it is about probiotics study...

2. Characterization & Encapsulation of Probiotic Lactobacillus Cells
Isolated from Fermented Curd and Evaluation of their Survival
under Simulated Gastrointestinal Conditions
Group Members
JYOTHI PALLANTI (BF 13-016)
SAISANKAR MUKKANTI (BF 13-035)
SAMEERA BANU SHAIK (BF 13-036)
USHA SRI TADEPALLI (BF 13-045)
ADVISORY
ANIL KUMAR VUNDRU
Teaching Associate
Dept. of Food & Industrial Microbiology,
CFST

3. CONTENTS
 INTRODUCTION
 JUSTIFICATION
 OBJECTIVES
 REVIEW OF LITERATURE
 MATERIALSAND METHODS
 RESULTSAND DISCUSSION
 CONCLUSIONS
 FUTURE STUDIES
 REFERENCE

4. INTRODUCTION
 Gut health products with probiotics.
 Minimum of 10⁶ cfu/g - optimum functionality.
 Encapsulation
 Probiotic lactic acid bacteria (LAB) - health benefits
• antimicrobial compounds, compete with pathogenic organisms for
adhesion to intestinal epithelium,
• Prevent infectious diseases and allergies,
• Manage lactose in tolerance,
• Reduce serum cholesterol,
• Provide anti-carcinogenic activity,
• Enhance immune function .

5.  Better survival rate of probiotic cells were gained with less
amount of whey powder incorporation.
 Least concentration of sodium alginate use in this study may
help in minimising health hazards.
JUSTIFICATION

6. OBJECTIVES
 To isolate and screen potent lactobacillus sps from curd
for the probiotic properties which viz. Resistance against
bile salts, acid pH values antibiotics, antibacterial
activity etc.
 To encapsulate probiotic lactobacillus cells with whey
powder – alginate hydrogel capsule beads via extrusion
technology.
 To examine the protective effect and release profile of
fresh capsules under simulated gastrointestinal
conditions

7. REVIEW OF LITERATURE
 Xu et al., ( 2016 ), reported the encapsulation process was done with
L. casei cells along with alginate - PPI is a compatible material for the
encapsulation with an yield of 85.69% ± 4.82 was observed. This high
encapsulation yield supports the idea that PPI is a compatible material for
the encapsulation of L. casei.
 Xu et al., ( 2016 ), also studied that the free cells showed a
significant reduction(5.22 log cfu/g) in the no.of viable cells after 2 h of
incubation in SGF and 0.7 log cfu/mL of viable cells in the 1st 30 min.
 Sandoval - Castella et al., ( 2010 ), said synthesized L. casei
encapsulated with in alginate – pectin microcapsule as the encapsulation
yield of this system was reported in the range from 54% to 79%.
 Hazal et al., (2014), studied the properties of different probiotic
organisms and also reported about the encapsulation technology and cell
life in the food matrices.

8.  Roy Fuller et al., (1989), studied that probiotic is a live microbial
feed supplement which beneficially affects the host animal by
improving its intestinal microbial balance. He emphasized on the
requirement of viability for probiotics and introduced the aspect of
a beneficial effect on the host.
 Ouwehand et al., (1999), recognised as functional food
components, probiotics should demonstrate the following
properties: acid and bile-stability, resistance to digestive enzymes,
adhesion to intestine surface, antagonistic activity against human
pathogens, anti-carcinogenic and anti-mutagenic activity,
cholesterol- lowering effects, stimulation of the immune system
without inflammatory effects, enhancement of bowel motility,
maintainance of mucosal integrity, improvement of bioavailability
of food components and production of vitamins and enzymes.
 Anderson et al., (1991), studied dietary effect of sodium alginate on
human body and concluded that the ingestion of sodium alginate at
a high level for 23 days caused no effects other than those normally
associated with a polysaccharide bulking agent; in particular, the
enzymatic and other sensitive indicators of adverse toxicological
effects remained unchanged.

10. Materials Required
• AMMONIUM SULPHATE
• DI POTASSIUM HYDROGEN PHOSPHATE
• POTASSIUM DI HYDROGEN PHOSPHATE
• TRI SODIUM CITRATE
• MAGNESIUM SULPHATE HEPTA HYDRATE
• SODIUM ALGINATE
• CALCIUM CHLORIDE DEHYDRATE
• PEPSIN
• SODIUM HYDROXIDE
• HYDROGEN CHLORIDE
• MRS AGAR
• MRS BROTH
• PANCREATIN
• WHEY PROTEIN
• SODIUM CHLORIDE

11. Isolation of bacterial strain
• Curd sample
• Serial dilution
• Plating
• Incubation
• Restreaking
• Conc. of viable cells were set to 9 log cfu/mL (109 cfu/mL)
using McFarland Standard
METHODOLOGY

12.  Phenotypic characterization
 Morphological
 Biochemical
 Physiological ( Temperature 25 – 42 °C, pH 5 – 9 and
2.5 - 5% (w/v) NaCl )
 Characterization of isolates for probiotic properties
 The turbidity of inoculum was continued until
reached to 107 – 108 CFU/mL (0.5 McFarland
standards)

13. BILE TOLARENCE TEST
Growth of lactobacillus isolates in MRS broth along with addition of bile salts %
( 0.15, 0.3, 0.5 & 1)
Incubation (24 hr – 37 °c)
Monitering growth of cells (at 560nm for 0 hr till 7 hr )
Measure of bile tolerance
% resistance =
increase of OD in MRS broth with Bile salt
increase of OD in MRS broth without Bile salt
x 100

14. ACID TOLERANCE TEST
1 ml of inoculum + 10 ml of MRS broth (PH-2, 3, 5 & 7 as control)
Incubation (24 hr at 37 °C)
OD is observed(530 nm)
 The isolates showing resistance more than 50% at pH 3 were considered to
be acid tolerant strains
 % Resistance =
𝑖𝑛𝑐𝑟𝑒𝑚𝑒𝑛𝑡 𝑜𝑓 𝑂𝐷 𝑖𝑛 𝑀𝑅𝑆 𝑏𝑟𝑜𝑡ℎ 𝑤𝑖𝑡ℎ 𝑝𝐻 2,3 & 5
𝑖𝑛𝑐𝑟𝑒𝑚𝑒𝑛𝑡 𝑜𝑓 𝑂𝐷 𝑖𝑛 𝑀𝑅𝑆 𝑏𝑟𝑜𝑡ℎ 𝑤𝑖𝑡ℎ 𝑝𝐻 7
x 100

15. Antibiotic susceptibility test
o Solidification of Agar plates
o Inoculation
o Addition of Antibiotics
o Paper discs were added
o Incubation
o Resistance/Sensitivity

16. Antimicrobial activity
 Preparation of MHA plates
 Inoculation ( S. aureus - 109 CFU/ml)
 Preparation of wells
 Lactobacillus cell suspension
 Incubation
 The inhibition zone diameter of 10 mm and
above was considered as positive
antimicrobial effect.

17. Lactobacillus sps dry pellet + 5 mL MRS broth
Incubation (24 h at 37 ᵒC)
Streaking (MRS agar plates)
Incubation (24 - 48 h at 37 ᵒC)
Inoculation (MRS broth – single colony )
Incubation (24- 48 h at 37 ᵒC)
Centrifugation (8000 rpm – 10 min at 20 ᵒC)
Resuspension of cells (modified phosphate buffer)
(Conc. of viable cells were set to 9 log cfu/mL (109 cfu/mL) using McFarland
Standards)
PREPARATIONOFBACTERIALCELLSFORENCAPSULATION

18. Encapsulation
Sodium alginate slurry preparation at 80 °C (0.5%, 1.0%, 1.5%, 2.0%)
Add whey powder (0.1% )
Add bacterial cells to sodium alginate slurry (1/10 v/v)
Extrusion of capsules (31 G needle)
Hardening of capsules (0.05 M CaCl₂ solution – 10, 20, 30, 60 min )
Collect capsules and rinse with sterile dist. water
Referred as ‘fresh capsules’

19. +
whey powder

20.  1 g of fresh capsules + 9 g of modified phosphate buffer
 Incubation (room temp) + Agitation
 Plating
 Incubation (37 ᵒC – 48 h)
Formula :
NE × ME/N0 × V
N0 (cfu = mL) is the number of viable cells in cell suspension,
V (mL) is the volume of cell suspension used for encapsulation,
NE (cfu = g) is the number of viable cells within 1 g of capsules and
ME (g) is the mass of capsules obtained from encapsulation.
= ( 294 × 10 ) / ( 336 × 10 )
= 87.50%
Free viable cells = 336 x 109 CFU/g = 9.52 log CFU/g
ENCAPSULATION YEILD

21. Scanning electron microscopy (SEM)
 Analysis of the fresh capsules with and with out bacteria
were examined by SEM (Ruska labs TSVU, Hyderabad,
Telangana, India) at an accelerate voltage of 10 kV.

22. SURVIVALOF LACTOBACILLUS SPS IN SIMULATED
GASTRIC FLUID
SGF (2.0 g NaCl + 6.0 g pepsin + 7 mL con. HCl, pH- 2)
9 mL SGF + 1g fresh capsules
Incubation (30, 60, 90 & 120 min at room temp.)
Transfer into 9 mL of MPB
Dissolution
Plating & Spreading
Incubation ( 24- 48 h at 37 °C )

23. RELEASE OF LACTOBACILLUS SPS IN SIMULATED
INTESTINALFLUID
SIF (6.8 g KH₂PO₄ in 250 mL sterile water + 77 mL 0.2 N NaOH
solution )
Addition of pancreatin powder (1% w/v)
pH - 6.8
9 mL SIF + 1g fresh capsules
Incubation ( 20, 40, 60 & 120 min at room temp.)
Dissolution
Plating & Spreading
Incubation ( 24- 48 h at 37 °C )

25. Morphology of probiotic cells & Culture and biochemical
characteristics of pure isolate:
 After incubation of curd sample inoculated MRS agar plates at
37˚ C for 36 h, their morphology was identified as small,
creamy white, shiny, entire raised, translucent.
S.No Culture and biochemical characteristics Lactobacillus
isolate
1 Gram Reaction Positive
2 Morphology Rods
3 Pigmentation Absent
4 Motility test Non motile
5 Endospore test Non spore forming
6 Catalase test Negative
7 Sugar Fermentation Positive

27. Bile tolerance
0
10
20
30
40
50
60
70
80
90
100
110Resistance(%)
Conc. of bile salt (%)
Resistance (%) at 0hr Resistance (%) at 1hr Resistance (%) at 2hr Resistance (%) at 3hr
Resistance (%) at 4hr Resistance (%) at 5hr Resistance (%) at 6hr Resistance (%) at 7hr

28. Tolerance to various pH
87.63
90.9
95.27
100
59.54
67.43
73.61
100
52.45
60.52
69.03
100
0
20
40
60
80
100
120
2 3 5 7(control)
Resistance(%)
pH
Resistance at 0hr Resistance at 2hr Resistance at 4hr

29. Antibiotic susceptibility test
Name of the Antibiotic Property of probiotic
Lactobacillus
Cell wall synthesis inhibitors
1. Penicillin (10μg)
2. Ampicillin (10μg)
Sensitive
Protein synthesis inhibitors
1.Tetracyclin (10 μg)
2. Gentamycin (10 μg)
Sensitive
Resistance
Nucleic acid inhibitors
1. Ciproflaxacin (10μg)
2. Norflaxacin (10μg)
Resistance
Resistance

30. Antimicrobial activity:
 Antimicrobial activity was determined against a potent
pathogenic bacterium Staphylococcus aureus in MHA
plates after 24 h of incubation at 37˚ C probiotic bacteria
showed an excellent antibacterial activity with IZD value
of 11.21 mm.

31. Encapsulation Yield
79.4 80.03
87.5
81
71
75.14
80.47 79.88
65.08
57.98
69.82 69.82
64.5
69.72
78.42 76.54
0
10
20
30
40
50
60
70
80
90
100
10 20 30 60
EncapsulationYield(%)
HardeningTime (min)
EY at 0.5% EY at 1% EY at 1.5% EY at 2%

32. SEMAnalysis
Fig: Scanning electron microscopy images of the internal structure of a
capsule without bacteria (1000x).

33. Fig: Scanning electron microscopy images of the internal structure of a capsule
with bacteria (500X) indicates probiotic cells.

34. Survival rate (log CFU/g) in SGF
9.02
8.67
8.18
7.61
9.14
8.94
8.39
8.01
9.28
9.13
8.85
8.24
9.29 9.18
8.93
8.78
8.32
6.75
5.38
4.164
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
10
30 60 90 120
SURVIVALRATE(LogCFU/g)
INCUBATION TIME (Min)
0.5% alginate at 30 min HT 1% alginate at 30 min HT 1.5% alginate at 30 min HT
2% alginate at 30 min HT Free cells

35. Release Rate (log CFU/g) in SIF
0
8.96 9.12
9.35 9.47
0
8.51
8.72
9.03 9.21
0
5.49
7.66
8.43
9.07
0
4.63
6.81
8.02
8.56
0
1
2
3
4
5
6
7
8
9
10
0 20 40 60 80 100 120 140
Releaserate(logCFU/g)
Incubation Time (min)
0.5% alginate at 30 min HT 1% alginate at 30 min HT
1.5% alginate at 30 min HT 2% alginate at 30 min HT

36. Conclusion
 The bacteria we have isolated have shown high potential
regarding to acid and bile tolerance, SGF, SIF ands hence
we concluded that the organism we had isolated was a
probiotic
 The matrix and the encapsulation process considered
compatible with the probiotic strain showed an
encapsulation yield of 87.50% .
 These capsules showed strong protective effect against
probiotic cells under strong gastric and intestinal
conditions.

37. Future studies
 Sequencing for the determination of species
 Incorporation of probiotics into functional
foods

38. REFERENCE
Meng Xu, Francois, G.B., Marie, J.D., Subha, J. 2016.
Encapsulation of Lactobacillus casei ATCC 393 cell and evaluation of their survival
after freeze-drying, storage and under gastrointestinal conditions. Journal of Food
Engineering 168, 52-59.
Vinderola, C.G., Reinheimer, J.A. 2003.
Lactic acid starter and probiotic bacteria: a comparative ” in vitro” study of
probiotic characterstics and biological barrier resistance. Food Research
International 36, 895-904
Saraniya, A., Jeevaratnam, K. 2015.
In vitro probiotic evaluation of phytase producing Lactobacillus species isolated
from uttapam batter and their application in soy milk fermentation. Journal of Food
Science and Technology 52(9): 5631-5640.
Rajam, R., Kumar, S.B., Prabhasankar, P. 2015.
Microencapsulation of Lactobacillus palantarum MTCC 5422 in
fructoligosaccharide and whey protein wall systems and its impact on noodle
quality. Journal of Food Science and Technology 52(7): 4029-4041.