The natural microbial starters for innovation and authenticity

STARTER CULTURES: LAB AND DAIRY PRODUCTS

The three main starters for making dairy products are: primary starters, secondary starters and adjunct starters.

Primary starters

Primary starters are selected lactic acid bacteria, which are deliberately added to the milk and they significantly contribute to the curd microbial biomass (in 6h >10 cfu g /1 day).

Their main role is the acidification so they ensure the decrease of the milk pH from 6.6 to 5.2-5.3 in about 6h and normally this acidification occurs at specific T (30-37°C).

Since their main role is the acidification, their selection should consider the carbohydrates metabolism.

Homofermentative LAB are selected as primary starters since we know that from 1 mole of lactose they're able to produce 4 moles of lactate [starting from lactose and not directly glucose] so they acidify faster the substrate compared to heterofermentative LAB which produce just 1 mole of lactate from 1 mole of lactose during the same time of fermentation.

Moreover, homofermentative compared to heterofermentative do not produce CO and acetic acid. The use of homofermentative LAB as primary starters in dairy products is important for acidification, aroma formation, and structure (rheology) of the final product due to the absence of CO2 and acetic acid.

In 6h from milk, there's the production of the curd. Among the primary starters, we can find:

• Lactococcus lactis
• Streptococcus thermophilus
• Lactobacillus delbrueckii subsp. lactis e subsp. bulgaricus
• Lactobacillus helveticus
• Leuconostoc sp.

The first and the last one are mesophilic primary starters, meanwhile the other ones are thermophilic primary starters. This definition does not come from the classification of LAB, but this definition was made based on the application point of view. Based on the optimal temperature of growth, mesophilic LAB grow at 30°C while thermophilic LAB grow at 37°C. But in this case, the definition is not related to their optimal temperature of growth.

From the applicative point of view,

Point of view: Mesophilic cultures are the ones producing lactate at an optimal level of T below 40°C, while thermophilic LAB produce lactate at higher temperatures (48-52°C). Furthermore, primary starters can be classified as:

1. Mesophilic starters (< 40°C).
2. Thermophilic starters (> 40°C).
3. Natural starters or artisanal starters like sieroinnesto or scotta-innesto; the microbial composition is not well defined and they can contain an undefined mixture of strains.
4. Complex starters are commercial primary starters, also with an undefined microbial composition.
5. Defined starters composed of a small number of selected strains, allowing greater control over the composition and properties of cultures.

Criteria for the selection of primary starters:

1. Carbohydrates metabolism: The main role for primary starters is to ensure a fast acidification of milk. See table below for information about the carbohydrates metabolism and focus on the production of lactate:

- Lactococcus lactis,

Mesophilic primary starter is an homofermentative LAB since 4 moles of lactate are produced starting from lactose (not glucose in this case).

Leuconostoc species, based on the production of lactate, we can affirm that is an obligate heterofermentative LAB since it produces moles of lactate + ethanol and CO2.

St. thermophilus e L. Delbrueckii produce 2 moles of lactic acid but in this case although they are homofermentative they do not have one enzyme so from the hydrolysis of lactose they're just able to metabolise the glucose and not galactose. For this reason they just produce 2 moles of lactate.

Based on this metabolic capability of primary starters we can select them. L. lactis and L. helveticus are primary starters with an high capability of producing lactate. The carbohydrates metabolism is the main criteria to consider for the selection but there are also other criteria.

2. Another criteria is the N metabolism. During cheese ripening there are 3 main chemical events characterising

proteolysis: primary proteolysis, secondary proteolysis and the catabolism of free aa.

Regarding the secondary proteolysis we know that during cheese ripening the secondary proteolysis is related especially to the activity of an high number of peptidases.

Different components characterise the proteolytic system including proteases which are located on the cell wall of LAB, then there are transporters of oligopeptides and inside the cell, into the cytoplasm there's a large number of peptidases.

Regarding the secondary proteolysis and the criterium for the selection of primary starters we can consider these activities when we select strains and primary starters. For sure the secondary proteolysis is carried out especially by NSLAB but primary starters can contribute as well.

Presence of lipase and esterase; we can say that these enzymatic activities in primary starters are marginal compared to carbohydrates metabolism and N metabolism since primary starters are not considered as lipolytic microbes.

cells are needed intact for the acidification of the substrate. Autolysis refers to the capability of primary starters of autolysis during cheese ripening: important for the release of peptidase.

Resistance to bacteriophages; infection of bacteriophages, especially in dairy processes, is a frequent event due to the environmental conditions: the persistence of whey, richness of the substrate, liquid matrix and so on. All these conditions promote the persistence of bacteriophages, so this criterium is also important for the selection of primary starters because they have to be resistant to infections. Another option is to rotate the primary starters. The drawback in this case is the not good standardisation of rheological and sensory properties of cheeses.

Synthesis of exopolysaccharides (EPS) is important especially in some cases for making low fat cheeses for instance.

Synthesis of bacteriocins; the capability to produce them can be an advantage for the cells although this phenomenon is

observed in vitro assays, in lab conditions, but there’re not somany studies showing that primary starters are able to produce bacteriocins during thecheese making process. Probably this is due to the conditions of the matrix (curd): complexsubstrate, rich in peptides, in real conditions they cannot produce them. The capability ofsynthesise the bacteriocins can be verified at lab level but then there’re problems in finding invivo the presence of bacteriocins.

Secondary starters

Microorganisms deliberately added to milk or favoured under particular environmentalconditions, are called secondary because they're only involved during the ripening phase of thecheese.

The source of secondary starters may be: starter, milk, processing tools, house microbiota.

Among secondary starters we can consider:

• Yeasts important for cheese like Brie, Camembert and Taleggio cheeses.
• Moulds such as Penicillium roqueforti (inside the cheese) and P. camamberti (on the surfaceof the cheese:

(Camembert, Roquefort, Gorgonzola cheeses).

Corineform bacteria - Artharobacter, Brevibacterium, Corynebacterium and Micro-bacterium(Limburger cheese or Taleggio cheese).

Propionic bacteria - Propionibacterium freudenreichii, P. jensenii and P. thoenii (Swisscheeses).

Mould selections

Visual appearance;

• De-acidifying activity; moulds breathe and as energy source they can use carbohydrates and alcohols but also organic acids like lactate. If they metabolise OA such as lactate, this one is transformed in CO + water. In the specific area where the mould is present, after this activity there will be less lactate and consequently there’s a de-acidification process. This process is important for some cheeses since the de-acidification activity promotes the further colonisation from acid sensitive bacteria which normally do not develop in such environment in presence of lactate. They can colonise cheeses just after the de-acidification process carried out.

adjuncts starters- Environmental adaptation: hostile conditions of cheese during the cheese ripening- Growth capacity during the cheese ripening- High peptidase activity

Normally these adjunct starters are added to increase the amount of the bacteria especially at the beginning of the process. The practice of isolating these adjunct starters from milk or a previous cheese making process, their cultivation at lab level and after the characterisation they can be added as adjunct starters. These process is very consolidated in diary process.

Evolution of primary starters (white) and NSLAB (black) during the cheese ripening step.

Primary starters show a high number after the first days of the cheese ripening process (10) meanwhile during the ripening process there's a decrease of their number (10), of course depending on the cheese and