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OCR for page 87
IV. PLANT
DT~ ~ TO ~ A STY T~ ~
OCR for page 88
OCR for page 89
12
Cassava Processing in Africa
Olusola B. Oyewole
Cassava is an important food crop in the tropics and many countries
in Africa. The crop contributes significantly to the diets of over 800
million people, with per capita consumption averaging 102 kilograms
per year. In some areas of Africa it constitutes over 50 percent of the
daily diets of the people.
Traditionally, cassava is processed before consumption. Processing
is necessary for several reasons. First, it serves as a means of removing
or reducing the potentially toxic cyanogenic glucosides present in fresh
cassava. Second, it serves as a means of preservation. Third, processing
yields products that have different characteristics, which creates
variety in cassava diets.
The objective of this paper is to detail the strategy and program being
followed in our laboratory to utilize the knowledge of biotechnology to
improve the processing of cassava in Africa.
TRADITIONAL PROCESSING
Processing of cassava for food involves combinations of fermenta-
tion, drying, and cooking. Fermentation is an important method
common in most processings. While there are many fermentation
techniques for cassava, they can be broadly categorized into solid-state
fermentation and submerged fermentation. Solid-state fermentation,
typified by gari production, uses grated or sliced cassava pieces that
are allowed to ferment while exposed to the natural atmosphere or
pressed in a bag. Submerged fermentation involves the soaking of
whole peeled, cut and peeled, or unpeeled cassava roots in water for
various periods, as typified by the production of fufu and lalun in
Nigeria. Traditionally, cassava is fermented for 4 to 6 days in order to
-
The support of the International Foundation for Science, Stockholm, Sweden, is
gratefully acknowledged.
89
OCR for page 90
JO
FERMENTED FOODS
effect sufficient detoxification of the roots. Some processors, out of
economic pressure, ferment cassava for less than 2 days. Some cases
of food poisioning have been attributed to this practice. Application
of biotechnology to traditional cassava processing has prospects for
producing safe and well-detoxified products.
RESEARCH APPROACH
Our approach on cassava processing research is divided into three
areas:
1. Investigating the science of the traditional submerged fermenta-
tion of cassava to fafu and lafun production;
2. Optimization of the processing through process controls; and
3. Improvement of traditional processing through application or
biotechnological techniques.
The microorganisms involved in the submerged fermentation process
have been isolated and found to include Bacillus subtilis, Klebsiella
sp., Candida tropicalis, C. krusei, and a wide spectrum of lactic
acid bacteria, major among which are Lactobacillus plantarum and
Leuconostoc mesenteroides. A microbial succession trend was found
with the starch degrading Bacillus subtilis, giving way to the lactic acid
bacteria and yeasts that dominate the latter part of the fermentation.
The pH and titratable acidity of the fermenting cassava roots increased
from 6.3+0.2, 0.08+0.03 percent to 4.0+0.3, 0.36+0.05 percent,
respectively, after the 96-hour fermentation period. Organic acids
produced during fermentation include lactic, acetic, propanoic, and
butanoic acids, among others, and these are believed to contribute to
the characteristic havor of fermented cassava products. Fermentation
causes release of some bound minerals, including calcium and magne-
sium. The most important contribution of fermentation is the release
from the plant tissues of the enzyme linamarase, which is involved in
the breakdown of the linamarin and lotaustralin (cyanogenic glucosides)
of cassava, which releases hydrogen cyanide and thus detoxifies the
product.
PROCESS OPTIMIZATION
Processing conditions for optimizing the fermentation process have
been investigated in our laboratory. We found that the temperature
range of 30° to 35°C, with a soaking period of 48 to 60 hours, is best
for submerged processing. The size to which the roots are cut, peeling
or nonpeeling of roots before processing, changing or nonchanging of
OCR for page 91
CASSAVA PROCESSING
91
fermentation water at intervals during processing, and the age of roots
all affect the characteristics of the final product. In addition, the protein
contents of products can be improved by cofermentation with legumes
such as soybeans and cowpea.
BIOTECHNOLOGICAL INVESTIGATIONS
The overall goal of our biotechnological investigations is to develop
an appropriate starter culture for cassava processing that will effectively
produce linamarase enzymes for detoxifying cassava, break down
starch to the simple sugars needed for acid production, improve the
protein content of the products, reduce processing time, and yield
products with stable desired qualities. The following summarizes our
current findings:
· The microorganisms involved in fermentation have been charac-
terized.
· Characterized isolates were used as single and multiple starter
cultures for cassava fermentation. This has made it possible to
understand the roles of each of the microorganisms implicated in the
natural fermentation process. Bacillus subtilis and Klebsiella spp.
contribute significantly to the rotting of cassava roots. In addition, B.
subtilis produces amylase enzymes that are necessary for the break-
down of starch to sugars, which are needed for the growth of other
fermenting microorganisms, including the tactics. Yeasts play a major
role in odor development and, where high yeast biomass is encouraged,
protein-enriched products are not. Lactic acid bacteria convert cassava
sugars to lactic and other acids that contribute to the flavor in addition
to having preservative effects.
· Appropriate starters have been developed that can produce amy-
lase and linamarase enzymes necessary for starch breakdown and
cyanogenic glucoside hydrolysis; two major biochemical processes
needed in cassava processing. For this, the lactic acid bacteria were
investigated since they were the predominant microbial group present
at the beginning of fermentation and which persist and survive the
acidic conditions that prevail in cassava fermentation. To date, we
have found strains of Lactobacillus plantarum that are capable of
producing amylase and linamarin. The linamarase produced has been
purified, and it exhibits optimal activity at pH 5 to 8 and temperature
of 30° to 40°C. Prospects for cassava processing using a selected single
culture with properties for starch hydrolysis, cyanide detoxification,
and acid production have thus evolved.
.. To initiate genetic manipulation of cassava lactic acid bacteria,
OCR for page 92
92
FERMENTED FOODS
the plasmid profiles of the lactobacilli isolated from cassava were
studied. The presence of plasmids among cassava lactobacilli has been
confirmed. Further research is needed to investigate the correlation
between possession of plasmids and linamarase production in order to
establish prospects for genetic manipulation.
FUTURE RESEARCH
Beyond the selection of appropriate starter cultures for cassava
fermentation, it will be necessary to improve the starter culture.
Genetic manipulation of the starter culture offers the best hope
for improved cassava processing, with higher economic returns and
improved stable qualities.
Cassava processing could also be enhanced by using biotechnological
principles to modify structural and processing characteristics of cassava
cultivars to meet specific product requirements.
The linamarase elaborated by cassava plant tissues and fermenting
microorganisms has been found to be unstable under high acidic
conditions characteristic of the latter part of natural fermentation.
Techniques for increasing the stability of linamarase enzyme to acidic
conditions could be investigated.
The usefulness of cassava fermenting microorganisms could be
further investigated for the production of other economically viable
products such as acidulants and antimicrobial agents.
A biotechnological approach could be investigated for the treatment
of odorous fermented cassava water and cassava root peels.
OCR for page 93
13
Improving the Nutritional Quality of
Ogi and Gari
T. G. Sokari
Ogi is a blancmange-like product processed by fermenting the slurry
from wet-milled maize (or sorghum or millet). Used as both a weaning
food for infants and as a breakfast food by adults, ogi is one of the
most important food items in Nigeria. Yet it is nutritionally inferior to
maize, which is deficient in certain essential amino acids, because of
the maize-milling process that is an integral part of ogi production.
Cassava, another very important food crop, has the problem of
possible nutritional complications because it contains the cyanogenic
glucosides linamarin and lotanstralin. Although the cyanogens in
cassava are hydrolyzed to hydrogen cyanide during processing by the
endogenous enzyme linamarase (1,2), not all processes are equally
effective. It has even been suggested that traditional processing
techniques are unlikely to remove all the cyanide from cassava (3,41.
In view of this, studies were undertaken to increase the protein
content of ogi relatively inexpensively and to develop a technique for
processing cassava into gari that would eliminate cyanogens from the
product or reduce them to innocuous levels.
PROCESSING OF OGI
The traditional technique for processing maize into ogi is summarized
in Figure 1. Also shown in Figure 1 is an alternative to this procedure;
a 20-minute boiling step is substituted for the normal 24- to 28-hour
steeping of maize prior to wet milling (51. Cowpea can be combined
with maize to increase the protein content of ogi.
Substituting 20 minutes of boiling for the traditional 24 to 28 hours
of steeping prior to wet milling maize reduced processing time from
72-76 hours to about 24 hours. There was, however, no significant
93
OCR for page 94
94 FERMENTED FOODS
Maize Cowpea
1 1
, Clean Clean and Dehull
,--"' 1 1
Boil ~ Steep
(20 min) (24-48h)
""" 1
Boil
(30 mint
1
1 ~ Wet Mill Wet Mill
Boil Water ~l it,
(discard)
Wet Sieve Wet Sieve
Slurry
Fermejtation
Ogi
-
~ Maize: Cowpea Slurry
(up to 30% cowpea w/w)
Fermentation
(24-48h)
Enriched Ogi
-
-
Slurry
FIGURE 1 Processing of maize into ogi including cowpea protein enrichment; dotted lines
show a bypass by which processing time can be reduced.
difference ~ p> 0.05) in the aroma, color, taste, and overall acceptability
between the products obtained by the short-time processing and
traditional processing (51. The same was also true for unenriched and
protein-enriched ogi except for color (61.
CYANIDE REDUCTION DURING CASSAVA PROCESSING
Two foods processed from cassava (gari and ijapu) were studied.
Adding water to grated cassava at the 75 percent (v/w) level and heating
at 50°C for 6 hours resulted in linamarin reduction of >99 percent
(Figure 21. The pH of the mash fell from 6.4 to 6.3 during the period
(71. After dewatering, the mash was adjusted to a pH below 4 by
equilibrating with a 3-day fermented cassava liquor (40 percent, v/w)
at 50°C for 12 to 18 hours. The equilibrated mash was then dewatered
and toasted (Figure 31. A panel of tasters who were familiar with gari
but otherwise untrained could not differentiate between the product
and traditionally processed gari. Both sets of products were equally
acceptable to the panel.
OCR for page 95
95
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96
Peels
(discard) ~
Cassava ~ Peel
1
Boil
(1 00°C/25 min)
1
Slice
Soak
(24-28h)
Rinse
Ijapu
FIGURE 3 Processing of ijapu and garifrom cassava.
FERMENTED FOODS
- ~ Grate ~ Add Water
(75% v/w, 50°C/6h)
Dewater/ferment
(3 days/30°C)
it\
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Gary Gary
( t r a d i t i o n a I p r o c e d u r e ) ( s e m i c o n t i n u o u s
process)
The modified procedure for processing cassava into gari reduced the
processing time from >96 hours to about 24 hours. Cyanogens were
not detectable in the product by the method of Cooke (8,91.
The study of the traditional production of ijapu (Figure 3) was
intended to aid in understanding the loss of cyanogens during cassava
processing. About 54 percent of the cyanogens in raw cassava were
lost after boiling peeled cassava, but a substantial proportion remained
in the water (Table 11. After slicing the boiled cassava and steeping
the slices, a substantial proportion of the cyanogens was again lost.
TABLE 1 Cyanide Content of Cassava During Processing into Ijapu
Cyanide Content (ppm)
Material analyzed pH Total Free HCN Bound Cyano
Unprocessed peeled cassava 6.3 76.1+15.3 5.5+2.2 2.9+0.4 70.6 2.6
Boiled cassava
Boil water
"Ijapu"
(after 24 hours steeping) ND
Steep water
6.0 35.1 +8.7
(53.9)
1 2.8+2.1
1 1.8+1.4
(84.5)
4.0 1 6.2+3.9
2.4+ 1.2 2.0+0.7
1.1 +0.2 0.5+0.2
4.2+1.4 3.1+1.4 7.6
(89.2)
7.8
5.7+3.4 5.6+3.4
32.7 0.4
(53.7)
11.6
0.6
Note: Cyano, Cyanohydrin; numbers in parenthesis, percent loss; ND, not determined
OCR for page 97
IMPROVING OGI AND GARI
97
Much of the loss could, however, be accounted for in the steep water,
and the proportion lost depended on the duration of steeping and the
cassava:water ratio (Tables 1 and 21.
ROLE OF FERMENTATION
The reduction of >99 percent in the linamarin content of grated
cassava within 6 hours of adding water, with little or no change in the
pH of the mash, would imply that fermentation had nothing to do with
the detoxication. Linamarin breakdown is essentially a hydrolytic
process catalyzed by the endogenous enzyme linamarase (1,21. The
results of the present study indicate that the addition of water aids in
the hydrolytic process. Apparently not all of the water normally in raw
cassava tuber is available for hydrolysis.
During the boiling of cassava for processing into ijapu, linamarase
would be inactivated. Yet a substantial proportion of the linamarin in
cassava was still lost, appearing to a large extent in the water used for
boiling and for steeping (Tables 1 and 2~. This would suggest that
leaching could be an important factor in cyanide loss during cassava
processing. This would be true not only during the boiling and steeping
of cassava for ijapu production but also during the dewatering of grated
cassava for gari production.
CONCLUSION
Cassava detoxication during processing is essentially an enzymic
hydrolysis of cyanogens in cassava (1,2,81. Fermentation has little role
TABLE 2 Effect of Sliced Boiled Cassava: Steep Water Ratio on Cassava Detoxification
.
Ratio
(w/v) Material analyzed pH Total Free HCN Bound Cyano
1:1 Unboiled cassava 6.4 90.2 4.5 3.0 85.7 1.5
Boiled cassavaa ND 51.7 1.5 0.5 50.3 1.0
(42.7) (41.3)
Boil water 6.1 20.2 0.5 0.3 19.7 0.2
-Sliced, boiled ND 5.6 1.1 0.6 4.5 0.6
cassavab (93.8) (94 7)
Steep water 4.3 25.5 5.7 4.5 19.7 1.3
1:2 Sliced boiled ND 5.7 1.7 0.6 4.0 1.2
cassavab (93.7) (95.3)
Steep water 4.3 21.3 1.9 1.4 19.4 0.5
1:3 Sliced, boiled ND 4.2 1.6 0.5 2.6 1.0
cassavab (95.3) (97.0)
Steep water 4.2 11.5 2.1 1.1 9.4 1.0
a Prior to slicing and soaking in water.
b after 24 hours soaking; other notes as in Table 1.
OCR for page 108
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110
FERMENTED FOODS
TABLE 3 Lactic Acid and Volatile Fatty Acid Content of Field Collected Kawal, Sigda, and Furundu
(Mean and Range in 9 100 9-' Dry Matter)
Acid Kawal Sigda
Lactic
Acetic
Propionic
Isobutyric
n-Butyric
Isovaleric
n-Valeric
Total VFA
Furundu
0.21
(0 03 0.51 )
5.08
(2.1 2~.75)
0.90
(0 51 - 1.59)
0.24
(0.04 0.38)
2.94
(1.18 4.73)
0.22
(0.06 0.60)
0.18
(0.01~.61 )
9.56
(4.7~1 2.1 )
3.07
(2 8~3.35)
1.10
(1 .0~1 . 19)
0.04
(0.03~.05)
<0.01
0.08
(0.02~. 1 5)
0.02
(0.01~.03)
<0.01
1.24
(1 .12 - 1 .38)
0.50
(0.0~1 .67)
1.59
(1 .22-2.05)
0.09
(0.02~.25)
<0.01
0.24
(0.05 0~73)
0.17
(0.02~.35)
0.01
(0.01~.02)
2.17
(1 .602.63)
the eleventh day of the latter fermentation, VFA mainly n-butyric (8
percent), acetic (5 percent) and n-propionic (2 percent) comprised 15
percent of the fermentation mixture. However, the pH had not changed
by more than 0.5 unit from the initial value. On the other hand, by the
fifth day of the sigda fermentation, when a total acid concentration of
6 percent had been attained, the pH of the fermentation mixture had
fallen to 4.0 from an initial value of about 6.0. This difference in the
course of fermentation is almost certainly attributable to the stronger
buffering capacity of the substrate of the kawal fermentation, C.
obtusifolia leaves, which possess approximately double the calcium
content of sesame seedcake. Conditions in kawal do not, therefore,
favor the selection of acidoduric lactic acid bacteria.
In addition to these bacteria, the two yeast species present in
unfermented sesame seedcake proliferated during the initial period of
fermentation. Concomitantly, starch levels were observed to fall rapidly
from 2 percent in the unfermented substrate to zero after the first two
days of fermentation. As the only amylolytic organisms present, the
yeasts presumably were responsible for degradation of starch, rendering
it available to the lactic acid bacteria. The poorly fermentative yeasts
Torulopsis sp. and Debaryomyces sp. isolated in the final stages of the
fermentation can utilize lactic acid aerobically and may cause the
decline in concentration of the compound during this period. The
addition of kambo did not appear to have any significant effect on the
course of the sigda fermentation, and it was concluded that this
supplementation was probably practiced mainly on organoleptic
grounds.
The VFA, primarily n-butyric and acetic acids, which are the
principal products of the kawal fermentation, are characteristic of
clostridial fermentation of plant material as is the accumulation of
OCR for page 111
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112
FERMENTED FOODS
ammonia nitrogen. However, all attempts to isolate clostridial species
from the fermentation mixture were unsuccessful, indicating that the
microbial origin of VFA must be sought elsewhere. The formation of
acetic acid can probably be ascribed to the heterofermentative B.
subtilis, the co-dominant microorganism, whereas propionic acid is
probably an end product of anaerobic fermentation by Propionibacte-
rium sp. for which lactate is a preferred substrate. Utilization of lactate
in this way could explain the low level of lactate in kawal, despite the
substantial population of Lactobacillus plantarum. The microbial
pathway leading to formation of n-butyric acid is difficult to define,
although its production may be characteristic of fermentation by this
type of mixed culture as a whole rather than that by any single
microorganism. n-Propanol (2.3 percent), n-butanol (0.1 percent), and
ethanol (0.1 percent) were also detected in kawal toward the end of
fermentation, though all were lost from the product during the drying
phase. Formation of such alcohols is probably due to anaerobic
fermentation of carbohydrate by the yeast species present.
The identification of Bacillus sp. as the principal microorganism in
furundu when considered in the context of a final pH of 6.2 and the
presence of both VFA and lactic acid in the fermentation mixture
suggest that the furundu fermentation may be intermediate in character
between those of sigda and kawal. Further investigation of the furundu
fermentation would be most instructive in this respect.
CONCLUSIONS
The sigda and furundu fermentations appear quite unlike the tradi-
tional oilseed fermentations practiced in Nigeria and elsewhere in West
Africa where foods such as ogili and ogiri are fermented from castor
oil seed (Ricinus communist and melon seed (Citrullus vulgarisJ. There
are even variations of the fermentation which use sesame seed and
karkade seed known as ogiri-sara and red sorrel, respectively. During
these West African fermentations, the pH increases to over 9.0 and
ammonia production is frequently observed in the later stages. The
fermentations are dominated by Bacillus sp., frequently Bacillus
subtilis, an organism associated with spoilt sigda in Sudan. The
principal reason for the difference would appear to be in the preparation
of the seeds prior to fermentation, which in West Africa involves
boiling for several hours in water until soft. Such pretreatment may
alter the course of fermentation by two mechanisms—first, by rendering
protein and polysaccharide more available for degradative attack by
microorganisms, and second, by effectively eliminating much of the
heat-sensitive indigenous microflora. The removal of amylolytic yeasts
OCR for page 113
FERMENTATIONS OF SUDAN
113
may well favor the selection of amylase-producing bacteria such as
Bacillus sp. rather than lactic acid bacteria incapable of utilizing starch.
The three fermentations studied appear to afford a route by which
unpalatable plant material or oilseed cake of little economic value can
be converted into acceptable meaty-tasting food that is particularly
rich in sulphur amino acids, which tend to be deficient in diets where
access to meat or fish is limited. Phytic acid present in seeds can
frequently hinder absorption of minerals in the gastrointestinal tract.
As fermentation of plant products has been shown to reduce physic
acid levels substantially, it is likely that the bioavailability of minerals
in both sesame and karkade seed is increased in sigda and furundu.
REFERENCES
1. Dirar, H. A. 1984. Kawal meat substitute from fermented Cassia
obtusifolia leaves. Economic Botany 38:342-349.
2. Dirar, H. A., D. B. Harper, and M.A. Collins. 1985. Biochemical
and microbiological studies on kawal, a meat substitute derived by
fermentation of Cassia obtusifolia leaves. Journal of the Science of
Food and Agriculture 36:881-892.
3. Elfaki, A. E., H. A. Dirar, M. A. Collins, and D. B. Harper. 1991.
Biochemical and microbiological investigations of sigda- a Sudanese
fermented food derived from sesame oilseed cake. Journal of the
Science of Food and Agriculture 57.
OCR for page 114
16
Continuous Production of Soy
Sauce in a Bioreactor
Takashi Hamada, Yaichi Fukushima, and Hiroshi Motai
Soy sauce is a traditional all-purpose seasoning with a salty taste
and sharp flavor. In the conventional method of brewing soy sauce
(Figure 1), cooked soybeans and roasted wheat are mixed with spores
of Aspergillus species and fermented in solid culture for 2 days to
produce koji. The koji is then mixed with brine to make moromi, the
mash that ferments to produce soy sauce. Over time the soybeans and
wheat are hydrolyzed by enzymes such as proteinases, peptidases, and
amylases. During the first stage of moromi fermentation, Pediococcus
halophilus grows and produces lactic acid, which lowers the pH.
Accompanying the decrease in pH, vigorous alcohol fermentation by
Zygosaccharomyces rouxii occurs. As a result, 2 to 3 percent ethanol
and many kinds of aroma components are produced by this yeast. At
the same time, phenolic compounds such as 4-ethylguaiacol (4EG) and
4-ethylphenol, which add characteristic aroma to soy sauce, are
produced by other types of yeasts such as Candida versatilis and
Candida etchellsii.
It takes over 6 months for the entire fermentation and aging of the
moromi mash. Therefore, shortening this period is important and new
processes for soy sauce brewing are desirable. This paper describes
the continuous production of soy sauce in a bioreactor system,
which consists of reactors containing immobilized glutaminase and
immobilized cells of P. halophilus, Z. rouxii, and C. versatilis.
MANUFACTURING PROCESSES
The processes for soy sauce production using the conventional and
bioreactor methods are shown in Figure 1. The bioreactor method
differs from the conventional one in the following ways: (a) proteases
114
OCR for page 115
CONTINUOUS PRODUCTION OF SOY SAUCE
Conventional Method
| Soybeans Wheat |
Steam Roast
1
~ Starter |
Koli
~ Salt Soln. |
| Morom i Mash |
! .
(n
c
o
Go
o
115
Bioreactor Method
I Defatted I [Wheat |
I Soybean I ' I
Steam Roast
Hydrolysis
and Fermentation
Lactic Acid
and
Alcohol
Fermentation
Aging
Culture LContinuous culture
Broth I of KoliMold
Hydrolysis (40° to 50°C for 3 days)
| MoromiMash |
Filtration
| Raw Liquid |
l Salt l
| Immol Sized |
| Glutaminase |
Immobilized Lactic
Acid Bacteria
P. halophilus
cat
Filtration ~ Immobilized Yeast
Z. rouxii, C. versatilis
Pasteurization
| Soy Sauce |
!
Pasteurization
| Soy Sauce |
FIGURE 1 Manufacturing processes for soy sauce by conventional
and bioreactor methods.
from continuous submerged culture are used (1), (b) fermentation is
carried out in the liquid state, and (c) the fermentation period is
considerably shorter. It takes several months for the conventional
fermentation but only about 2 days for the bioreactor method.
In the bioreactor method, raw liquid was successively passed
through, first, a glutaminase reactor to increase glutamic acid; second,
a P. halophilus reactor to carry out lactic acid fermentation; and, third,
a Z. rouxii reactor to carry out alcohol fermentation and a C. versatilis
reactor to produce phenolic compounds such as 4-ethylguaiacol. Two
reactors containing immobilized yeast cells were set in parallel, and
the flow rate of the feed solution to the Z. rouxii and C. versatilis
OCR for page 116
116
TABLE 1 Conditions of Fermentation in Each Reactor
FERMENTED FOODS
Column Packed Gel
Volume Volume Residence Tempera-
Reactor Carrier (L) (L) Time, hours sure, °C Aeration,vvm
Glutaminase Chitopearl 1.8 0.6 0.7 40
P. halophilus AS 7.5 5.0 6.1 27
Z. rouxii Al - 27.0 8.0 25.5 27 0.005
C. versatilis Al 1.0 0.2 10.7 27 0.08
AS, Alginate-colloidal silica.
Al, Alginate.
reactors was set in a ratio of 10 to 1. Carrier, packed gel volume, and
operating conditions such as residence time, temperature, and aeration
in each reactor are shown in Table 1.
CONTINUOUS FERMENTATION
A profile of continuous fermentation by immobilized cells of P.
halophilus, Z. rouxii, and C. versatilis is shown in Figure 2. The
fermentation continued for over 100 days without any microbial
contamination. A consistent increased level of glutamic acid (in the
range of 0.3 to 0.4 percent) was found in the effluent from glutaminase
reactor, with a residence time of 0.7 hours. Lactic acid was produced
by immobilized cells of P. halophilus in quantities of 0.7 to 1.0 percent
at a residence time of about 6 hours, and consequently the pH declined
to 4.9 to 5.0, similar to that of conventionally brewed soy sauce.
Ethanol was produced constantly by immobilized cells of Z. rouxii in
quantities of 2.5 to 2.7 percent at a residence time of about 26 hours.
This is the standard ethanol content in soy sauce. About 10 ppm (parts
per million) of 4-ethylguaiacol was produced by immobilized cells of
C. versatilis at a residence time of about 10 hours, and the final 4-
ethylguaiacol content after mixing the two fermented liquids from the
reactors of Z. rouxii and C. versatilis was about 1 ppm, which is the
optimum concentration in conventional soy sauce. The total residence
time for lactic acid and alcohol fermentation was about 30 hours in
this system. This was considerably shorter than the conventional
fermentation period of 3 to 4 months required to produce the same
amounts of lactic acid and ethanol.
High numbers of viable cells were present in the gel and liquid in
each reactor. The number was 10- to 100-fold higher in moromi mash.
The shortening of the fermentation period in the bioreactor method is
possibly due to the high density of immobilized cells in the gel and
free cells in the liquid.
The main chemical components of the fermented liquid from the
bioreactors were examined, including lactic acid, glucose, ethanol, and
nitrogenous compounds.
OCR for page 117
CONTINUOUS PRODUCTION OF SOY SAUCE
1.0
0.8
_ ~
~ ·C'
ct c, 0.6
Cat ·—
Ct
, '
0.4
0.2
o
3.0
^
— _
o ct5 2.0
_ ~
1.0
o
15
Q
Q
-
c~ 10
111
5
o
PROPERTIES
117
_ ~ _
t~ 5.0 I
_ ~ _
_
1 1 1 1 1
_
err
1 1 1 1 1
_
o 20
40 60 80 100
Time (days)
FIGURE 2 Profile of continuous fermentation of soy sauce by a
bioreactor system. (a), lactic acid; (a), glutamic acid (values
indicate the increase in the amount of glutamic acid); (v) pH;
(a), ethanol; (a), glucose; (a), 4-KG after passing through the
C. versatilis reactor; (a), 4-KG in the final product.
6.0
4.0
The organic acids and aroma components in the bioreactor soy sauce
were examined. The proportions of organic acids except citric acid
were not much different between the bioreactor soy sauce and the
conventional one, although the former was a little lower in acetic acid
and succinic acid. It appears that the high residual content of citric
OCR for page 118
118
FERMENTED FOODS
acid in the bioreactor soy sauce arises from the inability of P. halophilus
to utilize citric acid. Aroma components present in both the bioreactor
and conventional soy sauces were not qualitatively different. However,
the former was higher in isoamyl alcohol and acetoin and lower in
isobutyl alcohol, ethyl lactate, 4-hydroxy-2(or51-ethyl-5(or21-methyl-
3~2H)-furanone, and 4-hydroxy-5-methyl-3~2H)-furanone.
To evaluate the aesthetic qualities of the bioreactor-produced soy
sauce, sensory tests were carried out. For example, the intensity of
the alcoholic, fresh, sweet, acid, and sharp odors as well as the special
higa (baking aroma) and bushoshu (foul fermented aroma) were
compared between the bioreactor and conventional soy sauces. The
odors are important for the quality of soy sauce. Although the bioreactor
soy sauce was a little weaker in aroma and fresh odor than the
conventional soy sauce, the quality of the former was generally judged
to be similar to that of the latter.
The total time required for the production of soy sauce by the
bioreactor system, including enzymatic hydrolysis of the raw materials,
fermentation with immobilized whole cells, and the refining process,
is only about 2 weeks (24. This is considerably shorter than the 6
months with the conventional method of soy sauce brewing consisting
of koji making, fermentation and aging of moromi, and refining.
From these results we conclude that the quality of the bioreactor
soy sauce was very similar to that of the conventional soy sauce from
both chemical and sensory evaluations and that the bioreactor system
is practical for the production of soy sauce.
REFERENCES
1. Fukushima, Y., H. Itch, T. Fukase, and H. Motai. 1989. Applied
Microbiology and Biotechnology 30:60~608.
2. Hamada, T., M. Sugishita, Y. Fukushima, and H. Motai. 1991.
Process Biochemistry 26:39~5.
Representative terms from entire chapter:
lactic acid
