The natural microbial starters for innovation and authenticity

The natural microbial starters for innovation and authenticity

Fermented foods introduction

Fermentation can be defined as a fermentation process where different microbes, in anaerobic conditions, convert carbohydrates into alcohols or organic acids depending on the microbes involved (bacteria or yeasts). Microbes are naturally present in a substrate (autochthonous microbiota) but they also may be added microbes (starters).

There are seven main groups of fermented foods depending on the sources of substrates:

• Fermented milks and dairy products;
• Fermented cereals (sourdough baked goods);
• Fruit and fruit juices;
• Fermented vegetables;
• Fermented legumes and roots/tubers;
• Fermented meat and dried/smoked fish products;
• Beer, wine and other distillate beverages.

Fermented foods are widely spread in different parts of the world as traditional and typical fermented products but also as novel products. Each of these fermented foods is characterised by a specific microbiota depending on the substrate.

Nowadays fermented foods are very important because normally they are also included in different guidelines (Japanese, Indian, Mediterranean). In all these diets, we will find fermented foods and/or beverages because they’re very important, they have a beneficial role. The fermentation process is used to preserve foods, especially perishable materials but besides this, nowadays the fermentation may have different aims: to produce more digestible foods, to enrich products with desired microbial (GABA), to create new products but also to enhance dietary value and to develop characteristics and sensory properties (flavour, aroma, texture). Fermented foods, especially fermented beverages, are useful to introduce in our system probiotics.

Spontaneous fermentation

Spontaneous fermentation is a process guided by autochthonous microbes that are naturally present in a certain substrate. They can start this process if they’re under optimal conditions: temperature, salt concentration, pH, and O₂. There are three different types of microbes responsible for food fermentation:

• Bacteria: LAB, fructophilic lactic acid bacteria, acetic acid bacteria [Kombucha];
• Yeasts: different genera such as Saccharomyces, Brettanomyces, Pichia, Candida;
• Moulds: they’re a source of enzymes or they might be used for the degradation of antinutritional factors (e.g., genera Mucor, Neurospora).

Drivers affecting the spontaneous process of fermentation

The time and temperature of the fermentation process are the two main parameters to consider. The range of optimal temperature for LAB:

• Mesophilic bacteria: 25°C;
• Thermophilic bacteria: 30-35°C.

The parameter strictly related to the temperature is the time, we can play with the time: higher temperatures require shorter fermentation time. If the temperature is lower (e.g., 20°C), the time of fermentation increases. Other factors/parameters that can affect the fermentation are:

• Nutrients: The presence of fermentable carbohydrates (especially for LAB) and other nutrients such as amino acids. The concentration is important as well;
• Inhibitory/antimicrobial compounds: Presence of such as essential oils, phenolic compounds as well;
• pH: The pH is another driver affecting the fermentation, usually the fruit is acidic (except for bananas, coconuts…);
• Endogenous microbiota: The first driver is the endogenous microbiota (we’re speaking about spontaneous fermentation), if our target is to obtain a good product, it is really important the presence of proper microbes.

Sauerkraut

It’s a fermented food produced by spontaneous fermentation. It’s made with two main ingredients: cabbage (mainly green but also red) and salt.

In food, we may have three categories of microbes: spoilage, pathogens, and beneficial microbes. The addition of salt is important to obtain different fermented foods because it reduces the water activity (Aw). In this way, we can select the microorganisms. The water present in food can be divided into two fractions: free and bound. The water activity (Aw) is the partial vapour pressure of water in a substance divided by the standard state partial vapour pressure of water.

Aw = P/P₀. It ranges from 0 to 1. Most of the pathogens do not tolerate low value of Aw. Adding salt reduces the Aw so that the growth of most pathogens and spoiling micro-organisms will be inhibited. Salt acts as a preservative. Salt causes the cabbage cells to release fermentable sugars (plant juice) in the environment.

Another important factor is the presence or absence of O₂. During the spontaneous fermentation process, there’s a succession of different microbes. In fruit and vegetables, the presence of LAB is 10²-10³ but not more than 10⁴ for sure, so the cell density is very low meanwhile the cell density of spoiling bacteria and yeast is higher: 10⁵-10⁶.

At the beginning, the oxygen is present but after a few hours, the environment changes: there’s a decrease in oxygen. At this point, the spoiling bacteria decrease and at the same time, there is an increase of LAB. The endogenous microbiota can be divided into:

• Epiphytic: living on the surface of the vegetable - aerobic microbes;
• Endophytic: living inside the vegetable, within the plant tissues - anaerobic microbes.

LAB (endophytic) include different genera such as: Leuconostoc, which is a heterofermentative bacterium, and spoilage microbes:

1. Under normal conditions in fruit and vegetables, the concentration of spoilage bacteria is higher than that of LAB.
2. At the beginning of the fermentative process, there is an increase in Leuconostoc because the conditions of the environment (O₂, high salt concentration - it’s the only LAB genus that can tolerate high amounts of salt) promote its growth. At the same time, there’s a decrease in spoilage microbes because of the high concentration of salt. The same evolution is present in Kimchi and Pickles.
3. At this point, there is a shift in terms of microbes, but there are other changes: Leuconostoc, being a heterofermentative bacteria, produces as metabolites lactic acid and CO₂. CO₂ replaces the O₂ present and the pH begins to decrease. LAB are able to tolerate the presence of oxygen (microaerophilic) because they have an enzyme (pseudocatalase/superoxide dismutase) able to detoxify the ROS (reactive oxygen species).
4. During time, there are other modifications of the environment: Leuconostoc increases till 10⁸ and then begins to decrease but at the same time there’s the appearance of other genera of bacteria and in particular we may observe the presence of L. Brevis, Pediococcus meanwhile at the end of the process the environment is completely dominated by L. Plantarum.

Why does all of this occur?

Leuconostoc is the only genus of LAB which can tolerate high concentrations of salts (so it develops at the beginning), but it does not tolerate low pH values. L. Plantarum is the best acid-tolerant bacteria, so it will develop at the end of the fermentation process. The salt concentration changes because there are certain modifications in terms of material: there’s a release of carbohydrates and plant juices, so there’s a dilution of the salt content.

There are two methods to study (isolate, quantify, characterise) the microbes present in a substrate:

• Culture dependent methods;
• Culture independent methods: 16S rRNA sequencing.

Study of the microbial community

In the first sample (Day 0) we notice the autochthonous microbiota. Other factors affecting the fermentation process, besides pH, temperature, etc. may be: ingredients and the house microbiome. After 14 days of fermentation, the two main genera present are Lactobacillus and Leuconostoc. For sure there’s also a contribution from the environment, the house microbiota contributes to this kind of contamination. We should always consider the relationship between time and temperature.

In the first case, the traditional process requires a temperature of 18°C for 1 month. In the second case, the warm-temperature processing, the temperature used is higher so that the process will last fewer days. By comparing the microbiota, they observed a similar composition.

How to study the house microbiota

The technique adopted to collect the samples was the swab sampling technique. Each environment of the company was monitored in order to study the house microbiota. Microbiomes within sauerkraut production facilities were profiled and characterised to determine the sources of these bacteria. 32 swab samples were collected representing all the different rooms of the company.

Investigating the microbiome should combine traditional methods (culture-dependent) and culture-independent methods. It’s better to combine the two methods to have more accurate results and an overview of the microbiome but also cultivate microbes, especially LAB. The cultivation is important especially when we would like to select and propose starters. At the same time, pyrosequencing methods can give us an overview of all the microbes present. Culture-independent methods may have some limits, particularly for the subdominant microbes. Normally there’s a kit to indicate the presence of certain microbes. In the study, it was considered also the microbiome: sequencing the genes present in the environment. In this way, we may know also the functionality (metabolic potentiality of the microbiome) of the ecosystem.

Different techniques

• Through culture-dependent methods we can isolate different microbes using specific substrates. We can also have info about the subdominant species present.
• Cultural-independent methods to extract the total DNA, such as:
  • Metagenetics: provides information just at the taxonomic level, knowing the profile of all the microbes present in the ecosystem.
  • Metagenomics: through this technique we know the taxonomic information and all the genes present in our ecosystem. Once we have this information about the genes, through a database we can reconstruct all the potential metabolic pathways. Potential and not real, why? Potentially all the genes present can be identified so we can reconstruct all the pathways (carbohydrates, aa catalysis etc.) but we don’t know if microbes present in our ecosystem are using that specific pathway, they can potentially use it but we don’t know if that pathway is active so we have to check the expression of genes through transcriptomics. Thanks to transcriptomics we know which pathways are active. Through the list of genes, we may reconstruct the metabolic pathway and at the same time study the assembling of the autochthonous microbiome. If the sequencing results are good we can also annotate the genomes of all the microbes present.

Taxonomic levels: Domains, Kingdoms (Bacteria), Fila (firmicutes), Classes (bacilli), Orders (lactobacillales), Families (lactobacillaceae), Genera, and Species. Through metagenetics, we can get to the genera level, sometimes at the species level but generally at the genera one, meanwhile, through metagenomics we can get to the species level (thanks to the genome reconstruction). Through culturing, we may have info about the strain level as well. Sampling (swab), monitoring, extraction of DNA, after the amplification of the total DNA for each room of the company environment, we obtain a list of the sequences.

At this point, we can see that raw vegetables, cabbage in this case, and handling surfaces are rich in Proteobacteria (spoiling bacteria). The heat map is a map of different colours from a low abundance (clear colour) to the highest abundance (red colour) of microbes found in these environments. In the assessing room, the Proteobacteria number decreases and there’s an increase of Actinobacteria. In all the other rooms (fermentation room, vessels) and in sauerkraut, there’s an increase of Firmicutes. The map represents the evolution of microbes: at the beginning in the raw cabbage and on the handling surface there’s a high number of Proteobacteria which decreases over time but during the fermentation process, there’s an increase of Firmicutes. We can say that there’s also a contamination of LAB in the environment.

Cabbage and vegetable handling surfaces exhibit more similar microbiomes relative to the fermentation room, processing area, and dry storage surfaces. Leuconostoc and Lactobacillaceae dominated all surfaces where spontaneous fermentation occurs, as these taxa are associated with the process.

The same process was repeated at the laboratory level, the process lasted for 42 days. They used a culturing approach (culture dependent method) to isolate LAB and for the first 14 days just Leuconostoc was isolated, different species. After 14 days, Pediococcus appeared, and after 42 days, 9 different strains of L. plantarum were isolated and identified. Also, at the laboratory level, the same evolution was observed. Generally, Leuconostoc starts decreasing when the pH is more or less 4.6 (after 5-6 days).

Summary

Depending on the substrate, there’s a very specific microbial succession during the fermentation process. In the case of sauerkraut, in the first stage of fermentation, the environment is dominated by Leuconostoc. During the process, there’s an increase in other species of LAB but at the end of the process, the environment is dominated by L. plantarum because it is one of the LAB most resistant to very low pH values.

We can monitor the evolution of LAB by culture-dependent approaches (culturomics) but also by new culture-independent techniques. Through these analyses, we can say if there’s a good evolution of autochthonous microbes, but we can also check other parameters: cell density of LAB, to see if there is an increase in cell number by plate count, isolation, and identification of the LAB, but we always have to measure the pH value.

This is not enough, if the pH at the beginning is very low (e.g., pH 4.4), it is difficult to see variation of the pH because the environment is already very acid, for this reason, we also need to check the TTA (total titratable acidity) and this provides us info about the amount of organic acids present in the environment. During the fermentation, this parameter (TTA) can increase because it reflects the acetic acid and lactic acid production by LAB. Temperature in some cases could be checked: generally increases during time. From a metabolic point of view, the concentration of carbohydrates could be monitored: because of homo or heterolactic fermentation, the main sources of carbohydrates (glucose, fructose, sucrose) should decrease over time, so through chromatographic analysis we should be able to measure the content and the trend of carbohydrates over time. If we measure the decrease of carbohydrates at the same time we are able to measure the increase in the concentration of organic acids.

Parameters to check: pH, TTA (acidity), T (in some cases depending on the substrates), concentration of glucose and organic acids. During time there’s a decrease of the pH (from 6 to 4), decrease of carbohydrates, and increase of organic acids (lactic acid). All these parameters give us an indication regarding the fermentation evolution.

In order to understand which is the contribution of each microbe that we identified, we can also check other parameters, to go more in depth. In sauerkraut, during time, the decrease of fructose was noticed. With glucose and fructose decreased. Fructose can be used by LAB as a carbon source but also as an external electron acceptor. In the graph, we can see that there’s at the same time a decrease of fructose and an increase of mannitol.

During the heterolactic fermentation, there might be the production of ethanol or acetic acid. In presence of fructose as an external acceptor of electrons, the pathway that leads to the production of acetic acid is promoted. Leuconostoc was able to carry out such type of pathway in this case. Of course, we noticed an increase in acetic acid.

Every time we have to characterise a food substrate, we need to check: the cell density of LAB, pH, TTA, T (in some cases), concentration of carbohydrates and organic acids, and also other metabolites according to the aim of our studies.

Just a curiosity, not in the exam: during the spontaneous fermentation process as a consequence of the decrease of the pH there’s a modification of the internal redox state (it increases) which has a preservative effect on vitamin C but also on other antioxidant compounds. Generally, at the end of the process, it is useful to characterise the fermented foods by the total antioxidant activity, which normally increases during time throughout the fermentation process because there’s for sure an effect on the acidic condition.

There’s also an increase in bioavailability of antioxidant compounds of which the most important are phenolic compounds. Normally these compounds are present as bounded compounds, they’re not free but thanks to the acidification, most of them become free phenolic compounds, so we may notice an increase in the total phenolic compounds. Also, LAB with their enzymes may have an effect on phenolic compounds. Through a statistical approach they verified the abundance of LAB that they had identified during time and the concentration of all identified phenolic compounds. After this statistical analysis,