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Naturlige strategier til forbedring
af industrielle mælkesyrebakterier
Kim I. Sørensen
Senior Principal Scientist
Bacterial physiology and improvement, Discovery
Chr. Hansen A/S
_
Copyright © 2014 Chr. Hansen A/S. All rights reserved
New Product Development for the DAIRY Industry
Finding & Improving Lactic Acid Bacteria
Taste
Texture
Yield
Shelf life
Health benefits
Add in
Take
out
Sugar, Fat, Protein
Texturizers
Stabilizers
Preservatives
Copyright © 2015 Chr. Hansen A/S. All rights reserved
Chr. Hansen’s Strain Discovery platform
Combining LEAN principles and a State of the Art Automation & –Omics platform*
A platform designed to find and improve novel bacterial functionalities
2- Classical Strain Improvement
 Non-GMO
 Two approaches:
 High Throughput Screening
 Dominant Selection
Strain Supply Chain
1- Natural Diversity
 Wild type/natural isolates
 Chr. Hansen Culture Collection
 Robotized semi-automated setup
for characterization
* Johansen et al. in Advances in fermented food and beverages. 2013.
Copyright © 2015 Chr. Hansen A/S. All rights reserved.
Diversity Screening of Lactic Acid Bacteria
Chr. Hansen Culture Collection - CHCC
Genera-sorted MTP screening collection
 Chr. Hansen strains depository > 20.000 deposits
 Worldwide origin
 Sourcing in compliance with Convention on Biological
Diversity/Rio (1992)
Lactococcus
Streptococcus
Lactobacillus
……………
 >70% of species in CHCC = Lactic Acid Bacteria
Brevi 1%
Bifido 5%
Propioni 1%
Others 17%
Bacillus 4%






Single strain deposits (individually purified)
>25% of CHCC deposits available in MTP
96-Well MTP for fast retrieval and automation
Robotics to automate characterization
Datasets for each individual strain from >40 assays
Enabling fast new product development
Enterococcus
2%
Streptococcus
15%
Oenococcus
2%
Leuconostoc
3%
Lactobacillus
28%
Lactococcus
22%
Copyright © 2015 Chr. Hansen A/S. All rights reserved
Primary characterization of strains
Assay portfolio
Primary
characterization
Assay 1
Species identification
16S sequencing
Growth
M17/MRS + different C-sources
Fermentation media – upscaling
Temperature (30, 37, 40 & 43 ºC)
O2 tolerance
Bile / acid stress
Acidification speed
Milk acidification in microtiter plates
(100 to 2000 ul)
Assay 2
Salt Tolerance (0 vs. 4% NaCl)
Antibiotic resistance
Database
Erythromycin (2 µg/ml)
Tetracycline (2, 4, 8, 16 & 32 µg/ml)
Metabolite fingerprinting (typically milk)
Volatile Organic Compounds
Assay 3
Phage-HTS profiling
Milk acidification in microtiter plates
Mitomycin induction (prophage)
Enzymatic activities
Urease
Arginine deiminase
Peptidases (& proteinases)
Assay 4
Bacteriocin screen
Stress tolerance
Temp, pH, EtOH, SO32-, …
Copyright © 2015 Chr. Hansen A/S. All rights reserved.
Automated Characterization of LAB Collections
Assay Development - Acidification
Acidification - Color of pH
 MTP based method to measure pH in acidified milk*
 Bromocresol based pH indicators
 Measure “color” (hue angle) in flatbed scanner
Clustering of >800 Lactococci
 Clustering of >800 unique Lactococci based on their
ability to acidify milk under different conditions**
 Fast grouping, visualization and selection of strain leads
 Phenotypic overlays based on primary screening data
(species ID, antibiotic resistance profiles &
bacteriophage sensitivity, enzymatic activitities a.o.)
*: Houlberg et al. (2007) WO 2005/068982
**: Johansen et al. (2014) in Advances in fermented food and beverages: In Press
Copyright © 2015 Chr. Hansen A/S. All rights reserved
New Product Development for the DAIRY Industry
Finding & Improving Lactic Acid Bacteria
Example 1-Screening
Taste
Texture
Yield
Shelf life
Health benefits
Add in
Take
out
Sugar, Fat, Protein
Texturizers
Stabilizers
Preservatives
Copyright © 2015 Chr. Hansen A/S. All rights reserved
The needle in the Haystack
Lb. rhamnosus
- Giving low-fat yoghurts a good taste -
Low-fat yoghurt are healthy
Customers want low-fat, clean label & good taste
Screening of >250 Lactobacilli for the ability to produce
Acetaldehyde & Diacetyl/Acetoin to improve “fat
perception” in low-fat yoghurts
One new strain identified:
Lactobacillus rhamnosus
Diacetyl 
(Acetoin)
Acetaldehyde 
Temperature range
35-43ºC
Jiminez et al. patent WO2012/136832
Copyright © 2015 Chr. Hansen A/S. All rights reserved.
New Product Development for the DAIRY Industry
Finding & Improving Lactic Acid Bacteria
Example 2-Screening
Taste
Texture
Yield
Shelf life
Health benefits
Add in
Take
out
Sugar, Fat, Protein
Texturizers
Stabilizers
Preservatives
Copyright © 2015 Chr. Hansen A/S. All rights reserved
An Example
How some of the most effective bioprotective strains were
selected.
Searching in the Chr. Hansen strain
collection for LAB candidates
200 strains
55

Target contaminants isolated from
Russian Tvarog (fresh cheese) + library of spoilage
yeasts and molds

Screening of multiple combinations in laboratory
assays imitating conditions in the final application

Challenge tests
17
Secondary characterization
10
Copyright © 2015 Chr. Hansen A/S. All rights reserved.
Secondary characterization
Packages of six strains selected from primary characterization
showing the selected properties for the future product FreshQ
Question: do they resemble one another or strains already in
production?
11
100
80
60
Answer Yes or NO via DNA fingerprint comparison
PFGE1-AscI
.
Lactobacillus
paracasei L. casei 01
.
Lactobacillus
paracasei PDA10058
.
Lactobacillus
paracasei LBAca-14676 – the canidate is OK 
.
Lactobacillus
paracasei CRL-431
.
Lactobacillus
paracasei PDA10066
.
Lactobacillus
paracasei LBAca-3808
.
Lactobacillus
paracasei Contract strain
FreshQ® cultures
Currently available:
 F-DVS FreshQ®1 developed primarily for mesophilic applications
 FD-DVS FreshQ®2 and 4 developed primarily for thermophilic applications
The strains used in FreshQ® are from the species L. rhamnosus and L.
paracasei and both species are Qualified Presumed as Safe (QPS) by EU EFSA
and have been tested to confirm that they do not produce unwanted
metabolites or harbor any acquired antimicrobial resistance genes
Copyright © 2015 Chr. Hansen A/S. All rights reserved.
Different molds are strongly inhibition by FreshQ®1
- 18% sour cream
Ref.
Inhibition after 45 days
storage:
Top two rows: 7ºC/45ºF
Bottom rows: 12ºC/54ºF
FreshQ®1
Challenge moulds:
•
•
•
•
•
•
Ref.
208:
216:
151:
329:
416:
214:
P. crustosum
P. glabrum
P. brevicompactum
P. commune
P. solitum
A. versicolor
Contamination level:
1000 spores/spot,
The contamination level is
substantially higher than normal
industrial contamination.
FreshQ®1
208
216
151
329
416
214
Copyright © 2015 Chr. Hansen A/S. All rights reserved.
Mold inhibition of FreshQ® in sour cream
Challenge test on
sour cream
produced at Chr.
Hansen (6% fat;
0.01% DSG-200070)
Reference
Benchmark
30 days at 7°C
FreshQ 2
FreshQ 1
P. Commune
A. versicolorP. Brevicompactum
P. crustosum
P. glabrum
14
Copyright © 2015 Chr. Hansen A/S. All rights reserved.
Chr. Hansen’s Strain Discovery platform
Combining LEAN principles and a State of the Art Automation & –Omics platform*
A platform designed to find and improve novel bacterial functionalities
2- Classical Strain Improvement
 Non-GMO
 Two approaches:
 High Throughput Screening
 Dominant Selection (Work Smart)
Strain Supply Chain
1- Natural Diversity
 Wild type/natural isolates
 Chr. Hansen Culture Collection
 Robotized semi-automated setup
for characterization
* Johansen et al. in Advances in fermented food and beverages. 2013.
Copyright © 2015 Chr. Hansen A/S. All rights reserved.
New Product Development for the DAIRY Industry
Finding & Improving Lactic Acid Bacteria
Example 1- Selection
Taste
Texture
Yield
Shelf life
Health benefits
Add in
Take
out
Sugar, Fat, Protein
Texturizers
Stabilizers
Preservatives
Copyright © 2015 Chr. Hansen A/S. All rights reserved
Automated Characterization of LAB Collections
Assay Development - Texture
Texture – HTS
Pressure (Pa)
 Texture is a key parameter for fermented milks
 Development of a texture screening method based on back-pressure measurement during pipeting /96 channels
 Back pressure measurement of fermented milks correlates to conventional rheology/shear stress measurements (R2 = 0,92)
Time (mS)
 Fast & accurate deepwell-MTP based method to determine texturizing properties of LAB collections and mutant pools
 Proof of Concept: Characterization of >800 unique Lactococci for their ability to acidify & texturize milk*
*: Kuzina-Poulsen et al. (2014) EP14182194; Poster LAB11
Copyright © 2015 Chr. Hansen A/S. All rights reserved
Toolbox – selection: Texture improvement
• Galactose prototrophy – Dominant selection
• Texturizing lactic acid bacteria strains by selection of lactic acid
bacteria resistant towards D-cycloserine and/or functionally
equivalent antibiotics(WO 2012052557 A1)
• Lactic bacterium for texturizing food products selected on basis of
phage resistance (Patent Application WO/2011/092300)
Improving texture in yoghurt: Selection
Direct selection for mutants able to grow on galactose as sole carbon
source
Gal-medium
S. thermophilus 6008
Galactose prototrophy – Dominant selection
Yomax (CHCC11379)
Improved shear stress
Galactokinase (galK) promoter mutation in
CHCC11379
galK twofold up-regulated
20% reduction in galactose content in milk after 6h
fermentation
19
Copyright © 2013 Chr. Hansen A/S. All rights reserved.
Christiansen and Janzen EP 2473058 A1
Copyright © 2015 Chr. Hansen A/S. All rights reserved.
Chr. Hansen’s Strain Discovery Platform
Combining Lean Principles and a State of the Art Automation & –Omics Platform*
Example 2- Selection
A platform designed to find and improve novel bacterial functionalities
1- Natural Diversity
 Wild type/natural isolates
 Chr. Hansen Culture Collection
 Robotized semi-automated setup
for characterization
Taste
Texture
Yield
Shelf life
Health benefits
2- Classical Strain Improvement
 Non-GMO
 Three approaches:
 Random mutagenesis
 Directed Evolution
 Dominant Selection
Take
out
Add in
Sugar, Fat, Protein..
Texturizers,
Stabilizers
Preservatives
SWEETY™
Copyright © 2015 Chr. Hansen A/S. All rights reserved
Classical Strain Improvement Applied - Sweety
The Challenge
 Yoghurt is considered a healthy natural product; but
often contains added sucrose to boost sweetness
The Concept
 Enhancing the natural sweetness of yoghurt by designing
LAB mutants which, without the use of recombinant DNA
technology, excrete glucose & metabolize galactose
instead
 Pool et al. (2006): Lactococcus lactis**
 Not appropriate for yoghurt production
 Use of recombinant DNA technology to make strains
unable to grow on glucose
 Not all sugars are perceived equal, at least with regard
to sweetness *:
*: Robyt (1998) Sweetness, in Essentials of carbohydrate chemistry, Springer-Verlag, New York
**: Pool et al. (2006) Metab. Eng.
Copyright © 2015 Chr. Hansen A/S. All rights reserved
The Approach: A Multi Step CSI Process *
S. thermophilus
Step 1: Growth on Galactose
 Yoghurt starter cultures typically contain 90% S. thermophilus and 10% Lb. delbrueckii subsp. bulgaricus
 S. thermophilus grown on lactose, metabolize glucose and excrete galactose
**
 Enabling & boosting the fermentation of galactose by S. thermophilus***:
 Dominant selection using galactose as sole carbon source
 G to A mutation at -10 position in the galK promoter results in a 2.5 to 3.7 fold increase of transcription of the genes in the
galactose operon (galK, galT, galE, and galM)
*: Johansen et al. (2013) WO 2013/160413A1
**: Adapted from Hols et al. (2005) FEMS Microbiol Rev
***: Janzen & Christiansen (2011) WO 2011/026863
Copyright © 2015 Chr. Hansen A/S. All rights reserved
The Approach: A Multi Step CSI Process *
S. thermophilus
Step 2: Perturbed Glucose Metabolism
 Selection of mutants with impaired glucose metabolism resistant to 2-deoxy glucose***
 Glucokinase (glcK) negative mutants give resistance to 2-deoxy glucose
**
 To circumvent bypass of glcK, the glucose transporting PTS system has to be inactivated
 Selection of mutants resistant to higher levels of 2-deoxy-glucose gives mutations in PTS system transporting glucose
 Glucokinase-negative galactose-fermenting manPTS-negative S. thermophilus mutants (Sweety ST)
*: Johansen et al. (2013) WO 2013/160413A1
**: Adapted from Hols et al. (2005) FEMS Microbiol Rev
***: Youderian et al. (1999) J. Bacteriol
Copyright © 2015 Chr. Hansen A/S. All rights reserved
Glucokinase-negative Galactose-fermenting
manPTS-negative S. thermophilus Mutant (Sweety ST)
Sweety ST enhances sweetness of fermented milk by excretion of glucose
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑆𝑤𝑒𝑒𝑡𝑛𝑒𝑠𝑠 𝐼𝑛𝑑𝑒𝑥 =
16 ∗ 𝑙𝑎𝑐𝑡𝑜𝑠𝑒 + 33 ∗ 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑒 + 74 ∗ [𝑔𝑙𝑢𝑐𝑜𝑠𝑒]
750
Copyright © 2015 Chr. Hansen A/S. All rights reserved
Yoghurts made with Sweety ST & LB give limited
added Sweetness
Sweety ST enhances sweetness of fermented milk by excreting glucose which gets consumed
by Lb. delbrueckii subsp. bulgaricus
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑆𝑤𝑒𝑒𝑡𝑛𝑒𝑠𝑠 𝐼𝑛𝑑𝑒𝑥 =
16 ∗ 𝑙𝑎𝑐𝑡𝑜𝑠𝑒 + 33 ∗ 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑒 + 74 ∗ [𝑔𝑙𝑢𝑐𝑜𝑠𝑒]
750
Copyright © 2015 Chr. Hansen A/S. All rights reserved
Non-glucose fermenting Lb. delbrueckii
subsp. bulgaricus growing on Lactose *
Maintaining the sweetness in yoghurt
Carbohydrate profile & relative sweetness
 Lb. delbrueckii subsp. bulgaricus growing in milk:
 Prevent consumption of excreted glucose
 Selection of mutants resistant to 2-deoxy-glucose on
plates with lactose as sole carbon source to give
mutations in PTS system transporting glucose
 Mutants do not grow on glucose as sole carbon source
*: Johansen et al. (2013) WO 2013/160413A1
**: Adapted from Hols et al. (2005) FEMS Microbiol Rev
 Sweety Lb. delbrueckii subsp. bulgaricus mutants excrete
glucose and grow on lactose
**
 Non-glucose fermenting Lb. delbreuckii subsp.
bulgaricus mutant (Sweety LB)
Copyright © 2015 Chr. Hansen A/S. All rights reserved
A Combination of Sweety ST & Sweety LB enhances
Sweetness of plain Yoghurt by 3-fold
Yoghurt fermentations with both Sweety mutants
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑆𝑤𝑒𝑒𝑡𝑛𝑒𝑠𝑠 𝐼𝑛𝑑𝑒𝑥 =
16 ∗ 𝑙𝑎𝑐𝑡𝑜𝑠𝑒 + 33 ∗ 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑒 + 74 ∗ [𝑔𝑙𝑢𝑐𝑜𝑠𝑒]
750
Copyright © 2015 Chr. Hansen A/S. All rights reserved
Conclusions & Future Perspectives
Sweety
 Tailor made mutants without using of recombinant DNA technology which can be
used to:
 Make natural plain yoghurt with increased sweetnesss (2-3 x more sweet)
without increasing the caloric content
 Reduce the amount of added sucrose (by 10 g/l) while maintaining the
same sweetness (cost saving & calorie reduction)
 Significantly lower the levels of residual lactose after fermentation thus
making sweety yoghurts a better choice for people with lactose intolerance
Taste
Texture
Yield
Shelf life
Health benefits
Take
out
Add in
Sugar, Fat, Protein..
Texturizers,
Stabilizers
Preservatives
SWEETY™
Copyright © 2015 Chr. Hansen A/S. All rights reserved
Thank you for
your attention
Copyright © 2015 Chr. Hansen A/S. All rights reserved