CULTURES AND STARTER MANUFACTURE

Bacteria cultures, known as ‘starters’, are used in the manufacture of yoghurt, kefir and other cultured milk products as well as in buttermaking and cheesemaking. The starter is added to the product and allowed to grow there under controlled conditions. In the course of the resulting fermentation, the bacteria produce substances that give the fermented product its characteristic properties such as acidity (pH), flavour, aroma and consistency. The drop in pH, which takes place when the bacteria ferment lactose to lactic acid, has a preservative effect on the product, while at the same time the nutritional value and digestibility are improved.

Lactic acid bacteria

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Fig. 10.1

Bacteria in yoghurt: Lactobacillus bulgaricus and Streptococcus thermophilus, below.

Lactic acid bacteria are found on plants in nature, but some species occur in particularly large numbers in places where there is milk. Others are found in the intestines of animals. The group includes both bacilli and cocci, which can form chains of varying length but which never form spores.
Lactic acid bacteria are facultatively anaerobic. Most of them are killed by heating to 70 ºC, though the lethal temperature for some is as high as 80 ºC.
Lactic acid bacteria prefer lactose as a source of carbon. They ferment lactose to lactic acid. The fermentation may be pure or impure, i.e. the end product may be almost exclusively lactic acid (homofermentative fermentation), or other substances may also be produced, such as acetic acid, carbon dioxide and ethanol (heterofermentative fermentation).
Fermentation capacity varies according to species. Most lactic acid bacteria form between 0.5 and 1.5% lactic acid, but there are species that form up to 3%.
Lactic acid bacteria need organic nitrogen compounds for growth. They get them from casein in milk by breaking it down with the help of protein-splitting enzymes. However, the ability to split casein varies greatly from one species to another.
The most important types of lactic acid bacteria used in the dairy industry are listed in Table 10.1, which also gives the main data for the species mentioned. Some common species of mesophilic lactic acid bacteria have recently been renamed by substitution of Lactococcus (Lc.) for Streptococcus (Sc.) as the generic name. Thus, Sc. lactis, cremoris and diacetylactis have now become Lc. Lactis, cremoris and diacetylactis respectively.

Fermented dairy products and cheeses have different characteristics, and different starter cultures are therefore used in their manufacture. Starter cultures can be classified according to their preferred growth temperatures:

• Mesophilic bacteria – optimal growth temperatures of 20 to 30 °C
• Thermophilic bacteria – optimal growth temperatures of 40 to 45 °C

The cultures may be of:

• Single-strain type; containing only one strain of bacteria
• Multiple-strain type; a mixture of several strains, each with its own specific effect

Mesophilic bacteria cultures can be further divided into O and LD cultures. Table 10.2, reproduced from the Technology of Cheesemaking by Barry A Law, lists the new names of various cultures.
Some Streptococcus diacetylactis bacteria are such powerful acidifiers that they can be used alone as acidifying cultures, but they are used primarily together with Str. cremoris/lactis. However, it is not possible to use a pure Leuc. citrovorum culture, because growth of Leuc. citrovorum in milk is conditional upon the availability of nutrients produced by Str. lactis or Str. cremoris. Leuc. citrovorum grows very slowly in milk in the absence of acid-producing bacteria, and cannot produce aromatic substances in such conditions.
Bacterial characteristics such as optimum growth temperature and salt tolerance are very important in the composition of a culture. The purpose of the component strains is to produce the desired result in symbiosis, not to compete with each other. Their characteristics must therefore be complementary in this respect. Table 10.1 lists essential data for some important culture bacteria.
Dairies normally buy ready-mixed starters – commercial cultures – from special laboratories. These laboratories put a lot of effort into research and development to compose special cultures for a given product, e.g. butter, cheese and a large number of fermented milk products. Thus, the dairies can obtain cultures with selected properties for specific product characteristics such as texture, flavour and viscosity.

The dairies can buy the commercial cultures in various forms:

• Deep-frozen, highly concentrated cultures in readily soluble form, for direct inoculation of the product
• Freeze-dried, highly concentrated cultures in powder form, for direct inoculation of the product
• Deep-frozen, concentrated cultures for propagation of bulk starter
• Freeze-dried, concentrated cultures in powder form, for propagation of bulk starter
• Liquid, for propagation of mother culture (nowadays fairly rare)

The highly concentrated cultures are known as DVS (Direct Vat Set) or DVI (Direct Vat Inoculation).

Stages of propagation

In recent years, concentrated cultures have generally been used for direct manufacture of a bulk starter, (see Figure 10.2). This is a system used in cheese production, for example, where a lot of culture is used. But most cultures today are based on special highly concentrated cultures that can be used directly in production, without any further propagation at the dairy.
Some dairies, however, still propagate their own bulk starters in successive stages via a mother culture, as shown in Figure 10.3.

The process may involve two or more stages. Cultures in various stages of propagation are known by the following names:

• Commercial culture, master culture – the original culture that the dairy buys from the laboratory
• Mother culture – the culture prepared from master culture at the dairy. The mother culture is prepared daily and is, as the name indicates, the origin of all cultures made at the dairy
• Intermediate culture – an intermediate step in the manufacture of large volumes of bulk starter
• Bulk starter – the starter used in production

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Fig. 10.2

Bulk starter manufactured from freeze-dried or frozen commercial cultures.

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Fig. 10.3

Steps in the manufacture of starters.

1. Commercial culture
2. Mother culture
3. Intermediate culture
4. Bulk starter

Process technology

Starter manufacture is one of the most important and also one of the most difficult processes in the dairy. Production failures can result in heavy financial loss, as modern dairies process large quantities of milk.
Very careful attention must therefore be paid to the manufacturing technology and choice of process equipment. Starter production demands the very highest standard of hygiene. The risk of airborne infection by yeasts, mould fungi and bacteriophages must be reduced to an absolute minimum. Dairies that still propagate their own bulk starters should prepare their mother culture in a separate room supplied with filtered air at a pressure slightly above normal atmospheric pressure. The cleaning system for the equipment must also be carefully designed to prevent detergent and other CIP residues from coming into contact with the cultures and spoiling them.
Manufacture of intermediate culture and bulk starter can take place close to the point of production, or in the same room, where the mother culture is prepared.
The use of highly concentrated cultures will reduce the risks for reinfection, as less manual operations are required. This implies of course that the addition of the highly concentrated culture can be done in a hygienic way.

Stages in the process

The process, presented in Figure 10.4, is essentially the same for production of mother culture, intermediate culture and bulk starter.

It comprises the following stages:

• Heat treatment of the medium
• Cooling to inoculation temperature
• Inoculation
• Incubation
• Cooling of the finished culture
• Storage of the culture

Skim milk is the medium most frequently used for starter production, but reconstituted skim milk with 9 – 12 % dry matter (DM), made from top-grade skim milk powder, is another alternative.
The basic reason for using fresh or reconstituted skim milk is that anomalies in the flavour of the culture are much more readily apparent. Fresh milk from selected farmers is also used in some dairies.
A medium with constant composition, such as reconstituted antibiotic-free skim milk, is more reliable than ordinary skim milk.
The medium can also be modified by addition of growth factors such as Mn²⁺ (Manganese), e.g. 0.2 mg MnSO₄ per litre of culture, which is supposed to promote growth of Leuc. mesenteroides subsp. cremoris. Phage-inhibiting media (PIM) offer an alternative for production of single-strain or multi-strain starters. These media contain phosphates, citrates or other chelating agents which make Ca²⁺ (Calcium) insoluble. The reason for doing this is that most phages require Ca²⁺ for proliferation. Removing Ca²⁺ from the medium protects the lactic acid bacteria from being infected and thus avoids failure of starter activity. Skim milk powders with PIM are available in certain markets. Phage robust cultures are also available today.

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Fig. 10.4

Block diagram of starter manufacture.

Heat treatment of the medium

The first step in starter manufacture is heat treatment of the medium. It is heated to 90 – 95 °C and held at that temperature for 30 to 45 minutes. This heat treatment improves the properties of the medium through

• Destruction of bacteriophages
• Elimination of inhibitory substances
• Some decomposition of protein
• Expulsion of dissolved oxygen 
• Destruction of original living microorganisms

Cooling to inoculation temperature

After heat treatment, the medium is cooled to inoculation temperature, which differs according to the type of bacteria culture used. It is important that the temperatures recommended by the producer of the commercial culture, or empirically determined optimum temperatures, are maintained.
In propagation of multi-strain cultures, even small deviations from the proper incubation temperature may favour growth of one strain at the expense of the other(s), resulting in failure to obtain the desired typical characteristics of the end product. Figure 10.6 demonstrates what happens when typical yoghurt bacteria are incubated at a progressive temperature range.
Typical inoculation temperature ranges are 20 – 30 °C for mesophilic types of bacteria and 42 – 45 °C for thermophilic types.

Inoculation

For inoculation, a determined quantity of bacteria culture is transferred to the heat-treated medium after the temperature has been adjusted to the correct level. To prevent any deviations in the culture, it is most important that the starter dosage, the propagation temperature and the time are kept constant throughout all stages — mother culture, intermediate culture and bulk starter.
The amount of starter used can also affect the relative proportions of different bacteria which produce lactic acid and aroma substances. Variations in the amount of starter can consequently cause variations in the product. Each manufacturer must therefore determine which practical conditions suit his particular production process best. Figure 10.5 illustrates how the amount of starter used for inoculation affects the acidifying process in a culture. The curves represent dosages of 0.5% and 2.5% respectively. The inoculation temperature is 21 °C in both cases.

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Fig. 10.5

Acid development curves for inoculation with 0.5% and 2.5% of a mesophilic culture, incubating at 21 °C.

Incubation

As soon as inoculation has taken place and the starter has been mixed into the medium, the bacteria begin to multiply – incubation begins. The incubation time is determined by the types of bacteria in the culture, the inoculation dosage, etc., and can vary from 3 to 20 hours. It is most important that the temperature is carefully controlled and that no contaminants are allowed to come into contact with the culture.
During incubation, the bacteria multiply rapidly and ferment lactose to lactic acid. A culture containing aroma-producing bacteria will also produce aromatic substances such as diacetyl, acetic and propionic acids, ketones and aldehydes of various kinds, alcohols, esters and fatty acids, as well as carbon dioxide.
The importance of a correct incubation temperature is illustrated in the graph in Figure 10.6, which refers to a yoghurt culture. The culture contains two strains of bacteria, Str. thermophilus and Lb. bulgaricus, which coexist in symbiosis and together produce the desired characteristics of the yoghurt, such as pH, flavour, aroma and consistency. Most yoghurt has a ratio of cocci to bacilli between 1:1 and 2:1. The bacilli must never be allowed to gain the upper hand, as the flavour will then be too acidic.
An example of growth of Str. thermophilus and Lb.bulgarius with resulting aroma formation is demonstrated in Figure 10.7.
In this context, it may be mentioned that acetaldehyde is recognized (Pette and Lolkema, 1950 c; Schultz and Hingst, 1954) as the principal flavour component in the flavour of yoghurt. A principal role in acetaldehyde production is attributed to Lb. bulgaricus, although various strains of this species show considerable differences. In the associated growth of Str. thermophilus and Lb. bulgaricus, the rate of acetaldehyde production is considerably increased compared to the single Lb. bulgaricus species (Bottazzi & al., 1973).

Thus, the symbiotic relationships between these species favourably influence production of acetaldehyde in the manufacture of yoghurt. During production of yoghurt, formation of acetaldehyde does not become evident until a certain level of acidification, pH 5.0, has been reached. It attains a maximum at pH 4.2 and stabilizes at pH 4.0 (A.Y. Tamime & R.K. Robinson, Yoghurt - science and technology).
The optimum aroma and flavour of yoghurt are usually obtained with an acetaldehyde content ranging between 10 and 25 ppm and a pH value of between 4.4 and 4.0.
One of the factors affecting the ratio of cocci to bacilli is the incubation temperature. At 40 °C, the ratio is about 4:1, while at 45 °C it is about 1:2 (see Figure 10.6). The optimum temperature for inoculation (and incubation) in yoghurt manufacture is thus 43 °C, to achieve a cocci-to-bacilli ratio of 1:1, with a rate of inoculum of 2.5 – 3% and an incubation time of 2.5 – 3 hours.
During the incubation period, it is essential that the person responsible for production regularly checks acidity development and otherwise follows the routines found to give optimal results.
Careful handling of all starter cultures is a very important aspect of the processing of cultured milk products; this task should therefore always be given to skilled personnel.

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Fig. 10.6

Effect of incubation temperature on relative counts of cocci and bacilli at constant dosage and incubation time.

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Fig. 10.7

Growth of Str. thermophilus and Lb. bulgaricus with resulting aroma development, at 2.5 % inoculation. The curves are derived from information received from Chr. Hansen A/S.

Cooling the culture

Cooling is started at an empirically determined acidity to stop bacterial growth and thus to preserve the activity of the culture at a high level. Figure 10.8 demonstrates the course of events for an ordinary lactic-acid-forming culture inoculated with 1 % mother culture at 20 °C.
Cooling to 10 – 12 °C is often practised when the culture is going to be used within the next six hours. If the culture needs to be stored for an extended period, (more than six hours), it is advisable to cool it to about 5 °C.
In large-scale production, or production during more than one shift, it is more convenient to prepare starters at regular intervals of, say, four hours. This means that active cultures are available at all times, making it easier to follow the prescribed processing schedule and to assure consistently high quality in the end products.

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Fig. 10.8

Growth of lactic-acid producing bacteria with and without cooling at the end of incubation.

Preservation of starters

A great deal of research work has been done to find the best way to treat starters in order to preserve their activity during storage. One method is freezing. The lower the temperature, the better the cultures keep. Freezing with liquid nitrogen to –160 °C and storage below –45 °C preserves cultures very well.
Modern forms of starter cultures – concentrated, deep-frozen or freeze-dried (lyophilized) – can be stored for a considerable time provided that the manufacturers’ recommendations are followed.
Table 10.3 shows the recommendations issued by Chr. Hansen A/S of Hørsholm, Denmark.
It should be noted that deep-frozen cultures require lower storage temperature than lyophilized cultures. Moreover, the former are supplied in insulated polystyrene boxes packed with dry ice, and time in transit should not exceed 72 hours. The latter, on the other hand, can be transported at temperatures up to some 20 °C for up to 10 days without shortening the stated shelf life, provided that they are stored at the recommended temperature after arrival at the buyer’s premises.

Inoculation of highly concentrated cultures

Deep-frozen or freeze-dried super concentrated culture should be inoculated to the fermentation tanks or cheese vat in a hygienic way. Often the starter culture is added directly to the tank, through the man hole. Opening the tank for the addition does however also mean risking re-infection of the product. This risk can be reduced by for instance applying over pressure in the tanks, but there are also some alternative ways of inoculation.

In-line inoculation

The super concentrated culture can be inoculated direct in the milk stream prior to the incubation tank. A bypass line including a small container is connected to the milk pipe illustrated in Figure 10.9. The container is loaded with freeze-dried or deep frozen culture enough for inoculation of one incubation tank. When the operator decides to inoculate the tank during milk filling, he activates the valves for the bypass line and the milk will bring in the culture to the tank. After inoculation of the tank the bypass line and container are cleaned and sterilized. The container can then be loaded with culture again for inoculation of the second incubation tank. The small container can be built into a sterile air cabinet in order to minimize the reinfection risk.

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Fig. 10.9

In-line incubation with freeze dried culture.

1. Culture bag
2. Mixing container
3. Mixproof valves

Automatic Inoculation System (AISY)

In dairies or cheese factories with a lot of tanks to be inoculated, a special inoculation system, AISY, has been developed by the culture company Chr. Hansen and Tetra Pak. The AISY system combines the advantages of the highly concentrated cultures and an automatic inoculation system. The system is shown in Figure 10.10. Highly concentrated cultures are transferred into a buffer tank and diluted with cold water. After a few minutes of stirring, the diluted culture can be inoculated in-line to the milk stream or automatically pumped to fermentation tanks or cheese vats for inoculation of the milk.

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Fig. 10.10

The AISY inoculation system applied to cheesemaking tanks.

1. Culture bag
2. Mixing tank
3. Weighing cell
4. Feed pump
5. Cheesemaking tank