Probiotics and Prebiotics

The New Nutraceutical Stars for the 21st Century

Dr Nigel Plummer

The Normal Human Microflora - Where Is It?

The gastrointestinal tract
The genito-urinary tract The respiratory tract

The skin

The Normal Human Microflora - What Is It?

The commensal microbial population which resides within the particular human niche, which is both persistent and stable, and which does not elicit an acute immune response in normal circumstances

The Normal Human Microflora Numbers and Statistics

Total Numbers:

G.I. tract contains approximately 1014 or 100,000 billion viable bacteria Skin harbors approximately 1012 or 1,000 billion viable bacteria Total number of cells in the body is 1013 or 10,000 billion Between 33% and 50% of faecal mass is bacteria

Types:
Oral cavity Large intestine 200 species 400 - 500 species

The Normal Human Microflora Numbers and Statistics (cont)

Unknowns:
Between 20 - 40% of observed colonic microflora are unculturable and therefore unknown (Borriello, 2002) In human subjects over 50 years of age, the unculturable number of microflora rises to 80% (Collins & OMahony, 2002; Rowland, pers comm) Reasons for Inability to Culture:
1. 2. 3. 4. 5. Viable but stressed cells Obligate requirement for co-existing flora Obligate requirement for host derived products Lack of knowledge regarding culture and nutrient conditions Dead cells Microflora totals approximately 100 times total number of genes of the host Minimum 105 newly mutated viable bacteria every 2 hours in G.I. Tract (Hart et al, 2002)

Potential adaptability of microflora is vast:

The Normal Human Microflora - Birth

0 to 2 days: The newborn infant is microbiologically sterile. Its first flora is
acquired from vaginal, skin and rectal microflora of the mother

Dominant Types:
Lactobacilli: Mainly acidophilus types Streptococci: Non-haemolytic Enterobacteria: Various including E. coli

2 days to Weaning: Highly dependent on breast or bottle feeding

2 - 7 days Breast fed Bottle fed (log cfu/g) 9.0 (95%) 7.8 (22%) 7.3 (2%) 7.4 (8.7%) 3.5 (<1%) 7.5 (3%) 5.1 (<1%) 8.3 (69%) 1 19 weeks Breast fed Bottle fed (log cfu/g) 9.8 (97%) 9.7 (77%) 7.5 (0.5%) 9.0 (15%) 4.9 (<1%) 8.1 (2%) 6.6 (<1%) 8.7 (8%)

Bifidobacteria Bacteroides Clostridium Enterobacteria

120%

7 days (log cfu/g)

100%
9.0 9.8

19 weeks (log cfu/g)

9.7 8.3

percentage (%)

80% 60% 40%

7.8

20%
7.4

9.0 8.7 5.1 7.5 4.9 8.1 6.6 7.3 3.5 7.5

0%

Breast fed Bifidobacteria

Bottle fed Bacteroides

Breast fed Clostridium

Bottle fed Enterobacteia

Effect of Mode of Delivery on Gut Microflora of Children Aged 7 Years

Caesarean

section infants have profoundly different microflora in first months of life and abnormalities remain at 7 years and probably lifelong. C-section infants have increased risk of allergy compared to vaginal birth infants.

Effects of Formula Vs Breastfeeding Beyond Six Months of Age

In a recent trial, the gut flora of breast vs formula fed infants was still significantly different at 6 months. However, flora in formula supplemented with prebiotics ( 900mg GOS 100mg FOS) or feeding Bifidobacteria to the mother for 2 weeks prior and 2 months following birth have effect of increasing numbers of Bifidobacteria

Effect of supplement of probiotics to formula milk on population of Bifidobacteria in infants at 6 months

120 101 100 85
Counts (CFU/g faeces)

1- Formula milk FED 2- Breast FED 3- Formula milk + GOS/FOS

80 60 46 40 23 20 0 1 2 3 4 7.6 28 29 23

4- Breast FED + Probiotics (B. Lactis) given to mother 2 weeks prior to 2 months following birth

The Normal Human Microflora - Adult

The intestinal microflora acquires adult characteristics and is fully formed by two years of age It is important to note the variation in number and type of microbial species in the different parts of the intestine

Typical Microbial Flora of the Gastro-intestinal Tract of Man

Lactobacilli 1-100/g

Mouth

Lactobacilli Streptococci

1x102/g 1x104/g

Bifidobacteria Lactobacilli Stomach Streptococci Duodenum Bacteroides Enterobacteria

1x105 /g 1x108 /g

Jejunum
Lactobacilli Streptococci Enterobacteria Bifidobacteria Yeasts 1x105/g 1x106 /g

Ileum

Large intestine

Anus

Bacteroides Eubacteria Bifidobacteria Anaerobic cocci Clostridia Lactobacilli Enterobacteria Streptococci

1x1010 /g 1x1011 /g

FUNCTION OF NORMAL MICROFLORA

Summary of Functions
1) Essential to normal anatomical and physiological development of the intestinal mucosa:
Cell development and turnover, villous length, crypt depth Motility, secretion, and absorption

2) Provides non immunological protection against infection 3) Stimulates maturation and balancing of the immune system at birth and then stimulates and primes the immune system throughout life 4) Facilitates a wide variety of metabolic functions to the host. These have profound implications on human health

Non Immunological Protection Against Infection

The Protective Effect of the Human Microflora Prevention of Candidiasis

Normal condition
Occasional Candida cell Vaginal epithelial surface

Candida overgrowth

Numerous lactobacilli attached to epithelial surface prevent attachment and overgrowth of Candida albicans

Protective lactobacilli depleted Candida albicans overgrow and change to hyphal form some invade cells

Inflammatory symptoms

The Human Immune System and the Normal Flora - Summary Statement of Interaction
The colonisation of the human intestine by the normal microflora following birth:

Drives the development of the humoral, cell-mediated and immune tolerance systems during the neonatal period
Is compulsory for the normal development of the human immune system Enables our correct response to dietary and environmental antigens - and ensures our tolerance of the normal flora

Primes the immune system to react quickly to potentially harmful antigenic challenge

Initial Development of GALT in the Human Infant

Immature Peyers patches and lymphoid follicles together with low numbers of T-cells develop in the lamina propria during weeks 11 - 20 of gestation At this point the immature immune system is strongly biased towards the Th2 or antibody forming response. Development then ceases until birth and resumes on contact with enteric microflora and dietary antigens

Metabolic Activities of the Normal Flora

Synthesis of vitamins:
B-vitamins: B12

Folic acid Biotin Riboflavin

Vitamin K

Conservation of nitrogen:

30% of urea produced in liver is released into colon (70% urea released in urine) Bacteria recycle urea into amino acids - available to host

Metabolic Activities of the Normal Flora (cont)

Synthesis of short chain fatty acids (SCFAs):
The colonic epithelial cells deliver approximately 50% of their energy requirements from butyrate produced by the microflora. Estimated that 5-10% of the total body energy is from SCFAs

Colonic cells deprived of butyrate begin to atrophy within approximately 5 days. This decreases integrity of mucosal barrier and causes mucous permeability to increase dramatically
Relatively low numbers of colonic flora (notably eubacteria and peptococci) produce butyric acid: estimated production is 100mmol/day

Luminal Nutrition and Intestinal Conditioning

G.I tract mucosa is only body tissue with a systemic and luminal nutritional source Over 50% of nutrition of small and large intestinal mucosa is luminal
Energy Source
Glucose Glutamine Butyrate Aspartate Acetate Propionate Duodenum Jejunum Colon 30% 20% 5% 60% 70% 5% 60% 10% 10% 20% 10%

Metabolic Activities of the Normal Flora (cont)

Detoxification and toxin production:
The microbial flora can detoxify and positively transform many substances:

Heavy metals (by binding and by enhancing excretion from systemic circuit) Biotransformation of plant polysaccharides releasing lignases and phyto-estreogens (iso-flavones) by colonic flora (colon and breast cancer) Release of quercetin by bacterial -galactosidase from fruit carbohydrates. Quercetin & rutin have been shown to be powerful antimutagens and can also show cancer-promoting properties Plant polyphenols such as those contained in grape skin/seed extracts are only absorbed in the colon following interaction with microflora
(Lemaire, 2004 personal comm)

Evidence that depending on the type of flora, release of dietary glucosinolates( isothiocyanates) has effect of stimulating or repressing cytochrome P450 in liver (Nugon Bauden et al, 1998)

Microbial Flora and Production of Carcinogens.

The microbial flora are vigorous producers of carcinogens: Fecapentaenes: most potent carcinogens found in the intestine. Formed from derivatives of mainly saturated fats Heterocyclic amines: can be produced either from high temperature cooked fats or cigarette smoke interacting with protein. Potent tumour producer in prostate, colon and breast. One meal of high cooked fat/protein as in BBQ can produce 200 x normal level of heterocyclic amines in bowel

Bile acids, nitrosamines, diacylglycerol

Most active microbes are Bacteroides species Vegetarians have lower levels of Bacteroides, significantly lower levels of carcinogens and significantly lower levels of CRC.

Probiotics
Preparations of one or more components of the normal microbial flora designed to contribute to the population dynamics, or metabolic/immunological balance, of the intestinal flora, and so confer health benefits to the host

Some Historical Perspectives!

one of the founders of immunology, also advocated the potential for supplementing components of the microflora to provide health benefits. The earliest patient known to have shown resolution of eczema by using probiotics was Adolf Hitler Kaplan
Metchnikof
2000

Review of Micro-organisms Used as Probiotics

E.coli Conceptually sound, using commensal strain of E.coli to prevent overgrowth and colonisation of enteropathogenic strains. Current use - very little, due to safety consideration Clostridium difficile Conceptually sound, using commensal strain of C. difficile to prevent pseudomembraneous colitis. Current use - very little, due to safety consideration Bacillus subtilis Recent products which promote either Bacillus spp. or soil micro-organisms. Conceptually unsound. Potentially very dangerous in subjects who are immuno-compromised (Ogionni et al, 1998) Saccharomyces boulardii Good evidence of efficacy in antibiotic associated C.difficile and E.coli diarrhoea. Non- indigenous, non colonising yeast? Little evidence of immune stimulating effects in same way as LAB . Has caused fungaremia of catheterised patients ( over 30 cases) and question mark over safety with immunocompromised

Review of Micro Organisms Used as Probiotics Lactic Acid Bacteria

Enterococcus (Streptococcus) faecium Formally very popular due to long shelf life and sound concept. Pathogenic potential and antibiotic resistance issues have resulted in almost total withdrawal Lactobacillus casei var rhamnosus Conceptually sound, safe in normal use. Some recent data of bacteraemia in very premature infants Lactobacillus acidophilus Conceptually sound, totally safe organism with most compelling data. Major immunomodulation and disease antagonism function Bifidobacterium bifidum Major colon coloniser. Obligate anaerobe major function in microflora balancing and toxin/cancer potential

Selection Criteria for Probiotic Isolates

Must be considered totally non-pathogenic Should be species indigenous to the target host Should be capable of colonisation of digestive tract - resists stomach acid, bile salts, - capable of attachment to epithelial cells - capable of immunomodulation in-vitro Scientific evidence especially clinical data is desirable to support use

Commercial release of the product should be covered by thorough Quality Assurance programme

The Development of An Efficacious Probiotic - A Case History

The Lab4 Consortium

Advances in Probiotic Research

Other trial work recently published has shown probiotics at high potency to:

* General stimulation of immunity and maintenance of intestinal balance 4-8 billion

* Reduce incidence of neonatal allergy 2-6 billion * Reduce severity of established allergy 10-40 billion 100-400 billion

* Reduce severity of inflammatory bowel disease * Prevent dysbiosis from antibiotic use -

30-100 billion

Summary of Trials
THE TWO ESSENTIALS: Important to have functional effective strains Adequate numbers are essential Higher numbers produce a:

More profound therapeutic effect More consistently Faster!

New Consortium
Recent and current trials have been using the:

LAB 4 CONSORTIUM
CLT 21 Lactobacillus acidophilus Two strains CLT 60

CLT 20 Bifidobacterium bifidum Bifidobacterium lactis CLT 34

Following screening of numerous human strains, two new strains of L. acidophilus and B. bifidum were inseparable in terms of efficacy from the original two strains.

So..

Passages of Probiotics through the G.I. Tract

Log10 viable count (cfu/ml)

Bioacidophilus

Product A

Product C

Product B

Exposure time (hours)

The Acid Test: At a high pH of 4.2, Bioacidophilus remains

the most viable probiotic

Product C

Product B

Product A

Bioacidophilus

Passages of Probiotics through the G.I. Tract

Log10 viable count (cfu/ml)

Bioacidophilus

Product C

Product B

Exposure time (hours)

The Acid Test: At a low pH of 2.6, Bioacidophilus remains

the most viable probiotic

Product B

Product C

Product A

Product A

Bioacidophilus

Major Cytokine Influence on Immune Response/Tolerance

RA MS IBD

Inflammation TNF-

Allergy

TH1 Cell mediated immunity

TOLERANCE

TH2 Antibody mediated immunity

IFN-

IL-2 IL-12 TGF- IL-10

IL-4 IL-5

Effect of Different Probiotic Strains on In-vitro Stimulation of Cytokines in Peripheral Mononuclear Blood Cells (Cont..)
1200 1000 800

pg/ml

600 400 200 0 Strain 1 Strain 2

IL-10

Strain 3
IL-12

Strain 4

Strain 5

Strain 1 CLT 21 Strain 2 CLT 60

Strain 3 CLT 20 Strain 4 = CLT 89 L.salivarius Strain 4 = CLT 34

Biocares Clinical Trials

All trials carried out at Addenbrookes Hospital, Cambridge Gastroenterology Department in association with Dr John Hunter Consultant Gastroenterologist)
DBPC trial demonstrating significant competitive inhibition of Clostridium difficile in hospitalised patients:

67% of control patients with presence of C. difficile developed diarrhoea 18% of Bioacidophilus Forte treated patients with presence of C. difficile developed diarrhoea ( International Microbiology 2004)

DBPC trial demonstrating significant reduction of opportunistic pathogen regrowth following use of antibiotics:

Statistically significant reduction of Staphylococci, Enterococci and Enterobacteria (coliforms) in patients given Replete after antibiotics
Effect was more profound if Replete was given with and following antibiotic (J. of Medical Microbiology 2004)

Clinical Trials (cont)

DBPC trial demonstrating significant > 70% reduction in antibiotic resistance in coliforms, and enterococci when Replete was provided with and following antibiotic therapy. ( Journal of Medical Microbiology 2004)
(Resistance to antibiotics is probably the biggest problem facing human healthcare in the 21st century. This is the first time that any positive intervention has been demonstrated to impact this issue) Open trial demonstrating significant reduction in yeast notably Candida albicans overgrowth in human caecum following antibiotic use unique trial:

Administration of antibiotics causes intestinal overgrowth of yeasts in 50% of people, 33% of people with mucosal surface colonisation In up to 15% of people, this colonisation appears to be persistent, indicating continued mucosal colonisation and possibly continuous/intermittent low level inflammatory response ( International Microbiology 2004) Overgrowth and colonisation of yeast virtually eliminated with supplementation of Bioacidophilus Forte

Double-Blind Placebo-Controlled Trial Using Replete

To investigate the following:
The role of Replete on the re-establishment of the microflora post-antibiotic therapy intestinal

The incidence of antibiotic resistance within the re-growth population of the intestinal microflora

Replete DBPC Trial

Group
1 2 3

Days
17 8 15 17 8 15 17 8 15

Treatment
Antibiotic/Placebo Placebo Antibiotic/Placebo Replete Antibiotic /Replete /Replete

Samples: Days 1 (control), 7, 12, 17 and 28

Major Facultative Organisms Measured in Replete Trial

Enterobacteria Enterococci

Staphylococci

Replete DBPC Trial: Facultative Organism Population Shifts

Day 7
Placebo

Day 28

Day 1 Day 7
Placebo/Replete

Day 1
Replete/Replete

Day 28

Day 1

Day 7

Day 28

The Effects of Antibiotics on Populations of Candida albicans in the Intestine

The Effects of Antibiotics on Intestinal Microflora

Overgrowth by Candida albicans
Administration of penicillin, clindamycin, vancomycin and tetracycline allowed Candida albicans to proliferate in the intestine and to translocate extra-intestinally. This did not occur when antibiotics were not administered
(Kennedy & Voltz, 1985; Melstrom & Balish, 1979)

Supplementation of normal flora components following antibiotic treatment prevented multiplication of Candida albicans and its translocation
(Kennedy et al, 1988)

Examples of use of Bacteriotherapy to Ameliorate Intestinal Disease

Organism Amount Effect

Antibiotic-associated overgrowth: Candida albicans

Following clindamycin therapy, Candida albicans became an established part of flora in 100% of control patients but only in 25% of those treated (Lidbeck et al, 1988)

Lactobacillus acidophilus

2.5 x 1010/day

Effect of Bioacidophilus Forte on the Caecal Flora of IBS Patients Receiving Antibiotic Therapy
AIM: To investigate the role of antibiotics and Bioacidophilus on the: - caecal mucosal microflora - caecal lumen microflora - faecal flora

First ever microflora trial conducted on human caecal tissue.

1) Biopsy samples taken on day 0 and 14 2) Faecal samples taken on days 0,14, and 28 3) Antibiotics given days 7-14 4) Bioacidophilus Forte (1 cap) per day given from days 0 -14 5) 6 patients in each group- ethical approval!

Caecal Trial Design

DAY 0

DAY 7

DAY 14

DAY 28

Group 1 (6 patients)

B+F

B+F

No treatment

Antibiotics

Group 2 (6 patients)

B+F

B+F

No treatment + Bio Forte

Antibiotics + Bio Forte

Caecal Microflora Trial-Results

1) Before treatments 15% and 33% of patients in each group showed presence of C.albicans and total yeasts respectively. 2) All of this presence was luminal or faecal no mucosal colonisation. 3) Antibiotic treatment increased luminal presence of C. albicans to 50% and total yeasts to 50% 4) In 15% of antibiotic treatment patients C.albicans had colonised the mucosal surface. 5) In 33% of antibiotic treatment patients there was non C.albicans colonisation of the mucosal surface 6) At end of trial 33% of antibiotic treated patients had increased presence of yeasts in faeces at significantly higher levels indicating high liklihood of persistant mucosal colonisation along with luminal presence. 7) Administration of Bioacidophilus Forte reduced presence of C.albicans to 0% and the presence of non-albicans yeasts to 15%. 8) Bioacidophilus Forte prevented any colonisation of mucosal surfaces by either C.albicans or non-albicans yeast. 9) At the end of the trial, the faecal presence of C.albicans and other non albicans yeast was 0%

Effects of Antibiotics Alone and with Bioacidophilus Forte on the Incidence of Candida albicans in IBS Patients
6 5 4 Day 0 Day 14 Day 28 faecal sample

Antibiotics Only

3 2

1
0

Caecal Caecal mucosa lumen

Faeces Day 28 faecal

6 5

Antibiotics with simultaneous Bioacidophilus Forte supplementation

4 3 2 1 0

Day 0 Day 14 Day 28 faecal sample

Caecal Caecal mucosa lumen

Faeces Day 28 faecal

Effects of Antibiotics Alone and with Bioacidophilus Forte on the Incidence of Yeast Infection in IBS Patients
6 5 4

Antibiotics only

3 2 1 0

Day 0 Day 14 Day 28 faecal sample

Caecal Caecal mucosa lumen

Faeces

Day 28 faecal

5 4

Antibiotics with simultaneous Bioacidophilus Forte supplementation

3 2 1 0
Caecal mucosa Caecal Faeces lumen Day 28 faecal

Day 0 Day 14 Day 28 faecal sample

Caecal Trial - Conclusions

1) Administration of antibiotics induces intestinal overgrowth of C.albicans and total yeasts in 50% of people. 2) In a significant percentage of people this overgrowth progresses to become a mucosal colonisation indicating change of morphology of the yeast and induction of inflammatory response. 3) This mucosal colonisation is more likely in individuals with additional risk factors such as IRS and type 2 diabetics or high oestrogen loading ie pregnancy and contraceptive pill. 4) Mucosal colonisation is likely to be persistent and could cause intermittent immune challenge resulting in intermittent symptoms such as muscle/joint pain, slight/moderate fever, and fatigue. 5) Bioacidophilus Forte probiotic administered prior and during antibiotic therapy prevented luminal and faecal overgrowth and also prevented colonisation of mucosal surface. 6) Evidence suggests that Bioacidophilus Forte should be compulsory treatment alongside antibiotic therapy

Other Significant Results of the Caecal Trial

1) IBS patients have significantly lower levels of LAB and total anaerobes compared to healthy controls 2) The total caecal anaerobic population decreased in the antibiotic treated group but not in the probiotic treated group 3) The caecal and fecal Lactobacillus population increased in the antibiotic/probiotic treated group compared to the antibiotic alone group

Reduction in Clostridium difficile Infection in Hospital Patients on Antibiotic Therapy

Trial type: Location: Double blind placebo controlled Addenbrookes Hospital U.K

Aim: Use Bioacidophilus Forte to prevent or reduce Clostridium difficile infection and associated diarrhoea in patients receiving antibiotics.

Trial Design: 150 patients initiating antibiotic therapy were randomly assigned to probiotic 1 capsule daily or placebo group for 20 days with faecal sample taken at day 0 and 20 Incidence of the following were monitored: 1) Presence of C.difficile 2) Presence of C.difficile toxin 3) Incidence of diarrhoea

DBPC Clostridium difficile Trial Results.

1) Incidence of C.difficile: Placebo: 13.0% BF: 15.9% 2) Incidence of C.difficile toxin: Placebo: 78% of positives BF: 46% of positives (sig diff) 3) Development of C.difficile associated diarrhoea: Placebo: 67% of positives (7patients) BF: 18% of positives (2 patients)( sig diff)

Conclusions
1) Administration of Bioacidophilus Forte significantly reduced incidence of C.
difficile associated diarrhoea in antibiotic therapy patients. 2) Mechanism appears to be by neutralisation of C.difficile toxins rather reduction of organism number (subsequently confirmed by in-vitro study where L. acidophilus found to neutralise C. diff toxins A + B) 3) Estimated that C. difficile associated diarrhoea results in extension of hospital stay of average additional costs of $6000 (Wilcox 1996) * On basis of this trial the cost of placebo patients 6 x $6000 = $36000 whereas Bioacidophilus Forte treated group 2x $6,000 = $12,000 * Cost of treating all patients with Bioacidophilus Forte = $3,000, therefore saving of $21,000

4) Approx 75,000 cases of C.difficile/ year in USA. Potential saving $250 million
5) Evidence suggests that Bioacidophilus Forte should be given to all hospitalised patients receiving antibiotic therapy.

Advances in Probiotic Research

Other trial work recently published has shown probiotics at high potency to:

* General stimulation of immunity and maintenance of intestinal balance 4-8 billion * Reduce incidence of neonatal allergy 2-6 billion * Reduce severity of established allergy 10-40 billion 100-400

* Reduce severity of inflammatory bowel disease billion * Prevent dysbiosis from antibiotic use -

30-100 billion

Summary of Trials
THE TWO ESSENTIALS: Important to have functional effective strains Adequate numbers are essential Higher numbers produce a:

More profound therapeutic effect More consistently Faster!

Stability and Quality Assurance

Flow Diagram for Manufacture of Probiotic Cultures

Aseptic Processing (72 hours) Seed culture Non-Aseptic Processing (96 hours)

First stage Inoculum buildup (1 litre) Check Purity

Second stage Inoculum buildup (100 Litre) Production fermentation 5,000 50,000 Litre

Freeze Drying Drying at 30C under vacuum

Continuous centrifuge

Freeze dried probiotic powder

Mill

CONTROLS Agitation pH Temperature Aeration

Freeze drying formulants 50,000 litres addition 2,000 litres Finished freeze dried powder concentrate
50,000 litres

Probiotic soup Probiotic slurry

500kg powder

Manufacturing Technical Breakthrough

group continuously looking to improve:
1. Yield of LAB 4 organisms

100 80 Fermentation yield (%) 60 40 20

New yield Previous yield

0
Time

Effect on Different Freeze-dried Lactic Acid Bacteria of Long Term Storage at 300C
1012 1010

E. faecium B. bifidum L. casei var rhamnosus

108
106 104 102 0

L. acidophilus

20

40

60

80

100

120

140

160

180

Time (days)

Effect of Variable Temperature Storage on Freeze-Dried L. acidophilus

-200C
1010 108 106

+40C

+150C
104 102 0

+370C

+300C

20

40

60

80

100

120

140

160

180

Time (days)
All bacteria freeze-dried under same conditions in same formulation

Improvement in Stability of Probiotics

Previous
100 80 (Indefinite)

-200C

% survival

4 0C
60 40

20
0 0

200C 370C
1 2

300C
3 4 5 6 7 8 9 10 11 12

Months

Improvement in Stability of Probiotics (Cont..)

Now
100 80 (Indefinite)

-200C
4 0C

% survival

60

200C
40

20
0 0 1

370C
2 3 4 5 6 7 8 9

300C
10 11 12

Months

Bioacidophilus Shelf Life at Ambient 15-30 Celcius

Actual amount at time of manufacture 7 x 109 6 x 109 Minimum typical level if kept refrigerated Margin of laboratory variability

6 x 109/capsule (maximum) 4 x 109/capsule (minimum)

4 x 109 (Label claim)

3 months Bioacidophilus (capsules/powders); Neonate

6 months

Bioacidophilus Forte Shelf Life at Ambient 15-30 Celcius

Actual amount at time of manufacture
35 x 109 30 x 109

30 billion (maximum level after 3 months) Margin of laboratory variability

24 x (Label claim)

109 Minimum typical level if kept refrigerated Minimum level after 3 months

24 billion/dose

3 months

6 months

Improved Stability
Use of overage and improved stability now means that the range of probiotics has: Indefinite shelf life at 200C 12 months shelf life at 40C and still has 90% overage 3 6 months shelf life at ambient 300C and still reach label claim!

The New Specifications

Label claim Bioacidophilus Capsule
1 billion 4 billion 40C Minimum 6 billion

Bioacidophilus Forte

24 billion

30 billion

Parallel Development Pathway both Produce Remarkable Improvements

Clinical trials Demonstrable clinical effectiveness in human trials PLUS Manufacturing technology

Improved yield and stability

Review of Clinical Evidence for Effectiveness of Probiotics

Antibiotic Associated Diarrhoea

Antibiotic associated diarrhoea is attributable to imbalances in intestinal flora: Meta analysis showed four blinded placebo controlled trials with Lactobacilli and Bifidobacteria robust enough to be considered: Trial No. Subjects Relative Risk with Probiotic REF

1
2 3

60
188 119

0.13
0.28 0.30

Armuzzi et al 2001 Ali Pharm Ther 15

Vanderhoof et al 1999 J. Pediatr 135 Arvola et al 1999 Pediatrics 104

79

0.39

Gotz et al 1979 Am J. Hosp Pharm 36

Gastroenteritis, Travellers and Rotavirus Diarrhoea

Gastroenteritis is the major cause of acute diarrhoea, but is mainly self limiting within 5 days Six DBPC trials with Lactobacilli/Bifidobacteria have demonstrated significant reduction in duration of gastroenteritis in infants Five trials have shown significant reduction in duration of infant rotavirus diarrhoea Two trials have shown that Lactobacilli/Bifidobacteria significantly reduce risk of developing rotavirus diarrhoea in infants effect was 6.77% vs 31-34% in control Four studies have shown significant protection against travellers diarrhoea however three similar trials have shown no significant effect
(Mercenier et al, 2003; Marteau, 2002)

Probiotics and Helicobacter pylori

Lactobacilli, particularly L. acidophilus have been shown to inhibit H. pylori in vitro by pH reduction and in neutralised conditions In vivo administration of inactivated L. acidophilus alongside triple therapy significantly improved eradication rate of H.pylori. Eradication with triple therapy was 72% with probiotic 88%
Canducci et al 2000 Aliment.Pharm.Ther. 14

Probiotics in Inflammatory Bowel Disease (IBD)

E.coli
Non pathogenic E. coli (50 billion/day) fed for 12 weeks versus mesalazine in quiescent ulcerative colitis gave no difference in relapse rate (6/53 against 8/50). In active ulcerative colitis, E. coli provides same remission rate as mesalazine.
(Katz & Fiocchi, 2001)

Two similar trials over a period of 1 year also showed no difference in relapse rate of E.coli verses mesalazine (Hart et al, 2003)

Lactic Acid Bacteria

Mixture of lactobacilli and bifidobacteria fed for 12 weeks gave relapse rate of 15% in chronic pouchitis patients. In control (no treatment), relapse rate was 100%. Gionchetti et al 2000 Gastroenterology 119 Lactobacilli have also shown significant protection against inception of pouchitis following surgery for ulcerative colitis. 12 months after surgery 10% vs 40% controls had developed pouchitis (Gionchetti et al, 1999) Lactobacilli in combination with prebiotic(FOS) provided for 30 days, shown to produce complete suppression of symptoms in patients with refractory pouchitis (Friedman & George, 2000)

Irritable Bowel Syndrome

IBS is a multi-factorial GI tract disorder affecting 15-20% of the population in industrialized nations and provides between 25-50% of patient burden in hospital gastroenterology units (Mercenier et al., 2003
Curr Pharm Des 9)

IBS is often preceded by gastroenteritis or a course of antibiotics

Colonic mal-fermentation is hypothesized as a cause of IBS

Increased numbers of facultative microorganisms and decreased numbers of Lactobacilli and Bifidobacteria are associated with IBS

To date trials have been largely negative but justifiable reasons include : - trials of short duration
- insufficient potency of product.

Probiotics and Colon Cancer

Colorectal cancer (CRC) is the fourth highest cause of cancer mortality and morbidity, worldwide

8.9% of all new cancers are CRC Probiotics have been shown to prevent or cure colonic tumours in animal studies To date no robust randomized blinded trials have been carried out in humans but potential mechanisms of effect and in vitro evidence is compelling and includes:
Binding or degradation of potential carcinogens Stimulation of production of anti-tumorogenic compounds, e.g. short chain fatty acids Immune system modulation decreased inflammation, increased natural killer cell activity Modifying microfloral balance with resultant lower numbers of carcinogen-producing bacteria

Oral Tolerance, Probiotics and Allergy

Initiation of Type I Hypersensitivity Reaction (Allergy)

Allergens Outside body Epithelium Antigen-presenting cell Allergen is taken up by the antigen-presenting cell

Inside body

MHC class II Antigen Antigen receptor ACTIVATION

B cell

Immunoglobulin (IgE)

T lymphocyte (TH2)

Type I Immediate Hypersensitivity (Allergic) Response

Allergens Outside body Epithelium The feature of the allergen that the antibody recognizes IgE receptor Mast cell

IgE bound to mast cell

Inside body

Granules

INFLAMMATORY MEDIATORS

CLINICAL EFFECTS: e.g. asthma, hay fever eczema, upset stomach and urticaria

Allergy Recent Rise of an Epidemic

Allergic Asthma:
During the last 50 years, there has been a 40-fold rise in allergic asthma in the developed world. In the same period, there has been no significant rise in the developing world
(Strachen, 1989; Burr et al, 1988; von Matius et al, 1992; Bjorksten et al, 1999)

1 in 7 children born in the developed world will now develop allergic asthma
(Boner et al, 1998; Farooqi & Hopkin, 1998)

Environmental pollution now eliminated as cause

(Becker, 2000)

Allergy Development
Risk of Atopic Allergy Promoting Factors
(From Epidemiological study of 1,934 individuals)

% allergic No maternal atopy, pertussis immunisation or antibiotics Maternal atopy alone Pertussis immunisation alone Antibiotics <2 years old, alone 22 32 29 39

Maternal atopy and pertussis immunisation

Maternal atopy and antibiotics <2 years old Pertussis immunisation and antibiotics <2 years old Maternal atopy, pertussis immunisation and antibiotics <2years old

39
54 51 67
(Farooqi & Hopkin, 1998)

Allergic Development and Antigenic Challenge

Children with juvenile diabetes had fewer childhood infections Children with no infections during first year of life are 5 times more likely to develop diabetes or asthma

Every infection in first year of life correlates with 20% reduction in risk of developing diabetes
Measles sufferers proven to have 3-fold lower incidence of allergic disease Increasing use of antibiotics in first 2 years of life associated with significant increase in allergy in teenage years.

Environmental and Nutritional Trends Supportive of the Hygiene Theory, Notably in the Foetal/Neonate Period
* Increased levels of overall social hygiene in Westernised countries. * Introduction and increased use of antibiotics * Increased use of broad spectrum disinfectants * Early use of vaccination ( decreased viral challenge) * Inverse relationship between childhood infection and allergy * Reduced family size * Urbanisation ( in conjunction with 1) * Introduction of cows milk reduction of breast feeding.

All of the above result in reduced exposure to microbial challenge during the neonatal period

The Hygiene Theory

Children raised in less hygienic environments have significantly lower incidence of allergy than Western counterparts from a more hygienic environment Linkage with disease simply gives indication of exposure to a much wider library of antigens Microbial flora in G.I. tract known to be critically important in all aspects of immune system development Allergic infants have significantly different gut flora compared to non allergic infants Could manipulation/supplementation of gut flora safely optimise antigenic exposure and substantially reduce allergy?

The Neonate Immune System

The neonatal immune system is immature but biased towards Th2 cell types and cytokine profile. This dictates high antibody production capacity Contact with normal flora forces development of the immune system but is biased towards stimulation of Th1 and especially Th3 Contact with normal flora and dietary antigen has effect of balancing Th1 and Th2 and producing oral tolerance Allergic infants continue to demonstrate a dominant Th2 profile making the likelihood far higher that the response to an antigen will be Th2 mediated

Re-Directing the Neonate Immune System

Neonatal type I hypersensitivity is an effect of normal oral tolerance to allergens not being established.
Can induction and enhancement of oral tolerance to dietary and environmental allergens be produced by supplementing probiotics? The probiotic would provide sufficient antigenic stimulation for effective maturation of Th1/Th3 response without the danger of pathogenic response. The mechanism for this would be via bystander suppression. Deficiency of Th1/Th3 response cascade is due to deficiency in expression of IL-12 which stimulates Th1 response at start of cascade (MacDonald & Monteleone, 2002)

Human Peyers patches are strong stimulators of IL-12 but this depends on the quantity and type of flora present, e.g mice have much more limited flora with low potential to stimulate IL-12, so their immune system remains Th2 biased as in allergy.
If achievable, this would lead to reduction in prevalence and severity of allergic response.

Maturation of the Human Neonate Immune System by Acquisition of Normal Flora

Acquisition of sufficient microflora
Antibody Th2 mediated Cell Th1 mediated Oral tolerance Th3 mediated

Insufficient stimulation by microflora

Neonate at Birth

Normal development
Oral tolerance Th3

Abnormal development
Th2 Oral tolerance Th3

Th2

Th1

Th1

Different Likelihood of Response to First Experience of New Antigen (Allergen)

Processing by oral tolerance pathway (high likelihood)

Processing by antibody pathway (high likelihood)

Normal

Allergy

Lactic Acid Bacteria (LAB) and Allergy

Allergic (IgE/IgG) response in germ-free rats to ovalbumin, caused by lack of tolerance, is totally reversed by introduction of Bifidobacterium infantis (Sudo et al., 1997) Simultaneous dosing of bifidobacteria and b-lactoglobulin caused tolerance and increased levels of sIgA in rats. When bifidobacteria not fed, levels of IgE, and IgG were high risk of allergy. (Yoshino, 1998)
Long term consumption of yoghurt containing lactobacilli and

bifidobacteria reduced circulating IgE levels and clinical symptons in 3 clinical trials with asthma (Trapp et al, 1993; van der Water et al., 1999; Wheeler et al, 1997)
Milk hypersensitive infants given lactobacilli showed significant

decrease in inflammatory markers. This effect of reduced inflammation also seen in adults with milk hypersensitivity
(Majanaa & Isolauri, 1997; Pelto et al., 1998)

Reduced eosinophilia has been demonstrated in asthmatic adults

provided with L. acidophilus

(Wheeler et al., 1997)

Probiotics and Allergy

DBPC trial, providing 10 billion/day lactobacilli for 6 months to high risk atopic neonates, gave rate of eczema of 15/64 against 31/68 in placebo group. Result was significant (Kalliomaki et al., 2001

In DBPC trial with 43 children aged 1-13 years with nonspecific atopic dermatitis, administration of 40 billion Lactobacilli spp for 20 weeks caused a significant improvement in symptoms (56% improvement with probiotic, 15% with placebo) which was more pronounced with patients with more intense IgE response (Rosenfeldt et al, 2003)

Lactic Acid Bacteria and Allergy (cont)

Intestinal Microflora Differences in Allergic Infants High frequency of allergy in formula milk fed infants correlated with decreased
levels of Bifidobacteria and increased levels of Clostridia in feces. Rubatelli et al 1998 Allergic infants in countries of both high and low allergy frequency show low levels of lactobacilli and higher levels of aerobes notably coliforms and Staph.aureus compared to non-allergic infants. Bjorksten et al 1999 Intestinal microflora of infants at high risk of atopy were analysed at 3 weeks and 3 months for bacterial type. At 3 weeks the flora was significantly different in those that went on to become allergic with high numbers of Clostridia compared to Bifidobacteria the type of bacteria in neonate period can effect the outcome of the allergic response (Kalliomaki, 2001)

Evidence of Probiotics Causing Shift in Immune Balance.

Lactobacilli have been found to stimulate production of regulatory T cells producing TGF-B and IL-10 and suppressing Th2 cytokines on both animal and in human infant Haller et al 2000: van der Weid et al 2001; Pessi et al, 2000; Isolauri et al, 2000) Feeding lactobacilli to lactating mothers has been found to stimulate TGF-B in breast milk, suggesting probiotics exert direct effects in organs other than the intestine.

Lactobacilli have been found transiently stimulate Th1 and to down-regulate IL-4 (Th2) cytokine (Sutas et al, 1996)

Effect of probiotics on moderate/severe atopic dermatitis in children from 6-18 months

56 Subjects: 28 Placebo/28 Probiotics 8 Billion/day L. fermentum provided week 0-8 Normal drug use continues (Corticosteroid cream)
Time (weeks) 0

16

significant
improvement in probiotics treated (=0.03)

Scorad Index

-5

-4.2 -8.25

-3.9 -6.2

-10

-10.2 -15

placebo
-12

Change in SCORAD index

-18.2

improvement non significant

-17

-20

The Effect of Probiotic on the Severity of Allergy Following 8 Weeks of Supplementation and at 16 Weeks (Weston et al. 2005)
Mild (<25) 100% Moderate (25-50) Severe (>50)

percentage in each category

90% 80% 70% 60% 50% 40% 30% 20% 10% 0% placebo probiotic placebo probiotic placebo probiotic

Week 0

Week 8

Week 16

Possible Mechanisms for Probiotics Providing Preventative or Therapeutic Resolution of Allergy

Augmentation

of epithelial barrier Reduction of intestinally derived skin pathogens such as Staph aureus known to be implicated in eczema pathogenesis. Lack of immune tolerance induction either at the innate immune level or the adaptive immune function.

Probiotics Help Re-establish Tolerance in Allergy by Bystander Suppression

Soluble Lactobacillus antigen (diamond) is tolerised, stimulating TGF-B which suppresses processing of allergen (sphere) through the Th2 route

IEL /Th3
Blocking
Th2 cell

TgF -B
Th1 cell

or

TgF-

Blocks allergen from Being processed down Th2 pathway

Causes B-cell switching

From IgE and IgG to IgA

The South Wales Allergy Trial

Largest intervention trial in neonatal allergy ever set up Multi-centred across South Wales 3 major hospitals involved in patient recruitment

AIM: To evaluate the effectiveness of probiotic organisms and in the prevention of child onset type l hypersensitivity
Total of 3,000 individuals in 2 groups Outcome measured at 6 months (eczema) and 6 years of age (eczema, asthma and rhinitis) Sub-group evaluation of immune parameters First publication following first outcome Trial to commence in April 2005

Other Potential Uses of Probiotics 1) Autism 2) Protection from G.U.T infections 3) General protection in ICU 4) General protection against chronic inflammation syndromes.

The Gut-Brain Axis of Pathology in Autism

Opioid peptides excess

Dietary Protein

Defective enzyme/ Microbial environment

Opioid peptides excess

Intestinal permeability

Inflammation In autism patient

Low active tissue peptidases with antagonism endorphins

Systemic carriage

High TNF- titre High IL-1 and IL-6 titre Skewing to Th2 Depression of Th1 immunity
- Decreased NK cell - Decreased Th1 CD cell - Decreased Ig A

Opioid receptors in brain

Low sensitivity to pain

Repetitive behaviour

Social deficiencies

Some retardation in Cognitive development

Breakdown of wheat exorphin A5 by selected lactic acid bacteria

5.00E+06 4.50E+06
Peptide concentration

HMF 221 HMF 341

4.00E+06 3.50E+06 3.00E+06 2.50E+06 2.00E+06 1.50E+06 1.00E+06 5.00E+05 0.00E+00 0 20 40 60 80 100 120

140

160

Time (minutes)

Breakdown of -caseomorphin-7 by selected lactic acid bacteria

3.00E+06

HMF221
2.50E+06

HMF341

Peptide concentration

2.00E+06

1.50E+06

1.00E+06

5.00E+05

0.00E+00 0 20 40 60 80 100 120 140 Time (h)

A Probiotic Specifically Developed for Autistic Spectrum Disorder

Selection Criteria
1 Existing Strains L. acidophilus CLT 60 L. acidophilus CLT 21 B. bifidum CLT 20 B. lactis CLT 34 New Opiate Degrading Strains L. crispatus CLT 221 L. rhamnosus CLT 341
1 - survival through stomach 2 - resistant to bile salts 3 - attach to epithelial cells

2 Y Y Y Y

7 Y Y Y Y

Y Y Y Y

Y Y Y Y Y Y Y Y

N Y N Y N Y N Y

Y Y

Y Y

Y ? Y ?

Y Y Y Y

N N

4 - modulate immune system 7- Proven in human 5 - opiate peptide degradation DBPC trials 6 - industrial scale-up/stability

Effect of 21 days supplementation New Autism Specific probiotic on urinary excretion of opioid peptide exorphin B5 in six normal adult subjects

40000 35000 Before HMF supplementation After HMF supplementation

Relative size of LC/MS peak

30000 25000 20000 15000 10000 5000 0 0 2

6 Time (hours)

10

12

Are Probiotics Now an Essential Supplement?

Probiotics should now be considered essential post-antibiotic therapy Probiotics should now be considered essential daily supplements for all infants from birth to 12 months ( to prime the immune system and help prevent onset of allergy and intolerances Probiotics should be considered essential for anyone suffering IBD, IBS, rheumatoid arthritis or any other autoimmune disease

There is now building evidence that probiotics are beneficial in preventing CRC, in cholesterol lowering activity and possibly in helping sustain a competent immune system into old age

The emergence of the importance that the normal flora has in priming the immune system supports the concept of probiotics being a desirable dietary intervention for all individuals throughout our lives.

Prebiotics Good? Bad? Or Just Another Source of Fibre?

Definition
Non-digestible food ingredients that beneficially affect the gut by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, that can improve host health
(Gibson & Roberfroid 1995)

But in Practical Terms:

The function of prebiotics is to specifically promote the growth and/or activity of the desirable types ie. Bifidobacteria, Lactobacilli and others (SCFA producers), which consequently produces a relative reduction in undesirable types such as E coli, Klebsiella, Clostridium and Candida.

Types of Prebiotic
Classification
Disaccharides Lactulose Lacticol Oligosaccharides Fructooligosaccharides (FOS) Soybean oligosaccharides (Trans) Galactooligosaccharides Polysaccharides Inulin Resistant starches

Origin/Manufacturing Procedure
From Lactose Synthetic From Lactose Synthetic Legumes, vegetables, cereals Extraction/hydrolysis Soybean Extraction/hydrolysis From lactose Synthetic Legumes, vegetables, cereals Extraction Legumes, vegetables, cereals Extraction

Fructo-oligosaccharides Basic Facts

CONTENT OF FOS IN RAW COMMON FOODSTUFFS
Banana Asparagus Garlic Leeks Onions Chicory Wheat Flour
(Figures for Inulin are similar)
Moshfegh et a1, 1999

0.3 0.7g/100g 2.0 3.0g/100g 3.6 6.4g/100g 2.4 8.0g/100g 1.1 7.5g/100g 19.6 26.2g/100g 1.0 3.8g/100g

US intake range of FOS and Inulin 1-5g/day European intake range of FOS and Inulin 5-18g/day Northern Europe typically at low range Mediterranean Europe at high range

Physiological Effects of FOS

The specific stimulation of the probiotic component of the normal microflora, ie. Bifidobacteria and Lactobacilli, together with a concomitant reduction in less desirable microflora components, eg. Clostridia and Coliforms

The stimulation of production of lactate and short chain fatty acids, notably butyrate.
These effects and the resultant physiological sequelae are summarized on the next slide

Physiological Effects and Mechanism of Action of FOS

Reduced exogenous infections and dysbiosis Reduced levels of putrefactive bacteria Inhibited fermentation of protein Promotion of Bifidobacteria and Lactobacilli Stimulation of immune response and immune tolerance

FOS AND OTHER PREBIOTICS

Butyrate

Reduced ammonia, free amines, indoles and phenol

Production of lactic acid and SCFAs

Butyrate

Reduction in intestinal pH

1. Induced cell differentiation 2. Induced apoptosis Energy for colonocyte

Increased mineral solubility and absorption 1. Precipitation of deconjugated bile acids 2. Reduced conversion of primary to secondary bile acids

3. Stimulated immunogenicity Reduced intestinal permeability

Lower cholesterol recycling Lower serum cholesterol Reduction in risk of colorectal cancer

Reduction in risk of: Inflammation, IBD, IBS Allergy Toxin ingression Infection

Differential Effects on the Human Microflora The Bifodogenic Effect

Organism In Vitro Experiment Action and Animal In-Vivo Experimentation Bifidobacteria Lactobacilli Clostridia Effect of FOS Increase Increase Decrease E.coli

Decrease Other Coliforms Decrease

Wang & Gibson 1993 Davidson et al 1998 Rycroft et al 2001 Djouze&Andrieux 1997 Hussein et al 1999

Differentiation Effects on Human Microflora (Contd)

Human Trials Randomised, Blinded

Duration Significant Increase Significant Decrease No Change Reference
(Gibson et al, 1995)

Amount FOS Fed

15g/day

15 days

Bifidobacteria

Bacteroides Fusobacteria Clostridia Bacteroides

Lactobacilli Gram positive cocci Coliforms Bacteroides Clostridia E. coli Klebsiella Citrobacter Candida albicans

8g/day 10g/litre

35 days 28 days

Bifidobacteria Bifidobacteria

(Roberfroid et al, 1998) (Boehm et al, 2002) Pre-term infants

20-40g/day 18g/day 8g/day 5-20g 12 days 8 days

Bifidobacteria Bifido/Lactob Bifidobacteria Bifidobacteria

Enterococci

(Kleessen et al, 1997) (Bouhnik et al, 1996) (Menne et al, 1997) (Bornet et al, 2002)

Bacteroides

The Effects of FOS on Short Chain Fatty Acid (SCFA) Production

Fermentation of FOS and other Prebiotics result in the production of Lactate, Biomass, Gas and SCFA Ratio of SCFA production is relatively constant. Acetate Propionate Ratio 3 : 1 : Typical amount/day 18g 6g Butyrate 1 6g

Butyrate A Metabolic Powerhouse

Over the past 15 years the importance of butyrate to human metabolism has become clear, and is summarized below: Butyrate provides 70% of the energy for the colonic epithelial cells, or coloncytes (Roediger, 1980; Cummings & Macfarlane, 1997) Buyrate controls the turnover and differentiation of the colonic epithelial cells and is capable of inducing differentiation in colon carcinoma cells
(Smith et al, 1998; Velzquez et al, 1996)

Butyrate induces apoptosis (programmed cell death) in normal growing colonic cells and reverses resistance to apoptosis in colonic cancer cells (Bornet, 2002) Butyrate increases immunogenicity (susceptibility to immune cell policing eg. by NK cells) of cancer cells. Indeed, butyrate in combination with interleukin 2 caused complete clearance of induced colon carcinoma in rats
(Perrin et al, 1994; Bornet et al, 2002)

Does Supplementation with FOS Increase Production of SCFA?

In vitro models of colonic fermentation show 4-fold increase in butyrate production within 24 hours feeding FOS (Rycroft et al, 2001) Numerous studies in rats have shown substantial increases in SCFA concentration and decreased faecal pH, following administration of prebiotic oligosaccharides
(Djouze & Andrieux, 1997; Campbell et al, 1997; Younes et al, 1995)

In human studies the demonstration or significant levels of SCFA in the human colon was demonstrated using autopsy of sudden death victims
(Macfarlane et al, 1992)

Also increase in SCFA production following supplementation of FOS has been demonstrated in several human studies:
Gibson et al, 1995 Stowe et al, 1987 Rumessen et al, 1990

Do Prebiotics Decrease Risk of Colecterol Cancer?

FOS & Other Prebiotics Increased SCFA

Increased Butyrate

Physiological Impact

Decreased CRC

Any Evidence of Direct Link?

FOS & Mineral Absorption

Increasing acidity in large intestine increases mineral solubility magnesium, calcium, iron & zinc (Crittenden 1999; Trinidad et al, 1999) Dietary calcium absorption increases significantly by 26% from 47.8% to 60.1% in human trials where adolescents fed 5g/day FOS
(Van de Heuval, 1999)

In separate trial dietary calcium absorption increased 20% with adolescents fed 8g/day FOS (Van Poppel 2000)
Dietary magnesium absorption and plasma magnesium levels showed significant increase of 12% from 30.2% to 33.9% when human subjects supplemented with 10g/day FOS (Tahiri et al, 2001)

FOS & Insulin Resistance Syndrome (IRS)

Prevalence of IRS and type 2 diabetes increasing to epidemic levels - 3 to 5% of population now affected by type 2 diabetes
(Standl & Scholl, 2000)

Numerous studies have found FOS to reduce serum triglyceride, free fatty acid and cholesterol level in rats, and blood insulin levels
(Delzenne et al, 1993; Aghele et al, 1998; Trautwen et al, 1998; Jackson et al, 1999)

Possible Mechanism of FOS Effects on Plasma Lipid and Glucose Homeostasis

SCFA production effecting hepatic lipogenesis
Intestinal bile precipitation from decreased pH reduces recycling or bile from intestines to liver lower plasma cholesterol Direct reduction in plasma insulin levels (unknown mechanism)

Does FOS and Other Prebiotics Reduce Risk of Colorectal Cancer?

Studies in rats & mice show significant reduction in formation of abberant crypt foci (ACF) when FOS or inulin is fed
(Reddy et al 1997; Rowland et al, 1998)

Combining use of FOS with Bifidobacterium longum gave greater effect (in above) than either individual component.

In human trials 35.9% of patients with colonic adenomas removed has re-occurrence within 12 months, use of lactulose reduced this to 14.7% - a significant reduction.
Only butyrate generating prebiotics produce reduction in ACF, insoluble fibre such as starch free wheat bran does not reduce ACF
(Perrin et al, 2001)

Flatulence & Bloating Slight Breeze or Force 10 Gale!!!!

Consumption of FOS and other prebiotics causes increase in gas production in most people This is temporary and in a high majority of people subsides in 2-3 weeks, to level associated with relatively high fibre diet Lactobacilli & Bifidobacteria High acid producers and low gas producers Coliforms, Bacteroids etc Low acid producers and high gas producers Good regime is to start supplementation at 2g/day and build to 10g/day over a 2-3 week period

Flatulence & Bloating Slight Breeze or Force 10 Gale!!!!

Start FOS

FOS Supplementation

Probiotics and Prebiotics Combined Benefits

Conceptually, the desirable state or optimising intestinal flora can be achieved by either probiotics or prebiotics. Prebiotics help probiotics become established. Probiotics provide the most physiological beneficial strains.

Prebiotics help maintain high populations of probiotics.

Combination of prebiotics and probiotics will provide synergistic benefits (Gibson, 1998)

Summary
Key Features Key Clinical Indications

Low calorific sugar substitute

Bifidobacteria/Lactobacilli promoter

SCFA generator

Obesity Insulin resistance syndrome (IRS) and Type 2 diabetes Weight reduction program Tooth decay prevention Intestinal dysbiosis/overgrowth Leaky gut syndrome Intestinal inflammation and IBS Immune system stimulation Mineral absorption improvement Osteoporosis/osteoarthritis Cardiovascular disease Colorectal cancer Intestinal inflammation disease Irritable bowel syndrome Leaky gut syndrome