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Starter Cultures, Culture Media Preparation & Effect of Growth Inhibitors

Starter Cultures, Culture Media Preparation & Effect of Growth Inhibitors

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.

  1. 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). 
  2. In 1658, Athanasius Kircher defined the invisible organisms found in decaying bodies, meat, milk, and secretions as worms.
  3. 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.
  4. Antonie van Leeuwenhoek made powerful lenses in 1676 that could magnify up to 3000µm (*300) – 3mm size.
  5. 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.
  6. 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.
  7. 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 are mentioned, a bad thing is almost always implied. Bacteria are synonymous with diseases, poisoning, and even death. As studies on microorganisms continued, 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 are 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+
Str.thermophilus40-4520.8-1.0
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.

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 lactisStreptococcus 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.

Microbiology of Starter Culture Media

A culture media is either an organic or a synthetic substance that provides both the biophysical and the biochemical factors necessary for the growth of bacteria. Researchers have developed a variety of culture media to serve different needs/purposes. Each type of media serves the needs of a particular bacteria and/or the special requirements of the investigator.

One of the purposes of a culture media is to isolate and maintain pure bacterial strains. That is why the media is crucial in the identification of different bacteria.

The differentiating factors depend on the biophysical and biochemical properties of each bacteria. You can use liquid media to grow pure batch cultures and to estimate the bacterial populations. A gelling agent (usually nutrient agar), makes the media either solid or semi solid. Nutrient agar is a hydrocolloid of red algae.

Agar imparts unique physical characteristics to the culture media, which makes it suitable for its purpose. For instance, agar enables the media to melt at 100ºC and then cool back to less than 40ºC before re-solidifying. Additionally, very few organisms are capable of degrading nutrient agar.

Solid media are instrumental in isolation of pure cultures and determination of the number of viable bacterial populations.

Classification of culture media depends on:

  1. Their chemical composition, and
  2. The intended use of the media.

The classes of culture media include:

  • Chemically defined (synthetic) media: – have well defined chemical components and well-known proportions
  • Complex (undefined) media: – their exact chemical compositions are not known
  • Selective media: – contains components that will inhibit the growth of certain bacteria spp. but promote the growth of the desired species
  • Differential media: – allows the investigator to distinguish between different types of bacteria based on some observable patterns of growth in the medium. For instance, you can eliminate Staphylococcus aureus from a culture by increasing the salt concentration in the media
  • Enrichment media: – contains some components that permit the growth of specific bacteria spp. This is usually because only such species can utilize the nutrients from the media

Procedure for the preparation of the liquid media

To begin with, you will need a functional autoclave. You will use it to sterilize the media at high temperatures under pressure. After placing the media to be sterilized in the autoclave, tightly the equipment then heats it to a pressure of 15psi and temperature of 121ºC for 15 minutes. Read more about the operating pressures of an autoclave here.

You will also need the following:

  • Distilled water,
  • Clean 500 ml measuring cylinder,
  • Clean conical flask,
  • Electronic weighing scale,
  • Sterile media, and
  • Clean test tubes.

8-step-process for making culture media

  1. Weigh 6.5 grams of the sterile nutrient broth and transfer into the clean conical flask. The manufacturer recommends a dilution of 13 g/l but we need to make only 500 ml of the media.
  2. Add 500 ml of distilled water into the measuring cylinder and transfer into the conical flask to dilute the media.
  3. Put the conical flask with the media solution from step 2 into an autoclave basket. Ensure you properly secure the mouth of the flask with cotton wool before lowering it into the autoclave. Secure the autoclave and start the sterilization. It will take some time to attain sterilization temperatures. However, once you achieve the recommended temperature/pressure combinations, hold it there for the recommended 15 minutes. That time is adequate under these conditions to sterilize the media.
  4. Allow the autoclave to cool down.
  5. Put the petri dishes into a hot air oven at 80ºC for one hour to sterilize them.
  6. Remove the conical flask containing the now sterile media from the autoclave. Pour 15 ml into each petri dish and seal.
  7. Make sure your working bench is not only clean but also always sterile. Wipe the surfaces with 7% alcohol and keep the UV lamp on.
  8. Label the petri dishes and store in a refrigerator for later use.

Growth Inhibitors: The Effects of Adulterants on Milk Curdling

Adulteration of milk, as we have discussed, takes place through many ways, some of which can be intentional while others are non-intended. Growth inhibitors are very critical when it comes to milk contamination since they will impede the growth of culture bacteria needed for fermentation.

Common adulterants include water added to increase volume (baptizing the milk), preservatives added to improve the keeping quality of the milk (such as hydrogen peroxide, antibiotics, and sodium hydroxide). Sometimes, detergents may accidentally find their way into the milk and being bacteriostatic; they will inhibit bacterial activity in the milk and increase the keeping quality of the milk.

Antibiotics are used to treat mastitis and other common bacterial infections in lactating cows and the residue may find its way into the milk. They have a longer lasting effect on the milk. Antibiotics will kill the lactic acid bacteria (LABs) in milk; therefore, milk fails to curdle when you inoculate it with a starter culture.

An experiment to investigate the effect of inhibitory substances on milk curdling was done and the findings have been shared below.

The experiment

  • For this experiment, we partitioned five liters of heat-treated milk into five beakers and subjected to acidity and pH tests. We recorded the results we obtained from the tests.
  • After partitioning of the samples, we added adulterants including sodium hydroxide, antibiotic, detergent, and water into the beakers containing the milk samples.
  • We labelled the samples as A, B, C, D, and E in the order of the adulterants we added. We did not adulterate sample B with any substance; it was the control sample for the experiment.
  • Immediately after adding the adulterants, we determined the acidity and recorded the observations.
  • We then incubated the beakers containing the samples at 37ºC and measured the pH of the samples after every 15 minutes. We recorded all the observations.

The results

SamplesABCDE
Titratable Acidity (%)0.180.190.20.170.17
pH (on addition)6.46.376.333.216.51
pH (after 15 minutes)6.276.226.126.066.34
pH (after 30 minutes)6.436.356.356.176.76
pH (after 45 minutes)6.466.376.336.296.53

Discussion of the results

The acidity of the samples ranged fairly the same, indicating developing lactic acid in the sample. Addition of the adulterants seemed to have an effect on the acid development of the samples as the acidity of the adulterated sample seemed to stagnate showing no developed acidity.

The inhibitors affected the bacterial activity in the samples, impeding production of lactic acid in the milk samples during incubation. Some samples (B and E) did not show the downward trend in acid development observed in other samples.

The samples were left overnight and observed the following day. Only the control sample and samples B and E coagulated. Sample B formed a firm curd while E had curd with separated whey.

The conclusion

Sample B formed a firm curd because we did not add any adulterants into it. Its curd did not show any signs of syneresis. We adulterated sample E with water, which explains whey separation.

All the other samples contained inhibitory substances that inhibited bacterial activity in the milk and prevented acid development in the samples. As a result, we did not observe any curd formation in these samples on the following day.

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