EVALUATION OF

DEBITTERED AND

GERMINATED
FENUGREEK SEED FLOUR
ON THE CHEMICAL
CHARACTERISTICS,
BIOLOGICAL ACTIVITIES,
AND SENSORY PROFILE
OF FORTIFIED BREAD
Presented by kalaivani.D, PRN no. 18040343009
 JOURNAL PARTICULARS
NAME OF THE JOURNAL
IMPACT FACTOR
DATE OF PUBLICATION
REFERENCING STYLE
LITERATURE GENRE
TYPE OF STUDY
AUTHORS
RESEARCH FACILITY
NAME OF THE PRODUCT
HYPOTHESIS
FUNDING AGENCY
Journal of food preservation and processing
1.288
9 may, 2017
APA style
Original article
Experimental, quantitative
Pravesh S. Chaubey, Gauresh Somani,
Divya Kanchan, Sadhana Sathaye,
Sadineni Varakumar, Rekha S. Singhal
Institute of chemical technology, Mumbai
Wheat bread with 10% DGFSF (debittered
and germinated fenugreek seed flour)
Germination of fenugreek seed shows better
nutraceutical profile and yields
organoleptically acceptable fortified bread
Department of biotechnology (DBT),
government of India
ABSTRACT AND PRACTICAL APPLICATIONS
INTRODUCTION  India - world’s largest producer of fenugreek
 Fenugreek seeds possess hypocholestrolemic,
hypoglycaemic, anti-inflammatory, anti-
cancerous properties due to the presence of 4-
hydroxyisoleucine, trigonelline, saponins, and
flavonoids.
 It is used in the treatment of diabetes,
constipation, high blood pressure, bronchitis,
gastritis, atherosclerosis, rheumatoid arthritis
and is also an established galactogogue.
 But its bitter taste restricts the acceptability in
foods and overcoming this limitation is a major
challenge for food scientists.
 Germination of fenugreek seeds decreases
the bitterness, fat, dietary fibre content and
enhances the antioxidant activity and protein
content.
 Addition of fenugreek fibre (9% wt/wt) in wheat
flour has shown to improve storage quality due
to lowered starch retrogradation.
 Considering the nutritional benefits, low price,
and ready availability of fenugreek seeds, this
work was undertaken to develop value added
product from germinated fenugreek seeds.
 OBJECTIVES OF THE STUDY

 The determination of the chemical composition of germinated fenugreek

seed extract
 Study of In vivo anti-diabetic and anti-hypertriglyceridemia effects in rats
 Exploring sweeteners and curd for masking the bitterness of fenugreek
seeds
 Evaluate the effect of substituting wheat flour with 5, 10, 15 and 20%
(wt/wt) of debittered and germinated fenugreek seed flour (DGFSF) in the
fortified bread
 MATERIALS

MATERIAL SOURCE
Fenugreek seeds Local market of Mumbai city

α-glucosidase (from yeast, 5 U/mg) SRL pvt. Ltd

Linoleic acid
Acesulfame Advanced enzymes
Lipase (10 U/ mg)
Lipoxygenase (50,000 U/mg) Sigma aldrich
Amylase (10 U/mg)
Pepsin (5 U/mg)
DPPH (1, 1-diphenylpicryl-hydrazyl)
Curd Gowardhan brand

Sucrose Himedia pvt Ltd

Glucose oxidase–peroxidase (GOD–POD) kit Accurex biomedical pvt. Ltd

Glucometer and strips

AR - The standard Mallinckrodt grade of analytical reagents; suitable for laboratory and general
use. If the reagent meets the requirements of the American Chemical Society Committee on
Analytical Reagent, it will be labelled as AR (ACS) reagent
 METHODS

DEBITTERING & GERMINATION OF ETHANOLIC EXTRACT

SEEDS PREPARATION
Sterilization with 2.5% NaClO2 Sonication- Bath sonicator (50
solution for 15 min Hz) for 20 min
sterile water rinse
Debitteration in 1:4 (wt/vol) solution Centrifugation at 7,1703g for 20
ratio – 3 sets + I control min at 25°C
• Acesulfame (0.25%, 8 hr)
• Curd (1:1 in water, 8 hr)
• Sucrose (5%, 8 hr) supernatant collection
• control set plain distilled water

Evaporation - on a rotary
Drained & wrapped in muslin cloth vacuum evaporator under reduced
pressure at 50°C for 5 min
Germination - at 32°C for 48–72 hr

Drying – in tray drier at 40°C

for 4–6 hr Storage at 48°C

Sieving - 40 mesh sieve

ESTIMATION -TOTAL POLYPHENOLIC, FLAVONOID & SAPOGENIN CONTENTS
TOTAL C/O TECHNIQUE METHOD & YEAR UNITS

Polyphenols Spectrophotometry Modified folin–ciocalteu method of mg GAE/100 g

pandey & awasthi (2015)

Flavonoids Colorimetry Chlopicka, pasko, gorinstein, mg QE/g

jedryas, & zagrodzki (2012)
Sapogenins Spectrophotometry Method of baccou, lambert, and mg DE/g
sauvaire (1977)

DPPH (1,1-DIPHENYL-2-PICRYLHYDRAZYL) RADICAL SCAVENGING ACTIVITY

STOCK REAGENT SOLUTION (RS)
24 mg of DPPH in 100 mL methanol
and stored at- 20°C

UV-SPECTROPHOTOMETRY
2 mL of RS
At 517nm, absorbance of the
resulting solution is obtained and
0.5 mL seed extract expressed in ascorbic acid
equivalents (AAE)

(Ascorbic acid - standard solution,

methanol - blank)
 SONICATION

CENTRIFUGATION
 IN VITRO ACTIVITIES OF FENUGREEK SEED EXTRACTS

α-glucosidase inhibition assay Modified method of yoshida, hishida, iida,

hosokawa, and kawabata (2008)

Lipase inhibition assay Gu, hurst, stuart, and lambert (2011)

Lipoxygenase (LOX) inhibition assay Shinde et al. (1999) using linoleic acid

IN VIVO EXPERIMENTS WITH RATS

Sex : adult male rats

Strain : Wistar albino
Weight : 200–250 g
Sample number : five groups of six rats,
Housing : plastic cages in a well-ventilated
institutional animal house facility
Standard conditions : (30 ±2°C, 12 hr
light/dark cycle).
Acclimatization period: 10 days
Ethical approval : The institutional animal
ethics committee
ORAL GLUCOSE TOLERANCE TEST
(OGTT)
Fasting period: 16 hr
Group I - glucose at 2.5 g/kg BW orally
Groups II and III - extract from non-
germinated fenugreek seed flour (50 and 100
mg/kg, respectively) orally along with glucose
(2.5 g/kg BW)
Groups IV and V - extract from germinated
fenugreek seed flour (50 and 100 mg/kg,
respectively) orally along with glucose (2.5
g/kg BW)
Blood samples (20 mL) were drawn at 0,
30, 60, 90, and 120 min. Blood glucose
measured using AccuChek Glucometer.
ACUTE ANTIHYPERTRIGLYCERIDEMIA
Fasting period: 16 hr
Lipid emulsion : 4 mL consisting of olive oil
(50%, vol/vol), cholic acid (0.66%, wt/vol),
and deionized water (50%, vol/vol) orally to
all groups
Group I – only lipid emulsion
Groups II and III - extract from NGFSF (50
and 100 mg/kg, respectively)
Groups IV and V - extract from GFSF (50
and 100 mg/kg,respectively) orally along with
lipid emulsion
Blood was collected at the end of 0, 1, 2,
and 4 hr, and serum triglycerides were
analyzed using Accurex ELISA kits
 PREPARATION, ANALYSIS AND SENSORY
EVALUATION OF FUNCTIONAL BREAD

Wheat flour (maida) (200 g) was

supplemented with 5, 10, 15, and 20% of
NGSF, GFSF, and DGFSF separately
Hydration
Blending
Sensory evaluation was done using
9-point hedonic scale for color,
texture, flavor/taste, and overall
acceptability with 30 trained panellists
under white light, and water was used
as palate cleanser

Dough development, and dough

breakdown
Moisture, ash, crude fat, crude
First proofing under anaerobic protein, and total dietary fibre content
condition at 35 °C for 2 hr by AOAC methods analysis

Kneading and sheeting Cooling to room temperature, and

slicing

Incubation in moulds at 35 °C for 1.5

hr Baking at 240 °C for 20 min
 CHARACTERISTIC OF THE BREAD EQUIPMENT / FORMULA

Texture Ta-xt2i texture analyzer and P/36

cylindrical probe

Bulk density Weight of flour (g) divided by flour volume

(ml)

Color of bread Hunter lab colorimeter

IN VITRO STARCH DIGESTIBILITY OF BREAD

 In vitro starch digestibility of bread was determined according to goni, garcia-alonso,
and saura-calixto.
 The rate of starch digestion was expressed as % of bread hydrolysed at different
time intervals (30, 60, 90, 120, and 180 min).
 The areas under hydrolysis curves (AUC, 0–180 min) were calculated.
 The glycemic index (GI) was calculated as the relation between the AUC for the
sample and wheat bread

STATISTICAL ANALYSIS
 One-way ANOVA using SPSS, 20 for windows.
 The means were separated by fisher’s protected least significant difference (LSD).
 The significance level p < 0.05
RESULTS AND DISCUSSION
 PROXIMATE COMPOSITION OF FENUGREEK SEEDS

 Three varieties of fenugreek seeds (Rajwadi, CL, and A1) were screened for
proximate analysis
 Rajwadi and CL were found to contain significantly high protein, ash and moisture
content as compared to A1 but vice versa for fat content
 Total fibre contents of Rajwadi was significantly different from that of other two
varieties (further studies)

GERMINATED VS NON-GERMINATED SEEDS

 Moisture content of germinated seeds were significantly (p < 0.05) high compared to
non-germinated due to their hygroscopic nature
 Germinated seeds had decreased fat content as it was the source of energy in
germination process
 Increase in protein content after germination might be due to the reduction of seed
nitrates into protein or ammonium compound, or it might also be due to enzymatic
synthesis of proteins
 There was significant reduction in total dietary fibre (47.8 to 33.8%) , insoluble dietary
fibre, and soluble dietary fibre contents upon germination, which could be attributed
to enzymatic breakdown of galactomannan for their utilization by growing sprouts.
 DEBITTERING OF FENUGREEK SEEDS
 Soaking of the seeds in sucrose solutions above 5% (wt/vol) reduced the bitterness but
significantly affected the sprouting.
 Soaking in 1:1 dilution of curd with water was least bitter and hence was used for further
experiments.
 Although the exact mechanism of masking the bitterness is not known, we hypothesize
that saponins in the fenugreek seed may have formed a complex with one of the
curd constituent.

DPPH ANTIRADICAL SCAVENGING ACTIVITY

 Antiradical scavenging capacity was maximum for the extract from GFSF (7.85 ± 0.01
mg AAE/g) when compared to NGFSF (4.49 mg AAE/g).
 This is because of the increased total polyphenolic, flavonoid, and sapogenin contents in
germinated seeds
 The antiradical scavenging activity in DGFSF—curd was highest at 7.83 ± 0.07 mg
AAE/g
 The antioxidant activities depend on polyphenolic and flavonoid content of fenugreeks
seeds
 Owing to their high total polyphenolic, flavonoids, and DPPH antiradical activity, DGFSF
was used for further studies.
Inhibition assay of α-glucosidase activity
 INHIBITION ASSAY OF α-GLUCOSIDASE ACTIVITY

 Inhibitors of α-glucosidase delay the breaking down of carbohydrate in the small

intestine and reduce the postprandial blood glucose level.
 Germinated seeds had higher potential to inhibit a-glucosidase enzyme in a dose
dependent manner
 Among the extracts, DGFSE–curd had higher inhibition activity (173.16 mg/mL)

INHIBITION ASSAY OF PANCREATIC LIPASE ACTIVITY

 GFSF had high pancreatic lipase inhibitory activity compared to NGFSE which could
be attributed to increase in sapogenins and flavonoids content.
 IC50 value of orlistat was 16.4 µg/mL, which was significantly lower than GFSE (1.72
mg/mL)
 However this is due to the synergetic action of phytochemicals in GFSE and is safer
than orlistat.

INHIBITION ASSAY OF LIPOXYGENASE (LOX) ACTIVITY

 Inhibition of lipoxygenase (LOX) activities showed GFSE to have high anti-

inflammatory potential compared to NGFSE at all doses tested.
 Alkaloid, flavonoid and saponins fraction of fenugreek seeds were responsible for anti-
inflammatory activity.
 INHIBITION ASSAY OF
PANCREATIC LIPASE ACTIVITY

INHIBITION ASSAY OF LOX

ACTIVITY
 IN VIVO EXPERIMENTS IN RATS
ORAL GLUCOSE TOLERANCE TEST

 The extract from GFSF ( because of increase in hydroxyisoleucine) showed

significantly better ability than that from NGFSF at 50 mg/kg, demonstrating the
CEILINGpotential than non-germinated seeds at
germinated seeds to have higher anti-diabetic
low dosage. EFFECT
 At 100 mg/kg dosage, there were no significant (p < .05) differences in AUC values of
blood glucose in both NGFSF and GFSF.

ACUTE ANTITRIGLYCERIDEMIA
 Administration of fenugreek seed extracts significantly decreased the incremental plasma
triglyceride levels at 2 and 4 hr (p < .05).
 Triglyceride levels decreased significantly with increase dose of extracts and the AUC for
triglycerides were significantly lower in GFSE than in the control group.

Since postprandial hypertriglyceridemia and hyperglycemia independently induce

endothelial dysfunction through oxidative stress, intake of debittered and
germinated fenugreek seed flour (DGFSF) can alleviate the effects of both
hypertriglyceridemia and hyperglycemia.
ORAL GLUCOSE TOLERANCE TEST

ACUTE ANTITRIGLYCERIDEMIA
 PROXIMATE ANALYSIS OF BREAD

 The protein content in wheat bread increased significantly (p < .05) with the amount of
substitution.
 The bread fortified with DGFSF at 10% substitution had significantly higher protein
content.
 Fat content of wheat bread significantly increased on the substitution of seed flour.
 Moisture content increased on fortification with fenugreek flours which can be attributed
to the high water binding capacity of seed flour.
 However breads fortified with GFSF and DGFSF flours had lower dietary fibre content
which can be attributed to the breakdown of fibres during germination process.
 Substitution with 5 and 10% of all the fenugreek seed flours had a significant (p < .05)
effect on all the sensory parameters.
 Taste score and texture score decreased on increasing the level of substitution with
NGFSF flour which might be due to its bitter flavor.
 The crumb colour decreased with the level of substitution which was attributed to release of
water soluble pigments into the soaking medium and or due to Maillard reaction between
sugars and protein.
 The overall acceptability of fortified breads : control > GFSF (10%) or DGFSF (10%) >
NGFSF (5%).
 IN VITRO STARCH DIGESTIBILITY

 Maltose released after hydrolysis was calculated using maltose standard curve
 Rate of hydrolysis increased rapidly in wheat bread during the first 60 min of reaction
than in the fenugreek fortified breads.
 Hydrolysis of starch progressed slowly and reached a plateau in 120 min.
 Area under the curve was also reduced significantly on fortification with seed flour
compared to wheat bread.
 GI of wheat bread (94.61) > 5% NGFSF bread (75.02) > 10% GFSF bread (65.4)
> 10% DGFSF bread (63.3)
 Fenugreek seed flour fortified bread contain water-soluble galactomannan which coats
over the surface of starch granules and act as a barrier for the enzyme attack and the
subsequent release of hydrolyzed oligosaccharides.
 Hence, fortification of wheat flour with 10% DGFSF is recommended for positive health
effects on diabetic population all over the world.
 CONCLUSION
 Germination of fenugreek seed
increased its antihyperglycemic and
anti hyper triglyceridemic potential
 Fortification of wheat flour with
debittered and germinated fenugreek
seed at 10% (wt/wt) was
organoleptically acceptable, had
enhanced protein, fibre, total
polyphenol and flavonoid contents
than the control wheat bread, and
lower glycemic index.
 RATIONALE FOR CHOICE OF ARTICLE

1. It is quite a challenge to develop organoleptically acceptable value added products with

fenugreek seeds
2. Product development is not an isolated entity and this article establishes it.
3. In vitro inhibition assays are the initial stage of any functional product development to
be used for metabolic syndrome
4. Fenugreek is one among the most commonly used spice/herb/green vegetable in India
but also the most under-rated ingredient in product development. There are only a
handful of packaged food products that use fenugreek seeds. And in Indian market
there is no packaged food that uses sprouted fenugreek in the composition.
5. Fenugreek is less expensive, easily available and has numerous health benefits
6. Traditional foods made from fenugreek seeds are not being passed on, that creates a
avoid which needs to be filled.

FUTURE RECOMMENDATIONS

1. Diversification of value added products made from fenugreek is a need of the hour.
2. Any product development is successful only if the product reaches the market, satisfies
consumer and earns reasonable profit.
3. Also the studies which compare the galactogogue properties of germinated and non
germinated fenugreek needs will add to the knowledge of lactation enchancement.
 “Doubts are good. Confusion is
excellent. Questions are awesome.
All these are attempts to expand the
wisdom of mind.”
Any questions ?