Luc De Vuyst.pdf - ICoMST Contact Point

Research Group of Industrial Microbiology and Food
Biotechnology
Prof. Dr. ir. Luc DE VUYST
Probiotics in fermented sausages
Luc De Vuyst, Gwen Falony, Frédéric Leroy
54 th ICOMST
Cape Town, South Africa
August 10-15, 2008

Outline
• Introduction to functional/probiotic foods
• Probiotics
• Important considerations
• Probiotics in fermented meat products
• Conclusions

Outline
• Introduction to functional/probiotic foods
- Definition functional food
- Definition probiotics
- Definition probiotic meat products
• Probiotics
• Important considerations
• Probiotics in fermented meat products
• Conclusions

Introduction – Functional foods
A functional food is a food for which it has been satisfactorily
demonstrated that it beneficially affects one or more target functions in
the body, beyond adequate nutritional effects, in a way that is relevant to
either an improved state of health and well-being and/or reduction of risk
of disease (Diplock et al., 1999).
Relationship food-health:
Hippocrates – Nutrition (balanced diet) – Health (health-promoting diet) –
Nutrigenomics
Medicine or drug: something you take when you are ill
Functional food: the healthy food you eat throughout your life
and which in many cases will protect you from otherwise
life-threatening diseases

Introduction – Functional foods
A functional food is a food for which it has been satisfactorily
demonstrated that it beneficially affects one or more target functions in
the body, beyond adequate nutritional effects, in a way that is relevant to
either an improved state of health and well-being and/or reduction of risk
of disease (Diplock et al., 1999).
Relationship food-health:
First generation:
- focussed on the traditional, nutritional value of a food
- deal with the application of primary nutrients and nutritional claims
- aim is to avoid deficiencies and to optimise a normal diet
- e.g. natural foods (fruits and vegetables, dairy products),
diet margarine

Relationship food-health:
Second generation:
Introduction – Functional foods
A functional food is a food for which it has been satisfactorily
demonstrated that it beneficially affects one or more target functions in
the body, beyond adequate nutritional effects, in a way that is relevant to
either an improved state of health and well-being and/or reduction of risk
of disease (Diplock et al., 1999).
- focuses on effects beyond the traditional, nutritional value of a food
- deal with application of secondary nutrients and health claims
- aim is to optimise nutrition of a healthy diet and to focus on
health-promoting effects
- e.g. fibre-, calcium-, vitamin- and ω3-enriched foods
diet margarine with added phytosterols
hypoallergenic rice
gluten-free bread
probiotics, prebiotics, and synbiotics

Introduction - Probiotics
Probiotics (προ βίος, pro bios, ‘for life’) are live microorganisms that, when
consumed in an adequate amount as part of the food, confer a health
benefit on the host (FAO/WHO, 2001).
Long history:
Elias Metchnikoff (1907/1908): “The prolongation of life”
Minoru Shirota (1930): Lactobacillus casei Shirota
Parker (1974): animal feed supplements with a beneficial
effect on the host by contributing to its intestinal microbial
balance
Fuller (1989): live, microbial feed supplements, which
beneficially affect the host animal by improving its intestinal
microbial balance
Havenaar & Huis in ‘t Veld (1992): viable microorganisms that exhibit a
beneficial effect on the health of the host upon ingestion in sufficient
amounts by improving the properties of its indigenous microbiota
De Vuyst, Avonts & Makras (2004). Woodhead Publishing, Ltd., Cambridge, UK

Long history:
Introduction - Probiotics
Probiotics (προ βίος, pro bios, ‘for life’) are live microorganisms that, when
consumed in an adequate amount as part of the food, confer a health
benefit on the host (FAO/WHO, 2001).
… animals/humans … properties/functionalities … formulations
FAO/WHO (2001): live microorganisms that, when consumed in an
adequate amount as part of the food, confer a health benefit on the host
ILSI (2002): live, microbial food ingredient that, when taken up in
adequate amounts, confers health benefits on the consumer
EFFCA (2003): live microorganisms, which, when ingested or locally
applied in sufficient numbers, provide the consumer with one or more
proven health benefits
De Vuyst, Avonts & Makras (2004). Woodhead Publishing, Ltd., Cambridge, UK

Target organs:
Introduction - Probiotics
Probiotics (προ βίος, pro bios, ‘for life’) are live microorganisms that, when
consumed in an adequate amount as part of the food, confer a health
benefit on the host (FAO/WHO, 2001).
digestive tract (colon, small intestine, stomach)
other sites of the body (oral cavity, urogenital tract, skin)
immune system
cardiovascular system
Current delicate points of discussion:
Site of activity (foods or drugs, prevention or curing, …)
Viability (life or dead cells, alive upon ingestion, alive at the site of action, …)
Amount of cells (food product) necessary to exert a specified probiotic effect
Format of intake and carrier [mono- or mixed cultures, food (dairy) products,
food supplements, pharmaceutical preparations, …]
Requirement of oral administration for clinical efficacy
De Vuyst, Avonts & Makras (2004). Woodhead Publishing, Ltd., Cambridge, UK

Introduction - Probiotics
Probiotics (προ βίος, pro bios, ‘for life’) are live microorganisms that, when
consumed in an adequate amount as part of the food, confer a health
benefit on the host (FAO/WHO, 2001).
Micro-organisms (strains):
Lactic acid bacteria:
- Lactobacillus casei Shirota (YIT 9029, Yakult)
- Lactobacillus casei Imunitass (DN-114 001, Actimel)
- Lactobacillus rhamnosus GG (ATCC 53103, Gefilus, Vifit, …)
- Lactobacillus johnsonii La1 (NCC 533, LC1)
- Lactobacillus plantarum 299v (ProViva)
- Bifidobacterium animalis subsp. lactis Bb12 (various brand names)
- Bifidobacterium animalis subsp. lactis Actiregularis (DN 173-010, Activia)
- Bifidobacterium longum BB 536 (yoghurt, powder)
- VSL#3 (mixture of eight strains, biotherapeutic agent)
Other bacteria: E. coli Nissle 1917 (Mutaflor, biotherapeutic agent)
Yeasts:
- Saccharomyces boulardii (Enterol, biotherapeutic agent)
De Vuyst, Avonts & Makras (2004). Woodhead Publishing, Ltd., Cambridge, UK

Introduction - Probiotics
A probiotic food is a food that contains probiotics in sufficient quantities
and that exerts beneficial effects on the host beyond its primary
nutritional effect.
PROBIOTICS
as/via
Traditional
Functional
Food
Pharmaceutical
fermented foods
foods
supplements
preparations
Foods of animal or
vegetable origin
fermented with
lactic acid bacteria,
which are consumed
without heat treatment
New type of foods that
contain probiotic lactic
acid bacteria strains that
have been selected on
the basis of specific,
functional properties
(fermented dairy
products, fruit juices, ...)
Probiotic microorganisms that have been
selected on the basis of their specific,
functional properties (powders, tablets,
capsules, …)

Introduction - Probiotics
A probiotic food is a food that contains probiotics in sufficient quantities
and that exerts beneficial effects on the host beyond its primary
nutritional effect.
W. Kneifel

Introduction - Probiotics
A probiotic food is a food that contains probiotics in sufficient quantities
and that exerts beneficial effects on the host beyond its primary
nutritional effect.
Animal feed: 46 %
Dietary supplements:
44 %
Dairy products: 9 %
Other applications:
1 %

Introduction - Probiotics
A probiotic (fermented) meat product is a (fermented) meat product that
contains probiotics in sufficient quantities and that exerts beneficial
effects on the host beyond its primary nutritional effect.
Challenges:
Marketing and re-appreciation of fermented meat products as “healthy foods”
Microbiological, technological, and regulatory aspects
Steps to be taken:
Careful selection of probiotic strains:
- survival of the passage of the upper gastrointestinal tract
- cell viability in a fermented meat matrix
Meat as carrier for probiotic strains:
- resistance towards technological processing
Beneficial health effects of probiotic meat products:
- underlying mechanisms of action
- demonstration of human benefits
De Vuyst, Falony & Leroy (2008). Meat Science 80, 75-78.
A. Ouwehand

Introduction – Functional foods
A probiotic (fermented) meat product is a (fermented) meat product that
contains probiotics in sufficient quantities and that exerts beneficial
effects on the host beyond its primary nutritional effect.
And also:
- safe
- no disadvantageous effect on texture or taste
- no side effects when correctly used
- no disturbance of the normal eat pattern
- efficient production and payable by everybody
- scientific relevance of potential claims
De Vuyst, Falony & Leroy (2008). Meat Science 80, 75-78.
A. Ouwehand

Outline
• Introduction to functional/probiotic foods
• Probiotics
- Selection criteria
- Health effects
- Mechanisms of action
• Important considerations
• Probiotics in fermented meat products
• Conclusions

De Vuyst, Avonts & Makras (2004). Woodhead Publishing, Ltd., Cambridge, UK
Important criteria for probiotics
Probiotics: selection criteria
- Human origin and non-pathogenic behaviour
- Survive passage through the upper gastrointestinal tract
- resistance to gastric acid and bile toxicity, digestive enzymes, …
- active during transit from stomach to colon (expression of genes during intestinal
transit)
- Interact with the resident microbiota
- ability to transiently adhere to or interact with the intestinal epithelium and colonize the
colon
- do not affect the dominant microbiota significantly
- have an effect on the numbers and diversity of the endogenous Lactobacillus and
Bifidobacterium species
- Affect the function of the host:
- probiotic-host interactions (functional genomics, clinical trials and human intervention
studies)
- prevention of (the risk on) disease (meta-analyses)
- Resistant towards technological processing and storage
- importance of the food matrix and the applied process technology
- stable/alive till the moment of consumption (the number of live, active cells is
important)
- High safety profile and excellent tolerance (Lb. rhamnosus, D-lactic acid)

Probiotics: health effects
Classification according to scientific evidence
- improved food digestion (e.g. proteins, dietary polysaccharides)
- reduction of the risk of lactose maldigestion (β-galactosidase)
- supply and bioavailability of nutrients and growth factors (e.g. vitamins, minerals)
- maintenance and balancing of the colon microbiota
- reduction of (the risk of) intestinal disturbances (gastrointestinal infections,
constipation)
- reduction of the risk and duration of diarrhoea (rotavirus diarrhoea, acute
infectious diarrhoea, antibiotic-associated diarrhoea, Clostridium difficileassociated
diarrhoea, travellers’ diarrhoea)
- inhibition of undesirable and pathogenic bacteria (orogastrointestinal infections
caused by Streptococcus mutans, Helicobacter pylori, E. coli, Salmonella
Typhimurium, Clostridium difficile, Shigella flexneri, urinary tract infections,
respiratory tract infections)

Probiotics: health effects
- modulation/stimulation of the immune system (cell-mediated and antibodymediated
effects)
- reduction of the risk of atopic diseases and allergies (asthma, hay fever, food
allergy, eczema, dermatitis)
- beneficial effects on functional bowel disorders: irritable bowel syndrome
- beneficial effects on inflammatory bowel diseases: pouchitis, ulcerative colitis,
Crohn’s disease
- anticarcinogenic activities
- lowering of blood serum cholesterol levels
(De Vuyst, Avonts & Makras (2004). Woodhead Publishing, Ltd.; Servin & Coconnier (2004). Best Pract. Res.
Gastroenterol.; Servin (2004). FEMS Microbiol. Rev.; Rastall et al. (2005). FEMS Microbiol. Ecol.;
Saxelin et al. (2005). Curr. Opinion Biotechnol.; O’Hara & Shanahan (2007). Scientific World Journal;
Galdeano et al. (2007). Clin. Vaccine Immunol.)

Probiotics: mechanism of action
1. Probiotics influence microbe-microbe interactions
- Competition for nutrients available in the gut lumen
- Production of antimicrobial or toxic agents modifying the local
microenvironment and hence contributing to survival
2. Probiotics influence microbe-gut epithelium interactions
- Competitive exclusion along the epithelium
- Enhancement of the epithelial barrier function
- Suppression of intestinal inflammation
3. Probiotics influence microbe-immune system interactions and
modulate the host immune response
- Cell-mediated immune responses
- Humoral (antibody-mediated) and innate immune responses
(De Vuyst, Avonts & Makras (2004). Woodhead Publishing, Ltd.; Servin & Coconnier (2004). Best Pract. Res.
Gastroenterol.; Servin (2004). FEMS Microbiol. Rev.; Rastall et al. (2005). FEMS Microbiol. Ecol.;
Saxelin et al. (2005). Curr. Opinion Biotechnol.; O’Hara & Shanahan (2007). Scientific World Journal;
Galdeano et al. (2007). Clin. Vaccine Immunol.)

Outline
• Introduction to functional/probiotic foods
• Probiotics
• Important considerations
• Probiotics in fermented meat products
• Conclusions

Important considerations
Health-promoting properties/mechanisms are STRAIN-SPECIFIC and
MATRIX-DEPENDENT
Not all strains of a given species possess the health-promoting
properties/mechanisms of action of a probiotic strain of that species.
Also, not all probiotic strains possess all health-promoting properties.
However, mechanisms of action are often multifactorial and are not
mutually exclusive.
Good examples are (target applications, cf. Table 1):
- Lactobacillus casei Shirota (YIT 9029) Yakult
- Lactobacillus casei Imunitass (DN-114 001) Actimel
- Lactobacillus rhamnosus GG (ATCC 53103) Gefilus, Vifit, …
- Lactobacillus jonhsonii La1 (NCC 533) LC1
- Lactobacillus plantarum 299v Provie
- Bifidobacterium animalis subsp. lactis Bb12 several products
- Bifidobacterium animalis subsp. lactis DN 173-010 Activia
- Bifidobacterium longum BB 536 yoghurt, powder
- … De Vuyst, Avonts & Makras (2004). Woodhead Publishing, Ltd., Cambridge, UK

Important considerations
Health-promoting properties/mechanisms are STRAIN-SPECIFIC and
MATRIX-DEPENDENT
Efficacy of a probiotic strain depends on the food matrix (meat matrix?)
And further depends on:
- culture conditions of the strain
- dose and frequency of ingestion
of the strain/product
- target group characteristics
- children, adults, or elderly
- pregnant women
- healthy volunteers vs diseased people INDIVIDUAL-
- immunocompromised people DEPENDENT!
- normal gut transit vs constipated persons
- standard vs normal diet
- … De Vuyst, Avonts & Makras (2004). Woodhead Publishing, Ltd., Cambridge, UK

Important considerations
Health-promoting properties/mechanisms are STRAIN-SPECIFIC and
MATRIX-DEPENDENT
Unfortunately:
- misuse of the probiotic concept by food manufacturers, in particular by
not specifying the probiotic strain used, which indicates the need for a
correct typing and food labelling plus freely accessible easy-tounderstand
documentation
- no bacteria present, other bacteria present, or no viable bacteria
present at the time of consumption (Temmerman et al., 2003a,b)
- no safety evaluation (use of species known to be at the borderline of
causing disease)
- no demonstration of health-promoting effects
De Vuyst, Avonts & Makras (2004). Woodhead Publishing, Ltd., Cambridge, UK

De Vuyst, Avonts & Makras (2004). Woodhead Publishing, Ltd., Cambridge, UK
Important considerations
STRAIN IDENTIFICATION and SCIENTIFIC VALIDATION OF HEALTH-
PROMOTING EFFECTS
Health benefits of a certain probiotic strain belonging to a certain species
can not be extrapolated to strains of the same or another species:
importance of strain identification!
Health benefits should be scientifically supported:
- mutants and in vitro and in vivo models as tools
- well-designed, randomized, double-blind, placebo-controlled clinical trials
or human intervention studies: importance of multi-centre research, peerreviewed
publications
Health-supporting claims should be technically well characterized:
identified product (matrix dependency)
effect observed in humans (target group dependency)
dose/effect relationship (cell count plus amount of product)
relevant biomarkers to measure the efficacy for improved functions or for
risks of diseases.

Outline
• Introduction to functional/probiotic foods
• Probiotics
• Important considerations
• Probiotics in fermented meat products
• Conclusions

Probiotics: selection criteria
Fermented meat as a carrier for probiotic bacteria
+ - Usually not or only mildly heated
- Protection of the survival of probiotic lactic acid bacteria through
the gastrointestinal tract
- - High content in curing salt
- Low pH (acidification) → affects cell viability
- Low a w (drying) (strain-dependent!)
De Vuyst, Falony & Leroy (2008). Meat Science 80, 75-78.

Careful selection of strains
1. Screening natural sausage isolates
Probiotics: selection criteria
- Lb. gasseri JCM 1131 T Arihara et al., 1998
- Lb. sakei RM10, P. pentosaceus P2 Erkkilä & Petäjä, 2000
- Lb. sakei, Lb. curvatus, Lb. plantarum strains Papamanoli et al., 2003
- Lb. plantarum strains Pennacchia et al., 2004, 2006
- Lb. plantarum/pentosus Klingberg et al., 2005
- Staphylococcus spp. strains Villani et al., 2005
Paramithiotis et al., 2006
- Lb. casei, Lb. paracasei, Lb. rhamnosus, Lb. sakei Rebucci et al., 2007
2. Screening existing commercial meat starter cultures
- Lb. sakei Lb3, P. acidilactici PA-2 Erkkilä & Petäjä, 2000
- Lb. acidophilus LAFTI ® L10 Mahoney & Henriksson, 2003
+ - Demonstration of physiological requirements
- Demonstration of technological effectiveness
- - Demonstration of health-promoting properties
De Vuyst, Falony & Leroy (2008). Meat Science 80, 75-78.

Careful selection of strains
Probiotics: selection criteria
Survival of L. reuteri ATCC 55730 (SD2112) during the manufacture of
dry fermented sausages (● — unencapsulated L. reuteri, ■ — L. reuteri
microencapsulated by extrusion, and — L. reuteri microencapsulated by
emulsion).
Muthukumarasamy & Holley (2006) IJFM, 164-169

Careful selection of strains
1. Screening natural sausage isolates
Probiotics: selection criteria
2. Screening existing commercial meat starter cultures
3. Testing strains with documented health-promoting properties
e.g. Lb. rhamnosus GG (Erkkilä et al., 2001a,b)
- Competition with the natural meat microbiota in an environment which is not their
natural habitat
- Survival of the fermentation and drying process
- Survival of refrigeration and storage conditions
- Ability to grow to numbers that enable the display of health-promoting effects
+ - Survival of the added species in the meat matrix
- Influence on technological and sensory characteristics of the final
products
- - Demonstration of health effects
De Vuyst, Falony & Leroy (2008). Meat Science 80, 75-78.

Careful selection of strains
Probiotics: selection criteria
Cell count [log cfu/g]
8
6
4
2
0
HOWARU Rhamnosus
Staphylococcus xylosus
0 5 10 15 20
Ripening time [days]
A. Ouwehand

Careful selection of strains
Probiotics: selection criteria
Flavour profiles of dry sausages fermented by different strains.
Erkkilä et al. (2001). Meat Science 58, 111-116

Food safety enhancement
Probiotics: selection criteria
- Lb. reuteri ATCC 55730 + B. longum ATCC 15708
- Lb. rhamnosus GG
derease of E. coli 0157:H7
Muthukumarasamy & Holley, 2007
Erkkilä et al., 2000
- Lb. gasseri JCM 1131 T
- Lb. rhamnosus FERM P-15120 + Lb. paracasei subsp. paracasei FERM
P-15121
decrease of growth and enterotoxin production of Staphylococcus aureus
Arihara et al., 1998
Sameshima et al., 1998
- Lb. acidophilus LAFTI ® L10
anti-Listeria activity
Pidcock et al., 2002
Mahoney & Henriksson, 2003
De Vuyst, Falony & Leroy (2008). Meat Science 80, 75-78.

Probiotics: selection criteria
Human studies using probiotic fermented meat
products
- Lb. paracasei LTH 2579 immmunity +
blood serum lipids -
Design of the intervention study
Jahreis et al. (2002). FRI 35, 133-138.
CD4 (T-helper) lymphocytes in
volunteers in relation to the numbers
of Lb. paracasei in faeces (significant
increase for some but not all
volunteers).

Probiotics: selection criteria
Human studies using probiotic fermented meat
products
- Lb. paracasei LTH 2579 immmunity +
blood serum lipids -
Design of the intervention study
Jahreis et al. (2002). FRI 35, 133-138.
CD54 (ICAM-1, intercellular
molecule of adhesion for cell
cooperation) in volunteers with
different counts of Lb. paracasei in
faeces.

Outline
• Introduction to functional/probiotic foods
• Probiotics
• Important considerations
• Probiotics in fermented meat products
• Conclusions

Conclusions
• The health-promoting, functional food market is a growing
market.
• Probiotics play an important role in gut health.
• Health-promoting properties are strain-/product-specific,
matrix-dependent, and individual-dependent.
• Food manufacturers should apply a careful strain selection
and correct labelling.
• Mechanisms of action are becoming elucidated.
• Health claims should be supported by scientific studies
(mechanistic studies, human trials, ...).
• More research is needed to justify the launch of new
probiotics in general and of probiotic fermented sausages in
particular, particularly with respect to human studies.

Acknowledgements
• Research Council of the Vrije Universiteit
Brussel (OZR, BOF)
• Fund for Scientific Research – Flanders
• Institute for the Promotion of Science and
Technology in Flanders (IWT-GBOU-010054,
IWT-040815, IWT-070029)
• Ministry of the Flemish Community (AWI)
• European Commission (QLK1-CT-2001-
01179-PROPATH)
• National and international food companies
(Imperial Meat Products, Danone, Orafti,
Yakult, ...)

Thank you !