73

Chapter 6. Fermented foods

All food raw materials are contaminated by microorganisms, which take part in
the mineralisation of organic materials in Nature. Therefore, Man had early to
learn to live with microbially infected food. The microbial reactions mostly
resulted in spoilage of the food. However, Man learnt to handle some foods in
ways that extended their shelf-life. These preservation methods were mainly
based on drying or fermentation. Food fermentations are still used to produce so
called fermented food, but today preservation is not the main objective of the
fermentation, but it is rather the specific taste and texture that is the goal of the
fermentation. Food fermentation is applied to a all main types of food, as meat
(sausage), milk (cheese and yoghurt), grains (beer and bread), fruit juice (wine)
and vegetables (sauerkraut and pickles). In Africa and Eastern Asia many other
types of food fermentation are applied. For a European, the most well-known of
these products is soy sauce, which is produced by fermentation of soy, sometimes
supplemented with rice. Table 6.1 lists the main types of fermented food in the
Western world together with the main biochemical reactions employed in these
fermentations.
In most food fermentation the basis of fermentation control is inoculation and
adjustment of the oxygen concentration and the water activity:
1) Inoculation with a microflora. In traditional fermentations the inoculum was a
contamination from earlier production via the equipment or addition of some
product that had already been fermented. Some processes still rely on the
spontaneous natural microflora. This method is now gradually replaced by the use
of pure starter cultures, as the production becomes more industrial, since
inoculation increases the control and reproducibility of the process.
2) Adjustment of the oxygen concentration. Ethanol fermentations are inhibited by
oxygen and therefore require un-aerated conditions. Lactic acid bacteria are
independent on the oxygen concentration, but since anaerobic metabolism of
competing organisms is slower than aerobic metabolism, also lactic acid
fermentation is favoured by anaerobic conditions. Acetic acid fermentations
require oxygen. Also moulds, which are important producers of hydrolytic
enzymes in some food fermentation, are obligately aerobic organisms.
3) Reduction of water activity. Several food fermentation processes are controlled
by reduction of the water activity by addition of salt. This is the case in
fermentation of meat, fish, vegetables and soy sauce (the lactic acid stage). The
background to this is that lactic acid bacteria, which are active in these
fermentations, are relatively resistant to reduced water activity and therefore are
favoured in this environment. In sausage fermentation the salt is mixed with the
minced meat and in the other cases the raw material is placed in a salt brine.
 6. Fermented foods 74

Table 6.1 Fermented foods, their raw materials and main biochemical reactions
Raw material Products Main type of reaction
Meat Sausages Lactic fermentation
Fish Sour herring Enzymatic hydrolysis and lactic
fermentation
Milk Cheese Enzymatic hydrolysis and lactic
fermentation and (sometimes mold)
fermentation
Yoghurt Lactic (thermophilic) fermentation
Fermented milk Lactic (mesophilic) fermentation
Butter Lactic fermentation1)
Vegetables Sauerkraut Lactic fermentation
Pickles Lactic fermentation
Cereals Bread Ethanol fermentation
Beer Enzymatic hydrolysis and ethanol
fermentation
Soy sauce Enzymatic hydrolysis by moulds, lactic and
ethanol fermentation
Fruits Wine Ethanol (and malo-lactic) fermentation
Cider Ethanol fermentation
Vinegar Ethanol and acetic acid fermentation
Cocoa Ethanol and acetic acid fermentation
Coffee Microbial pectin hydrolysis
Olives Lactic fermentation
1) In some countries the cream is fermented before the churning of butter to provide
diacetyl as aroma compound.

6.1 Beer brewing

Production of beer by ethanol fermentation of grains dates back to at least 4000
BC, when it was applied in Egypt. In the ancient beer production lactic acid
fermentation probably played a role, and certain beer types are still produced with
mixed cultures of yeast and lactic acid bacteria. Hops was introduced as an aroma
compound and preservative during the 15th century. Around 1840 the lager type
of beer was introduced in Bavaria, characterised by slow fermentation at low
temperature (below 10 °C) and maturation before bottleing.
 6. Fermented foods 75

The beer brewing process is outlined in Fig 6.1. It contains a large number of
biochemical reactions. The raw materials of beer are malt, sometimes supplied
with other grains called adjunct, hops and water. Yeast, either Saccharomyces
cerevisiae or Saccharomyces uvarum, is added as a biocatalyst and sometimes
also additional enzymes of microbial origin are added to improve the enzymatic
reactions.

Fig 6.1 Summary of the beer production process.

Malting. The first stage of beer production is the malting of barley. The barley
should be of low nitrogen type, as opposite to the fodder barley. The grains are
first soaked in water in a steeping process during about two days to raise the
water content to 45%, which initiates sprouting of the grains. The grain content of
giberellic acid is important for the resulting germination. This germination
involves respiration, and the grains must be aerated to provide oxygen and remove
the carbon dioxide. Since the reaction is exothermic cooling must also be provided
and the grains are mechanically turned to provide homogeneous conditions.
During the malting process many of the barley enzymes are activated and start to
 6. Fermented foods 76

hydrolyse the grain: Hemicellulases, proteinases, α- and ß- amylases. Roots also

develop from the grain during the germination, which may take about 4-6 days to
be completed.
The germination and the emerging enzymatic reactions are interrupted by the
kilning, in which the temperature is gradually raised to 65-85 °C. During the
kilning, the high temperature results in Maillard reactions between reducing
sugars and amino-groups, that colour the malt, darker the higher the temperature is
used. This is the main way of controlling the beer colour. Maillard reaction
products also contribute to the taste of the malt and the beer. During the kilning
the water content is reduced so the malt can be stored for later use. Thus, malt can
be considered as a package of hydrolytic enzymes, notably α- and ß-amylases,
packed with the enzyme substrates, mainly starch. The last stage of the malting is
the removal of the rootlets which, like most other by-products from the beer
production, are used as fodder. Malt is not always produced by the brewer, but
often obtained from specialised malting companies.

Table 6.2 Composition of barley grains before

and after malting
Compound % in barley % in
malt
Starch 64 59
Sugar 2.5 9
ß-glucans 9 7
Cellulose 5 5
Amino acids and
peptides 0.5 1.5

Mashing. The malt is milled, coarsely to facilitate the later separation of the husk.
The milled malt is mixed with hot water to extract starch and enzymes from the
grains in the mashing process at about 65 °C. Some brewers supply additional
starchy materials, adjuncts, that are cheaper than malt, like maize, wheat or rice.
Even sugar may be used. This also reduces the protein concentration of the wort,
which may be an advantage if the malt is too protein rich, since proteins may
cause problems with precipitations in the beer. On the other hand, the use of
starchy adjuncts requires higher enzyme activity in the malt.

Starch is composed of amylose, that is a straight chain of α-1,4-linked poly-

glucose, and amylopectin which besides α-1,4 bonds also contains branching
points with α-1,6 bindings (Fig 6.2). During starch hydrolysis α-amylase
randomly hydrolyses α-1,4 bonds between the glucose units in the starch, which
results in smaller poly-glucose molecules called dextrins. Thus, hydrolysis by α-
 6. Fermented foods 77

amylase gradually reduces the mean molecular weight and the viscosity of the
starch solution but little fermentable sugar is produced in this reaction.

Fig 6.2. Hydrolysis of amylopectin to dextrins, maltotriose and maltose by α-

amylase and ß-amylase. Both enzymes hydrolyse at the α-1,4 site leaving the
branching α-1,6 sites in low molecular weight dextrins. Oligosaccharides larger than
maltotriose are not fermented by the yeast.

The ß-amylase hydrolyses α-1,4 bindings two glucose units from the non-
reducing terminal of amylopectin, amylose or dextrin to produce the disaccharide
maltose, which is the main fermentable sugar in the wort (Table 6.4). Thus, the
longer the mashing continues the higher becomes the concentration of fermentable
sugar. However, these enzymes can not hydrolyse the branching points (α-1,6
bonds) of the amylopectin and therefore small branched dextrins are left. These
dextrins are not fermentable and they remain in the beer and contribute to
sweetness and viscosity of the product.
Additional enzymes like proteases or ß-glucanases, may also be added to improve
the proteolysis or the ß-glucan hydrolysis. Pullulanase, a debranching enzyme that
hydrolyses α-1,6 bonds in the amylopectin, may also be used to increase the
concentration of fermentable sugar from the starch.
 6. Fermented foods 78

Table 6.3 Temperature and pH optima

of the main malt enzymes
Enzyme pH Temperature
α-amylase 5.7 70
ß-amylase 5.5 60
ß-glucanase 5.1 57
proteinase 4-5 40-50

These enzymes have different temperature optima (Table 6.3). During the
mashing different temperature programmes can therefore be used to control the
hydrolysis of the macromolecules. The proteolysis should furnish the wort with
amino acids for the growth of the yeast during the fermentation but it should also
degrade proteins that would otherwise precipitate in the beer. Likewise, the ß-
glucanolysis is important to reduce later precipitations and it yields
oligosaccharides. The main reaction during mashing is the degradation of starch to
fermentable sugars and non-fermentable dextrins. A typical composition of the
wort is shown in Table 6.4

Table 6. 4. Components of starch hydrolysis in wort.

Product % of total starch
Maltose 51
Maltotriose 12
Glucose 9 Fermentable
Fructose 2
Sucrose 2
Maltotetrose 3 Non-fermentable
Dextrins 21

The enzymatic hydrolysis is interrupted by boiling of the wort for 1-2 hours. pH
has then dropped from 5.8 to 5.4. Before this, the husks and precipitated proteins
are removed from the wort and hops are added. It is the dried non-fertilized female
flower of Humulus lupulus that is used. Today also pelleted hops and even hops
extract is used by the brewer. During the subsequent wort boiling, aromatic
compounds are extracted from the hops, some unwanted aroma compounds are
evaporated, all enzymatic activity ceases and the wort becomes essentially sterile.
Hops contain two main types of flavour compounds: humulones (the so called
alpha acids) and lupulones (called beta acids).

Fig 6.3 Basic structure

of the humulones of hops.
 6. Fermented foods 79

The molecules isomerise during the wort boiling which makes them more water
soluble and more bitter. Negatively charged tannins are also extracted from the
hops and they form precipitate with proteins. After the wort boiling the hops
residuals are separated off together with the precipitated proteins and used as
fodder. The so clarified wort is cooled and inoculated with yeast.

Fermentation. The fermentation process is performed in a batch according to

either of two principles. In top fermentation Saccharomyces cerevisiae is used.
This yeast flotates to the top when the fermentation has ceased due to lack of
fermentable sugar. The bottom fermentation processes utilise Saccharomyces
uvarum (carlsbergensis) which sediments to the bottom after the fermentation.
Bottom fermentation is typical for lager beer and pilsner and it is performed at
low temperature: 5-10 °C for about one to two weeks, until all visible
fermentation has ceased. Top fermentations is applied to produce the beers of ale,
stout and porter type and this fermentation is made at higher temperature, around
20°C, which results in more ester production.

N*10-6/mL EtAc (mg/L)

E (%)
60 5 50

E
EtAc

0 0 0
0 50 100 150
Time (hrs)
Fig 6.4 Progress of a lager beer fermentation at 10°C. N = yeast cell
number; E = ethanol concentration; EtAc = concentration of ethyl acetate.

During the fermentation, the yeast biomass concentration increases about four
times (Fig 4.4). Cells separated from the beer after fermentation are partly used to
inoculate next batch and partly used as fodder. To permit growth of the yeast
during the conditions in the wort, oxygen must be available for synthesis of cell
membrane constituents. Therefore the wort is saturated with oxygen from air
before inoculation. This oxygen is quickly consumed by the cells and then the
process is strictly anaerobic. From this time in the process much effort is focused
on keeping the beer free from oxygen since the shelf-life is strongly reduced by
 6. Fermented foods 80

oxidations in the beer. All fermentable carbohydrates (Table 6.4) are converted
during the fermentation to biomass carbon dioxide, ethanol and other organic
compounds that contribute to the taste. Since the yield coefficient for ethanol from
maltose is about 0.5 g/g, the final alcohol concentration can be predicted from the
concentration of wort used to start the fermentation. However, it depends also on
the extent of the starch hydrolysis to fermentable sugars. To make a low-caloric
beer there is only one way: reduce the wort concentration, since most of the
energy of the sugar is preserved in the ethanol. Depending on the extent of starch
hydrolysis, the low caloric beer can either be a low alcohol beer with a normal
alcohol to dextrin ratio or a low dextrin beer with normal alcohol content.
Ethanol is a major contributer to the taste of beer, but minor quantities of organic
acids, higher alcohols, esters and other aroma compounds are also produced and
make important contributions to the taste of the beer. However, also less pleasant
compounds are produced and for this reason a post-fermentation process is
included. One of these unwanted compounds is diacetyl. It is not produced
directly by the yeast cells, but α-acetolactate is secreted by the cells during the
later phase of the primary fermentation (see the ethyl acetate curve in Fig 6.4) and
then spontaneously decarboxylated to diacetyl.
The main fermentation results in a "green" beer which must be matured in a post-
fermentation process at 0 - 10 °C before use. Lager beer is generally matured for
a longer period, 2 weeks to 2 months at a temperature close to 0 °C, while ale is
stored at higher temperature for a much shorter period of time. Many less
characterised reactions takes place during the post- fermentation. One of the
products from the main fermentation, α-acetolactic acid, spontaneously
decarboxylates to diacetyl, which is considered unpleasant in beer. However,
during the late stage of the fermentation, and further during the post fermentation,
this diacetyl is resorbed by the remaining yeast cells, and the concentration of
remaining diacetyl is sometimes used as a measure of the post-fermentation
progress. A problem in this process is that it is the decarboxylation of the α-
acetolactate that is the rate limiting step. New technology has been developed to
achieve the postfermentation by means of an accelerated decarboxylation induced
by continuous heat treatment in a heat exchanger followed by diacetyl removal by
immobilised yeast in a packed bed column. In this way, the post fermentation
reactions can be accomplished with about 2 hours residence time during which
almost all diacetyl is resorbed by the cells.
After the post-fermentation the beer is clarified by centrifugation or filtration. To
reduce effects of microbial infections, the beer is often pasteurised or sometimes
sterile filtered. It is mainly other yeasts and lactic acid bacteria that can interfere
with beer during storage, due to the low pH, the alcohol content and the high
partial pressure of carbon dioxide. As long as these infections can be avoided the
 6. Fermented foods 81

shelf life of some 3-6 months is mainly limited by oxidation reactions. To reduce
these reactions ascorbic acid is commonly added as an anti-oxidant in beer.

6.2 Fermented milk products

Lactic acid bacteria is a group of species that are characterised by fermentation of
sugar to lactic acid. The group is divided into two categories, homofermentative
and heterofermentative lactic acid bacteria, depending on whether the metabolism
yields mainly lactic acid (homofermentative) or also considerable amounts of
acetic acid, ethanol and carbon dioxide is formed (heterofermentative). This
classification is not strict, since cultivation conditions may influence the product
pattern. Table 6.5 lists typical representatives of lactic acid bacteria in these
groups.
Table 6.5 Lactic acid bacteria classification according to
the product pattern
Homofermentative Heterofermentative
Lactococcus spp . (all) Leuconostoc spp .(all)
Pediococcus spp. (all) Lactobacillus spp. (some)
Lactobacillus spp . (some)

The lactic acid bacteria play an important role in fermentation of food. Table 6.1
shows that they are involved in fermentation of milk, meat, fish and vegetables. In
these cases the lactic acid fermentation plays an important role to stabilise the
product against microbial spoilage. The mechanism of this food preservation
effect is not at all generally known. It is well known, however, that many lactic
acid bacteria, when grown in mixed culture in the laboratory, are very
competitive. This competitiveness has been ascribed a number of factors like
production of inhibitors and resistance against low pH and low water activity (aw)
as depicted in Table 6.6.
Table 6.6 Competition advantages associated with lactic acid bacteria
Antagonistic products
Lactic acid
Acetic acid
Hydrogen peroxide
Antibiotics, e.g. nisin and reuterin

Properties of the bacteria

Ubiquitous on food raw materials (inoculum)
Oxygen indifferent
Relatively fast growing
Tolerant to carbon dioxide
Tolerant to low pH
Tolerant to low aw
 6. Fermented foods 82

6.2.1 Fermented milk and yoghurt.

Fermentation of milk with lactic acid bacteria is probably the oldest method to
preserve milk. It is widely used all over the world, probably because it has been
the safest way to consume milk. Milk that is not quickly fermented with lactic
acid bacteria soon becomes infected with a number of potentially pathogenic
bacteria. Only lately has it become possible to store non-fermented milk safely for
several days in refrigerators. Milk is fermented with lactic acid bacteria in many
different ways in different countries. Here only two types of fermentation will be
considered: a mesophilic fermentation employing a mixture of Lactococcus spp.
and yogurt, that is a thermophilic fermentation employing Lactobacillus spp. as
well. These two types are summarised in Table 6.7 and Fig 6.4.
Note that the Lactococcus genus in older literature is called Streptococcus. Only
the so called lactic streptococci are re-named Lactococcus. Streptococcus of the
enteric, viridans and pyogenes types are still classified in the Streptococcus genus.
The mesophilic fermentation employs two types of Lactococcus spp.; the
acidifiers Lactococcus lactis and Lactococcus cremoris, which are
homofermentative and have the task to quickly reduce pH and produce lactic acid,
and the heterofermentative aroma bacteria Lactococcus diacetilactis and
Leuconostoc cremoris, which are slow fermenters but produce diacetyl, which is a
desired aroma contributor in dairy products. The species mentioned in Table 6.7
are used by Swedish dairies, but many variants of this concept may be utilised.
The American fermented buttermilk, Swedish filmjölk, Danish ymer and Finnish
villi belong to this category . Villi is, however, also inoculated with a surface
growing mould, Geotrichium candidum, that contributes to the flavour and the
surface crust.
Lactobacillus spp. are generally slower to initiate the lactic acid fermentation, but
they are more resistant to low pH. Reduction of pH inhibits the glycolysis in all
starter organisms but Lactococcus spp stop the fermentation at about pH 4.5,
while the Lactobacillus fermentation continues to pH 3.9. Thus, pH in the
Lactococcus fermented milk is higher than in yogurt.

Table 6.7 Examples of two starter cultures for

fermentation of milk
Mesophilic (20°C) Thermophilic (44°C)
"Filmjölk" Yogurt

Lactococcus lactis Lactococcus thermophilus

Lactococcus cremoris Lactobacillus bulgaricus
Lactococcus diacetilactis
Leuconostoc cremoris
 6. Fermented foods 83

Another difference between the two types of fermented milk is the consumption of
lactose. The starter culture is inoculated to a concentration of about 106-107
cells/ml which grow to about 108-109 cells/ml. For this purpose lactose is used as
the energy source. The organisms of the yogurt starter culture do hydrolyse
lactose to glucose and galactose, but only glucose is consumed leaving the
galactose. Since the total biomass produced is similar or even higher in yoghurt,
the result is that yoghurt has lower concentration of lactose than the common
mesophilically fermented milk (Fig 6.5).This may be of significance in many parts
of the world, since adults generally do not accept too much lactose. The so called
lactose intolerance among adults, expressed as abdominal pains and diarrhoea
because of inability to hydrolyse the lactose in the intestines, is unevenly
distributed over the world. Generally, North Europeans and the white population
in America have a large tolerance to lactose while Asians and Africans generally
have very low lactose tolerance.

Many alternative species of lactic acid bacteria are used for fermentation of milk,
sometimes with the claim to give a more healthy product. The basis of these
properties would be that the cells colonise the intestine. Examples of such starter
organisms are Lactobacillus acidophilus, which grow very slowly compared to
other starter bacteria, and Bifidobacterium spp., which is frequently isolated from
the gastrointestinal tract. Other fermented milk types, like kefir and koumiss
contain yeast species, e.g. Candida spp and Saccharomyces spp , which contribute
to the flavour by production of alcohols and esters in very small quantities.

Fig 6.5 Schematic presentation of the lactose consumption in a fast thermophilic yoghurt
fermentation and mesophilic 'filmjölk' fermentation with a Lactococcus based starter culture.
 6. Fermented foods 84

6.2.2 Cheese. Like beer production, manufacturing of cheese is a combination of

enzymatic and microbial processes and the origin of the product dates back to
prehistoric times. The main steps of hard cheese production is outlined in Table
6.8. However, the variety of cheeses available on the market is reflected by a large
number of process variations. Only some common features and examples from
two main types of hard cheeses and the mould fermented cheeses will be treated.

The milk selected for cheese production is pasteurised (with some exceptions) at
for instance 72°C for 15 seconds. It is extremely important that it is antibiotic free,
since the starter cultures used are very sensitive to antibiotics. Especially
penicillin, which is often used to treat mastitis, may accidentally be present in the
milk. Lactic acid bacteria are extremely sensitive to penicillin. Antibiotics in the
milk may delay the lactic fermentation and it gives the opportunity for
Clostridium spores to germinate. Especially Cl. tyrobutyricum is a problem and a
spore concentration below 10 spores per 100 ml milk is required. Clostridial
growth in cheese may cause excessive gas production, butyric acid off-flavour and
even health hazards. Thus, special quick-test kits have been developed to analyse
the presence of antibiotics in the milk before cheese production.

Table 6.8 Main stages of cheese production

Action Main purpose
Pasteurisation Inactivate pathogenic and
competing organisms

Fermentation Reduce pH.

Produce lactic acid
Produce cells for later function

Addition of rennet Hydrolyse and precipitate

casein to a curd

Cutting and pressing Formation of the cheese

of curd, whey
separation

Storing Maturation of the cheese

Cow's milk contains about 87% water. The main ingredients of the dry matter are
shown in Table 6.9. Cheese is composed mainly of the caseins, except for part of
the κ-casein that is removed by enzymatic hydrolysis, the fat and part of the salts.
 6. Fermented foods 85

Table 6.9. Main ingredients of cow's milk

Component Concentration (%)
Water 87
Lactose 5
Fat 3.8
Protein 3.4
Caseins 2.8
α- 1.7
β- 0.6
γ- 0.1
κ- 0.4
Whey proteins 0.6
albumins
globulins
Salts 0.9
Calcium-
Citrates-

The milk is inoculated with starter cultures that have much concordance with
those used to produce fermented milk. Two main types may be distinguished for
hard cheese production: The Emmentaler and Gruyère type of cheese is based on
thermophilic Lactobacillus and Propionibacterium mixture while the Cheddar
and Gouda type is based on a mesophilic Lactococcus mixture (Table 6.10). the
purpose of the fermentation is to initiate the casein precipitation by reduction of
pH and to provide cells which are entrapped in the precipitated curd to take part of
the later maturation process.
Also the soft cheeses like Camembert, Brie, Roquefort, Stilton and Gorgonzola
are started with Lactococcus mixtures but they are also inoculated with a mould
species before the maturation and the action of these organisms takes place during
the maturation(Table 6.11). Since moulds are obligate aerobes, they grow only on
the surface, unless the cheese is perforated by holes.

Table 6.10 Examples of starter cultures for cheese production

Cheddar / Gouda
Lactococcus cremoris
Lactococcus lactis
Lactococcus diacetylactis
Leuconostoc spp.
Emmentaler / Gruyère
Lactococcus. thermophilus
Lactobacillus helveticus
Lactobacillus lactis
Lactobacillus bulgaricus
Propionibacterium shermanii

The declining pH during the fermentation contributes to precipitation of casein at

its isoelectric point 4.6, as in the case of milk fermentation. However, proteases
 6. Fermented foods 86

are also added to the milk during cheese manufacturing, and these enzymes
contribute to an efficient precipitation of the main part of the casein. The major
protease preparation is calf-rennet, which is an enzyme extract from young
calves. The proteases of rennet are mainly chymosin (rennin) and pepsin. When
the calf grows older the proportion of pepsin increases, which makes the extract
less useful for cheese production, since pepsin hydrolysis is too extensive which
reduces the curd yield. A relative lack of calf rennet has provoked the
development of microbial proteases for cheese production. Such microbial rennet
is in extensive use in some countries. Calf chymosin has been cloned to a yeast,
Kluyveromyces sp., to produce chymosin in bioreactors. The process has been
scaled up and introduced on the market.

Table 6.11 Mould species used for maturation cheeses

Cheese type Example Mold specie (example)
White moulded cheeses Camembert Penicillium camemberti
Brie

Blue-vein cheeses Roquefort Penicillium roqueforti

Gorgonzola
Stilton

The casein is present in milk as colloidal micelles of very complex structure as

illustrated in Fig 6.6. The micelles with a diameter around 100 nm, are composed
of submicelles. The inner part of the submicelle is composed of α- and ß- caseins
which interact by their hydrophobic parts and via Ca2+ ions also between their
hydrophilic parts. The stabilisation of the submicelle is achieved by a surface
layer of κ-casein which is divided into a very hydrophilic part, turned outside, and
a hydrophobic part turned inwards the submicelle.
The main action of the rennet enzymes is a selective cleavage in the region
between the hydrophobic and hydrophilic parts of κ-casein. This removes the
hydrophilic surface layer from the micelle which, by hydrophobic interactions,
start to aggregate and precipitate as the cheese curd. Thus, the hydrophilic parts of
the κ-casein make up the whey proteins together with the globulins and the
albumins. The whey also contains the lactic acid and most of the remaining
lactose. It is important that the cells and some of the rennin enzymes and a little
lactose are entrapped in the curd, since they form the basis for the maturation
process.
 6. Fermented foods 87

Fig 6.6 Schematic illustration of the composition of a casein submicelle in milk. The casein
molecules have characteristic hydrophobic (dotted) and hydrophilic (white) regions. ß- casein
forms chains which are interlinked by hydrophobic interaction. α-casein binds to the
hydrophobic areas of this chain and Ca2+ ions stabilises the complex by ionic bindings between
the hydrophilic parts. Finally, the submicelle increases its hydrophilicity of the surface by
attracting κ-casein units which bind their hydrophobic ends inwards against the hydrophobic
sites. Chymosin and pepsin act by specific hydrolysis in the region between hydrophilic and
hydrophobic parts of κ-casein, thus exposing a hydrophobic surface. The degraded micelles
start to interact by hydrophobic binding and precipitate as a cheese curd.

The precipitated curd is cut in pieces, separated from the whey, washed and
pressed etc., according to different procedures for the different types of cheeses.
During the subsequent storing for some months, a large number of biochemical
reactions takes place to give the product its special texture and taste. First the
starter culture cells resume a slow growth, since the pH, that had declined to stop
the glycolysis during the initial fermentation, is increased after removal of most of
the lactic acid with the whey. During this stage heterofermentative lactic acid
bacteria or propionic bacteria produce gas that is entrapped in the cheese to give
the characteristic holes. Heterofermentative lactic acid metabolism also results in
diacetyl formation which is important for the flavour, as is the lactic acid and in
some cases propionic acid and other microbial products of the primary
metabolism.
Eventually the microorganisms die and lyse, thus releasing proteases and lipases.
These enzymes, together with the traces of the rennet proteases and the low
activity extracellulary cell bound proteases of the lactic acid bacteria induce a very
slow proteolysis and lipolysis that produce peptides and fatty acids to contribute
to the flavour. Furthermore, in mould inoculated cheeses, extracellular proteases
and lipases gradually diffuse from the mycelium to slowly soften and mature the
cheese. Moulds also produce lipoxydases, enzymes that catalyse oxidative
degradation of fatty acids which results in methylketones. Among these
 6. Fermented foods 88

degradation product are 2-heptanone and 2-nonanone considered to be especially

important for the cheese flavour.

6.3 Fermented meat products

In Europe, fermented meat is mainly found in some types of sausage, like the
salami and many other of the hard, often smoked sausages. Meat is normally
contaminated by an aerobic psychrotrophic flora dominated by Pseudomonas spp.
that normally spoil the meat by growth on the surface. When meat is minced this
flora is mixed into the product and it is furnished by a surplus of nutrients from
the damaged meat cells. Thus, minced meat is extremely sensitive to microbial
spoilage. However, since long time ago, Man learnt that if the minced meat was
salted and stuffed in a gut it did not develop the unpleasant odour but stabilised
and could be used as food for very long time. This is still the basic procedure in
production of fermented sausages.
There are several mechanisms that stabilise the meat in a fermented sausage:
Addition of salt reduces the water activity to prevent the Pseudomonas spp. to
develop. These organisms are especially sensitive to reduced water activity, while
lactic acid bacteria are especially tolerant in this respect. It is also essential that
lactic acid bacteria are present in the mixture so that lactic acid is quickly
produced and pH declines. This prevents organisms of the Enterobacteriaceae
family, Clostridium spp. and Bacillus spp., which are normally present at low
concentrations, to develop. Traditional formulations often included garlic or
spices with antimicrobial compounds that further increased the stability. To
increase the safety with respect to the dangerous Clostridium botulinum , also
nitrite is added nowadays, since the undissociated acid, HNO2, is known to be
very efficient in preventing endospore germination. Actually, the name botulinum
comes from Latin botulus = sausage, since botulism was formerly often
associated with infected sausages. The package of the sausage, originally guts
from animals but nowadays often synthetic materials, also protects the meat from
infection during the storage. It is quite common that the surface becomes covered
with certain species of mould during the storage. This growth of moulds is even
utilised for the processing in certain case, like the production of Salami.
In the traditional procedures the inoculum was obtained either automatically from
the not too clean vessels used to mince and mix the meat. Some formulations also
contain milk or other sources of lactic acid bacteria. It is also common to mix old
product into the fresh unfermented mixture to inoculate the meat. Today it has
been common practice to use starter cultures to make the process more safe and
reproducible. Lactobacillus plantarum, Pediococcus spp , Lactococcus spp. and
in some cases even Micrococcus spp are used as starter cultures for fermentation
 6. Fermented foods 89

of sausages. After the fermentation the sausages are often smoked, which further
contributes to the preservation of the product.

6.4 Fermented vegetables

Vegetables are not fermented to a large extent in Europe. Some common products
are sauerkraut, that is fermented cabbage, pickles, that is a mixture of fermented
vegetables and fermenter cucumber. The history of fermented vegetables is,
however, probably as old as that of the other fermented foods. It is documented
that large scale fermentation was applied to furnish the workers with food during
construction of the Great Chinese Wall during the third century BC.
The microbiology of fermented vegetables is not so well documented as that of
beer, wine or cheese production. It is also just recently that starter cultures has
been adopted. The common method is still to place the vegetables in a 3-6% salt
brine and wait till the natural flora starts the fermentation. This takes some time
since the lactic acid bacteria are present only at very low concentrations.
Meanwhile, the main flora that may be Enterobacteriaceae members and Bacillus
spp. are retarded by the salt and they gradually die. Typically, this fermentation
starts with Leuconostoc mesenteroides and is followed by Lactobacillus brevis,
that can ferment pentoses and Lactobacillus plantarum that is the main acid
producer. Also Pediococcus cerevisiae is commonly found in fermented
vegetables. After the main fermentation a slow post-fermentation by yeasts is
common
During the fermentation, that may take a couple of weeks, the low concentration
of fermentable sugars is further reduced, which is part of the stabilisation of the
product against other microorganisms. Lactic acid is also produced at
concentrations determined by the available sugar concentration. 1-2% lactic acid
is achievable. The product obtains special characteristics not only by the taste of
the acid produced, but also by the effect of the low pH that makes the vegetable
crispy. Another important function of the fermentation is that it may inactivate
some of the plant enzymes, like pectinases, that otherwise would hydrolyse
pectins to soften the product.