
Project Officer
Charles J. Rogers
Solid and Hazardous Waste Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views and
policies of the U.S. Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorsement or recommendation
for use.
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FOREWORD
The Environmental Protection Agency was created because of -increasing
public and government concern about the dangers of pollution to the health
and welfare of the American people. Noxious air, foul water, and spoiled
land are tragic testimony to the deterioration of our natural environment.
The complexity of that environment and the interplay between its compon-
ents require a concentrated and integrated attack on the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact, and
searching for solutions. The Municipal Environmental Research Laboratory
develops new and improved technology and systems for the prevention,
treatment, and management of wastewater and solid and hazardous waste
pollutant discharges from municipal and community sources, for the pres-
ervation and treatment of public drinking water supplies, and to minimize
the adverse economic, social, health, and aesthetic effects of pollution.
This publication is one of the products of that research', a most vital
communications link between the researcher and the user community.
The object of this study was to determine if fungal protein produced
from the bio trans formation of organic wastes could be substituted for con-
ventional protein in chick starting diets. Results of the feeding trial
clearly showed that fungal protein when added as a replacement for soybean
protein, supported chick body maintenance but was not as effective as the
standard protein diet in stimulating growth. Palatability of ration and
poor feed consumption were factors contributing to slow growth.
Francis T. Mayo
Director
Municipal Environmental Research
Laboratory
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ABSTRACT
The objective of this study was to investigate the use of micro-
bial protein produced from organic wastes, as a source of protein in
chick starting rations.
Microbial protein recovered from the mold mycelia was produced
when potato waste materials were homogenized and added to a mineral
salt containing a culture medium suitable for the action of a strain
of Aspergillus niger.
The new product containing approximately 29.0 percent protein was
added to chick starting rations, replacing the soybean protein. Sixty-
two (62) percent of the protein in the ration were supplied by soybean
meal. The length of the feeding trial was four (4) weeks.
Results of the feeding trials showed that fungal protein was effec-
tive in supporting body maintenance but was ineffective in stimulating
growth. Palatability of rations and poor feed consumption were factors
contributing to slow growth. However, feed utilization was less of a
problem as shown by nitrogen retention trials.
Carcass quality, pigmentation, deposition of fat and uniformity in
size were similar in all groups.
Fungal protein has the potential for use as a source of protein in
chick rations. However, additional research is needed to further study
such factors as feed consumption, feed (amino acids) utilization and
palatability.
This report is submitted in fulfillment of Grant Number R802679-01
by Tennessee State University, Nashville, Tennessee, under the sponsor-
ship of the Environmental Protection Agency. Work was completed
October 1974.
^v
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CONTENTS
Sections Page
I Conclusions 1
II Recommendations 2
III Introduction 3
IV Experimental Procedure 6
V Discussion 11
VI References 16
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TABLES
No. Page
I Ami-no Aoid Analysis of Fungal Strain Compared to
Several Standard Proteins 9
II Composition of the Rations 10
III Effect of Fungal Protein on Chick Growth and
Feed Utilization (in grams) 13
IV Effect of Fungal Protein on the Retention of
Dietary Nitrogen by the Chick (in grams) 15
v^
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FIGURE
No. Page
I Effect of Fungal Protein on Growth Rate of Chicks
(Four Weeks) 14
•011
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ACKNOWLEDGMENTS
Special thanks are due to Cliarles J. Rogers, Senior Research
Chemist, Solid and Hazardous Waste Laboratory for his technical sugges-
tions 3 literature and arranging for contacts with professional
colleagues working in this field.
Indebtedness is also acknowledged to Phillip 0. Meachem, Marvin
Jones and Mozell Byars, student assistants, Tennessee State University,
for their able research assistance.
z;^^^
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SECTION I
CONCLUSIONS
Microbial (fungal) protein produced from pota^^ waste materials
was effective in supporting body maintenance in growing chicks but was
ineffective in stimulating growth.
The data also showed that feed consumption was a major factor
contributing to poor growth. Feed utilization was less of a problem
since there was little difference in nitrogen retention between the
chicks fed the rations containing all soybean protein when compared to
the chicks fed rations containing the fungal protein. Palatability of
the diets seemed to have been another factor contributing to slow
growth.
The overall carcass quality of the chicks fed the basal and experi-
mental rations was good, even though the chicks fed high levels of
fungal protein were small -when compared to those fed the basal ration.
Pigmentation, deposition of fat and uniformity in size were similar in
all groups.
The data indicated that fungal protein produced from potato waste
materials has the potential for use as a source of protein in chick
starting rations.
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SECTION II
RECOMMENDATIONS
It is recommended that additional microbial protein be produced
from potato waste materials and in combination with other high and low
starch waste materials.
It is also recommended that additional research be conducted to
further study such factors as toxicity, palatdbility3 feed consumption,
feed utilization, protein quality and the cost factor.
In addition to chicle studies, other types of poultry such as
turkeys, as well as small animals (rats), should be used in feeding
trials.
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SECTION III
INTRODUCTION
The disposal of sol-id waste, whether1 from industrial, agricultural
or other sources, continues to be a major problem in most areas in the
United States, The traditional waste disposal methods of landfills,
incineration and dumping into flowing streams have not only been unsat-
isfactory from a resource recovery standpoint, but are becoming less
feasible as more stringent regulations concerning air and water pollu-
tion are put into effect. Technological development for acceptable
disposal methods and the economical recycling of organic wastes into
usable products has been and remains the primary objective of the
National Environmental Research Center, Cincinnati, Ohio 45256.
In the past many proposals have been made for the conversion of
organic waste materials into useful products ranging from utilization
of their intrinsic fuel value, anaerobic conversion to methane,
production of chemicals including ethyl alcohol, and the utilization
of fermentable substrates to produce edible protein. After evaluating
the many recycling alteratives, the Solid & Hazardous Waste Research
Division has identified, as one promising option, the biological
conversion of organic wastes into foodstuff.
Some of the processes developed for the utilization of agricultural
and industrial wastes are based on their conversion to single-cell
protein (1,2) by using the wastes as substrates on which to grow yeasts
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and other fungi and bacteria for animal or human consumption. Food or
animal feed is thus produced from a renewable resource without inter-
fering with the primary productivity of agricultural land. Torula
yeast (Candida utilis) grown on molasses or sulfite liquor has been
long used in animal feed—but in small quantities, primarily as a vita-
min source. Processing to lower its nucleic acid content will be
required and has been researched (3) to make it acceptable as a signi-
ficant protein source for human consumption. The same limitation
applies to all other single-cell proteins, because rapidly growing cells
have a high content of nucleic acid that man can excrete only in the
form of uric acid. Too much uric acid leads to its deposit in the
joints, causing gout, or in the kidneys, where it may lead to stone
formation.
As in the case of higher plants, each new yeast, fungus, or bacte-
rium species proposed for human use will have to be carefully screened
for toxicity and allergenicity in man. Careful processing will be
required to render them suitable for food use.
There is now much interest in the production of single-cell protein
from petroleum hydrocarbons, and their use as food will similarly depend
greatly on the ability of the food technologist to render them safe and
palatable, and on the food industry to incorporate them into attractive
food products. T'hci. • can be little doubt that single-cell protein, in
the broad sense in which the term is currently used to include multi-
cellular lower plants such as filamentous fungi as well as yeasts and
bacteria, will make an increasingly important contribution to animal and
human feeding. However, the use of single-cell protein, grown on rela-
tively pure substrates and processed for human consumption, seems almost
4
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certain to ~be initially limited to the processed foods of the industrial-
ized countries because of its high cost compared with that for protein
from legumes and oilseeds.
As an alternative to direct human use of single cell protein, a
short-term preliminary feed trial with fungal protein produced in the
SHWRD laboratory was conducted at Oregon State University Food Science
and Technology Laboratory. The fungal protein (36 j. ~L
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SECTION IV
EXPERIMENTAL PROCEDURE
The microbial (fungal) protein used in this study was prepared
when potato waste or starchy material was homogenized and added to a
mineral salt containing a culture medium suitable to the action of a
strain of Aspergillus niger. The protein material containing approxi-
mately 29.0 percent, was recovered from the mold mycelia3 Rogers et al.
(1972) (2).
The amino acid composition of the fungal protein compared with
several standard proteins is shown in Table I. The nutritional value
of proteins depends upon a complement of essential amino acids present.
It is apparent from Table I that the fungal protein developed compares
favorably with other standard proteins. The product contains all of
the essential amino acids required by the chick.
Day-old broiler type chicks were used in this study. The chicks
were wing-banded, weighed and distributed at random into five experi-
mental groups (12 chicks per group). All groups were maintained in
electrically heated, thermostatically controlled battery brooders
equipped with raised wire floors. The chicks received constant light-
ing and the room in which the brooders were located was heated to
provide a minimum temperature of 70°F. Ventilation was provided by
thermostatically controlled exhaust fans. Individual chick weights and
group feed consumption were recorded at bi-weekly intervals during the
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experimental period and feed efficiency was calculated at the end of
the experiment. Feed and water were supplied ad_ libitum. Mortality
was recorded on the day of occurence, and only surviving chicks were
included in the statistical analysis. The length of the feeding period
was four weeks. Management of birds in the battery was in keeping with
good poultry practices.
Experimental Rations
One basal ration of the corn-soybean meal type in combination with
other ingredients commonly employed in poultry feeding was computed.
Approximately 62 percent of the total protein in the basal ration was
supplied by soybean meal. Four subsequent rations were calculated
replacing 253 50, 75 and 100 percent of the soybean meal protein with
fungal protein (Table II).
Analysis of Data
The criteria used for determining the effectiveness of the new
protein were (1) average body weight; (2) average gain; (3) feed
efficiency., freedom from diseases; (4) mortality and (5) nitrogen
balance trials.
The body weight data were subjected to statistical treatment by
the analysis of variance, Snedecor (1956) (4). When a significant
difference due to treatment was made by computing the L.S.D. (least
significant difference). The L.S.D. represents the minimum increase
or decrease in body weight required for significance at the five
•percent level of probability.
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Nitrogen Balance Trials
> Nitrogen retention tests, conducted to measure the utilization of
the experimental diets by the chicks, were performed by the indirect
method involving the analysis of diet and excreta. Special wire cages
holding six to eight birds were placed on battery wire floors. Porce-
lain trays measuring 12" x 18" x 2%" that fitted conveniently under
each cage were used as receptacles for the feces. In order to prevent
the loss of nitrogen to the atmosphere, 1.5 liters of a 1 normal
solution of sulfuric acid were placed in each receptacle. At the end
of the two-day test period, the acid mixtures were transferred to
tared gallon jars and weighed. The total nitrogen excreted was deter-
mined by mechanically stirring the acid mixture to form a uniform
slurry from which a sample of approximately 500 ml. was transferred to
a Waring blender and completely homogenized, after which a 20 to SO
ml. aliquot was weighed into a digestion flask, and. the nitrogen was
then determined by the AOAC*method. The individual chick weights and
the feed consumption were recorded for the test period. Kjeldahl
nitrogen (AOAC) values were also obtained on the samples of feed used.
The nitrogen retained by the birds was calculated as the difference
between the nitrogen values in the feed samples and the total nitrogen
content of the feces excreted during the test period.
Due to the amount of material available, the study was limited to
broiler type chicken rations.
*AOAC - Association of Official Agricultural Chemists
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TABLE I
A14INO ACID ANALYSIS OF A FUNGAL STRAIN
COMPARED TO SEVERAL STANDARD PROTEINS
(Grams/16 Grams Nitrogen)
Aspergillus
Amino Aaid Niger on Casein
Potato Waste
Lysine
Histidine
Arginine
Aspartic Acid
Threonine
Serine
Giutamic Acid
Pro line
Glycine
A lanine
Cystine
Methionine
V aline
Isoleucine
Leuoine
Tyrosine
Pheny la lanine
Tryptophan
4.2
1.8
4.7
9.4
4.8
4.7
10.5
4.4
5.0
4.3
0.8
3.2
6.5
5.3
8.6
4.4
7.7
1.2
8.0
3.0
4.0
7.0
4.7
6.7
25.0
11.0
2.5
3.0
1.0
3.5
7. 7
6.5
9.7
6.5
5.9
1.2
Soybean
Meal
6.6
2.5
7.0
8.3
3.9
5.6
18.5
5.0
3.8
4.5
1.2
1.1
5.2
5.8
7.6
3.2
4.8
1.2
Opaque- 2
Corn
4.2
3.5
6.8
10.0
3.3
4.3
18.7
8.6
4.8
6.5
1.7
1.4
4.9
3.2
8.4
3.9
4.4
1.3
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TABLE II
COMPOSITION OF THE RATIONS
Ingredients (in percentage)
Ground yellow corn
Soybean oil meal (44% protein)
Fungal protein (28,88% protein)
Meat and bone scraps (50% protein)
Menhaden fish meal (60% protein)
Dried whey product
Dehydrated alfalfa meal
(17% protein)
Dicalcium phosphate
Limes -tone , ground
Salt, iodized
Premix
Percentages, Crude Protein
Basal
56.50
SO. 00
-0-
3.00
3.00
2.00
2.00
1.50
1.00
0.50
0.50
21.44
25% Fungal
Protein
52.575
22.50
11.425
3.00
3.00
2.00
2.00
1.50
1.00
0.50
0.50
21.50
50% Fungal
Protein
48.650
15.00
22.850
3.00
3.00
2.00
2.00
1.50
1.00
0.50
0.05
21.125
75% Fungal
Protein
45.725
7.50
33.275
3.00
3.00
2.00
2.00
1.50
1.00
0.50
0.50
21.125
100% Fungal
Protein
40.80
-0-
45.70
3.00
3.00
2.00
2.00
1.50
1.00
0.50
0.50
20.70
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SECTION V
DISCUSSION
The results of the treatments are shown in Table III and Figure I.
The basal ration contained 21.44 percent crude protein. Of this amount,
62 percent of the protein was supplied by soybean meal. Treatment ra-
tions were calculated replacing the soybean meal protein in the rations
t,
with equivalent fungal protein at 25, 50, 75 and 100 percent levels.
The data show that the average body weight of the birds in the
experimental groups was significantly less than those in the control
group (P ^.05). When the average body weight of the birds in the
experimental groups was compared a significant difference -was observed
between the groups fed rations in which the soybean protein was replaced
by 25 and 50 percent fungal protein and those fed rations containing 75
and 100 percent fungal protein. However, there was no significant
difference between the average body weight of the birds fed the rations
containing the 25 and 50 percent fungal protein.
The data also show that the birds fed the ration containing 100
percent fungal protein made the poorest gain in body weight. However,
mortality was low and the marketing qualifies of the birds were good.
Pigmentation, deposition of fat and uniformity in body size were also
good. These observations indicate that the ration provided the essen-
tial nutrients to support body maintenance and fat production but not
growth. Adequate utilization of the fungal protein by the chicks seemed
11
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not to have been a problem. On the other hand, feed consumption and/or
absorption may have been problems. The diet in whioh 62 percent of the
total protein was supplied by fungal protein also contributed to the
production of body fat as well as the general overall appearance of the
dressed birds.
Palatability of the rations was another factor to consider in the
evaluation of chick growth. Chicks fed rations in which the soybean
protein was replaced by 50, 75 and 100 percent fungal protein consumed
considerably less feed.
Particle size was a factor that may have affected feed consumption.
The texture of the fungal protein supplement resulted in a powder form
when ground and was very light in weight. The chicks had a tendency to
pick out the large feed particles and to leave the finely ground
materials.
Nitrogen Retention Trials
A criterion for the evaluation of the nutritive value of a feed is
the efficiency of its utilization when fed to growing chicks. The
efficiency of utilization is affected by the amount of nutrients re-
tained in the body of the birds. The results of the nitrogen retention
trials, shown in Table IV, indicate little variation in the retention
of nitrogen in chicks fed the basal of the experimental rations. How-
ever, much more variation existed in feed consumption. Feed consumption
decreased proportionally as the level of fungal protein increased in the
rations. In view of these findings, factors affecting feed consumption
may have contributed more to poor growth than feed utilisation.
Due to the limited amount of available fungal protein materials,
palatability and toxicity were not studied.
12
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TABLE III
EFFECT OF FUNGAL PROTEIN ON CHICK GROWTH
AND FEED UTILIZATION (In Grams)
Weight
Average ^e Difference Required
Rations Weight Ga^n Due to L.S.D.
4-Weeks Treatment
Basal 401.6 271. 0
Fungal Protein 25% 319.6 290.11 - 82.0 31
Fungal Protein 50% SOS. 09 273.46 - 96.51 31
Fungal Protein 75% 211.3 180.51 -190.3 31
Fungal Protein 100% 113.97 82.19 -287.63 31
Commercial Chick Starter 334.8 302.22 - 66.8 31
Feed Survivors
Conversion at 4-Weeks
2.14 11-12
2.47 11-12
2.84 11-12
3.60 9-12
5.46 12-12
2.22 12-12
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FIGURE I
EFFECT OF FUNGAL PROTEIN ON
GROWTH RATE OF CHICKS (Four Weeks)
400
300
200
100
Contra I
25% Fungal Protein
50% Fungal Protein
75% Fungal Protein
100% Fungal Protein
WEEKS OF AGE
14
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TABLE IV
EFFECT OF FUNGAL PROTEIN ON THE
RETENTION OF DIETARY NITROGEN BY THE CHICK (In Grams)
Rations
Basal
Fungal Protein 25%**
Fungal Protein 50%
Fungal Protein 75%
Fungal Protein 100%
Total Gain
Four Weeks
371.0
290.0
273.46
180.51
82.19
Gain, N. R.
Trials*
41.6
23.0
21.0
1.2
0. 7
Total
Nitrogen
in Feed
36.4
36.6
36.1
35.6
34.5
Total
Nitrogen
in Feed
16.5
16.4
16.6
16.4
16.4
Percent
Nitrogen
Retained
54.67
55.19
54.02
53.93
52.46
*Gain during nitrogen retention trials'
**Replaeing 25, 50, 75 and 100 percent of protein supplied by soybean meal in
basal diet
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SECTION VI
REFERENCES
Callihan, C. D. "Construction of Continuous Chemical-Microbial Pilot
Plant to Produce Edible Proteins from Cellulosic Waste," Contract
No. PH-86-68-152. Baton Rouge: Louisiana State University, 1968.
Rogers, C. J. et al. "Production of Fungal Protein from Cellulose and
Waste Cellulosic," Environ. Sci. Technol. No. 63 8 (August 1972).
Daly, W. E. "Fabrication of Single Cell Protein from Cellulosic
Waste," Grant No. EP-OOS24. Baton Rouge: Louisiana State Univer-
sity, 1970.
Snedecor, G. W. Statistical Methods, 5th ed. Ames: Iowa State
University Press, 1965.
Horwitz, William (ed.). AOAC Methods, llth ed. Washington: Associa-
tion of Official Analytical Chemists, 1970.
16
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-76-074
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
5. REPORT DATE
GROWTH RESPONSES OF CHICKS FED MICROBIAL PROTEIN
PRODUCED FROM ORGANIC WASTES
August 1976 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Ozie L. Adams and Erly J. Thornton
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Tennessee State University
Nashville, Tennessee 37203
10. PROGRAM ELEMENT NO.
1DB314; ROAP 21BFS
NO.
R802679-01
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
Project Officer: Charles J. Rogers
16. ABSTRACT
The objective of this study was to investigate
produced from organic wastes as a source of protein
the use of microbial protein
in chick starting rations.
Microbial protein recovered from the mold mycelia was produced when potato
waste materials were homogenized and added to a mineral salt containing a culture
medium suitable for the action of a strain of Aspergillus niger.
The new product containing approximately 29.0 percent protein was added to
chick starting rations, replacing the soybean protein. Sixty-two percent (62%)
of the protein in the ration were supplied by soybean meal. The length of the
feeding trial was four (4) weeks.
Results of the feeding trials showed that fungal protein was effective in
supporting body maintenance but was ineffective in stimulating growth. Palat-
ability of rations and poor feed consumption were factors contributing to slow
growth. However, feed utilization was less of a problem as shown by nitrogen
retention trials.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COS AT I Field/Group
Organic wastes
Utilization
Biological products
Aspergillus
Bacterial proteins
Growth curves
Chicks growth responses
Microbial protein
Aspergillus niger
Palatability
Feed consumption
Fungal protein
6F
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RELEASE TO PUBLIC
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UNCLASSIFIED
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25
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22. PRICE
EPA Form 2220-1 (9-73)
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