Fermentations as tools for making traditional and innovative foods and beverages

Fermentations as tools

for making traditional

and innovative

foods and beverages

Introduction

biotechnology process

Fermentation is a for converting carbohydrates into alcohol or organic acids using

microorganisms - yeasts or bacteria - under almost anaerobic conditions. Fermentation usually implies the

action of desirable microorganisms naturally present or added in form of selected starters.

It’s a biotechnology because essentially is a technological process but that implies the use of m.o. such as

bacteria are prokaryotes.

Fermentation is an anaerobic process that takes place without air and oxygen.

Starters are responsible for starting the fermentations.

spontaneous controlled

Fermentation can be if it's based on microorganism that are present in food or if I'm

using\adding my microorganism\starters.

Lactic acid bacteria

LAB are the most important microbes for industry purposes.

lactic acid.

The main waste\end product is They produce mainly lactic acid but not just that.

We have three kingdoms Bacteria, Archea (old microbes) and Eukarya.

Lactic acid bacteria are fastidious (they eat different nutrients and they can populate a lot of ecosystem if

gram-positive

these are rich enough of nutrients) and they’re bacteria.

They can be found everywhere: all the kinds of food and beverage, animals and humans.

Generally non-spores forming (pasteurisation: they have to be added after this process otherwise they will be

killed since they are not spore forming), non-mobile (no flagella present), non-respiring, either rod-shaped

(bacilli) or spherical (cocci) bacteria.

catalase-negative.

They’re Catalase it's an enzyme that they can use to detoxify the oxygen. If they don't

have it, oxygen will be toxic for them. So that means they leave in anaerobic conditions.

They could be divided into two different categories according to the their optimal growth:

- Mesophilic → optimum growth at 30°C.

- Thermophilic → optimum growth at 37°C.

Food processing take place at temperatures not higher than 40°C if LAB are involved.

They can use a lot of different soluble carbohydrates as a source of energy. Soluble carbohydrates are

generally monosaccharides (glucose, fructose which are hexoses but also pentoses can be used) and

disaccharides (lactose, maltose, sucrose).

Carbohydrates may be also complex carbohydrates such as starch and cellulose; to use them specific

enzymes are required but LAB don’t have them. When we speak about LAB and carbohydrates we do not

speak about complex carbohydrates because these are not an energy source for the LAB. Complex

carbohydrates can be used by most of the fungi.

auxotrophs:

They are they can't synthesise themselves certain amino acids and vitamins so they need to find

them in the environment\ecosystem otherwise they won't grow.

categorisation based on the lifestyle.

Recent LAB Three main “lifestyle” categories:

Free-living: they can be found everywhere; in all the ecosystem.

• Nomadic: they can jump from an ecosystem to another. They need all the pool of genes to be able to do

• that (eg. Lactobacillus Plantarum).

Host adapted: they can be found in a specific ecosystem (if they live in humans is difficult to find them in

• food, maybe in animals). If they live in one ecosystem, they don't need all the pool of genes but just the

genes to live in that one. Microbes during evolution eliminate all the genes that they don't need anymore

(genes loss) so they have a smaller genome compared to other categories. They have the capability to

colonise just a specific host.

The size\dimension of the genome, gene contents depends on what type of microorganism are. This is the

main difference among them.

What a microbe needs for growing: water, carbon source, energy source, nitrogen source (LAB need

organic nitrogen), mineral salts and growth factors (free amino acids, vitamins and nitrogen bases).

In most of the cases the energy and carbon source are the same (eg. glucose).

Phototrophy: m.o. that make energy from light.

• Chemotrophy: m.o. that make energy from chemicals

• (e.g. carbohydrates):

- Organic chemicals: chemoorganotrophs.

- Inorganic chemicals: chemolithotrophs.

The two main LAB metabolisms that are important for us are:

→

A. Fermentation of carbohydrates acidification is the main repercussion;

→

B. Proteolysis variation of proteins.

[Synthesis of bacteriocins, EPS and so on are important as well]

Fermentation is a metabolic pathway for getting energy and to keep\store the energy they transform it in ATP.

The fermentation is an oxidative reducing process where an organic compound acts both as donor and

acceptor of electrons within a chain of reactions, which promotes the synthesis of ATP through the

mechanism of phosphorylation at the substrate level.

Metabolism of carbohydrates

The homolactic fermentation is a chain of reactions, it’s an example of energy metabolism. The most common

example of homolactic fermentation is the one which starts from glucose.

The green part is the cell wall and the glucose, which is outside, needs to enter in the cell to get energy from

it.

The glucose has 6 atoms of carbon.

I. The glucose undergoes phosphorylation and we get glucose six phosphate (we spend energy, - 1 ATP);

II. Isomerization of 6-P-glucose to 6-P-fructose (we spent again energy, - 1 ATP)

III. 6 atoms of carbons are split in half:

1. 3 atoms of carbon (Glyceraldehyde-3-P) +

2. 3 atoms of carbon (Dihydroxyacetone-phosphate) are in equilibrium.

→

IV. Glyceraldehyde-3-P [oxidation, electrons move to the glyceraldehyde]→Glycerate.(The transporter is

reduced). → →

V. Glycerate [dephosphorylation] 2 mole of ATP this reactions takes time 2 times because of the 2

compounds are in equilibrium (no step in between, he won’t ask them at the exam).

→ →

VI. Phospoenolpyruvate [dephosphorylation] Pyruvate + 2 ATP.

VII. Pyruvate is reduced to lactate or lactic acid by the enzyme lactate dehydrogenase.

energy yield 2 moles of ATP

The from glucose: (synthesis of 4 moles of ATP but at he beginning 2 moles of

2 moles of lactic acid

ATP were consumed). The end product is just (lactate).

The microbe has to regenerate the transporter (NAD) and that happens in the last step (from pyruvate to

lactate).

The pH value of the milk is 6.6-6.7 →

The pH value of the yoghurt is less than 4.6 lactose lactic acid as a metabolite: increase the acidity that

prevents the growing of other unwanted bacteria.

Fermentation is the most green, sustainable, natural way to increase the shelf-life of products.

If the level of lactic acid is too high (low pH) that can lead to the inhibition of the same m.o. When enzymes

are inhibited, the activity of the microbes will stop if the pH is too low.

Heterolactic fermentation (not just lactic acid as a end product):

→ →

I. Glucose [phosphorylation (-1 ATP)] 6P-Glucose.

⇨

→ →

II. 6P-Glucose [oxidation (NAD NADH)] 6P-Gluconate.

→ →

III. 6P-Gluconate [oxidation + decarboxylation] 5P-Ribulose + CO 2.

IV. Isomerization of 5P-Ribulose to 5P-xilulose.

V. We have a split:

⇨

Acetyl-P If nothing happens it follows the path to generate ethanol (we MUST regenerate NAD).

⇢ Acetate pathway: we can get an extra mole of ATP; BUT it needs a different reaction to regenerate

NAD.

3-P-Glyceraldehyde (same as the homolactic but with one exception, just for 1 mole because we

don't have the equilibrium with the dihydroxyacetone-phosphate): we get two moles of ATP at the end

(just 1 because one was spent for the phosphorylation of glucose at the beginning).

1 mole of CO 1 mole of lactic acid 1 mole

The products of heterolactic fermentation are: , and which is a

2

ethanol acetic acid.

mixture of and

The decreasing of the pH will not be that high as in the homolactic fermentation (as end products: 2 moles of

lactic acid meanwhile in the heterolactic is just 1).

energy yield one mole of ATP

The is lower as well: just if the acetate pathway does not occur. In the best

2 moles of ATP.

cases it can be

To increase the energy yield generally microbes have to find other ways. There are two possibilities: spending

less energy or find other pathways to get more energy. fructose external acceptor of

One way to increase the energy yield is using as an

electrons. Fructose can be found in fruits, vegetables, flours etc.. It's not used to make

energy but just as an acceptor of electrons.

Fructose is oxidised to mannitol to have the regeneration of NAD.

Key enzymes that are important: aldolase (homolactic) and phosphoketolase

(heterolactic). The speed of the all reaction depends on these enzymes.

The reaction take place in the cytoplasm.

The reactions take place inside the cell (cytoplasm) but the glucose is outside. The cell need energy in form of

ATP.

Phospholipids are present on the membrane so energy and transporters\carriers (proteins in most of the

cases) are needed to cross the membrane.

For some mo. the energy yield of a fermentation is really low so they need to find different biological solutions

to save energy. Using a secondary transport system: combining the transport so that while one compound

goes inside the other one goes outside (anti porter), two components are going outside/inside together in the

same direction (symporter) is a way to save energy.

Facultative heterofermentative microbes in presence of hexoses (glucose, fructose) behave like

• homofermentative microbes but in the presence of pentoses they act as heterofermentative. [pentoses

→

advantage in sourdough increase of acetic acid, faster growth that means better flavour]. L. plantarum is

a typical representative of this category and is nomadic because its metabolism is versatile.

Homofermentative microbes are: Lactobacillus delbrueckii subs. bulgaricus, S. thermophilus and L.

• helveticus (important for Cheeses, in particular Grana Padano).

Heterofermentative microbes are: L. sanfranciscensis, L. rossiae.

•

The nitrogen metabolism

Proteins, oligopeptides and free amino acids (AA) are the source of nitrogen in a food matrix.

A microbe needs to hydrolyse proteins into

oligopeptides, after that it needs to hydrolyse

oligopeptides into free aa. proteolysis.

The phenomenon is called In order to do

that they need a proteolytic system (a series of

enzymes).

In these reaction there are a substrate (protein) and an

enzyme (proteinase) that takes the name from the

substrate (e.g. protein - proteinase PrtP, peptide -

peptidases ).

proteinase

The is associated to the cell wall (it’s not in

it but it’s not even free) and it hydrolyses the protein in

oligopeptides. transporter

Oligopeptides are still out of the cell but when their dimension is between 8-40 aa the can carry

the oligopeptides inside the cell. This transportation needs energy (ATP) so the oligopeptides can be realised

peptidases

in the cell. Inside the cell hydrolyses oligopeptides into free aa.

The microbe does It to have free aa that represent a source of nitrogen.

The liberation of free aa is at the basis of food flavour and taste. Some of the free aa are used for metabolic

reason but others are released to give us taste and flavour (e.g. Maillard reaction when we bake a loaf of

bread). Amino acids are also precursors of some molecules that give flavours (chetons, aldehydes, alcohols).

Proteolysis is useful to make cheeses, breads, yoghourts etc.

Peptidases are enzymes that hydrolysed peptides. The microbe to hydrolysed

peptides needs a pool of enzymes located in the cytoplasm. The main

peptidases are:

Amino-peptidase (towards the N end of the chain, N-terminus);

• Carboxy-peptidase (towards the C end of the chain, C-terminus);

• Endopeptidase (In the middle of the aa chain);

•

For each transfer (from the outside to the inside of the cell) the microbe needs one mole of ATP.

If a microbe wants to transfer 8 AA inside the cell, it needs 8 moles of ATP; if the same microbe

wants to transfer one peptide made of 8 AA it needs only one mole of ATP. So it’s easier (less

expensive in term of energy) for the microbe to transport one oligopeptide (8 AA) from the outside to

the inside and then hydrolyse it into 8 free AA.

Arginine deaminase pathway

The pathway (3 enzymes + 1 transporter) starts from arginine (AA).

It does not require any energy. It’s an antiport system because

while the arginine is going inside the ornithine is going out. ATP

Thanks to this pathway the microbe can get an extra mole of

starting from a aa (so they don’t need to start from glucose).

That’s important because some of the microbes have a low yield

of energy (homolactic fermentation).

The end products are : ornithine, 2 moles of NH , CO and ATP.

4 2

They try to buffer the pH. If the pH is too low is not good for them.

NH can buffer the pH so this pathway occurs when the pH is

4

decreasing too much.

This pathway is really important for bread making. Ornithine is a precursor of a chemical compound

generated during baking and this chemical compound (2-Acetyl-1-pyrroline) is responsible for the bread

crust.

We have to look for natural methods for food processing: we should not use nor antibiotics (antimicrobial to

stop the growth or to kill the microbe) not chemicals.

To increase the shelf-life we use acidification (lactic acid bacteria).

We’re looking for microbes that have an antagonistic effect on other negative microbes (pathogens and

spoiling). Lactic acid bacteria are used because they can synthesise organic acids that decrease the value

pH.

Exo-polysaccharides (EPS) from lactic acid bacteria.

EPS are complex carbohydrates. Exo means external with respect to the cell, these are synthesised outside/

around the wall of cell from lactic acid bacteria. They can be a defect for some beverages (wine, beer)

because of their texture (they increase the viscosity and elasticity) but for other ones is a positive attribute

(yoghurt, beer).

The EPS can be homo-polysaccharides or hetero-polysaccharides

Homo-polysaccharides are more important (high yield) and are made of just one type of monosaccharide

• (fructose or glucose).

LAB synthesise glucans and fructans from sucrose (glucose+fructose) thanks to enzymes

(glucosyltransferases and fructosyltransferases). They are glucans if they are made of glucose Fructan if

they are made of fructose.

Hetero-polysaccharides are formed by at least 4 monosaccharides, including unusual monosaccharides,

• acids and esters.

Bacteria replication

Starting from one cell you get 2 cells (it’s called generation). The time of generation is important for a

microbiologist, we try to shorten the time of generation if we have positive microbes and we try to extend the

time of generation for negative microbes. The time of generation can be calculated experimentally.

YEAST (eukaryotic)

Comparison between prokaryotic and eukaryotic cells:

They differ in sizes, the eukaryotic ones are bigger, and in complexity, the eukaryotic ones have more organs

inside the cells.

Eukaryotic cells are more sensitive to heat because of the presence of so many organs within the cells.

If they’re in competition the winner are the small ones. They’ve a higher ratio of surface/volume that leads to a

better intake/uptake of nutrient, they’ve a higher surface to intake nutrient. The smallest microbes will be

always winner. They grow faster, consume nutrients and occupy the space so no chance for the bigger

microbes to grow.

Alcoholic fermentation Same reactions as the homolactic fermentation until the

synthesis of pyruvate.

From pyruvate a decarboxylation takes place

acetaldehyde and from that through the enzyme called

alcohol dehydrogenase we get ethanol.

2 CO + 2 Ethanol.

End products: They produce more

2

CO than the lactic acid bacteria, yeasts have a better

2

capability to produce CO .

2

Yeast are eukaryotic, they are single-cell fungi (moulds

are multi cellular). The multiplication of fungi is different

from the bacteria on: it’s mainly through budding or also

fission sometimes (from 1 cell we can get 2 cells).

Buddings is a mechanism in which new cell appears in the inside of yeast and they’ll come free. From one

yeast you can get more than 2 cells.

Yeasts are defined as alcohol fermenting microorganism but numerous yeasts have respiratory activity.

Yeasts can be called Blastomyecets as well.

We have several yeasts of interest for foods and beverages:

Hanseniaspora (Kloeckera), Saccharomycodes, Nadsonia Metschnikowia pulcherrima;

• Schizosaccharomyces;

• Saccharomyces (cevisiae: they have both the fermentative and respiratory capability);

• Zygosaccharomyces;

• Dekkera;

• Pichia.

•

The taxonomy has changed through the years.

Nowadays when we speak about saccharomyces 2 different groups are distinguished:

- Saccharomyces sensu stricto cervisiae,

in which there’re 4 species: padoxus, bayanus, pastorianus.

- Saccharomyces sensu lato.

Bacteriophages Bacteriophages are viruses that infect bacteria, they have bacteria as

target. entities made of genetic material

Never say that viruses are cells but

(DNA, RNA or DNA+RNA).

The ecological meaning of a virus is to keep the equilibrium among

bacteria population to avoid that a certain population will strongly

predominate on others.

In food processes (eg. beer, yoghurt) if a virus infect the starters the process won’t take place.

A feature of the viruses is the extreme specificity of the infection: if one virus has the capability to infect a

species, the same virus won’t be able to infect other species (extreme specificity at the level of species).

The virus infection is extremely specific at not just the level of species but strains (have different capabilities

that have repercussion on food processes).

capsid

A virus is genetic material within a (made of capsomers, proteins).

attachment;

The first step is the viruses can attach or adsorb to

recognise only specific cells (species or strains); a virus has to

recognise the external part of the cell.

Penetration (injec