1. Vidya Pratishthan’s
VSBT College of Agricultural Biotechnology
Hands on Training (HOT 481)
Solid state fermentation of Bee Collected Pollen for
value addition
Submitted by:
Dhananjay Vitthal Shirsat
Reg No. VSBT-37/2010
Research Guide:
Dr. D. M. Wakhle
Department of Post Harvest and Food Biotechnology (FB)

2. Abstract
This project aimed to establish the principle conditions for obtaining a novel
natural food product from pollen, which will be safe and with improved
nutritional value, to be used as a dietary supplement or a functional ingredient for
formulating other foods. Bee-collected pollen subjected to lactic acid
fermentation using lactic acid bacteria Lactobacillus lactis, as a starter isolated
from whey and their effect on some of the natural characteristics of the pollen
were studied.
The optimum conditions for the pollen fermentation were provided that are 20%
inoculums volume, anaerobic condition for solid state fermentation, incubation at
350
C for first 96 hrs, then 200
C for next 72 hrs, and optimum moisture content was
35-40%. The process was characterized by production of lactic acid and decrease
in pH and sugar content.
(Key words: Solid state fermentation, Bee collected pollen, Pollen nutrition,

3. Introduction
 Pollen:
• A male reproductive cell of the flowering plants
• Rich in proteins, sugars, essential amino acids, fatty acids,
minerals and abundant in vitamins.
 Honeybees collect pollen to fulfillment of their protein
requirement
• Bee Pollen: It is refer to the pollen collected by the honey
bees in their hind legs (in pollen baskets) while foraging on
flowers
• Bee bread: It is refer to the stored pollen load in honey combs
by adding honey and bees saliva prior to feed to the other bees
and brood

4. Introduction
Figure 1: Honey bee (Apis mellifera) having pollen load in pollen basket on their hind
legs

5. Introduction
Bee Pollen Bee Bread
(Bee collected pollen) (Stored pollen load in combs)

6. Introduction
• Lactic acid bacteria (LAB) involve in the production of Bee
Bread
• It is demonstrated for first time that bee bread probably
fermented by the Honey stomach LAB flora that has been
added to the pollen via regurgitated nectar from the honey
stomach (Alejandra & Tobias, 2009)
• Recently the large flora of Lactic acid bacteria was identified
in honey stomach of Apis mellifera, Apis dorseta.
• In this study the lactic acid bacteria are used to ferment bee
collected pollen in control environmental conditions in
laboratory and the chemical changes were studied.

7. Objectives:
• To collect bee pollen and bee bread from honey bee colony.
• To separate whey from curd to use as a starter
• Solid state fermentation of bee collected pollen by Lactic acid
bacteria from whey.
• To estimate and compare nutritive value of fermented, non-
fermented pollen.

8. Materials
• The bee collected pollen is used as a substrate for fermentation
• The whey drained from curd and purified LAB culture used as
a starter( inoculums ) culture
• 250 ml Glass conical flasks are used as a fermentation vessels
for solid state fermentation process

9. Sample Collection
Pollen Collection:
Pollen collected from Apis mellifera bee hives located in area of
Jalgoan Village of Baramati Taluka
This hives are used for pollination of Helianthus annuus
(Sunflower) crop
 GPS Location of the site: N 180
23.275’
E
0750
02.592’
Collection of Whey from Curd:
• The overnight draining of whey using muslin cloth results in
complete separation of whey from curd

10. Pollen Collection
Fig. 1: Flow diagram of pollen collection method

11. Collection of Whey from Curd
• Whey is the liquid part of the milk which produce after
precipitation by acid or enzyme
• Whey is chosen as starter for fermentation because it contain
LAB as well as 20% of whey proteins which helps in
enrichment of the products nutritive value
• The overnight draining of whey using muslin cloth results in
complete separation of whey from curd
• 62 ml volume of whey is drained from 200 g of curd

12. Isolation of Lactic acid bacteria
• The lactic acid bacteria Lactobacillus lictis were isolated from
whey using MRS agar medium by spread plate technique.
• The Lactobacillus lictis were purified by streak plate
technique on MRS agar plate.
• The pure culture were grown in the MRS broth by incubating
for 48 hours to use as a inoculum for pollen fermentation.

13. Solid state fermentation of bee collected
pollen
The 3 sets of bee collected pollen are fermented.
• Set A: Bee collected pollen fermented by adding whey
directly
• Set B: Bee collected pollen fermented by pure culture of
Lactic acid bacteria (The LAB was isolated from curd using
selective MRS medium )
• Set C: The control set without any inoculation

14. Fermentation Conditions
• Inoculums volume: 20%,
• Environmental condition: Anaerobic condition
• Incubation period: 350
C for first 96 hrs.,
then 200
C for next 72 hrs.
• Moisture content: 35-40%.
• Process Characterization: The process was characterized
by production of lactic acid and
decrease in pH and sugar
content.
• Storage temperature: 40
C in refrigerator

15. Solid state fermentation of bee collected
pollen
Fig. 2: Sets of bee pollen fermentation

16. Methods
• Estimation of total acidity was done by acid-base titration with 0.1 N
Sodium hydroxide and phenolphthalein indicator (AOAC,1990)
• Protein content was determine by by Folin-Lowary method (Loway 1951).
• Sugar content was determine by Anthrone method.(Hedge J.E & Hofreiter
B.T, 1962)
• Determination of water content in samples by Method 935.29 (AOAC,
1997)
• Total poly-phenol content was estimated by using Folin-Ciocalteau reagent
(Carpes 2009)
• Total amino acid content estimated by Acetone-Ninhydrin reagent method
(Sadashivam 1992)
• Free radical scavenging activity of pollen was determined by ABTS assay
(Thaipong 2006)
• Estimation of mineral content by automic absorption spectrophotometry
(Method 986.11)(AOAC,2000)

17. Results
Total 320 gm pollen was collected in 4 days period in the month of December
and characterized by microscopic examination
Table 1: Pollen collection report of individual Bee hive for each day
Fig : Helianthus annuus (Sunflower) pollen under 400X microscopic zoom

18. Lactic acid fermentation:
Process characterization
• In all sets on first day the
acid level decreased and
sudden increased in two
sets inoculated with Whey
and LAB
• In control set the acid
content remains constant
• The acid production
indicated the active
fermentation processFig. 3: Lactic acid content in pollen during fermentation
process

19. Lactic acid fermentation:
Process characterization
• In all sets of fermentation
the sugar level found
decreased however time
period of fermentation
increased.
• In control set the sugar
content remains constant
with slight decreased.
Fig. 4: Effect of fermentation on sugar concentration

20. Lactic acid fermentation:
Process characterization
• After every 24 hrs
interval the pH of the
pollen were determined it
found decreased in both
whey and LAB
inoculated pollen
fermentation sets
• In control it remains
constant
• The fall in pH indiacates
acid production
Fig. 4: Effect on pH of pollen during fermentation process

21. Water content of the pollen
Table : Water content of the bee collected and fermented pollen
Pollen type Water content % Average(AM) SD
Bee collected pollen
(Control) 33.6 34.66 1.10
34.6
35.8
Whey fermented pollen 44.23 42.66 1.54
42.62
41.14
LAB fermented pollen 47.4 46.66 0.80
46.8
45.8

22. Protein content of the pollen
Table : Protein content of the bee collected and fermented pollen
(All analytical data are the mean values of triplicate analysis ± standard deviation)
Pollen type Protein content (mg/g)
Bee collected pollen (Control) 26.80 ±0.2 mg/g
Whey fermented pollen 42.66 ±0.28 mg/g
LAB fermented pollen 41.33 ±2.30 mg/g

23. Total sugar content of the pollen
Table : Total sugar content of the bee collected and fermented pollen
(All analytical data are the mean values of triplicate analysis ± standard deviation)
Pollen type
Total Sugar content
(g/100g)
Bee collected pollen (Control) 43.00 ±0.40 g/100g
Whey fermented pollen 16.01 ±0.27 g/100g
LAB fermented pollen 10.40 ±0.52 g/100g

24. Lactic acid content of the pollen
Table : Lactic acid content of the bee collected and fermented pollen
(All analytical data are the mean values of triplicate analysis ± standard deviation)
Pollen type Lactic acid content (%)
Bee collected pollen (Control) 4.68 ±0.38 mg/g
Whey fermented pollen 5.31 ±0.75 mg/g
LAB fermented pollen 6.03 ±0.65 mg/g

25. Total free amino acid content of the pollen
Table : Total free amino acid content of the bee collected and fermented pollen
(All analytical data are the mean values of triplicate analysis ± standard deviation)
Pollen type Total free amino acid content (mg/g)
Bee collected pollen (Control) 80.76 ±0.20 mg/g
Whey fermented pollen 88.07 ±0.02 mg/g
LAB fermented pollen 99.95 ± 0.13 mg/g

26. Determination of amino acids present in pollen
Table 4.13 Amino acids present in bee pollen and fermented pollen
[(+) :Amino acid Present and (-) : Amino acid absent]
Amino acids Detected in
pollen by TLC
Bee collected
pollen
Whey fermented
pollen
LAB fermented
pollen
Asparagine (Asn) + + +
Alanine (Ala) + + +
Threonine (Thr) + + +
Arginine (Arg) + + +
Proline (Pro) - + +
Methionine (Met) - - +
Unknown (Rf 0.07) + + +

27. Determination of amino acids present in pollen
Figure: TLC plate showing spots of amino acids separated from pollen samples.
Where, A: Bee collected pollen; B: Whey fermented pollen; C: Lactic acid bacteria
fermented pollen

28. Total poly-phenol content of pollen
Fig. : Total poly-phenol content in pollen

29. Mineral content of the pollen
Table : Mineral content of the bee collected and fermented pollen
Minerals In Pollen (mg/g)
Pollen type Ca Fe Mg Mn Cu Zn
Bee Collected pollen 0.154 0.116 7.032 0.017 0.003 0.171
Whey fermented pollen 0.423 0.146 7.612 0.035 0.021 0.532
LAB fermented pollen 0.362 0.96 9.403 0.205 0.071 1.032

30. Mineral content of the pollen

31. Radical scavenging activity of the pollen
Table : Radical scavenging activity of the bee collected and fermented pollen
(All analytical data are the mean values of triplicate analysis ± standard deviation)
Pollen type Radical scavenging activity (%)
Bee collected pollen (Control) 67.27 ±0.2%
Whey fermented pollen 80.00 ±0.3%
LAB fermented pollen 86.06 ±0.3%

32. Results & Discussion
• After fermentation of bee collected pollen the Protein content
found increase by 16.20 mg/g in Whey inoculated set and
11.20 mg/g in LAB pure culture inoculated set.
• The sugar content found decreased by 26%, 31.60% and
21.20% in Whey inoculated, LAB inoculated, Heat treated and
whey inoculated pollen respectively in 7 days fermentation
process.
• The lactic acid content found increased by 0.63%, 1.35% in
Whey inoculated and LAB pure culture inoculated sets
respectively.

33. Conclusion
As a result of this project the proteins were increased by
1.53%, total sugars were decreased by 32.6 %, lactic acid
content increased by 1.35%, total free amino acid content
increased by 1.99%, total poly-phenol content decreased by
1.8%, increment in all minerals and radical scavenging activity
increased by 18.86% in lactic acid bacteria fermented pollen.
So it is suggested that the lactic acid bacteria is a suitable
starter culture for the fermentation of the bee collected pollen.
The solid state fermentation of the bee pollen by LAB is
effective to increase its nutritional value.

34. Acknowledgement
I wish to thanks to Dr. Sushama Chaphalkar, Director,
Dr. A.S. Patil , Principal and Dr. Dhananjay Wakhle Research
guide to give me access to the laboratory for project work and
their guidance for my research project.
I also wish to thanks all staff members and laboratory
assistants and my friends for helping me in my laboratory
work

35. References
• Stefan Bogdanov (2012) ; Pollen: Production, Nutrition and Health: A
review ; Bee product science ,1-34 (www.bee-hexagon.net)
• Roulstone T H; Cane J H (2000); Pollen nutritional content and
digestibility for animals; Plant Systematics and Evolution 222(1-4),187-
209.
• Schmidt J O; Schmidt P J (1984); Pollen digestibility and its
potentialnutritional value; Gleanings in Bee Culture, 320-322.
• Jorde W; Linskens H F (1974); Persorption of pollen and diet intake; Acta
allergologica 29; 165-175.
• Baltrusaityte V; Venskutonis P R; Ceksteryte V (2007); Antibacterial
activity of Honey and bee bread of different origin against S.aureus &
S.epidermidis; Food technology and Biotechnology 45(2), 201-208.

36. References
• Koc A N; Silici S; Kasap F (2011); Antifungal activity of Honeybee
products against Candida spp. & Trichosporon spp. ; Journal of Med.
Food (7),1-7.
• Ceylan D A; Yetisir R (2003) Antifungal effect of pollen and propolis
extracts collected from different regions of Turkey 1132, Uludag Bee
Journal 3(3); 27-34.
• Value added products from beekeeping: Food and Agriculture
Organization (1996) FAO Agriculture service bulletin 126
(http://www.fao.org/docrep/w0076E/w0076E00.htm )
• Jolene (2007) Study on Nutritional Reinfrocer preparation of the Bee
Pollen by Solid State Fermentation; Tianjin University of Science and
Technology; Master degree thesis.

37. THANK YOU!