Zymomonas mobilis in drinks (beer, cider, and other alcoholic beverages obtained by fermentation) – Culture; Molecular diagnosis (PCR); Identification (PCR and sequencing)
Zymomonas mobilis is a gram-negative, mobile, not sporulated, facultatively anaerobic bacillus isolated from alcoholic beverages throughout the world (Americas, Europe, Asia, and Africa), and in many fermentation juices obtained from various plants (palm, cane sugar, pears, apple, agave, ...), in which there is a high concentration of glucose. In our environment, it is important because of the decomposition that can be generated in beverages such as beer, apple cider, or pear cider -performed-. Its origin is unknown, although in the case of the degradation of beer it is attributed to contamination from soil or water.
The ability to survive in liquids with a high concentration of sugars (glucose and / or fructose) and in ethanol concentrations, higher than those tolerated by yeast, is due, in part, to their ability to perform an anaerobic metabolism of glucose to through the production of acetaldehyde, and this is then converted into ethanol and CO2. To do this, use the Entner-Doudoroff path. It is the only known microorganism capable of using this anaerobic metabolism, with a single substrate, glucose, fructose or sucrose. This ethanologenic pathway is similar to that used by Saccharomyces cerevisiae, with the implication of two enzymes, pyruvate decarboxylase that converts pyruvate into acetaldehyde and CO2, and the alcohol dehydrogenase that converts acetaldehyde into ethanol. Thanks to this metabolic pathway it allows them to grow and produce ethanol up to concentrations of 12%, at a rate twice as fast as yeasts, which makes it a good candidate for the synthesis of ethanol fuel. In addition, it is useful to produce acetaldehyde used as a flavor, or as a precursor for the synthesis of acetic acid, acetic anhydride, butanol, etc.
Zymomonas mobilis is the only species and in 1976 it was subdivided into two species: Z. mobilis subsp. mobilis and Z. mobilis subsp. pomaceae, to which Z. mobilis subsp. francensis was subsequently added.
Normally, this bacterium tolerates a certain acidity, but does not develop in products with a pH lower than 3.75, so in 92% of the cases in which it has been found decomposing ciders had a pH equal to or greater than 3.90. For this reason, English ciders have a pH lower than 3.5, thus preventing their proliferation. In addition, it tolerates ethanol concentrations higher than those tolerated by Saccharomyces cerevisiae, being able to tolerate up to 12% ethanol. The microorganism responsible for the degradation of beer, Zymomonas mobilis subsp. mobilis, is able to grow at a pH of 3.5 to 7.5, and in concentrations up to 16% ethanol. The growth produces large amounts of acetaldehyde and / or sulfide (H2S) and considerable turbidity. In the English cider houses, the subspecies Z. mobilis subsp. pomaceae is responsible for the alteration of cider, while in French cider houses, Z. mobilis subsp. francensis, is responsible for degradation known as "framboisé" for the aroma that cider acquires.
The alterations of the cider are correlated with some aromas, described as the smell of rotten lemons ("rotten lemon skin"), "raspberry-like", herbaceous, "banana-like", due to the accumulation of acetaldehyde (150-400 mg/L). In addition, gas accumulates in the bottles, and the content becomes cloudy, which becomes whiter and more dense, due to the accumulation of phenolic products. In France the maximum limit of acetaldehyde is 100 mg/L in bottled ciders, although for some types of ciders up to 1,000 mg/ L is allowed.
In England, to avoid degradation, ciders have a pH below 3.5. On the contrary, in France, natural fermentations are carried out keeping the ciders at pH above 3.7. These natural fermentations, provide nutrients, such as sugars, and nitrogen elements, favorable for use by bacteria, including Zymomonas mobilis. As a result, it is estimated that between 5% and 17% of annual production may suffer from "framboise", with its corresponding economic consequences.
Tests carried out in IVAMI
- Culture in liquid and solid media to detect their presence.
- Molecular detection by PCR (polymerase chain reaction).
- Identification of the species by sequencing.
Delivery of results
- Culture with identification: 7 working days.
- Molecular detection with identification: 48 to 72 working hours.
Type of sample:
- Container with altered drink.
Conservation and shipment of the sample:
- Refrigerated (preferred) for a maximum time of 24 to 48 hours.