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AgriQuora . 27th Jul, 2021

Microbiology Of Starter Cultures: Types Of Bacteria, Their Growth & Inhibition

A starter culture is a bacteria culture that you can use to manufacture fermented products such as yoghurt, kefir, cheese, salami and butter among many other cultured dairy and non-dairy products.

You inoculate ("seed") the starter culture into the milk/dairy product and allow it to grow/multiply under controlled conditions. This controlled environment will allow the bacteria to multiply and impart the characteristic features of the resultant cultured dairy product such as acidity (or pH), aroma, consistency, and flavor.

Bacteria break down lactic acid in the dairy product, resulting into increased acidity (or low pH). The low pH imparts a preservative effect to the product and improves its nutritive and digestive quality.

What is microbiology?

Microbiology is the study of microscopic organisms such as bacteria, fungi, algae, and the infectious agents at the borderline of life such as viruses. The study encompasses microbiological characteristics such as form, structure, reproduction, physiology, classification, and metabolism.

Microbiology further looks at:

  • their distribution in nature,
  • relationships with each other and other living organisms, and
  • their effect on plants and animals.

For the sake of this brief history of microbiology, we will look at microorganisms related to the food industry.

History of Microbiology

Microbiology is a very old subject. The first person to postulate the existence of microorganisms was Aristotle in 4 B. C. He suggested that living organisms are made up of cells.

It was only until 13th century when people realized that ground pieces of glass provided a greater magnifying power. They were able to see tiny objects that they could otherwise not see through their naked eyes.

Following these developments, Roger Bacon postulated that invisible living creatures cased diseases.

In 1530, Fracastoro of Verona coined the term syphilis to describe an outbreak that ravaged Europe in the 1400's when the returning French soldiers spread the disease.

He called the disease agent 'seminaria morbi' (living germs) that spread 'contagium vivum' (via contact with an individual with the germ).

In 1658, Athanasius Kircher defined the invisible organisms found in decaying bodies, meat, milk, and secretions as worms.

In 1665, Robert Hooke made a powerful compound microscope that he used to confirm Aristotle's postulate that living beings comprise repeating units of cells. That same year, an Italian named Francisco Redi confirmed that maggots are the larval stage of flies.

Antonie van Leeuwenhoek made powerful lenses in 1676 that could magnify up to 3000µm (*300) - 3mm size.

In 1765, Lazzaro Spallanzani boiled meat in hay infusion and covered the broth in an air-tight container. Bacteria could not grow for a longer time.

Later in 1810, Nicholas Appert discovered that bacteria could not grow in foods in air-tight cans. His method of preservation became popularly known as appertization and later as canning.

In 1861, Louis Pasteur confirmed that air contains microorganisms when he cultured cotton wool that he had used to filter air. Louis also discovered the widely popular pasteurization method of food preservation.

Here is the timeline infographic showing a brief history of microbiology.

Whenever bacteria is mentioned, a bad thing is almost always implied. Bacteria are synonymous with diseases, poisoning, and even death.

As studies on microorganisms continued, the scientists discovered that not all bacteria were harmful. There are also very many beneficial bacteria.

Some of the benefits of microorganisms include:

  1. Production of antibiotics to treat diseases affecting man, animals, and plants. Bacteria can also act as biological pesticides in organic farming.
  2. Probiotics are used to create longevity products such as yogurts and other fermented products.
  3. Bacteria can produce enzymes used in food production/processing. Some cleaning products and dyes rely on bacterial enzymes.
  4. In the food processing industry, bacteria is used to make vinegar from alcohol.
  5. Bacteria play a vital role in maintaining a balanced environmental ecosystem. For instance, nitrogen fixing bacteria help plants harvest nitrogen from the air to improve productivity.

Differentiating D, L, and DL Cultures

Different dairy products have different qualities and distinctive characteristics, which are dependent on the type of culture you use to make that specific product. The cultures may contain a pure strain of bacteria (single strain) or multiple strain type (with many species of bacteria; each strain has its specific role to play in the mixture).

Some starter culture bacteria only ferment lactose into lactic acid. Such strains include Streptococcus lactis, Streptococcus cremoris, and Streptococcus thermophilus. They are majorly used to make acidified dairy products. Other strains such as Streptococcus diacetylactis and Leuconostoc citrovorum produce flavor and aroma as well.

About D, L, and DL cultures

Starter cultures that contain the three stains of Streptococcus lactis, Streptococcus cremoris and Streptococcus diacetylactis to produce both acid and flavor in the dairy product are classified as D cultures (D is for the diacetylactis).

On the other hand, if you opt to use Leuconostoc citrovorum to produce the aroma and flavor in the dairy product, you will end up with an L culture (L is for the leuconostoc).

A combination of both D and L cultures produces a DL culture, which has the qualities of all the bacteria used.

  • D culture: - contains Streptococcus lactis, Streptococcus cremoris and Streptococcus diacetylactis
  • L culture: - contains Streptococcus lactis, Streptococcus cremoris and Leuconostoc citrovorum
  • DL culture: - contains Streptococcus lactis, Streptococcus cremoris, Streptococcus diacetylactis and Leuconostoc citrovorum

List of bacteria present in the starter culture
Bacterial strainWhat it doesUsed to make
Propionic bacterium shermaniiFlavor/aroma, eye formationEmmental cheese
Lactobacillus bulgaricusAcidity, aroma/flavorKefir, yoghurt
Lactobacillus lactis,Flavor/aromaCheese
Lactobacillus helveticusFlavor/aromaCheese
Lactobacillus acidophilusAcidityAcidophilus milk, cultured milk
Streptococcus thermophilusAcidityYoghurt, cheddar and emmenthal cheese
Streptococcus diacetylactisAcidity, flavor/aromaButter, cultured cream, cultured milk
Streptococcus lactis, Streptococcus cremorisAcidityCheese, butter, cultured cream, cultured milk
Leuconostoc citrovorum, Leuconostoc dextranicumFlavor/aromaCheese, butter, cultured cream, cultured milk
Streptococcus durans, Streptococcus faecalisAcidity, flavor/aromaCheddar cheese, Italian soft cheese

Symbiotic Relations of Starter Culture Bacteria

Some Streptococcus diacetylactis bacteria produce concentrated acid in the product that they do not need the help of Streptococcus lactis/Streptococcus cremoris in acidifying the culture. The bacterial strains are combined because they have a mutual benefit in the culture.

For instance, Leuconostoc citrovorum needs the nutrients (metabolites) produced by Streptococcus lactis/Streptococcus cremoris for its growth. It means that if you eliminate these, the Streptococcus citrovorum will not grow properly in the culture.

The slow growth in the acid-less environment will affect aroma production. Consequently, the quality of the final product will not be consistent. You will fail to achieve a similar product as the one you produce in the presence of acid-producing bacteria.

Bacterial tolerance towards temperature, pH, or salt concentration in the medium affects the products you produce. Control the process to produce the results you want.

The purpose of mixing the strains is to impart the symbiotic advantages to the culture. This will reduce competition among the bacteria in the starter culture. The bacteria characteristics are to complement one another in the process of product formation.

The table below lists some of the tolerance levels for the culture bacteria

Bacterial strainOptimum temp (°C)Max % salt tolerance% acid formationFerments citric acid (- no; + yes)
Str. lactisAbout 304-6.50.8-1.0
Str. cremoris25-3040.8-1.0
Str. diacetylactisAbout 304-6.50.8-1.0+
Leuc. citrovorum20-25small+
Lb. helveticus40-4522.5-3.0
Lb. lactis40-4521.5-2.0
Lb. bulgaricus40-5021.5-2.0
Lb. acidophilus35-401.5-2.0

Where to get starter culture to make fermented dairy products

Various laboratories that specialize in manufacturing starters make special cultures for the production of the various dairy products. These starters can be obtained by special orders form the manufacturers or their vendors. These cultures have been specially mixed to produce the desired effect on the processed dairy products.

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 exceptStreptococcus themophilus, which is thermoduric
  2. 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.
  3. 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.

#Startercultures are carefully selected MO deliberately added to #milk to make desired products.

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.

In short, the 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)

Starter Culture Growth Inhibitors: Antibiotics and Phages

Dairy starter cultures are applicable as single strains, in pairs, or as a mixture. Whatever the case may be, it is important to consider the starter culture growth inhibitors that may impede the activity of the starter culture bacteria.

Mesophilic lactic starter cultures (whose optimum temperatures range between 20-30ºC) are widely used to make many fermented dairy products. In the cheese industry, they are found in three categories (as single, multiple or mixed strains).

Thermophilic LABs (whose optimum temperatures range between 37-45ºC) are used to manufacture yoghurt, acidophilus milk, and high temperature scalded cheese (e.g. Swiss varieties). These bacteria include Streptococcus thermophilus and all Lactobacillus spp.

Single Strain and Multiple Strain Cultures

In theory, a single strain starter should consist of only one type of organism but this is very rare in practice. However, you can pair up single strain cultures to safeguard against bacteriophage attack, intolerance of salt or cooking temperature, and to vary in the quality of the end product.

Multiple strain starter cultures consist of known numbers of single strains developed for lengthy use during the cheese-making season. These mixed strain starters consist of a combination of Streptococcus lactis, Streptococcus cremoris, and other gas and aroma producing mesophilic LABs.

The aroma producing lactic starters are essential for the production of buttermilk, sour cream, cultured butter, and some fermented milk products.

The starter culture growth inhibitors

There are many factors that can cause inhibition or reduction of the activity of a starter culture. The resultant effect would be poor quality fermented dairy products reaching the consumer and financial loss to the producer.

These factors include:

1. Antibiotics

Residues of antibiotics in milk result from mastitis therapy in dairy cows. Some unscrupulous milk traders intentionally add penicillin and other antibiotics in milk to preserve its quality.

Starter cultures are susceptible to very low concentrations of the antibiotic residue.

2. Bacteriophages

Some viruses (also known as phages) can attack bacteria and destroy starter cultures. The result is a failure to produce lactic acid after inoculation. The lactic streptococci and lactobacilli are the most vulnerable microorganisms in the dairy starter cultures.

You can reduce the effect of the phages in the dairy industry by:
  • Propagating starter cultures in very aseptic conditions i.e. adopt aseptic technique in handling dairy products and processes
  • Proper heat treatment (temperature/time combination) of bulk starter milk to destroy the viruses in milk
  • Daily rotation of phage-resistant strains
  • Effective filtration of air in the starter room
  • Proper sanitation of the equipment and premise
  • Location of starter room far away from production area
  • Personnel, especially those from cheese room should NOT enter the starter room
  • Propagate starter culture in phage inhibitory medium
  • Develop phage-resistant strains
  • Use mixed strain starter cultures.

3. Detergent and disinfectant residues

Detergents and disinfectants for cleaning and sanitization in the dairy plant may cause contamination. The residues of these compounds (alkaline detergents, chlorine based materials, iodophors, quaternary ammonium compounds and ampholytes) do affect the activity of the starter culture.

Yoghurt cultures are more tolerant to the activities of these residues at the inhibitory levels (mg/l) of culture compounds. Contamination of starter milk with these compounds is majorly due to human error, or malfunction of the automatic chain cycle.

4. Miscellaneous starter culture growth inhibitors

Natural antibodies (such as lactelins/agglutinins) that are present in milk can inhibit the growth of the starter cultures. These antibodies are heat sensitive, and heat treatment of bulk starter milk ensures their destruction.

Leucocytes in mastitis milk can cause phagocytosis of the starter microorganisms. Thiocyanates present in late lactation milk may also inhibit the growth of starters. Heating of the starter results in no significant improvement of the end product

You can attribute other inhibitors to environmental pollution factors, such as insecticides, volatile and non-volatile compounds.

Such volatile compounds may include fatty acids, formic acid, formaldehyde, acetonitrile, chloroform, and ether. When their concentrations reach 100ppm, they will inhibit growth of Streptococcus spp. and Lactobacillus cremoris.

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