T3 Conducting Malolactic fermentation in wine

Question 1

In a spontaneous MLF, what factors does the winemaker have influence over?

Answer 1

• pH- 3.2-3.4 is ideal. high values increase the risk of unfavourable strains. Low pH increase mSO2.
• Temperature- 20-22C ideal (16-25 limits). Affected by EtOH concentration.
• SO2- ideally zero, aim for less than 40 ppm bound, very low free.
• Lees contact- Autolytic breakdown products stimulate LAB growth.
• Clarification- excessive clarification removes indigenous LAB, removes suspended solids that are a source of nutrients.
• Contact with skins- extraction of stimulatory substances from skins.

Question 2

Lees Contact, non-excessive clarification and skin contact have what effect on MLF?

Answer 2

all provide a source of nutrients to LAB and stimulate growth.
Over-clarification removes tannin-protein complexes that protect against bacteriophage.

Question 3

Scenario - pH 3.7, temperature 26C, EtoH 15%, Free SO2 of 22ppm.

What is the chance of a successful MLF? what conditions would be more ideal?

Answer 3

pH too high to select favoured O.oeni. Temperature too high. Free SO2 too high. Successful MLF is unlikely.

Ideal conditions- ph 3.2-3.4, temp 20-22C, free SO2=0ppm.

Question 4

What are the benefits of using a LAB starter culture?

Answer 4

• reliability increased

- large starter culture that does not need to increase in cell numbers for an effective MLF.

Question 5

What properties of a LAB strain make it suitable for use in a starter culture?

Answer 5

• Tolerance of inhibitory compounds- SO2 (up to 50ppm bound), ethanol (up to 15%)
• Ability to grow at low temperatures down to 15C.
• Ability to grow at low pH.
• Resistant to phage infection
• Has a positive effect on the sensory properties of the wine- produces no spoilage compounds.

Question 6

Why do starter cultures need to be prepared from commercial LAB cultures?
How should these cultures be stored?

Answer 6

They are very susceptible to damage during the rehydration step and they are costly.
Starter cultures give the opportunity for adaption to wine conditions.
Direct inoculation usually results in a large drop in viability.

Question 7

What is a typical commercial LAB reactivation protocol?

Answer 7

• Dilute grape juice to half strength with filtered water.
• adjust Malic acid to 2 g/l. adjust pH to 3.2-3.6 (as close to wine pH to reduce lag time).
• Inoculate with LAB 1E6-10E7 cells/ml, and yeast, 1E4 cells/ml.
• incubate at 20-25C until LAB cells density reaches 1E8-1E9.

This can be scaled up with more of the above media.
Seeded into wine at 1:100-1000.

Question 8

Why are LAB often rehydrated with yeast present?

What are the potential downsides to this approach?

Answer 8

-Allows gradual buildup of ethanol allowing LAB to adapt.

The downside is that S.cerevisiae may slow LAB growth due to inhibitors (medium-chain fatty Acids, SO2) and nutrient competition. Makes microscopy to determine LAB numbers more difficult.

Question 9

What is the procedure for maintaining an in-house LAB strain from vintage to vintage?

Answer 9

• Bacterial strains kept on agar slants.
• Extensive subculturing and scaling up of growth medium volume.
• increase proportion of wine in the growth medium slowly to acclimatise strain to wine conditions.

Needs trained staff, tank space and time, as it can take 2-3 weeks to complete.

Question 10

What does cross-inoculating wines with LAB involve?

Answer 10

-portions of a wine already undergoing MLF are stored at 2-4C. Warmed up just before seeding into a new wine that is yet to undergo MLF.

Question 11

Failed MLF is still an issue. What are some possible causes of this?

Answer 11

• Inadequate adaption of starter culture (often due to attempts to save money and time)
• Strain inoculated may be inactive and have a low viability
• SO2, pH and ethanol conc may be outside acceptable parameters.
• Nutrient deficiency.
• Improper choice of MLF strain.

Question 12

What are the three options for the timing of LAB culture addition?

Answer 12

• Prealcoholic inoculation
• concurrent alc and MLF
• Post alcoholic MLF.

Question 13

What are the Pros and Cons of Pre-alcoholic MLF?

Answer 13

Pros

• Shortening of the winemaking process
• no warming of the tank is usually required.
• no adaption of the culture is required.

Cons

• difficulties due to SO2 applied during harvest.
• acetic acid production if the heterofermentative O. oeni is used.
• uncontrolled growth of wild yeasts.

Question 14

What bacteria is usually used for pre-alcoholic LAB inoculation? what are some considerations when using this strain?

Answer 14

Lactobacillus plantarum.
Homofermentative so no acetic acid production.

-not tolerant of etoh >8% so MLF must conclude before this level is reached.

Question 15

What are the Pros and Cons of concurrent alcoholic fermentation and MLF?

Answer 15

Pros
-winemaking process accelerated
-reduced wine handling- less racking and racking losses.
-more reliable than pre-alcoholic malo.
cons
-AA production with heterofermentative strains. can inhibit yeast growth.
-LAB may be inhibited by yeast growth (inhibitors and nutrient competition).
-No nutrient stimulation due to yeast autolysis.
-if fermentation becomes stuck then heterofermentative strains may consume a considerable amount of glucose and produce a lot of acetic acid.