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Bio-protection with Viniflora® malolactic cultures

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2015 Chr. Hansen.

Some of the highly innovative features include:

• Managing MLF in grape juice with NoVA™, the new generation of selected Lactobacillus

• Increasing fruit flavour intensity through MLF with CH16 or CiNe™ • Modulating diacetyl production to maximise complexity with CH35, or conversely to reveal primary and secondary flavours with the exclusive strain CiNe™. • Managing MLF in white & red wines to lower the final total sulfur content by utilising MLF with NoVA™ before AF, or using CiNe™ for MLF in white or rosé wines without dairy flavour production. This bio-chemical stabilisation helps decrease the total level of sulfites in the wine.

Wine Technical Services are locally available to help you select the best strains and optimise their use with the latest application protocol

Things to keep in mind when selecting MLB solutions:

• M: Magic Number (1E+06 cfu/ml) is the key parameter to understand malolactic fermentation management.

• Commercial MLB products should be assessed according to their concentration of active cells after inoculation. This value should be expressed in number cells per bag or pouch available divided by 1E+06 cfu/ml, giving the concentration factor for the product or ‘M’.

• The first level for quality products corresponds to the ability to perform malic acid conversion and strictly requires more than 1M of viable, active cells, directly inoculated. If the strain has been selected correctly, then MLF can be managed with 1.1 to 1.4 M, in easy winemaking conditions: medium to high pH, moderate alcohol concentration (co-inoculation to is a way secure this point)…

• The higher the concentration factor (number of M per bag) the better will be the bio-protective effect of the commercial product and its ability to perform MLF in various conditions.

• Viniflora® products are available in very high concentration through the FroZen™, Freasy™ and Freeze-dried range (> 2.4 M). They can therefore all handle MLF in easy, medium and difficult conditions, or when conditions are changing while ensuring maximal bio-protection. The Viniflora® Freeze-Dried Expert range is also available and offers adjusted concentration and standardized weight...

• All our Viniflora® oenological bacteria are suitable for sequential inoculation, early or late co-inoculation and some – like NoVA™– for reverse inoculation where MLF is managed before AF! • Our portfolio of strains covers all kind of winemaking situations from high alcohol to low pH wines.

What is Bio-protection? The use of a biological control to protect grapes, must or wine.

Where a high concentration of identified and living microorganisms is used instead of preservatives. This mitigates against the development of spoilage microorganisms (yeasts, moulds, bacteria) that could degrade microbial quality, organoleptic characteristics and overall wine quality, thus spoiling a wine or rendering it unsafe.

Two mechanisms explain Bio-protection applications:

1. Competitive exclusion: The culture or the blend of cultures selected can overcome other species or strains of microorganisms.

An example of Bio-protection by competitive exclusion is the direct inoculation of wine with high concentration of selected strains of Oenococcus oeni -in the tail of alcoholic fermentation- to manage malolactic fermentation (MLF) and avoid the

development of contaminants such as Brettanomyces.

2. Production of compounds able to inactivate other microorganisms: Alcohols, organic acids, peptides like mycocins or bacteriocins.

An example of Bio-protection by production of mycocins and bacteriocins is the direct inoculation of Viniflora® NoVA™ a pure Lactobacillus plantarum into the grape juice to protect against moulds and acetic-acid bacteria development once grapes are harvested.

How to select the right product for a given wine application?

Among commercial lactic acid bacteria products, Viniflora® is the benchmark Viniflora® FroZen™, Freasy™ and Freeze-Dried ranges contain 2,4 to2,6 M. These products are by far the most concentrated available on the market and they bring two value propositions to winemakers:

1. ‘Policy insurance’ fermentation tool to manage malic acid conversion in most winemaking situations. The high concentration factor is a clear benefit compared to wild flora or other commercial products; Viniflora® will be more resistant. The speed of the process is then strain and wine related.

2. High bio-protection activity, the high concentration factor helps to maximize competitive exclusion. Viniflora® Expert products have been designed to bring >1.2 M in a standardized in weight product to perfectly adjust the dosage to some customers´ applications (standardised pH, co-inoculation, …) when bio-protection against Moulds, Acetobacter/Gluconobacter, wild O. oeni or Brett. infection is not the primary objective.

Viniflora® solutions are based both on Lactobacillus and Oenococcus strains able to cover the vast majority of winemaking situations.

Viniflora® strains have a wide diversity of organoleptic impact on flavour and mouthfeel: from NoVA™, to CH16, CH35 or CiNe™, there are multiple options available.

This tri-fold represents the state-of-the-art, regarding bio-protection in winemaking using lactic acid bacteria to protect grapes, musts and wines. Another document is available about new usages of specialty yeasts (Torulaspora, Kluyveromyces, Pichia…) as bio-protective solutions for winemakers.

Product Logistic/ Standardized Suitable for Suitable No. of Strain related featuresType Storage in weight Organic for bio strains Speed Flavor impact (EU&NOP) protection available selection options

F-DVS® -45°C/ No Yes Yes, ideal 7 High High Fruit IntensityFroZen™ -45°C Pure culture Medium High/med diacetyl Slow No diacetyl

F-DVS® -45°C/ No Yes Yes, ideal 3 High High Fruit IntensityFreasy™ -18°C* Pure culture Medium Medium diacetyl No diacetyl

FD-DVS® Ambient/ No Yes Yes 6 Medium Good Fruit Intensity -18°C Pure culture Slow High/med diacetyl +5°C** No diacetyl FD-DVS® Ambient/ Yes Yes No 1 High Medium/Expert -18°C Culture Low diacetyl +5°C** & support

*: best before date is 12 weeks once the product starts to be stored in a freezer, at min. -18°C**: best before date is 12 weeks once the product starts to be stored in a fridge, at max. +5°C

Merlot grape juice at pH 3.5 with sulfites (>10 ppm)

NOT PROTECTED with NoVa™(L. plantarum, for bio-protection)

Merlot grape juice at pH 3.8 without sulfites

PROTECTED with NoVa™(L. plantarum, for bio-protection)

MOULD DEVELOPMENT NO MOULD DEVELOPMENT

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Lag phase Simultaneous deceleration(Lag and μmax similar)

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Contaminants(molds, yeasts, bacteria)

Contaminants (molds, yeasts, bacteria) Contaminants contained by Protective culture

Protective culture

Understanding the ‘’Magic Number ‘’ M – to manage MLF, bio-protect wines and compare ML products between each other.

Magic Number M: 1E+06 cfu/ml is the number of active Oenococcus oeni cells needed per ml of wine to trigger MLF. Below this nothing happens. The bacteria will only start converting malic acid into lactic acid once the quorum sensing level is reached. This data is specific to MLF management and central in the way we have designed the Viniflora® oenological bacteria range, defined their specifications and developed innovative applications.

The Bio-protective effect increases with inoculation rate; the higher the amount of cells inoculated, the faster they affect other microorganisms like moulds, yeasts like Brett., lactic acid bacteria or acetic acid bacteria. High concentration (> 2M) means faster impact and therefore a higher level of bio-protection.

As a consequence:

1. Viniflora® products are ready for direct inoculation (DVS®); no need to rehydrate or acclimatize. Products are ready to be used through an easy procedure.

2. Viniflora® products deliver the highest concentration of active cells per bag of products; this parameter is available on our specifications and certificate of analysis; cfu/bag or cfu/pouch.

3. These cells are alive and active and Chr. Hansen monitors this activity through an exclusive, proprietary test, named ‘MACC test’ (for Malic Acid Conversion Capacity) to secure every batch performance.

very low

Cell countper ml of wineinoculatedimmediatelyafter inoculation

Why Viniflora® is really differentFrom MACC test to our high quality specifications

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Average activity per cell in wine. (Measured thanks to ‘MACC’)

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