Evolution of the Lactic Acid Bacterial Community during Malt Whisky Fermentation: a Polyphasic Study

Abstract

The development of the lactic acid bacterial community in a commercial malt whisky fermentation occurred in three broad phases. Initially, bacteria were inhibited by strong yeast growth. Fluorescence microscopy and environmental scanning electron microscopy revealed, in this early stage, both cocci and rods that were at least partly derived from the wort and yeast but also stemmed from the distillery plant. Denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA genes and sequence analysis revealed cocci related to Streptococcus thermophilus or Saccharococcus thermophilus, Lactobacillus brevis, and Lactobacillus fermentum. The middle phase began 35 to 40 h after yeast inoculation and was characterized by exponential growth of lactobacilli and residual yeast metabolism. Lactobacillus casei or Lactobacillus paracasei, L. fermentum, and Lactobacillus ferintoshensis were detected in samples of fermenting wort examined by DGGE during this stage. Bacterial growth was accompanied by the accumulation of acetic and lactic acids and the metabolism of residual maltooligosaccharides. By 70 h, two new PCR bands were detected on DGGE gels, and the associated bacteria were largely responsible for the final phase of the fermentation. The bacteria were phylogenetically related to Lactobacillus acidophilus and Lactobacillus delbrueckii, and strains similar to the former had previously been recovered from malt whisky fermentations in Japan. These were probably obligately homofermentative bacteria, required malt wort for growth, and could not be cultured on normal laboratory media, such as MRS. Their metabolism during the last 20 to 30 h of fermentation was associated with yeast death and autolysis and further accumulation of lactate but no additional acetate.Scotch malt whisky is distilled from the fermented hot-water extract of malted barley. The malted cereal is milled and infused with water (mashed) at about 63°C. After about 30 min, the first wort is removed, cooled, and pumped to the fermentation vessel. The second water, which is conducted at a higher temperature (about 75°C) to effect the maximal extraction of carbohydrate from the grist, is cooled and added to the first wort to fill the fermentation vessel. The wort is not boiled, as it is in a brewery in order to retain the activity of the soluble enzymes from the malt during the fermentation and to maximize alcohol yield. Consequently, bacteria from the malt that can survive mashing enter the fermentation, resulting in a mixed yeast-bacterial fermentation (11,19). If large numbers of lactobacilli enter the fermentation (more than 10 6 cells/ml), they compete for nutrients with yeast cells and reduce the ethanol yield (9,11,20). In well-operated distilleries, however, the lactobacilli flourish after the yeast cells have reached stationary phase and grow on residual nutrients and autolysing yeast cells. This "late lactic fermentation" is encouraged by many distillers, since it is thought to have a beneficial effect on the flavor of the final spirit (13,24).Littl...

Indigenous Microbial Community of Barley Greatly Influences Grain Germination and Malt Quality

“…Denaturing gradient gel electrophoresis (DGGE) is perhaps the most commonly used culture-independent fingerprinting technique for studying the response of community dynamics to environmental variations. This technique has also demonstrated its potential in food-related ecosystems 12,19 and has been applied in beverage fields such as whisky 54,55 and wine fermentations 34 . PCR-DGGE detects the predominant species of a community without discriminating living from dead cells or cells in a non-culturable state.…”

Lactobacillus suntoryeus sp. nov., isolated from malt whisky distilleries

“…The name Lactobacillus suntoryeus sp. nov. is proposed for this novel taxon; the type strain is strain SA T (=LMG 22464 T =NCIMB 14005 T ).Lactic acid bacteria comprise a natural component of the microflora of malt whisky fermentation (Simpson et al, 2001;van Beek & Priest, 2002). In well-maintained distilleries their numbers are low at the beginning of the fermentation, but, once the yeast has completed the alcoholic fermentation, they grow prolifically during the 'late lactic fermentation' and are considered to confer positive flavour notes to the spirit (Takatani & Ikemoto, 2004).…”

“…Phylogenetic studies based on the 16S rRNA gene sequence of strain Y10 suggested that this bacterium was a member of the Lactobacillus acidophilus group (van Beek & Priest, 2002). This group comprises L. acidophilus sensu stricto, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus johnsonii (Johnson et al, 1980;Fujisawa et al, 1992;Gancheva et al, 1999), with three relatively recent additions: Lactobacillus amylolyticus, isolated from malt and beer wort (Bohak et al, 1998), Lactobacillus iners, associated with human sources (Falsen et al, 1999) and Lactobacillus kitasatonis, from chicken intestines (Mukai et al, 2003).…”

Methods to assess lactic acid bacteria diversity and compatibility in food

“…These fragments are separated using a denaturing gradient gel -containing a mixture of urea and formamide -based on their differential melting profi le. The technique was profi tably used to identify LAB in mixed culture, thus monitoring LAB dynamics during fermentation of pozol (AMPE et al, 1999), sour cassava starch (AMPE et al, 2001), soy sauce (TANAKA et al, 2012), sausage (KESMEN et al, 2012), sourdough (MEROTH et al, 2003), malt whisky ( VAN BEEK & PRIEST, 2002), probiotic products (TEMMERMAN et al, 2003) and, primarily, dairy products (APONTE et al, 2008). Temporal Temperature Gel Electrophoresis (TTGE), or Temperature Gradient Gel Electrophoresis (TGGE), is also able to separate DNA fragments of the same length but with different sequences.…”