This presentation includes the details about probiotics, its mechanism of action, its assessment and manufacturing process of supplements.

1. DEVELOPING A NEW PROBIOTIC
&
DETERMINING ITS ACTIVITY
Miss Ankita G. Chaudhari
B. Tech IIIrd yr (Food Tech)
Email ID: harshada.23aug@gmail.com
Contact No.: +91-9423545582
Guided by:
Dr. P.A.Pawar
(Associate Professor &
Guide)

2. Probiotics: Definition
• World Health Organization:
• “live microorganisms which when administered
in adequate amounts confer a health benefit on
the host”
• They:
• Survive stomach acid and bile
• Establish residence in the intestines
• Impart health benefits

3. • First identified in 1995
• Non-digestible food ingredients that stimulate the growth
and/or activity of probiotics
• Typically oligosaccharides: galacto-oligosaccharides
(GOS), fructo-OS (FOS), xylo-OS (XOS), Inulin
• Adding Prebiotics to Probiotics increases production of gut
Short-Chain Fatty Acids (SCFA)
Prebiotics
(Functional Food)

4. Synbiotics
• Products containing BOTH
– Prebiotics
– Probiotics

5. Gut Microflora(Human Microbiota)
• Microbiologically, the gut has three principal regions: the
stomach, small intestine, and colon.
• The stomach has very low bacterial numbers
• Facultative anaerobes such as lactobacilli, streptococci, and
yeast are present at ;100 colony forming units (CFU) per
millilitre due to the low environmental pH .
• The small intestine has a larger bacterial load that consists
of facultative anaerobes such as lactobacilli, streptococci,
and enterobacteria as well as anaerobes such as
Bifidobacterium spp., Bacteroides spp., and clostridia at
levels of ;104–108 CFU/ml.
• However, the colon, has a total population of 1011–1012
CFU/ml of contents

9. History
Joseph Lister-1878
Isolation of
Lactobacillus
Louis Pasteur- 1857
Potential Benefits of
Lactobacillus
Ellie Metchnikoff- 1905
Concept of Probiotics
Henry Tissier-1899
Isolation of
Bifidobacterium
Dr. Minoru Shirota-1930
First stable culture

10. Benefits
Probiotics
Stronger
Immunity
Improved
Digestion
Production
of vitamin
B12
Reduces
Cold and
Flu
Better
Breath
Healthier
Skin
Weight
Loss
Healing
from
Leaky Gut
and IBS

11. Diarrhea
Gas
Bloating
Acne
Cramps
Rashes
Side Effects

12. Types of Probiotic Strains
Lactobacillus
Bifidobacterium
Streptococcus
Sacchromyces

13. P
r
o
b
i
o
t
i
c
F
o
o
d
Kefir
Yogurt
Sauerkraut
Coconut
Kefir
Kombucha
Kvass
Natto
Raw
Cheese

14. Mechanism of Action
Enhancement of Epithelial Barrier
Increased Adhesion to Intestinal Mucosa
Competitive Exclusion of Pathogenic
Bacteria
Production of Antimicrobial Substances
Modulation of Immune System

15. Enhancement of Epithelial Barrier
• The intestinal barrier is a major
defense mechanism
• Defenses of the intestinal barrier
consist of the mucous layer,
antimicrobial peptides, secretory IgA
and the epithelial junction adhesion
complex
• Once this barrier function is
disrupted, bacterial and food antigens
can reach the sub-mucosa and can
induce inflammatory responses
• Probiotics may promote mucous
secretion as one mechanism to
improve barrier function and the
exclusion of pathogens
• Mucous production may be increased
by probiotics in vivo

16. Increased Adhesion to Intestinal
Mucosa
• Adhesion to intestinal mucosa is
prerequisite for colonization
interaction between probiotic strains
and the host
• Adhesion of probiotics to the
intestinal mucosa is important for
modulation of the immune system and
antagonism against pathogens.
• Probiotics also cause qualitative
alterations in intestinal mucins that
prevent pathogen binding
• Probiotic strains can also induce the
release of defensins from epithelial
cells. These small peptides/proteins
are active against bacteria, fungi and
viruses. Moreover, these small
peptides/proteins stabilize the gut
barrier function.

17. Competitive Exclusion of Pathogenic
Bacteria
• The ‘competitive exclusion’ term is
used for the scenario in which one
species of bacteria more vigorously
competes for receptor sites in the
intestinal tract than another species
• The mechanisms includes:
• creation of a hostile microecology
• elimination of available bacterial
receptor sites
• production and secretion of
antimicrobial substances and
selective metabolites
• competitive depletion of essential
nutrients
• Probiotic strains are able to inhibit the
attachment of pathogenic bacteria by
means of steric hindrance at
enterocyte pathogen receptors.

18. Production of Antimicrobial
Substances
• Probiotics form the LMW compounds
(< 1,000 Da), such as organic acids,
and produces antibacterial substances
termed bacteriocins ( > 1,000 Da)
• Organic acids, in particular acetic
acid and lactic acid, have a strong
inhibitory effect against Gram-
negative bacteria
• The undissociated form of the organic
acid enters the bacterial cell and
dissociates inside its cytoplasm
eventually lowering the intracellular
pH or accumulation of the ionized
acid can lead to the death of the
pathogen
• Probiotic bacteria are able to produce
so-called de-conjugated bile acids,
which are derivatives of bile salts
which show a stronger antimicrobial
activity

19. Modulation of Immune System
• Probiotic bacteria can exert an
immunomodulatory effect
• The immune system can be divided
between the innate and adaptive
systems
• The adaptive immune response
depends on B and T lymphocytes,
which are specific for particular
antigens
• The innate immune system responds
to common structures called
pathogen-associated molecular
patterns (PAMPs)
• The host cells that interact most
extensively with probiotics are
intestinal epithelial cells (IECs) and
can encounter DCs

20. Screening For
Acid And Bile
Tolerance
Tolerance To
Other
Inhibitory
Substances
Adhesion
Assay
Treatment Of
Bacteria With
Various Agents
Scanning
Electron
Microscopy
(SEM)
Antimicrobial
Activity Assay
Antibiotic
Resistance
Study
Hydrophobicit
y Cell Surface
Test
Transit
Tolerance In
Gastrointestina
l Tract
Identification
Of The Isolates
Probiotic Properties

21. Screening For Acid And Bile Tolerance
• Tolerance for pH is studied by incubating the isolates in
appropriate medium adjusted to pH 2.0 and 3.0. One
milliliter of overnight bacterial suspension is adjusted to 0.6
OD at 620 nm using a UV-Visible spectrophotometer, then
inoculated into 10 ml sterile medium and incubated at 37°C.
Samples are withdrawn periodically (at 0, 30, 60, 90, and
120 min) to determine the cell concentration by measuring
OD at 620 nm.
• The most pH tolerant isolates, based on survival rate, are
further studied for tolerance to bile salt concentration (0.3,
0.5, and 0.8% of bile salt in BHI/MRS broth) by
determining the cell concentration at the same time intervals
as above (measuring OD at 620 nm).

22. Tolerance To Other Inhibitory
Substances
• The best isolates of the tested, which were tolerant to both
pH and bile, are selected, based on their overall ranking
from the pH and bile testing and are tested for tolerance to
NaCl (3, 6, 9, and 12%) and phenol (0.2, 0.4, 0.6%) as
before.
• Overall ranking of the isolates is performed using the
average of survival rate and stability to 120 min.

23. Adhesion Assay
• For each adhesion assay, 0.5 ml of bacterial suspension is
mixed with DMEM medium (0.5 ml) and the final
concentration of bacteria was 2×108 bacteria/ml.
• The bacterial suspension is added to each well of the tissue
culture plate, which is then incubated at 37°C in 5 % CO2.
• After incubation for 1h, cells are washed five times with
sterile PBS, fixed with methanol, Gram stained and
examined microscopically under oil immersion.
• For each glass coverslip monolayer of Coca-2 cells, the
number of adherent bacteria is counted in 20 random
microscopic areas and expressed as number of bacteria
adhering to 100 Caco-2 cells.

24. Apical Media
CaCo-2 Monolayer
Basolateral Media

25. Treatment Of Bacteria With Various
Agents
• Bacterial cells are incubated with trypsin, lipase and pepsin
(2.5 mg/ml, Himedia) for 60 min at 37°C and with sodium
metaperiodate (10 mg/ml, Himedia) for 60 min at room
temperature.
• These bacteria are then used for the adhesion assay as
described above.

26. Scanning Electron Microscopy (SEM)
• Cells for scanning electron microscopy are grown on glass
coverslips.
• The specimen is then examined with a scanning electron
microscope to confirm the adhesion of the isolates to the
Caco-2 human intestinal cells

27. Antimicrobial Activity Assay
• The inhibitory potential of the isolated strains is
investigated using a modified agar well assay method.
• Indicator organisms such as Salmonella, Escherichia coli,
Klebsiella etc. are used. Overnight cultures of these
pathogens are swabbed on nutrient agar plates in which
wells are cut.
• Supernatants (50 μl) of 12 h isolated cultures grown in
BHI/MRS broth are added to the wells. They are further
incubated for 24 h at 37°C.
• Activity is quantified by measuring the diameter of any
clear zone.
• Supernatant from medium broth without inoculum is used
as control.

29. Antibiotic Resistance Study
• Antibiotic resistance patterns of the strains are determined
by a disk diffusion method using the Kirby-Bauer
technique.
• Muller-Hinton agar plates are plated evenly with 50 μl of
isolates using a sterile swab.
• Antibiotic discs are placed over the plates, which are then
incubated for 24 h–48 h at 37°C.
• The susceptibility and resistance of the strains are
determined as per recommendation of NCCLS.

31. Hydrophobicity Cell Surface Test
• The degree of hydrophobicity of the strains is determined
based on adhesion of cells.
• Cultures are grown in 10 ml of appropriate broth,
centrifuged at 6,000g for 5 min and the cell pellet is washed
and re-suspended in 10 ml of Ringers solution (6% NaCl,
0.0075% KCl, 0.01% CaCl2, and 0.01% NaHCO3).
• The absorbance at 600 nm is measured (ODA). Then 4 ml
of cell suspension is mixed thoroughly by vortexing with an
equal volume of n-hexadecane, chloroform and ethyl
acetate.
• The two phases were allowed to separate for 30 min and the
absorbance of the aqueous phase (ODB) was read at 600
nm.

33. Transit Tolerance In Gastrointestinal
Tract
• A simulated gastric juice is prepared by suspending 3mg/ml
pepsin (1:3,000) in sterile saline and adjusted the pH to 3.0
with 1.0 M HCl.
• 1.0 ml of 24 h old cultures is subjected to centrifugation
(10,000 rpm, 10 min) and washed twice with sterile saline
before being re-suspended in simulated gastric juice.
• Resistance is assessed in terms of viable colony count and
enumerated after incubation at 41°C for 2 h.
• After 120 min of gastric digestion, cells are harvested and
suspended in simulated intestinal fluid which contained 1
mg/ml pancreatin and 7% fresh chicken bile at pH 8.0.
• The suspension is incubated at 41°C for 6 h and the viable
count is determined.

35. Identification Of The Isolates
• The isolates with the greatest probiotic effect are identified
by biochemical and molecular characterization.
• The DNAs are isolated, amplified by PCR and then the 16S
rRNA genes in the PCR products are sequenced. The
primers used were universal primers 8F 5’-
AGAGTTTGATCCTGG CTCAG-3’and 1492R 5’
GGTTACCTTGTTACGACTT-3’.
• The sequences are submitted to the GenBank database and
accession numbers were obtained.

37. ALTERATION IN COMPOSITION AND
INTESTINAL MICROFLORA
• The intestinal microflora within a given individual is
remarkably stable
• Administration of probiotics to either newborns or
adults results in certain changes in the microbial
profiles and metabolic activities of the feces
• Changes are minor
• Probiotic administration results in an increase in fecal
counts of bifidobacteria and lactobacilli
• A decrease in fecal pH
• A decline in those bacterial enzyme activities that are
associated with the development of colon cancer.

39. INDUSTRIAL MANUFACTURING OF
PROBIOTICS

40. SOURCING OF PROBIOTIC
• Storage Stability: -
• comprehensive series of testing and quality criteria
• data proving 100% survival through the date of expiration at the specified
storage temperature
• Survival in The Upper Digestive Tract: -
• stomach, where it dissolves, releasing the organisms into the acidic, pepsin-
rich gastric fluid
• small intestine, where they encounter bile and pancreatic enzymes
• careful evaluation of in vitro research data on specific strains
• selects strains that exhibit natural tolerance to the conditions of the digestive
tract.
• Adhesion to The Intestinal Lining: -
• Colonization is a requisite for proliferation and production of beneficial
metabolites that mediate local and systemic health benefits
• Only the strains that exhibit a natural ability to adhere to intestinal cells are
selected as raw ingredients

41. TRIAD OF PROBIOTIC QUALITY
Viability
Triad
Timing
Temp.Moisture

42. VIABILITY TRIADS
• Temperature Control: - The most familiar variable affecting probiotic
viability is temperature. An ambient temperature must be maintained
within a specific range throughout all stages of receiving, handling and
manufacturing. An equally important consideration is the control of
temperature fluctuations. Freezing and thawing probiotics too quickly
causes lethal condensation. This fluctuation in temperature is a major
cause of potency reductions during standard manufacturing processes.
• Moisture Control: - Control of moisture in the air and in the probiotic
material itself is even more important to survival than control of
temperature. One of the advantages of low relative humidity is that
manufacturing temperatures can be higher without loss of viability.
• Timing: - Operating efficiently within strict time limits reduces
incidental exposure to both humidity and temperature. Accordingly,
timing is incorporated into relevant standard operating procedures
(SOP).

43. PROBIOTIC MANUFACTURE
• Arrival: - Attention to timing begins when raw material is
purchased. Ordering small volumes minimizes the time required to
adjust the material from storage conditions to manufacturing
temperature.
• Prior to Manufacturing: - Opening the package immediately
would expose cold material to moisture in the air, resulting in lethal
condensation. This is a common cause of potency reductions during
typical manufacturing processes. Conversely, tempering effectively
minimizes condensation and improves survival.
• Manufacturing: - Adherence to time limits is particularly important
at stages prior to encapsulation, such as weighing, blending and
encapsulation. These operations expose a large surface area of
powdered material to air without the protection afforded by capsules
and bottles. These sensitive steps must be regulated such that the
powder remains as cool and dry as possible.

44. MOISTURE CONTROL
• Humidity Control
• Tempering: - In this process, stored raw material is gradually
adjusted to the manufacturing temperature prior to blending
and encapsulation.
• Low-exposure Blending: - During the blending process,
compressed, dry air is used at a low, tightly controlled dew
point, such that condensation is nearly impossible.
• Efficient Timing: - While systemic control of ambient
conditions is fundamental, this practice alone does not always
guarantee finished product viability. Moisture within the
blended material itself is an equally imperative consideration.
To minimize moisture inside the product itself, probiotics are
formulated with minimal water activity (Aw).

45. MOISTURE METER

46. TEMPERATURE CONTROL
• Proper temperature control encompasses two requirements:
1. a cool ambient temperature
2. minimal temperature fluctuations
• Sourcing and Formulation: - Anticipation of occasional lapses in proper
storage on the part of the consumer are taken into consideration at this
stage, and the amount of live cells per capsule may be increased slightly to
ensure that labeled potency is maintained.
• Receipt of Raw Ingredients: - Raw material is received in refrigerated
packaging or in unheated trucks during the cold months. Quality inspectors
have less than 20 minutes to enter the stock into the system, and material is
kept on ice as samples are collected for independent potency and purity
testing.
• Tempering: - In addition, pre-tempered probiotics are maintained at 4°C,
instead of freezing temperatures (~-20° C). This allows for a milder shift in
temperature, further protecting the cells from condensation. Tempering
protocols are fine-tuned according to the package weight and the stage of
manufacturing.

47. • Blending: - The blending procedure is executed using compressed dry air
and maintaining a low dew point, making condensation nearly impossible.
Keeping probiotics dry in this manner increases their tolerance to any
unfavorable temperature changes.
• Encapsulation and Bottling: - Encapsulation and bottling runs are kept
small to minimize any untoward effects of ambient moisture and
temperature. The use of low-moisture transfer bottles and desiccants also
helps protect probiotics from moisture, a factor that can sensitize them to
temperature.
• Shipping: - Following shipment to hot climates, independent laboratory
potency testing confirms that these practices maintain label claim potency,
even if the product arrives warm.

50. STRAIN AUTHENTICATION
• The Human Microbiome Project, has identified more than 10,000 microbial
species in the human body.
• Since probiotic chemical makeup can change spontaneously, conventional
ingredient test methods are not definitive.
• The sequence of probiotic DNA is different for every genus and species,
and is the blueprint from which its functional health benefits materialize.
Therefore, proof of genetic identity is indispensable in the sourcing of these
organisms for clinical use.
• However, a small strip of DNA in the genome, known as the ribosomal
RNA gene, bears a sequence that typifies genus and species, providing a
convenient “bar code” for identification. Microbial genetics laboratories
have carried the routine sequencing of this gene to methodological
perfection, enabling precise identification.
• Within a species, many unique strains may exist. However, bacterial strains
display unique patterns of lipids in their membranes, forming a signature
that can be detected by chromatography. Known as fatty acid methyl ester
analysis (FAME), this analysis can reveal a strain-specific signature that
matches an authoritative reference.

51. PROBIOTIC POTENCY TEST
• Enumeration of bacteria has been a routine practice in microbiology
for over 100 years.
• The gold-standard method, known as viable plate count, is used as
exclusive quantification of live bacteria. In this method, a known
weight of probiotic material is suspended in culture, applied to a
nutrient plate, and incubated. Each living cell develops into a single,
discrete colony that is visible to the naked eye.
• A colony, technically referred to as a colony forming unit (CFU), is
equal to one viable cell. Repetition of this test with multiple samples
and plates maximizes accuracy. On probiotic labels, results are
expressed in CFU per serving.
• Since probiotic cells are sensitive to their environment, potency is
subject to change. Therefore, potency must be determined after
manufacturing, shipping and storage.
• Potency refers to the number of viable cells at a single point in time.

52. PROBIOTIC SHIPPING
• Strategic Formulation: - To maximize stability, select hardy
strains. If necessary, capsules may be overfilled to ensure that
potency is maintained in the event of prolonged exposure in
shipment.
• Temperature and Humidity Control during Manufacturing: - As
handling and manufacturing practices impact the stability of the
finished product, a comprehensive dedication to protecting each
ingredient and product, from start to finish, remains a fundamental
aspect of probiotic quality assurance promise.
• Timely Shipping
• Cold Packaging: - All probiotics, including shelf-stable products,
are shipped in insulated coolers with foam refrigerant bricks.
• Independent Laboratory Testing: - All finished products are tested
for potency following shipping to 3rd party laboratories without
cold packs.

54. PROBIOTIC DELIVERY INNOVATION
• To reach the destination, probiotics must prevail over multiple challenges in
the upper digestive milieu. The first and most formidable adversity is the
acidic environment of the stomach. Thus far, these studies have clearly
asserted that probiotic organisms exhibit varying degrees of natural acid
resistance, and the magnitude of this fortuitous attribute is dependent upon
genus, species and strain.
• Natural Acid Tolerance: - Members of the genus Lactobacilli express a
transporter that readily pumps acid protons across membranes. This
maintains intracellular pH, lending intrinsic acid tolerance to most species
of Lactobacilli. However, many Bifidobacteria lose viability at acidic pH
values below 3.0. The extent of vulnerability varies considerably across
species and strains.
• Hypoallergenic Intestinal Delivery: - Intestinal delivery technology
remains an area of interest in probiotic product development. However, a
recent innovation in acid-resistant capsule technology has provided a
delivery option that meets the highest standards of purity.

56. EVIDENCE-BASED PROBIOTICS
• Verifying Identity: - While DNA sequencing can precisely
verify the genus and species, strains are difficult to
distinguish through common genetic methods. Strains vary
in the fatty acid composition of their membranes, and
differences in these patterns are amenable to comparative
analysis. When conducted in combination with DNA
sequencing, this method, known as Fatty Acid Methyl Ester
(FAME) analysis, affords accurate identity verification.
• Verifying Potency: - While verifying colony forming units
(CFU) number upon completion of manufacturing is
straightforward, ensuring its accuracy at the time of patient
consumption requires careful attention to manufacturing
practices.

57. VERIFYING PROBIOTIC STABILITY
• Sourcing: - Selects probiotic raw ingredients
that exhibit natural hardiness through their
dates of expiration.
• Formulation: - Formulate probiotics to
minimize contact of bacteria with water
molecules.
• Manufacturing: - Operating within an
appropriate temperature and humidity range is
essential to maintaining viability and stability.

59. CONCLUSION

60. Probiotics, perhaps in combination with
prebiotics, may become an important means of
preventing and treating disease. In fact, several
types of diseases have been successfully treated
with probiotics. This practice, however, may
represent only the “tip of the iceberg” because
the potential benefits of probiotic therapy
promise to be almost limitless.

62. All probiotic supplements are
basically the same…
• TRUE • FALSE

63. Number of probiotic strains live in our
body…

64. Do supplement probiotic strains stay
in our body…
TRUE FALSE

65. Weight and number of count of
probiotic cells in our body….
10 times cells in our
body
3.5lbs.=1.58gm