Food Analytical Methods

https://doi.org/10.1007/s12161-020-01831-2

Rancidity Matrix: Development of Biochemical Indicators

for Analysing the Keeping Quality of Pearl Millet Flour
Suneha Goswami 1 & Purva Asrani 1 & T. P. Ansheef Ali 1 & R. Dinesh Kumar 1 & T. Vinutha 1 & K. Veda 1 & Sweta Kumari 1 &
Archana Sachdev 1 & Sumer P. Singh 2 & C. Tara Satyavathi 3 & Ranjeet R. Kumar 1 & Shelly Praveen 1

Received: 6 March 2020 / Accepted: 29 July 2020

# Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Millets are known for its nutritional excellence because of its high nutrient density. Among all the millets, pearl millet is
considered the most important nutricereals, having 3–8% lipids. Rancidity or off-odour development in flour is one of the major
limiting factors in the popularization of pearl millet all over the world. In the present investigation, we have evaluated the flour
(freshly milled and 10 days after milling) of diverse genotypes of pearl millet for their hydrolytic and oxidative degradation of
lipids using different biochemical parameters such as comprehensive acid value (CAV: the number of milligrammes of potassium
hydroxide required to neutralize the free fatty acids present in 1 g of flour), comprehensive peroxide value (CPV: determination
of the primary oxidation product i.e. hydroperoxides in the flour) and activities of lipase and lipoxygenase (LOX). We have
classified pearl millet genotypes into low, medium and high rancid groups and developed a rancidity matrix (RM) with 3 groups
and 6 classes; the matrix was validated using 93 diverse pearl millet genotypes. RM will be of great help to the pearl millet
breeders for developing low rancid pearl millet lines.

Keywords Storage quality . Lipase . LOX . Comprehensive acid value . Comprehensive peroxide value . Rancidity matrix

Abbreviations Introduction
CAV Comprehensive acid value
CPV Comprehensive peroxide value Pearl millet (Pennisetum glaucum) accounts for almost half of
LOX Lipoxygenase global millet production. It is the sixth most important cereal
PM Pearl millet crop in the world, next to maize (Zea mays L.), rice (Oryza
RM Rancidity matrix sativa L.), wheat (Triticum aestivum L.), barley (Hordeum

* Suneha Goswami Archana Sachdev

suneha08@gmail.com arcs_bio@yahoo.com
* Shelly Praveen
shellypraveen@hotmail.com Sumer P. Singh
sumerpalsingh@yahoo.com
Purva Asrani
purvaasrani@gmail.com C. Tara Satyavathi
csatyavathi@gmail.com
T. P. Ansheef Ali
anshifali47@gmail.com Ranjeet R. Kumar
R. Dinesh Kumar ranjeetranjaniari@gmail.com
dineshkarthick575@gmail.com
1
T. Vinutha Division of Biochemistry, ICAR-Indian Agricultural Research
vinuthabiochem@gmail.com Institute, New Delhi, India
2
K. Veda Division of Genetics, ICAR-Indian Agricultural Research Institute,
vedabiochem@gmail.com New Delhi, India
3
Sweta Kumari All India Coordinated Research on Pearl Millet, Jodhpur 342304,
sweta381@gmail.com India

vulgare L.) and sorghum (Sorghum bicolor L.) (FAOSTAT
2014). Global warming is the real threat to the agricultural
system, as evident from the decrease in the production of
important crops like cereals, legumes and horticultural crops
(Kumar et al. 2019). Pearl millet is a boon crop having the
inherent capacity to grow under adverse environmental con-
ditions without compromising with the quality and quantity of
the grains (Singh et al. 2017). The nutritional composition of
the grains provides a balanced nutritional diet to the down-
trodden burgeoning population. Pearl millet is well adapted to
cropping systems grown under unfavourable climatic condi-
tions such as low rainfall (200–600 mm), low soil fertility and
high temperature, and thus can be grown as substitute crops
for the staple crops such as wheat, rice and maize (Shivhare
and Lata 2017). In India, wheat and rice are the major cereals
consumed in the routine diet. Substituting the wheat-rice
cropping system with millets and providing the same in the
diet will help in combating the various health-related issues
such as hidden hunger, malnutrition, micronutrient deficiency
(especially iron, zinc and calcium), obesity, celiac disease and
diabetes (Chandrasekara and Shahidi 2012). According to the
latest data available on National Family and Health Survey
(2015–2016) by the Indian Government, in rural India, about
27% women and 23% men are malnourished (Verma and
Kumar 2019). Comprehensive National Nutrition Survey
(2016–2018) data showed 34.7% of children under 5 years
of age are still low height for age (stunted growth) and
33.4% are low weight for age (underweight) (Kumar and
Kumar 2020). Among millets, pearl millet is one of the best
options, having unlimited health benefits and is considered the
powerhouse of nutrition (Nambiar et al. 2011) and can be a
solution for nutritional security. Nature has provided huge
diversity in pearl millet germplasm based on the colour and
nutritional composition; it varies from ivory, cream, yellow,
grey, deep grey, greyish brown, purple and purplish-black
(Yadav et al. 2012).
Nutritionally, pearl millet is a good source of energy, qual-
ity protein (9–13%; with more balanced amino acid profile),
vitamins, minerals (such as iron, zinc, calcium, magnesium,
phosphorous), crude fibres (including resistant starch, soluble
and insoluble dietary fibres), antioxidants and fat (3–8%) with
better fat digestibility than other cereals (Uppal et al. 2015). It
contains up to 74% polyunsaturated fatty acids (PUFA), out of
total fat content with higher nutritionally important omega-3
fatty acids such as oleic acid (25%), linoleic acid (45%) and
linolenic acid (4%), incredibly important and vital for optimal
health especially for the brain (Rooney 1978; Nantanga 2006;
Singh et al. 2018; Dyall 2015). Due to the nutritional superi-
ority and climate-resilient nature of pearl millet over other
crops, it has given the tag of “nutricereals” by the Ministry
of Agriculture and Farmers Welfare, Government of India.
Despite nutritional superiority, the utilization of pearl millet
flour is limited to few specific pockets and regions all around the
Food Anal. Methods
world due to the poor keeping quality of the flour and develop-
ment of off-odour during storage (Rani et al. 2018). The poor
keeping quality of pearl millet flour is due to the oxidative/
hydrolytic rancidity caused by enzymes like lipase and
lipoxygenase (LOX). Lipase catalyses the hydrolysis of triacyl-
glycerol (TAG) to diacylglycerols, mono glycerol, glycerol and
free fatty acids (Manley and Mayer 2012). Lipase belongs to the
class of serine hydrolases. It has an affinity for short-chain fatty
acids, unsaturated fatty acids (oleic, linoleic, linolenic, etc.). In
pearl millet, the lipase enzyme is present in the pericarp and
aleuronic layer of the grain.
Lipoxygenase is a non-heme, iron-containing dioxygenase
that catalyses the oxygenation of polyunsaturated fatty acids con-
taining a cis, cis-1,4-penta diene moiety to form conjugated diene
hydroperoxide (Anthon and Barrett 2001). The substrates for the
enzymes are 18 carbon fatty acids, linoleic (18:2) and linolenic
acids (18:3). The action of lipoxygenase generates very reactive
compounds like free radicals, which can react with chlorophylls,
carotenoids, ascorbic acid and phenols and cause alteration of
organoleptic properties and the colour. Pearl millet consists of
an abundance of linoleic, linolenic and oleic acids, which acts as
substrate for lipoxygenase (LOX) enzyme. The primary oxida-
tion product of free fatty acids i.e. hydroperoxides leads to the
production of secondary oxidation products such as aldehydes
and ketones, which is responsible for the production of off-
flavour and off-odour to the pearl millet flour. Due to the devel-
opment of off-flavour in the flour within 10 days after milling, it
delimits the nutritional potential of pearl millet. Although differ-
ent organizations are working in this direction to develop a
variety/hybrid having enhanced keeping quality of flour, the lim-
ited success is achieved. The biggest problem in pearl millet
breeding and processing is the non-availability of desirable mo-
lecular and biochemical markers or matrix indicating their possi-
ble rancid behaviour.
In the present investigation, we have identified and evalu-
ated key biochemical parameters associated with the rancidity
in the flour of 93 diverse lines of pearl millet collected from
different agro-climatic zones of India. Based on the data, a
rancidity matrix (RM) has been developed to be used by the
breeders and researchers for direct analysis of germplasm for
its flour keeping quality.
Experimental
Reagents and Methods
Collection of Pearl Millet Genotypes and Standardization
of Crucial Stage of Pearl Millet Flour for Rancidity Measuring
Tests
Mature harvested grains of 93 pearl millet genotypes procured
from different climatic zones of India were used in the present
Food Anal. Methods
investigation. The grains (100 g) were milled to get fine flour
using a mini-miller machine. The fine flour was sieved with
0.2-mm sieve, to get uniform flour particle size and it was
stored in airtight plastic zip bags for 1 month under dark con-
dition. A pilot experiment was performed to standardize the
critical stage for rancidity measuring tests. In the pilot exper-
iment, flour stored for 1 month and CPV were estimated at 5-
day intervals for 30 days. Further for rancidity evaluation in
diverse genotypes of pearl millet, we made two groups of
flour. One group of flours was used as freshly milled (group
I) for the estimation of comprehensive acid value (CAV),
comprehensive peroxide value (CPV) and the activities of
rancidity causing enzymes like lipase and lipoxygenase. The
other group of flours was stored for 10 days (group II) at room
temperature in airtight zip bags under dark condition, estimat-
ing the biochemical parameters (as mentioned above) for the
development of rancidity matrix (RM). Hence, we studied all
the rancidity causing parameters in freshly milled flour and
10-day stored flour to develop a rancidity matrix.
Analysis of Biochemical Parameters
Estimation of Comprehensive Acid Value
Since in the flour, lipids are present in matrix with other bio-
molecules such as carbohydrate or protein, and hence we are
using the term comprehensive acid value (CAV) in place of
acid value. The comprehensive acid value (CAV) is defined as
the number of milligrammes of potassium hydroxide required
to neutralize the free fatty acids present in 1 g of flour. The
comprehensive acid value in the flour was estimated using the
standard methods of acid value estimation of the Association
of Official Analytical Chemists (AOAC 1990) with little mod-
ification. In brief, 1 g of flour taken in a 25-mL conical flask
and 10 mL of freshly neutralized hot ethyl alcohol was added
and kept on a hot plate until the sample started to boil. Then,
100 μL of phenolphthalein indicator solution was added. The
mixture was titrated against the alkali solution of 0.1 N NaOH.
The CAV (mg NaOH g−1flour) was calculated using the for-
mula:
CAV ¼ 40  A  N =W
where CAV is the comprehensive acid value, A denotes the
volume of NaOH (mL), 40 is the constant value equivalence
of mass of 0.1 N NaOH, W is the weight of the sample and N is
the normality of the standard NaOH solution.
Estimation of Comprehensive Peroxide Value
Similar to the CAV, in place of peroxide value, the term com-
prehensive peroxide value was used. Peroxide value is the
term commonly used for oil/fat samples. The comprehensive
peroxide value (CPV) determines the concentration of
hydroperoxide—the primary oxidation product in the flour
and is an indication of the extent of oxidation suffered by
the flour. The CPV was estimated as per the method
mentioned in AOCS (1994) with some modifications. In brief,
1 g of flour sample was taken in a 25-mL glass conical flask,
and further 6 mL of acetic acid-chloroform solution (3:2 ratio)
was added. Flask content was mixed vigorously and kept at
room temperature for 5 min. Freshly prepared saturated potas-
sium iodide (KI) solution (100 μL) was added into it. Flask
content was vortexed and kept at room temperature for
10 min. Six millilitres of distilled water was added and
shacked vigorously. Then, 200 μL of freshly prepared 1%
starch solution was added. The solution was turned blue-
black colour. The mixture was titrated against 0.1 N sodium
thiosulphate until the blue-black colour disappears. The same
preparation without a flour sample served as a blank experi-
ment. The CPV (milli equivalent peroxide kg−1 of the flour)
was calculated using the formula:
CPV ¼ ðA−BÞ  N  1000=W
where CPV is the comprehensive peroxide value, A and B
denote the titration volume of Na2S2O3 (mL) in the sample
and blank, respectively, W is the weight of flour in gramme
and N is the normality of the standard Na2S2O3 solution.
Activity Assay of Rancidity Causing Enzymes
Preparation of Enzyme Extract
A pilot experiment was conducted to optimize the extraction
conditions concerning pH, molarity and type of buffer. One
gramme of flour was homogenized in a pre-chilled pestle and
mortar in 10 mL of 0.2 M potassium phosphate buffer
(pH 7.5). The homogenate was centrifuged at 10,000 rpm
for 20 min in a refrigerated centrifuge. The supernatant was
carefully filtered through four layers of cheesecloth and used
as an enzyme extract for activity assay. The extract was dilut-
ed to a suitable extent to obtain the linear response of each
enzyme for time and concentration of respective substrates.
The total soluble protein of extract was estimated using the
method of Bradford (1976).
Activity Assay of Lipase
Lipase activity was assayed as per the method of Itaya and Ui
(1966) with some modifications. To 10 μL of enzyme extract,
1 mL of the substrate (0.98% (w/v) NaCl, 200 μL Tween 20,
mix olive oil and water in 1:3 ratio) was added and kept at
37 °C for 15 min, afterward, placed it in the water bath (90 °C)
for 5 min to stop the reaction. Further, 5 mL chloroform was
added to the reaction mixture. Thereafter, in the bottom layer,

2 mL copper triethanolamine reagent (1 M triethanolamine,
1 N acetic acid, 6.45% (w/v) copper nitrate) was mixed prop-
erly. Two colour layers, upper blue and lower yellow colour,
were formed. Later, in the lower part, 100 μL of 11 mM
diethyldithiocarbamate (DDC) was added; absorbance of the
yellow colour layer was recorded at 440 nm. The standard
curve was prepared using oleic acid and enzyme activity
was expressed as mMol free fatty acids min−1 mg−1 soluble
protein.
Activity Assay of Lipoxygenase
LOX activity assay was carried out spectrophotometrically at
234 nm. Linoleic acid was used as a substrate for determining
LOX activity. The substrate was prepared by the method of
Surrey (1964). In brief, the substrate was prepared as Tween
20, 0.5 mL was dissolved in 10 mL of borate buffer of pH 9.0
and 0.5 mL of linoleic acid was added drop by drop. The
contents were thoroughly mixed so as to disperse the acid into
a fine emulsion. Then, 1.3 mL of 1 N NaOH was added and
the mixture once again agitated until a clear transparent solu-
tion was obtained. To this solution, 90 mL of the borate buffer
was added, and the final volume made up to 200 mL with
water. The resulting solution was approximately 7.5 ×
10−3 M (7.5 mM) in linoleic acid.
The reaction mixture in a final volume of 2.66 mL was
prepared using 2.5 mL of 0.05 M acetate buffer (pH 4.2),
60 μL of 7.5 mM linoleic acid substrate solution and 25 μL
of enzyme extract, and the progress of the reaction was mon-
itored by recording increase in absorbance at 234 nm for 60 s
against the blank. The LOX activity was calculated as
nanomole hydroperoxide/min/mg soluble protein (nM
HPOD min−1 mg−1 soluble protein), using the molar extinc-
tion coefficient of linoleic acid (25 mM−1 cm−1).
Statistical Analysis
The biochemical analysis was carried out using three repli-
cates. The analyses of variance of different parameters were
calculated using the one-way ANOVA for statistically signif-
icant differences (P ≤ 0.05). The mean sum of squares
(ANOVA) due to genotypes was significant for all the param-
eters studied. This indicates the prevalence of enough genetic
variability in the genotypes.
Results and Discussions
In this study, a rancidity matrix has been developed, which
will help in the estimation of rancidity probability in any line
of pearl millet and for the selection of low rancid lines for
further variety development. Through pilot experiment, CPV
was estimated in 10 pearl millet genotypes at 5-day interval
Food Anal. Methods
for 30 days. It was observed that the maximum CPV reaches
on the 10th day and thereafter its value gradually decreases
(Fig. 1). Hence, it was concluded that the 10th day is a crucial
stage for measuring rancidity status in pearl millet flour. Datta
Mazumdar et al. (2016) reported that the peroxide value in
pearl millet fat increases up to the 10th day after milling.
Pearl millet fat oxidation has major contribution in rancidity
development. Varsha and Narayanan (2017) had also reported
initially increase and thereafter decrease in peroxide value in
pearl millet flour.
Lipase Activity and Comprehensive Acid Value
Correlation
In the present investigation, we have developed an assay to
estimate the acid value directly in the flour, rather than frac-
tionating the flour and extracting the lipids to be used for the
measurement and given the term comprehensive acid value
(CAV). Lipase enzyme, which is responsible for hydrolysing
fat/lipids into free fatty acids and contributes towards compre-
hensive acid value (CAV), gives an estimation of free fatty
acids liberated in the flour (Fig. 2a). Hence, both lipase activ-
ity and CAV showed a direct correlation with each other. Here
we compared the lipase activity with CAV in 93 diverse ge-
notypes of pearl millet grown in different zones of India
(Table 1). On comparing CAV with lipase activity in group
I (freshly milled flour), we observed a parallel increase in
CAV and lipase activity. In group I, we observed CAV value
(CAV0) ranges from 1.2 to 2 mg NaOH g−1flour for low
CAV0 type, having lipase activity vary from 17 to 20 mMol
free fatty acids min−1 mg−1 soluble protein. Ten genotypes
were identified and categorized having low CAV0. Thirty-
two genotypes belong to medium CAV0, ranges from 2.1 to
4.5 mg NaOH g−1 flour and lipase activity varies from 21 to
34 mMol free fatty acids min−1 mg−1 soluble protein, and the
remaining fifty-one genotypes having CAV0 more than
4.5 mg NaOH g −1 flour and lipase activity more than
37 mMol free fatty acids min−1mg−1soluble protein were cat-
egorized having high CAV0 (Fig. 2b).
Further, we have estimated both lipase activity and CAV in
group II (10-day stored flour: CAV10), and it was found to be
increased on the 10th day (Table 1). Genotypes having CAV10
ranges from 1.5 to 2.8 mg NaOH g−1flour and lipase activity
varies from 20 to 23 mMol free fatty acids min−1 mg−1 soluble
protein were considered under low CAV10. Fifteen genotypes
were categorized in low CAV10 type. Twenty-five genotypes
having CAV10 2.9–6 mg NaOH g−1flour and lipase activity
24–45 mMol free fatty acids min−1 mg−1 soluble protein were
categorized as medium CAV10 and the remaining fifty-three
genotypes having CAV10 more than 6 mg NaOH g−1 flour and
lipase activity, more than 45 mMol free fatty acids
min−1mg−1soluble protein, were placed in high CAV10 cate-
gory (Fig. 2c). Lipase activity is confined to the aleurone layer
Food Anal. Methods

Fig. 1 CPV was estimated at 5-day interval for 30 days in 10 genotypes of pearl millet and the 10th day was optimized as critical time for CPV estimation
(CPV reaches to its maximum value) to measure rancidity status in pearl millet flour

Fig. 2 a Correlation between lipase activity and comprehensive acid value (CAV). b Correlating the lipase activity and CAV of 93 pearl millet genotypes
in group I (freshly milled flour). c Correlating the lipase activity and CAV of 93 pearl millet genotypes in group II (10-day stored flour)
 Food Anal. Methods

Table 1 Comparison of comprehensive acid value (CAV) and lipase enzyme activity of 93 pearl millet genotypes in fresh milled flour and 10-day
stored flour

S No. Pearl millet verities CAV0 (mg CAV10 (mg Lipase0 (mMol free fatty acids/min/mg Lipase10 (mMol free fatty acids/min/mg
NaOH/g flour) NaOH/g flour) soluble protein) soluble protein)

1. 86M88 1.20 ± 0.2 2.00 ± 0.5 17.99 ± 0.4 20.11 ± 0.2

2. 86M38 1.20 ± 0.3 2.40 ± 0.4 17.78 ± 0.4 21.23 ± 0.2
3. Nandi 72 1.60 ± 0.1 2.00 ± 0.2 18.72 ± 0.3 20.98 ± 0.2
4. HHB 234 1.60 ± 0.1 2.40 ± 0.2 18.78 ± 0.1 21.33 ± 0.2
5. NBH 4903 1.60 ± 0.3 3.20 ± 0.1 17.72 ± 0.4 25.74 ± 0.2
6. Nandi 70 1.80 ± 0.2 2.40 ± 0.3 20.78 ± 0.2 21.31 ± 0.4
7. HHB 67 2.00 ± 0.1 2.40 ± 0.3 20.92 ± 0.2 20.97 ± 0.3
(improved)
8. HHB 226 2.00 ± 0.2 2.80 ± 0.2 20.98 ± 0.2 24.10 ± 0.3
9. MP 7792 2.00 ± 0.2 2.80 ± 0.3 20.11 ± 0.5 22.87 ± 0.4
10. GHB 732 2.00 ± 0.2 2.80 ± 0.3 20.71 ± 0.2 23.98 ± 0.3
11. NBH 5767 2.20 ± 0.2 2.40 ± 0.3 21.73 ± 0.2 22.98 ± 0.2
12. Nandi 75 2.20 ± 0.2 2.80 ± 0.1 21.23 ± 0.2 22.44 ± 0.2
13. 86M13 2.40 ± 0.1 3.60 ± 0.3 21.44 ± 0.5 26.91 ± 0.4
14. KBH 108 2.40 ± 0.2 2.8 ± 0.2 21.89 ± 0.3 22.76 ± 0.1
15. Pratap 2.20 ± 0.4 2.80 ± 0.3 20.16 ± 0.4 22.47 ± 0.2
16. HHB 223 2.40 ± 0.3 2.80 ± 0.4 21.78 ± 0.2 22.76 ± 0.5
17. MP 7872 2.40 ± 0.3 4.00 ± 0.3 21.32 ± 0.1 40.87 ± 0.2
18. 86M82 2.40 ± 0.4 4.40 ± 0.1 21.88 ± 0.3 43.22 ± 0.3
19 GHB 744 2.80 ± 0.1 3.60 ± 0.4 22.77 ± 0.1 26.87 ± 0.09
20. PAC 909 2.80 ± 0.1 5.20 ± 0.1 22.65 ± 0.4 41.33 ± 0.3
21. XMT 1497 2.40 ± 0.5 6.92 ± 0.2 21.89 ± 0.4 62.56 ± 0.4
22. PHB 2168 2.60 ± 0.4 2.60 ± 0.3 21.76 ± 0.2 21.34 ± 0.1
23. GHB 558 2.80 ± 0.2 2.90 ± 0.3 22.98 ± 0.3 24.89 ± 0.5
24. 86M89 2.80 ± 0.2 4.80 ± 0.1 22.77 ± 0.02 37.19 ± 0.5
25. 86M86 2.80 ± 0.2 6.62 ± 0.3 22.77 ± 0.1 48.87 ± 0.2
26. 86M64 3.20 ± 0.2 6.80 ± 0.1 27.77 ± 0.2 57.71 ± 0.3
27. GHB 905 3.20 ± 0.4 5.40 ± 0.2 27.87 ± 0.3 40.44 ± 0.2
28. 86M84 3.60 ± 0.1 6.80 ± 0.5 29.87 ± 0.1 57.99 ± 0.2
29. GHB 757 3.60 ± 0.1 7.20 ± 0.3 29.11 ± 0.4 64.22 ± 0.3
30. RHB 223 3.60 ± 0.2 5.90 ± 0.2 30.44 ± 0.1 26.96 ± 0.2
31. MP 7171 3.60 ± 0.2 6.40 ± 0.3 29.13 ± 0.2 55.12 ± 0.5
32. Dhodhsar local 3.60 ± 0.3 5.30 ± 0.1 29.72 ± 0.03 42.67 ± 0.01
33. GHB 538 3.60 ± 0.4 4.90 ± 0.1 29.87 ± 0.1 40.75 ± 0.2
34. Nandi 65 3.60 ± 0.4 4.80 ± 0.3 29.66 ± 0.5 36.54 ± 0.3
35. Anmol 3.86 ± 0.2 6.04 ± 0.5 29.78 ± 0.3 48.16 ± 0.2
36. HHB 299 4.04 ± 0.1 6.40 ± 0.2 30.97 ± 0.3 50.32 ± 0.1
37. RHB 173 3.80 ± 0.4 6.12 ± 0.4 29.32 ± 0.4 50.45 ± 0.3
38. AHB 1200 4.00 ± 0.3 4.80 ± 0.1 30.98 ± 0.01 35.22 ± 0.4
39. Nandi 61 4.32 ± 0.1 6.40 ± 0.2 34.74 ± 0.2 51.76 ± 0.2
40. HHB 50 4.04 ± 0.4 6.00 ± 0.2 30.50 ± 0.4 45.66 ± 0.4
41. HHB 197 4.28 ± 0.2 4.92 ± 0.3 31.03 ± 0.3 43.88 ± 0.4
42. Nandi 81 4.32 ± 0.2 5.00 ± 0.1 34.25 ± 0.5 55.13 ± 0.2
43. Eknath 301 gold 4.52 ± 0.3 5.40 ± 0.3 37.61 ± 0.3 41.60 ± 0.04
44. ICMH 356 4.56 ± 0.3 5.64 ± 0.1 36.86 ± 0.4 44.22 ± 0.5
45. Pusa 23 4.68 ± 0.2 5.50 ± 0.1 37.65 ± 0.2 44.99 ± 0.2
46. RHB 177 4.56 ± 0.4 5.60 ± 0.2 37.73 ± 0.4 44.43 ± 0.5
Food Anal. Methods

Table 1 (continued)

S No. Pearl millet verities CAV0 (mg CAV10 (mg Lipase0 (mMol free fatty acids/min/mg Lipase10 (mMol free fatty acids/min/mg
NaOH/g flour) NaOH/g flour) soluble protein) soluble protein)

47. 9001 4.90 ± 0.1 5.62 ± 0.1 37.17 ± 0.2 40.77 ± 0.5
48. RHB 233 4.72 ± 0.3 6.12 ± 0.2 36.46 ± 0.3 49.15 ± .5
49. AHB 1269 4.88 ± 0.2 7.16 ± 0.3 36.18 ± 0.1 64.03 ± 0.1
50. HHB 67 4.86 ± 0.3 6.92 ± 0.5 36.63 ± 0.4 61.71 ± 0.3
51. RHB 234 5.08 ± 0.1 6.14 ± 0.4 42.79 ± 0.2 49.91 ± 0.02
52. SulkhaniyaBajra 5.12 ± 0.1 6.90 ± 0.4 42.63 ± 0.5 62.16 ± 0.4
53. 7686 4.84 ± 0.4 6.22 ± 0.1 36.93 ± 0.5 47.47 ± 0.3
54. HHB 146 4.96 ± 0.3 6.88 ± 0.2 37.70 ± 0.1 57.04 ± 0.5
55. KBH 3580 5.28 ± 0.1 6.76 ± 0.3 41.71 ± 0.1 54.98 ± 0.3
56. DamodaraBajri 5.12 ± 0.3 6.08 ± 0.2 42.35 ± 0.2 48.57 ± 0.2
57. Nandi 52 5.26 ± 0.2 6.20 ± 0.3 41.95 ± 0.3 63.79 ± 0.5
58. JKBH 1486 5.44 ± 0.1 5.68 ± 0.2 45.94 ± 0.4 44.62 ± 0.5
59. ICMH 423 5.16 ± 0.4 5.88 ± 0.3 42.18 ± 0.4 45.78 ± 0.3
60. Kaveri super 5.48 ± 0.1 6.10 ± 0.3 43.31 ± 0.1 46.59 ± 0.4
boss
61. Pusa purple 1 5.20 ± 0.4 8.90 ± 0.5 41.39 ± 0.4 76.77 ± 0.5
62. MP 7878 5.42 ± 0.2 7.64 ± 0.1 43.23 ± 0.1 91.58 ± 0.5
63. Pusa 605 5.34 ± 0.3 5.96 ± 0.2 42.36 ± 0.03 45.82 ± 0.04
64. RHB 58 5.48 ± 0.2 6.16 ± 0.3 43.12 ± 0.4 40.20 ± 0.2
65. MBH 2210 5.36 ± 0.4 6.58 ± 0.3 42.70 ± 0.1 52.70 ± 0.5
66. MH 179 5.46 ± 0.3 7.54 ± 0.2 43.58 ± 0.3 66.25 ± 0.3
67. Pusa 322 5.64 ± 0.2 6.36 ± 0.3 44.97 ± 0.01 53.30 ± 0.03
68. HB 3 5.66 ± 0.2 6.12 ± 0.3 43.22 ± 0.3 46.10 ± 0.4
69. DedhaBajri 5.84 ± 0.1 6.10 ± 0.3 41.42 ± 0.2 48.79 ± 0.2
70. Ajeet 42 5.74 ± 0.3 6.32 ± 0.4 43.00 ± 0.4 53.20 ± 0.3
71. Gadhwalki 5.64 ± 0.4 6.90 ± 0.6 44.13 ± 0.2 59.87 ± 0.1
Dhani-1
72. 1827 5.68 ± 0.4 6.06 ± 0.1 43.09 ± 0.3 48.84 ± 0.5
73. MPMH17 5.60 ± 0.5 7.20 ± 0.4 44.78 ± 0.3 85.76 ± 0.4
74. Chanana 6.00 ± 0.1 7.36 ± 0.3 47.10 ± 0.4 62.06 ± 0.5
Bajra-2
75. Gadhwalki 5.92 ± 0.2 7.20 ± 0.4 45.86 ± 0.4 58.66 ± 0.3
Dhani-3
76. Pusa composite 6.04 ± 0.1 6.96 ± 0.1 47.05 ± 0.2 57.33 ± 0.4
443
77. MBH 2232 5.68 ± 0.5 6.44 ± 0.4 42.38 ± 0.3 55.81 ± 0.2
78. RHB 121 5.90 ± 0.3 6.32 ± 0.4 44.93 ± 0.5 51.33 ± 0.5
79. MPMH 21 6.00 ± 0.2 6.80 ± 0.3 47.30 ± 0.1 75.67 ± 0.1
80. Pro agro 9444 6.00 ± 0.2 6.94 ± 0.4 47.21 ± 0.2 55.62 ± 0.4
81. 86M01 5.92 ± 0.3 7.08 ± 0.3 43.74 ± 0.3 63.22 ± 0.2
82. JKBH 26 5.96 ± 0.4 6.52 ± 0.2 43.30 ± 0.3 56.87 ± 0.5
83. HHB 311 6.00 ± 0.4 9.10 ± 0.3 47.52 ± 0.3 81.75 ± 0.2
84. Bio 70 6.50 ± 0.1 7.26 ± 0.1 53.74 ± 0.2 65.44 ± 0.3
85. Dhanshakti 6.20 ± 0.4 10.00 ± 0.3 49.00 ± 0.4 83.53 ± 0.3
86. PB 1705 6.40 ± 0.3 6.64 ± 0.5 50.85 ± 0.3 79.25 ± 0.4
87. BJ 104 6.50 ± 0.2 10.00 ± 0.4 52.46 ± 0.2 83.51 ± 0.3
88. JKBH 676 6.44 ± 0.3 7.20 ± 0.4 50.45 ± 0.5 63.47 ± 0.2
89. HTBH 4201 6.36 ± 0.4 7.44 ± 0.2 49.02 ± 0.5 63.01 ± 0.6
90. MLBH 308 6.28 ± 0.5 6.76 ± 0.3 49.32 ± 0.1 54.69 ± 0.3
91. Chadi Bajri 6.48 ± 0.3 8.10 ± 0.2 52.96 ± 0.4 74.24 ± 0.2
 Food Anal. Methods

Table 1 (continued)

S No. Pearl millet verities CAV0 (mg CAV10 (mg Lipase0 (mMol free fatty acids/min/mg Lipase10 (mMol free fatty acids/min/mg
NaOH/g flour) NaOH/g flour) soluble protein) soluble protein)

92. WGI 100 6.40 ± 0.4 9.00 ± 0.2 51.89 ± 0.4 81.68 ± 0.2
93. Super boss 6.76 ± 0.1 7.06 ± 0.2 54.30 ± 0.1 67.83 ± 0.3

and embryonic tissues, whereas lipids are localized into the
germ of the pearl millet grain. Milling of pearl millet grains
leads to tissue damage and accessibility of lipase with lipids
(Dvoracek et al. 2010). To conclude, lipid hydrolysis is the
first step towards flour quality deterioration pathway.
Genotypes having higher lipase activity lead to more produc-
tion of free fatty acid and hence more CAV and as its result the
flour becomes more susceptible for quality degradation.
Various reports state that the pearl millet lipase activity is
relatively higher than the other cereal grain lipase activity
(Galliard 1999; Rani et al. 2018) which is responsible for lipid
hydrolysis in pearl millet flour. Many investigators reported
increase in free fatty acid content in pearl millet flour during
storage for different periods (Nantanga et al. 2008; Yadav
et al. 2012; Cepkova et al. 2014; Tiwari et al. 2014; Goyal
et al. 2017), which implies an increase of CAV of pearl millet
flour during storage.
Lipoxygenase Activity and Comprehensive Peroxide
Value Correlation
Different biomolecules present in the grains of pearl
millet also affect the peroxide value of flour. Due to the above
reason, instead of peroxide value, we used the term compre-
hensive peroxide value (CPV), which gives the primary oxi-
dation estimation of the flour fatty acids. Lipoxygenase

Fig. 3 a Correlation between LOX activity and comprehensive peroxide value (CPV). b Correlating the LOX activity and CPV of 93 pearl millet
genotypes in group I (freshly milled flour). c Correlating the LOX activity and CPV of 93 pearl millet genotypes in group II (10-day stored flour)
Food Anal. Methods

Table 2 Comparison of comprehensive peroxide value (CPV) and lipoxygenase activity of 93 pearl millet genotypes in fresh milled flour and 10-day
stored flour

S Pearl millet CPV0 (meq peroxide/ CPV10 (meq peroxide/ LOX0 (nM HPOD min−1 mg−1 LOX10 (nM HPOD min−1 mg−1
No. varieties kg flour) kg flour) soluble protein) soluble protein)

1. Gadhwalki 6.50 ± 0.2 14.00 ± 0.2 39.62 ± 0.1 45.50 ± 0.2

Dhani-1
2. Kaveri super 12.00 ± 0.1 18.00 ± 0.2 44.25 ± 0.3 47.75 ± 0.1
boss
3. HHB 311 11.50 ± 0.5 19.00 ± 0.6 43.87 ± 0.2 53.62 ± 0.2
4. Chadi Bajri 10.00 ± 0.4 20.00 ± 0.2 41.87 ± 0.3 56.87 ± 0.2
5. Chanana 20.00 ± 0.1 30.00 ± 0.3 53.37 ± 0.1 66.12 ± 0.2
Bajra-2
6. Dhodhsar local 22.50 ± 0.1 30.00 ± 0.4 53.62 ± 0.3 66.50 ± 0.4
7. DamodaraBajri 6.00 ± 0.2 30.00 ± 0.5 38.12 ± 0.2 71.37 ± 0.2
8. Pusa composite 31.00 ± 0.4 35.50 ± 0.2 64.00 ± 0.1 75.87 ± 0.3
443
9. Gadhwalki 30.00 ± 0.6 40.00 ± 0.1 56.12 ± 0.2 78.12 ± 0.3
Dhani-3
10. GHB 732 16.00 ± 0.01 40.00 ± 0.4 47.87 ± 0.1 79.62 ± 0.2
11. DedhaBajri 27.00 ± 0.2 40.00 ± 0.5 59.12 ± 0.2 80.62 ± 0.3
12. RHB 121 48.50 ± 0.1 49.00 ± 0.1 75.37 ± 0.3 81.12 ± 0.3
13. HHB 67 (imp) 20.00 ± 0.3 50.00 ± 0.1 55.87 ± 0.2 82.62 ± 0.1
14. Super boss 29.50 ± 0.4 50.00 ± 0.2 59.75 ± 0.3 81.12 ± 0.2
15. 86M82 30.00 ± 0.1 50.00 ± 0.2 54.25 ± 0.2 82.75 ± 0.2
16. AHB 1200 30.00 ± 0.2 50.00 ± 0.3 57.37 ± 0.2 76.00 ± 0.3
17. KBH 108 40.00 ± 0.5 50.00 ± 0.4 66.25 ± 0.1 85.37 ± 0.2
18. GHB 558 30.00 ± 0.4 50.00 ± 0.5 60.50 ± 0.2 79.0 ± 0.3
19. HHB 146 44.00 ± 0.5 57.00 ± 0.1 69.25 ± 0.3 85.75 ± 0.2
20. Pusa purple 1 52.00 ± 0.1 58.00 ± 0.2 86.75 ± 0.2 86.87 ± 0.4
21. RHB 223 50.00 ± 0.1 58.00 ± 0.6 83.37 ± 0.2 86.62 ± 0.4
22. RHB 177 50.00 ± 0.4 60.00 ± 0.2 78.25 ± 0.2 94.62 ± 0.1
23. MH 179 27.00 ± 0.5 65.00 ± 0.5 58.12 ± 0.3 96.12 ± 0.2
24. WGI 100 65.00 ± 0.4 67.00 ± 0.3 95.62 ± 0.1 96.25 ± 0.1
25. Pusa 322 19.00 ± 0.3 75.00 ± 0.1 53.25 ± 0.5 110.00 ± 0.3
26. JKBH 26 71.00 ± 0.5 75.00 ± 0.4 123.87 ± 0.3 108.12 ± 0.4
27. HHB 50 47.50 ± 0.4 77.00 ± 0.5 73.87 ± 0.2 111.25 ± 0.3
28. HHB 226 20.00 ± 0.1 80.00 ± 0.3 52.12 ± 0.3 120.62 ± 0.3
29. MP 7792 30.00 ± 0.4 80.00 ± 0.5 60.87 ± 0.2 121.25 ± 0.3
30. ICMH 356 63.00 ± 0.1 80.50 ± 0.2 99.37 ± 0.2 128.75 ± 0.3
31. RHB 233 50.00 ± 0.5 81.00 ± 0.1 81.87 ± 0.3 136.62 ± 0.2
32. ICMH 423 63.00 ± 0.1 81.00 ± 0.1 94.50 ± 0.3 134.37 ± 0.4
33. SulkhaniyaBajra 19.00 ± 0.1 82.00 ± 0.5 52.87 ± 0.3 142.00 ± 0.1
34. Nandi 52 21.00 ± 0.2 82.50 ± 0.1 54.37 ± 0.2 154.00 ± 0.3
35. Pusa 605 21.00 ± 0.3 82.50 ± 0.2 55.53 ± 0.1 154.50 ± 0.2
36. MBH 2210 67.00 ± 0.6 82.50 ± 0.5 106.87 ± 0.3 147.87 ± 0.1
37. HB 3 81.00 ± 0.4 88.00 ± 0.3 137.12 ± 0.2 153.75 ± 0.2
38. MBH 2232 61.00 ± 0.3 89.00 ± 0.5 91.12 ± 0.3 159.25 ± 0.1
39. 86M88 10.00 ± 0.4 90.00 ± 0.1 44.12 ± 0.2 155.50 ± 0.3
40. HTBH 4201 69.00 ± 0.1 90.00 ± 0.1 111.75 ± 0.4 157.37 ± 0.2
41. GHB 757 50.00 ± 0.5 90.00 ± 0.2 85.50 ± 0.5 155.12 ± 0.3
42. Dhanshakti 70.00 ± 0.1 90.00 ± 0.2 114.00 ± 0.2 156.62 ± 0.4
43. Nandi 72 20.00 ± 0.2 90.00 ± 0.3 60.53 ± 0.4 155.75 ± 0.5
44. NBH 5767 20.00 ± 0.4 90.00 ± 0.3 60.12 ± 0.6 155.50 ± 0.5
 Food Anal. Methods

Table 2 (continued)

S Pearl millet CPV0 (meq peroxide/ CPV10 (meq peroxide/ LOX0 (nM HPOD min−1 mg−1 LOX10 (nM HPOD min−1 mg−1
No. varieties kg flour) kg flour) soluble protein) soluble protein)

45. Nandi 70 30.00 ± 0.6 90.00 ± 0.3 61.5 ± 0.2 157.12 ± 0.4
46. HHB 299 50.00 ± 0.4 90.00 ± 0.3 79.00 ± 0.2 157.00 ± 0.1
47. HHB 223 70.00 ± 0.3 90.00 ± 0.3 115.50 ± 0.3 156.12 ± 0.4
48. Pratap 40.00 ± 0.2 90.00 ± 0.4 65.87 ± 0.1 154.50 ± 0.2
49. PB 1705 16.50 ± 0.3 90.00 ± 0.6 47.92 ± 0.3 152.87 ± 0.2
50. RHB 234 71.00 ± 0.3 91.50 ± 0.5 120.00 ± 0.1 159.62 ± 0.2
51. 86M01 76.00 ± 0.1 92.50 ± 0.1 116.87 ± 0.3 162.25 ± 0.2
52. Ajeet 42 29.00 ± 0.2 94.00 ± 0.1 61.58 ± 0.3 174.75 ± 0.3
53. Nandi 81 78.00 ± 0.2 95.50 ± 0.3 121.87 ± 0.1 169.87 ± 0.2
54. AHB 1269 29.00 ± 0.2 96.50 ± 0.1 60.25 ± 0.1 166.25 ± 0.1
55. Bio 70 25.50 ± 0.3 96.50 ± 0.2 57.37 ± 0.3 164.87 ± 0.5
56. RHB 58 85.00 ± 0.4 96.50 ± 0.2 136.62 ± 0.4 171.75 ± 0.4
57. 7686 88.00 ± 0.3 96.50 ± 0.2 147.87 ± 0.2 165.37 ± 0.1
58. JKBH 1486 24.50 ± 0.4 97.00 ± 0.3 56.75 ± 0.3 172.62 ± 0.2
59. Pro agro 9444 82.00 ± 0.2 97.00 ± 0.3 136.87 ± 0.1 174.25 ± 0.3
60. HHB 197 78.00 ± 0.1 98.00 ± 0.5 121.25 ± 0.2 17.50 ± 0.1
61. 9001 42.00 ± 0.3 99.00 ± 0.3 67.12 ± 0.4 184.00 ± 0.4
62. KBH 3580 79.00 ± 0.2 99.00 ± 0.3 117.25 ± 0.5 187.12 ± 0.4
63. 86M89 40.00 ± 0.2 100.00 ± 0.2 65.37 ± 0.5 182.12 ± 0.5
64. MPMH 17 50.00 ± 0.1 100.00 ± 0.2 84.62 ± 0.3 178.87 ± 0.2
65. 86M64 30.00 ± 0.1 100.00 ± 0.3 61.37 ± 0.1 183.50 ± 0.2
66. Nandi 65 40.00 ± 0.1 100.00 ± 0.3 65.12 ± 0.4 185.50 ± 0.4
67. 86M84 10.00 ± 0.1 100.00 ± 0.4 42.75 ± 0.5 186.75 ± 0.4
68. PAC 909 50.00 ± 0.3 100.00 ± 0.5 80.21 ± 0.3 185.25 ± 0.3
69. MP 7872 90.00 ± 0.6 100.00 ± 0.5 153.75 ± 0.2 184.87 ± 0.1
70. NBH 4903 10.00 ± 0.2 100.00 ± 0.6 42.25 ± 0.6 186.37 ± 0.4
71. MLBH 308 26.00 ± 0.3 101.00 ± 0.4 57.62 ± 0.2 184.00 ± 0.2
72. HHB 67 51.50 ± 0.3 101.00 ± 0.4 82.25 ± 0.3 178.75 ± 0.4
73. Pusa 23 82.00 ± 0.4 101.00 ± 0.5 137.25 ± 0.2 187.25 ± 0.2
74. 1827 23.00 ± 0.5 102.00 ± 0.6 52.25 ± 0.1 180.87 ± 0.1
75. JKBH 676 66.00 ± 0.5 103.00 ± 0.2 99.37 ± 0.5 180.75 ± 0.4
76. MP 7171 62.50 ± 0.1 109.00 ± 0.09 90.75 ± 0.5 184.87 ± 0.5
77. Eknath 301 gold 89.00 ± 0.1 109.00 ± 0.2 139.25 ± 0.2 179.37 ± 0.1
78. Nandi 75 40.00 ± 0.2 110.00 ± 0.1 66.62 ± 0.3 186.125 ± 0.4
79. XMT 1497 40.00 ± 0.3 110.00 ± 0.1 67.37 ± 0.4 175.37 ± 0.5
80. Nandi 61 50.00 ± 0.2 110.00 ± 0.1 84.75 ± 0.2 185.50 ± 0.1
81. MPMH 21 50.00 ± 0.1 110.00 ± 0.2 84.62 ± 0.3 184.87 ± 0.4
82. RHB 173 50.00 ± 0.1 110.00 ± 0.2 83.87 ± 0.2 183.37 ± 0.1
83. GHB 538 50.00 ± 0.5 110.00 ± 0.2 79.50 ± 0.4 179.25 ± 0.2
84. GHB 905 40.00 ± 0.5 110.00 ± 0.3 68.87 ± 0.2 184.75 ± 0.3
85. HHB 234 20.00 ± 0.2 110.00 ± 0.4 54.75 ± 0.5 179.25 ± 0.6
86. MP 7878 19.00 ± 0.1 110.50 ± 0.4 49.81 ± 0.4 185.75 ± 0.2
87. BJ 104 40.00 ± 0.5 112.00 ± 0.1 65.25 ± 0.6 180.87 ± 0.7
88. 86M38 30.00 ± 0.3 120.00 ± 0.1 62.12 ± 0.5 185.75 ± 0.4
89. 86M86 80.00 ± 0.5 120.00 ± 0.4 134.37 ± 0.5 187.62 ± 0.6
90. Anmol 70.00 ± 0.1 120.00 ± 0.5 114.37 ± 0.4 192.75 ± 0.3
91. 86M13 70.00 ± 0.08 130.00 ± 0.2 113.75 ± 0.2 193.87 ± 0.4
92. PHB 2168 80.00 ± 0.4 130.00 ± 0.2 133.75 ± 0.4 194.62 ± 0.5
Food Anal. Methods

Table 2 (continued)

S Pearl millet CPV0 (meq peroxide/ CPV10 (meq peroxide/ LOX0 (nM HPOD min−1 mg−1 LOX10 (nM HPOD min−1 mg−1
No. varieties kg flour) kg flour) soluble protein) soluble protein)

93. GHB 744 90.00 ± 0.3 140.00 ± 0.2 156.12 ± 0.2 196.62 ± 0.4

(LOX) enzyme converts free fatty acids into hydroperoxides
and comprehensive peroxide value is the measure of primary
oxidation products in the flour such as hydroperoxides
(Fig. 3a). Hence, LOX and CPV are directly correlated with
each other. On comparing LOX and CPV in group I, both
were showing a similar pattern and the genotypes having
higher LOX activity, and they also have higher CPV
(Table 2). In group I (freshly milled flour), CPV (CPV0)
ranges from 6 to 30 meq peroxide kg−1 flour for low CPV0
type, having LOX activity vary from 38 to 62 nM HPOD
min−1 mg−1 soluble protein and 39 pearl millet genotypes
were observed in this category in group I. Twenty-six geno-
types belong to medium CPV0 type having CPV0 ranges from
31 to 60 meq peroxide kg−1 flour and LOX activity varying
from 64 to 87 nM HPOD min−1 mg−1 soluble protein, and the
remaining 28 genotypes having CPV0 more than 60 meq

Fig. 4 Rancidity model developed based on the tenth day CAV and CPV
 Food Anal. Methods

Table 3 Correlation of
comprehensive peroxide value S No. Pearl millet varieties Comprehensive peroxide value (CPV) Comprehensive acid value (CAV)
and comprehensive acid value.
CPV and CAV belong to the 0 day 10th day 0 day 10th day
same group in rancidity matrix
(CPV α CAV) 1. 1827 23.00 ± 0.5 102.00 ± 0.6 5.68 ± 0.4 6.06 ± 0.3
2. 7686 88.00 ± 0.3 96.50 ± 0.2 4.84 ± 0.4 6.22 ± 0.1
3. 86M01 76.00 ± 0.1 92.50 ± 0.1 5.92 ± 0.3 7.08 ± 0.3
4. 86M64 30.00 ± 0.1 100.00 ± 0.3 3.20 ± 0.2 6.80 ± 0.1
5. 86M84 10.00 ± 0.1 100.00 ± 0.4 3.60 ± 0.1 6.80 ± 0.5
6. 86M86 80.00 ± 0.5 120.00 ± 0.4 2.80 ± 0.2 6.62 ± 0.3
7. AHB 1269 29.00 ± 0.2 96.50 ± 0.1 4.88 ± 0.2 7.16 ± 0.3
8. Ajeet 42 29.00 ± 0.2 94.00 ± 0.1 5.74 ± 0.3 6.32 ± 0.4
9. Anmol 70.00 ± 0.1 120.00 ± 0.5 3.86 ± 0.2 6.04 ± 0.5
10. Bio 70 25.50 ± 0.3 96.50 ± 0.2 6.50 ± 0.1 7.26 ± 0.1
11. BJ 104 40.00 ± 0.5 112.00 ± 0.1 6.50 ± 0.2 10.00 ± 0.4
12. 86M82 30.00 ± 0.1 50.00 ± 0.2 2.4 ± 0.4 4.40 ± 0.1
13. GHB 732 16.00 ± 0.01 40.00 ± 0.4 2.00 ± 0.2 2.80 ± 0.3
14. HHB 50 47.50 ± 0.4 77.00 ± 0.5 4.04 ± 0.4 6.00 ± 0.2
15. HHB 67 51.50 ± 0.3 101.00 ± 0.4 4.86 ± 0.3 6.92 ± 0.5
16. HHB 67 (imp) 20.00 ± 0.3 50.00 ± 0.1 2.00 ± 0.1 2.40 ± 0.3
17. ICMH 356 63.00 ± 0.1 80.50 ± 0.2 4.56 ± 0.3 5.64 ± 0.1
18. ICMH 423 63.00 ± 0.1 81.00 ± 0.1 5.16 ± 0.4 5.88 ± 0.6
19. JKBH 676 66.00 ± 0.5 103.00 ± 0.2 6.44 ± 0.3 7.20 ± 0.4
20. KBH 108 40.00 ± 0.5 50.00 ± 0.4 2.40 ± 0.2 2.80 ± 0.2
21. KBH 3580 79.00 ± 0.2 99.00 ± 0.3 5.28 ± 0.1 6.76 ± 0.3
22. MLBH 308 26.00 ± 0.3 101.00 ± 0.4 6.28 ± 0.5 6.76 ± 0.3
23. MP 7171 62.50 ± 0.1 109.00 ± 0.09 3.60 ± 0.2 6.40 ± 0.3
24. MP 7878 19.00 ± 0.1 110.50 ± 0.4 5.42 ± 0.2 7.64 ± 0.1
25. MPMH 17 50.00 ± 0.1 100.00 ± 0.2 5.60 ± 0.5 7.20 ± 0.4
26. MPMH 21 50.00 ± 0.1 110.00 ± 0.2 6.00 ± 0.2 6.80 ± 0.3
27. Nandi 61 50.00 ± 0.2 110.00 ± 0.1 4.32 ± 0.1 6.40 ± 0.2
28. Pro agro 9444 82.00 ± 0.2 97.00 ± 0.3 6.00 ± 0.2 6.94 ± 0.4
29. Pusa 605 21.00 ± 0.3 82.50 ± 0.2 5.34 ± 0.3 5.96 ± 0.4
30. RHB 173 50.00 ± 0.1 110.00 ± 0.2 3.80 ± 0.4 6.12 ± 0.4
31. RHB 234 71.00 ± 0.3 91.50 ± 0.5 5.08 ± 0.1 6.14 ± 0.4
32. RHB 58 85.00 ± 0.4 96.50 ± 0.2 5.48 ± 0.2 6.16 ± 0.3
33. Super boss 29.50 ± 0.4 98.50 ± 0.2 6.76 ± 0.1 7.06 ± 0.2
34. XMT 1497 40.00 ± 0.3 110.00 ± 0.1 2.40 ± 0.5 6.92 ± 0.2

peroxide kg−1 flour and LOX activity varying from 91 to
154 nM HPOD min−1 mg−1 soluble protein (Fig. 3b) were
considered under high CPV0.
Further, we have estimated both LOX activity and CPV in
group II (10-day stored flour: CPV10) (Table 2), and it was
found to be maximum on the 10th day as confirmed from pilot
experiment. Genotypes having CAV10 ranges from 14 to
60 meq peroxide kg−1 flour and LOX activity varying from
45 to 95 nM HPOD min−1 mg−1 soluble protein were consid-
ered under low CPV10 type. Twenty-two genotypes were
categorized in the low CPV10 category. Twenty-seven geno-
types having CAV10 varying from 61 to 90 meq peroxide kg−1
flour and LOX activity 96 to 157 nM HPOD min−1 mg−1
soluble protein were categorized as medium CPV10 type and
the remaining forty-four genotypes having CAV10 more than
90 meq peroxide kg−1 flour and LOX activity varying from
159 to 197 nM HPOD min−1 mg−1 soluble protein were con-
sidered under high CPV10 type (Fig. 3c). There was an in-
crease in the LOX activity observed in group II (stored flour)
as compared with group I (freshly milled flour). From the
Food Anal. Methods
Fig. 5 Correlation of CPV and CAV, where both belong to the same
group in rancidity matrix. a Freshly milled flour. b Ten-day stored flour.
c CPV belongs to higher group than CAV in the rancidity matrix in
above analysis, we can conclude that LOX activity is the ma-
jor contributor in off-odour development in the pearl millet
flour. LOX activity can be used as a biochemical marker for
the evaluation of pearl millet genotypes for its rancid behav-
iour and since CPV is the measure of LOX activity product,
hence only by measuring the CPV, pearl millet genotypes can
be screened for its rancid category. Various reports state that
LOX is an important factor responsible for the deterioration of
corn and soybean flour during storage (Orak et al. 2010;
Mandal et al. 2014).
Development of Rancidity Model
Relation Between CAV and CPV
In the process of rancidity development in pearl millet flour,
both lipase and LOX enzyme played an important role and
significantly contributed to CAV and CPV respectively. Here
we have evaluated 93 pearl millet genotypes using different
freshly milled flour. d Ten-day stored flour. e CPV belongs to
lower group than CAV in the rancidity matrix in freshly milled flour. f
Ten-day stored flour
biochemical markers such as CPV and CAV along with the
activity of the enzymes in group I (fresh flour) and group II
(10-day stored flour). It was observed that different pearl mil-
let genotypes behave differently for CPV and CAV both in
group I and group II. We have classified both CAV and CPV
in both group I and group II in three categories as low, medi-
um and high CAV and CPV. Since the 10th day is crucial for
rancidity measurement, we found that some of the genotypes
showed both CPV10 and CAV10 belong to the same category
(26 genotypes), while some of the genotypes have CPV10 in
higher category (29 genotypes) and others have CAV10 in
higher category (38 genotypes). The relationship between
CPV10 and CAV10 makes the basis for the development of
rancidity matrix model (Fig. 4).
CPV and CAV Belong to the Same Group in Rancidity
Matrix
On comparing CAV and CPV in group I (freshly milled flour:
CAV0 and CPV0), in the case where both belong to the same
 Food Anal. Methods

Table 4 CPV belongs to the

higher group than CAV in the S No. Pearl millet varieties Comprehensive peroxide value (CPV) Comprehensive acid value (CAV)
rancidity matrix (CPV > CAV)
0 day 10th day 0 day 10th day

1. 86M13 70.00 ± 0.08 130.00 ± 0.2 2.40 ± 0.1 3.60 ± 0.3

2. 86M38 30.00 ± 0.3 120.00 ± 0.1 1.20 ± 0.3 2.40 ± 0.4
3. 86M88 10.00 ± 0.4 90.00 ± 0.1 1.20 ± 0.2 2.00 ± 0.5
4. 86M89 40.00 ± 0.2 100.00 ± 0.2 2.80 ± 0.2 4.80 ± 0.1
5. 9001 42.00 ± 0.3 99.50 ± 0.3 4.90 ± 0.1 5.62 ± 0.1
6. Eknath 301 gold 89.00 ± 0.1 109.00 ± 0.2 4.52 ± 0.3 5.40 ± 0.3
7. GHB 538 50.00 ± 0.5 110.00 ± 0.2 3.60 ± 0.4 4.90 ± 0.1
8. GHB 744 90.00 ± 0.3 140.00 ± 0.2 2.80 ± 0.1 3.60 ± 0.4
9. GHB 905 40.00 ± 0.5 110.00 ± 0.3 3.20 ± 0.4 5.40 ± 0.2
10. HHB 197 78.00 ± 0.1 98.00 ± 0.5 4.28 ± 0.6 4.92 ± 0.3
11. HHB 223 90.00 ± 0.3 90.00 ± 0.3 2.40 ± 0.3 2.80 ± 0.4
12. HHB 226 20.00 ± 0.1 80.00 ± 0.3 2.00 ± 0.2 2.80 ± 0.2
13. HHB 234 20.00 ± 0.2 110.00 ± 0.4 1.60 ± 0.1 2.40 ± 0.2
14. JKBH 1486 24.50 ± 0.4 97.00 ± 0.3 5.44 ± 0.1 5.68 ± 0.2
15. MP 7792 30.00 ± 0.4 80.00 ± 0.5 2.00 ± 0.2 2.80 ± 0.3
16. MP 7872 90.00 ± 0.6 100.00 ± 0.5 2.40 ± 0.3 4.00 ± 0.3
17. Nandi 65 40.00 ± 0.1 100.00 ± 0.3 3.60 ± 0.4 4.80 ± 0.3
18. Nandi 70 30.00 ± 0.6 90.00 ± 0.3 1.80 ± 0.2 2.40 ± 0.3
19. Nandi 72 20.00 ± 0.2 90.00 ± 0.3 1.60 ± 0.1 2.00 ± 0.2
20. Nandi 75 40.00 ± 0.2 110.00 ± 0.1 2.20 ± 0.2 2.80 ± 0.1
21. Nandi 81 78.00 ± 0.2 95.50 ± 0.3 4.32 ± 0.2 5.00 ± 0.1
22. NBH 4903 10.00 ± 0.2 100.00 ± 0.6 1.60 ± 0.5 3.20 ± 0.1
23. NBH 5767 20.00 ± 0.4 90.00 ± 0.3 2.20 ± 0.2 2.40 ± 0.3
24. PAC 909 50.00 ± 0.3 100.00 ± 0.5 2.80 ± 0.1 5.20 ± 0.1
25. PHB 2168 80.00 ± 0.4 130.00 ± 0.2 2.60 ± 0.4 2.60 ± 0.3
26. Pratap 40.00 ± 0.2 90.00 ± 0.4 2.20 ± 0.4 2.80 ± 0.5
27. Pusa 23 82.00 ± 0.4 101.00 ± 0.5 4.68 ± 0.2 5.50 ± 0.1

category, twenty-seven genotypes observed to have CPV0
were proportional to CAV0 (Table 3). Here, we found 7 ge-
notypes were in low category, 14 genotypes were observed in
medium and 6 genotypes were observed in high CAV0 and
CPV0 category (Fig. 5a) which indicates that in such cases,
CAV0 and CPV0 are directly proportional to each other and
the rate with which free fatty acids were formed, with the same
rate, free fatty acid converted into hydroperoxides. A similar
pattern was also observed on the 10th day in group II (Fig. 5b),
although the value of CAV10 and CPV10 was more in group II
compared with group I. Those genotypes having very low
CAV10 (less than 2.8 mg NaOH g−1flour) and CPV10 (less
than 60 meq peroxide kg−1flour) were considered better geno-
types while genotypes having very high CAV10 (more than
6 mg NaOH g−1 flour) and CPV10 (more than 90 meq perox-
ide kg−1 flour) were considered high rancid types. Out of 93
genotypes, 26 genotypes showed their CAV10 and CPV10 in
the same category (Table 3). Among 26 genotypes, 3 geno-
types were in low CAV10 and CPV10 category, 5 genotypes in
medium and 18 genotypes were in the high CAV10 and CPV10
category. Datta Mazumdar et al. (2016) reported that fat ex-
tracted from pearl millet flour showed an increase in acid
value and peroxide value up to the 10th day and then gradually
decrease and it is the basis for rancidity measurement in pearl
millet flour.
CPV Belongs to the Higher Group than CAV in the
Rancidity Matrix
Some of the pearl millet genotypes showed variation in CPV
and CAV, as having higher CPV than CAV (Table 4). In
group I (fresh flour), 21 genotypes have CPV0 which was
more than CAV0. Here, it was observed that 3 genotypes were
in low, 1 genotype was in medium and 17 genotypes were in
the high CPV0 category (Fig. 5c). In group II (10-day stored
flour), 29 genotypes were observed in this category having
higher CPV10 than CAV10. Here, 8 genotypes were in medi-
um and 21 genotypes were in the higher CPV10 category (Fig.
Food Anal. Methods

Table 5 CPV belongs to lower

group than CAV in the rancidity S No. Pearl millet varieties Comprehensive peroxide value (CPV) Comprehensive acid value (CAV)
matrix (CPV < CAV)
0 day 10th day 0 day 10th day

1. DamodaraBajri 6.00 ± 0.2 30 ± 0.5 5.12 ± 0.3 6.08 ± 0.2

2. Gadhwalki Dhani-1 6.50 ± 0.2 14.00 ± 0.2 5.64 ± 0.4 6.90 ± 0.6
3. Chadi Bajri 10.00 ± 0.4 20 ± 0.2 6.48 ± 0.3 8.10 ± 0.2
4. Chanana Bajra-2 20.00 ± 0.4 30.00 ± 0.5 6.00 ± 0.1 7.36 ± 0.3
5. Dhodhsar local 22.50 ± 0.1 30.00 ± 0.4 3.60 ± 0.3 5.30 ± 0.1
6. DedhaBajri 27.00 ± 0.2 40.50 ± 0.5 5.84 ± 0.1 6.10 ± 0.3
7. AHB 1200 30.00 ± 0.2 50.00 ± 0.3 4.00 ± 0.3 4.80 ± 0.1
8. Dhanshakti 70.00 ± 0.1 90.00 ± 0.2 6.20 ± 0.4 10.00 ± 0.3
9. Gadhwalki Dhani-3 30.00 ± 0.6 40.00 ± 0.1 5.92 ± 0.2 7.20 ± 0.4
10. GHB 558 30.00 ± 0.4 50.00 ± 0.5 2.80 ± 0.2 2.90 ± 0.3
11. GHB 757 50.00 ± 0.5 90.00 ± 0.2 3.60 ± 0.1 7.20 ± 0.3
12. HB 3 81.00 ± 0.4 88.00 ± 0.3 5.66 ± 0.2 6.12 ± 0.3
13. HHB 146 44.00 ± 0.5 57.00 ± 0.1 4.96 ± 0.3 6.88 ± 0.2
14. HHB 299 50.00 ± 0.4 90.00 ± 0.3 4.04 ± 0.1 6.40 ± 0.2
15. HHB 311 11.50 ± 0.5 19.00 ± 0.6 6.00 ± 0.4 9.10 ± 0.3
16. HTBH 4201 69.00 ± 0.1 90.00 ± 0.1 6.36 ± 0.4 7.44 ± 0.2
17. JKBH 26 71.00 ± 0.5 75.00 ± 0.4 5.96 ± 0.4 6.52 ± 0.2
18. Kaveri super boss 12.00 ± 0.1 18.00 ± 0.2 5.48 ± 0.1 6.10 ± 0.03
19. MBH 2210 67.00 ± 0.6 82.50 ± 0.5 5.36 ± 0.4 6.58 ± 0.3
20. MBH 2232 61.00 ± 0.3 89.00 ± 0.5 5.68 ± 0.5 6.44 ± 0.4
21. MH 179 27.00 ± 0.5 65.00 ± 0.5 5.46 ± 0.3 7.54 ± 0.2
22. Nandi 52 21.00 ± 0.2 82.50 ± 0.1 5.26 ± 0.2 6.20 ± 0.3
23. PB 1705 16.50 ± 0.3 90.00 ± 0.6 6.40 ± 0.3 6.64 ± 0.5
24. Pusa 322 19.00 ± 0.3 75.00 ± 0.1 5.64 ± 0.2 6.36 ± 0.3
25. Pusa composite 443 31.00 ± 0.4 35.50 ± 0.2 6.04 ± 0.1 6.96 ± 0.1
26. Pusa purple 1 52.00 ± 0.1 58.00 ± 0.2 5.20 ± 0.4 8.90 ± 0.5
27. RHB 121 48.50 ± 0.1 49.00 ± 0.1 5.90 ± 0.3 6.32 ± 0.4
28. RHB 177 50.00 ± 0.4 60.00 ± 0.2 4.56 ± 0.4 5.60 ± 0.2
29. RHB 223 50.00 ± 0.1 50.00 ± 0.6 3.60 ± 0.2 5.90 ± 0.5
30. RHB 233 50.00 ± 0.5 81.00 ± 0.1 4.72 ± 0.3 6.12 ± 0.2
31. SulkhaniyaBajra 19.00 ± 0.1 82.00 ± 0.5 5.12 ± 0.1 6.90 ± 0.4
32. WGI 100 65.00 ± 0.4 67.00 ± 0.3 6.40 ± 0.4 9.00 ± 0.2

5d). The pearl millet genotypes having very high CPV10
(more than 90 meq peroxide kg−1 flour) and low CAV10 (less
than 4.5 mg NaOH g−1 flour) were placed in the most rancid
category in the rancidity matrix. In this case, the rate of free
fatty acid formation is relatively lower than the rate with
which hydroperoxide was formed. As a result, when the rate
CPV was increased, CAV was not increased. In other words,
in such genotypes, their LOX enzyme activity was higher than
lipase activity. Since, the LOX activity is dependent on avail-
able free fatty acids and once the free fatty acid is produced by
lipase, it is smoothly available to LOX and hence the CPV
was higher than CAV. Here we also conclude that the reaction
catalysed by LOX is the rate-limiting step in rancidity devel-
opment in pearl millet flour.
CPV Belongs to Lower Group than CAV in the
Rancidity Matrix
Some of the pearl millet genotypes showed higher CAV than
their CPV (Table 5). In group I (fresh flour), a total of 45
genotypes showed their CPV0 lower than CAV0. Among 45
genotypes, 29 genotypes were in low, 11 genotypes in medi-
um and 5 genotypes having high CPV0 (Fig. 5e). In group II
(stored flour), 38 genotypes showed their CPV10 was lower
than CAV10, out of which 15 genotypes were in low, 17 ge-
notypes were in medium and 6 genotypes were in the high
CAV10 category (Fig. 5f). The lipase activity in such geno-
types was observed higher than LOX activity and hence, the
rates with which free fatty acids were formed that was

Fig. 6 Rancidity matrix developed to classify pearl millet genotypes into low, medium and high rancid categories and each category is further divided
into two classes
oxidized into hydroperoxides with a comparatively slower
rate. Although availability of more free fatty acids in such
genotypes makes them pro-oxidant but since the flour was
not exposed to light and having lower LOX activity, it lowers
the oxidation rate of free fatty acids present in the flour.
Development of Rancidity Matrix
Rancidity development in pearl millet flour is the major problem
that limits the popularity of pearl millet as a staple food. The pearl
millet flour produces bitterness in taste after 10 days of milling
into flour. It was observed that different pearl millet genotypes
behave differently for CPV and CAV both in freshly milled flour
and stored flour. Based on their extent to be rancid, all the pearl
millet genotypes evaluated here were classified into three cate-
gories, namely low, medium and high rancid categories. Further,
each category was divided into two classes; hence, the total six
classes were formed (Fig. 6). All the 93 pearl millet genotypes
were placed in different classes based on their CPV on the 10th
Food Anal. Methods
day (CPV10). In low rancid type, the CPV10 limit set was 14 to 30
as class I, 31 to 60 as class II, for medium rancid type; 61 to 75 as
class III, 76–90 as class IV, and for high rancid type 91 to 110 as
class V and more than 110 as class VI. We have taken the 10th
day CPV as the main rancidity criteria because it gives the infor-
mation of the primary oxidation product of fat, i.e. hydroperox-
ides. The CPVs estimated in freshly milled flour (CPV0) and
10 days after milling (CPV10) in 93 genotypes of PM were
mapped to characterize the diversity in the pattern of increase
of CPV. We observed significant variations in the pattern of
increase of CPV in diverse genotypes of PM and concluded that
lines showing minimum slope are best suited to be used as breed-
ing material for low rancidity. Based on the above hypothesis,
out of 93 pearl millet genotypes, 7.52% were placed in class I,
16.12% in class II, 4.3% in class III, 24.73% in class IV, 37.63%
in class V and 8.6% in class VI. This matrix further confirms the
existence of huge diversity in the rancidity profile of pearl millet
genotypes as reported by various researchers (Datta Mazumdar
et al. 2016; Goyal and Chugh 2017) and first time we have
Food Anal. Methods
classified diverse genotypes of pearl millet into the different ran-
cid categories (low, medium and high rancid) based on their
rancidity development efficiency.
Conclusions
The keeping quality of flours of 93 diverse genotypes of PM,
using different biochemical parameters like CPV, CAV and ac-
tivities of rancidity causing enzymes like lipase and LOX, in
fresh flour and 10 days after milling was characterized. The
10th day CPV (CPV10) was observed to be the most appropriate
criterion for the development of the rancidity matrix (RM). The
93 diverse genotypes of PM were classified into three
categories—low rancid, medium rancid and highly rancid
type—each having two classes with specific ranges. The RM
developed in the present investigation will help the PM breeder
to screen large germplasm for low rancidity and better keeping
quality within a short duration of time and millet processors for
millet-based processed food. There is a need to further character-
ize a large subset of PM using the matrix developed in the present
investigation to pace up the PM breeding programme for the
development of cultivars having a balanced nutritional composi-
tion with high keeping quality—the need of hours.
Acknowledgments The genotype shared by IARI and AICRP on Pearl
Millet, Jodhpur is highly acknowledged.
Funding Information The authors received external funding from the
Agricultural Education Division, Indian Council of Agricultural Research
(ICAR) under the Niche Area of Excellence (NAE) Programme (Scheme-
Strengthening and Development of Higher Agricultural Education in India)
(Project Sanction no. Edn. 5(22)/2017-EP&HS, 2019; IARI code: 12/223).
Compliance with Ethical Standards
Conflict of Interest Suneha Goswami declares that she has no conflict of
interest. Purva Asrani declares that she has no conflict of interest. Ansheef Ali
T.P declares that he has no conflict of interest. R. Dinesh Kumar declares that
he has no conflict of interest. Vinutha T. declares that she has no conflict of
interest. Veda K. declares that she has no conflict of interest. Sweta Kumari
declares that she has no conflict of interest. Archana Sachdev declares that she
has no conflict of interest. Sumer P. Singh declares that he has no conflict of
interest. C. Tara Satyavathi declares that she has no conflict of interest.
Ranjeet R. Kumar declares that he has no conflict of interest. Shelly
Praveen declares that she has no conflict of interest.
Ethical Approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed Consent Not applicable.
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