Trends in Society re Food

1
Trends in Society re Food

• The function of Food is changing
• what we eat caloriesgt experience gt
  nutrition health, variety
• when we eat regular meals gt grazing snacking
• where we eat in-home gt out-of-home
• with whom we eat social gt individual
• how we prepare our food from scratch gt
  ready-to-eat heat eat
• A wealth of choice
• primary production year round, global supply
• food industry preserved, frozen, chilled,
  freshly prepared
• role of retail
• out-of-home

2
The starting pointConsumer Needs
Trust
Can I trust this company?
Is it good for me?
Honesty
Can I trust this brand?
Is it fresh?
Does it do what the advertising claims?
Is it natural?
Naturalness
Familiarity
What was added?
What does the label mean?
Food Safety
Quality
Is it safe to eat?
Does it taste good?
Trust
Courtesy to the ETP Food http//www.etp.ciaa.be/a
sp/press.asp
3
Challenges for Food Industry

• Consumer preferences

• Clean label / preservative-free products
• Milder processing
• Lower salt and sugar content
• Convenience

Microbiological safety is a must have!

4
Functional genomics of food fermentation derived
preservatives weak organic acids as a case study

The Food Industry
Courtesy to the ETP Food http//www.etp.ciaa.be/a
sp/press.asp
5
Sequencing genomes in days
Key events in molecular biology genetics
molecular biological techniques
discoveries
1859
Origin of species -Charles Darwin
1866
Mendel's model of inheritance
1953
DNA double helix -Watson Crick
1961
discovery of restriction-enzymes
1967
sequencing technology
1977
PCR technique
1980
microbial sequencing total yeast DNA sequence
known
1996
1997
development micro-array technology
1998
total human DNA sequence known over 300 microbial
genomes known
2001
6
Present concept of Food Fermentation
Bioprocess by micro-organisms and their enzymes
Desirable product properties
A food is considered fermented when one or more
of the constituents of its substrate have been
acted upon by selected microorganisms or their
enzymes to produce a significantly altered final
product desirable for human consumption
7
Range of fermented foods
8
Desirable properties
Appeal
Exterior Texture Odour Taste
9
Desirable properties
Appeal
Utility
Volume reduction Cooking time reduction Shelf-life
Nutrient retention Valorisation of by-products
10
Desirable properties
Appeal
Utility
Functionality
Safety Digestibility Probiotic effect Other
physiological effects
11
Preservative effects of fermentation
Antimicrobial metabolites
MWlt1000 Da
Acids Alcohols Carbon dioxide Diacetyl Hydrogen
peroxide Reuterin, etc.
MWgt1000 Da
Bacteriocins Killer-toxins
Microbial competition
12
Preservation

• Lowering pH by production of lactic acid prevents
  growth of food-spoilage and pathogens
• LABcompete out other bacteria for available
  nutrients
• Production antibacterial and anti-mold/fungi
  substances special protective cultures
• Examples
• Acidification (all cultures)
• Nisin-producing starter for cheese (CSK)
• Holdbac (Danisco)

13
Yeasts response to weak organic acids?
14
Abbott et al. 2007 FEMS Yeast Research. Anaerobic
chemostats
medium pump
F
Gas in
Sr
s
Gas in
V
DF/V
Sampling
15
Expression profiles of cells responding to 4
different weak organic acids
16
Defined and controlled fermentor setup

• Saccharomyces cerevisiae strain CEN.PK 113-7D
• 500 ml batch fermentors
• defined mineral medium, glucose excess
• 30C
• pH controlled (no buffers)
• steady airflow
• easy sampling
• high reproducibility

17
The transient effect of weak acids sorbic acid in
yeast a case study
See also Papadimitriou et al. 2007 Int. J. Food
Micro.
18

• Cell wall (protein)
• adaptation

• Membrane homeostatis

• gt Mitochondrial activity

• Energy depletion

• Membrane damage

HA
HA
HA
Fps1

• Oxidative damage

• gt ROS damage repair
• systems

?
Pma1

• Intracellular acidification

• Enhanced Pma1
• activity

?
Pdr12

• Anion accumulation

OUT
IN

• Membrane pumps

19

• Intracellular acidification

vacuole
Pma1
Measure intracellular pH accurately in vivo
without perturbing the cells in different
organelles.
Pdr12
mitochondrion
OUT
IN
20
The need to measure intracellular pH accurately

• Measure in vivo
• Measure different compartments
• Available methods give large variation
• Handling cells influence measurements

21
Handling cells influence measurements
Karagiannis et al, Journal of Cell Science, 2001
22
Measure pH in vivo with pHluorin GFP
Wild type GFP
pHluorin E132D, S147E, N149L, N164I, K166Q,
I167V, R168H and L220F.
Miesenböck et al, Nature, 1998
23
Target pHluorin to different organelles in the
cell
ACT
pHluorin
mit.
-
URA
24
GFP expression in single cells
25
Initial results show stable pHi
26
Stress with sorbic acid results in drop of pHi
27
Intracellular pH effects correlate with growth
effects
28
Possible components of sorbic acid stress

• Intracellular acidification

?

• measurable in independent
• cell compartments

• Anion accumulation ?

• Energy depletion ?

• Membrane damage ?

29
Assumptions.
Rate equations.

• Outside pH is kept constant.

• Total intracellular cell volume is very small
• compared to extracellular volume.

• Free diffusion of HA over the membrane.

Pma1
Could also be exported to vacuole or other cell
compartments

• Dissociation rate much higher than other
  constants.

• Active, ATP dependent transport for A- and H.

For other organisms a degradation
component could be added
Pdr12
OUT
IN
30
How does Bacillus subtilis counteract sorbic acid
stress?

• Gram-positive model organism
• Responsible for food spoilage (endospores!) ?
  problem for food industry
• Bacillus subtilis has highly developed adaptation
  mechanisms towards starvation/stress

• Transcriptional Programs
• General Stress Response (SigB)
• Stringent Response (preventing waste of
  nutrients RelA dependent)
• Sporulation (Spo0A)
• Competence (ComK)
• Specific Stress Responses

End Goal -Understand the mode of action of weak
organic acids. -This can lead combined with the
insight in thermal stress on spores to the
development of process-ingredient minimal
preservation conditions.
31
Transcriptome analysis (microarray) was performed

• All treated samples were compared to untreated
  (control) samples of the same time-points

32
B .subtilis oligonucleotide slides (Sigma
genosys) 4,128 oligos 4,106 oligos for
Bacillus genes one oligo per gene 65-mer oligo
plus linker, spotted in duplo
Ambion/stratagene spike controls
33
Data Analysis
Hierarchical Clustering Bioinformatics tool that
identifies groups of genes with similar
transcription profiles
T-Profiler Bioinformatics tool (Boorsma et al.,
NAR, 2005) that determines significantly
regulated groups of genes

• Need multiple experiments
• Need cut-off
• Focus on individual interesting (unknown) genes

• One experiment
• Insight in biological processes
• No cut-off needed
• Need predefined groups

(DBTBS (TF SF), KEGG Pathways, SubtiList
Functional Categories)
34
Induction of stationary phase genes
stringent-type response
No induction of Sporulation and General Stress
Response!
35
Energy related functions
The cell experiences a possible nutrient
mineral limitation
36
Induction of membrane related functions
Sorbic acid treated cells are more resistant to
cerulenin a fatty acid biosynthesis inhibitor
Repair and/or remodelling (longer more branched
chains)
37
Data Analysis
Hierarchical Clustering Bioinformatics tool that
identifies groups of genes with similar
transcription profiles
T-Profiler Bioinformatics tool (Boorsma et al.,
NAR, 2005) that determines significantly
regulated groups of genes

• Need multiple experiments
• Need cut-off
• Focus on individual interesting (unknown) genes

38
Susceptibility of selected mutants
mdrA unknown similar to multidrug resistance
protein Sensitive mdrB unknown similar to trp
repressor binding protein Resistant
fabHB beta-ketoacyl-acyl carrier protein
synthase WT susceptibility ureC urease (alpha
subunit) WT susceptibility yxkJ unknown
similar to metabolite-sodium symport
Resistant ycsF unknown similar to lactam
utilization protein WT susceptibility sigB gen
eral stress sigma factor WT
susceptibility sigL involved in utilization of
arginine and ornithine WT susceptibility and
transport of fructose sigM RNA polymerase
ECF-type sigma factor WT susceptibility
Little correlation between transcriptome patterns
and behavior of mutant strains
39
Long term stress survival of lipophilic weak acid
resistance mutant on plates
mut
mut
mut
40
From cell to molecule and back a systems approach
Major targets in cells for the weak
organic acid sorbic acid
A. Ter Beek et al. 2006 PATENTED J.
Bacteriol. 2008

• Major facilitator superfamily multidrug
  resistance (mdr) protein homologue. The deletion
  mutant responds to sorbic acid depending on the
  nutrient richness of the media. In rich media
  sensitive but in defined media resistant!

• Phospholipids with longer tails and more branching

• Glucose uptake
• perturbation

A-
A-
Mdr

• Regulatory Processes

41
Bacillus spore germination and molecular
mechanisms involved
Keijser et al. (2007) J. Bacteriology 189,
3624-3634
42
Model for the transcriptional systems during B.
subtilis spore germination
antimicrobial targets
43
Proteome verification in B. subtilis spores
from lab-strains (168) and true wt (Hr and Hs)
cells
96 identity
85 identity
pH 4 pH 7
pH 4 pH 7
pH 4 pH 7
B. subtilis Hr
B. subtilis Hs
B. subtilis 168
2.11 fold increased expression
44
Peptide identification of induced protein spots
in A163 Hr versus A 163Hs summary

• B. subtilis superoxide dismutase (required for
  the assembly of CotG into the insoluble matrix of
  the spore)
• B. subtilis endopeptidase Clp (involved in
  stationary phase adaptative responses good
  indicator for protein stress in the cell)
• B. subtilis general stress protein (sB dependent
  protein)
• B. subtilis yvaB homolog (NADPH dehydrogenase
  with an unknown function)
• B. subtilis dapB protein (dihydrodipicolinate
  reductase a shunt in dipicolinic acid synthesis)

Spores derived from knock-outs strains are to be
assessed for their thermal resistance
45
Stability by Design
Consumer Use
Microbial Communities Prediction of behaviour
Intervention strategies
Supply Chain
Metabolism Physiology
Product Manufacture
Raw Materials
Process Design
Gene-expression
Product Design
46
Systems (micro)biology the way forward
cell
Results
Hypotheses Predictions
New validation experiments
Bioinformatics
(Food) Environment
47
Systems biology in microbial food
stability
48
Systems biology in food fermentation
49
Acknowledgements
University of Amsterdam (molecular biology) Bart
Keijser until march 2005 Luc Hornstra Muus de
Haan Hans van der Spek Alex Ter Beek Andrea
OBrien (STW)
Unilever Suus Oomes Jody Herkenkamp Elaine
Vaughan Marcel van der Vaart Gary
Mycock European Sourcing Unit Oss Andre van
Zuylen Piet Klapwijk
Checkpoints Pieter Vos Joost Thijssen
UvA departments Molecular Cytology Microarray
division (MAD) Bioinformatics (Han Rauwerda/Asli
Umur)
TNO Frank Schuren Jos van der Vossen Roy Montijn
Bart Keijser as of april 2005
50
ETP Food core team

• Mrs. Daniela Israelachwili, CIAA, B
• Dr. Jan Maat Unilever RD Vlaardingen, NL
• Dr. Jacqueline Castenmiller, Wageningen Centre
  for Food Sciences, NL
• Dr. Fred Beekmans, Wageningen University and
  Research Center, NL
• Dr. Roger Fenwick, Institute for Food Research,
  UK
• Mr Daniele Rossi, Federazione Italiana
  dellIndustria Alimentare, CIAA, I
• Dr. Josef Haber, BASF, D
• Mrs. Beate Kettlitz, CIAA, B
• Dr. Kerstin Lienemann, German Initiative
  Agro-Food-Research, D
• Prof. Dr. Gerhard Schiefer, University of Bonn, D
• Prof. Dr. Brigitte Petersen, GIQS, D
• Prof. Dr. Andrzej Babuchovski, University of
  Warmia Mazury, PL
• Prof. Dr. Tim Hogg, Universidade Catolica
  Portuguesa, PT
• Prof. Dr. Didier Majou, ACTIA, F
• Prof. Dr. Francisco Tomas-Barberan, CEBAS, E

Courtesy to the ETP Food http//www.etp.ciaa.be/a
sp/press.asp