Microbiology of Starter Cultures: Why Bacteria Matter

Understanding the microbiology of starter cultures is the first step in understanding how to deal with fermented dairy products. Several microorganisms including bacteria, yeast, moulds, and/or combinations thereof are necessary for milk fermentation during manufacturing of cheese and other fermented milk products.

Understanding the #microbiology of starter cultures is key to understanding fermented foods. By far the most important group is the bacteria, mostly the lactic acid bacteria (LABs) e.g. Streptococcus, Leuconostoc and Lactobacillus. These three genera stand out as the most important in the dairy industry.

Why you should understand the microbiology of starter cultures:

1. Streptococcus spp. are widely used in the cheese manufacturing industry, especially; Streptococcus lactis, Streptococcus lactis sub-specie diacetylactis, Streptococcus cremoris, and Streptococcus themophilus

• The entire group of starter culture bacteria are homofermentative, i.e. they produce only lactic acid from glucose.
• All starter culture bacteria are mesophilic except Streptococcus themophilus, which is thermoduric

Of the genus Leucorostoc, only Leuconostoc cremorisand Leuconostoc dextranicum are used in dairy starter cultures. They are heterofermentative organisms, i.e. they produce lactic acid, carbon (IV) oxide (CO₂), and aroma (volatile) compounds. The genus Lactobacillus has both homofermentative and heterofermentative species.

The most common starter culture bacteria spp. include:

• Lactobacillus bulgaricus
• Lactobacillus lactis
• Lactobacillus acidophilus
• Lactobacillus helveticus
• Lactobacillus casei
• Lactobacillus plantarum

The other not very common starter culture bacterial spp include:

• Streptococcus faecium: – mostly used in the manufacture of modified cheddar cheese in the USA
• Brevibacterium linens: – (related to Arthrobacter globiformis) used to impart a distinctive, reddish-orange colour to the rind of brick and limburger cheese.
• Propionibacterium freudenreichiishermanii: – manufacturers widely usethis in Swiss cheese varieties due to its ability to produce large gas holes (eyes) in cheese during the curing period.
• Bifidobacterium bifidium: – (previously known as Lactobacillus bifidus) reside in the gut of infants. It is used together with yoghurt or acidophilus milk starter culture to manufacture bioghurt, which is a therapeutic fermented milk.

It is important to note that the study of the microbiology of starter cultures also branches out to include even molds. Dairy manufacturers mainly use molds (moulds) to manufacture some semi-soft cheese varieties. They not only enhance the flavor and aroma but also modify (slightly) the body and texture of the curd.

Types of molds based on their color and growth characteristics:

1. White molds include Penicillium camemberti, Penicillium caseicolum, and Penicillium candidum, which grow externally on the cheese e.g. Camembert and Brie.
2. Blue molds such as Penicillium roqueforti, which grows internally in the cheese to produce blue cheeses such as Roquefort, Blue stallion, Danish blue, Gorgonzola, and Mycella

Other genera of molds with limited application include:

• Mucor rasmusen, used to manufacture ripened skim-milk cheese in Norway
• Aspergillus oryzae, used to make various varieties of Soya milk cheese in Japan.

The presence of yeasts in milk, besides the LAB cultures, results in a lactic acid/alcohol fermentation. This type of fermentation is limited to the manufacture of Kefir and Kumis in the dairy industry. Kefir starter culture contains Saccharomyces kefir and Torulopsis kefir.

Scientists have also isolated the following yeast spp. from Kefir:

• Mycetoma kefir
• Cryptococcus kefir
• Mycetoma lactosa
• Candida pseudotropicalislacosta

Starter Culture Preparation: Different Types & Sources of Cultures

Starter culture preparation is a critical step in quality control during production. Starter culture bacteria have the ability to influence various processes during incubation by the means of their metabolites.

We have already seen that starter cultures are carefully selected microorganisms deliberately added to milk to initiate fermentation to produce the desired products. They are majorly lactic acid bacteria (though other bacteria types also apply), yeasts, and/or moulds.

You can opt to use these microorganisms either singly or in groups. Those cultures that have only one type of bacteria are called single strain cultures while those that have a mixture of microorganisms are called multiple (mixed) strain cultures.

Starter culture selection depends on the following three determinants:

1. The conditions of production
2. The availability of different forms of the starter to be used
3. One’s knowledge about the starter to be used

Choose the correct starter for a given job and subject it to optimum production conditions. This is because starters are the chief determinants of the quality (and the characteristics) of the fermented milk products.

Bulk starter culture preparation

Milk is the best medium for inoculating starter bacteria due to its unique composition, which enables it to nurture bacteria. You should blend the bulk milk for starter inoculation to reduce the effects of contaminants that may be present in milk from a single farm. Blending also improves the quality of the bulk.

Milk used for producing the starter should possess the following qualities:

1. First grade
2. Free from inhibitors/antibiotic
3. Able to form a smooth and homogenous coagulum
4. Clean flavour and odour
5. Free from microorganisms that produce compounds that affect lactic fermentation
6. Less than 10 cells per gram of spore-forming bacteria
7. Milk whose citric acid content is about 2.2 grams per litre (citric acid affects formation of diacetyl)
8. Adequate amounts of minerals, especially manganese and vitamins
9. Relatively high solid non-fat (SNF) content
10. Milk with relatively low content of free fatty acids

Forms of Starter Cultures

Starter cultures generally come in three forms, namely:

1. Frozen starters
2. Liquid starters
3. Dried starters

It is now a common practice in most dairies to use concentrated starters for production purposes.

Advantages of concentrated starters:

1. Ease of use at the dairy
2. Allows for easy management of the bacteriophage
3. Facilitaes production scheduling for a fixed number of production days
4. Easy to monitor and control before usage
5. High quality and well preserved
6. They have very good activity

Disadvantages of concentrated starters:

1. High quality starters are not suitable for the production of certain fermented milk products
2. They need adequate storage space with well-monitored temperatures to preserve quality
3. They are delicate and quite expensive to ship because of the equipment and conditions involved. If these are not properly observed, they risk losing activity
4. The dairies lose control over which starters are selected for producing certain products

Liquid starter culture preparation

The dairies receive the starter in liquid form from the supplier and has to propagate it for about three cycles before using in the production process. These starters are very delicate to handle and require professional manipulation to avoid messing with the end product. Phages can easily attack them; therefore, they need extremely hygienic environment for production/handling.

Some specialty fermented dairy products still require the use of liquid starters. Liquid starters are also instrumental in some cases where the concentrated cultures are not readily available. It offers the dairy an opportunity to be independent of the starter producers and the ability to determine the quality of its own products.

The dairy can obtain a seed freeze-dried starter from a producer for producing bulk liquid starter for its internal use. The operator mixes different freeze-dried strains in the batch and varies the growth temperature to differentiate the culture to suit the production needs.

Starter culture preparation by freezing

The dairy can use a concentrated frozen culture for either bulk culture preparation or directly on the milk for production. Direct application will save the dairy some production costs, as it eliminates the bulk preparation process and its associated expenses in equipment installation.

Even though it saves the dairy the cost of production, the dairy becomes dependent on the starter manufacturer for its own operations.

Steps to follow for concentrated starter culture preparation:

1. Prepare the inoculum
2. Prepare the starter media
3. Inoculate the fermenter
4. Incubate at constant pH and keep neutralized
5. Harvest the bacteria cells
6. Suspend the harvested bacteria cells in cryo-protectant solution
7. Freeze the concentrate
8. Package the concentrate aseptically
9. Store at low temperatures

These starters are majorly grown in milk treated with proteinase, whey permeate, or whey-based media. Make sure to sterilize these media to eliminate any other kind of bacteria, which may adversely affect the culture bacteria and lower the final product quality.

Sterilization also helps in achieving a constant pH of the media. A constant pH is necessary for the growth of the starter bacteria (resulting into higher yield of the cells). You can harvest the cells by centrifugal separation of the growth media. To protect these cells against damage that may result from the freezing process, it is advisable to use a cryo-protectant solution, which majorly contains milk solids, glutamic acid, and/or lactose.

Freeze the media by using liquid nitrogen because it acts very fast and inflicts minimum damage to the cells. You can keep the frozen product for a minimum of six months at -196ºC without quality deterioration. It is preferable to transport these starters using liquid nitrogen to assure quality. However, if you are to use the product in the short term, you can keep it in a deep freezer that achieves low freezing temperatures of -40°C for up to several months.

Starter culture preparation by drying/lyophilization

Freeze-dried concentrates have been in the market for quite sometime now. They have a technically similar production method as the frozen starters; only that the producer freeze-dries them (lyophilization). Lyophilized cultures have very high cell count per unit volume of the starter than the frozen culture. They are very active such that only about 10 grams of the culture is sufficient to inoculate up to 1000 liters of milk.

These cultures do not need the extreme storage temperatures. You can store them in the ordinary home deep freezers (or fridges) at a maximum temperature of -20ºC for at least five months. They are therefore much safer to use and very easy to transport to the dairies from the manufacturer as compared to the liquid cultures.

The use of these starters eliminates the need for sophisticated storage systems at the dairies, which drives down production costs at these dairies. However, adoption of these starters make the dairy dependent on the starter producer for its operational needs.

Some Starter Culture Manufacturers

1. Chr-Hansen Laboratories (Denmark, USA, France)
2. Centro Sperimentale del Latte (Italy)
3. Eurozyme (France)
4. Lactolabo (France)
5. Mauri Foods (Australia, UK)
6. Microlife Technics (USA)
7. Miles/Marshall (USA,France)
8. Scandinavian Dairy Associations (Sweden, Norway, Finland)
9. Wiesby (West Germany, Denmark)
10. CISRO (Australia)
11. Jouy-enJosas (France)
12. Liebefeld (Switzerland)
13. New Zealand Dairy Research Institute (New Zealand)
14. NIZO (Netherlands)