2.4 Hygiene

Question 1

Gram stain

Answer 1

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

Question 2

Acetobacter

Answer 2

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

Question 3

Gluconobacter

Answer 3

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

Question 4

E. coli and enterobacter

Answer 4

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.

Question 5

Obesumbacterium

Answer 5

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

Question 6

Lactobacillus

Answer 6

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

Question 7

Pediococcus

Answer 7

possible contaminant, but less common –> diacetyl= problem

Question 8

Leuconostoc mesenteroides

Answer 8

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)

Question 9

Leuconostoc oenos

Answer 9

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

Question 10

Zymomonas

Answer 10

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.

Question 11

2 main types of Wild yeast

Answer 11

1. Facultative anaerobes 2. Aerobic wild

Question 12

facultative anaerobic wild yeast

Answer 12

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

Question 13

Aerobic wild yeast

Answer 13

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.

Question 14

Yeasts stain gram- or +

Answer 14

gram positive

Question 15

Culture methods for detection of contaminants

Answer 15

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)

Question 16

testing methodology for grape juice or wort prior to innoculation

Answer 16

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

Question 17

testing methodology for culture yeast or an active fermentation

Answer 17

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

Question 18

testing/ incubation conditions for anaerobic MOs

Answer 18

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.

Question 19

Selective media for cultured yeast vs wild yeast?

Answer 19

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

Question 20

Instant methods for contamination detection

Answer 20

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

Question 21

Rapid methods for contamination detection

Answer 21

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

Question 22

ATP method for rapid detection of contamination

Answer 22

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.

Question 23

Conductance method for rapid detection of contamination

Answer 23

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

Question 24

2 basic types of CIP system

Answer 24

1. 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

Question 25

Cleaning cycle

Answer 25

pre-rinse to remove loose soil, detergent re-circulation to clean vessel and pipework, then after discarding or recovering dilute detergent, vessel is rinsed with clean water, disinfectant may e added to rinse water…

Question 26

Burst rinsing

Answer 26

highly efficient use of water during CIP

Question 27

propagation, rehydration, fermentation, pumps, and pipework associated CIP?

Answer 27

cleaning and sterilization

Question 28

still charger vessel/ beer well and associated pipework and equipment CIP?

Answer 28

also be considered for CIP, to lower risk of further MO growth in beer

Question 29

all other stages of process CIP?

Answer 29

physical cleanliness that is required for a food product is sufficient

Question 30

How often is cleaning required?

Answer 30

1. preparation of fermentatble extracts: molasses/ grape–> daily cleaning, cereal –> very susceptible to soiling, cleaning (not sterilization) after each use is essential, or various bacteria can grown producing off flavors 2. fermentation = every use…clean and sterilization 3. Distillation –> occasional, yet regenerative cleaning is necessary; steam coils/ heat exchangers: soil build-up can effect heat transfer; sieve plates: solids can clog perforations, is not efficient liquid/volatile contact pts and pressure build-up, or back flow…also heated deposits could cause off notes.

Question 31

Material types/ surface effect cleaning

Answer 31

stainless and cu –> smooth vs cast Fe, mild steel, or wood (extremely hard to clean and disinfect, and complete sterilization is not possible…steam is your friend); joints –> between plates or planks

Question 32

Four distinct stages are involved in the actual cleaning

Answer 32

1. wetting of the surface to allow intimate contact b/e detergent and soil 2. chemical action on the soil (i.e. solution of mineral scale by acids, hydrolysis of protein by acid or alkali, saponification of lipid material by caustic detergents 3. dispersion of large particles as finely-divided soil 4. suspension in solution of any removed soil,,,also a good detergent allows efficient and complete removal of detergent and suspended soil by final rinsing

Question 33

NaOH meets many of the requirements for cleaning, and 2% used hot at >70C is also an efficient disinfectant…but there are 2 main disadvantages

Answer 33

1. precipitation of mineral salts (Ca, Mg associated with hard water which is prevented by addition of sequestrants Na gluconate or Na hexametaphosphate to detergent formulas…forms chelate complexes with metal ions 2. reaction with CO2, forming much less efficient Na2CO3, requires fermentation vessels to be drained of CO2 prior to cleaning

Question 34

Acid detergents?

Answer 34

to avoid lengthy delay…additional advantage of action on mineral scale; phosphoric acid with added surfactants makes a good CIP detergent; some formulations also include nitric for greater detergency, also for disinfectant effect, but HNO3 mixtures are destructively incompatible with Cu or Cu-containing alloys. However, the stainless used in the fermentation industries are resistant. Various organic acids are also possible but have no advantage over H3PO3 and are more expensive.

Question 35

Quaternary Ammonium and amphoteric detergents

Answer 35

surfactants; they combine sergent and disinfectant acitivity; their powerful surface-active effect lyses microbial, particularly bacterial, cell walls and membranes; disadvantages–> foaming during CIP, difficulat to rinse off, and residues cause foaming during ferm, therefore they are not ideal for distilling use. On the other hand, that residual biocide on food processing surfaces is an advantage.

Question 36

Killing of MOs by a disinfectant is influenced by:

Answer 36

concentration of disinfectant, temp of disinfectant, # of MOs, time of contact b/e disinfectant and MOs. and amt of inert organic soil

Question 37

2 most useful biocides 4 distillery fermentation vessels

Answer 37

steam and peracetic acid

Question 38

Saturated steam use for biocide activity

Answer 38

1. saturated (wet) steam –> very effective for sterilizing cleaned equipment only; steam bakes some soil onto surfaces, making it more difficult to remove and can protect MOs; kills all types of MOS, although to achieve sterility at ATM pressure requires continuous steaming for 1-5 hrs; steam is freely available in a distillery, and does not need to rinse; however, radiated ht from vessel being other viable option;

Question 39

Peracetic acid benefits

Answer 39

breaks down first to acetic acid and H2O2 and then the latter to water and oxygen; the small amt of acetic acid formed at use-dilution is insufficient to cause flavor taint so a final rinse is not required…and is best avoided, or UV water should be sued.

Question 40

Chlorine as hypochlorite NaOCl use

Answer 40

Chlorine as hypochlorite, iodine as iodophor (solution of I2 in a surfactant); must be rinsed off (base brand risks; most NaOH detergent formulations give effective disinfection when used hot, but the alkaline residue must be rinsed off, and they are incompatible with residual CO2 in ferm vessels.