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A handful of unmilled malted barley can be added to the kettle for inoculation instead of a pure culture, since the husks of grain carry many microorganisms (not just Lactobacillus; the temperature and low level of oxygen in the wort however favors Lactobacillus). If unmilled grain is added, it is recommended to fill the head space of the kettle with CO2 because oxygen can encourage off flavors such as rancid cheese from [[Butyric Acid]] and/or [[Isovaleric Acid]], which is produced by microbes that are naturally found on the grain. Keeping the temperature between 113°F-120°F (45°C-48.8°C) will encourage naturally found Lactobacillus on the grain, and will discourage other bacteria. Lowering the pH of the wort to under 4.5 will also discourage many other bacteria from thriving in the wort during the incubation period.
If a pure culture of Lactobacillus bacteria is used it is ideal but not necessary to fill the head space of the fermenter with CO2 gas (some brewers have reported that this will help reduce sulfur in the finished beer). The kettle should be held at the desired temperature for 24-72 hours (in some cases longer, but no longer than 5 days). Depending on the strain of Lactobacillus, and the desired sour level, the time of incubation is ultimately a variable that is up to the brewer. The kettle lid should be firmly in place and optionally sealed with plastic wrap so that other microorganisms do not get in. Potential formation of [[Butyric Acid]] and [[Isovaleric Acid]] when using only a pure culture is extremely slight to noneassuming no other microbes are allowed inside the kettle.
Once the level of acidity is reached (this can be tested with a reliable pH meter, or in the case of using a pure culture can safely be taste tested), the wort is brought to a boil. ''Caution: the wort gravity may also be tested to check for alcohol presence. Alcohol can be more easily boiled off since it boils at 173.1°F or 78.37°C <ref>[http://chemistry.about.com/od/moleculecompoundfacts/f/What-Is-The-Boiling-Point-Of-Alcohol.htm Boiling temperature of ethanol]</ref>.'' The wort may be boiled normally in the case of any style of beer that requires a longer boiling process, or it it may not be boiled for no more than a minute or two in the case of making a [[Berliner Weissbier]]. Technically speaking, the wort doesn't need to be boiled at all, as long as it is heat pasteurized <ref name="pasteurization" />, so if alcohol is present then the brewer can choose to heat pasteurize the wort at 170°F (76.6°C) for 15 minutes.