DEVELOPING A NEW PROBIOTIC &

DETERMINING ITS ACTIVITY

Miss Ankita G. ChaudhariB. Tech IIIrd yr (Food Tech)Email ID: harshada.23aug@gmail.comContact No.: +91-9423545582

Guided by:Dr. P.A.Pawar(Associate Professor & Guide)

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

• 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)

Synbiotics• Products containing BOTH– Prebiotics– Probiotics

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

Human Microbiome Project

History

Joseph Lister-1878Isolation of Lactobacillus

Louis Pasteur- 1857Potential Benefits of Lactobacillus

Ellie Metchnikoff- 1905Concept of Probiotics

Henry Tissier-1899Isolation of Bifidobacterium

Dr. Minoru Shirota-1930First stable culture

Benefits

Probiotics

Stronger Immunity

Improved Digestion

Production of vitamin

B12

Reduces Cold and

FluBetter Breath

Healthier Skin

Weight Loss

Healing from

Leaky Gut and IBS

Diarrhea

Gas

Bloating

AcneCramps

Rashes

Side Effects

Types of Probiotic StrainsLactobacillus

Bifidobacterium

Streptococcus

Sacchromyces

Probiotic Food

Kefir

Yogurt

Sauerkraut

Coconut Kefir

Kombucha

Kvass

NattoRaw

Cheese

Mechanism of ActionEnhancement of Epithelial Barrier

Increased Adhesion to Intestinal Mucosa

Competitive Exclusion of Pathogenic Bacteria

Production of Antimicrobial Substances

Modulation of Immune System

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

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.

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.

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

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

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

Hydrophobicity Cell Surface

Test

Transit Tolerance In

Gastrointestinal Tract

Identification Of The Isolates

Probiotic Properties

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).

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.

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.

Apical Media

CaCo-2 Monolayer

Basolateral Media

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.

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

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.

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.

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.

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.

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.

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.

INDUSTRIAL MANUFACTURING OF PROBIOTICS

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

TRIAD OF PROBIOTIC QUALITY

Viability Triad

Timing

Temp.Moisture

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).

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.

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).

MOISTURE METER

TEMPERATURE CONTROL• Proper temperature control encompasses two requirements:

1. a cool ambient temperature2. 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.

• 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.

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.

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.

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.

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.

Seeds

Pre-Culture

Fermentation

Centrifuge

Drying

Lyophilisate

Product

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.

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.

CONCLUSION

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.

All probiotic supplements are

basically the same…

• TRUE • FALSE

Number of probiotic strains live in our body…

400+

Do supplement probiotic strains stay in our body…

TRUE FALSE

Weight and number of count of probiotic cells in our body….

10 times cells in our body

3.5lbs.=1.58gm