REVIEW ARTICLE

J. Food Sci. Technol. Nepal, Vol. 6 (1-9), 2010

ISSN: 1816-0727

Bacillus Fermentation of Soybean: A Review

ASHOK KUMAR SHRESTHA1*, NAWA RAJ DAHAL2 and VEDASTE NDUNGUTSE1
1

Centre for Nutrition and Food Sciences, The University of Queensland, St Lucia, Queensland 4072,
Brisbane, Australia
2
Department of Food Technology and Quality Control, Kathmandu, Nepal

Soybeans in its natural form have a little direct use as a food due to its poor digestibility as well as beany taste and flavour.
Fermentation; however, can improve the eating and nutritional qualities of soybeans. Fermented soybean foods have been an
intricate part of oriental diet for a long time. Bacillus subtilis dominated traditionally fermented soyfoods have typical taste,
texture and aroma which is popular in Asian and African countries. B. subtilis fermentation of soaked and cooked soybeans
brings many physico-chemicals and sensory changes that make it highly digestible and nutritious. This paper reviews various
facets of B. subtilis fermented traditional foods, properties of fermenting organisms, preparation of such fermented foods,
changes in chemical composition and nutritional properties and improving the quality of these foods.
Keywords: Soyabean, Bacillus fermentation, Health benefits, Acceptability, Commercialization
Introduction
Soybean (Glycine max L.) is one of the nutritionally richest
natural vegetable foods known to human kind, records of its
food usage dates back to 2838 BC in China. Although having
high protein content, minerals, vitamins and bio-actives, it
has little direct use because of high satiety value caused by
high oil content, poor digestibility, green beany taste, long
cooking time and persistent bitterness. Soybeans as a food is
used as soymilk, soy flour, soy oil, feed for livestock and
poultry, soy concentrate, protein isolates, soy yoghurt, tofu
and fermented foods such as Tempeh, soy sauce, Miso , Natto
and Sufu (Beuchat, 1984). Fermentation is a proven method
to improve flavour, texture and nutritional quality of the
soybeans. Besides bringing physico-chemical and sensory
quality changes, fermentation contributes towards the
preservation of food due to release of metabolites that
discourage the growth of pathogenic bacteria in foods.
Fermentation involves a range of microorganisms such as
lactic acid bacteria, acetic acid bacteria, yeasts, moulds and a
range of bacteria. It also covers wide range of products such
as staples, adjuncts to staples, condiments and beverages
that use substrates such as cereals, pulses, soybeans, flowers,
milk, meat etc. (Tamang and Sarkar, 1988).
There are many traditional B. subtilis fermented soybeans
foods in various parts of the world. One of the common
examples is Kinema which is traditionally consumed by the
non-Brahmin Nepali inhabiting Nepal; Darjeeling and Sikkim
of India; and some parts of Bhutan. It is popular among
Lepchas and Bhutias who call it Satlyangser and Bari,
respectively (Sarkar et al., 1994). Yoshida (1988) reported the
origin of Kinema in Southern part of China. While it spread,
this food settled into a niche as seasonings in East Nepal,
North East India, Burma, Thailand and in Japan. Some Kinema
*Corresponding author, E-mail: a.shrestha@uq.edu.au

like fermented soy foods reported in literature are: Natto in

Japan, Thua-Nao in Thailand, Douchi in China, Chungkook-jong in Korea, Tao-si in Philippines, Dawadawa in
Africa (Sarkar et al., 1994; Shrestha and Noomhorm, 2001; Hu
et al., 2010). With its powerful odour and slimy appearance
like a rotten food, large number of population do not like it,
whereas other finds eating Kinema (and similar products) a
delightful experience. These foods are eaten in the fresh form
or as a fried curry dish along with boiled rice and sometime
with boiled rice, and sometimes as soup, pickle or mixed with
other vegetables (Shrestha, 1989).
Bacillus spp. is the most dominant naturally fermenting agents
in soybeans. These hydrolytic bacteria are associated with
utilization and reduction of indigestible oligosaccharides and
polysaccharides. The organism has also shown to reduce the
activity of anti-nutrients that hinders availability of proteins
and phytochemicals present in soybeans (Sarkar et al., 1997;
Esteves et al. 2010). B. subtilis fermentation is accompanied
by covering intact granules with white-coloured viscous
substance, slimy appearance, softer texture, and unique
rotten flavour. It also completely removes the beany odour
of raw soybeans and increases sensory quality of the product
(Sarkar and Tamang, 1994; Shrestha and Noomhorm, 2001;
Hu et al., 2010). The current paper will review various aspects
of Bacillus fermentation of soybeans, particularly chemical
changes that lead to improvement of nutritional and sensory
qualities of soybeans. It will also investigate the potential
benefits of Bacillus fermentation on human health, storability
and other aspects as a food.
Soybean fermentation and fermenting Bacilli
Fermenting bacilli encompasses a group of bacteria with
various similarities, or dissimilarities (Neway, 1989). They are
mostly gram positive, aerobic, rodshaped, heat resistant and
spore forming bacteria. Bacillus can produce different variety
1

Shrestha et al., : J. Food Sci. & Technol. Nepal, 6 (1-9), 2010

of enzymes including polysaccharides, proteins and nucleic

acid hydrolysing enzymes during fermentation (Neway, 1989).
B. subtilis is a group of related strains under the family
Bacillaceae. It is also called hay Bacillus because it is easily
isolated from hay by enrichment culture. This bacteria is largely
non pathogenic and grows quickly in inexpensive media.
When cultivated in liquid medium, organisms often
accumulate on the surface, giving rise to a skin or pellicle.
The colonies are irregular and have a curled or hair like
structure giving what is sometimes called "Medusa head"
appearance. On microscopic examination tangled coils of long
chains of bacilli may be found (Burrows, 1973). Priest (1989)
reported the amylolytic, proteolytic and lipolytic (deesterification) activity of this organism. It has ability to secrete
high levels of protein into the growth medium. Marshall (1990)
reported B. subtilis as one of the few organisms that has
been used to remove the bitter taste of soybeans. The lack of
pathogenecity among strains of B. subtilis and any of its
close relatives has resulted Food and Drug Administration
granted the organism as GRAS (Generally Regarded as Safe).
The genus B. subtilis is the most dominant bacterium
naturally present in raw soybeans (Sarkar and Tamang, 1995).
There are a number of naturally fermented soybeans foods
where this species grow without adding inoculum. Some
examples of these soy based foods are: Japanese HikiwariNatto, Itohiki-Natto; Thai Thua-Nao; Chinese Shui-Toushi;
Nigerian Daddawa or Dawadawa; and other legume/oilseed
based fermented products from different regions of Africa
(Shrestha, 1997). These traditionally fermented soyfoods are
littered with several other microbial species such as
Enterococcus faecium, Candia parapsilosis, Geotrichum
candidum etc., but Bacillus remains the sole organism
carrying out the fermentation (Sarkar and Tamang, 1994).
Because soybeans fermentation is still mainly based on
traditional way of preparation where it is spontaneous, end
products will be different depending on microbial species
that are predominant during fermentation. It will also be
different from one region to another and country-to-country
depending on fermentation methods. However, where a single
species have been used either for experimental or for
standardisation purposes, production of consistent products
have been obtained and this will be considered in the next
sections.
Methods of soybeans fermentation
Most of the Bacillus fermented soy products are still prepared
by traditional methods. So the method of preparation depends
on countries/locality, family convenience, and materials
available. The basic steps of product preparation such as,
soaking, boiling, fermentation, incubation and finally packing
remain more or less same. Figure 1 shows a typical example of
B. subtilis fermented soy product, as Kinema fermentation.

Figure 1. Flow chart of traditional fermentation of B.

subtilis fermented soybeans (Shrestha, 1989)
Method involves wrapping soaked-boiled soybeans by
banana (Musa paradisica L.) or Smith leaves (Leucosceptrum
canum) or even polythene bags in bamboo lined basket which
is covered with sack clothe and fermented in warm place for 2
to 3 days (Tamang and Sarkar, 1988).
Often a little bit of firewood ash is added to boiled and crushed
soybeans before final fermentation commences. The
fermentation method also varies, for example, in more
sophisticated methods pure culture is used to inoculate the
boiled soybeans. Shrestha and Noomhorm (2001) isolated B.
subtilis from Kinema and hygienically prepared Kinema by
using the pure culture. In this method soaked beans were
autoclaved at 121C for 35 min, cooled to 30 to 35C, inoculate
with pure culture, ferment at 37C for 48 hours at 85% RH. Hu
et al. (2010) and Chukeatirote et al. (2010) also prepared ThuaNao using pure culture of B. subtilis. Efforts have been made
to increase the shelf life (2-5 days) by drying fresh product
into small balls or ground these dry beans into powders
(Shrestha and Athapol, 2001; Chukeatirote et al., 2010). Sarkar
and colleagues have extensively studied the influence of
process variables and inoculum composition on the sensory
as well as product qualities (Sarkar et al., 1993; Sarkar et al.,
1994; Sarkar et al., 1994; Sarkar and Tamang, 1995).
Ikemnebonech, (1986) reported a number of factors such as,
water activity, salt concentration, pH and temperature have
impact on Bacillus activity in Dawadawa preparation. Uyar
and Baysal (2004) reported that optimum inoculum
concentration is important as low concentration of inocula
does not grow fast at desirable rate and at high concentration
nutrients are exhausted before enzymes are produced.

Shrestha et al., : J. Food Sci. & Technol. Nepal, 6 (1-9), 2010

Chemical/nutritional properties of soybean and Bacillus

fermented foods
Fermentation brings substantial chemical changes in
fermented soybeans (Sarkar and Tamang, 1995; Chukeatirote
et al., 2010; Hu et al., 2010). The nature of fermenting
organisms and preference of substrate determines the
evolution of pH in Bacillus fermentation. Traditional B.
subtilis fermentation showed initial decrease in pH from 6.94
to 6.64 (16hr) due to fermentation of carbohydrate into acids
by microbial enzymes (Sarkar et al., 1993; Odunfa, 1985).
Hydrolysis of protein to produce amines and ammonia through
peptides and amino acids is reported to be responsible for
initial change in pH (Odunfa, 1985). There is an increase in
titratable acidity from 0.02 to 0.09% (as lactic acid) during the
course of fermentation. At later stages proteolysis prevail
with subsequent production of ammonia and pH reaches up
to 8.34 at 48hr (Sarkar et al., 1993; Sarkar and Tamang, 1995).
At the end of fermentation there is a slight increase in moisture
level (from initial 60.5 to 64.8%) (Sarkar and Tamang, 1995; Hu
et al., 2010). Sarkar and Tamang (1995), reported that protease
from B. subtilis degrades soy proteins resulting in significant
increase in non-protein and soluble nitrogen, whereas protein
nitrogen decline e.g., from 7.7% N (47.8% protein) to 6.3% N
(39.4% protein). However, most of the literatures have reported
a general increase in protein content during B. subtilis
fermentation, e.g., 44.1 to 46.2% (traditional) and 45.1% (pure
culture) fermentation on dry basis (Shrestha and Noomhorm,
2001); 35.7 to 40.3% (Hu et al., 2010); and overall 7% increase
(Nikkuni et al., 1995) and 14% increase over raw soybeans
(Hayashi, 1974). It is believed that increase in protein content
is associated with the increase in microbial synthesis of protein
or enzymes or rearrangements of compounds followed by
degradation of other compounds (Hayashi, 1974; Hu et al.,
2010).
Literature data showed B. subtilis fermentation cause very
little change in lipid content in soybeans (Shrestha and
Noomhorm, 2001; Hu et al., 2010). This is despite strong
lipolytic activities of microorganisms degrading a quarter of
the fat content as reported by Sarkar et al. (1994). The level of
titrable free fatty acid of soybeans significantly increased
from 0.7 to 6% during Kinema fermentation (Shrestha and
Noomhorm, 2001). In another study, Sarkar et al., (1994)
reported an increase in titratable acidity from 0.01 to 0.1% (as
lactic acid) and free fatty acid acidity from 0.08 to 0.3% (as
linoleic acid) during Kinema preparation, suggesting release
of lipase during the fermentation process. The total energy of
the soybeans, however, remains unchanged during
fermentation (Shrestha and Noomhorm, 2001). The effect of
fermentation on ash content is inconclusive: decrease from
4.8 to 2.3% (Hu et al., (2010); remain unchanged (Shrestha
and Noomhorm, 2001); and increase from 5.0 to 5.6-7.4%
(Sarkar et al., 1994). Carbohydrates are reported to decrease
due to their utilisation by microbes for energy supply (Hu et
al., 2010), however, chemical analysis showed no marked

change (Sarkar et al., 1994; Shrestha and Noomhorm, 2001).

Kinema fermentation reduces total sugar level of soybeans
but increases the reducing sugar level (Shrestha and
Noomhorm, 2001). Crude fiber level of soybeans also
decreased during fermentation due to hydrolysis by
carbohydrate splitting enzymes from B. subtilis (Shrestha and
Noomhorm, 2001).
Proteolytic activity of B. subtilis fermented soybean
B. subtilis has high proteolytic activity that is evident by
progressive increase in trichloroacetic acid-nitrogen (TCAN) and ammonia nitrogen with Natto fermentation, almost
doubled after 48 h incubation. Viessanguan et al., (2005) also
observed similar trend where B. subtilis during Thua Nao
fermentation releases proteinases that play important role in
proteolysis of soy proteins. These proteolysis reactions are
reported to be responsible for the characteristic flavour of
Thua Nao. It is reported three types of extra cellular enzymes
namely: neutral proteases, alkaline protease and esterase play
a major role during this activity and it was observed that most
proteases are produced during exponential growth of
microbes (Hu et al., 2010). Several proteolytic enzymes have
been known to play regulatory roles in various physiological
roles. Certain regulatory proteases such as fibronolytic and
caseinolytic showed higher activities in Natto prepared with
black soybeans (than soaked beans). These enzymes are
capable of dissolving fibrin clots that can be used to cure
diseased vascular system. B. Natto in soybeans fermentation
is capable to produce (or enhance) -glucosidase in soybeans
may promote cleavage of -glucosyl bond in black soybeans
glucoside isoflavones to form aglycones (Hu et al., 2006; Lee
and Chou, 2006). Furthermore, the fermentation of black
soybeans with the starter organisms examined caused a marked
increase in the content of aglycone and total anthocyanin
(Kuo et al., 2006).
Amino acids and fatty acid profiles
Soybeans contain all essential amino acids but low in sulphur
containing amino acids (Ang et al., 1999). B. subtilis
fermentation of soybeans resulted in proteolysis of its major
proteins. Sarkar et al., (1997) reported the acidic, basic and
aromatic amino acids form 20.8, 15.1 and 13.0% of the total
amino acid residues in Kinema. Unfermented soybeans
contained only 0.2% free amino acids which increased by 60
folds in fermented product. B. subtilis fermentation also alters
the amino acid profiles as there is relative increase in free
acidic, hydrophobic and apolar amino acid but depletion of
basic, hydrophilic and sulphur containing amino acids (Sarkar
et al., 1997; Ang et al., 1999). Sarkar et al., (1997) also compared
the essential amino acid (EAA) profiles of Kinema with that
of the reference patterns established by FAO/WHO/UNU.
The result indicated the Kinema proteins are a good source
of almost all EAAs and that their score is as high as that of
egg and milk proteins.

Shrestha et al., : J. Food Sci. & Technol. Nepal, 6 (1-9), 2010

Soybeans oil contains good quality fatty acid e.g., 53% linoleic
acid, 23% oleic acid, 11% palmitic acid, 8% linolenic acid, and
4% stearic acid (Ang et al., 1999). Lipase produced during
fermentation breakdown triglycerides into assimilated fatty
acids (Mital and Garg, 1990). Sarkar et al., (1996) identified the
linoleic acid as the major fatty acid in soybeans as well as in
Kinema fermented with B. subtilis and other mixed cultures.
Free fatty acids liberated from soybeans during fermentation
play an important role in imparting typical flavour in these
products. They further reported a significant increase in chain
length content of fatty acid during fermentation, a
phenomenon also reported by Kiuchi et al., (1976) in Natto.
However, one of the negative aspects of Kinema, as reviewed
by Sarkar et al., (1996), is that accumulation of free fatty acids
can inhibit glycolytic and glucogenic enzymes; some
hydrolyses including trypsin; and also affect fatty acid
synthesis.
Vitamins and mineral profiles
Microorganisms are highly versatile and have ability to
synthesize various micronutrients such as vitamins, and alter
the mineral profiles (Wang, 1986). Sarkar et al., (1998) reported
the preliminary steps of Kinema preparation such as soaking
and cooking lower thiamine (Vitamin B1) and riboflavin (Vitamin
B2) content. However, B. subtilis fermentation of cooked
soybeans causes an increased concentration of thiamine (5.8
to 8.4 mg/kg), riboflavin (6.8 to 11.6 mg/kg) and niacin (36.4 to
4.8 mg/kg). They further added that the presence of
Enterococcus faecium in Kinema significantly reduce the level
of all three vitamins. Literature is scarce on the effect of B.
subtilis fermentation of other vitamin levels of soybeans.
Tempeh (Mold) fermentation reported to reduce thiamine
content almost by half but other B-vitamins level increases
(Murata, 1985). Ginting and Arcot (2004) reported a significant
increase in total folate content of soaked-boiled soybeans
from 72 g to 416 g in fermented Tempeh. Interestingly,
Kinema fermentation significantly decreases most of the
minerals e.g., calcium, copper, iron, magnesium, manganese,
phosphorus, potassium and zinc (Sarkar et al., 1998). It is
found that the water used for soaking and cooking leach a
significant amount of minerals causing a heavy loss. Despite
of that Kinema still contains appreciable amount of minerals.
Phenolic compounds, anthocyanins and other compounds
Soybeans have various seed coat pigmentations, such as
yellow, green, brown and black, due to presence of
anthocyanin, chlorophyll and various combinations of
breakdown compounds from these pigments. It has been
reported that black soybeans in particularly contains
numerous bioactive compounds which inhibit oxidation of
low density lipoprotein oxidation (Takahashi et al., 2005),
scavenge free radicals (Ignasius et al., 2009), and reduce the
incidence of DNA damage by cyclophosphamide (Ribeiro and
Salvadori, 2003). Most antioxidant nutrients are polyphenolic
compounds acting as reducing agents, metal chelators and
4

singlet oxygen quenchers. Many traditional fermented foods,

including Natto, reported to have elevated amount of these
compounds. Moktan et al., (2008) evaluated the antioxidant
activities of Kinema and cooked non-fermented (CNF)
soybeans by four in vitro methods: stable free radical 2,2diphenyl-1-picrylhydrazyl (DPPH) scavenging activity; Fe3+
-reducing power; Fe2+ chelating power; and activity in linoleic
acid emulsion system. The total phenol content of Kinema
was 144% higher than raw soybean; proven better free radical
scavenger; greater reducing power; and better metal chelating
power than CNF soybeans. They suggested Kinema could
be exploited as a functional food to alleviate oxidative stress.
It has been observed that soaking as well as steaming reduce
the polyphenol as well as anthocyanin content of soybeans
(Hu et al., 2010). However, B. subtilis fermentation tends to
increase total polyphenolic compound and anthocyanins,
upto 10 and 250%, respectively during Natto fermentation of
black soybeans. Moreover, phenolic compounds increased
with fermentation time due to glucosidase activities. In
natural form, phenolic compounds are combined with sugar
which reduces their availability to organism. During
fermentation, this enzyme from the starter organism
hydrolyses glucosyl bonds in soybeans that cause total
increase of aglycone and anthocyanin. Hu et al., (2010) also
reported DPPH radical scavenging activities of the fermented
black soybeans increased linearly with fermented time and
concentration.
Phytosterols are another group of compounds that is naturally
present in plant foods including soybeans. It has been linked
to decreased absorption of cholesterols if incorporated to
human diets, therefore, regarded as one of the means of
decreasing plasma cholesterol (Mattson et al., 1977). Sarkar
et al., (1996) identified three phytosterols, campesterol,
stigmasterol and -sitosterol. Kinema fermentation resulted
in 58% increase in overall phytosterol concentration.
Isoflavones and related compounds
Soybeans are associated with number phytochemical
compounds with high biological value which tends to increase
during fermentation (Wei et al., 2008). Isoflavones are class
of chemical compounds related to flavonoids which act like
phytoestrogen and are associated with many health benefits.
In their natural form isoflavone exists as isoflavone glucose
(Glucoside conjugate) which is converted into their accessible
form as isoflavone aglycone under acidic or alkaline condition
by -glucosidase. Setlow et al., (2004) reported that B. subtilis
contains -glucosidase, which catalyses the hydrolysis of
glucoside isoflavones to aglycone forms. Wei et al., (2008)
reported that fermentation of cooked soybeans with B. subtilis
BCRC 14718 at 37C for 48 h effectively convert glucosides to
aglycones than with other strains of B. subtilis, increasing
the proportion of isoflavone aglycones from 12 to 68% of the
total isoflavones in the fermented Natto. The proportions of
the isoflavone aglycones daidzein and genistein in cooked

Shrestha et al., : J. Food Sci. & Technol. Nepal, 6 (1-9), 2010

soybeans fermented with B. subtilis BCRC 14718 for 48 h is

reported to increase from 6 to 54% and from 5 to 13%,
respectively. Hu et al., (2010) reported the genistin and daidzin
(-glucoside) of soaked and cooked soybeans decreased as
fermentation time increased whereas genistein and daidzein
(aglycone) tend to increase. The proportion of isoflavone
glucosides decreased significantly due to -glucosidasecatalyzed hydrolysis during with incubation with B. subtilis
during Natto fermentation (Yin et al., 2004; Kuo et al., 2006).
Likewise Chukeatirote et al., (2010) reported increase in
isoflavone aglycone content in fermented soybeans and many
other authors reported its increase in fermented soybean (Wei
et al., 2008).
Aromatic compounds and flavouring agents
Microbial fermented soybeans, particularly B. subtilis, have
a distinct flavour. The microbial activity during fermentation
of soybeans which is linked to extra cellular hydrolase enzymes
including amylase, proteases, lipases and sucrose is mostly
responsible for the change in volatile compounds,
contributing the typical flavour. Leejeerajumnean et al., (2001)
reported a significant increase in concentration of volatile
compounds, up to 100times in concentration, during
fermentation of cooked soybeans within 72 hours. The major
components of these volatiles are 3-hydroxybutanone
(acetoin), 2-methylbutanoic acid, pyrazines, and dimethyl
sulfides together with other aromatic compounds. Similarly,
Owens et al., (1997) reported that under anaerobic condition
B. subtilis secretes -acetolate which is decarboxylated by
-acetolate decarboxylase (extra cellular enzyme) to acetoine.
Acetoine is then oxidised to diacetyl which is an aromatic
compound linked in to flavour of many foods, such as yogurt.
Production of ketone compounds is due to degradation of
soy lipids and amino acids (Owens et al., 1997). Owens et al.,
(1997) listed changes in a number of aliphatic compounds;
aliphatic aldehydes; aliphatic ketones; aliphatic acids and
esters; pyrazines; furans; sulphur compounds; aromatic
compounds; and other miscellaneous compounds during the
B. subtilis fermentation. In particular, concentration of
pyrazines compounds increase well above their initial level
which contributing significantly to the aroma of fermented
soybeans. However, drying of Thua-Nao drastically lower
the concentration of volatile compounds in commercial Nao
(fermented soybeans), reducing level of aliphatic acids, esters
and sulphur compounds, but enhancing hexanol and
trimetylpyrazine level. The loss of volatile compounds can be
minimized by improving drying method or by alternative of
preservation method (Owens et al., 1997).
Physico-chemical properties
Fermentation reduces firmness of cooked soybean and
increases viscosity of the food matrix due to formation of
mucilaginous materials. Firmness of B. Natto black fermented
soy bean reduced gradually decrease from 45.6 kg (force) in
steamed bean with incubation time, 36.7 kg after 48 h

fermentation (Hu et al., 2010). Viscosity measurement of Natto

extract showed the viscosity of the cooked soybeans
increased with increasing incubation time. Shrestha and
Noomhorm, (2002) dried the Kinema, prepared by traditional
as well as pure culture method, in hot air oven, ground into
fine powder and anlaysed for their physico-chemical properties
(Table 1).
Table 1. Chemical and physical properties of raw and
soybeans B. subtilis fermented soybeans (Kinema)1
Raw
Kinema
Kinema
Properties2
oybeans (traditional)
(pure culture)
Moisture, %
6.6
7.2
6.8
Protein, %
44.0
46.2
45.1
Lipid, %
21.8
22.2
23.0
Ash, %
4.7
4.8
4.5
Total carbohydrate, %
29.8
26.3
27.4
Crude fiber, %
6.5
4.2
4.8
Total sugar, %
21.0
14.5
16.5
Reducing sugar%
3.1
5.1
4.7
Energy, MJ/100g
1.9
1.9
1.9
Free fatty acid, %
0.7
3.1
6.2
Bulk density, g/mL
0.6
0.8
0.7
Water binding capacity, g/g
3.5
3.5
2.5
Oil binding capacity, g/g
2.4
1.9
1.9
Wettability, min
0.4
0.4
1.1
2
4.7
5.7
8.5
Emulsifying activity index, m /g
Nitrogen solubility index, %
17.1
29.2
66.0
PH
6.93
6.9
6.5
Foam volume increase, %
8.1
22.00
6.1
LGC*, %w/v
22
20
NG
84.2
76.7
58.7
L
0.54
2.32
13.3
a
26.5
27.44
35.9
b
Hue
88.84
86.16
69.66
1
Shrestha and Noomhorm, (2001). 2Values are in moisture free basis

They reported bulk density of the Kinema powders is only

slight higher than soyflour. Formation of gummy materials on
the surface of the beans due to Bacillus fermentation could
have contributed greater inter-particle adhesion in Kinema
flour and hence the density. The wettability of Kinema
prepared by pure culture method was much higher than
soyflour, most likely to be contributed by autoclaving as it
can affect the surface activity and porosity of particles,
causing higher wettability.
The emulsifying activity indices showed Kinema powder has
higher values compared to soyflour. Nitrogen solubility index
of protein powders is much higher than soyflour which is
also linked to emulsifying capacity of the soy protein (Liu,
1999). Higher protein solubility of Kinema powder should be
helpful in incorporation of this nutritious product into other
food formulations. The nitrogen solubility index of Kinema
flours is higher than soyflours. B. subtilis is known to produce
a number of proteolytic enzymes which hydrolyze protein to
smaller peptides and finally ammonia (Priest, 1989). These
chemicals are responsible for increase in water-soluble
nitrogen and flavour of Kinema (Sarkar et al., 1994; Nikkuni
et al., 1995).
Data on the foaming capacity shows Bacillus fermentation
did not affect foaming capacity of soyflour. Yatsumatsu et

Shrestha et al., : J. Food Sci. & Technol. Nepal, 6 (1-9), 2010

al., (1972) reported the denaturation of protein during

processing and also the lipid materials in soy preparations
are very detrimental to foaming because they destabilized
protein films. The stability of foam resulting from soyflour
and Kinema powder was almost same, total collapse occurred
within 2hr (Shrestha and Noomhorm, 2001). Water retention
capacity is an index of the amount of water retained within a
protein matrix or other system under certain conditions. Water
retention by the Kinema flours is lower than that by soyflours
which might have been caused by physical and chemical
modifications of the protein structure caused by heating and
fermentation. However, there was no marked difference in oil
binding capacity of these three flours. Kinema flours were
significantly darker, more reddish (a) and more yellowish
(b) than soyflours. The dark and brown colours of Kinema
flours, particularly PKF, were probably due to a greater degree
of heat treatment during processing, resulting in a Maillard
type browning reaction.
Health benefits of B. subtilis fermented soybeans
Fermentation of soybeans, particularly with B. subtilis, lead
to extensive hydrolysis of all the macromolecules such as
proteins to smaller peptides, amino acids and simple sulphur
and nitrogen compounds; starch into oligosaccharides and
simple sugars and lipids into simple fatty acids. One of the
key health benefits of B. subtilis fermentation is improved
digestibility of normally indigestible soybeans (and its
components). It reported to improve the quantity of highly
digestible soluble matters by almost three times (22 to 65%)
and at the same time almost 40% total defatted dry matter
becomes dialyzable due to degradation of high molecular
weight compounds (Kiers et al., 2000). One of the most
reported health benefits of B. subtilis fermentation is almost
complete removal of indigestible oligosaccharides such as
sucrose, trisaccharides (raffinose), tetrasaccharides
(stachyose) which are responsible for indigestion and
flatulence in humans and in monogastric animals (Sarkar and
Tamang, 1995; Esteves et al., 2010). Fermentation reduces
anti-nutrients level in soybeans which hinder the availability
of proteins and phytochemicals. It does help in improving
the protein digestibility (Esteves et al., 2010). Shrestha and
Noomhorm, (2001), found the urease activity of Kinema (dried)
is close to zero and concluded that steps Kinema preparation
lead to inactivation of anti-nutrients such as trypsin inhibitors
and other biologically active enzymes in naturally present in
raw soybeans.
In developing countries enteric diseases caused by strains
of Enterotoxigenic E. coli (ETEC) are common type of
colibacillosis (cause several diarrhoea) in pigs, calves as well
as in human beings (Nagy and Fekete, 1999). It has been
realized that antibiotics and anti-microbial compounds given
to starter piglet diets for their health and growth-promoting
properties, but the continued use of sub-therapeutic levels
of antibiotics in animal feeds may contribute to antibiotic
resistance in humans. Kyriakis et al., (1999) suggested that
6

probiotics from strains of lactic acid bacteria and Bacillus

spp. could be used as alternative feed additives to piglets so
as to replace antibiotics and antimicrobial compounds in their
feeds. Kalavi et al., (1996) claimed Rhizopus fermented
soybeans (Tempeh) reduces the duration of diarrhoea when
added to the diet of malnourished children. Kiers et al., (2003)
showed that while fermented soybeans inhibits adhesion of
ETEC K88 in Rhizopus fermented soybeans, Bacillus acting
as probiotics allow Bacillus fermented products to inhibit
proliferation of pathogens in gut. Furthermore, piglets fed
with B. subtilis fermented soybeans showed increased feed
intake by 12%, average daily weight gain by 21% and feed
efficiency by 8%. They further confirmed that fermented
soybeans could be used as source of food for weaned pig to
reduce their death rate and use of antibiotics and promote
growth for either pigs or under nourished people.
It is already mentioned that B. subtilis fermented soybeans
are richer in isoflavin aglycone and phenolic compounds.
Wei et al., (2008) reported that isoflavone by acting like
oestrogen is able to reduce risks related to cardiovascular
diseases, lower rate of prostate, breast and colon cancers
and improve health benefits. Increase of total phenol and
aglycone isoflavone enhances the ability of DPPH (2,2diphenyl -1 picrylhydrazyl) which acts as antioxidant due to
its scavenging ability of free radicals by hindering oxidation
of low density lipoprotein, reduces DNA damage by
cyclophosphamide and potentially carcinogenic compounds
(Omoni and Aluko, 2005; Hue et al., 2010). Similarly, Kon et
al., (2010) linked consumption of fermented soybeans with
reduction of diabetes type-2 where improve of glucose control
and insulin resistance were observed. They emphasised that
occurrence of cardiovascular diseases and other organic
diseases are rare in Asian countries where consumption of
fermented soybeans is consumed at rate of 9 to 30g per day
compared to western countries where consumption of
fermented soybeans is rare. They further attributed this ability
not only to isoflavone, but also to some other peptides that
might be produced during fermentation which are still
understudy (Kon et al., 2010).
Methods to improve quality of B. subtilis fermented foods
The products like Kinema, Thua-Nao and Dawadwa are still
prepared in primitive way as it was done centuries ago. There
is a great health risk in eating these products as it is largely
prepared in unhygienic way (Shrestha, 1997). There is now
an increased scientific knowledge on different products of
fermented soybeans in order to improve their quality,
standardisation and its availability throughout the year. It
mainly consists of identification and isolation of
microorganisms which pre-dominate the fermented product
and used as a specific inoculum for fermentation. There have
been several studies where pure B. subtilis (Natto) (isolated
or from stock culture) was used as a primary inoculum to
produce hygienic, safe and better quality fermented soy
products (Shrestha, 1997; Hu et al., 2000; Chukeatitrot et al.,

Shrestha et al., : J. Food Sci. & Technol. Nepal, 6 (1-9), 2010

2010). Commercial production of Natto in Japan is very

common. In Nigerian, B. subtilis inoculated soybeans
(Dawadawa) is fermented in shorter incubation time than
traditional ones, with consistent quality, properly packaged
and marketed (Iwuoha and Eke, 1996). Natto production using
B. Natto at fermentation time of 18 hr, as compared to 48 h in
traditional method, reported to give equally good quality
product (Hu et al., 2010). However, fermented products are
still largely produced by traditional method, therefore, it was
suggested to establish control measures and follow good
manufacturing practice (GMP) for people involved in its
production and commercialisation to ensure its safety (Iwuoha
and Eke, 1996).

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Fermented soy based foods such as Natto, Kinema, ThuaNoa, Dawadawa and many other similar foods are very good
source of high quality protein source to the poor masses of
developing countries who cannot afford the expensive source
of proteins such as meat, fish, egg and milk. However, all
these B. subtilis fermented soy products possess
characteristic odour, slimy appearance and low shelf-life and
which make it not so popular among the larger population. In
order to increase usefulness and acceptability, diversification
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prepared in many cultures around Asian and Africa. This
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