An introduction to probiotics and prebiotics and their role in immunity.

1. Mathew Rogers Muyinda
BSc. (Hons) Human Nutrition
Makerere University

2. Probiotics
The term probiotic is derived from Greek
 literally means “for life.”
It was first coined in 1965 by Lilley and
Stillwell.
Currently defined as by FAO and WHO as:
“live microorganisms which when administered in adequate amounts
confer a health benefit to the host”
Élie Metchnikoff first suggested the possibility
of colonizing the gut with beneficial flora in
the early 20th century.

3. Properties of Probiotics
Generally accepted x-tics of probiotics:
 they are microbial organisms
 they remain viable and stable after culture manipulation, and
storage before consumption
 they survive gastric, biliary, and pancreatic digestion
 able to adhere to mucosal surfaces

4. Properties continued
 they are able to induce a host response once they enter the
intestinal microbial ecosystem and they yield a functional and
clinical benefit to the host when consumed.
 they should be of “human origin”
 they should “colonize” the intestine

5. Mechanisms of Action of Probiotics
Although not well documented, some mechanisms probiotic
action include (also see Figure 1):
 Competition for adhesion sites with pathogens
 adhere to the epithelium and act as “colonization barriers” by preventing
pathogens from adhering to the mucosa.
 synthesis of antimicrobial compounds
 produce bacteriocins or and other antimicrobial compounds such as
hydrogen peroxide, diacetyl, and short-chain fatty acids

6. Mechanisms continued
 Stimulate the immune response
 increased secretion of IgA, elevated numbers of NK cells, or enhanced
phagocytic activity of macrophages, enhanced lymphocyte cytokine response,
enhancing intestinal mucin production & secretion.
 Probiotics may also compete for nutrients that would otherwise be
utilized by pathogens
 Probiotic organisms in sufficient numbers can utilize most of the available
monosaccharides, which results in the inhibition of Clostridium difficile

7. Mechanisms continued
 Transform and promote excretion of toxic substances such as bile
acids, nitrosamines, heterocyclic amines, and mutagenic
compounds.
 Enhance fecal bulk production which may decrease transit time and
thereby lower colon’s exposure to toxic substances.
 Acidify colonic pH by producing fermentation products such as
SCFAs

8. Figure 1. Mechanisms of probiotic function

9. Classification of Probiotics
Table 1. Classification of probiotics
Genus Species Strain
Lactobacillus lactis
plantarum
rhamnosus
johnsonii
reuteri
casei
GG, HN110
LJ-1 (LA-1), NCFB 1748
ATCC 55730
Shirota
Bifidobacterium bifidum
longum
breve
infantis
lactis
adolescentis
Bb-12
HN019
Streptococcus thermophilus
Enterococcus faecalis
faecium
Eschericia coli
Bacillus cereus

10. Commercial forms of Probiotics
There are two main forms in which probiotic organisms can
be ingested—
 Fermented foods
 dairy e.g., yogurt
 vegetable origin e.g., sauerkraut.
 Probiotic supplements
 freeze-dried (lyophilized) bacteria in powder, capsule, or tablet form.

11. Prebiotics
Term coined by Gibson & Roberfroid, who define a
prebiotic as
 “a non‐digestible food ingredient that confers beneficial effects in
the host by selectively stimulating the growth and/or activity of
one or a limited number of bacteria in the colon and thus improves
host health and well‐being.”

12. Prebiotics continued
Prebiotics are essentially fiber from plants (fruits,
vegetables or whole grains)
Figure 2. Food sources of prebiotics

13. Prebiotics continued
Prebiotics transit through the stomach and small intestines
undigested.
 They make their way intact to the colon, where they will be
bacterially fermented, together with the unabsorbed nutrients.
Lactobacilli and Bifidobacterium more likely to benefit
from prebiotics

14. Prebiotics continued
Examples of prebiotics
 Oligosaccharides (such as inulin and its derivatives),
 fructooligosaccharides
 Other oligosaccharides such as raffinose, stachyose, and verbascose
 Polysaccharides (such as cellulose, hemicellulose, pectin)
 Guar gum
 soy bean oligosaccharides, etc.

15. Quintessential benefits of prebiotics
The most recognized physiological and beneficial effects of
the prebiotics are as follows:
 improving laxation or regularity by increasing stool bulk;
 reducing blood glucose and/or low‐ density lipoprotein
(LDL)‐cholesterol levels;
 increment of high‐density lipoprotein (HDL)‐ cholesterol;
 reducing post‐prandial blood glucose and/or insulin levels;

16. Benefits continued
 providing energy‐ yielding metabolites through colonic
fermentation;
 enhancing feeling of satiety;
 reducing energy intake (which results in weight management
especially in combination with probiotics);
 having positive effects on immune system (e.g., less risk for
allergy in both infants and adults especially in combination with
probiotics), and others

17. Figure 3. A classical model of the well-documented physiological and beneficial effects of adequate and
continued intake of prebiotics in individuals

18. Intestinal Microflora
Most human mucosal surfaces colonized by bacteria.
Colonization of the gut with healthy normal microflora
begins immediately after birth increasing throughout
infanthood (See Figure 4).

19. Intestinal microflora continued
Figure 4. Succession flows of gut microbiota in a healthy infant from birth

20. Intestinal microflora continued
At birth, the gut is sterile, but it is rapidly colonized by bacteria
from mother’s birth canal and the environment.
 Other sources include equipment, air and other infants in case of
caesarian birth.
Some bacteria are got from breastmilk during breastfeeding.
 Breastfed infants have higher numbers of Bifidobacterium, lower
Bacteriodes than formula-fed infants, and do not have clostridia.

21. Intestinal microflora continued
 Overall, the microflora of breastfed infants appears to be less
complex than formula-fed infants.
 Differences in microflora account for the lower incidence of
infections in breastfed compared with formula-fed infants.
With the introduction of solid foods, microflora becomes
more complex and resembles that of adults by the second
year of life.

22. Intestinal microflora continued
 The adult human intestinal microflora comprises of approximately
100 trillion microbes.
 Microflora from numerous genera mostly anaerobic and smaller
numbers of aerobes.
 Clostridium, Bifidobacterium, Fusobacterium, Eubacterium, Bacteriodes,
Ruminococcus, Peptococcus, and Peptostreptococcus.
 The density of colonization increases from the stomach to the
distal colon.

23. Intestinal microflora continued
Subsequently, the species composition of the intestinal microflora
generally appears to be quite stable in
the absence of disturbing factors:
1. Intake of antimicrobial agents
2. Changes in dietary habits
3. Stress
4. Age
5. Infections & diseases
6. Ingestion of certain pro- and prebiotics Figure 5. Increased use of antibiotics reduces intestinal
microflora which increases susceptibility to Clostridium
dificile. C. dificile causes CDI characterized by these
pseudomembranes.

24. Mucosa-Associated Lymphoid Tissues
(MALT)
Mucosal surfaces are protected by an extensive system of
lymphoid tissues known generally as the mucosa-associated
lymphoid tissues (MALT).
MALT include specialized cells in the;
 gut (Gut-Associated Lymphoid Tissue)
 respiratory tract
 nasal-associated lymphoid tissue (NALT)
 bronchus-associated lymphoid tissue (BALT)

25. Gut-Associated Lymphoid Tissue (GALT)
GALT refers to
 the collections of lymphocytes and APCs within the mucosa of the
gastrointestinal tract where adaptive immune responses to
intestinal microbial flora and ingested antigens are initiated.
GALT is the largest lymphoid tissue of the human body.
GALT includes the tonsils, adenoids, append, and Peyer's
patches.

26. GALT continued
Functions of GALT
 collect antigen from the epithelial surfaces of the GI tract.
 production of secretory IgA

27. Commensal Bacteria & Immune System
Commensal bacteria provide benefits to hosts (humans)
whilst they are less if at all affected by the host.
Immunity benefits of commensal bacteria
 Initial colonization is a vital stimulus for the synthesis of
substances that fortify the mucosal barrier and decreases intestinal
permeability.

28. Commensal Bacteria continued
 Commensal bacteria play a role in colonization
resistance by;
1. production of antimicrobial substances and toxic metabolites;
2. competitively inhibiting adhesion of pathogens;
3. modification of toxins or toxin receptors;
4. stimulation of immune system

29. Commensal Bacteria continued
 Colonization essential for development of a fully functional and
balanced immune system by;
1. development and maturation of IgA plasmocytes
2. stimulate secretion of IgA
3. development of tolerance towards food antigens and intestinal microbes.
 Interactions between commensal bacteria and GALT help maintain
the intestinal immune system in a state of permanent low-level
activation that is considered to be important in host defense againt
pathogens.

30. Probiotics in Immunomodulation
Effect of Nonspecific Immune Responses
1. First Line of Defense
 compete with and inhibit growth of potential pathogens
 promote mucin production
 decrease gut permeability

31. Immunomodulation continued
2. Second Line of Defense
 some strains  NK cell numbers and activity
• daily consumption of yogurt for 28 days progressively increases peripheral
blood NK cell count
  phagocytic action
• by L. acidophilus, B. bifidus, L. rhamnosus, and B. lactis
  ability of neutrophils to produce oxygen radicals
  bactericidal activity

32. Immunomodulation continued
Effect on Specific Immune Responses
 Lactobacillus species stimulate adaptive immunity
1. Cell mediated Immunity
 Increased production of cytokines by T cells
 modulate inflammatory gut immune responses
2. Humoral Immunity
  total and specific sIgA in serum and intestinal lumen
  IgA-, IgG-, and IgM-secreting cells

33. Probiotics in Allergy and Atopic Disease
Allergy is a state/disorder in which a symptomatic
immediate hypersensitivity immune reaction is made to a
normally innocuous environmental antigen.
It involves the interaction between the antigen and
antibody or primed T cells produced by
earlier exposure to the same antigen.

34. Probiotics and Allergy continued
Atopy is a genetically based propensity of an individual to
produce lgE-mediated allergic reactions against innocuous
substances.
 People who have allergies to environmental antigens, such as
pollen or house dust, are said to be atopic.
Anaphylaxis is a related but not an identical condition.

35. Figure 6. How allergy arises
Vasoactive amine,
lipid mediators
Cytokines
Late phase
Reaction
(6-24 hrs)
Immediate
hypersensitivity
reactions within
minutes
First exposure
to allergen Antigen activation of Tfh and
Th2 cells and stimulation of
IgE class switching in B cells
Production of IgE
Binding of IgE to
FcεRI on mast cells
Repeat exposure
to allergenActivation of mast cell:
release of mediators
1
2
3
4
5
6

36. Probiotics and Allergy continued
Allergic diseases have greatly increased over the last
decades.
The hygiene hypothesis tries to explain this as follows:
 Increased hygiene reduces exposure to microbes
 Microbial stimulation of immune system reduced
 As a result, there is a shift from Th1- to Th2-type immune
responses that favors development of IgE-mediated allergies

37. Probiotics and Allergy continued
Effect of Probiotics on allergy diseases
 Supplementation with LGG results in improvements in atopic
dermatitis
 LGG ingestion alleviates intestinal inflammation as evidenced by 
1-antitrypsin and (TNF)-. LGG acts by:
 normalization of intestinal permeability
 modification of degradation, permeation, and targeting of food antigen to
Peyer’s patches
 direct anti-inflammatory effects

38. Probiotics and Allergy continued
Evidences for probiotic role in allergy
 Children from traditional environments with low antibiotic use and
a high consumption of lactobacilli-containing fermented products
exhibited a lower incidence of allergic disease than those from the
westernized environments.
 Studies show that children who are nonallergic are more likely to
have lactobacilli as part of their normal microflora than their
allergic counterparts.

39. Probiotics in Prevention & Treatment
of disease
probiotics have a wide range of beneficial effects and numerous
indications of use in pediatric populations, such as;
Acute diarrhea
Antibiotic-Associated Diarrhea
Allergy prevention
Necrotizing enterocolitis

40. 1. Diarrhea
A. Acute Infectious Diarrhea
 Probiotics reduce diarrheal disease by 57%
 L. rhamnosus (GG) reduces the duration of rotaviral diarrhea
 B. lactis and LGG reduce the incidence or severity of acute
diarrhea
 both LGG and L. reuteri (during treatment) and B. lactis (used
prophylactically) reduce rotaviral shedding

41. Diarrhea continued
B. Antibiotic-associated diarrhea (AAD)
 AAD is an acute inflammation of the intestinal mucosa caused by the
administration of a broad spectrum of antibiotics.
 Several probiotic bacteria reduce risk from 28.5% to 11.9%.
 Most important are B. lactis & S. thermophilus and L. rhamnosus.
C. Nosocomial Diarrhea
 any diarrhea contracted in health care institution
 LGG reduces risk of nosocomial diarrhea
 B. bifidum and S. thermophiles reduce its prevalence

42. 2. Vaginal Microflora & Bacterial
Vaginosis
Lactobacillus normally inhabit the vagina.
Lactobacilli bacteria produce H2O2 which keeps the healthy
balance of vaginal microorganisms.
H2O2 interacts with peroxidase present in vaginal fluid
Disruption of microfloral balance in vagina results in disease

43. Figure 7. Normal Vaginal microflora

44. Vaginal Microflora continued
Bacterial vaginosis (BV)
 The most common urogenital disease in women, affecting
19-24% women in reproductive ages.
 BV is believed to be caused by an imbalance in the normal vaginal
microflora.
 Lactobacilli are replaced by anaerobic bacteria such as Gardnerella
vaginalis, Mycoplasma hominis, Prevotella and Peptostreptococcus.
“Bad” bacteria
Lactobacilli

45. Vaginal Microflora continued
 Signs of bacterial vaginosis
 foul, fish-like or musty odor which is stronger after sex
 watery or foamy vaginal secretions
 milky or gray vaginal secretions
 itching on the outside of the vagina
 burning or discomfort during urination

46. Vaginal Microflora continued
Factors associated with upset the balance in BV include;
 having new sex partners
 multiple sex partners
 douching
Probiotics and Bacterial Vaginosis
 probiotics cure and reduce recurrence of BV
 can be administered orally or vaginally

47. Vaginal Microflora continued
Orally consumed probiotics ascend into the vaginal tract after
they have been excreted from the rectum (see Figure 8).
Mechanisms of Action
 Occupation of specific adhesion sites at the epithelial surface of the
urinary tract
 Maintenance of low pH
 Production of antimicrobial substances like acids, H2O2 and bacteriocins
 Production of surfactants with antiadhesive properties

48. Figure 8. Capability of pathogenic and probiotic bacteria to ascend the vagina after being
excreted from rectum

49. 3. Diabetes Mellitus
Diabetes is a disease in which the body cannot regulate the
amount of sugar in blood.
It is divided into 2 types:
 Type 1: there is little or no insulin production
 Type 2: insulin resistance
Diabetes is increasing alarmingly & it is estimated that 342
million people will be suffering from it by 2030.

50. Mechanisms by which probiotics improve diabetes
Improve lipid profile by the production of SCFA which act as
inhibitors of hepatic lipogenesis.
Modulate Th1 and Th2 proinflammatory responses, aiding in
prevention of development of T2DM.
 inflammation is an important event in the pathogenesis of
autoimmune diseases such as T1D

51. Mechanisms continued …
Prebiotics (inulin) controls glycemic index by reducing the
absorption rate of glucose.
Inulin also controls lipid profile by inhibiting glycerol-3-
phosphate acyltransferase and fatty acid synthase as well as
key enzymes in de novo lipid genesis.

52. Mechanisms continued …
Probiotics (Lactobacillus acidophilus, L.fermentum, L gasseri
and L rhamnosus);
 modulate the expression of genes encoding junction and adhesion
proteins E-cadherin and β-catenin
 reduce the expression of protein kinase C-δ (PKC-δ).
 PKC-δ activation results in dispersion of adherence junctions, increasing
intestinal permeability.
Increased intestinal permeability may facilitate the absorption
of antigens which can injure pancreatic β cells.

53. Probiotics
TLR-4
 Bifidobacterium
 Permeability
Antigen
 Adhesive proteins in
intestinal mucosa:
(catenin, occludin, E-
cahedrin, claudin)
 Tight junction
Lumen
Basolateral  Permeability  Th17 and activation
 Pancreatic  cell destruction
Figure 9. Mechanism by which probiotics may prevent diabetes from pancreatic cell destruction

54. Evidence of probiotic role in diabetes
Children with T1D showed higher counts of Clostridium, Bacteroides
and Veillonella, followed by lower counts of Bifidobacterium and
Lactobacillus, than healthy children.
T1D children have lower counts of bacteria producing butyrate w/c,
 has anti-inflammatory actions
 reduces bacterial translocation,
 improves the organization of tight junctions and
 stimulates the synthesis of mucin, a glycoprotein maintaining the integrity of
the intestinal epithelium.

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