Kavli - Norge

Lactic acid bacteria from an industrial perspective

Mette Øhrstrøm Runge – Chr. Hansen A/S Specialist Application Manager Fermented Milk and Probiotics

Chr. Hansen A/S - Where do we come from? Founded in 1874 by a very innovative and creative pharmacist, Dr. Christian Hansen… in Denmark … who developed the first standardized animal rennet solution for cheese makers to ensure quality, reliability and safety to its customers – a breakthrough a this time! 135 years later the company is: One of the top players in the food & health ingredients industry A worldwide leader in its 3 main activities: Cultures for direct inoculation: bacteria used to produce cheeses, yogurts, meat, wine, supplements for human & animals… Enzymes for dairies (still the worldwide leader in coagulants for cheese makers our historical activity) Natural extracts to produce natural colors, phytonutrients…

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Chr. Hansen in brief Fermenting bacteria cultures for more than 100 years Since 2005 owned by French capital fund PAI partners Global presence 2350 employees in 32 countries 85 distributors & agents around the world State-of-the-art production facilities on five continents Turnover of 500M EUR with double digit organic growth 6 % of turnover spent on R&D 120 employees in R&D Strong partnerships with customers Everyday over 500 million people consume products containing Chr. Hansen Ingredients

USA Mexico Panama Colombi a Peru

Latvia DenmarLithuani Netherl k a Russia ands German Ireland France Italy y Spain UK Poland China Japan Greece Ukraine TuCzech U.A.E rk Republi India eyc Malaysia Hungary Romania Brazil Argentin a

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Australi a

New Zealand

CH provides ingredient solutions to selected industries

Cultures

Dairy

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Food

Natural Colors and phytonutrients

Enzymes

Beverages

Meat & Prepared Foods

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Human Health and Nutrition

Animal Health and Nutrition

Lactic acid bacteria - description A large and diverse group of beneficial bacteria that all produce lactic acid as the major metabolic end product of carbohydrate fermentation Homofermentative: Lactic acid main product Heterofermentative: lactic acid and a number of secundary metabolites like organic acids, carbondioxide and alcohols Gram-positive, non-respiring, non-spore forming, low-GC, acidtolerant, katalase negative rods or cocci Widespread in nature and are found in our digestive system Genera that comprise the LAB Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Enterococcus, Tetragenococcus, Carnobacterium, Weisella

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Lactic acid bacteria - description Widespread in nature and are found in our digestive system Most important bacteria in spontaneous and desirable food fermentations Lactic acid bacteria have for thousands of years been used to produce cultured/fermented foods with improved preservation properties and with characteristic flavours and textures different from the original food. Acidification inhibits the growth of spoilage agents Bacteriocins produced by several LAB strains provide an additional hurdle for spoilage and pathogenic microorganisms Lactic acid and other metabolic products contribute to organoleptic and textural profile of the fermented food product

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Examples of daily life products containing cultures s ure es tur c cult l u dc oti Foo Probi

Wine Starter cultures - Malolactic fermentation Meat (sausages/dried meat) Starter cultures – Protection cultures Cheese Starter cultures – Ripening cultures Fermented milk (yogurts…) Starter cultures

Cheese, Fermented milk, juices, tablets… with probiotics for Gut & Immunity applications

Human Health Animal Health

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Wine cultures

Use of lactic acid bacteria in wine making Lactic Acid Bacteria are naturally found in the winery and on the grapes (Oenococcus oeni, and various species of Lactobacillus and Pediococcus) Malolactic fermentation (MFL) is accomplished by lactic acid bacteria During MFL malic acid is converted to lactic acid plus carbon dioxide Lowering of overall acidity The tart tasting malic acid which is naturally present in grape must is converted to softer tasting lactic acid. Adds mouthfeel (hard and metallic edged malic acid => softer lactic acid) Malolactic fermentation can occur naturally In commercial wine making, malolactic conversion is typically initiated by an inoculation of desirable species of LAB Prevention of undesirable bacterial strains from producing off-flavors

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Use of lactic acid bacteria in wine making Malolactic conversion can take place at any time during or after alcoholic fermentation Fruit juice must

Grapes

Alcoholic ferm.

Malolactic ferm.

Fining/ Elevage

Metabolism of Oenococcus oeni in grape juice Glucose/fructose → Fructose → L-malic acid →

D-lactic acid, acetic acid, ethanol, CO2 Mannitol L-lactic acid + CO2

Metabolism of Oenococcus oeni in wine L-malic acid Citric acid

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→ →

L-lactic acid + CO2 acetic acid, acetoin/2.3-butandiol, diacetyl

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Faster Malolactic fermentation

MLF achieved in 2 weeks through co-inoculation or sequential inoculation

Data from an Australian winery

30 tanks

9 weeks

(250 hl)

spontanious tank

Mar-15

Apr-01

Apr-17

May-01

May-15

Jun-01

Jun-15

Jul-01

1

Malolactic fermentation Shiraz 2006 (Australia)

2 3 4 5 6 7

pH 3.43-3.45, Total SO2 18-22 ppm, EtOH 13.2-13.7 vol%

8 9 10 11 12 13 14 15 16 17 18 19 20

2

21 22 23 24 25

Malic acid (g/L)

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Long LongMLF MLF (4 to (4 to99weeks) weeks) without without Viniflora Viniflora

1,5 1

26 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

0,5

55 56 57 58 59 60 61 62 63 64 65

0

66 67 68

Tanks Tanksnon nonavailable available represent an important represent an important issue issuefor forthe thewinery winery manager manager

69 70 71

0

10

20

30

40

50

Time (days) Viniflora LS

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Cross seeded Viniflora oenos

Spontaneous MLF

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72 73 74 75

Short ShortMLF MLF (3 (3weeks) weeks) with with Viniflora Viniflora

76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97

Problem Problemsolved solved

Jul-15

Get organoleptic consistency color, flavour, mouthfeel…

Different strains for different purposes: pH, T, SO2, ethanol content? Red, rosé or white wine? Co-inoculation or sequential inoculation?

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VINIFLORA® offers:

Advantages of MLF Process control Accelerate Accelerate MLF* MLF*

Optimize Optimize wine quality wine qualitypotential potential

Ensure Ensure Quality & Quality &Food Foodsafety safety

Be ready first

Control MLF as well as AF

Know & manage your inoculums

Save energy

Get consistency (flavor…)

Use documented ingredients

Optimize tank/ cask management Use manning for higher value operations

Avoid biogenic amines production Reduce SO2 levels

Get traceability

Maximise Maximiseprofitability profitabilitythrough throughan anexcellent excellentinvestment investment * : Process control = Malo-lactic Fermentation (MLF) control 13

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Meat cultures Starter cultures for acidification Bioprotective cultures

Starter cultures for meat fermentation Acidification Lactic acid bacteria Homofermentative lactobacilli (L.sake, L.curvatus, L.pentosus) Pediococci (P.acidilactici, P.pentosaceus)

Flavour and color Staphylococci Kocuria (previously Micrococci)

Yeast and molds Debaryomyces Penicillium

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Acidification of meat How does acidification take place Microbiological by Natural contaminating flora of lactic acid bacteria Starter culture of lactic acid bacteria Added sugar => lactic acid

Importance of acidification Inhibits pathogens and spoilage bacteria Affects drying profile Controlled Promotes texture formation acidification is Affects color formation essential for uniform Affects flavour formation production 16

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Regional differences in selection of starter culture North-Europe

South-Europe

USA

Fermentation time

40 hours

32°

Texture

Soft

Dry and firm

Soft

Time from meat mince to final sausage

< 3 weeks

> 3 weeks

2 -3 weeks

Other

Often smoked before fermentation

Nitrate often added Often use of mould cultures on surface

Sausages are heat treated

Selection of culture

Traditional culture – Lactobacilli

Traditional culture – Lactobacilli

Fast culture – pediococci

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≥

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Use of lactic acid bacteria for bioprotection Application of lactic acid bacteria to a product in order to control the contaminating flora

Improving quality by delaying growth of spoilage bacteria Increasing safety by suppressing and reducing pathogens At the same time keeping the sensorial freshness of the product

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How do bioprotective cultures work as a hurdle ? Compete with the indigenous flora Grow well at the storage conditions Use easily fermentable nutrients Take up space for growth Remove oxygen, i.e. lower redox potential

Inhibit the indigenous flora Produce inhibitory organic acids Produce bacteriocins

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Application of bioprotective cultures Cured whole muscle products bacon, cured meats, filet, … In the brine

Minced products fermented sausages, non-cooked sausages, … Directly into the mince

Ready to eat products Cooked sliced ham, Mortadella, hotdog sausages, toppings, .… After cooking and cooling By spraying during slicing or during transportation on the conveyor belt

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Control of spoilage flora with Lactobacillus sake (B-2) 1.E+06

Control + B-2



 

Brochothrix thermosphacta suppressed Sensory quality improved Shelf life prolonged

Brochothrix (CFU/g)

Smoked sliced filet stored at 7°C 1.E+04

1.E+02

1.E+00 0

2

4

Weeks

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SafePro® B-LC-48 – L.curvatus Listeria / log10 (CFU/g)

B-LC-48 kills and controls Listeria monocytogenes 10

Hot-dogs + B-LC-48

8

Control

6 4 2 0 0

5

10

15

20

25

Time at 7oC (days)

B-LC-48 enhances the fresh perception of RTE-meat products

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Cultures for Cheese

24 hours

4 weeks to 2 years

Cheese manufacture

Cheese ripening Fresh curd

Milk

Renneting, stirring, whey draining, moulding and pressing

Primary starter cultures

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Mature cheese

Storage and ripening

Ripening cultures

Cultures for cheese Effect of Culture (24h) Lactose Lactic acid

Culture Groups Mesophilic O, L, D and LD

Thermophilic

pH drop

Improved renneting Improved syneresis Influence of the final product e.g. texture and flavor Abbreviations: O cultures contains only acid-producing strains of bacteria LD cultures contain citrate fermenting bacteria resulting in flavor and aroma components

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St. thermophilus Lb. helveticus Lb. bulgaricus Single strain Multiple strains Combination of above incl. ripening cultures

Mesophilic Homofermentative Cultures O culture consists of Lactococcus lactis subsp. cremoris Lactococcus lactis subsp. lactis

Activity Aroma Gas

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Application in Cottage cheese Cheddar Feta

Demands Fast acidification Phage robust

Mesophilic Aromatic Cultures LD consists of: Lactococcus lactis subsp. cremoris Lactococcus lactis subsp. lactis Lactococcus lactis subsp. diacetylactis Leuconostoc species

Activity Aroma Gas

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++ ++ +++

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Application in Continental cheeses Soft cheeses

Demands moderate acidification controlled CO2 development

Thermophilic Cultures

Thermophilic Cultures consist of: Streptococcus thermophilus Lactobacillus delbruecki subsp. bulgaricus Lactobacillus helveticus

Application in

Demands

Emmental Mozzarella Pizza cheese Hard cheeses like Grana

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Very fast acidification Growth at high temperature, 50ºC phage robust positive interactions

Effect of Cheese Ripening Cultures

Sensoric profile of reduced fat Gouda cheese: 8% total fat/15% fat in dry matter evaluated after 7 weeks maturation starter culture in all cheeses were CHN-19 Sample

Flavor profile

Reference

Bitter notes, simple flavor

CR-520

No bitterness, high intensity, complex cheese flavor

CR-540

No bitterness, high intensity, sweet and nutty flavor

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Bioprotection in cheese Danbo was produced from organic milk contaminated with spores 5 weeks old Danbo

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Culture for fermented milk

Use of lactic acid bacteria for fermented milk Effect of lactic acid bacteria in fermented milk Preserve Acidification of milk, by formation of lactic acid from lactose

Flavour Formation of flavour components such as diacetyl, acetaldehyde, acetic acid dependent of culture

Formation of lactic acid

Texture Coagulation of milk Formation of exopolysaccharides

Health Probiotic effect

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Lactic acid bacteria for fermentered milk Mesophilic cultures

Lactococcus lactis subsp lactis Lactococcus lactis subsp cremoris Lactococcus lactis subsp lactis biovar diacetylactis Leuconostoc subsp.

Fermentation temperature 22 – 30 °C

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Thermophilic cultures Lactobacillus bulgaricus Streptococcus thermophilus

Fermentation temperature 35 – 43 °C

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Lactic acid bacteria for fermented milk

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Mesophilic cultures

Thermophilic cultures

Flavour characteristics Diacetyl (buttery)

Flavour characteristics Acetaldehyde (fruity)

Products Buttermilk, Thickemilk Ymer, Sour cream, Quarg

Produkter Stirred or set yoghurt Drinking yoghurt Yoghurt ice

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From milk to yoghurt Mainly LB-strains

Lactose Lactic acid bacteria

Liquid milk

Casein

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H+

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Exopolysaccharides Mainly ST-strains

-

Lactic acid

Flavour

Viscosity

Decrease pH to the iso electric point

Casein Network

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Yoghurt

Symbiotic growth of yoghurt bacteria – Streptococcus thermophilus and Lactobacillus bulgaricus milk proteins - peptides - amino acids proteinase/ peptidase (enzyme)

Streptococcus thermophilus

Lactobacillus bulgaricus formic acid CO2 puryvate HCO3

Shorter fermentation time and different characteristics than product fermented with a single species 36 32009 March 2009 03 March

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Performance of cultures for cultured milk Lactococcus lactis -

Lactococcus lactis -

Lactococcus lactis -

subsp. lactis

subsp. cremoris

subsp. lactis biovar

Leuconostoc spp.

Streptococcus thermophilus

diacetylactis Type of culture Speed of lactic acid production

O

O

D

L

ST

xxx

xxx

xx

x

xx xxx

xxx xxx

x x xxx

xx (with/without)

Production of exo-polysaccharides Citrate fermentation Diacetyl production

x (from citrate) xx

Production of CO2 Acetaldehyd production Acetaldehyd reduction Fermentation temperatur/optimal Homo./Hetero fermentativ

To ethanol 30-33°C

30-33°C

26-32°C

24°C (7.5°C)

35-43°C

Homofermentativ

Homofermentativ

Homofermentativ

Heterofermentativ

Homofermentativ

XPL-1, XPL-2 XPL-20 XT-202, XT-204 XT-302, XT-303 XT-312, XT-313, XT-314 - with EPS from L. cremoris CH-N-12, CHN-13, CHN-14, CHN-22 CH-BAN-1 with BB-12 DSG-2000 R-603, R-604, R-607, R-608 R-703, R704, R-707, R-708, DSG-HB DSG-FLVR1

x : moderate influence, xx: high influence, xxx: very high influence 37

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xx

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Probiotics

Defining Probiotics Probiotics means ”for life” in Latin “Live microorganisms which when administered in adequate amounts confer a health benefit on the host” FAO / WHO 2002 Most commonly used are: Lactobacilli L.acidophilus, L.paracasei subsp. paracasei L.rhamnosus L.plantarum L.reuteri L.gasseri L.johnsonii

Bifidobacteria B.animalis subsp. lactis B.longum B.infantis

But also: Streptococcus, Enterococcus, Bacillus, Saccharomyces boulardi 39

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Over the years, research in effects of probiotics on many health indications…… Colds & flu

Stomach

Immune modulation

Cancer

Skin health

Stress Small intestine

Oral health

Vaginal health Heart health

Colon

Gastrointestinal health

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Atopic ezcema

….however, most significant documentation is within gastrointestinal and immune areas

Benefits of probiotics – Identified mechanisms (not complete) Inhibit adhesion of pathogen cells Produce surfaceactive substances

Modulates cytokine production

Probiotic strain Produce acids

Produce bacteriocins

Inhibit growth of pathogens 41

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Strengthen the immune system Produce hydrogen peroxide Inhibit growth of pathogen cells

Work as co-aggregation molecules Block the spread of pathogens

Number of new probiotic product variants launched pr. year (1997 – 2008)

Probiotics have been a success in dairy Now it will be taken to other products High end beverages such 100 % fruit Juices / smoothies / Nectars Sweet milk, Cheese, Ice cream, Cereals, Dietary supplements, Others 42

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Specific probiotic strains promoted on consumer products in EU L. paracasei subsp. paracasei Shirota, L. casei immunitas, L. casei 431, F-19 Bifidobacterium BB-12, Bifidobacterium Essensis, BB-536, DR-10 Lactobacillus acidophilus LA-5 Lactobacillus johnsonii Lc-1 Lactobacillus rhamnosus LGG Others: Reuteri, L. plantarum 299v

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Human health Probiotic strains for dietary supplements

Probiotics for supplements Primary strains L acidophilus, LA-5®

Products Quatro-cap-4 (BB-12, LA-5, ST, Lb)

Bifidobacterium, BB-12®

AB-caps

L rhamnosus, LGG®

AB-blends

L rhamnosus, GR-1® L reuteri, RC-14® L paracasei ssp. paracasei,CRL431 Streptococcus thermophilus, TH-4

Single strain powders Single strain powders, infant formula (BB-12, TH-4, CRL-431) UREX-cap-5 (GR-1, RC-14) Sticks Customized blends

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Animal health Probiotic feed additives Natural growth promoters Silage inoculants

Silage - Use of bacteria Why use bacteria? Direct and secure fermentation in the crop Rapid pH-reduction reduces protein degradation and the possibility for competing microorganisms to grow Living organisms that increase in number after being applied to the crop

What do the bacteria require? Anaerobic conditions Presence of substrate for growth Temperatures below ca. 50 °C

Examples of species used for different types of silage (Grass, maize, whole

crop cereals and alfalfa) Pediococcus acidilactici, Pediococcus pentosaceus Lactobacillus plantarum, Lactobacillus casei, Lactobacillus buchneri, Lactococcus lactis Enterococcus faecium 47

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Production of lactic acid bacteria

Culture production line Raw materials

Media

Media

UHT

UHT

Culture bank Hørsholm PIM

IM

PFM ~170 l

”Sterile” CIP/SIP

Fermentation

By-product

Concentration Concentration

Clean rooms

500 – 40.000 l

Cryoprotectants

Pelletizing

F-DVS FD-DVS

Freeze-drying

Packaging

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Packaging

Quality and safety in culture production Fermentation Freeze-drying

Packing

Storage/logistics

Certified GMP pharma & food, Kosher, QC, HACCP, ISO 9002

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Stringent Quality Control throughout the value chain

QC Laboratory Reliable, traceable analytical results and release of products The QC laboratory analyses and evaluates the quality of: Raw materials Process samples Frozen & freeze-dried bulk before packaging Final products before shipping Samples are examined for: Cell counts, Composition, Performance and Purity Wide range of microbiological and chemical methods Close communication and cooperation: With production, GPQ, RDA, marketing and other Culture Production sites

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DVS Concept DVS is a highly concentrated and standardized frozen or freezedried dairy culture used for the direct inoculation of milk DVS cultures need no activation or other treatment prior to use and offer a number of advantages over conventional bulk starter cultures: Convenience and flexibility of use in the production process Consistent performance Culture is tested before use Eliminate bulk starter production Reduce risk of phage attack Reduce risk of contaminations

Possibility of using customized culture blends

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Thank you for Your Attention!

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