2.4 Hygiene Flashcards
Gram stain
gram+ stain violet due to the presence of thick layer of peptidoglycan in their cell walls, which retain crystal violet the cells are stained with; negative = red
Acetobacter
strictly aerobic gram-, oxidizes ethanol to acetic acid in such great quantities that flavor of acid and ethyl acetate via subsequent esterification would be a change, but eventually can oxidize to CO2; yeast growth is inhibited by acetic acid (decrease in pH and abundance of acetate ion); can spoil up to 12% ABV, but because strict aerobe, cannot grow with heavy CO2 during ferm; can thrive on sugary fruits
Gluconobacter
strictly aerobic gram-; oxidizes sugars to acetic acid in great quantities, cannot ox to CO2; can spoil up to 12% abv, and can thrive on sugary fruits
E. coli and enterobacter
able to grow at ~2% abv and pH<4,6, but in short periods of growth in fresh molasses or cereal wort, they produce neg aromas which can taint final spirit.
Obesumbacterium
named for unusually fat cells; is related to Escherichia and enterobacter, but not known to have an intestinal habitat; it is assumed to live on plant surfaces; has similar off defects but is sl more resistant to ethanol and low pH
Lactobacillus
most likely contaminant; gram+, non spore forming, and common on plants; tolerant in air, but grow anaerobic ferm to lactic acid and possible to ethanol and CO2 depending on species (homo vs heterolactative); troublesome strains can grow through ferm and even in beer well, being unaffected by anaerobic conditions, ethanol or low pH, and able to utilize a wide range of simple sugars and dextrins; some contamination is good in some cases for rum and whisky, but needs to be controlled to <10 to the 6th –> not only causes potential off notes/ shift in flavor profile, but also loss on yield; some spp could potentially convert di- or tri-carboxylic acids to lactic, resulting in a more mellow spirit of prolonged rum or whisky fermentations
Pediococcus
possible contaminant, but less common –> diacetyl= problem
Leuconostoc mesenteroides
capable of growth in concentrated molasses or syrup stocks of rum, using glucose half of the sucrose molecules to synthesize the viscous polymer dextrin which blocks pipework and pumps (ropey)
Leuconostoc oenos
is not a harmful contaminant in malo-lactic, but on the contrary improves flavor of cider and wine –> converts malic acid with its 2 -COOH groups to lactic acid with only 1
Zymomonas
live on plant surfaces as their natural habitat, Gram- rods, tolerant of atm O2 but grow only by anaerobic fermentation (fruc or gluc to ethanol and CO2); is not affected by final pH and alc conc. and more tolerant of the anaerobic conditions vs. S cer, so capable of growth throughout ferm; however, its production of flavor congeners is different from cultured yeast.
2 main types of Wild yeast
- Facultative anaerobes 2. Aerobic wild
facultative anaerobic wild yeast
can grow during part or all of a ferm (depending on alc tolerance) certainly affecting flavor but possible spirit yield; metabolic products are important, being different from distillation yeast, but distillation of wild yeast cells of different chem structure could also affect the flavor of the distillate; other Sacch spp, Hanseniaspora (Kloeckera if non-sporing) and Schizosaccharmoyces are perhaps the most important
Aerobic wild yeast
restricted to growth early in fermentation, but some (Hansenula and Pichia spp) are capable of producing sig amts of esters in that time; a few wild yeasts produce zymocin (killer factor against culture yeasts) is a possible hazard of natural mixed cultures for grape and molasses fermentations, but the risk is low.
Yeasts stain gram- or +
gram positive
Culture methods for detection of contaminants
require 2-3d incubation at 25C (1-2d at 30C); different methods are required for testing a. graped juice or wort before inoculation (std nutrient media) and b. culture yeast or an active fermentation (requires selective medium)
testing methodology for grape juice or wort prior to innoculation
std nutrient media and a spread-plate count with a 0.1 ml sample on malt extract agar can detect contaminants down to 10 cells/ml; using an indicator medium (WLD…MEB with added pH indicator bromocresol green) can distinguish color and shape of colonies of diff yeasts and bacteria (I.e. distinguish that they are different, it is impossible to give a definitive ID by colony morph). The same media can be used to confirm effectiveness of sterilization of a vessel, to grow contaminants trapped on a 0.45 after filtration of 250 ml of last rinse
testing methodology for culture yeast or an active fermentation
requires selective medium to allow any contaminants to grow byt inhibit growth of the culture yeast that is known to be there. For detection of LAB and Zymomonas, and other usual bacterial suspects, actidione (i.e. WLD+100g/ml of cycloheximide) ia xommonly uaws, but various other media on the same principle are available; both LAB and Zym. grow by anaerobic metabolism and may require a reduced O2, high CO2 atm for first isolation
testing/ incubation conditions for anaerobic MOs
incubating plates in a sealed can/ chamber, with a lit candle right prior to closing lid…completely anaerobic conditions are only required for Clostridium spp, which may be interest to rum distillers.
Selective media for cultured yeast vs wild yeast?
none exists; lysine agar is most useful –> gluc+lysine+salts+vitamins, which depends on inability of Sacc spp to grown on lysine as sole source of N, most other yeast genera, and certainly all of the common non sacch. contaminants anre able to utilize lysine and grow to normal colonies; one problem with this media is that it depends on starvation of cultured yeast, but even after aseptic washing and centrifugation of sample, sugg intracellular N remains to allow growth of small colonies, wild sacc yeasts cannot be detected though, but modified actidione agar works well, with only 5-10 g/ml of antibiotic since many wild yeasts have sufficient antibiotic resistance
Instant methods for contamination detection
no growth is required; two methods which involve brief incubation, but not for growth of the MOs of concern –> 1. Immunofluorescence for detection of contaminants in yeast culture 2. PCR (polymerase chain reaction): capable of amping minute mats of DNA from a specific contaminants, although PCR can detect contam after a cleaning cycle, it can not confirm the effectiveness of sterilization since the DNA may be from killed organisms, so its usefulness is limited; disadvantages of both is the inability to distinguish b/e live and dead cells
Rapid methods for contamination detection
i.e. shorter incubation time vs traditional methods; does distinguish b/e live and dead cells, but possibilities for distinguishing diff types is limited; ATP by bio-luminescence and detection of microbial metabolism by conductance, impedance, or micro-calorimetry
ATP method for rapid detection of contamination
light emission by fireflies require ATP and the light intensity from purified extract of insects in commercial kits of the luceferin/ lucefirase system is proportionate to amt of ATP; ATP is extracted from the sample and light emitted by its rxn w/ firefly extract is compared with known amts of ATP which can in turn be related to #s of MOs.
Conductance method for rapid detection of contamination
and its reciprocal measurement, impedance, change in culture media during lag phase with ion efflux, and change is proportionate to # of living cells; electrical effects is temperature sensitive, so incubation of samples requires water bath accurately attemperated to +/- 0.1C. Cells of the instrument are each fitted with a pair of probes to detect and record electrical activity, comparing graphs with those from calibration cells with known numbers of commonly encountered yeasts or bacteria gives a reasonable accurate measure of #s.; commonly available selective media allows diff types of bacteria or yeasts to be recognized; alternative instrumentation is available to detect the heat produced by MO growth but for rapid method to detect sm #s of MOS
2 basic types of CIP system
- total loss–> best suited for plant with high soiling where recovered detergent would be too contaminated for re-use 2. partial recovery 3. full recovery
2&3 –> ,save detergent and water –> detergent collected is topped up and reused (2.5% NaOH, ie), H2O –> final H2O is recycled and used a a pre-rinse in next cycle
