3.2.1. introduction: fermented food -...

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,


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.


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.


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


Lactobacillus delbrueckii

Actinomucor elegans

Aspergillus oryzae,

Rhizopus spp.

Saccharomyces sp.

Paecilomyces, Aspergillus

Saccharomyces cerevisiae,


Bacillus natto


Saccharomyces spp.

Molds, yeasts, bacteria

Leuconostoc mesenteroides,

Strepromyces faecalis, yeasts

Penicillium acidilactici,

Bacillus, Micrococcus

Lactic acid bacteria, yeasts


Aspergillus oryzae


Rhizopus oligosporus,

Aspergillus oryzae

Endomycopsin fibuliger

Hansenula anomala,Candida

quilliermondii, C. tropicalis,

Geotrichum Candidum

Pediococcus, Streptococcus,

Leuconostoc

Candida spp., Saccharomyces

Spp


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


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.


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


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


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


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).


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).


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


�

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



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%


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


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.


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:


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.


250

�

MALDI-TOF MS-MS spectrum of 1936 Da signal identified from peptide enriched

fraction. For clarity only b and y ions is labeled.

�

�

�

�

�

�

�

�

�

�

�

�

�


251

�


fraction(PepM). For clarity only b and y ions is labeled.

�

�

�

�

�

�

�

�

�

�

�

�

�


252

�


fraction(PepM). For clarity only b and y ions is labeled.

�

�

�

�

�

�

�

�

�

�

�

�

�


253

�


fraction (PepM). For clarity only b and y ions is labeled.

�

�

�

�

�

�

�

�

�

�

�


254

�

Mascot database search result for 1849 and 1936 Da signal

�

�

�

�

�


255

�

Mascot database search result for 1901 Da signal

�

�

�

�

�

�

�

�

�

�

�

�


256

�

Mascot database search result for 2114 Da signal

�

3.2.1. introduction: fermented food -...

Documents