International Baccalaureate Diploma Programme

Biology Extended Essay 2023

Title: Sprouting duration on seed nutrients.

Research Question: To what extent does the increasing duration ( 0,12,24,36,48 hours ) of
sprouting affect the total protein and fat content (%) in Vigna radiata and Cicer arietinum
measured through absorbance of 400 nm and 450 nm through biuret and emulsion test of
nutrients respectively at 25℃?

Candidate Number: kcw042

Word Count: 3998
Contents

1. Introduction __ 1

2. Literature Review 2
2.1 Physiological process investigated___________ 2
2.2 Plants Used 3
2.3 Test for presence of protein: Biuret test 4
2.4 Test for presence of lipids: Emulsion test 4
2.5 Use of Spectrophotometer 4

3. Hypothesis 5
3.1 Null Hypotheses 5
3.2 Alternative Hypotheses 5
4. Variables of the experiment 5
4.1 Independent variables 5
4.2 Dependent variables 5
4.3 Controlled variables 6
5. Procedure 6
5.1 Seed Selection 6
5.2 Soaking of Seeds 6
5.3 Sprouting Incubation 6
5.4 Seed Processing 6

5.5 Protein test and it’s quantification 7

5.6 Lipid test and its quantification 7
5.7 Data Processing 7
5.8 Seed Shape and Colour 7
6. REPRESENTATION OF DATA 8
6.1 Qualitative Analysis- Protein 9
6.2 Quantitative Analysis- Lipid 9
6.3 Quantification of Proteins and Lipids 12
7 Results 14
8 Discussion 15
9 Quantitative and Qualitative Evaluation 16
10 Conclusion 17

11 Limitations and achievable improvements 18

12 Further Studies 18
13 Bibliography 19
14 Appendix 21
1. Introduction:
Sprouting and soaking of seeds/legumes have been demonstrated to lower phytic acid by enabling it to
leach into the soaking water, which we then pour down the drain. Additionally, sprouting and soaking
may stimulate the production of phytic acid-degrading enzymes. The process by which seeds or spores
naturally germinate and send out branches, mature plants develop new leaves or buds, or other
structures continue to expand is known as sprouting. However, sprouts are typically richer in protein,
folate, magnesium, phosphorus, manganese, and vitamins C and K than non-sprouted plants since the
sprouting process raises nutritional levels for consumption.1For instance, numerous research indicates
that sprouting contributes to a higher protein content.

Additionally, sprouts typically have larger concentrations of critical amino acids, with some individual
amino acids rising by as much as 30%2. Additionally, sprouts' proteins can be simpler to digest. This is
probably because sprouting appears to remove up to 87% of the antinutrients, which are substances that
hinder your body's capacity to absorb nutrients from plants.3 According to various studies and articles,
the optimum duration for sprouting is between 24 hours to 36 hours.4 Thus the present investigation
emphasises on change in nutrition value of sproutings seeds. Since on a daily basis legumes are
consumed as sprouts, this investigation will use Vigna radiata and Cicer arietinum. Major nutrients in
seeds can be categories as carbohydrates, proteins and fats. Among these proteins are admired, hence
total protein content is estimated and to support the hypotheses that sprouting alters nutritional value
fat is also investigated. Both biomolecules can be tested with specific reagents and their absorbance
value (%) is recorded. Thus the following research question is framed:
“To what extent does the increasing duration ( 0,12,24,36,48 hours ) of sprouting affect the total
protein and fat content (%) in Vigna radiata and Cicer arietinum measured through absorbance of
400 nm and 450 nm through biuret and emulsion test of nutrients respectively at 25℃?”

1
"Raw Sprouts: Benefits And Potential Risks". Healthline, 2022, https://www.healthline.com/nutrition/raw-
sprouts#TOC_TITLE_HDR_3. Accessed 30 Sept 2022.
2
Singh, A. K., Jagbir Rehal, Amarjeet Kaur, and Gagan Jyot. "Enhancement of Attributes of Cereals by
Germination and Fermentation: A Review." Journal of Food Science,
3
https://pubmed.ncbi.nlm.nih.gov/24915317/
4
Johnston, Cassie. "Sprouting 101". Wholefully, 2020, https://wholefully.com/sprouting-101/.

1
2. Literature Review:

2.1 Physiological process investigated- Sprouting and it effect on protein and fat content:
Germination is the sprouting of a seed, spore, or other reproductive body, usually after a period of
dormancy5(image 1.1). A viable seed's temporary incapacity to sprout in a favourable environment is
known as dormancy (Simpson, 1990). Dormancy aids in both seed distribution and seed survival in
challenging environmental conditions (Snape et al., 2001).6 Environmental factors that can influence
seed germination include temperature, light, pH, and soil moisture (Chachalis and Reddy 2000;
Taylorson 1987).7 Light aids in photosynthesis, whose end result, upon hydrolysis, yields energy
molecules (ATP), which then aid in protein biosynthesis, gluconeogenesis, the conversion of oil stores
into starch, and the mobilisation of lipid reserves up until the cotyledons turn photoautotrophic. In this
investigation, just the impact of sprouting duration on the biochemical content of the seeds was
examined while holding all other variables constant. The sprouting incubation period for this
investigation will be 0, 12, 24, 36, and 48 hours respectively. This is based on the actual duration of
seeds being left to sprout at our house.

Image 1.1: Germination5

5
"Germination | Description, Process, Diagram, Stages, Types, & Facts". Encyclopedia Britannica, 2023,
https://www.britannica.com/science/germination. Accessed 1 Oct 2022.
6
Kant, Lakshmi et al. "Barley". Genetic And Genomic Resources For Grain Cereals Improvement, 2016, pp.
125-157. Elsevier, doi:10.1016/b978-0-12-802000-5.00003-4. Accessed 7 Oct 2022.
7
Ars.Usda.Gov,2022,https://www.ars.usda.gov/ARSUserFiles/64022000/Publications/Reddy/Kogetetal04WS5
2-6-989.pdf. Accessed 7 Oct 2022.

2
2.2 Plants used:

2.2.1 - Vigna radiata:

Mung bean (Vigna radiata L.), a widely consumed pulse in Asia, is known for its high nutritional value,
majorly Lysine amino acid and bioactive compounds such as polyphenols, polysaccharides, and
peptides that contribute to its health benefits. The bean has been found to improve conditions such as
high blood sugar, high cholesterol, and high blood pressure, as well as protect against cancer,
melanogenesis, and liver damage, and boost the immune system. During the process of soaking the
seeds in water, enzymes get activated and seeds undergo cellular respiration to produce energy and
metabolic processes that lead to the mobilization of fat (crude fat 2.26%)8 and production of proteins
(19.05 – 23.86%)8 through transcription and translation during interphase.

2.2.2 - Cicer arietinum:

The chickpea is a type of legume that belongs to the family Leguminosae. It is also known by its
scientific name, Cicer arietinum. It is a type of pulse that is characterised by a high nutritional value
and an abundance of protein (12.6-30.5%)9, dietary fibre, general fat content (3.8-10.2%)10 and dietary
minerals in its composition.11The chickpea is a grain legume that is utilised all over the world for a
variety of purposes, most notably as a source of protein (Bejiga et al., 2006). It is common practise to
eat dried chickpea seeds of the local variety as a form of pulse, either whole, split, or crushed as dhal
or flour. They can also be found in soups and sauces like hummus (Bejiga et al., 2006; van der Maesen,
1989). There are many different applications for Kabuli varieties, including salads, vegetable mixtures,
and even canning. Fresh consumption is possible for both the seeds and the pods. In addition, chickpeas
can be roasted, salted, and consumed in the form of snacks (Bejiga et al., 2006).12 Cells in these legumes

8
Ullah Riaz et al.”Nutritional Assessment and antioxidant activities of different varieties of Vigna radiate”.
The scientific world journal. Accessed 17 Nov 2022.
9
Oar.Icrisat.Org, 2023, http://oar.icrisat.org/3436/1/JA_431.pdf. Accessed 3 Dec 2022.
10
Madurapperumage, Amod et al. "Chickpea (Cicer Arietinum L.) As A Source Of Essential Fatty Acids – A
Biofortification Approach". Frontiers In Plant Science, vol 12, 2021. Frontiers Media SA,
doi:10.3389/fpls.2021.734980. Accessed 3 Dec 2022.
11
Singh, Ajey et al. "Circadian Regulation Of Abiotic Stress Tolerance In Legumes". Abiotic Stress And
Legumes, 2021, pp. 105-136. Elsevier, doi:10.1016/b978-0-12-815355-0.00008-4. Accessed 17 Nov 2022.
12
"Chickpea (Cicer Arietinum) | Feedipedia". Feedipedia.Org, 2022, https://www.feedipedia.org/node/319.
Accessed 17 Nov 2022.

3
also undergo transcription and translation. Transcription is the transfer of genetic instructions in DNA
to mRNA in the nucleus. It includes three steps: initiation, elongation, and termination.13

2.3 Test for presence of protein: Biuret test

The biuret test is a method of qualitative protein analysis that confirms the presence of proteins in a test
solution. To conduct this test, sodium hydroxide solution (NaOH) and copper sulphate solution
(CuSO4), known as biuret reagents, are employed.14 The principle behind the Biuret test is that when
one or more peptide bonds in a test solution contact copper ions, a complex of violet colour results; the
darker the colour, the more protein is present. Colour intensity corresponds to absorbance through
spectrophotometer. Because the biuret reagent can analyse even two amino groups present in the
sample, enabling analysis of even extremely minute amounts of protein or amino acids, the test is
excellent for selection.

2.4 Test for presence of lipid: Emulsion test

Lipids are chemical compounds that are liquid at normal temperature because they contain a number
of unsaturated acids. Lipids are most soluble in non-polar organic solvents, particularly alcohol,
because they are non-polar and organic by nature. The Ethanol Test is a qualitative lipid assay that
establishes the presence of lipids in a test sample by using the solubility of organic molecules in ethanol.
When an ethanol test is positive, the "Milky" colour indicates that lipids were present in the test sample.
Colour intensity corresponds to absorbance through spectrophotometer. The test was selected because
it solubilizes the lipids quickly and yields results right away. During germination total triglycerides
decrease as lipid mobilisation takes place, however phospholipids and glycolipids increase gradually,
thus these can be tested through the ethanol test.

13
Miller, Christine. "5.7 Protein Synthesis". Thompson Rivers University, 2020, p. .,
https://humanbiology.pressbooks.tru.ca/chapter/5-6-protein-
synthesis/#:~:text=Protein%20synthesis%20is%20the%20process,initiation%2C%20elongation%2C%20and%
20termination. Accessed 23 Dec 2022.
14
"Food Test 4 - Biuret Test For Proteins". Biology Notes For IGCSE 2014, 2023, https://biology-
igcse.weebly.com/food-test-4---biuret-test-for-proteins.html. Accessed 1 Sep 2022.

4
2.5 Use of Spectrophotometer:
The Beer-Lambert law states that the absorbance of a solution is directly proportional to the total
amount of solute that is present in the solution.15 This law is the foundation for the spectrophotometer.
Because every solute absorbs a distinct wavelength of light, this property contributes to the
measurement of the solute. According to S. Nakai et Al16, the wavelength of light at 400 nm can be
used to assess both proteins and lipids. Quantification of the amounts of protein and lipids found in
both types of seeds was performed with the help of the appropriate standard plots 40.17 Protein and
lipid content increases during sprouting after which the absorbance value (%) will also change.

3. Hypothesis:

3.1 Null Hypothesis: The increase of duration (0,12,24,36 and 48 hours) in the sprouting of Vigna
radiata and Cicer arietinum will have no effect on the Percentage Absorbance (%) (400 nm and 450
nm ) as well as the protein content and lipid content.

3.2 Alternative Hypothesis: The increase of duration (0,12,24,36 and 48 hours) in the sprouting of
Vigna radiata and Cicer arietinum will have an effect on the Percentage Absorbance (%) (400 nm and
450 nm ) as well as the protein content and lipid content.

15
"Beer Lambert Law | Transmittance & Absorbance | Edinburgh Instruments". Edinburgh Instruments, 2023,
https://www.edinst.com/blog/the-beer-lambert-law/. Accessed 18 Nov 2022.
16
Nakai, S., and Anh Chi Le. "Spectrophotometric Determination Of Protein And Fat In Milk Simultaneously".
Journal Of Dairy Science, vol 53, no. 3, 1970, pp. 276-278. American Dairy Science Association,
doi:10.3168/jds.s0022-0302(70)86197-5. Accessed 17 Nov 2022.
17
Okoronkwo, Nnenna. “Estimation of Protein Content and Amino Acid Compositions in Selected Plant
Samples
Using UV-Vis Spectrophotometric Method.” American Journal of Food Science and Health, 2017, 2 Oct. 2019
*www.researchgate.net/publication/319057476_Estimation_of_Protein_Content_and_Amino_Acid_Compositi
ons_i
n_Selected_Plant_Samples_Using_UV-Vis_Spectrophotometeric_Method.

5
4. Variables:

4.1 Independent Variables: The increase of duration (0,12,24,36,48 hours) in the sprouting of Vigna
radiata and Cicer arietinum.
4.2 Dependent Variables: The variation in absorbance (%) of 400 nm to 450 nm wavelength of light
and its effect on the biomolecules (protein, lipid) content (%) of Vigna radiata and Cicer arietinum.
4.3 Controlled Variables:
The investigation ensured that seeds of similar types and species were studied by using a fixed seed
source. A soaking temperature of 25℃ was selected as it enhanced the proximate composition and
functional qualities of the seeds. To ensure identical Protein and Lipid composition per mass of the
seeds, 50 gm of seeds were soaked in each incubation trial. In order to provide sufficient water for seed
soaking and to maintain a stable pH of 7, which prevents damage to the enzyme system involved in
seed metabolism, 500 cm³ of distilled water was used in each incubation trial. Uniform crushing of
seeds to obtain extract for protein and fat estimation. Vernier spectrophotometer is used to measure
absorbance.

5. Procedure:

5.1 Seed Selection:

50 mg of Vigna radiata and Cicer arietinum each was used for this investigation. These seeds were
used considering their popularity and their part in the daily diet of South Asian individuals etc.

5.2 Soaking of Seeds:

After soaking the seeds in distilled water for 0, 12, 24, 36, and 48 hours, the sprouting process was
timed. In order to accomplish this, Vigna radiata and Cicer arietinum were separated into 5 sets, and
each Sprouting incubation duration was given 5 individual trials to test out. Using the weighing
balance, 50 gm of seeds were measured out, in cups that were then filled with water to a volume of 75
cm3, seeds were soaked.

6
5.3 Sprouting Incubation:
To remove excess water, each sample of Vigna radiata and Cicer arietinum seeds were pat dried on a
blotting sheet. Incubation times for soaked seeds were 0, 12, 24, 36, and 48 hours.

5.4 Seed Processing:

To begin the experiment, 50mg of Vigna radiata and Cicer arietinum were taken out from the muslin
cloth after 0 hours of incubation. These were then mixed with 50 cm³of distilled water separately and
ground to form a fine paste. The resulting paste was divided into two separate beakers labelled "VR0"
for Vigna radiata and "CA0" for Cicer arietinum, indicating an incubation time of 0 hours. This same
process was repeated for varying incubation periods (12, 24, 36, and 48 hours) and trials. The filtrates
were collected separately into beakers labelled VR12, VR24, VR36, VR48 for Vigna radiata, and
CR12, CR24, CR36, CR48 for Cicer arietinum. The Lipid and Protein content of each seed type was
evaluated daily, taking into consideration the necessary Sprouting incubation period prior to processing
the seeds.

5.5 Protein test and it’s quantification:

2 cm3 of the seed filtrate were utilised to measure the protein content in Vigna radiata (VR0) and Cicer
arietinum (CA0) by distributing them into 25 labelled test tubes each (VR0PT and CA0PT). The
experiment involved testing the protein content of different samples using the Biuret reagent. To begin,
the reagent was added to each test tube, followed by manual shaking. After five minutes, the resulting
colour gradient was noted, and the liquid was transferred to a cuvette to determine its protein content
absorbance. Prior to analysis, the spectrophotometer was calibrated using the Biuret reagent. The
experiment was repeated for other samples, namely VR12PT, VR24PT, VR36PT, VR48PT for Vigna
radiata and CA12PT, CA24PT, CA36PT, and CA48PT for Cicer arietinum.

By following the same procedure, the protein content of each sample was assessed and compared. This
method of protein analysis using the Biuret reagent is a commonly used technique in the field of
biochemistry. By calibrating the spectrophotometer using the same reagent, accurate measurements of
protein content can be obtained.

7
5.6 Lipid test and it’s quantification:
The experiment for estimating the lipid content followed a similar process to that of protein estimation,
with the exception of using 2 cm³ of 100% ethanol instead of biuret reagent. The resulting liquid was
then moved into a cuvette using a measuring cylinder to measure the absorbance of its lipid content.
This method ensured uniformity in approach for both protein and lipid estimation while changing only

the reagent.
Figure 1: Materials and apparatus used for the experiment of Protein and Lipid estimation in
sprouting of Vigna radiata and Cicer arietinum

5.7 Data processing:

The results of the Spectrophotometric Analysis for both seeds were summarized. The standard
deviation was computed after calculating the average absorbance for all 5 trials of each incubation time
(appendix 1a, 1b, 2a, 2b). Following that, the data's Percentage Change and Percentage Affectivity
were determined. Average absorbance value were placed in equations for standard curve for both
protein and lipid (sample calculations in appendix) to obtain total protein and total lipid for both the
plants (table 3 and 4). The Pearson correlation 'r' value and ANOVA was conducted in order to
statistically analyse the data.

5.8 Seed Shape and Colour:

Vigna radiata generally have ellipsoid or cube-shaped seeds. The seeds vary in colour, being green
most of the time but occasionally yellow, olive, brown, purplish brown, or black, mottled and/or

8
ridged.18 Whereas Cicer arietinum have a coloured (usually brown), angular-shaped seed with a
noticeable, distinctive "beak" that contains the embryonic axis. Seeds typically weigh between 0.1 and
0.3 g depending on genotype.

6. Representation of Data

6.1 Qualitative Analysis:

Morphological changes found in seeds included linear increases in seed length(turgid) until 36 hours
of incubation.

6.1.1 Analysis- Protein:

The Biuret test yields a dark blue colour upon detecting the presence of Proteins, with the intensity of
the colour deepening in proportion to the amount of Proteins detected. The results of the experiment
revealed that the seeds which underwent longer Sprouting incubation times (ranging from 0 to 36 hours)
exhibited a steadily increasing gradient of blue colour, with the deepest colour being observed at the
36-hour mark. Subsequently, the colour began to lighten gradually during the 48-hour incubation
period. These findings highlight the relationship between the duration of Sprouting incubation and the
amount of Proteins present in the seeds, as evidenced by the intensity of the resulting blue colour in the
Biuret test.

18
Agritrop.Cirad.Fr, 2023, https://agritrop.cirad.fr/582490/7/ID582490_ENG.pdf. Accessed 15 Dec 2022.

9
 Figure 2: Change in colour intensity of Protein- Biuret in Vigna radiata with increasing duration of
sprouting. ( 0, 12, 24, 36 and 48 hours ). ( Left to right )

6.1.2 Analysis- Lipid:

A positive outcome in the Ethanol test is characterised by the appearance of a Milky colour. The
experimental results revealed that the seedlings subjected to longer periods of Sprouting incubation
demonstrated a progressively increasing gradient of colour development, which was observed to be
linear in nature, from 0 to 36 hours. The most significant colour development was observed at the 36-
hour mark, and the colour subsequently began to fade and stabilise during the 48-hour incubation
period. These findings highlight the correlation between the duration of Sprouting incubation and the
extent of colour development in the Ethanol test, with the emergence of a Milky colour indicating the
presence of desirable qualities in the seeds being tested.

10
Figure 3: Change in colour intensity of Lipid- Emulsion in Vigna radiata with increasing duration of
sprouting. (0, 12, 24, 36 and 48 hours). (Left to right)

11
6.2 Quantitative Analysis:

Graph 1: The Impact of Prolonged Sprouting Duration on Protein Content Percentage of Vigna
radiata and Cicer arietinum. (Error Bars = 5%)

Graph 2: The Impact of Prolonged Sprouting Duration on Lipid Content Percentage of Vigna
radiata and Cicer arietinum. (Error Bars = 5%)

12
6.3 Quantifying Proteins and Lipids in Vigna radiata and Cicer arietinum at Varied Sprouting
Durations:
The Percentage Affectivity graph demonstrated the relationship between the duration of incubation and
the levels of Protein and Lipid present. The experiment utilized a standard graph to determine the
quantity of Protein and Lipid present in Vigna radiata and Cicer arietinum after various sprouting
incubation periods. By calculating the slopes of the standard graphs, the quantity of lipids and proteins
both in Vigna radiata and Cicer arietinum were accurately determined.
Table 1: Contrast between the Sprouting Time in Lipid and Protein content Variation in Vigna
radiata. 19

Duration ( hrs ) Total Protein (mg/ml) Total Lipid (mg/ml)

0 0.107 0.0137

12 0.121 0.0162

24 0.163 0.0181

36 0.164 0.0175

48 0.162 0.0176

Table 2: Contrast between the Sprouting Time in Lipid and Protein content Variation in Cicer
arietinum.

Duration (hrs) Total Protein (mg/ml) Total Lipid (mg/ml)

0 0.076 0.0155

12 0.084 0.0198

24 0.112 0.0252

36 0.111 0.0245

48 0.107 0.0243

19
Tools.Thermofisher.Com, 2023, https://tools.thermofisher.com/content/sfs/brochures/TR0057-Read-std-
curves.pdf..

13
7. Results:

The purpose of this research was to determine the ideal amount of time to allow seeds to germinate in
order to maximize the nutritional value of the end product for human consumption. During the course
of this investigation, the protein and lipid content of the sprouted seeds were measured in a laboratory
setting. Observations indicated that the average absorbance values of lipid and protein content in the
seeds demonstrated a linear rise from 0 to 36 hours, and the maximum levels of biochemical content
were detected after 36 hours. Subsequently, a slight decline in the values was noticed at 48 hours, which
continued until 48 hours. This pattern was seen initially. After 36 hours of incubation, the Cicer
arietinum sample had an absorbance value of 0.084% for its protein content, whereas the Vigna radiata
sample had an absorbance value of 0.053% (Tables 1 and 2). After 36 hours of incubation, the Vigna
radiata sample exhibited a maximum absorbance of 0.140% for its lipid content, whereas the Cicer
arietinum sample exhibited a maximum absorbance of 0.193% (Tables 1 and 2).

Both Cicer arietinum and Vigna radiata exhibited a similar pattern with regard to the percentage
affectivity of their protein content. In Cicer arietinum, the percentage of affectivity at 0 hours, 12 hours,
and 24 hours was determined to be 100%, 110.61%, and 147.70%, respectively; this demonstrates a
linear increase in the quantity of protein present, as shown in Graph 1. Following 24 hours of
incubation, a slight decline in the trend for protein content was observed at 48 hours of incubation. This
tendency continued till the conclusion of the incubation period. After 36 hours of incubation, the
percentage of affectivity was found to be 146%, which decreased slightly to 140.15% after 48 hours of
incubation. The protein content of Vigna radiata showed a linear increase in percentage affectivity,
starting from 100% at 0 hours, then rising to 113% at 12 hours, and reaching 152% at 24 hours (as
shown in Graph 1). Sprouting legumes reduces the amount of antinutrients, which may improve protein
and mineral absorption, such as iron, zinc, calcium, magnesium, and manganese.20 After 24 hours of
incubation, there was a slight increase in protein content in Vigna radiata, which peaked at 153.03%
at 36 hours and then decreased to 151.46% at 48 hours. The error bars did not overlap until 36 hours,

20
"7 Interesting Types Of Bean Sprouts". Healthline, 2023, https://www.healthline.com/nutrition/bean-sprouts-
nutrition#:~:text=Sprouting%20grains%20and%20legumes%20decreases,%2C%20calcium%2C%20magnesiu
m%20and%20manganese. Accessed 14 Dec 2022.

14
indicating a significant difference in protein content. The rise in protein content was due to the rapid
synthesis of proteins during the peak phase of metabolism, followed by a constant level of protein after
36 hours, as there was no further biosynthesis of molecules due to the utilisation of reserve food and
newly synthesised molecules in radicle growth. The lipid content in Vigna radiata and Cicer arietinum
followed a similar linear trend. After 36 hours of incubation, there was a slight decline in lipid content
in both seeds, with the percentage affectivity at 36 and 48 hours being 127% and 126% in Vigna radiata
and 158.91% and 156.09% in Cicer arietinum, respectively. There was no overlap of the error bars,
indicating a changing trend in lipid content in both seeds with increasing duration.

8. Discussion:

The process of germination is essential for the growth and development of seeds into mature plants.
During germination, various metabolic activities occur, which lead to changes in the nutritional
properties of the seeds. In the case of both types of seeds studied, a steady increase in lipid content was
observed during the post-soaking stage of germination, which is attributed to lipid biosynthesis21. The
lipids produced during this stage are then utilized in the formation of the radicle later. It was determined
that the optimal sprouting period for obtaining the highest amount of lipids was 24 hours. Soaking the
seeds plays a crucial role in the germination process. It results in the hydration of the seeds and
decreases lipid peroxidation, which in turn increases the metabolic activity of the lipase enzyme.

This increase in metabolic activity leads to starch mobilization, which supplies energy to the expanding
embryo. Enzymes that break down proteins, lipids, and carbohydrates stored in the seeds facilitate
germination by producing simpler biomolecule units. After 12 hours of sprouting incubation, the radicle
begins to protrude, and cellular protein and lipid content in both types of seeds increase due to metabolic
activities.22 The relationship between sprouting length and protein and lipid content is significant, as
indicated by the substantial 'r' values. These findings suggest that the nutritional properties of the seeds
change significantly as the sprouting length increases.23

21
Agritrop.Cirad.Fr, 2023, https://agritrop.cirad.fr/582490/7/ID582490_ENG.pdf. Accessed 15 Dec 2022.
22
https://pubmed.ncbi.nlm.nih.gov/24915317/

23
Okoronkwo, Nnenna. “Estimation of Protein Content and Amino Acid Compositions in Selected Plant Samples

15
The ANOVA test was conducted to confirm the significant changes in nutritional properties of the
seeds, which vary as sprouting length increases, with statistical significance at p<0.5. The results of
this test indicate that the nutrient concentrations change as the sprouting length increases, and these
changes are significant at a confidence level of 95%. Thus, it is important to consider the length of the
sprouting incubation period when determining the nutritional properties of sprouted seeds.

Test Seed Pearson coefficient ANOVA Test value

'r'

Proteins Vigna radiata 0.8545 p<0.00001

Cicer arietinum 0.8008 p<0.00001

Lipids Vigna radiata 0.8342 p<0.00001

Cicer arietinum 0.8702 p<0.00001

Table 5: Comparison of ANOVA Test and Pearson Coefficient 'r' for Protein and Lipid
Content Measured by Biuret and Ethanol Tests in Vigna radiata and Cicer arietinum Seeds.

9. Qualitative and Quantitative Evaluation:

Conducting experiments on two different plant species helps to elucidate the impact of prolonged
sprouting on nutritional content. Simple techniques such as the Biuret test for proteins and the Ethanol
test for lipids were utilised to measure the biomolecules and track their development. To collect enough
data for analysis, five trials of five different lengths of Sprouting were undertaken for each of the two
seeds. The experiment aimed to establish a clear relationship between the independent and dependent
variables, and to achieve this, certain variables were kept constant throughout the experiment. The
purpose of the experiment was to collect data, which would be used to determine whether the
hypothesis put forth could be accepted or rejected.

16
To determine the number of bio-molecules present at different incubation durations, analyses of the
collected data in the form of percentage absorbance. Standard curves were used for proteins and lipids
to quantify their respective quantities. To gain a deeper insight into the nutritional content of the
samples, the use of trendlines and error bars were employed, which helped them identify any variability
and changes in the nutritional content. By doing so, they were able to gain a more profound
understanding of the relationship between the independent and dependent variables, and how they
impacted the nutritional content of the samples over time. The use of standard curves, trendlines, and
error bars helped in gaining a more in-depth understanding of the nutritional content of the samples
over time.

From this research, it can be inferred that 36 hours of sprouting is the optimal duration to attain
maximum Protein and Lipids in the two chosen seeds. Additionally, exposing the germinating
seeds to light after 36 hours might have contributed to an increase in the biomolecule content in
both seed types.

10. Conclusion:

The purpose of the research was to investigate the effect of increasing sprouting duration on the quantity
of proteins and lipids in two types of legumes, Vigna radiata and Cicer arietinum. I aimed to determine
the optimal sprouting duration that would result in the highest protein and lipid content in the seeds.
This experiment was carried out in order to observe the results to the research question:
“ To what extent does the increasing duration ( 0,6,12,24,48 hours ) of sprouting affect the protein
and fat content (%) in Vigna radiata and Cicer arietinum measured through absorbance of 400 nm
and 450 nm through biuret and emulsion test of nutrients respectively at 25℃? ”
After collecting data from the experiment, we analysed it to investigate how increasing sprouting
duration affected the protein and lipid content of the seeds. Our findings showed that the quantities of
both biomolecules increased linearly up to 36 hours of sprouting incubation. However, after this period,
the protein and lipid content started to decline slightly. Based on these results, the researchers

17
concluded that there is a positive correlation between increasing sprouting duration and protein and
lipid content in the seeds, thus accepting the alternative hypothesis. The correlation between sprouting
duration and protein and lipid content was further supported by the high R² values obtained in the
experiment. The R² values of 0.784 and 0.695 in Graph 1, and 0.590 and 0.722 in Graph 2 indicate a
strong positive correlation between sprouting duration and protein and lipid content in Vigna radiata
and Cicer arietinum. In conclusion, the research findings suggest that increasing sprouting duration up
to 35 hours can result in a higher quantity of proteins and lipids in Vigna radiata and Cicer arietinum
seeds. This information may be useful in developing optimal sprouting conditions for legumes and
other seeds to increase their nutritional value.

11. Limitations and achievable improvements:

Even though the seeds were used from the same packet, seed dormancy of Vigna radiata and Cicer
arietinum might have varied as it is genetically controlled. As a result, even if the length of sprouting
is increased, the seeds' enzyme system may not be triggered. Furthermore, in addition to proteins and
lipids, compounds such as starch may have been released while crushing the seeds. In the inquiry, some
purification procedures for proteins, such as affinity chromatography, and for starch, such as Silica gel
chromatography, can be applied. Additionally, the research was conducted under controlled laboratory
conditions and may not reflect the impact of environmental factors on sprouting and nutrient content
in natural settings.

One possible improvement could be to include a wider range of seed types to obtain a more
comprehensive understanding of the effect of sprouting duration on nutrient content. Another
improvement could be to conduct the research under more diverse environmental conditions to
determine the effect of natural factors such as temperature and light on sprouting and nutrient content.
Future research could expand on this research by measuring the content of other nutrients to provide a
more complete picture of the nutritional value of sprouted seeds. Finally, more in-depth research on
the impact of different sprouting methods on nutrient content could be conducted to identify the most
effective and efficient methods of sprouting for maximum nutrient content.

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12. Further Studies:

The topic of the effect of duration of sprouting on nutrient content of seeds is an exciting field for future
research that holds great potential for gaining valuable insights into human nutrition and agriculture.
To fully explore this area, researchers could delve into a range of potential avenues of investigation.
For example, they could examine how sprouting affects the bioavailability of nutrients in seeds, which
would shed light on how the human body processes and absorbs these essential components.
Additionally, they could explore the impact of various sprouting conditions on nutrient content,
including factors such as light exposure, temperature, and humidity. By studying these variables,
researchers could gain a more complete understanding of how to optimize sprouting processes to
produce seeds with maximum nutritional value.

Another area of potential study involves examining the potential health benefits of consuming sprouted
seeds. Research in this area could help to elucidate the mechanisms through which sprouted seeds may
promote health and prevent disease. Additionally, researchers could investigate the differences in
nutrient content and health effects between different types of seeds, providing valuable insights into
which varieties may be most beneficial for human consumption.

One specific avenue of research that could be explored in conjunction with investigating the duration
of sprouting is reducing the quantity of anti-nutritional content of seeds, such as phytic acid, tannins,
lectins, and other compounds, could assist in identifying the proper length for soaking and sprouting.
Ultimately, a deeper understanding of the effects of sprouting on seed nutrient content and health
benefits could have important implications for both human nutrition and agriculture.

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13. Bibliography:
13.1 Websites:
● Hou, Dianzhi et al. "Mung Bean (Vigna Radiata L.): Bioactive Polyphenols, Polysaccharides,
Peptides, And Health Benefits". Nutrients, vol 11, no. 6, 2019, p. 1238. MDPI AG,
doi:10.3390/nu11061238. Accessed 17 Nov 2022.
● Nakai, S., and Anh Chi Le. "Spectrophotometric Determination Of Protein And Fat In Milk
Simultaneously". Journal Of Dairy Science, vol 53, no. 3, 1970, pp. 276-278. American Dairy
Science Association, doi:10.3168/jds.s0022-0302(70)86197-5. Accessed 17 Nov 2022.
● Benincasa et al. “Sprouted Grains: A Comprehensive Review.” MDPI. Multidisciplinary
Digital Publishing Institute, 17 Feb. 2019. Web. 14 October 2022.
<https://www.mdpi.com/2072-6643/11/2/421/htm>
● Phillips, F. “Vegetarian Nutrition.” Wiley Online Library, John Wiley & Sons, Ltd
(10.1111), 26 May 2005, onlinelibrary.wiley.com/doi/full/10.1111/j.1467-
● Johnson, Mary. "Protein Quantitation." Materials and Methods. N.p., 11 Nov 2022. Web.
12 Sept. 2019. <https://www.labome.com/method/Protein-Quantitation.html>.
● Mayer, Poljakoff. “The Science of Sprout Nutrition.” Default Store View, 2003,
sproutpeople.org/sprouts/nutrition/science/#Proteins.
● Li, Ying-Chao, et al. “The Effects of Germination on Chemical Composition of Peanut
Seed.” Food Science and Technology Research, Japanese Society for Food Science and
Technology, 20 Sept. 2014, www.jstage.jst.go.jp/article/fstr/20/4/20_883/_html/-char/en.

13.2 Journal And Articles:

● "Raw Sprouts: Benefits And Potential Risks". Healthline, 2022,
https://www.healthline.com/nutrition/raw-sprouts#TOC_TITLE_HDR_3. Accessed 30 Sept
2022.
● Singh, A. K., Jagbir Rehal, Amarjeet Kaur, and Gagan Jyot. "Enhancement of Attributes of
Cereals by Germination and Fermentation: A Review." Journal of Food Science,
● Kant, Lakshmi et al. "Barley". Genetic And Genomic Resources For Grain Cereals
Improvement, 2016, pp. 125-157. Elsevier, doi:10.1016/b978-0-12-802000-5.00003-4.
Accessed 7 Oct 2022.
● Ars.Usda.Gov, 2022,
https://www.ars.usda.gov/ARSUserFiles/64022000/Publications/Reddy/Kogetetal04WS52-6-
989.pdf. Accessed 7 Oct 2022.

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 ● Singh, Ajey et al. "Circadian Regulation Of Abiotic Stress Tolerance In Legumes". Abiotic
Stress And Legumes, 2021, pp. 105-136. Elsevier, doi:10.1016/b978-0-12-815355-0.00008-4.
Accessed 17 Nov 2022.
● "Chickpea (Cicer Arietinum) | Feedipedia". Feedipedia.Org, 2022,
https://www.feedipedia.org/node/319. Accessed 17 Nov 2022.
● Okoronkwo, Nnenna. “Estimation of Protein Content and Amino Acid Compositions in
Selected Plant Samples
● Using UV-Vis Spectrophotometric Method.” American Journal of Food Science and Health,
2017, 2 Oct. 2019
● *www.researchgate.net/publication/319057476_Estimation_of_Protein_Content_and_Amino
_Acid_Compositions_i
● n_Selected_Plant_Samples_Using_UV-Vis_Spectrophotometeric_Method.
● Tools.Thermofisher.Com, 2022,
https://tools.thermofisher.com/content/sfs/brochures/TR0057-Read-std-curves.pdf. Accessed
9 Oct 2022.
● Eva, Darko et al. “Photosynthesis under Artificial Light: the Shift in Primary and
● Secondary Metabolism.” Philosophical Transactions of the Royal Society B: Biological
● Sciences. N.p., 19 Apr. 2014.
● <https://royalsocietypublishing.org/doi/full/10.1098/rstb.2013.0243>
● ● Ferguson, J. M., and R.D Keys. "Seed and Seed Quality." NC State Extension. N.p., 1 Jan.
● 1991. Web. 11 Sept. 2022. <https://content.ces.ncsu.edu/seed-and-seed-quality>.
● ● Forcato, Diego. "Milk Fat Content Measurement by a Simple UV Spectrophotometric
● Method: An Alternative Screening Method." Journal of Dairy Science. N.p., 4 Oct. 2004.
● Web. 16 Sept. 2022.
● <https://www.researchgate.net/publication/8076957_Milk_Fat_Content_Measurement_b
● y_a_Simple_UV_Spectrophotometric_Method_An_Alternative_Screening_Method>.
● ● G, Rogério, et al. “Soaking Curve and Effect of Temperature on the Germination of Daisy
● Seeds.” Horticultura Brasileira, Associação Brasileira De Horticultura, June 2012,
● www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-05362012000200021.
● ● Ghani, Mirwais, and Krishnanand Kulkarni. “Glycine max Sprouts: A Review of Nutrient
● Composition, Health Benefits and Genetic Variation .” Plant Breed. Biotech, 2022

13.3 Books
Tools.Thermofisher.Com, 2022, https://tools.thermofisher.com/content/sfs/brochures/TR0057-Read-
std-curves.pdf.

14. Appendix

14. 1 Material required

Vigna radiata

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Cicer arietinum
Vernier spectrophotometer
Biuret reagent
Ethanol
Sodium hypochlorite

14.2 Raw Data Tables

Appendix Table 1a: Average absorbance for Protein content in Vigna radiata
Hours of Trials
Average% SD
sprouting 1 2 3 4 5 Difference to control %
0 0.055 0.055 0.056 0.055 0.054 0.055 0.006 0.000
12 0.061 0.062 0.062 0.062 0.063 0.062 0.006 12.727
24 0.08 0.082 0.08 0.082 0.081 0.081 0.008 47.273
36 0.082 0.084 0.085 0.084 0.085 0.084 0.001 52.727
48 0.083 0.08 0.082 0.083 0.082 0.082 0.001 49.091

Appendix Table 1b: Average absorbance for Protein content in Cicer arietinum
Hours of Trials
Average% SD
sprouting 1 2 3 4 5 Difference to control %
0 0.036 0.038 0.038 0.036 0.037 0.037 0.008 0.000
12 0.041 0.042 0.042 0.041 0.044 0.042 0.001 13.514
24 0.057 0.059 0.058 0.056 0.052 0.056 0.002 52.432
36 0.06 0.056 0.056 0.057 0.056 0.057 0.001 54.054
48 0.053 0.055 0.054 0.058 0.055 0.055 0.001 48.649

Appendix Table 2a: Average absorbance for lipid content in Vigna radiata
Hours of Trials Average
SD
sprouting 1 2 3 4 5 % Difference to control %
0 0.109 0.108 0.111 0.109 0.108 0.109 0.001 0.000
12 0.128 0.126 0.129 0.126 0.127 0.127 0.001 16.697
24 0.141 0.143 0.143 0.142 0.141 0.142 0.008 30.275
36 0.14 0.139 0.141 0.141 0.139 0.140 0.008 28.440
48 0.138 0.14 0.141 0.138 0.139 0.139 0.001 27.706

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Appendix Table 2b: Average absorbance for lipid content in Cicer arietinum
Hours of Trials Average
SD
sprouting 1 2 3 4 5 % Difference to control %
0 0.122 0.124 0.123 0.121 0.125 0.123 0.002 0.000
12 0.153 0.154 0.156 0.153 0.154 0.154 0.001 25.203
24 0.195 0.19 0.193 0.195 0.192 0.193 0.002 56.911
36 0.195 0.196 0.196 0.198 0.195 0.196 0.001 59.350
48 0.191 0.192 0.192 0.194 0.191 0.192 0.001 56.098

Table 3: Effect of sprouting on nutrient content in Vigna radiata. 24

Hours of sprouting Total Protein (mg/ml) Total Lipid (mg/ml)

0 0.107 0.0137

12 0.121 0.0162

24 0.163 0.0181

36 0.164 0.0175

48 0.162 0.0176

24
Tools.Thermofisher.Com, 2022, https://tools.thermofisher.com/content/sfs/brochures/TR0057-Read-std-
curves.pdf.

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 Table 4: Effect of sprouting on nutrient content in Cicer arietinum.

Hours of sprouting Total Protein (mg/ml) Total Lipid (mg/ml)

0 0.076 0.0155

12 0.084 0.0198

24 0.112 0.0252

36 0.111 0.0245

48 0.107 0.0243

Table 5: Percentage Fats

Hours of sprouting Vigna Cicer

0 100 100
12 118 127
24 132 162
36 127 158
48 126 156

Table 6: Percentage Proteins

Hours of sprouting Vigna Cicer

0 100 100
12 113 110
24 152 147
36 153 146
48 151 140

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14.3 Sample Calculations

1. After conducting the Biuret test on Vigna radiata after 24 hours of sprouting incubation,
the quantity of protein present was determined to be y = 0.0006x, where y represents the
absorbance rate and x represents the concentration of protein. The standard curve
equation for proteins used was y = 0.0005x + 0.0006, with the value of y at zero
concentration of protein subtracted from the equation. The absorbance rate recorded
was 0.081.

0.081
Therefore, 0.006
= 135 micrograms/ml
= 0.135 mg/ml

2. Based on the ethanol test conducted on Cicer arietinum after 24 hours of sprouting
incubation, the quantity of lipid present is 0.025079 mg/ml. This was calculated using the
standard curve equation for lipids, which is y = 127.95 x, where y represents the
concentration and x represents the absorbance. The absorbance value recorded was
0.196, which was multiplied by the slope of the standard curve (127.95) to obtain the
lipid concentration in micrograms/ml (25.079) before converting it to milligrams/ml.

3. ANOVA Test

An ANOVA test was conducted on the protein levels in Vigna radiata using the Biuret test. The f-
ratio value obtained from the test was 538.0141, with a p-value of less than 0.00001. This result
indicates that the difference in protein levels among the samples tested is significant at a confidence
level of 95%, with a p-value threshold of 0.05.

4. Average Protein and Lipids absorbance=

Example- Average absorbance in the amount of Protein in Vigna radiata at

Sprouting incubation of 36 hours (Biuret test)

0.080 + 0.820 + 0.080 + 0.820 + 0.081

5

= 0.081%

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