STUDIES ON THE PHYSIOLOGY OF THE MUCOR MUCEDO AND ITS ROLE IN THE FERMENTATION OF SOYBEAN CURD

112 Pages·05.57 MB·English

by  STERN, ARTHUR MARVIN

'COPYRIGHTED
BY
ARTHUR MARVIN STERN
1952
STUDIES ON THE PHYSIOLOGY OF THE
MUCOR MUCEDO AND ITS ROLE IN
THE FERMENTATION OF
SOYBEAN CURD
BY
ARTHUR MARVIN STERN
B.S., University of Illinois, 1948
M.S., University of Illinois, 1949
THESIS
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BACTERIOLOGY
IN THE GRADUATE COLLEGE OF THE
UNIVERSITY OF ILLINOIS, 1951
URBANA. ILLINOIS
Acknowledgements
The author wishes to express his appreciation
to Dr. J. Ordal for suggesting this problem and for
his patient supervision and advice. He also wishes
to extend his thanks to Dr. H. 0. Halvorson for the
many helpful suggestions offered during the course
of this study.
Table of Contents
Page
Introduction 1
Survey of the Literature 3
A. History 3
B. Chemical Composition of Soybeans . . .. lO
1. Introduction 10
2. General 10
3. Vitamins 11
h. Carbohydrates and carbohydrate-like
materials 11
5. Proteinaceous materials 12
6. Amino acids 13
7. Enzymes 1"+
8. Minerals ih
9. Lipid Materials 15
Development of a Fermented Soybean Curd . .. 16
Physiology of Mucor Mucedo 27
A. Introduction • 28
B. Growth Studies 28
1. Introduction 28
2. Effect of various carbohydrates . .. 29
3. Effect of amino acids 32
4-. Effect of complex nitrogenous sub
stances 35
5. Effect of variation of types and con
centrations of carbon sources along
with variation of nitrogen sources . 36
6. Interrelationship of nitrogen source,
method of cultivation, growth and pH
changes 37
Table of Contents (cont'd)
Page
7. Ability of soybean oil to support
growth kh
8. Ability of glycerol and fatty acids
derived from soybean oil to support
growth 4-5
9. Ability of various fatty acids to
support growth k-7
C. Enzyme Studies 50
1. Introduction 5°
2. Amylase studies 51
a. Introduction 51
b. Adaptive nature of amylases . 52
c. Effect of nitrogen source and
pH on activity as tested by
plate-disc technique 53
d. Colorimetric procedure for
determining optimum pH of
activity 67
e. Colorimetric procedure for
determining rate of activity . 73
3. Lipase studies 75
a. Introduction 75
b. Method and preliminary work . 75
c. Influence of growth medium on
enzyme production 77
d. Influence of pH on lipase
activity 81
e. Influence of growth tempera
ture on enzyme production . . 85
f. Influence of culture age on
lipase production 88
h. Protease studies 92
V. Summary and Conclusions 98
VI. Bibliography 101
VII. Vita 107
I. Introduction^
In recent years, soybeans have become very important
in the American economy. Although various functions have been
found in this country for the bean, its use as a human food
source has been rather limited. Abundance of other edibles
along with a natural prejudice against introducing a source
of "animal feed" into the human diet have probably been the
chief factors leading to this state of affairs.
For many years the Orientals have utilized legumes
and legume products as the chief source of protein in their
diet. Of these, soybeans have been the most important. Per
haps one reason for this has been the fact that the Chinese
have been able to produce an amazing number of food variants
from this single crop which introduced variety into what other
wise could be considered an uninteresting diet. Among the
foods the Orientals have obtained from soybeans are a group
of fermented products resembling, in their physical appearance
and mode of manufacture, dairy cheeses with which most people
in this country are familiar. Of special interest in this
study are those food products in which the fermenting agents
are fungi—particularly those dependent upon Mucor mueedo (3D
for ripening. One of the purposed of this investigation has
been to duplicate and, if possible, to improve on Oriental
methods of production of a fermented soybean curd, in which
the active biological agent is Mucor mucedo. Another objective
has been to characterize some of the physiological properties
°f Mucor mucedo. The mechanism of the ripening process cannot
be fully appreciated without first acquiring some understanding
of these properties. Such information would aid in establish
ing production conditions and in the control of the quality of
the finished product.
II. Survey of the Literature
A. History;
Many years before soybeans were introduced to the
Western World they constituted a major part of the Oriental
diet. The ease with which they are grown and their minimal
demands upon the soil contributed to their early extensive use
in this area. Other factors, such as overpopulation, small
ratio of cultivatable land to total area, and primitive agri
cultural methods also predestined the Orient as the locale of
soybean origin.
The cultivation of the bean had been concentrated in
three regions—the eastern part of China including what were
then three provinces of Manchuria, Japan, and Korea and scat
tered sections of Inner Mongolia. Although the soybean had
been cultivated by the Chinese for centuries, the real credit
for capitalizing on its commercial potentialities belongs to
the Japanese. It wasn't until after the opening of the Newchwang
Railway in 1900, connecting Manchuria with the sea, that the bean
became known outside of the Orient. Since that time the soybean
has become a major crop in the United States and many industrial
uses have been found for it. The availability of animal milk
and meat products in the United States have been the chief
If
reasons why the soybean has never taken its place as a normal
constituent of the American diet. Another reason for this,
of course, is the fact that little attempt has been made to
improve the quality of soybean food derivatives to the point
where they would be readily acceptable to the American palate.
Soy milk was supposed to have been originated by the
Chinese philosopher Whai Naln Tze long before the Christian era
and was known to the Chinese as fu Chiang or bean curd sauce (53)«
For many years the Orientals had prepared this material by soak
ing the beans in water, crushing them, boiling the mash, and
finally filtering the "milk" through cloth. Variations of this
general technique are still being used to produce soy milk of
different qualities. The milk thus obtained could then be used
per se or as a base for the manufacture of other food products.
One of the products obtained from soy milk is teou fu
or bean curd.(68), The Chinese prepared this by precipitating
the protein from soybean milk with salts such as MgCl^ and CaSO^
or with acid. This process was also carried out by using rennet
(in place of the salts and acid) in 1911 by Li Yu Ylng and Grand-
voinnet. It had the added advantage of eliminating the bitter
taste which is characteristic of curds made by the former methods.
Once the protein is precipitated, it is separated from the liquid
and pressed into cakes. Curds of varying consistencies can be
made by altering the amounts of coagulating material and pres
sures applied. Tofu nao or bean curd brains is another product
made by the Chinese from soy milk. It has a more fluid consistency
than the bean curd and is made by adding very little MgCl2 or
gypsum to the milk, the resulting precipitate not being pressed
at all. Other similar products manufactured by the Chinese are:
tofu kahn (dry bean curd), chien chang tofu (thousand folds),
tza tofu (fried bean curd) and hsiang kahn (fragrant dry bean
curd). All of these products are the result of slight varia
tions in the precipitating process or of the additions made to
the coagulated protein after precipitation.
Another group of soybean derivatives are fermented curds
which resemble, to some extent, some of our more exotic dairy
cheeses. One of the most common constituents of the Oriental
diet is a type of soybean cheese called miso. It is a mold
derivative, the fermenting agent of which is known as kojii—
a non-descript mixture of fungi containing Aspergillus oryzae
as the principle biological component. The kojii contains or
ganisms which have the ability to produce enzymes capable of
breaking down both complex proteins and sugars of the soybeans
to simpler and more soluble substances. In preparing miso, the
beans are first steamed for 25 hours. The initial heating is
a vigorous process which slowly tapers off in violence. The
cooked beans are then rubbed into a thick homogenous paste to
which are added proper amounts of powdered kojii, salt and water.
After thorough mixing, the entire mass is stored in a special
vat where the temperature is kept at about 15°C to 20°C during
the "curing" process. The difference between the types of miso
depends upon the use of barley or rice kojii, the amount of salt