Yoghurt and Other Fermented Milks | Microbiology

In this article we will discuss about fermentation process of yoghurt and other fermented milks.

Fermentation Process of Yoghurt:

Fermentation to extend the useful life of milk is probably as old as dairying itself. The first animals to be domesticated are thought to have been goats and sheep in the Near East in about 9000 BC. In the warm prevailing climate it is likely that their milks furnished the first fermented milks and only some time later, between 6100 and 5800 BC in Turkey or Macedonia, was the cow first domesticated.

Fermented milks which include yoghurt, buttermilk, sour cream, and kefir differ from cheese in that rennet is not used and the thickening produced is the result of acidification by lactic acid bacteria.

Yoghurt whose name comes from the Turkish word ‘Jugurt’ is the most widely available fermented milk in the Western world today where its popularity derives more from its flavour and versatility than from its keeping properties.

It is made from milk, skimmed milk or fortified milk usually from cows but sometimes from other animals such as goats or sheep. The production process most commonly applied commercially is outlined in Figure 9.4.

The first prerequisite of any milk to be used in a fermentation process is that it should be free from antimicrobials. These could be antibiotic residues secreted in the milk as a result of mastitis chemotherapy or sanitizers carried into the milk as a result of inadequate equipment cleaning regimes at the farm or dairy.

Inhibition of the starter culture would result not only in economic losses but could potentially allow pathogens to grow.

In commercial practice it is usual to supplement the solids content of the milk to enhance the final texture of the product. The SNF (solids not fat) content is increased to between 11 and 15%, compared with a level of around 8.5% in fresh milk.

The simplest way of achieving this is by addition of skim- or whole-milk powder depending on whether a conventional or low-fat product is required. The properties of the product may also be improved and stabilized by the addition of small amounts of natural or modified gums which bind water and thicken the product.

If left to stand, the milk fat would separate out to form a cream layer. To prevent this, the milk is homogenized by passing it through a small orifice under pressure, typically 100-200 kg cm^-2 at 50-60 °C, to reduce the size of the fat globules to below 2 µm. This improves the product’s stability, increases the milk’s viscosity, and also makes it appear whiter as the number of light-reflecting centres is increased.

Before addition of the starter culture, the milk is heated at 80-90 °C for about 30 min. Being well in excess of the normal pasteurization requirements for safety, this has a substantial lethal effect on the microflora. All but heat-resistant spores are eliminated so that the starter culture encounters little by way of competition.

The heat process also improves the milk as a growth medium for the starter by inactivating immunoglobulin’s, expulsion of oxygen to produce a microaerophilic environment, and through the release of stimulatory levels of sulfydryl groups.

Excessive heating can however lead to the production of inhibitory levels of these compounds. Heating also promotes interactions between whey or serum proteins and casein which increase the yoghurt viscosity, stabilize the gel and limit syneresis (separation of whey).

The heat-treated milk is cooled to the fermentation temperature of 40-43 °C which is a compromise between the optima of the two starter organisms Strep, salivarius subsp. thermophilus (39 °C) and Lb. delbrueckii subsp. bulgaricus (45 °C). The starter culture is added at a level of about 2% by volume to give an initial concentration of 10⁶— 10⁷ cfu ml^-1 composed of roughly equal numbers of the two organisms.

The fermentation can be conducted in the retail pack to produce a firm, continuous coagulum, which is known as a set yoghurt, or in bulk tanks to produce a stirred yoghurt where the gel has been broken by mixing in other ingredients and by pumping into packs.

The fermentation takes about 4 h during which the starter bacteria ferment lactose to lactic acid decreasing the pH from its initial level of 6.3-6.5. The lactic acid helps solubilize calcium and phosphate ions which destabilize the complex of casein micelles and denatured whey proteins.

When the pH reaches 4.6-4.7, the isoelectric point of the casein, the micelles aggregate to produce a continuous gel in which all the components are entrapped with little or no ‘wheying-off’.

During fermentation growth of the streptococci is fastest in the early stages, but as the pH drops below 5.5 it slows and the lactobacilli tend to predominate. By the end of fermentation the product has a total acidity of 0.9-0.95% and the populations of the two starter organisms are roughly in balance again with levels in excess of 10⁸ cfuml^-1.

The relationship between the two starter organisms is one known as pro-to-cooperation, that is to say they have a mutually favourable interaction but are not completely interdependent.

Both will grow on their own in milk but will grow and acidify the product faster when present together. Growth of the streptococcus in milk is limited by the availability of peptides and free amino acids which are present in relatively low concentrations (≈ 50 mg kg^-1).

The lactobacillus is slightly proteolytic and liberates small amounts of these, particularly valine, which stimulate streptococcal growth. In its turn the streptococcus produces formate, pyruvate and carbon dioxide all of which stimulate the lactobacillus.

Formate is used in the biosynthesis of the purine base adenine, a component of RNA and DNA and Lb. delbrueckii subsp. bulgaricus tends to grow poorly in milk with low levels of formate, forming elongated, multinucleate cells.

Acetaldehyde (ethanal) is the most important flavour volatile of yoghurt and should be present at 23-41 mg kg^-1 (pH 4.2-4.4) to give the correct yoghurt flavour. Its accumulation is a consequence of the fact that both starter organisms lack an alcohol dehydrogenase which would otherwise reduce the acetaldehyde to ethanol.

Both will produce acetaldehyde from the glucose portion of lactose via pyruvate and through the action of threonine aldolase. The latter activity (Figure 9.5) is more pronounced in the lactobacillus but in the streptococcus methionine has been shown to increase levels of acetaldehyde via threonine.

Diacetyl, an important flavour compound in many dairy products, is present at very low levels (≈0.5 mg kg ^-1) but is thought to make a contribution to the typical yoghurt flavour.

When the fermentation is complete the yoghurt is cooled to 15-20 °C before the addition of fruits and flavours and packaging. It is then cooled further to below 5°C, under which conditions it will keep for around three weeks. Yoghurt is not usually pasteurized since chill storage will arrest the growth of the starter organisms. The acidity will however continue to increase slowly during storage.

Because of its high acidity and low pH (usually 3.8-4.2), yoghurt is an inhospitable medium for pathogens which will not grow and will not survive well. It is unusual therefore for yoghurt to be involved in outbreaks of foodborne illness, although the hazelnut yoghurt botulism outbreak in the UK in 1989 is a notable exception. Yoghurts are spoiled by acidoduric organisms such as yeasts and moulds.

Yeasts such as the lactose-fermenting Kluyveromyces fragilis and, in fruit-containing yoghurts, Saccharomyces cerevisiae are particularly important but the yeast-like fungus Geotrichum and surface growth of moulds such as Mucor, Rhizopus, Aspergillus, Penicillium, and Alternaria can also be a problem.

Advisory guidelines for microbiological quality have suggested that satisfactory yoghurts should contain more than 10⁸ cfu g^-1 of the starter organisms, < 1 coliform g^-1, <1 mould g^-1 and < 10 yeasts g^-1 (fruit-containing yoghurts may contain up to 100 yeasts g^-1 and remain of satisfactory quality).

Other Fermented Milks:

The popularity of acidophilus milk is largely due to health-promoting effects which are claimed to stem from the ability of Lactobacillus acidophilus to colonize the gut. It is a thermophilic homo-fermenter but is slow fermenting and a poor competitor and is easily outgrown.

As a result, the fermentation takes longer than for yoghurt and great care must be taken to avoid contamination. In the original process whole or skimmed milk was sterilized prior to fermentation by a Tyndallization process. This involved two heating stages of 90-95 °C for up to an hour separated by a holding period of 3-4 h to allow spore germination to occur.

Nowadays the same effect can be achieved more swiftly and economically by UHT processing. The milk is then homogenized, cooled to the fermentation temperature of 37-40 °C and inoculated with 2-5% of starter culture. It can take as long as 24 h to produce the required acidity of about 0.7%, after which the product is cooled to 5°C.

In addition to the extra care required in its production, acidophilus milk suffers from a number of other drawbacks. In particular, it lacks the sensory appeal of yoghurt being restricted to a rather sour, acidic taste. Also, the Lb. acidophilus cells do not survive well in the acid product, dying out after about a week’s storage at 5°C.

To avoid these problems, a non-fermented sweet acidophilus milk is produced in the United States where large numbers of Lb. acidophilus are simply added to pasteurized milk without incubation.

In an attempt to combine the supposed virtues of acidophilus milk with those of yoghurt, a number of ‘bio-yoghurts’ are now produced. These contain a mixture of organisms, those able to colonize the gut such as Lb. acidophilus and Bifidobacterium spp. with Strep, salivarius subsp. thermophilic to provide the characteristic yoghurt flavour.

However, because of its poor survival at acid pH, it is unlikely that the Lb. acidophilus will survive in large numbers throughout the normal shelf-life of a yoghurt.

Kefir and koumiss are distinctive fermented milks produced by a mixed lactic acid bacterial fermentation and an alcoholic yeast fermentation. Kefir is further distinguished by the fact that the microflora responsible is not dispersed uniformly throughout the milk but is added as discrete kefir ‘grains’.

These are in fact sheets composed largely of a strong polysaccharide material, kefiran, which folds upon itself to produce globular structures resembling cauliflower florets. The outside of the sheets is smooth and is populated by lactobacilli while the inner, rougher side of the sheet carries a mixed population of yeasts and lactic acid bacteria.

A large variety of different organisms have been reported as being associated with the fermentation, probably reflecting the widespread and small-scale nature of production. The morphology of the grain itself suggests that the lactic acid bacteria are responsible for its production and a capsular, homofermenter Lactobacillus kefiranofaciens has been shown to produce kefiran.

A heterofermentative lactobacillus Lb. kefir is numerically very important in many grains and plays a key role in the fermentation, probably among other things contributing to the required efferves­cence in the product. Although less significant numerically, several yeasts have been reported including Candida kefir, Saccharomyces cerevisiae and Saccexiguus.

The latter is particularly interesting because it was shown to utilize galactose preferentially in the presence of glucose and this may confer an advantage when growing in a mixed culture of organisms most of which will preferentially metabolize the glucose portion of lactose.

Kefir is produced commercially in a number of countries, most importantly in Russia and those states which comprised the old Soviet Union. In the mid-1980s production of kefir reached 12 million tonnes representing 80% of all dairy products, excluding soft cheese and sour cream.

In commercial practice, milk for kefir production is homogenized and heated to 85-95 °C for between 3 and 10 min. It is cooled to 22 °C before addition of kefir grains at a level of up to 5%. The fermentation itself lasts for 8-12 h but is sometimes followed by slow cooling to around 8° C over 10-12 h to allow for the required flavour development.

Kefir has an acidity of about 0.8% and an alcohol content which has been reported as varying between 0.01% and 1%. Ethanol levels tend to be lower in commercial products than domestically produced kefir and increase with the age of the product. In addition to the character imparted by the ethanol, lactic acid and carbon dioxide, acetaldehyde (ethanal) and diacetyl are also present as flavour components.

Koumiss is a fizzy, greyish white drink produced traditionally from mare’s milk in eastern Europe and central Asia. It can have an acidity up to 1.4% and an ethanol content up to 2.5%. A mixed yeast/LAB flora is responsible for the fermentation comprising Lb. delbrueckii subsp. bulgaricus and a number of lactose fermenting yeasts.

These are dispersed throughout the product and do not form discrete particles as in kefir. Cow’s milk is a more convenient raw material to use nowadays and this is usually modified to resemble more closely the composition of mare’s milk which has a lower fat content and higher carbohydrate levels.

Strictly speaking, buttermilk is the liquid which separates from cream during the churning of butter. However to achieve a consistent quality product most buttermilk today is produced directly by the fermentation of skimmed or partially skimmed milk. Cultured buttermilk is an acidic refreshing drink with a distinctive buttery flavour.

A mixture of starter organisms are required to produce these attributes; Lactococcus lactis produces most of the lactic acid, while the buttery flavour is the result of diacetyl production by so-called flavour bacteria such as Lactococcus lactis subsp. diacetylactis and Leuconostoc mesenteroides subsp. cremoris.

Most bacteria produce diacetyl and acetoin from carbohydrate via pyruvate. However, because of the key role pyruvate plays as an electron acceptor in LAB, it cannot usually be spared for this purpose unless an additional source other than carbohydrate or an alternative electron acceptor is available.

Citrate metabolism can provide this extra pyruvate and lead to the accumulation of diacetyl as indicated in Figure 9.6. Fresh milk contains citrate but levels decline during storage so that, for the production of cultured buttermilk, the milk is often supplemented with 0.1-0.2% sodium citrate to ensure good flavour development.

In the production process, pasteurized, homogenized milk is fermented at 22 °C for 12-16 h. The product contains 0.7-0.9% lactic acid and will keep for two weeks at 5 °C. Another property of LAB valued in some fermented milks is their ability to produce a glycoprotein slime which provides a characteristic texture and viscosity to products such as Swedish langfil and Finnish villi.

Like several other properties of LAB important in dairy fermentations such as the ability to ferment citrate, slime production is a plasmid-mediated characteristic and the ease with which this ability can be lost by the ‘ropy’ strains of Lactococcus lactis used in these fermentations can cause serious problems in commercial production.