Acetic acid bacteria

Question 1

Incentives of food fermentation

Answer 1

–Improves the organoleptic profile of food material

–Shelf life is extended

–Availability of nutrients is improved

–Health-promoting properties may develop (probiotic)

Question 2

Three ways of starting fermentation

Answer 2

Spontaneous

Inoculated

Back slopping

Question 3

What is spontaneous fermentation

Answer 3

–Which is based on the action of wild microorganisms that are already naturally on the food or in the area. You encourage the growth of certain microbes by creating optimal conditions for them (sauerkraut).

Change the environment so it is selective for desired microorganisms, high salt, anaerobic (lactic acid bacteria) : this organism is naturally present in the food

Question 4

What is inoculated fermentation

Answer 4

–Which is based on introducing a single pure culture or several (mixed culture) into an environment that is favorable to their proliferation. The advantage is that it stabilizes fermentations which are sensitive to contamination (wine).

Question 5

what is back slopping

Answer 5

Which is based on adding the product of a previously successful fermentation reaction to start a new reaction (friendship bread in the 90’s)

Question 6

Five types of fermentation

Answer 6

1- Acetic, 2- Lactic, 3- Alcoholic, 4- Amylolytic, 5- Proteolytic

Question 7

Acetic fermentation:

what products, substrates, required conditions

What happens if acetic acid bacteria get into products like wine or beer?

A role of acetic acid bacteria in kombucha

Answer 7

• Acetic acid bacteria
• Produces acetic acid products such as kombucha or apple cider vinegar
• Alcohol and oxygen must both be present (basically alcohol is being oxidized)
• If acetic acid bacteria find their way into a container that is no-longer airtight they will convert wine, beer, cider or any other alcoholic product into vinegar
• In Kombucha (a symbiotic fermentation) the yeasts and bacteria work together to make alcohol (yeast) and convert it to vinegar (bacteria). This is why kombucha has a low alcohol content and a vinegary flavor.

Question 8

Lactic fermentation:

what products, substrates, required conditions

Do you need a starter culture for lactic acid bacteria for vegetable fermentation?

Why need extra for milk products?

Answer 8

• Bacteria feed on sugars and produce lactic acid which quickly acidifies the environment
• This is the most common type of fermentation and is used for vegetables (sauerkraut, kimchi, pickles, capers, miso), meat (dry sausage), and milk (yogurt, kefir, cheese)
• Lactic acid bacteria are robust and are naturally present in the environment. For vegetable fermentations, you don’t generally need a starter culture, you can just create favorable conditions for the right bacteria
• It’s hard to make the dairy based fermentations attractive enough for lactic acid bacteria so that they overcome other bacteria in the mix, so starter cultures are usually used for dairy fermentations.

Question 9

Alcoholic fermentation: type of organisms, substrates, products, conditions, why beneficial

Answer 9

• Uses pure yeast cultures (or wild yeast)
• Yeast convert sugar into alcohol and produce beer, wine cider, sake, whiskey, or bread
• Alcoholic fermentation is an effective means of preservation since alcohol has a toxic effect on many other microorganisms
• Alcoholic fermentations must be carried out in an anaerobic environment, therefore airlocks are required

Question 10

What organisms are used in amylolytic fermentation

Answer 10

•Uses molds (Aspergillus oryzae)

Question 11

The principle of amylolytic fermentation

Answer 11

• Starch is a polymeric carbohydrate consisting of a large number of glucose units joined by glycosidic bonds
• Amylolytic fermentation simplifies these complex carbohydrates into more simple sugars, generally before they are used in a secondary fermentation to produce alcohol

Question 12

In what products amylolytic fermentation is used?

Answer 12

• This is not a common fermentation reaction in North America, but it is popular in Asia where it is used to make koji and nuruk
• Amylolytic fermentation can transform cooked rice into a sweet syrup in approximately a day and then the syrup is used in an alcoholic fermentation to make sake

Question 13

Proteolytic fermentation: what organisms, what foods, starter culture or spontaneous

two organisms should be mentioned for two products

Answer 13

• Mainly uses molds on high protein foods
• Requires a starter culture
• Tempeh is a good example where Rhizopus oryzae is used to ferment soybeans into a kind of pancake
• Penicillium grows on the rinds of soft cheeses and digests the proteins to make a runnier cheese with very complex flavors

Question 14

Common abbreviation for acetic acid, formula, another name

Answer 14

•Acetic acid is the most commonly used name for CH3CO2H. But ethanolic acid can also be used. A common abbreviation is AcOH or Hac.

Question 15

The difference between vinegar and glacial acetic acid

Answer 15

• Vinegar is the most common name for acetic acid in the food industry
• Glacial acetic acid is a name for water free acetic acid (laboratory grade).

Vinegar 5% acetic acid the rest is water, always made of fermentation because of food laws. Glacial 100% acetic acid

Question 16

The difference between strong and weak acid, and their effect against bacteria

Answer 16

Strong acid-> readily dissociates, a lot of hydrogen ions

lipophilic properties that allow the uncharged form of weak acids to diffuse freely across the bacterial cell membrane into the cytoplasm until reaching an equilibrium. There is also evidence indicating that weak acids result in anions accumulating inside the cytoplasm, which may have an osmotic effect and alter metabolic processes within the cell.

• Acetic acid is a weak lipophilic acid that can readily diffuse though the cytoplasmic membrane of bacteria (we will talk about the effects of organic acids on bacteria in detail later in the course)
• Concentrations as low as 0.5% can readily kill some bacteria
• The toxic effect is caused by the dissociation of the proton when it encounters the higher pH of the cytoplasm
• The release of the proton decreases the internal pH in the cytoplasm and causes the uncoupling of oxidative phosphorylation resulting in disruption of the proton gradient (Disrupts protein folding, disrupts hydrogen pump), resulting in poisoning of the cell
• Lower pH can also cause protein mis-folding
• Obviously, AAB must be resistant to acetic acid

Question 17

How acetic acid can be synthesized? What is the predominant way and why

Answer 17

• Acetic acid can be produced both synthetically and by bacterial fermentation.
• The biological route accounts for only about 10% of world production of acetic acid, but it remains important since many food purity laws require vinegar (the most popularly consumed acetic acid) to be of biological origin.

Question 18

What products can be made into vinegar?

Answer 18

Any product that has enough sugars to produce alcohol can be carried through to vinegar

Question 19

Acetic acid bacteria: gram positive or gram negative? What type of fermentation they carry out? What is the reaction equation

Answer 19

Acetic Acid Bacteria (AAB) are a group of Gram-negative bacteria that carry out oxidative fermentation to produce acetic acid from ethanol in a fermentation process.

Question 20

bacteria that synthesize acetic acid during anaerobic fermentation can be considered acetic acid bacteria?

Answer 20

No

Question 21

Most vinegar is produced by ___

Answer 21

Acetobacter sp

Question 22

Common sources of ethanol to make vinegar

Answer 22

–Wine

–Apple Cider

–Fermented grain, malt, rice, or potato mashes

Question 23

The difference between artisanal and industrial methods of vinegar production

Answer 23

• A dilute alcohol solution is inoculated with Acetobacter sp. and kept in a warm place for a few months.
• Industrial vinegar making plants accelerate this process by improving the supply of oxygen to the bacteria

Question 24

Where Acetobacter sp. will be found during vinegar production?

Answer 24

On the top as the film so they have access to oxygen

Question 25

What does acetogenic bacteria mean and what do they do?

Answer 25

Some anaerobic bacteria including Clostridium and Acetobacterium can convert sugar to acetic acid without an ethanol intermediate

C6H12O6 → 3 CH3COOH

•These acetogenic (bacteria that produce acetic acid anaerobically) bacteria produce acetic acid from one carbon compounds including methanol or carbon dioxide, and hydrogen:

2 CO2 + 4 H2 → CH3COOH + 2 H2O

Question 26

How AAB compared to acetogenic in terms of their tolerance to acetic acid

Answer 26

• Intuitively it would seem that this a quicker process since there is no ethanol intermediate, but these bacteria cannot tolerate the accumulating acetic acid, thus the process is much slower
• Acetobacter sp. is very tolerant to acetic acid

Question 27

4 generas of acetic acid bacteria that we need to know

Answer 27

Acetobacter, Gluconobacter, Gluconacetobacter, and Komagataeibacter

Question 28

Where they naturally live, what environments they can live in, and what they take from fruits

Answer 28

• AAB live in sugar-rich niches, especially fruits and flowers, and can utilize either sugar or sugar alcohol for their energy sources and produce the corresponding sugar acids
• They appear to be very adapted to environments with high concentrations of sugar, alcohols, or sugar alcohols exist in highly aerobic conditions

Question 29

Why acetic acid bacteria metabolism can be considered somewhat unique?

Answer 29

• Their metabolism (oxidative fermentation) is somewhat unique, they oxidize substrates, and then use the accumulated products later
• In their early growth they accumulate sugar acids in the growth media

Question 30

How gluconobacter is different from acetobacter and Komagataeibacter

Answer 30

–Gluconobacter can use several sugars or sugar alcohols such as D-glucose, D-sorbitol, and glycerol in addition to ethanol

–Acetobacter and Komagataeibacter are only able to use ethanol

Question 31

Sugar alcohols: what is it, common sugars, glycemic acid and where it is applied

Answer 31

Sugar Alcohol: Are organic compounds that are typically derived from sugars and comprise a class of polyols. They are widely used in the food industry as thickeners and sweeteners. They are sweet but have fewer calories when consumed.

Sugar Alcohol: Sugar:

HOCH2(CHOH)nCH2OH HOCH2(CHOH)nCHO

Common sugar alcohols include: Glycerol, Xylitol, Mannitol, and Sorbitol

Sugar alcohols affect blood sugar levels, but much less than sucrose by glycemic index. Therefore, they are commonly used in “sweets” for diabetics.

Question 32

Describe Oxidative fermentation

Answer 32

• AAB are obligate aerobes!
• They use oxygen to oxidize ethanol, sugars, and sugar alcohols to produce the corresponding sugar acids.
• At the periplasmic side of the cytoplasmic membrane AAB partially oxidize (don’t produce CO2) EtOH by two successive catalytic reactions:

–Alcohol dehydrogenase (ADH) takes electrons from the ethanol (or other sugar alcohol) and then transfers them to the terminal oxidase via ubiquinone.

–The oxidized alcohol become acetylaldehyde.

–Aldehyde dehydrogenase (ALDH) transfers electrons via ubiquinone (UQ) to oxygen as a final electron acceptor and the acetylaldehyde becomes acetic acid.

• AcOH accumulates in the media
• Membrane bound ADH and ALDH are linked to the respiratory chain which transfers electrons via ubiquinone (UQ) and terminal ubiquinol oxidase to oxygen as the final electron acceptor (generating water as a biproduct)
• The complete oxidation of EtOH can occur in the cytoplasm
• Acetic acid produced in the cytoplasm can be utilized by acetyl CoA synthase and feed the tricarboxylic acid (TCA) cycle

Question 33

What is overoxidation? How to recognize when it starts?

Answer 33

• When strains oxidize acetic acid this is called overoxidation (the same strains can also oxidize lactic, pyruvic, malic, succinic, citric, and fumaric acid). When acetic acid is not produced any more, but when only CO2
• The pH optimum for the oxidation of organic acids is near 6.0 but there is evidence that it can occur at pH 3.5

To stop it: Pasteurize or alcohol content high enough in the beginning of fermentation so they produce a bunch of acetic aicd and restrict their growth

Question 34

What is diauxic growth curve, how is it related to overoxidation? How to avoid diauxic growth?

Answer 34

• AAB can have a diauxic growth curve when the initial ethanol concentration is below 1%
• The first log phase is due to oxidation of ethanol to acetic acid
• The bacteria then transition to a second growth phase that relies on using the acetic acid as energy (overoxidation) via the TCA cycle
• This results in decreasing acetic acid concentrations outside of the cell
• If the starting ethanol concentration is above 3% this does not happen, since the products of the fermentation (acetic acid) is too high and kills the cells

Question 35

what strains can oxidize acids in TCA cycle

Answer 35

• Acetobacter and Gluconacetobacter strains can both oxidize acetic acid via the TCA cycle
• Gluconobacter do not have a functional TCA cycle since they are missing key enzymes, therefore they are unable to metabolize organic acids

Question 36

How overoxidation is connected to butter flavor in spoiled wine (mention strains)

Answer 36

•Lactic acid can also be oxidized via the same pathway by Acetobacter and Gluconacetobacter. In this case the lactic acid is oxidized to acetoin which smells like butter. This sometimes leads to a butter-like flavor and smell to spoiled wine.

Question 37

Two ways of acetic acid fermentation?

Answer 37

Surface static processes

submerged processed

Question 38

Describe surface static processes and what strains

Answer 38

•Surface Static Processes commonly use Acetobacter, and final concentrations can reach 8-9%. In this process, AAB forms a thin film on the surface of the growth media, which becomes thicker and more gelatinous with time. Bacteria (mother of vinegar)are embedded in this layer

5% a standard vinegar

Question 39

What is submerged processes? what strain, acetic acid concentration

Answer 39

•Submerged Processes commonly use Komagataeibacter (commercial strain strains and may have acidities up to 15-20%. This involves rapid mixing with forced aeration in a bioreactor

And then you dilute it to 5%

Question 40

There are four primary mechanisms responsible for AAB resistance to acetic acid ___

Answer 40

1. Prevention of acetic acid influx into the cell (CPS, EPS, and LPS). Polysaccharide can stop it from coming back to the bacteria, when exported. Capsular Polysaccharides and Exopolysaccharides, lipopolysaccharides
2. Acetic acid assimilation (overoxidation)
3. Acetic acid efflux (efflux pumps)
4. Protection of cytoplasmic proteins against denaturing (general stress proteins)

Question 41

•Ethanol and acetic acid are stressors, AAB need mechanisms to survive ethanol at the beginning of a fermentation process and acetic acid at the end. They are ___

Answer 41

Molecular chaperons

Question 42

Two classes of molecular chaperons and their function

Answer 42

–Those involved in the restoration of denatured/aggregated proteins.

–Those involved in the degradation of denatured/aggregated proteins.

•The function of molecular chaperones are to

(1) Prevent denatured proteins from aggregating
(2) Resolubilize aggregated proteins
(3) Facilitating the degradation of seriously damaged proteins.

Question 43

Names of restoration and degradation chaperon systems in AAB

Answer 43

•The GroES-GroEL system and DnaK-DNAJ-GrpE system are each representative molecular chaperones in bacterial cells

Question 44

Describe GroES-GroEL: their structure, function, when expressed, in all bacteria?

Answer 44

• GroES-GroEL is a chaperone that is representative of heat-shock proteins that are found in all bacteria
• This chaperone prevents the aggregation of denatured proteins that provides the space and time required for proteins properly fold
• GroEL forms a heptamer ring structure, and GroES forms heptamer lid-like structure.
• Together they make a cage like space, referred to as a central cavity in which denatured proteins are separated from outer conditions so that unfolded proteins can be safely re-folded into proper conformations
• When AAB are suddenly exposed to ethanol or acetic acid GroES-GroEL is expressed, and therefore it is considered an important factor which allow AAB to perform acetic acid fermentation

Question 45

DnaK-DnaJ-GrpE: describe this chaperone, components, function, by what substance it is triggered

Answer 45

• DnaK-DnaJ-GrpE This chaperone has various functions including folding nascent proteins, refolding denatured proteins, and degrading seriously denatured proteins
• DnaJ binds to improperly folded proteins and targets them to DnaK
• DnaK places the protein into a cleft, when in it’s open conformation
• DnaJ binds to DnaK and promotes the hydrolysis of ATP, which causes the DnaK to close and then the substrate protein is tightly bound
• While being held by DnaK the protein is prevented from aggregating with other proteins, which could be fatal for the cell
• GrpE can release proteins from DnaK by removing the ADP from it and providing it with an ATP
• Expression of this system is linked to ethanol and not acetic acid

Question 46

How Komagateibacter spp. can withstand 20% of acetic acid and thus become very handy in industrial production of acetic acid?

Answer 46

• These bacteria have efflux-pumps which elimination excess dissociated acetate and protons from the cytoplasm
• Synthesis of EPS has been observed in submerged fermentation. These likely serve as a physical barrier to acetic acid diffusion
• The composition of the lipid membrane is shifted to decrease the area available for passive transport of lipophilic molecules like acetic acid. The amount of glycolipids also increased which probably strengthens the hydrophobic barrier.

Question 47

Summary of physiology of AAB (fermentation, protection from acetic acid/ethanol, overoxidation)

Answer 47

Question 48

How traditional vinegars are produced?

Answer 48

• Vinegars are produced globally, most of which are of plant origin, except those from whey or honey
• All traditional vinegars are produced via a two-step process the first is production of ethanol from a carbohydrate by a yeast (if starches (i.e. rice) are used, the starch is broken down by a fungi first), the second step is oxidation of ethanol to acetic acid using Acetobacter sp., Gluconobacter sp., or Gluconacetobacter sp.

Question 49

How AAB stay floating during fermentation on top of the water? What other applications it can have

Answer 49

• AAB produce large amounts of cellulose that has a very unique, ultrafine structure
• The biological function of this cellulose appears to be the formation of biofilms that allow the retention of bacterial cells on the culture surface to that they have access to the large amounts of oxygen that they need for oxidative fermentation
• This cellulose has interesting features and has become an important commodity…
• Biofill is using bacterial cellulose for artificial skin applications

Question 50

What is Nata de Coco?

Answer 50

• is composed of bacterial cellulose by Komagataeibacter xylinus (do not need the name) at the liquid-air interface of coconut water
• It was originally developed in the Philippines
• It consists of ~90% water imbedded in bacterial cellulose and is commonly used in desserts, fruit cocktails and fruit jellies

Question 51

What is combucha, what bacteria is used, only bacteria?, why alcohol content is very low

Answer 51

• Kombucha is a fermented, lightly effervescent sweetened black or green tea drink
• It is produced by fermenting tea with a symbiotic culture of acetic acid bacteria and yeast
• Generally Gluconacetobacter xylinus is the culture
• Since the AAB is converting the alcohol produced by the yeast into acetic acid the alcohol content of the beverage is very low

Question 52

How vitamin C can be synthesized with AAB (name of bacteria and the pathway)

Answer 52

• Currently each year, 100000 tons of pure vitamin C are produced globally
• Originally the Reichstein process was used to generate vitamin C, where most of the reactions were chemical but one microbial step (M1) the conversion of Sorbitol to sorbose was preformed by Komagataeibacter xylinus
• Now a microbial process is also available (M2 and M3) using AAB
• Microbial production of vitamin C is more ecofriendly, but is still under development commercially

Question 53

AAB as a spoilage organism in wine

Answer 53

• During wine production yeast is used as a starter culture and sulfur dioxide is used as an antibacterial
• Neither intervention has an affect on AAB, and we rely on cleanliness (and Campden Crush) to keep AAB out of wine fermentations
• Wine spoiled by AAB have a vinegar like sourness, or can taste buttery, nutty or like bruised apples.
• If wine was improperly bottled (into non-sterile bottles) and bottles are stored vertically, it can spoil with AAB

Question 54

AAB only a spoilage organism in beer, or can be beneficial?

Answer 54

•Acetic acid is generally considered an off-flavor in beer

–With the notable exception of Flanders Red Ale and Oud Bruin (which use AAB)

•Lambic beer is produced by spontaneous fermentation (rather than using a brewer’s yeast inoculation), and AAB have been found in these fermentations