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Chapter-III, Section-B�
233
3.2.1. Introduction: Fermented food
Any process by which large organic molecules are broken down to simpler molecules as
the result of the action of microorganisms is called fermentation. For example, conversion
of sugars and starches to alcohol by enzymes in yeast and conversion of proteins to
peptides/amino acids by microorganisms. Fermented foods are those, by the action of
micro-organisms/enzymes on food ingredients make desirable biochemical changes,
which cause significant modification to the food.
Fermentation is one of the oldest, safest, and most economical techniques in food
manufacture, and preservation (Billings, 1998; Chavan and Kadam, 1989; Blandinob et
al., 2003). In addition, fermentation provides a natural way to enhance essential amino
acids, proteins, vitamins, to destroy anti-nutrients, to enhance aroma, flavours and
appearance of the food, to reduce the energy required for cooking and to make a safer
product (Simango, 1997; Hamad, and Fields, 1979; Kitts and Weiler 2003;). Since the
dawn of civilisation, methods for the fermentation and preparation of fermented foods
from milk, vegetables, meat and cereals has been known. In older days, the preparation of
these fermented foods and beverages was in an artisan way without any prior knowledge
of the role of the microorganisms or enzymes involved in fermentation. After 19th
century, microbiology developed as a science and the fermentation process was
understood for the first time (Caplice and Fitzgerald, 1999). After the development of
microbiology, many new technologies have been developed for the industrial production
of fermented products from milk, meat, fruits, vegetables and cereals (Hirahara, 1998;
Pagni, 1998). Nowadays fermented food business has become multi-billion dollar
business and it provides livelihood to hundreds of thousands of people worldwide. A
range of fermented foods prepared from milk, cereals, vegetables and meat in different
parts of the world are listed in Table-1.
3.2.2. Classification of fermented foods
Fermented foods can be classified in many different ways, based on the kind of
microorganisms involved or fermentation process, based on substrate and based on
function of the food. There are four main fermentation processes: alcoholic, lactic acid,
acetic acid and alkali fermentation (Soni and Sandhu, 1990). Alcohol fermentation results
in the production of ethanol, and yeasts are the predominant organisms here (e.g. wines
and beers). Lactic acid fermentation (e.g. fermented milks and cereals) is mainly carried
out by lactic acid bacteria. Acetic acid fermentation results in the production of acetic
acid, and acetobacter species are the predominant organisms (e.g. palm wine vinegar,
Chapter-III, Section-B�
234
apple cider vinegar, wine vinegar and coconut water vinegar). Acetobacter convert
alcohol to acetic acid in the presence of excess oxygen. Alkali fermentation often takes
place during the fermentation of fish and seeds, popularly used as condiment (McKay and
Baldwin, 1990).
3.2.3. Bioactive peptides from fermented foods
Fermented foods are good source of bioactive peptides and essential amino acids (Table-
2). Fermentation is consider to be one of the best way to produce bioactive peptides and
several fermented foods based on milk, soybean, fish etc. has been studied extensively
and identified several peptides having anticancer (Song et al., 2000), antimicrobial
(Rizzello et al., 2005; McCann et al., 2006), antihypertensive (Hata et al., 1996; Jae-
Young et al., 2005a, 2005b), immunomodulatory (Kitts and Weiler, 2000), antioxidant
(Amadou et al., 2009) and opioid activities (Zioudrou et al., 1979).
3.2.4. Fermented food of India: Idli and dosa
India has several traditional fermented food and beverages like idli, dosa, lassi, dahi,
naan, dhokla, uthappam, jann, daru etc. Among these idli and dosa are very much popular
and has great history. The term “idli” mentioned in the Kannada writing of
Shivakotiacharya in 920 A.D. In 1025 A.D., the poet Chavundaraya described idli
preparation in his literature. Dosa mentioned in Sanskrit book “Manasollasa” written in
1051 AD by Western Chalukya king Somesvara III. Dosa is also described in Tamila
Sangam literature about the sixth century A.D. A fermented, thick suspension made of a
blend of rice and black gram is used in the preparation of idli and dosa. Nutritive value
and health benefits of these traditional foods have been well documented. Particularly
fermented foods like ‘idli’ is considered not only as whole food but also believed to
improve the disease resistance in humans and particularly recommended for routine
consumption by woman and children apart from patients suffering from various ailments.
Today several food industries are producing idli and dosa in tonnage scale and offering
ready mix and several thousands of people benefiting from this industry daily. The
microbiological, physical and biochemical changes of idli and dosa during fermentation
and its nutritive values are almost same (Chavan and Kadam, 1989; Purushothaman et al.,
1993; Ramakrishnan, 1993; Sands and Hankin, 1974; Shortt, 1998).
The lactic acid bacteria Streptococcus faecalis, Leuconostoc mesenteroides, Lactobacillus
delbrueckii, Lactobacillus fermenti, Lactobacillus lactis and Pediococcus cerevisiae have
been found to be responsible for the fermentation process.
Chapter-III, Section-B�
235
Table-1: Few Fermented Foods
Product Geography Substrates Microorganism(s)
Ang-kak
Banku
Bonkrek
Bouza
Braga
Burukutu
Busa
Chee-fan
Chicha
Dawadawa
Gari
Hamanatto
Idli & Dosa
Jalebies
Jamin-bang
Kanji
Katsuobushi
Kimchi
Kishk
China, Southeast
Asia, Syria
Ghana
Central Java
Egypt
Romania
Savannah regions
of Nigeria
Syria, Turkestan,
Egypt
China
Peru
Africa,
West Africa
Japan
India
India, Nepal,
Pakistan
Brazil
India
Japan
Korea
Egypt, Syria,
Arab world
Rice
Maize, cassava
Coconut press cake
Wheat
Millet
Sorghum and
cassava
Rice or millet
Soybean wheat curd
Maize
African locust bean
Cassava root
Whole soybeans,
wheat flour
Rice and black gram
Wheat flour
Maize
Rice and carrots
Whole fish
Vegetables, some-
times seafoods, nuts
Wheat, milk
Monascus purpureus
Lactic acid bacteria, yeast
Rhizopus oligosporus
Unknown
Unknown
Lactic acid bacteria,
Candida spp., Saccharomyces
cerevisiae
Lactobacillus and
Saccharomyces
Mucor sp., Aspergillus
glaucus
Aspergillus, Penicillium
spp., yeasts,bacteria
Lactic acid bacteria, Yeasts
Corynebacterium manihot,
Geotrichum candidum
Aspergillus oryzae,
Streptococcus, Pediococcus
Lactic acid bacteria, yeast
Saccharomyces bayanus
Yeasts and bacteria
Hansenula anomala
Aspergillus glaucu
Lactic acid bacteria
Lactic acid bacteria,
Bacillus spp.
Chapter-III, Section-B�
236
Lafun
Mahewu
Meitauza
Meju
Merissa
Minchin
Nan
Natto
Ogi
Papadam
Pozol
Puto
Rabdi
Sorghum
Soya milk
Tao-si
Tarhana
Tauco
Thumba
Torani
Vada
Waries
Africa
South Africa
China, Taiwan
Korea
Sudan
China
India, Pakistan,
Afghanistan, Iran
Japan
Africa
India
Mexico
Philippines
India
South Africa
China, Japan
Philippines
Turkey
West Java
India
India
India
India
Cassava root
Maize
Soybean cake
Soybeans
Sorghum
Wheat gluten
Unbleached wheat
flour
Soybeans
Maize
Black gram
Maize
Rice
Maize and
buttermilk
Sorghum, maize
Soybeans
Soybeans, wheat
Parboiled wheat
meal and yoghurt
Soybeans, cereals
Millet
Rice
Cereal/legume
Black gram flour
Lactic acid bacteria
Lactobacillus delbrueckii
Actinomucor elegans
Aspergillus oryzae,
Rhizopus spp.
Saccharomyces sp.
Paecilomyces, Aspergillus
Saccharomyces cerevisiae,
Lactic acid bacteria
Bacillus natto
Lactic acid bacteria
Saccharomyces spp.
Molds, yeasts, bacteria
Leuconostoc mesenteroides,
Strepromyces faecalis, yeasts
Penicillium acidilactici,
Bacillus, Micrococcus
Lactic acid bacteria, yeasts
Lactic acid bacteria
Aspergillus oryzae
Lactic acid bacteria
Rhizopus oligosporus,
Aspergillus oryzae
Endomycopsin fibuliger
Hansenula anomala,Candida
quilliermondii, C. tropicalis,
Geotrichum Candidum
Pediococcus, Streptococcus,
Leuconostoc
Candida spp., Saccharomyces
Spp
Chapter-III, Section-B�
237
Table-2: Some bio-active peptides from fermented food
Activity Origin Name/remarks/sequence
ACE inhibitory
Immunomodulatory
Cytomodulatory
Opioid agonist
Opiod antagonist
Antimicrobial
Antithrombotic
Mineral binding,
Hypocholesterolemic
Antioxidant
Soy
Fish
Meat
Milk
Egg
Wine
Wheat
Brocoli
Rice
Egg
Milk
Wheat
Milk
Wheat
Milk
Milk
Egg
Milk
Milk
Milk
Soy
Milk
Fish
Wheat
Milk
NWGPLV
LKP, IKP, LRP, EVMAGNLYPG
IKW, LKP
LRP, LKP, VPP, IPP, FFVAP, WLAHK, FALPQY
KVREGTTY, FRADHPPL, KVREGTTY
AWPF, SWSF, YYAPF, WVPSVY, IPPGVPY,
YYAPFDGIL
IAP
YPK
Oryzatensin (GYPMYPLR)
Peptides not specified
Immunopeptides(TTMPLW)
Immunopeptides
� -Casomorphin HIQKED(V), �-casomorphin-7
(YPFPGPI)
Gluten-exorphins A4, A5 (GYYPT), B4, B5, and C
(YPISL)
� -Lactorphins, �-lactorphins, casomorphins
Lactoferroxins, Casoxins
OTAP-92 (f109–200)a
Lactoferricin, Casecidins, isracidin, kappacin
k-CN (f106–116)a, casoplatelin
Caseinophosphopeptides
LPYPR
IIAEK
MY
Peptides not specified
MHIRL, YVEEL, WYSLAMAASDI
Chapter-III, Section-B�
238
Particularly the microorganisms Leuconostoc mesenteroides and Streptococcus faecalis
are essential for leavening of the batter and for the production of amino acids in idli and
dosa (Purushothaman et al., 1993; Ramakrishnan, 1993). The yeasts Geotrichum
candidum, Torulopsis holmii, Torulopsis candida and Trichosporon pullulans have also
been identified in idli and dosa fermentation (Chavan and Kadam, 1989; Shortt, 1998).
Fermentation of idli and dosa batter also increases all essential amino acids and reduces
anti-nutrients, enzyme inhibitors and flatus sugars (Steinkraus et al., 1993).
3.2.5. Rationale behind the identification of novel bioactive peptides from Indian
fermented food derived from Oryza sativa�
Even though, the degradation of several proteins into smaller bioactive peptides by the
action of microbial enzymes during fermentation of milk and other food products are
known in literature, till date no reports are available revealing the presence of bioactive
peptides available in rice based fermented food idli and dosa. The identification of novel
bioactive compounds will contribute towards better understanding of the benefits of these
food items to enhance health and quality of life. In view of the importance of these
bioactive peptides, we planned to undertake the MALDI mass spectral analysis of the
fermented raw material used in the preparation of these fermented foods with a initial
simple hope to identify such bioactive peptides.
3.2.6. Enrichment of peptides
Polished rice (Ratna cultivar, 250 g) and black gram (62.5 g) soaked in 200 mL and 100
mL of tap water each separately for 6 hrs and coarse grinded separately. The two
suspensions are then blended together and allowed to undergo natural fermentation for 48
hrs at room temperature (about 30 ºC). After 48 hrs of fermentation additional 200 mL of
tap water was added to the batter and allowed to stand for 1 hr till the supernatant water
layer separates from the suspension. Then supernatant water layer was decanted and
lyophilized to a sticky mass to which 100 mL ethanol was added and undissolved starch
particles were filtered off. The clear filtrate was lyophilized again. The residue obtained
was washed with portions of ethyl acetate (3 x 35 mL) in order to remove small organic
molecules if any and was subjected to (NH4)2SO4 precipitation at 80% saturation (Huynh
et al., 1996). The precipitated solid was further subjected to ultra filtration (cut-off: 3
kDa) using an Amicon 810 system (Millipore Corp., Bedford, USA) in order to remove
larger protein fragments and the filtrate containing peptides enriched fraction (PepM) so
obtained was lyophilized.
Chapter-III, Section-B�
239
3.2.7. Identification of peptides
Bradford protein assay has shown 2.1-2.4 mg/mL of protein/peptides content in the
lyophilized final product (Bradford, 1976). MALDI mass spectral analysis of peptide
enriched fraction derived from fermented food batter used for the preparation of idli and
dosa has been undertaken to examine the presence of novel peptides possibly arising out
of enzymatic cleavage of proteins present in the feed stock. The MALDI results of the
compound have shown strong signals at 2114, 1936, 1901 and 1849 Da along with some
other small mass peaks shown in Figure-1. These peaks were taken up for further
exploration and characterization.
3.2.8. MALDI-TOF/TOF MS, MS-MS analysis of PepM
All MS, MS-MS experiments were performed on a MALDI-4800 analyzer, Applied Bio-
systems. Matrix used in MALDI analysis was alpha-cyano-4-hydroxy cinnamic acid
(CHCA). PepM (1 mg) was dissolved in 10 µl acidified water-acetonitrile buffer (80:20,
0.1% TFA in water). From this 2 µl was taken and mixed with 2 µl of the matrix solution.
The matrix solution used was 0.05 M alpha-cyano-4-hydroxy cinnamic acid in
water/acetonitrile (1:1). Then 2 µl was transferred onto the plate and allowed to dry. The
peptides were desorbed and ionized by laser and acquired in reflector positive mode and
processed using the 4000 Series Explorer software. The MALDI MS spectrum of PepM is
shown in Figure-1 revealing intense signals at 2114, 1936, 1901, 1849 Da accounting for
the presence of low molecular weight peptides apart from low intensity peaks. These
major peaks were taken for further sequencing and characterization. These peaks having
molecular masses of 1936, 1849, 1901, 2114 Da were used for the MS-MS analysis. The
amino acid sequences of the peptides from their MS/MS-MS was done using MASCOT
(Matrix science, http://www.matrixscience.com, London, UK), NCBInr, SWISS-PORT
and MSDB database as well as by manual calculations, and they corresponded to the
following plausible sequences SRLEKNSTTSDSSPSLRA (1936 Da) and
RLEKNSTTSDSSPSLRA (1849 Da) derived from the cleavage by the action of lactic
acid bacteria on Oryza sativa based 138 kDa protein as well as
TPRRLSPLPSVAPLSAEPLL (2114 Da) and VDDVIPESFTAGSEYKSG (1901 Da) are
attributed to 99 kDa and 12 kDa protein respectively derived from Oryza sativa. MS/MS
of 1936 and 1849 Da revealed similar fragmentation pattern indicating the possibility of
similar sequences for these peptides probably originating from the same protein source.
The Mascot database results also shown the presence of similar sequence for these two
Chapter-III, Section-B�
240
peptides, the peptide with molecular mass 1849 Da have been generated by the loss of
serine from the N-terminal of peptide having mass 1936 Da.
3.2.9. Biological studies
The PepM was analyzed for the anticancer and immunomodulatory activity.
3.2.9.1. In vitro anticancer activity
For anticancer activity peptide enriched fraction PepM was screened against a panel of
six human cancer cell lines viz., IMR-32 (neuroblastoma), PC-3 (prostate), Colo-205
(colon), SiHa (cervical), HOP-62 (non small cell), Hep-2 (liver), A549 (lung) and MCF-7
(breast). IC50 values against these cell lines were found to be 86, 105, 122, 174, 300, 317,
605, 923 µM respectively. PepM exhibited moderate anticancer activity at higher
concentrations.
3.2.9.2. Immunomodulatory studies
PepM was analyzed for the immunomodulatory activity. For immunomodulatory
response, HA titre, DTH reaction, splenocyte proliferation and NO production were
measured (shown in Figure 2-6). The findings outlined in results have demonstrated that
PepM possesses a potent immunostimulant activity. The immune response of the body is
mainly composed of specific and non-specific immunity. The specific immune response
includes humoral and cellular immunity. Humoral immunity via the antibody response is
regulated by B cells and other immune cells involved in antibody production. The
stimulation of the humoral response against SRBCs by PepM was evidenced by the
increase in HA titer shown in Figure-2. A DTH reaction is an expression of cell-mediated
immunity and plays a role in many inflammatory disorders. Such reactions are
characterized by large influxes of non-specific inflammatory cells, of which the
macrophage is a major example. It is a type IV hypersensitivity reaction that develops
when antigen activates sensitized T cells. These cells generally appear to be a Th1
subpopulation although sometimes TC cells are also involved. Several lines of evidence
suggest that DTH reactions are important in host defence against parasites and bacteria
that can live and proliferate intracellular. Treatment with PepM enhanced the DTH
reaction, as reflected by the increased footpad thickness compared to the control group
shown in Figure-3, suggesting heightened infiltration of macrophages to the
inflammatory site. This study may support a possible role of PepM in assisting the cell-
mediated immune response. In our present investigation, we also found that PepM
augmented Con-A and LPS induced splenocyte proliferation shown in Figure-4. In view
of the pivotal role played by macrophages in coordinating the processing and presentation
Chapter-III, Section-B�
241
of antigen to B-cells, PepM was evaluated for its effect on NO production from
macrophages. In this study, we found that PepM could significantly increase the NO
production from macrophages shown in Figure-5. We also determined the possible effect
of PepM on soluble mediators of Th1 and Th2 response. PepM enhanced the Th1 and Th2
immune responses as shown in Figure-6 by significantly increased the production of Th2
(IL-4) and Th1 cytokines (IL-2 and IFN-�) as compared with control. Investigation of the
balance of Th1 and Th2 cytokine production should be helpful to understanding the
outcomes of different immune responses and are clinically useful in treating
immunologically deregulated states (Fang et al., 2005). PepM up-regulated the
production of IL-4 via a significant release of IFN-� by regulating the shift of Th1 to Th2.
The HA titre, DTH reaction, splenocyte proliferation, NO production and cytokine
production Th2 (1L-4) and Th1 cytokines (IL-2 and IFN-�) suggested that bioactive
peptides in PepM enhance both humoral and cellular immunity in a mouse model.
3.2.9.3. Protein contents determination
The protein content of PepM was determined by Bradford method (Bradford, 1976)
against bovine serum albumin (BSA) as standard.
3.3.0. Conclusion
This work describes the discovery of new peptides in fermented batter of Oryza sativa,
used in the preparation of Indian food items. Immune potentiating activity of peptide
enriched fraction indicates possible health benefits offered by these traditional food items.
Furthermore, the moderate cytotoxicity exhibited by the peptide enriched fraction
indicates the possible chemo protective effect of these food items from various cancers.
3.3.1. Experimental
3.3.2. Evaluation of in vitro cytotoxicity
The effect of PepM on the growth of cancer cell lines was evaluated according to the
procedure adopted by the National Cancer Institute for in vitro anticancer drug screening
that uses the protein-binding dye sulforhodamine B to estimate cell growth (Skehan et al.,
1990). The detailed experimental procedure was discussed in chapter-2, section-1,
experimental section.
3.3.3 In vivo and ex vivo immunomodulatory studies of PepM
3.3.4. Animals
The study was conducted on male Balb/c mice (18-22 g). The Ethical committee of the
Indian Institute of Integrative Medicine (IIIM, CSIR) instituted for animal handling
approved all the protocols. The animals were housed under standard laboratory conditions
Chapter-III, Section-B�
242
Figure-1: MALDI-MS spectrum of PepM.
�
Figure-2. HA titre: The animals were immunized by injecting 0.2 mL of 5 x 109 fresh
SRBC suspension intraperitonially on day 0. Blood samples were collected in
microcentrifuge tubes from individual animals by retro-orbital plexus on day 7 for
primary antibody titre and day 15 for secondary antibody titre. Data are mean ± S.E. of
six animals. * p < 0.05 ,** p < 0.01, ***p < 0.001 when compared with control group
determined by one-way ANOVA (Bonferroni correction multiple comparison test).
Chapter-III, Section-B�
243
Figure-3. DTH response: Balb/c mice were challenged with 20 �L of 5 × 109 SRBC in
the right hind footpad. The control lateral paw received equal volume of saline. Data are
expressed as mean ± S.E. of five observations of right hind foot pad thickness measured
at 24, 48 and 72 hrs. *P < 0.05; **P < 0.01; ***P < 0.001 as compared to control
determined by one-way Anova (Bonferroni correction multiple comparison test).
�
Figure 4. Splenocyte proliferation: Splenocyte proliferation expressed as the absorption
at 570 nm. Data are mean ± SE of six animals. * p < 0.05 and ** p < 0.01 and *** p <
0.001 compared with control group determined by one-way ANOVA (Bonferroni
correction multiple comparison test).
Chapter-III, Section-B�
244
�
Figure-5. NO production: Results are expressed in �M. Data are mean ± SE of six
animals. * p < 0.05 ,** p < 0.01 and *** p < 0.001 compared with control group
determined by one-way ANOVA (Bonferroni correction multiple comparison test).
�
Figure-6. Determination of IFN-�, TNF-� and IL-4: Concentration of Th1 (IL-2 and
IFN-�) and Th2 (IL-4) cytokine production in mouse serum. The IL-2, IFN-gamma, and
IL-4 concentration in pg/mL were determined using ELISA. Data are mean ± SE of six
animals. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with control group
determined by one-way ANOVA (Bonferroni correction multiple comparison test).
Chapter-III, Section-B�
245
(temperature 25 ± 2 °C and 12 hrs dark/light cycles) and fed with commercial standard
pellet diet and tap water ad libitum.
3.3.5. Treatment
SRBC collected in Alsever’s solution, were washed three times in large volumes of
pyrogen free 0.9% normal saline and adjusted to a concentration of 5 x 109 cells/mL for
immunization and challenge. The animals were divided into five groups of six animals
each. (Group I) control, received 1% gum acacia; (Group II) positive control received
levamisole (2.5 mg/kg body weight); (Group III) received 0.2 mg/kg of PepM; (Group
IV) received 1 mg/kg of PepM; (Group V) received 5 mg/kg of PepM. PepM was
dissolved in 1% gum acacia and were administered orally for 14 days. The dose volume
was 0.2 mL.
3.3.6. HA titre
Blood was collected on days 7 and 15 from the retro-orbital plexus of each mouse for
serum preparation. Serial two-fold dilutions of serum samples were made in 50 �L of
PBS (pH 7.2) in 96-well micro titre plates and mixed with 50 �L of 1% SRBC suspension
in PBS. After mixing, the plates were kept at room temperature for 2 hrs. The value of the
antibody titre was assigned to the highest serum dilution showing visible
haemagglutination (Mungantiwar et al., 1999).
3.3.7 DTH reaction
PepM was administered 2 hrs after SRBC injection and once daily on consecutive days.
Six days later, the thickness of the right hind footpad was measured with a
spheromicrometer (pitch, 0.01 mm) and was considered as the control. The mice were
then challenged by injecting 20 µL of 5 x 109 SRBC/mL intradermally into the right hind
footpad. The foot pad thickness was measured again after 24, 48 and 72 hrs (Bafna and
Mishra, 2006).
3.3.8 Splenocyte proliferation assay (ex-vivo)
Spleen collected from untreated and treated groups under aseptic conditions in HBSS,
was minced using a pair of scissors and passed through a fine steel mesh to obtain a
homogeneous cell suspension and the erythrocytes were lysed with ammonium chloride
(0.8%, w/v). After centrifugation (380 x g at 4 °C for 10 minutes), the pelleted cells were
washed three times with PBS and re-suspended in complete medium [RPMI 1640
supplemented with 12 mM HEPES (pH 7.1), 0.05 mM 2-mercaptoethanol, 100 IU/mL
penicillin, 100 �g/mL streptomycin and 10% FCS]. The cell number was counted with a
haemocytometer by the trypan blue dye exclusion technique. Cell viability exceeded 95%
Chapter-III, Section-B�
246
(Wang and Li, 2002). To evaluate the effect of the PepM on the proliferation of splenic
lymphocytes, the spleen cell suspension (1 x107 cell/mL) was pipetted into 96-well plates
(200 �L/well) and cultured at 37 °C for 72 hrs in a humid saturated atmosphere
containing 5% CO2 in the presence of Con-A (5 �g/mL) and LPS (10 �g/mL). After 72
hrs, 20 �L of MTT solution (5 mg/mL) was added to each well and incubated for 4 hrs.
The plates were centrifuged (1400 x g, 5 min) and the untransformed MTT was removed
carefully by pipetting. To each well, 100 �L of a DMSO working solution (192 �L
DMSO with 8 �L 1 M HCl) was added and the absorbance was evaluated in an ELISA
reader at 570 nm after 15 min.
3.3.9 Nitric oxide assay
The amount of stable nitrite, the end product of NO generation by the activated
macrophages, was determined by a colorimetric assay. Briefly, 50 �L of culture
supernatants was mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1%
naphthylethylenediamine dihydrochloride, 2.5% H3PO4). This mixture was incubated at
room temperature for 10 minutes. The absorbance at 540 nm was read on a
spectrophotometer. The nitrite concentration was determined by extrapolation from a
sodium nitrite standard curve.
3.3.10 Determination of IFN-�, TNF-� and IL-4 by ELISA method
Serum was collected 4 hrs after the final oral administration of PepM. The interleukin-2
(IL-4), interferon-gamma (IFN-�) and tumor necrosis factor-alpha (TNF-�)
concentrations were measured with an enzyme-linked immunosorbent assay (ELISA kit,
R & D Systems) according to instructions of manufacturer (Ferrar and Schreiber, 1993).
3.3.11. References
Amadou, I., Yong-Hui, Sun, S. and Guo-Wei, L. (2009). Fermented soybean products:
some methods, antioxidants compound extraction and their scavenging activity.
Asain Journal of Biochemistry 4: 68-76.
Bafna, A.R. and Mishra, S.H. (2006). Protective effect of bioactive fraction of
Sphaeranthus indicus Linn against cyclophosphamide induced suppression of
humoral immunity in mice. Journal of Ethnopharmacology 104: 426-429.
Billings, T. (1998). On fermented foods. Available: http://www.livingfoods. com.
Blandinob, A., Al-Aseeria, M.E., Pandiellaa, S.S., Canterob, D. and Webba, C. (2003).
Cereal-based fermented foods and beverages. Food Research International 36:
527-543.
Bradford, M.M. (1976). A rapid and sensitive method for the quantisation of microgram
Chapter-III, Section-B�
247
quantities of protein utilizing the principle of protein dye binding. Analytical
Biochemistry 72: 248-254.
Caplice, E. and Fitzgerald, G. F. (1999). Food fermentations: role of microorganisms in
food production and preservation. International Journal of Food Microbiology,
50, 131–149.
Chavan, J.K. and Kadam, S.S. (1989). Nutritional improvement of cereals by
fermentation. Critical Reviews in Food Science and Nutrition 28(5): 349-400.
Fang, S.P., Tanaka, T., Tago, F., Okamoto, T., & Kojima, S. (2005). Immunomodulatory
effects of Gyokuheifusan on IFN /IL-4 (Th1/Th2) balance in ovalbumin (ova)
induced asthma model mice. Biological and Pharmaceutical Bulletin
28: 829-833.
Ferrar, M.A. and Schreiber, R.D. (1993). The molecular cell biology of interferon-gamma
and its receptor. Annual Reviw of Immunology 11: 571-611.
Hamad, A.M. and Fields, M.L. (1979). Evaluation of the protein quality and available
Lysine of germinated and fermented cereals. Journal of Food Science
44: 456-459.
Hata, Y., Yamamoto, M., Ohni, M., Nakajima, K., Nakamura, Y. and Takano, T. (1996).
A placebo-controlled study of the effect of sour milk on blood pressure in
hypertensive subjects. American Journal of Clinical Nutrition 64: 767-771.
Hirahara, T. (1998). Functional food science in Japan. In: Mattila-Sandholm, T and
Kauppila, K. (eds.). Functional food research in Europe pp. 19–20.
Julkaisija-Utgivare, Finland.
Huynth, Q.K., Borgmeyer, J.R., Smith, C.E., Bell, L.D. and Shah, D.M. (1996).
Isolation and characterization of 30 kDa protein with antifungal activity from
leaves of Engelmannia pinnatifida. Biochemical Journal 316: 723-727.
Jae-Young, J., Ji-Young, P., Won-Kyo, J., Pyo-Jam, P. and Kim, S. (2005a).
Isolation of angiotensin I converting enzyme (ACE) inhibitor from fermented
Oyster sauce, Crassostrea gigas. Food Chemistry 90: 809-814.
Jae-Young, J., Pyo-Jam, P., Hee-Guk, B., Won-Kyo, J. and Se-Kwon, K. (2005b).
Angiotensin I converting enzyme (ACE) inhibitory peptide derived from the sauce
of fermented blue mussel, Mytilus edulis. Bioresource Technology 96: 1624-1629.
Kim, S.E., Kim, H.H., Kim, J.Y., Kang, Y., Woo, H.J. and Lee, H.J. (2000). Anticancer
activity of hydrophobic peptides from soy proteins. Bio Factors 12: 151-155.
Kitts, D.D. and Weiler, K. (2003). Bioactive proteins and peptides from food sources.
Chapter-III, Section-B�
248
Applications of bioprocesses used in isolation and recovery. Current
Pharmaceutical Design 9:1309-1323.
McCann, K.B., Shiell, B.J., Mischalski, W.P., Lee, A., Wan, J., Roginski, H. And
Coventry, M.J. (2006). Isolation and characterisation of novel antibacterial
peptide from bovine �s1-casein. International Dairy Journal 16: 316-323.
McKay, L.L. and Baldwin, K.A. (1990). Applications for biotechnology: present and
future improvements in lactic acid bacteria. FEMS Microbiology Reviews
87: 3-14.
Mungantiwar, A.A., Nair, A.M., Shinde, U.A., Dikshit. V.J., Saraf, M.N., Thakur, V.S.
and Sainis, K.B. (1999). Studies on the Immunomodulatory effects of
Boerhaavia diffusa alkaloidal fraction. Journal of Ethnopharmacology 65: 125-
131.
Pagni, J. (1998). Demonstrating bacteria for health. VTT Biotechnology and Food
Research (Catalogue 5).
Purushothaman, D., Dhanapal, N. and Rangaswami, G. (1993). Indian idli, dosa, dhokla,
khaman, and related fermentations. In: Steinkraus, K.H. (eds). Handbook of
indigenous fermented food pp.149-165. Marcel Dekker, New York.
Ramakrishnan, C.V. (1993). Indian idli, dosa, dhokla, khaman, and related fermentations.
In Steinkraus, K.H. (Eds). Handbook of indigenous fermented food, p.149-165.
New York: Marcel Dekker.
Rizzello, C.G., Losito, I., Gobbetti. M., Carbonara, T., De Bari, M.D. and Zambonin, P.
G. (2005). Antibacterial activities of peptides from the water soluble extracts of
Italian cheese varieties. Journal of Dairy Science 88: 2348-2360.
Sands, D.C. and Hankin, L. (1974). Selecting lysine-excreting mutants of lactobacilli for
use in food and feed enrichment. Journal of Applied Microbiology 28: 523-534.
Shekhan, P., Storeng, R., Seudiero, D., Monks, A., Memahon, J., Vistica, D., Warren,
J.T., Bokesch, H., Kenney, S., Boyd, M.R. (1990). New colorimetric cytotoxicity
Assay for anticancer-drug screening. Journal of Natural Cancer Institute 82:
1107-1112.
Shortt, C. (1998). Living it up for dinner. Chemistry and Industry 8: 300-303.
Simango, C. (1997). Potential use of traditional fermented foods for weaning in
Zimbabwe. Journal of Social Science and Medicine 44: 1065–1068.
Song, E.K., Hyuck, H.K., Yeon, K.j., Young, I.K., Hee, J.W. and Hyong, J.L. (2000).
Anticancer activity of hydrophobic peptides from soy proteins. Bio Factors 12:
Chapter-III, Section-B�
249
151-`55.
Soni, S.K. and Sandhu, D.K. (1990). Indian fermented foods: microbiological and
biochemical aspects. Indian Journal of Microbiology 30: 135–157.
Steinkraus, K.H., Ayres, R., Olek, A. and Farr, D. (1993). Biochemistry of
Saccharomyces. In: Steinkraus, K.H. (eds). Handbook of indigenous fermented
food pp 517–519. Marcel Dekker, New York:
Wang, Y.P. and Li, X.Y. (2002). Effect of astragaloside IV on T, Blymphocyte
proliferation and peritoneal macrophage function in mice. Acta Pharmacologica
Sinica 3: 263-266.
Zioudrou, C., Streaty, R.A. and Klee, W.A. (1979). Opioid peptides derived from food
proteins-exorphins. Journal of Biological Chemistry 254: 2446-2449.
Chapter-III, Section-B�
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