Sourdough basics


Sourdough basics

From a biochemical point of view, fermentation is a metabolic process of deriving energy from organic compounds without the involvement of an exogenous oxidizing agent. Fermentation plays different roles in food processing. Major roles considered are:

Italian sourdoughs

The lactic acid bacteria and yeast microbiota of eighteen sourdoughs used for the manufacture of some traditional Italian sweet leavened baked goods were studied through culture-dependent method and pyro-sequencing. Flours used for back slopping and sourdoughs were also biochemically characterized. Principal component analysis was applied to explore eventual correlations between process parameters applied during back slopping, some flour nutrients, profile of microbiota, and biochemical characteristics of sourdoughs. The median values of the cell density of lactic acid bacteria and yeasts were 8.05 and 7.03 log CFU/g, respectively. As shown by culture-dependent method, Lactobacillus sanfranciscensis was identified in all the sourdoughs, except for Panaredda and Torcolo di San Costanzo. For eleven sourdoughs, all the lactic acid bacteria isolates were allotted to this species. For Buccellato di Lucca, Mbriagotto, Pandoro, and Nadalin sourdoughs, at least 80% of the isolates were allotted to this species. Other lactic acid bacteria isolated with a relatively high frequency were Lactobacillus plantarum and Leuconostoc citreum. Pyro-sequencing confirmed and complemented the culture-dependent approach, detecting L. sanfranciscensis also in Panaredda and Torcolo di San Costanzo sourdoughs. Saccharomyces cerevisiae was identified in all the sourdoughs, except for Mbriagotto, Ciambella di Mosto and Pandolce Genovese. These latter sourdoughs harboured strains of Candida humilis, whereas five sourdoughs combined the presence of both yeast species. Positive correlations were found between time of back slopping and cell density and main metabolites of lactic acid bacteria. Percentage of sourdough used as inoculum was mainly correlated with the cell density of yeasts and the concentration of ethanol.

This study provided a comprehensive and comparative approach to highlight the dominant microbiota of Italian sourdoughs, which could be exploited further to guarantee a highly reproducible quality of the Italian sweet goods studied, while preserving their traditional trait.

Sourdoughs from buckwheat and teff

In this study the suitability of commercial starters for the production of gluten free sourdoughs was investigated. For this purpose, four different laboratory scale sourdoughs were developed from the flours buckwheat or teff. Two different starters (SA, SB) were used to start the fermentations, which were carried out using two technological conditions. Back slopping propagated sourdoughs until stability was reached. The composition of the stable sourdoughs was investigated by culture dependent techniques and the development of the dominant biota was monitored by PCR-DGGE. Unique and complex LAB and yeasts communities were detected in each sourdough, comprising strains, which originated from the flours. The competitiveness of the starter LAB varied according to the substrate and the fermentation conditions applied. Among the LAB present in both SA and SB, L. helveticus and L. paracasei strains did not persist in buckwheat or teff sourdoughs. Lc. argentinum was competitive only in buckwheat sourdoughs, whereas L. reuteri persisted only in teff sourdough. L. fermentum and L. helveticus present in both starters dominated only the sourdoughs fermented at the higher temperature. Remarkably, the starter yeasts were out-competed by spontaneous yeast strains, i.e. Kazachstania barnetti and Saccharomyces cerevisiae in teff sourdoughs, whereas no yeasts were isolated from buckwheat sourdoughs. The isolation of autochthonous LAB and yeasts from the stable teff and buckwheat sourdoughs indicates that both flours represent an important reservoir for the isolation of novel and competitive starters for the production of gluten free sourdough bread.

Traditional Turkish fermented non-alcoholic beverages

I mention these products, although they have nothing to do with sourdough as such, because they can be used as a started for sourdoughs based on cereals. Shalgam juice, hardaliye, boza, ayran (yoghurt drink) and kefir are the most known traditional Turkish fermented non-alcoholic beverages. The first three are obtained from vegetables, fruits and cereals, and the last two ones are made of milk. Shalgam juice, hardaliye and ayran are produced by lactic acid fermentation. Their microbiota is mainly composed of lactic acid bacteria (LAB). Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus paracasei subsp. paracasei in shalgam fermentation and L. paracasei subsp. paracasei and Lactobacillus casei subsp. pseudoplantarum in hardaliye fermentation are predominant. Ayran is traditionally prepared by mixing yoghurt with water and salt. Yoghurt starter cultures are used in industrial ayran production. On the other hand, both alcohol and lactic acid fermentation occur in boza and kefir. Boza is prepared by using a mixture of maize, wheat and rice or their flours and water. Generally previously produced boza or sourdough/yoghurt are used as starter culture, which is rich in Lactobacillus spp. and yeasts. Kefir is prepared by inoculation of raw milk with kefir grains, which consists of different species of yeasts, LAB, acetic acid bacteria in a protein and polysaccharide matrix. The microbiota of boza and kefir is affected from raw materials, the origin and the production methods.

Classification of sourdoughs

Sourdough bread making is an ancient biotechnological process and various protocols for it use are applied in many countries. On the basis of the technology applied, sourdoughs can be grouped into 4 types.

Type I sourdough

Type I or traditional sourdoughs are manufactured by continuous daily back slopping at ambient temperature (30C), to keep the microorganisms in an active state. Therefore mother doughs are used as an inoculum for subsequent doughs by addition of he desired amount of dough to a fresh flour-water batch according to defined cycles of preparation. These small-scale sourdough productions are used in traditional home-made sourdough and frequently harbour Lb. sanfranciscensis, C. humilis and K. exigua as prevalent LAB and yeast species.

Traditional sourdoughs whose microorganisms are kept metabolically active through daily refreshments are included in this group. These sourdoughs are generally suitable for achieving dough leavening without the addition of yeast. Generally a 3-stage protocol is applied relying on 3 refreshments over 24 hours in order to obtain the leavened dough to bake. Each step is characterised by a given dough yield as well as technological conditions of time and temperature. At the end of the last step of fermentation the sourdough is used as the leavening agent. It can thus be considered as a natural starter culture containing many microbial strains. Normally following dominating strains are found: L. sanfranciscensis that can co-exist with other obligate heterofermentative lactic bacteria such as L. pontis, L. brevis, L. fermentum, L. fructivorans and yeasts such as Candida milleri. C. holmii, S. cerevisiae, S. exiguus (new name Kazachstania exigua). Candida milleri is a species of the genus Candida. It is present, along with the bacterium Lactobacillus sanfranciscensis, in the production of sourdough bread.

The French system of making sourdough produces type I sourdoughs. The mother sponge preparation begins with a quite firm wheat flour dough (dough yield of 150 – 152: this means 100 kg of flour + 50 to 52 kg of water). At this stage some salt and malt can be added. This dough undergoes a first fermentation step lasting about 24 hours. This corresponds to the early fermentative activity of flour-resident yeast and lactic acid bacteria, which results in a low CO2 and organic acid production. The decrease in pH induces the activity of flour endogenous proteases, which, together with the bacterial hydrolytic enzymes, act on the gluten, and leads to a lower dough firmness.

The second step begins with the first refreshment, which is aimed at introducing oxygen and new fermentable carbohydrates into the mixture to stimulate microbial growth and activity. The refreshment is obtained by adding a quantity of flour and water to bring the dough yield down to 148.

Example: starting mother sponge: 100 kg flour + 50 kg of water (DY = 150). After the first refreshment the dough yield must become 148 i.e. 200 kg of flour + 96 kg of water. So to the original mother sponge one has to add 100 kg of flour and 46 kg of water.

This dough ferments quickly and represents the starting dough for the next refreshment. By applying such a procedure a sourdough with a steady fermentative and leavening capability is obtained. During the last step, each refreshment is carried out at a regular interval of time (e.g. 7 to 8 hours) with the aim to maintain equilibrium in the ratio between microbial communities.

According to prof. Calvel, when the dough volume increases by 3 or 4 fold with respect to the initial dough a new refreshment should be performed. The mother sponge, which is obtained following the above procedure, represents the dough used to prepare the full sour needed to leaven the bread dough.

Type II sourdough

Type II or industrial sourdoughs are produced through one-step propagation processes of long duration (typically 2 - 5 days) at a fermentation temperature above 30C and with high water content. These large-scale sourdough productions result in semi-fluid preparations, which are used as dough acidifiers or flavour ingredients. Lb. amylovorus, Lb. fermentum, Lb. pontis and Lb. reuteri are commonly found in type II wheat or rye sourdoughs.

Sourdoughs obtained through a unique fermentation step of 15 - 20 hours followed by storage for many days belong to this group. Type II sourdoughs are generally not suitable for achieving dough leavening but are used for dough acidification an as dough improvers. These sourdoughs are generally liquid (DY ≈ 200). They are produced at industrial level using bioreactors or tanks at a controlled temperature that exceeds 30C. Such a protocol aims at shortening the fermentation process. During storage a portion of the mature sourdough can be used as the inoculum with the aim of acidifying the dough and enriching it with aroma and flavour compounds, which are characteristic of sourdough bread.

On the basis of the high DY, the long fermentation time and temperature of fermentation, lactic acid bacteria such as L. panis, L. reuteri and L. pontis which are resistant to low pH, dominate these sourdoughs. Spontaneous flour yeasts are inhibited and consequently, the leavening of the final dough is obtained by adding commercial yeast.

Notes

Type III sourdoughs

Type II sourdoughs are liquid. After drying and stabilising, they are named type III sourdoughs. They are mainly used at the industrial level as their quality is more constant compared to type I and II sourdough, and they are simpler to manage.

On the basis of the preparation method (drum drying or spray drying), drying-resistant lactic acid bacteria such as Pediococcus pentosaceus, L. plantarum and L. brevis can dominate type III sourdoughs.

Type III sourdoughs are prepared in dried form to be used as non-living acidifier supplement and flavour carriers for (sourdough) bread production. In contrast to type I sourdoughs, doughs of type II and III require the addition of baker's yeast for leavening.

Commercially available bulk starter cultures to prepare type II and type III sourdoughs aim at standardising the end products through acidification of and flavour formation in the dough. New trends tend to develop starter cultures that lead to improved functional properties other than acidification and flavour formation, such as texture improvement, antibacterial and anti-fungal activities and health promoting effects.

Sponge and dough

This method is sometimes considered the 4th type of sourdough although the aim of this protocol is to acclimatising the bakers’ yeast and improving swelling of the flour components. As a result one obtains a better loaf volume, taste and flavour of the bread as well as an improvement in he shelf life of the bread.

It can be considered as an intermediate procedure between straight dough and sourdough. Sponge dough is obtained in two steps: in the first one (pre-dough) the yeast is mixed with a part of the flour and water of the recipe, while the second dough is obtained by adding the rest of the flour, water and possibly other ingredients. Depending on the type of bread, the length of the pre-dough fermentation can vary from 3 to 20 hours and as a consequence various percentages of flour and yeast, besides different combinations of DY and temperature can be applied in this step.

As in the case of longer fermentations, lactic acid present as contaminants from either baker’s yeast or flour, grow in the dough reaching typically more than 108 cfu/g and contribute to the overall quality of baked goods. Therefore the sponge and dough method can be included in the category of sourdough.

Stability of sourdough

In order to keep microorganisms active, type I sourdoughs are generally propagated daily. So problems of microbiota stability during propagation may arise.

In general, it is recognised that in a sourdough ready to be used for bread the following basic characteristics occur:

  1. The prevalence of a microbial community different from that of the flour used as raw material.
  2. A stable ratio between lactic acid bacteria and yeasts (in the order of 100:1)
  3. A predominance of obligate heterofermentative lactic acid bacteria over other lactic acid bacteria (although for certain types of sourdough it is preferred to have a predominance of homofermentative lactic acid bacteria. Italian type of sourdough cannot be as acid as German sourdoughs where the predominance of acetic acid is preferred)

As a stable microbiota during sourdough propagation and use is essential in order to obtain standard and repeatable final products, the problem of microbial stability in terms of species and strain composition in type I sourdough propagated by applying different endogenous (e.g. type of flour, quantity of water) and exogenous (e.g. temperature, time of fermentation) parameters should be carefully addressed both in terms of identification and typing of dominant and sub-dominant microorganisms. Equally important is the selection of robust, well-adapting and competitive starter strains.

The robustness of sourdough lactobacilli varies depending on the species and on the strains. While the majority of L. sanfranciscensis strains showed quite a low robustness during daily back-slopping performed at laboratory level, selected strains of L. plantarium seemed to share several phenotypic traits that determined the capacity to out-compete the contaminating lactic acid bacterium biota.

Methods to evaluate the performance of sourdough

Both microbiological and physic-chemical parameters are used to evaluate the performance of a sourdough.

The microbiological aspect essentially deals with the assessment of the community of lactic acid bacteria and yeasts, as those microorganisms are dominant in a good quality sourdough and are generally present at a ratio of approximately 100:1.

Nevertheless, an array of both phenotypic and genotypic methods is necessary to identify the species/strain composition of the dominant and sub-dominant microbiota of the sourdough.

The standard plate count is used to estimate the cell density of both lactic acid bacteria and yeasts. Both lactic acid bacteria and yeasts can be identified and monitored during sourdough fermentation by culture-independent systems. A similar approach was applied by Meroth et all to evaluate the effect of type I and type II sourdough fermentation on microbial population dynamics.

The balance between the communities of lactic acid bacteria and yeasts and the composition of the microbial communities in terms of strains and species markedly influences the sourdough performances and the overall quality of related leavened baked goods. The microbial community is influenced by some sourdough characteristics and, in turn, it modifies the chemical composition and physical parameters of the sourdough, which is reflected in the overall quality of the products.

An overview of the physico-chemical characteristics of the sourdough, which are related to its performance, is determined through the evaluation of the following parameters

Dough yield: the ratio between water and flour in the dough is indicated as dough yield and it deals with the dough consistency. Considering that different flours have different capabilities to absorb water, doughs of various consistencies are obtained having the same DY. The DY is calculated as follows:

(flour weight + water weight) x 100/flour weight.
Example: a mix of 60 kg of flour + 50 kg of water has a dough yield of (60 + 50) x 100/60 = 183,33
In other words, for 100 kg of flour, 83,33 kg of water has been used.

Overall, a firm wheat flour sourdough has a DY of about 150 – 160. A liquid sourdough shows values close to 200. Intermediate values indicate soft dough. It has been observed that both DY and temperature of fermentation markedly influence the aroma of the sourdough, and especially, the molar ratio between lactic acid and acetic acid (FQ). Overall more acetic acid is present in firm dough fermented at 25 – 30°C, while lactic acid is found in soft dough fermented at 35 – 37°C. Obviously, on the basis of microbial adaptation to the various environmental factors, the combination of DY and temperature during refreshments markedly influences sourdough microbiota and its performance.

Acetic acid plays an important role by influencing many bread properties (flavour, rope inhibition, shelf life), and its content in sourdough can be increased by fructose addition or by aeration in the presence of heterofermentative lactobacilli. In many cases, the effect of temperature on the acetic acid content of type I sourdoughs (generally propagated at a temperature of about 25C), can strictly depend on the yeast activity: at low temperature, yeasts grow and hydrolyse kestose and other fructo-oligosaccharides, with the fructose being available as electron acceptors for a higher production of acetic acid by L. sanfranciscensis via the acetate-kinase pathway.

At a high temperature (more than 32C), growth of yeasts is inhibited, fructo-oligosaccharides remain unhydrolysed, and L. sanfranciscensis has less fructose available for acetate production.

The final pH, which ranges from 3,5 to 4,3, is usually considered as an index of well-developed sourdough fermentation. The pH of the sourdough influences the values of the pH of the final dough and bread, depending on the amount of full sour that is used as the inoculum. In the case of a standard inoculum of 20 % (referred to dough weight), values of pH that range from 4,7 to 5,4 are usually found in the final dough.

The value of TTA is a measure of the total organic acids synthesised during sourdough fermentation. The TTA is expressed as ml of NaOH 0,1 N per 10 g of dough. The values of TTA range from 30 to 150 ml for liquid sourdoughs and to 40 – 220 ml for dried sourdoughs. Nevertheless, the optimal value of TTA for the sourdough depends on the type of bread. Overall, sourdough with high TTA are preferred for bread making with rye flour.

The acidity of the sourdough depends on lactic acid and acetic acid production by lactic acid bacteria. Usually low temperatures of sourdough fermentation delay the lactic acidification and decrease the time of yeast exposure to high acidity. A low fermentation temperature was suggested as a means of improving the synthesis of CO2 by yeasts.

Because only the non-dissociated acids diffuse into the cell, the type of acid more than the pH determines yeast inhibition. Mainly the level of dissociation of acetic acids affects the leavening capacity. The non-dissociated form of the acetic acid inhibits yeasts by acidifying the cytoplasm, which causes physiological stress or suppresses metabolic activity. This especially happens when the pH of the sourdough is below the pKa of acetic acid (4,76). Non-dissociated acetic acid decreases the leavening capacity of C. milleri when grown together with the heterofermentative L. brevis compared to homofermentative species. C. milleri adapts to a wide range of pH (3,5 – 6) and has a good inherent acid tolerance, but the leavening activity is obviously affected by the fermentation process.

Method for the determination of TTA

There is more then one method to determine the total titratable acidity of a sourdough. I report here a common method which differs slightly from the AACC method

1) Scale 10 g of dough, sourdough or bread
2) Add 5 ml acetone
3) Add 95 ml distilled water
4) Mix it with a magnetic mixing system for lab analysis
5) Add your pH measurement to the liquid suspension
6) Titrate 0,1 n NaOH as long you reach and hold a pH value of 8,5
7) The used NaOH is the TTA level of the sourdough

German reference values for bread

type of bread

pH

TTA

rye meal bread

4,7 – 4,8

12 – 14

rye bread

4,5 – 4,6

8 – 10

rye mixed bread

4,6 – 4,8

7 – 8

mixed bread

4,8 – 4,9

6 – 7

wheat mixed bread

4,9 – 5,1

5 – 7

wheat bread

5,4 – 5,6

3 - 4

The fermentation quotient

The fermentation quotient, FQ indicates the molar ratio between lactic and acetic acids during sourdough fermentation. After the determination of the concentration of lactic and acetic acids by enzymatic or chromatographic methods, the FQ is calculated as follows:

(g of lactic acid in 100 g of dough/molecular weight of lactic acid) / (g of acetic acid in 100 g dough/molecular weight of acetic acid)

The molecular weight of lactic acid = 90,08 g/mol and the molecular weight of acetic acid = 60,05 g/mol.
To calculate the molecular weight of a substance such as acetic acid, you have to start from the chemical formula

Acetic acid = CH3COOH
Molecular weight of carbon = 12,0107
Molecular weight of oxygen = 15,9994
Molecular weight of hydrogen = 1,00794

(2*12,0107) + (4*1,00794) + (2*15,9994) = 60,052 g/mol

This parameter is strictly related to the type of lactic acid bacteria dominating the fermentation and markedly varies depending on the balance between homo- and fermentative lactobacilli. In turn this balance depends on exogenous and endogenous factors that prevail during fermentation. And although there is a common belief that this quotient cannot be more than 1, it is possible to have a result higher than 1. It all depends on the type of lactic acid bacteria. In the "hypothetical" case that the sourdough is made exclusively with homofermentative lactic acid bacteria (i.e. practically only lactic acid is formed and a low amount of acetic acid), the result of the above quotient would be more than 1.

As endogenous factors there are: carbohydrates, nitrogen sources, minerals, lipids, free fatty acids and enzymatic activity (amylases, proteases), environment microbiota (from raw materials for instance).

As exogenous factors there are: the inoculum, temperature, dough yield (aw), oxygen (redox potential), and fermentation time, number of refreshments.

Importance of temperature

It is of course well known that the fermentation temperature affects the ratio of lactic acid to acetic acid. In general homofermentative LAB starter cultures are used at high temperature and for short fermentation times (e.g. 37°C for 36 hours) and heterofermentative LAB starter are used at low temperatures for a longer fermentation time (e.g. 25°C for 48 hours), resulting in sourdoughs with mainly lactic acid and acetic acid respectively.

The fermentation temperature, one of the criteria to distinguish types I and II sourdoughs, is essential of a sourdough microbiota. For instance, spontaneous wheat sourdough back slopping fermentations (type I) carried out at 23°C for 10 days select for Le. citreum instead of Lb. fermentum that prevails at 30°C and 37°C.

Whereas Lb. sanfranciscensis prefers long fermentation times at relatively low temperature, conditions that often prevail during type I sourdough preparations, this species grows optimally at 32°C. However, whereas C. humilis grows optimally at 27 – 28°C, but does not grow above 35°C, the association of Lb. sanfranciscensis – C. humilis grows optimally at 25°C and 30°C and may explain its stability between 20°C and 30°C.

The abundance of Lb. sanfranciscensis in wheat sourdoughs made at ambient temperature indicates a low competitiveness of other LAB species such as Lb. fermentum that prefers higher temperatures for optimal growth. Similarly, temperature may be responsible for a selection toward Lb. helveticus during Sudanese sorghum sourdough fermentations, which are carried out at 37°C.

Influence of pH and ionic strength

For the growth of sourdough LAB, also the pH plays an important role. For instance Lb. sanfranciscensis cannot grow below pH 3,8 – 4,0 whereas C. humilis is not influenced by the pH. An optimal pH for growth around 5,0 has been found for Lb. sanfranciscensis. This pH value corresponds approximately to that observed during the first stage of dough fermentation. However, the growth of lactobacilli is favoured over yeast growth at pH values above 4,5. Hence, the rate of acidification of the dough may determine the level of Lb. sanfranciscensis in the dough.

Natural sourdough fermentations displaying higher pH values are often dominated by a different microbiota, encompassing Enterococcus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus and Weissella, which are commonly present in cereal flour or during the early fermentation process but die off when a significant pH decrease occurs upon fermentation.

Although sourdough fermentation happens anaerobically, the presence of oxygen in the beginning of the fermentation and when small amounts of dough (high ration of surface to volume) are used may favour certain LAB and yeast species.

Further, the ionic strength and salt concentration of the dough affects microbial growth. Similarly, the presence of organic acids in and the buffering capacity of the flour, influence the growth of both yeasts and LAB.

In general LAB species are acid-tolerant and their growth is favoured in the presence of salt. Alternatively, the growth of C. humilis and S. exiguus is completely inhibited by 4 % NaCl. Also the growth of these yeasts is strongly inhibited in the presence of acetic acid and to a lesser extend to lactic acid.

Whereas back slopping practices select for mainly heterofermentative LAB, the amounts of dough used for back slopping and the frequency of the refreshments determine the community dynamics and stability of the sourdough microbiota as well.

The amount of back slopping dough defines the initial pH and in this way influences the growth and acidification rates of the LAB species involved. Also the amount of back slopping dough determines the dough yield and hence the availability of water (water activity of the dough). Short refreshments times may select for rapidly growing LAB species, which in turn depends on the fermentation temperature and influences the acidification rate. In this respect Lb. fermentum is most competitive at 30°C and 37°C with back slopping every 24 h, while a mixture of Lb. fermentum and Lb. plantarum prevails at 30°C with back slopping every 48 hours. Also short refreshment time seems to favour C. humilis during sourdough fermentation compared to S. cerevisiae.

Finally, interactions between LAB and yeasts are an important aspect for the community dynamics and stability of the sourdough microbiota. Interactions encompass both cooperative and antagonistic ones. During some sourdough fermentations processes yeasts cannot develop at all, perhaps because of inhibition of yeast growth by nutritional competition or the presence of inhibitory compounds. In other processes mutualistic interactions lead to stable associations, not only between LAB species and yeasts (well know example is Lb. sanfranciscensis and C. humilis, but there are others such as Lb. plantarum and S. cerevisiae) but also between LAB species (e.g. Lb. sanfranciscensis and Lb. plantarum). Nevertheless the competitiveness of LAB and yeasts in sourdough systems seems to be strain-specific and not species-specific.

The use of stabilised sourdoughs versus active sourdoughs

Different types of stabilised sourdough are obtained starting from an accurate selection and mix of raw materials (e.g. wheat flour, durum, rye or other cereals) and microbial strains. Dried sourdough is a type of stabilised product used for making traditional breads that is based on a mix of tailor-made aroma compounds.

Different drying protocols can be applied such as freeze-drying, spray granulation, fluidised bed drying, spray drying and drum drying.

The later two techniques are most common for type III sourdough production. In both cases, the higher the DY of the starting type II sourdough the higher the resulting TTA value of the derived type III sourdough, which also increases due to water evaporation.

In the spray-drying process, liquid sourdough is dried by using a warm air flux that removes water until the humidity becomes less than 10 %

In the drum drying system, the vapour of the warmed drums removes water from the thin-layered liquid sourdough during contact. On the basis of various combinations of time and temperature and on the extent of the Maillard reaction, different type III sourdoughs are obtained, which show different degrees of caramelisation or toasting and, as a consequence different flavouring profiles.

Since many volatile compounds, in particular acetic acid, are missing due to the evaporation (even to a different extent depending on the drying technique), pasteurisation, cooling or salting can be applied to obtain a stabilised liquid or pasty sourdough. With the exception of cooling, all other stabilisation systems lead to microbiota inactivation and stop gas and/or acid production. Especially liquid sourdough gives an advantage for industrial applications because it can be pumped and easily dosed, while showing constant quality.

Generally, the use of a stabilised liquid sourdough is simple. It can be stored at room temperature for a long time (30 - 60 days) and directly be added to the final dough at a proportion of 5 – 10 %.

Because of the low cell density of lactic acid bacteria and yeasts, baker's yeast is generally added to leaven the dough for bread production.

Examples of applications of type III sourdough are

Microbial strains are evaluated on the basis of

Recently also improving the biosynthesis of specific chemical compounds by careful selection of particular strains of lactic acid bacteria.

The capacity to adapt to the sourdough ecosystem seems to be an essential trait for selecting strains in order to obtain a sourdough with a constant microbial composition and performance. Currently this seems the main limitation in the use of pure cultures as sourdough starters and the most innovative applications mainly deal with the use of stabilised sourdoughs.

Taxonomy and biodiversity of sourdough yeasts and LAB

Taxonomy of sourdough yeasts

Yeasts are microscopic fungi that undergo typical vegetative growth by budding of fission resulting in an unicellular appearance and sexual reproduction upon which the resulting spores are formed.

Yeasts are amongst the most important eukaryotes (an organism consisting of a cell or cells in which the genetic material is DNA in the form of chromosomes contained within a distinct nucleus. Eukaryotes include all living organisms other than the eubacteria and archaea). Yeast species found in sourdough microbial communities share an adaptation to the specific and stressful environment created mainly by low pH, high carbohydrate concentrations and high cell densities of lactic acid bacteria.

The major sourdough yeasts belong to the genera that are currently placed in the family Saccharomycetaceae. The genus Saccharomyces has been limited to the group of species known as Saccharomyces sensu stricto including the type species of the genus Saccharomyces cerevisiae. The group of species formerly often addressed as Saccharomyces sensu lato has been divided in several genera. The new genus Kazachstania is accommodating the former Saccharomyces exiguus, Saccharomyces unisporus and Saccharomyces barnettii as K. exigua, K. unispora and K. barnettii. Other new geniuses are Lachancea (L. kluyveri), Pichia (P. fermentans, P. membranifaciens. Pichia occidentalis) and Wickerhamomyces (W. anomalus, W. subpelliculosus).

Most of the six regularly encountered species are:

  1. Saccharomyces cerevisiae
  2. Candida humilis
  3. Pichia kudriavzevii
  4. Kazachstania exigua
  5. Torulaspora delbrueckii
  6. Candida colliculosa
  7. Wickerhamomyces anomalus

Taxonomy of sourdough lactic acid bacteria

LAB comprise a heterogeneous group of gram-positive, nonsporulating, strictly fermentative lactic acid producing bacteria that play an important role in the organoleptic, health promoting, technological and safety aspects of various fermented foods.

As a result of natural contamination through the flour or the environment or by deliberate introduction via dough ingredients, a wide taxonomic range of LAB has also been found in sourdoughs.

In sourdough environments, LAB live in association with yeasts and are generally considered to contribute most to the process of dough acidification, while yeasts are primarily responsible for the leavening. Although also obligate homofermentative LAB have been isolated from sourdoughs, obligate or facultative heterofermentative LAB species have the best potential and competitiveness to survive and grow in this particular food environment.

Obligate heterofermentative

Facultative heterofermentative

Obligate homofermentative

Lb. acidiforinae

Lb. alimentarius

Lb. acidophilus

Lb. brevis

Lb. casei

Lb. amylolyticus

Lb. buchneri

Lb. kimchi

Lb. amylovorus

Lb. cellobiosus

Lb. pentosus

Lb. crispatus

Lb. collinoides

Lb. perolens

Lb. delbrueckii

Lb. crustorum

Lb. plantarum

Lb. helveticus

Lb. curvatus

Lb. sakei

Lb. mindensis

Lb. fermentum

P. acidilactici

Lb. nagelii

Lb. fructivorans

P. dextrinicus

Lb. salvarius

Lb. frumenti

P. pentosaceus

Lc. lactis

Lb. kefiri

Lb. paralimentarius

S. constellatus

Lb. lindneri

 

S. equinus

Lb. panis

 

S. suis

Lb. pontis

 

E. durans

Lb. reuteri

 

Lb. pentosus

Lb. sanfranciscensis

 

 

Le. mesenteroides

 

 

W. cibaria

 

W. confusa

W. hellenica

W. kandleri

E = Enterococcus, Lb = Lactobacillus, Lc = Lactococcus, Le = Leuconostoc, P = Pediococcus, S = Streptococcus, W = Weissella.

It is not clear whether or not the species in blue are really typical for sourdough environments, while Lb. sanfranciscensis and Lb. paralimentarius (in blue) seem to be optimally adapted to this specific environment. Also Lb. brevis and Lb. plantarum are frequently isolated from fermented sourdoughs.

Although the LAB microbiota of sourdoughs is clearly dominated by lactobacilli, other less predominant or subdominant LAB species may also be found, including members of the genera Weissella, Pediococcus, Leuconostoc, Lactococcus, Enterococcus and Streptococcus.

In sourdoughs the dextran producing species Weissella cibaria and Weissella confusa are most frequently found.

Within the facilitative heterofermentative pediococci, the species Pediococcus acidilactici and Pediococcus pentosaceus are most commonly found in sourdoughs.

In the obligate heterofermentative genus Leuconostoc, the majority of sourdough isolates so far identified belong to Leuconostoc mesenteroides and Leuconostoc citreum.

Microbial species: diversity of sourdoughs

It was only from the late nineteenth century onwards that yeast starter cultures were introduced for bread production from wheat flour, first by using brewing yeasts as remnants of beer production followed by intentionally produced commercial bakers’ yeast, Saccharomyces cerevisiae. Consequently, sourdough must have been consumed for a long time. Also afterwards, bread production in countries relying on other cereals other than wheat such as rye, had still to be supported by lactic acid fermentation. Rye bread baking requires dough acidification to inhibit the abundant α-amylase in the rye flour and to make rye starch and pentosans more water retaining to form a good dough texture since not enough gluten in present in the rye.

San Francisco sourdough bread is commercially produced in the San Francisco area and remains a part of the culture of the San Francisco bay area. In the early 1970’s the responsible sourdough bacterium was identified as Lb. sanfranciscensis named according to the area where it was discovered. Notice that this LAB species is actually identical to Lb. brevis var. lindneri, which was found to be responsible for various sourdough breads produced in Europe. It was also found that these sourdough LAB species occur in a stable association with the yeasts C. humilis (synonym C. milleri) and K. exigua (synonym S. exiguus).

Nowadays sourdough is used for its technological and nutritional effects. Moreover, sourdough products are appreciated for their traditional value, gastronomic quality and natural and healthy status.

It turned out that traditional sourdoughs (for Panettone, Altamura, pane pugliese, colomba etc.) harbour a mixture of distinctive yeasts and LAB strains, which may be held responsible for the typical organoleptic quality of the breads made thereof, as back slopping results in a prevalence of the best-adapted strains.

Sourdough starter cultures comprise one or more defined strains. In addition to Lb. sanfranciscensis strains, commercially available strains of other LAB species include Lb. brevis, Lb. delbrueckii, Lb. fermentum and Lb. plantarum, albeit that not all strains in use are competitive enough to dominate the sourdough fermentation processes that have to be started up.

In Italy several studies have been focused on region-specific sourdoughs. However, no clear-cut relationship could be shown between the region typical sourdoughs and their associated microbiota.

In contrast Italian sourdoughs harbour simple to very complex communities of LAB species depending on the final products examined, among which Lb. brevis, Lb. paralimentarius, Lb. plantarum, Lb. sanfranciscensis, Lb. fermentum, P. pentosaceus and W. confusa are widespread.

Belgian bakery sourdoughs have been analysed and are characterised by LAB consortia of Lb. brevis, Lb. hammesii, Lb. nantensis, Lb. paralimentarius, Lb. plantarum, Lb. pontis, Lb. sanfranciscensis and P. pentosaceus.

Also Lb. rossiae seems to have a widespread distribution in sourdoughs, as it has been found in sourdoughs in Central and Southern Italy, Belgium and elsewhere.

LAB species are responsible for the acidification of the dough and contribute to flavour formation. Besides LAB species, a large variety of yeast species are found in sourdough ecosystems. S. cerevisiae has been found in almost every sourdough regardless as to whether or not bakers’ yeast is added. Occur also frequently is C. humilis, K. exigua and P. kudriavzevii. Yeasts are responsible for the leaving of the dough and also contribute to flavour formation.

For an extensive table of which LAB species are found in various sourdoughs according to the type of substrate (wheat, rye, spelt, teff, rice, amaranth, barley, sangak, oat, buckwheat, maize, sorghum, kisra) and according to geographical origin.

The dedicated use of basic raw materials of basic raw materials as well as the technological procedures applied determines the stability and persistence of the yeast and LAB communities involved in the sourdough fermentation process. The presence of certain LAB strains can be ascribed to its selection by the type of technology applied i.e. back slopping practices, temperature of incubation, pH of the dough and/or microbial interactions. However the use of certain raw materials, encompassing cereal types and other ingredients such as adjunct carbohydrates, salt, yoghurt, herbs etc. and operational practices, such as dough yield and refreshment times, most of time linked to local traditions, may favour particular microorganisms as a result of trophic and metabolic relationships and interactions, and hence may associate specific LAB and/or yeast species with specific geographical regions.

For instance, the use of rye may select for amylase positive homofermentative Lb. amylovorus, although higher temperatures cause a shift toward the predominance of heterofermentative lactobacilli. The dominance of heterofermentative LAB species in traditional sourdoughs is caused by a highly adapted carbohydrate metabolism, dedicated amino acid assimilation and environmental stress responses. In particular, maltose, as the most abundant fermentable energy source in dough, is metabolised via the maltose phosphorylase pathway and the pentose phosphate shunt by strictly heterofermentative LAB species such as Lb. sanfranciscensis, Lb. reuteri and Lb. fermentum.

Maltose positive LAB species such as Lb. sanfranciscensis often form a stable association with maltose negative yeast species such as C. humilis, thereby preventing competition for the same carbohydrate sources.

Also the production of specific inhibitory compounds, maintained through back slopping, such as antibiotic reutericyclin produced by Lb. reuteri, may favour the dominance of this LAB species, as is the case for certain German type II sourdoughs.

Besides the cereals and other dough ingredients, which are mainly responsible as the source of metabolic activity in the form of flour enzymes and endogenous microorganisms, specific technological process parameters determine the species diversity, number and metabolic activity of the microorganisms (whether or not added) present in the stable, ripe sourdough. These process parameters include

Process parameters and metabolic response of microorganisms

Process parameters, including temperature, dough yield, oxygen, pH as well as the composition of starter cultures, determine the quality and handling properties of sourdough and the metabolic response of microorganisms responsible for the fermentation process. The exposure of microbial cells to stressful and fluctuating conditions during fermentation involves a broad transcriptional response with many induced or repressed genes. The selective pressure exerted by environmental conditions encountered by yeast cells during sourdough fermentation, accounts for the consolidated dominance of selected yeast species.

Within the sourdough ecosystem there are numerous mechanisms whereby one species may influence the growth of another. Although autochthonous bacteria and yeasts are adapted and competitive in their respective environment, the dough environment can be described as a stressful environment for microorganisms.

The conditions of the sourdough micro-environment that principally affect yeast responses and growth rate are:

In bakery practice, refrigeration of dough or sourdough is used to control fermentation. Under the refrigerated conditions, yeasts have to maintain and then to recover their fermentative capacity in a very short period of time. At 4 – 8°C, many yeasts strains continue to ferment at a slow rate and induce a slow increase of the dough volume. The fermentation stops at 4°C. Only selected strains recover their leavening capacity when the dough temperature is raised to 28 – 35°C.



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