Starter Culture: Types, Examples in Food Fermentation

Leave milk out and it may spoil, or it may turn into yogurt. The difference is which microbes take over. For thousands of years, fermented foods depended on whatever organisms happened to land in the food. A starter culture removes that luck: it is a known microbial preparation, added on purpose, so the fermentation goes the same way every time.

A starter culture is a preparation of one or more selected microorganisms added to a food to drive and control its fermentation. These microbes produce enzymes (such as proteases, peptidases, and lipases) and acids (mainly lactic acid) that change the food in predictable ways. Louis Pasteur's work in the 19th century first explained how these microbes drive fermentation, and in 1892 Christian Hansen sold the first commercial dairy starter. Since then, defined starters have been developed for nearly every fermented food and drink.

A starter culture does four things a chance fermentation cannot guarantee: it makes the product safe (by rapidly lowering pH), consistent (same result every batch), better tasting (controlled flavor compounds), and better preserved (acid and bacteriocins hold back spoilage organisms). This article covers how starter cultures are classified, which microbe ferments which food, and how a culture is chosen, prepared, and preserved.

Figure: Source: Slow food

Characteristics of Starter Culture

The ideal starting culture has the qualities listed below:

• Starter culture produces strong and consistent lactic acid.
• It quickly grows when supplied with the proper organic ingredients.
• It tolerates changing conditions such as shifts in temperature, salt, and pH.
• Viable during production and storage.
• It can use lactose and rapidly acidify the product.
• It possesses stable genetic traits.

Types of Starter Culture

Starter cultures are classified into various categories based on different requirements and characteristics like strain, state, temperature requirement, producing flavor, and end products produced.

Based on the strain

Starter cultures are categorized based on strain into the following groups:

1. Single-strain culture: One pure strain, such as Lactococcus lactis subsp. lactis. Its weakness is phage sensitivity: because there is only one strain, a single bacteriophage attack can wipe it out and the fermentation fails. Single strains are used where one defined activity is needed, and are often rotated or combined to reduce phage risk.
2. Multi-strain culture: Two or more defined strains combined on purpose, such as Lactococcus lactis with Lactococcus lactis subsp. diacetylactis. The classic example is yogurt, made by the fixed pairing of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, where the two strains help each other grow. Defined multi-strain cultures give predictable, repeatable results and are used for yogurt and many cheeses.
3. Mixed strain culture: It is a composite of more than two types of a strain belonging to unrelated species. The microbes used in this culture are Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and gas and aroma-producing mesophilic LAB (Lactic Acid Bacteria) such as Leuconostoc spp. Mixed starters are regarded as safe since they continue to function even if a phage attacks one strain due to their high phage specificity. A further benefit would be a greater tolerance for other conditions, such as variations in temperature and pH. Used for production of sauerkraut, white brine cheese.

Based on the state

Depending on the culture’s state, such as liquid, powder, or frozen, starter cultures are categorized into three types.

1. Liquid culture: Starting culture created in a liquid form is known as liquid culture. It is prepared using one of the two different liquid media, i.e., Skim milk/ litmus milk and Non-fat dry milk. Drawbacks- high risk of contamination, rapid strain degradation in mixed cultures, and viability is roughly for a week at 5°C.
2. Powder culture: These culture classes are produced by lyophilizing or spraying the liquid cultures to dry them off. Powder cultures can be kept for up to 6 months at 5°C and up to 1 year at -20°C and contain significantly more microbe cells per unit mass than liquid cultures.
3. Frozen culture: The culture is deep-frozen (typically -40°C or lower) to hold the cells inactive until use. Note that freezing is not the same as freeze-drying: frozen cultures stay wet and cold, while powder cultures are dried. A frozen culture keeps for a few months at -40°C and up to about a year in liquid nitrogen at -196°C.

Based on the growth temperature

The organisms can multiply their cells number at various temperatures. Based on temperature, the starter culture is divided into the following categories:

1. Mesophilic culture: Grows best at moderate temperatures, roughly 22–40°C with an optimum near 30°C. Includes Lactococcus species. Used for mesophilic cheeses (such as cheddar and gouda), cultured butter, and buttermilk.
2. Thermophilic culture: These cultures have a growth temperature range of 32–45°C, with 40°C being the ideal temperature for growth. This culture contains microbes such as Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.. It is suitable for products like yogurt, and mozzarella cheese.

Based on the flavor producer

The organism in the starter culture produces different types of flavors like diacetyl, acetaldehyde, and acetoin. On the basis of the formation of flavor, the starters are grouped into the following types.

1. L type: These consist of Leuconostoc spp. as a flavor producer (such as diacetyl). This type of species is typically employed as a starter culture for fermenting products like cheese and kimchi.
2. D type: D type contains microbes Lactococcus lactis subsp. diacetylactis strain as both an acid (lactic acid) and flavor producer (diacetyl or its precursor α-acetolactate). This type is mostly used in fermenting dairy products like cheese.
3. LD type: It is a mixture of both L and D-type organisms. It is used in the production of gouda cheese
4. O type: Contains no flavor (aroma) producer. These strains mainly produce acid, and some also produce bacteriocins that suppress spoilage and pathogenic bacteria. Example: Lactococcus lactis subsp. cremoris. Used where clean acidification without strong aroma is wanted, such as some cheeses.

Based on the End Product: Homofermentative and Heterofermentative

Lactic acid bacteria are grouped by what they make when they ferment glucose. This is one of the most important distinctions in food microbiology, because it decides whether a product is simply sour or also gassy and aromatic.

Homofermentative culture: Ferments glucose almost entirely to lactic acid, with little or no gas. Examples include Lactobacillus delbrueckii and Lactococcus lactis. Because they produce acid without gas, homofermenters are used where a clean sour taste and firm texture are wanted, such as yogurt, curd, and cheddar-type cheese.

Heterofermentative culture: Ferments glucose to a mixture of lactic acid, carbon dioxide, and ethanol or acetic acid. Examples include Leuconostoc mesenteroides and Lactobacillus brevis. The carbon dioxide and aroma compounds they make matter in products that need gas or a complex flavor, such as sauerkraut, kefir, and the eyes (holes) in some cheeses.

A quick way to hold the difference: homofermenters make one thing (lactic acid); heterofermenters make many (acid plus gas plus alcohol). Note: Bifidobacterium is sometimes grouped with heterofermenters, but it uses a distinct route called the bifid shunt (the fructose-6-phosphate pathway), which is why it is often treated as a separate category.

Starter Cultures in Common Fermented Foods

This table maps the fermented food to the organism that makes it and the conditions used. It answers the most practical question in the topic: which microbe ferments which food.

How to Choose a Starter Culture

Picking a starter culture is a series of decisions, not one choice. Each classification axis above answers one question:

1. What is the end product? Decide first whether you need only acid (homofermentative) or acid plus gas and aroma (heterofermentative). Yogurt needs clean acid; sauerkraut and kefir need gas and complexity.
2. What temperature will the fermentation run at? A hot process (yogurt, mozzarella, ~40–45°C) needs a thermophilic culture. A cool or room-temperature process (most cheeses, cultured butter) needs a mesophilic one.
3. How much flavor do you want? Choose L, D, LD, or O type by how much diacetyl and aroma the product should have. O type for clean acid, LD type for full buttery flavor.
4. How much phage protection do you need? For industrial-scale, continuous production, a mixed-strain culture survives phage attack better than a single strain. Single strains give precision but are fragile.
5. What form suits your storage and scale? Frozen and freeze-dried (powder) direct-vat cultures suit large industry; liquid cultures suit small or traditional operations but spoil within about a week.

Read top to bottom, these five questions take you from "I want to ferment this food" to a specific culture choice. That reasoning, not the definition, is what separates someone who knows the topic from someone who has only memorized it.

Preparation of Starter Culture

A fine quality can be achieved with the basic guidelines of preparing a starter culture. The nutrient requirement depends on the type of organism used. The nutritional medium often contains beet molasses or cane as a carbon source, ammonium salt, urea, malt sprouts as a nitrogen source, vitamin precursor, inorganic salts, and growth factors.

Temperature and pH may differ depending on the organism used. Unquestionably, the microorganisms of starter culture are chosen based on the product and its properties.

An overview of the preparation of starter culture is shown in the below flowchart:

![Preparation of starter culture

• Preparation of starter culture](</preparation-of-starter-culture-1.png)

Figure: Commercial preparation of starter culture Source: Ravindra P (2015)

Preservation of Starter Culture

Starter cultures are preserved in liquid, frozen or dried forms. The following approaches can be made to preserve the starter culture:

1. Sub-culturing: The starter culture can be preserved by periodic transfer of organisms in a sterile new media in 2- 3 months intervals. The primary issue with this method is contamination during cell transportation and mutation during cell multiplication.
2. Storage at reduced temperature: The cultures are inoculated on agar slopes and kept at -20°C coated with sterile medicinal-grade mineral oil or at -196°C in liquid nitrogen and re-suspended in cryo-protectant (10% glycerol) to prevent cell damage brought on by the development of ice crystals. These cells can be preserved for six months to 1 year.
3. Storage in the dehydrated form: The inoculated culture in sterile, moist soil is left to grow for a few days and then to dry at room temperature for about two weeks. This method has been widely used to store and preserve fungi and actinomycetes
4. Lyophilization: This method involves growing the culture to its maximal stationary phase. Cells are re-suspended using a protective media such as milk, serum, or sodium glutamate. An ampoule containing a small amount of the suspension is then put in a high vacuum and frozen until the sublimation is finished. Next, the ampoule is sealed. If maintained in a refrigerator, the cells in the ampoules survive for up to ten years.

Application of Starter Culture

Starter culture has several uses in various industries. Following are some of the applications of starter culture.

• Starter cultures enable us to manage the fermentation process and produce predictable results since they are tailored to the substrates.
• The use of starter culture as a probiotic is currently given equal priority.
• Starter cultures are used across food industries: dairy (cheese, yogurt, and other fermented dairy products) and meat (fermented sausages such as salami). In brewing and winemaking the primary fermenter is yeast (Saccharomyces cerevisiae), which serves the same starter role for those products.
• It aids in the improvement of flavor, texture, and lactose utilization.
• It can be used as a preservative agent (García-Díez & Saraiva, 2021).

How to Remember

Homo makes one, hetero makes many. Homofermentative = one product (lactic acid only). Heterofermentative = many products (acid + gas + alcohol). "Hetero" means different, so different products.

The flavor types spell what they carry. L = Leuconostoc (aroma only). D = diacetylactis (acid and aroma). LD = both. O = zero flavor. So L, D, LD, O runs from aroma-only to acid-only.

Temperature picks the culture: hot yogurt, cool cheese. Thermophilic cultures (near 40–45°C) make yogurt and mozzarella. Mesophilic cultures (near 30°C) make most cheeses and cultured butter. If the process is hot, think thermophilic Streptococcus thermophilus.

Yogurt is a duo, not a solo. Yogurt is always the pair Streptococcus thermophilus plus Lactobacillus bulgaricus. Two organisms that help each other grow. If an exam asks the yogurt organisms, name both.

Single strain, single point of failure. One strain means one phage can end the batch. Mixed strains survive because if a phage takes one, the others carry on.

Key exam facts

Where Students Get Confused

Freezing versus freeze-drying. These are different preservation methods. Frozen cultures stay wet and cold (-40°C or in liquid nitrogen). Freeze-dried (lyophilized) cultures are dried to a powder. Do not call a frozen culture "lyophilized."

Yogurt is not a single-strain product. Yogurt is the defined pair Streptococcus thermophilus and Lactobacillus bulgaricus. Naming only one is a common exam mistake.

Homofermentative does not mean "one organism." It means one main product (lactic acid). Heterofermentative means several products. The "homo/hetero" refers to the end products, not the number of microbes.

Starter cultures do not make beer and wine ferment. Beer and wine are fermented by yeast (Saccharomyces cerevisiae), not by lactic acid bacteria. LAB in beer or wine are usually spoilage organisms, not the intended starter.

The flavor letters are not random. L, D, LD, O describe which flavor pathway is present. O type has no aroma producer at all; it is not "the best" type, just the one used when clean acid is wanted.

A "culture" here is not a lab culture on a plate. In this topic, a starter culture is a food-grade microbial product added to food, not a diagnostic culture grown to identify a pathogen. Same word, different world.

Ringkasan dalam Bahasa Indonesia: kultur starter

Kultur starter adalah sediaan mikroorganisme terpilih (satu jenis atau lebih) yang sengaja ditambahkan ke dalam makanan untuk mengendalikan proses fermentasi. Mikroba ini menghasilkan enzim dan asam laktat yang membuat produk lebih aman, konsisten, tahan lama, dan beraroma khas.

Kultur starter dikelompokkan berdasarkan: jenis strain (tunggal, multi, campuran), bentuk (cair, bubuk, beku), suhu pertumbuhan (mesofilik ~30°C, termofilik ~40–45°C), penghasil rasa (tipe L, D, LD, O), dan hasil akhir (homofermentatif menghasilkan asam laktat saja; heterofermentatif menghasilkan asam laktat, karbon dioksida, dan alkohol).

Contoh: yogurt dibuat oleh Streptococcus thermophilus dan Lactobacillus bulgaricus; tempe oleh Rhizopus oligosporus; natto oleh Bacillus subtilis; kimchi dan sauerkraut oleh Leuconostoc dan Lactobacillus.

References

• Taskila, S. (2017). Industrial production of starter cultures. In Starter Cultures in Food Production, 79–100. https://doi.org/10.1002/9781118933794.ch5
• García-Díez, J. and Saraiva, C. (2021). Use of starter cultures in foods from animal origin to improve their safety. International Journal of Environmental Research and Public Health, 18(5), 2544. https://doi.org/10.3390/ijerph18052544
• Sulieman, A.M.E. (2018). Microbial Starter Cultures. (Reference text on starter culture types and preparation.)
• Ravindra, P. (2015). Advances in Bioprocess Technology. Springer.
• Adams, M.R. and Moss, M.O. (2016). Food Microbiology, 4th ed. Royal Society of Chemistry. (Fermented foods and lactic acid bacteria chapters.)