This is all about the development of low-calorie jam by replacing sugar with stevia utilizing the dried seabuckthorn berries.

1. DEVELOPMENT OF LOW CALORIE JAM USING DRIED
SEABUCKTHORN BERRIES
A DISSERTATION SUBMITTED TO
I.K. GUJRAL PUNJAB TECHNICAL UNIVERSITY
BY
Sakshi Thakur
Regd. No. 1732065
Masters of Science in Food Technology

2. INTRODUCTION
OBESITY
• Global epidemic affecting public health.
• Non-communicable disease.
Obesity prevalence among adults and youth (Hales, 2017)

3. • Many factors responsible for obesity.
• Also the cause of many diseases.

4. OXIDATIVE STRESS
• Obesity also contribute to oxidative stress (Marseglia, 2015; Styskal, 2012).

5. DIET AND OBESITY
• Increased consumption of high fat diet, fast food.
• Overconsumption of refined sugar, sugar-sweetened beverages (Yach, et al., 2006; Popkin, 2001).
• “Added sugar increase excess energy and reduce nutrient density in our diets, often contributing to
weight gain and obesity”- Elyse Powell.
Consumption of sugar in food and beverages (Source: NHANES)
228
308
275
390
325

6. • Many products high in sugar content – Jam, Jellies, Sweetened beverages etc.
• Jam only allows fruit to be preserved.
• Jam contains 40% fruits and 68-70% sugar.
• Jam is thought to be a contributor for obesity and type 2 diabetes (Baker, et al., 2005; Besbes, et al.,
2009).
• Replacement of sugar with low calorie sweetener can be beneficial.
• Stevia has zero calorie.
• Stevia is “Generally recognised as safe” by FDA.

7. SEABUCKTHORN BERRIES (Hippophae sp.)
• Best remedy for oxidative stress – Antioxidants
• Fruits and vegetables are rich in antioxidants.
• Seabuckthorn, a thorny shrub, belongs to Hippophae sp. meaning, “hippo” – horse and “phaos” –
shiny.
• Also called “ magic plant”.
• Seabuckthorn berries are excellent source of antioxidants (Khan, et al., 2010).
• Rich source of various vitamins such as B1, C, K etc.

8. • Seabuckthorn berries are delicate and highly perishable.
• Market linkage for seabuckthorn berries is poor.
• Many products of seabuckthorn berries are commercialised – jam, juice, squash.
• Seeds of seabuckthorn are used for oil extraction.

9. AIMS AND OBJECTIVES
AIM:
To evaluate the antioxidant properties of dried seabuckthorn berries and utilization of berries
for formulation of low calorie jam.
OBJECTIVES:
• Evaluation of proximate composition of dried seabuckthorn berries.
• To prepare hydro-alcoholic extracts of dried seabuckthorn berries and evaluation of
antioxidant properties using different antioxidant assays.
• To develop low calorie jam using dried seabuckthorn berries.
• To analyse the antioxidant properties of developed jam.
• To perform the sensory evaluation of developed jam.
• To evaluate the glycemic index of low calorie seabuckthorn jam.

10. METHODS
PROXIMATE ANALYSIS OF DRIED SEABUCKTHORN BERRIES:
• MOISTURE CONTENT - AOAC 2000, 934.06
• TOTAL ASH CONTENT - AOAC 2000, 900.02
• CRUDE FAT CONTENT - AOAC 1995, 920.39
• CRUDE FIBER CONTENT - AOAC 1990, 962.09
• REDUCING SUGAR - Schoorl method, 1929.

11. PREPARATION OF DRIED SEABUCKTHORN BERRY EXTRACT (Anish, 2014)
5g dried berry sample+200 ml of solvent (methanol/ethanol: water)
Continuous stirring in incubator shaker (180 rpm, 40˚C for 12 hrs.)
Centrifuge to obtain supernatant (20 minute at 7500 rpm)
Concentrated under vacuum (45˚C) and freeze dried
Stored in air tight container

12. In-Vitro ANTIOXIDANT ASSAY:
TOTAL PHENOLIC CONTENT ASSAY (Ferreira, et al., 2007)
Mixed properly 200 μl sample or standard and 4 ml 2% Na2CO3
Incubated for 2 minutes at room temperature
Added 200 μl of 50% of FCR into the test tube and vortex
Incubated in dark for 30 minutes at room temperature
Again vortex the test tubes
Measured absorbance at 720 nm

13. TOTAL FLAVONOID CONTENT ASSAY (Xu & Chang, 2007)
Mixed properly 500 μl sample or standard and 2 ml distilled water 150 μl 5% NaNO2
Incubated for 6 minutes at 25˚C
150 μl of 10% of AlCl3 was added
Again Incubated for 6 minutes at 25˚C
2 ml 1 mM NaOH and 200 μl distilled water was added
Measured absorbance at 510 nm

14. FERRIC REDUCING ANTIOXIDANT POWER ASSAY (Benzie, 1996)
Pipetted 100 μl sample in test tubes
2.9 ml of FRAP reagent was added
Incubated at 37 ˚C for 30 minutes
Measured absorbance at 593 nm
• Determination of lycopene content of seabuckthorn dried berries – Fish, 2002
• Determination of Vitamin C content of seabuckthorn dried berries – Dashman, 1996

15. PREPARATION OF DRIED SEABUCKTHORN BERRY JAM
SUGAR JAM STEVIA JAM
Weigh 6 g of seabuckthorn berries, 3 g of grapes and 3 g of apple Weigh 6 g of seabuckthorn berries, 3 g of grapes and 3 g of apple
Wash and grind the fruits Wash and grind the fruits
Add 13.75 g sugar & Mixed 0.3 g pectin and 0.075 g citric acid Add 1.375 g stevia & Mixed 2.2 g pectin and 0.075 g citric acid
added mixed content with grinded fruits and heat at 105 ˚C with continuous stirring
Heated till TSS of jam 66˚ Brix Heated till sheet test was positive
Filled hot into the sterilized bottle Filled hot into the sterilized bottle
Cooled Cooled
Stored in refrigerator Stored in refrigerator
Srivastava & Kumar, (2010)

16. PHYSICOCHEMICAL ANALYSIS OF JAM:
• Determination of pH - AOAC, 981.12
• Titrable acidity - AOAC, 2000
• Total soluble solid - ISO 2173 (1978)
PREPARATION OF JAM EXTRACT:
10 gm of Jam sample + 100 ml of solvent ( 100% methanol/70% ethanol: water)
Stir continuously on magnetic stirrer in dark (500 rpm for 3 hrs.)
Filtered to obtain supernatant
Supernatant stored in air tight container
• In-Vitro antioxidants of jam
• Determination of Total sugar content of jam – Aloh, et al., 2015
• Sensory Evaluation of Jam – Meilgaard, et al., 1999
• Glycemic index estimation – Marques, 2007

17. 9- POINT HEDONIC SCALE
9 Like Extremely
8 Like Very Much
7 Like Moderately
6 Like Slightly
5 Neither Like or Dislike
4 Dislike Slightly
3 Dislike Moderately
2 Dislike Very Much
1 Dislike Extremely
PARAMETER SAMPLE
A B C
Jam Colour
JamAroma
JamTaste:
a) Flavour
a) Sweetness
a) Sourness
a) Aftertaste
JamTexture
a) Spreadability
a) Smoothness
a) Consistency
Over all acceptability
SENSORY EVALUATION FORM
NOTE:
•The following sensory evaluation of jams is being performed as a part of M.Sc. dissertation
•Kindly report in case of any medical condition/allergies in the space provided below.
•Please provide your independent views without discussion with others.
•Kindly follow the instructions provided below carefully, for performing the sensory evaluation
NAME: …………………………………… PARTICIPANT NO: …………
PROFESSION: ………………………………... AGE: …………………
MEDICALCONDITION/ALLERGY: ………………………………………
(E.g., Diabetes, allergy etc.)
INSTRUCTIONS:
•Taste the coded samples provided to you and rate them based on the scale given in the table (on the right) depending on how much you like or dislike them.
•Rinse mouth with water before starting the evaluation and after tasting each sample and wait for 30 seconds to allow for the taste of the previous sample to disappear.
I affirm that the above performed sensory evaluation was conducted with my concept. I have followed the above stated instructions carefully and presented independent views.
Signature: Date: ………………..

18. PATIENT CONSENT FORM
Title of the study: A randomized, crossed over, open label study to determine the glycemic index of seabuckthorn low calorie jam in healthy volunteers.
Name of the participants:
Name of the Principal investigator: Dr. Shabir Sidhu
Name of the Co-investigator: Ms Sakshi Thakur
Name of the institution: IKG-PTU Main Campus, Kapurthala
Documentation of the informed consent
I,………………………………………. , exercising my free power of choice, hereby give consent to be included as a participant in “ A randomized, crossed over, open label study to determine the
glycemic index of seabuckthorn low calorie jam in healthy volunteers”.
I am over 18 years of age and am freely making a decision to be in this research study.
1. I have a read and understood this consent form and the information provided to me.
2. I have had the consent document explained to me.
3. I have been explained about the nature of the study.
4. My rights and responsibilities have been explained to me by the investigator.
5. I have been advised about the risks associated with my participation in the study.
6. I understand that my participation in this study is voluntary.
7. I understand that I am completely free at any time to refuse to participate.
8. I understand that there is no guarantee that this study will provide any benefits to me.
9. I consent to participate in this study.
I am aware, that if I have any question during this study, I should contact at one of the addresses listed above.
For Participants
Name:
Signature:
Date:
Investigator’s Certificate
I certify that all the elements including the nature, purpose and possible risks of the above study as described in this consent document have been fully explained to the subject. In my judgement, the
participant possess the legal capacity to give informed consent to participate in this research and is voluntarily and knowingly giving informed consent to participate.
Name: Dr. Shabir Sidhu Signature:
Name: Sakshi Thakur Signature:
Date: ………

19. Statistical Analysis
• Data presented as Mean ± S.D, except for sensory evaluation i.e. median.
• Statistical analysis used for FRAP, TPC , TFC, total sugar - Unpaired t test
• Statistical analysis used for Sensory evaluation - Wilcoxon Sign Rank test
• Statistical analysis tool used for Glycemic index – Paired t test
• p value ≤0.05 considered statistically significant

20. RESULTS
PROXIMATE ANALYSIS OF DRIED SEABUCKTHORN BERRIES:
(Data presented as mean percentage ± SD)
Material Moisture
(%)
Ash
(%)
Fat
(%)
Reducing sugar
(%)
Crude fiber
(%)
Dried
Seabuckthorn
Berries
16.35±2.36 2.46±0.11 20.9±9 1.3±0.00 5.33±0.55

21. In-Vitro ANTIOXIDANT ASSAY:
TOTAL PHENOLIC CONTENT ASSAY (Ferreira, et al., 2007)
(Data presented as mean ± SD; p≤0.05; a significant difference vs 70% methanol extract at 3 mg/ml; b significant
difference vs 70% methanol extract at 3 mg/ml; csignificant difference vs 70% methanol extract at 10 mg/ml;
dsignificant difference vs 70% methanol extract at 25 mg/ml)
Concentration
(mg/ml)
70% Ethanol extract
(μg GAE)
70% Methanol extract
(μg GAE)
3 37.744±0.007a 6.016±0.001
5 79.437±0.008b 20.000±0.003
10 142.917±0.028c 74.901±0.007
25 340.854±0.030d 278.619±0.01
50 497.873±0.028 494.676±0.08
0
100
200
300
400
500
600
3 5 10 25 50
μgGAE
Extract concentration (mg/ml)
Ethanol Extract
Methanol Extract
a
b
c
d

22. Concentration
(mg/ml)
70% Ethanol extract
(μg QE)
70% Methanol extract
(μg QE)
3 15.677± 0.002a 7.599±0.004
5 56.069±0.004b 38.200±0.006
10 172.588±0.008c 131.923±0.020
25 402.546±0.010d 359.348±0.003
50 907.373±0.076 848.291±0.052
TOTAL FLAVONOID CONTENT ASSAY (Xu & Chang, 2007)
(Data presented as mean ± SD; p≤0.05; asignificant difference vs 70% methanol extract at 3 mg/ml, bsignificant
difference vs 70% methanol extract at 5 mg/ml, csignificant difference vs 70% methanol extract at 10 mg/ml,
dsignificant difference vs 70% methanol extract at 25 mg/ml)
0
100
200
300
400
500
600
700
800
900
1000
3 5 10 25 50
μgQE
Extract concentration (mg/ml)
Ethanol Extract
Methanol Extract

23. FERRIC REDUCING ANTIOXIDANT POWER ASSAY (Benzie, 1996)
(Data presented as mean ± SD; p≤0.05; asignificant difference vs 70% methanol extract at 1 mg/ml,
bsignificant difference vs 70% methanol extract at 3 mg/ml, csignificant difference vs 70% methanol
extract at 5 mg/ml, dsignificant difference vs 70% methanol extract at 10 mg/ml, esignificant
difference vs 70% methanol extract at 25 mg/ml, fsignificant difference vs 70% methanol extract at
50 mg/ml)
Concentration
(mg/ml)
70% Ethanol extract
(mM FSE)
70% Methanol extract
(mM FSE)
1 0.248±0.084a 0.012±0.013
3 0.749±0.012b 0.116±0.018
5 1.097±0.048c 0.452±0.038
10 2.582±0.038d 1.234±0.020
25 4.579±0.030e 3.473±0.195
50 7.079±0.041f 6.237±0.184
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
1 3 5 10 25 50
mMFSE
Extract concentration (mM/ml)
Ethanol Extract
Methanol Extract
a
b
c
d
e
f

24. Vitamin C content and Lycopenene Content of Dried Seabuckthorn Berries
MATERIAL VITAMIN C CONTENT LYCOPENE CONTENT
SEABUCKTHORN 0.153±0.003 mg AAE/g 133.40±4.92 mg/g.
Proximate analysis of Dried Seabuckthorn Berry Jam
PROXIMATE ANALYSIS SUGAR JAM STEVIA JAM
FAT CONTENT (%) 0.65±0.12 0.66±0.11
FIBER CONTENT (%) 1.08±0.23 1.84±0.23
REDUCING SUGAR (%) 18.64±1.31 13.96±1.12

25. PHYSICOCHEMICAL ANALYSIS OF JAM
Jam TSS˚ Brix pH
Titrable acidity (%) w.r.t.
Citric acid Malic acid
Sugar Jam 66.3±1.15 3.23±0.05 0.120±0.17 0.126±0.17
Stevia Jam 57.3±3.05 3.16±0.05 0.124±0.01 0.130±0.11
(Data presented as mean ± SD)

26. SUGAR JAM STEVIA JAM

27. TOTAL PHENOLIC CONTENT OF DRIED SEABUCKTHORN BERRY JAM
Jams Total Phenolic content (mg
GAE/g)
Sugar Jam 4.68±0.95
Stevia Jam 12.52 ±1.83@
0
2
4
6
8
10
12
14
16
Sugar Jam Stevia Jam
mgGAE/g
@
(Data presented as mean± SD; p≤0.05; @significance difference vs sugar jam)

28. TOTAL FLAVONOID CONTENT OF DRIED SEABUCKTHORN BERRY
JAM
Jams Total Flavonoid content (mg
QE/g)
Sugar Jam 5.42±0.56
Stevia Jam 6.17±0.17@
0
1
2
3
4
5
6
7
8
Sugar Jam Stevia Jam
mgQE/g
@
(Data presented as mean ± SD; p≤0.05; @ significant difference vs sugar jam)

29. FERRIC REDUCING ANTIOXIDANT POWER ASSAY OF DRIED
SEABUCKTHORN BERRY JAM
0
50
100
150
200
250
300
350
Sugar Jam Stevia Jam
mMFSE/g
@
Jams
Ferric reducing antioxidant power
assay (mM FSE/g)
Sugar Jam
118.085±11.173
Stevia Jam 294.609±7.5358@
(Data presented as mean± SD; p≤0.05; @significance difference vs sugar jam)

30. TOTAL SUGAR CONTENT OF DRIED SEABUCKTHORN BERRY JAM
Jams Total Sugar Content
(mg/dL)
Sugar Jam 658.57±14.94
Stevia Jam 398.29±6.11@
0
100
200
300
400
500
600
700
800
Sugar Stevia
Totalsugarcontent(mg/gJam)
@
(Data presented as mean ± SD; p≤0.05; @ significant difference vs sugar jam)

31. DETERMINATION OF LYCOPENE CONTENT OF JAM
(Data presented as mean ± SD; p≤0.05)
Jams Lycopene content (mg/g)
Sugar Jam 18.06±0.60
Stevia Jam 20.07±2.66

32. GLYCEMIC INDEX EVALUATION OF JAM
Time
(minutes)
Sugar Jam
(mg/dl)
Stevia Jam (mg/dL)
0 88.9±12.644 80.8±13.693
30 110.4±11.824 99.3±30.324
60 80.8±11.545 73.3±19.183
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70
Bloodglucoselevel
Time
SUGAR STEVIA
(Data presented as mean ± SD)

34. SENSORY EVALUATION OF JAM
Parameters Sugar Jam
(A)
Stevia Jam
(B)
Control Jam
(C)
Colour 8 (7-9) 8 (7-9) 9(6-9)
Aroma 8(2-9) 8(4-9) 9(7-9)
Flavour 8(2-9) 8(5-9) 9(4-9)
Sweetness 7(3-9) 8(5-9) 9(3-9)
Sourness 8(3-9) 8(4-9) 9(1-9)
Aftertaste 8(3-9) 8(6-9) 9(4-9)
Spreadability 8(3-9) 8(5-9) 9(4-9)
Smoothness 8(2-9) 8(7-9) 9(7-9)
Consistency 8(7-9) 8(6-9) 9(7-9)
Overall
acceptability
8(4-9) 8(4-9) 9(4-9)
0
1
2
3
4
5
6
7
8
9
Colour
Aroma
Flavour
Sweetness
Sourness
Aftertaste
Spread
Smooth
Consistent
Overall
A B C
(Data presented as median (range). A-sugar jam, B-stevia jam and C- control jam

35. (A) Sugar Jam, (B) Stevia Jam (C) Control Market
Jam for Sensory Evaluation
A
B
C

36. • Dried berries good source of natural antioxidants for utilization as food additive
• Lycopene content of berries are higher than jam
• Stevia jam has higher antioxidant properties than sugar jam
• Lower glycemic index of stevia jam compared to sugar jam
• All the parameters in sensory evaluation were comparable in sugar jam and
stevia jam except for sweetness
• This study shows that the replacement of sugar with stevia can have beneficial
effect
CONCLUSION

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