probiotic products, factors affecting probiotics viability: microbiological parameters, food, processing and storage parameters, factors affecting viability of probiotics in gut, regulations for probiotics ( FSSAI regulations, FAO/WHO regulations), comparison between regulations of different countries, regulatory framework: issues of concern, future recommendations

1. SURVIVAL OF PROBIOTICS:
EFFECT OF PROCESSING AND
STORAGE CONDITIONS
Submitted by:
Neha Mahto
M.Tech FTM

2. CONTENTS
1. Probiotic products
2. Factors affecting probiotics viability
a) microbiological parameters
b) food parameters
c) processing parameters
d) storage parameters
2. Factors affecting viability in gut
3. Regulations for probiotics
a) FSSAI regulations
b) Labelling requirements
c) FAO/WHO guidelines
4. Comparisons between regulations
5. Regulatory framework : issues of concern
6. Future recommendations

3. DISSCUSION TILL NOW...
Introduction to probiotics: its definition
“live microorganisms, which when administered in adequate
amounts confer a health benefit to the host”
a) Most common probiotic strains - Bifidobacterium and
Lactobacillus.
b) Desirable properties of probiotics, mode of action of probiotics
c) Health benefits of probiotics
d) viability significance

4. PROBIOTIC FOODS

5. Few Probiotic products in India...

6. PROCESSING OF YAKULT

7. Factors affecting probiotics viability
Storage
parameters
Processing
parameters
Food
parameters
Microbiological
parameters

8. Microbiological parameters
strain
Inoculation
Rate
Inoculation
Proportion
PROBIOTICS
STRAIN
1. Selection according to
compatibility with and
resistance to the product and in
vivo conditions
2. compatibilities between the
strains and the food production
steps, food matrix and storage
conditions.
3. should also result in adequate
sensory characteristics of final
product.

9. Food parameters
FOOD INGREDIENTS 5
OXYGEN 3
pH 1
.
TITRATABLE ACIDITY2
WATER ACTIVITY4
Low pH restricts the growth
and stability of probiotic
bacteria in fermented products.
H-ions damage probiotic cells
Optimal pH :5.5-6.0
final acidity and the
concentration of lactic acid
and acetic acid of the
product
molecular oxygen is
detrimental to probiotic
growth and survival
Food additives : salt, sugar,
flavoring agents
Growth and protective factors:
vitamins, NPN The amount of water
remaining after drying affects
not only viability of bacteria,
but also the rate of loss of
viability during storage.

10. Presence of oxygen
directly toxic to some cells
toxic peroxides produced in
presence of oxygen
free radicals produced from the
oxidation of components (e.g., fats)
are toxic
Oxygen affects probiotics in three ways:
level of oxygen
within package
during storage of
probiotic should
be as low as
possible to avoid
toxicity and death
of microorganism

11. Food ingredients
Ingredients in food can be :protective, neutral, or
detrimental
Growth promoters ( glucose, vitamins, minerals, casein,
WPH) added in dairy products to increase the growth of
probiotics
Viability of L. acidophilus and Bifidobacteria was improved by
addition of cysteine, WPC & tryptone (Dave & Shah, 1998).
Higher levels of few ingredients can inhibit the growth.
Ex- Curing agents such as sodium nitrite (meat)

12. Processing parameters
FERMENTATION
CONDITIONS
Temperature
Exposure to oxygen
Resistance to heat
stress
DRYING
(freeze/ spray/
microwave/ vacuum)
 spray drying can
lead to loss of viability
due to high
temperature,
dehydration, osmotic
pressure, mechanical
stress
FREEZING AND
THAWING OPERATIONS
Cells wall damage by
mechanical stresses of
the ice crystals,
by condensation of
solutes, or by
dehydration of the cells

13. Continued…
REHYDRATION OF DRIED
PROBIOTIC PRODUCTS
The rate of recovery of the
probiotics to viable state is
influenced by
rehydration conditions
(time, temperature, volume
of media)
physical properties of the
material, osmolarity, pH
MICROENCAPSULATION
entrapment/enclosure of
microorganisms cells by means
of coating them with proper
hydrocolloid(s); in a way that
results in appropriate cell
release in the intestinal
medium

14. Storage parameters
Increasing the RH of the environment at which
the samples are stored cause an increase in water
mobility and the rate of loss in viability
.
Probiotic food products should preferably be stored at a
temperature of 4–5 °C
Use of plastic films with high oxygen barrier
properties and active packages with
oxygen absorbers
321
TEMPERATURE
RELATIVE HUMIDITY
PACKAGING

15. FACTORS AFFECTING VIABILITY IN GIT
• resistance to lysozyme is a criterion for the selection
of new probiotic strains.
Saliva
• the degree of stomach acidity,
• the length of exposure to acid,
• Generally, pH 1 -3 and mean exposure times 90 min
Gastric acidity
• the concentration of and length of exposure to bile
salts,
• the level of bile salt hydrolase activity,
Bile acids and enzymes in intestine

16. Continued...
• Competitive exclusion of pathogens
Colonic environment
• Fresh dairy products are the most common
product forms of probiotic delivery
• the presence of a fermentable carbohydrate also
improves a culture’s ability to survive simulated
gastric environment
Food matrix

17. REGULATIONS FOR
PROBIOTICS

18. The above regulation came on the date of
publication (23 Dec 2016).
The obligation of Food Business Operator to comply
with the regulation is effective from 1st Jan 2018.
Food Safety and Standards (Health Supplements,
Nutraceuticals, Food for Special Dietary Use, Food for
Special Medical Purpose, Functional Food and Novel
Food)Regulations,2016.
Probiotics regulations in the section of ‘Food with
added probiotic ingredient’
REGULATION IN INDIA

19. FSSAI REGULATIONS
01
02
03
04
No food business operator shall use probiotic ingredients in
food except the probiotic culture of microorganisms
approved
Probiotic preparations may contain added prebiotics
permitted under these regulations.
The viable number of organisms in food with added probiotic
ingredients shall be ≥108 CFU/g
The labelling, presentation and avertisement shall not claim
that the probiotic food has property of preventing, treating
or curing a human disease

20. LABELLING REQUIREMENTS
01
02
03
The words “PROBIOTIC FOOD”
genus and species including strain
designation or culture collection
number
viable numbers at the end of the shelf-life
of probiotic strain corresponding to the
level at which efficacy is claimed

21. 04
05
06
Recommended serving size which shall
deliver effective viable dose of probiotics
related to health claims
An advisory warning ‘NOT FOR
MEDICINAL USE’ prominently written
07
A warning or any other precaution to be
taken while consuming, known side effects
Proper storage conditions, and time
limit for ‘Best Use’ after opening the
container

22. Label of Yakult

23. FAO/WHO GUIDELINES FOR
EVALUATION OF PROPERTIES OF
PROBIOTICS

24. Strain identification by phenotypic and
genotypic methods: Genus, species, strain
Safety assessment
• In vitro and/or animal
• Phase 1 human study (safety)
phase 2 human trial (efficacy) or other
appropriate design to determine if
strain/product is efficacious
Probiotic Food
Labelling

25. In vitro tests
Resistance to gastric
acidity
Adherence to mucus &/or
human epithelial cells
Ability to reduce
pathogen adhesion to
surface
Bile acid resistance
Bile salt hydrolase
activity
3
1 2
4
5 6
Antimicrobial activity

26. Resistance to acidity
Strain growth in MRS broth at 37°C/48 h
Centrifugation ( 8,000 rpm for 10 min/4°C)
pellets washing in PBS & resuspension
studying survival at low pH (pH 1, 2 and 3 prepared in
PBS)
suspensions incubation at 37°C samples removed after
every 1 h up to 4 h.
Counting of surviving cells (by plating on MRS agar)

27. Bile acid resistance
culture growth in MRS broth (2% (w/v) of bile salts mixture) at
37°C for 24 and 48 h.
1 ml of culture (different dilutions) on MRS agar.
Incubation (37°C/48 h)
cell count comparison with control MRS agar plates (without bile
salts mixture).

28. Antimicrobial activity
culture growth in MRS
centrifugation (20 min/4°C, 8,000 rpm)
pH of supernatant adjusted to 5.5
Nutrient agar inoculation with indicator organisms
(E.coli, B.aureus)
Punch well into solidified plates
pour cell free extract (100 μl) in wells
measure zone of clearance around wells (well
diameter + ZoI)
Zone of
inhibition

29. Bile salt hydrolase activity
Bile salt plates preparation with sodium salts of
taurocholic acid (TC), taurodeoxycholic acid etc.
& MRS agar.
Streak strain on plates & incubate (37°C/72 hrs)
Presence of precipitated bile acid around
colonies is a positive result. Fig: BSH activity in L.salivarius

30. Comparison between Regulations
S.No. PARAMETERS INDIA MALAYSIA
1. Regulatory
category of
probiotics
Drugs and functional
foods.
Drugs/biologics/die
tary supplements or
food ingredient/live
biotherapeutic
products.
Functional foods.
2. Regulatory
authority for
approval
1. Functional food
regulated by FSSA
2. Drugs regulated by
FDA.
1. Drugs/ biologics
need approval
from FDA
2. Dietary
supplements
and food
ingredients are
approved by
Dietary
Supplement
Health and
Education Act
(DSHEA).
Functional foods
regulated by
1. FSQD (Food
Safety & Quality
Division).
2. NPCB (National
Pharmaceutical
Control Bureau)

31. S.No. PARAMETERS INDIA USA MALAYSIA
3. Recommended
regulatory
guidelines
Guidelines are
recommended for
probiotics and major
stress is on
identification and
evaluation of probiotic
strain along with health
& labeling claims.
Guidelines are
different for dietary
supplements and
drugs based on their
intended use. For
LBP’s, major stress is
on manufacturing
controls and the
extent of such
manufacturing
controls.
No official
definition and
exclusive
guidelines for
functional foods.
They comply with
food regulations
only which
include nutrition
labeling and
covering claims.
4. Assessment of
safety
parameters
FDA is responsible for
the safety of drugs and
food safety is
responsibility of FSSA.
ICMR guidelines can
also be used to identify
and evaluate safety.
USFDA is responsible
for the safety of
drugs, LBPs, biological
products. Food safety
is responsibility of
DSHEA.
FSQD is
responsible for
the safety of
functional food.

32. S.No. PARAMETERS INDIA USA MALAYSIA
5. Premarket
Approval
Required for both
drugs and functional
foods.
Required for drugs,
biologics and live
biotherapeutic
agents. Not required
for dietary
supplements and
food ingredients.
Required for
functional foods
by FSQD.
6. Structure/funct
ion claim
Not allowed. Allowed only for
Functional foods but
not for the drug.
Nutritional/functi
on claim referring
physiological role
can be mentioned
on food products.

33. .
Regulatory Framework for Probiotics:
Issues of Concerns
Catogerisation
and regulations
vary from
country to
country
No universally
accepted
definition &
harmonised
regulation
Status of probiotic
as a component of
food is not
established on
international basis
Confusion among
stakeholders
about distinction
between
nutraceuticals &
their health claims
Unavailability of
diagnostic tools
to determine
probiotics to use,
safety and
efficacy

34. FUTURE RECOMMENDATIONS
Comprehensive guidelines
Adoption of unified
definition of probiotics
Suggestive catogerisation
of probiotics

35. REFERENCES
• Tripathi, MK & Giri, SK 2014, ‘Probiotic functional foods: Survival of
probiotics during processing and storage’, Journal of Functional
Foods, vol.9, pp. 225-241.
• Baldi, A 2017, ‘ Probiotics for Health benefits: the regulatory
concerns and suggestive roadmap’, Journal of Scientific and
Technological Research, vol.1, no. 5.
• Joint FAO/WHO Working Group Report on Drafting Guidelines for
the Evaluation of Probiotics in Food London, Ontario, Canada, April
30 and May 1, 2002.
• Arora, M, Sharma, S and Baldi, A 2013, ‘Comparative Insight of
Regulatory Guidelines for Probiotics in USA, India and Malaysia: A
Critical Review’, International Journal of Biotechnology for Wellness
Industries, vol.2, pp. 52-64

36. THANK YOU !!