Monitoring of starter culture-initiated liquid wheat and teff sourdough fermentations by selected ion flow tube-mass spectrometry.

BACKGROUND: Selected ion flow tube-mass spectrometry (SIFT-MS) is a direct-injection mass spectrometric technique that has been introduced recently into the field of food and flavor analysis. It also shows potential for use in the monitoring of food fermentations. Therefore, this study aimed at the online monitoring of different volatile compounds produced during starter culture-initiated liquid sourdough fermentations by SIFT-MS, for which a new workflow was developed. RESULTS: The online monitoring of the volatile sample compounds acetoin and ethyl acetate, diacetyl, and ethanol was made possible during the production of sourdoughs obtained through fermentation with several interesting strains belonging to the species Lactobacillus crustorum, Lactobacillus fermentum, Lactobacillus hilgardii, Lactobacillus nagelii, Lactobacillus sakei, and Gluconobacter oxydans. Acetoin and ethyl acetate could not be distinguished based solely on SIFT-MS data. Diacetyl production was monitored in the case of Lb. crustorum LMG 23699 as a starter culture strain, thereby making the distinction between those volatiles produced in sourdough without extra ingredients added or after the addition of citrate or malate. CONCLUSION: Starter culture-initiated liquid sourdough fermentations were monitored successfully. The volatile compound production of the different starter culture strains tested reflected differences in their metabolism and/or competitiveness in a sourdough matrix. © 2018 Society of Chemical Industry.

Microbial Ecology and Process Technology of Sourdough Fermentation.

From a microbiological perspective, sourdough is to be considered as a specific and stressful ecosystem, harboring yeasts and lactic acid bacteria (LAB), that is used for the production of baked goods. With respect to the metabolic impact of the sourdough microbiota, acidification (LAB), flavor formation (LAB and yeasts), and leavening (yeasts and heterofermentative LAB species) are most noticeable. Three distinct types of sourdough fermentation processes can be discerned based on the inocula applied, namely backslopped ones (type 1), those initiated with starter cultures (type 2), and those initiated with a starter culture followed by backslopping (type 3). A sourdough-characteristic LAB species is Lactobacillus sanfranciscensis. A sourdough-characteristic yeast species is Candida humilis. Although it has been suggested that the microbiota of a specific sourdough may be influenced by its geographical origin, region specificity often seems to be an artefact resulting from interpretation of the research data, as those are dependent on sampling, isolation, and identification procedures. It is however clear that sourdough-adapted microorganisms are able to withstand stress conditions encountered during their growth. Based on the technological setup, type 0 (predoughs), type I (artisan bakery firm sourdoughs), type II (industrial liquid sourdoughs), and type III sourdoughs (industrial dried sourdoughs) can be distinguished. The production of all sourdoughs, independent of their classification, depends on several intrinsic and extrinsic factors. Both the flour (type, quality status, etc.) and the process parameters (fermentation temperature, pH and pH evolution, dough yield, water activity, oxygen tension, backslopping procedure and fermentation duration, etc.) determine the dynamics and outcome of (backslopped) sourdough fermentation processes.

Microbiota and metabolites of aged bottled gueuze beers converge to the same composition.

Gueuze beers are prepared by mixing young and old lambic beers and are bottle-refermented spontaneously for aging. The present study analyzed the microbiota and metabolites present in gueuze beers that were aged between a few months and up to 17 years. Yeasts were cultivated from all beers sampled, but bacteria could not be grown from beers older than 5 years. Lactic acid and ethyl lactate concentrations increased steadily during aging, whereas ethanol concentrations remained constant. The concentrations of isoamyl acetate and ethyl decanoate decreased during the aging process. Hence, ethyl lactate and ethyl decanoate can be considered as positive and negative gueuze beer-aging metabolite biomarkers, respectively. Nevertheless, considerable bottle-to-bottle variation in the metabolite profiles was found, which hindered the generalization of the effects seen during the aging of the gueuze beers examined, but which illustrated the unique character of the lambic beers. The present results further indicate that gueuze beers are preferably aged for less than 10 years.

Production of new-to-nature sophorolipids by cultivating the yeast Candida bombicola on unconventional hydrophobic substrates.

The naturally occurring sophorolipids synthesized by Candida bombicola possess--despite their overall heterogeneity--little variation in the length of the lipid tail. The range is limited to C16-C18 fatty acids and is governed by the specificity of a cytochrome P450 monooxygenase. However, incorporation of fatty acids differing from the conventional C16-C18 range could broaden up the application potential of sophorolipids. The incorporation of medium-chain fatty acids should render the molecules more hydrophilic and consequently improve their water solubility. Two strategies to circumvent this C16-C18 preference are described in this paper. The first one skips the controlling action of the cytochrome P450 enzyme by supplying the yeast with already hydroxylated substrates, while the other method is based on the deception of the enzyme by presenting it substrates structurally resembling stearic acid. This later strategy can be applied to create very specific tailor-made sophorolipids when combined with post-fermentive modification.

Lemon juice and apple juice used as source of citrate and malate, respectively, enhance the formation of buttery aroma compounds and/or organic acids during Type 2 and Type 3 sourdough productions performed with Companilactobacillus crustorum LMG 23699.

Extra ingredients are often used in traditional sourdough production recipes by artisan bakeries. These ingredients may be the source of microorganisms or stimulate the growth and/or the metabolic activities of the microorganisms added to or naturally present in the flour-water mixture. The present study examined the influence of the addition of lemon juice or apple juice as source of citrate or malate, respectively, on the growth and activity of the citrate- and malate-positive Companilactobacillus crustorum LMG 23699 strain (formerly known as Lactobacillus crustorum LMG 23699), used to initiate firm (dough yield of 200) wheat sourdough productions, and on the flavour of the baked goods produced. Three fermentation strategies were applied, namely one-step long fermentation sourdough production processes with the addition of juice at the start (Type 2) and backslopped fermentations with the addition of juice either only at the start of the sourdough productions or at the start of the sourdough productions and at the beginning of each subsequent refreshment step during the whole backslopping process (both Type 3). It turned out that the starter culture strain used prevailed during all sourdough productions performed. Yeasts were particularly present in Type 3 sourdough productions, although lemon juice retarded their growth. Due to high yeast activity, high concentrations of ethanol and glycerol were produced toward the end of the sourdough productions. Addition of lemon juice stimulated the production of lactic acid, acetic acid, and the buttery flavour compounds acetoin and diacetyl, because of citrate conversion, during the Type 2 and Type 3 sourdough productions. In Type 3 sourdough productions, these compounds were found in higher concentrations only when lemon juice was added at each backslopping step. Alternatively, the addition of apple juice led to high concentrations of lactic acid because of malolactic fermentation in both Type 2 and Type 3 sourdough productions. Moreover, the addition of apple juice increased the initial concentrations of the carbohydrates (fructose, glucose, and sucrose) and sugar alcohols (mannitol and sorbitol), which were exhausted upon backslopping or accumulated in the sourdough matrix, respectively. Baked goods produced using sourdoughs obtained from the Type 2 and Type 3 sourdough productions with the addition of juice at each backslopping step were significantly different in flavour from doughs supplemented with the respective juices and lactic acid and/or Type 3 sourdough productions with the addition of juice only at the start.

Diverse Microbial Composition of Sourdoughs From Different Origins.

Hundreds of sourdoughs have been investigated in the last decades. However, many studies used a culture-dependent and/or culture-independent microbiological approach [mainly based on denaturing gradient gel electrophoresis (DGGE) of PCR amplicons], seldomly combined with a metabolite target analysis, to characterize the microbial species communities of the sourdoughs examined. Moreover, attention was mainly paid on lactic acid bacteria (LAB) and yeast species. In the present study, distinct household-scale (including an artisan lambic brewery) and artisan bakery-scale backslopped sourdoughs (17 in total), obtained from different regions (Belgium, France, United Kingdom, and USA), were examined through a multiphasic approach, encompassing a culture-dependent analysis [targeting LAB, acetic acid bacteria (AAB), and yeasts], different culture-independent techniques [rRNA-PCR-DGGE, metagenetics, and metagenomics (four bakery sourdoughs)], and metabolite target analysis. It turned out that the microbial species diversity of the sourdoughs was influenced by the house microbiota of the producer. Further, when the producer made use of different flours, the sourdoughs harbored similar microbial communities, independent of the flour used. AAB were only present in the Belgian sourdoughs, which might again be related to the processing environment. Fructilactobacillus sanfranciscensis (formerly known as Lactobacillus sanfranciscensis) was the prevalent LAB species of the eight sourdoughs produced by two of the three bakeries of different countries analyzed. These sourdoughs were characterized by the presence of either Saccharomyces cerevisiae or Kazachstania humilis. Moreover, the presence of Fl. sanfranciscensis was positively correlated with the production of mannitol and negatively correlated with the presence of other LAB or AAB species. Sourdoughs produced in an artisan lambic brewery were characterized by the presence of the yeast species Dekkera anomala and Pichia membranifaciens. One household sourdough was characterized by the presence of uncommon species, such as Pediococcus parvulus and Pichia fermentans. Metagenomic sequencing allowed the detection of many more LAB and AAB species than the other methods applied, which opened new frontiers for the understanding of the microbial communities involved during sourdough production processes.

Potential of Bacteria from Alternative Fermented Foods as Starter Cultures for the Production of Wheat Sourdoughs.

Microbial strains for starter culture-initiated sourdough productions are commonly isolated from a fermenting flour-water mixture. Yet, starter culture strains isolated from matrices other than sourdoughs could provide the dough with interesting metabolic properties and hence change the organoleptic properties of the concomitant breads. Furthermore, the selection of sourdough starter cultures does not need to be limited to lactic acid bacteria (LAB), as other food-grade microorganisms are sometimes found in sourdoughs. Therefore, different strains belonging to LAB, acetic acid bacteria (AAB), and coagulase-negative staphylococci (CNS) that originated from different fermented food matrices (fermenting cocoa pulp-bean mass, fermented sausage, and water kefir), were examined as to their prevalence in a wheat sourdough ecosystem during 72-h fermentations. Limosilactobacillus fermentum IMDO 222 (fermented cocoa pulp-bean mass isolate) and Latilactobacillus sakei CTC 494 (fermented sausage isolate) seemed to be promising candidates as sourdough starter culture strains, as were the AAB strains Acetobacter pasteurianus IMDO 386B and Gluconobacter oxydans IMDO A845 (both isolated from fermented cocoa pulp-bean mass), due to their competitiveness in the wheat flour-water mixtures. Wheat breads made with G. oxydans IMDO A845 sourdoughs were significantly darker than reference wheat breads.

Omics approaches to understand sourdough fermentation processes.

The application of omics methodologies helps to further unravel sourdough fermentation processes. Of all methodologies applied, metagenetics is the most used one to analyse sourdoughs, allowing to elucidate their microbial structure, albeit that it is based on the sequencing of a very small part of whole-community DNA. Although shotgun metagenomics, metatranscriptomics, metaproteomics, and meta-metabolomics are very promising to analyse sourdough fermentations and the terminology is often used in a confusing way, they have not been fully used sensu stricto yet. For instance, up to now, metatranscriptomics is restricted to the use of a functional gene microarray for lactic acid bacteria. Further, meta-metabolomics often deals with metabolite target analysis of sourdough fermentation samples to determine the actual concentrations of residual substrates and metabolites produced as well as to list their volatilome solely. In contrast, genomics has been applied several times, albeit that the whole-genome sequence of only one yeast strain and of 41 lactic acid bacterial strains, originally isolated from sourdoughs, is available. However, the genomics data accessible in public databases should be considered with caution because of inaccurate gene annotations, due to automated annotation pipelines, thereby possibly overruling original, high-quality, well-curated gene annotations.

Monitoring of volatile production in cooked poultry products using selected ion flow tube-mass spectrometry.

Cooked poultry products are nutritious and economically valuable products that are at risk of bacterial spoilage, which can be postponed by cooling and modified-atmosphere-packaging (MAP). In this study, a cooked chicken product was stored at three different temperature ranges (4-6 °C, 7-9 °C, and 11-13 °C) and volatile production was measured over time using selected ion flow tube-mass spectrometry (SIFT-MS). The identities of the volatiles formed were confirmed by headspace solid-phase microextraction coupled with gas chromatography time-of-flight mass spectrometry (HS-SPME-GC-TOF-MS) analysis. In total, 33 volatiles were proposed using the latter technique and their concentrations were calculated using product ion counts after assignment of these counts to specific volatiles. The results indicated that 1-octen-3-ol, 2,3-butanediol, acetoin, benzaldehyde, ethanol, methylbutanal, and methylbutanol may serve as biomarkers for bacterial growth and/or chemical degradation of cooked poultry products. In parallel, the bacterial loads of the product samples were determined on selective agar media. A total of 495 bacterial isolates was classified and identified by (GTG)5-PCR fingerprinting, followed by gene sequencing of representative cluster isolates. Carnobacterium divergens, Carnobacterium maltaromaticum, Rahnella aquatilis, and Serratia proteamaculans were the most commonly found species, besides minor contributions of Lactobacillus sakei and Hafnia alvei. Differences in volatile profiles could thus be ascribed to variations in bacterial loads and storage temperatures.

The application of selected ion flow tube-mass spectrometry to follow volatile formation in modified-atmosphere-packaged cooked ham.

Cooked pork products, i.e., sliced cooked hams maintained under modified-atmosphere-packaging (MAP), were analysed both microbiologically and with respect to volatile levels during storage. Three storage temperature ranges were compared (4-6 °C, 7-9 °C, and 11-13 °C), representing different refrigeration conditions at household level. The microbial loads were determined by plating samples on six different agar media, followed by (GTG)5-PCR fingerprinting of genomic DNA of selected isolates, and identification of representative isolates by 16S rRNA, pheS, and rpoA gene sequencing. Carnobacterium maltaromaticum, Lactobacillus sakei, and Serratia proteamaculans were the major bacterial species found among the 619 isolates identified. The volatiles produced during storage were followed by selected ion flow tube-mass spectrometry (SIFT-MS) and the identity of the volatiles was confirmed by headspace solid-phase microextraction combined with gas chromatography and time-of-flight mass spectrometry (HS-SPME-GC-TOF-MS). SIFT-MS analysis showed that volatiles, such as 2,3-butanediol, acetoin, and ethanol, may serve as potential markers for spoilage development. Differences in volatile production between samples were likely due to discrepancies in the initial microbial load and the effect of storage conditions. In conclusion, this study combines the use of new mass spectrometric techniques to examine volatile production during spoilage as an additional source of information during microbiological community analysis.

Impact of process conditions on the microbial community dynamics and metabolite production kinetics of teff sourdough fermentations under bakery and laboratory conditions.

Teff and teff sourdoughs are promising ingredients for bread production. Therefore, this study aimed at the characterization of spontaneous and flour-native starter culture-initiated teff sourdough productions under bakery and laboratory conditions. Backslopped laboratory and bakery teff sourdough productions were characterized by different lactic acid bacteria (LAB) and yeast species, but were both characterized by a pH below 4.0 after five backslopping steps. The sourdough-associated Lactobacillus sanfranciscensis was isolated for the first time from backslopped spontaneous teff sourdoughs. The autochthonous strain L. sanfranciscensis IMDO 150101 was tested as starter culture during laboratory teff sourdough fermentations. Its prevalence could be related to the process conditions applied, in particular the ambient temperature (below 30°C). Breads made with 20% teff sourdough (on flour basis) displayed interesting features compared with all-wheat-based reference breads. Teff sourdoughs were characterized as to their pH evolution, microbial community dynamics, and microbial species composition. Representative strains of the LAB species isolated from these sourdoughs, in particular L. sanfranciscensis, may be selected as starter cultures for the production of stable teff sourdoughs and flavorful breads, provided they are adapted to the environmental conditions applied.

Impact of starter culture, ingredients, and flour type on sourdough bread volatiles as monitored by selected ion flow tube-mass spectrometry.

This study deals with the detection of volatile compounds originating from the crumb of breads made with sourdoughs obtained through starter culture-initiated fermentations, which differed in flour type (wheat and teff), ingredients (citrate and malate), fermentation time (24h or 72h), and starter culture strains (homo- and heterofermentative lactic acid bacteria species and acetic acid bacteria species) applied. Therefore, selected ion flow tube-mass spectrometry (SIFT-MS) was used. SIFT-MS is an easy-to-use and promising technique in the field of food and flavor analysis. Volatile compounds of crumb samples from the breads with sourdough were measured and compared with those of reference bread crumb samples. In general, sourdough addition had a positive effect on the concentrations of the volatile compounds measured by SIFT-MS. Furthermore, a trend toward higher concentrations of several volatiles was seen upon the addition of sourdoughs that were fermented up to 72h, compared to the addition of sourdoughs that were fermented for a shorter time. Ethanol was the major volatile compound identified tentatively, next to alcohols, aldehydes, esters, terpenes, and heterocyclic compounds. Also acetoin/ethyl acetate could be identified, but these compounds could not be distinguished. Higher alcohols showed an increase upon the use of sourdough fermented for a long time. High concentrations of acetic acid were found in breads made with Gluconobacter oxydans IMDO A845-initiated sourdough, indicating its potential for sourdough production. Breads made with teff sourdoughs were distinct from wheat-based sourdough breads as to their volatile compound profiles.

Community dynamics and metabolite target analysis of spontaneous, backslopped barley sourdough fermentations under laboratory and bakery conditions.

Barley flour is not commonly used for baking because of its negative effects on bread dough rheology and loaf volume. However, barley sourdoughs are promising ingredients to produce improved barley-based breads. Spontaneous barley sourdough fermentations were performed through backslopping (every 24h, 10days) under laboratory (fermentors, controlled temperature of 30°C, high dough yield of 400) and bakery conditions (open vessels, ambient temperature of 17-22°C, low dough yield of 200), making use of the same batch of flour. They differed in pH evolution, microbial community dynamics, and lactic acid bacteria (LAB) species composition. After ten backsloppings, the barley sourdoughs were characterized by the presence of the LAB species Lactobacillus fermentum, Lactobacillus plantarum, and Lactobacillus brevis in the case of the laboratory productions (fast pH decrease, pH<4.0 after two backslopping steps), and of Leuconostoc citreum, Leuconostoc mesenteroides, Weissella confusa and Weissella cibaria in the case of the bakery productions (slow pH decrease, pH4.0 after eight backslopping steps). In both sourdough productions, Saccharomyces cerevisiae was the sole yeast species. Breads made with wheat flour supplemented with 20% (on flour basis) barley sourdough displayed a firmer texture, a smaller volume, and an acceptable flavour compared with all wheat-based reference breads. Hence, representative strains of the LAB species mentioned above, adapted to the environmental conditions they will be confronted with, may be selected as starter cultures for the production of stable barley sourdoughs and flavourful breads.

A low pH does not determine the community dynamics of spontaneously developed backslopped liquid wheat sourdoughs but does influence their metabolite kinetics.

This study dealt with the influence of a crucial pH value of 4.0 on the microbiota of spontaneously fermented backslopped liquid wheat sourdoughs. Two spontaneously fermented wheat sourdough fermentation experiments were carried out, one without control of the pH and one with the pH kept constant at pH4.0, both during nine backslopping steps. In each case, two additional backslopping steps were carried out, with the pH kept constant at 4.0 and with free pH, respectively. Keeping the pH constant at 4.0 changed the microbial community dynamics and metabolite kinetics of the sourdough fermentations. A slower prevalence of sourdough-specific Kazachstania yeasts occurred. Nevertheless, in both experiments, Lactobacillus fermentum, Lb. plantarum/pentosus/paraplantarum, and Kazachstania exigua/bulderi/barnettii prevailed ultimately. The lactic acid and ethanol concentration profiles were affected positively by keeping the pH constant at a minimum of 4.0 as well as the l- and d-lactic acid ratio profile, a potential biological marker for sourdough stability and maturity. Also, the concentration and diversity of acetate esters and their precursors, in particular isoamyl acetate and isoamyl alcohol, were affected negatively by the pH control, indicating the role of pH stress in the sourdough aroma formation.

Yeast diversity of sourdoughs and associated metabolic properties and functionalities.

Together with acidifying lactic acid bacteria, yeasts play a key role in the production process of sourdough, where they are either naturally present or added as a starter culture. Worldwide, a diversity of yeast species is encountered, with Saccharomyces cerevisiae, Candida humilis, Kazachstania exigua, Pichia kudriavzevii, Wickerhamomyces anomalus, and Torulaspora delbrueckii among the most common ones. Sourdough-adapted yeasts are able to withstand the stress conditions encountered during their growth, including nutrient starvation as well as the effects of acidic, oxidative, thermal, and osmotic stresses. From a technological point of view, their metabolism primarily contributes to the leavening and flavour of sourdough products. Besides ethanol and carbon dioxide, yeasts can produce metabolites that specifically affect flavour, such as organic acids, diacetyl, higher alcohols from branched-chain amino acids, and esters derived thereof. Additionally, several yeast strains possess functional properties that can potentially lead to nutritional and safety advantages. These properties encompass the production of vitamins, an improvement of the bioavailability of phenolic compounds, the dephosphorylation of phytic acid, the presence of probiotic potential, and the inhibition of fungi and their mycotoxin production. Strains of diverse species are new candidate functional starter cultures, offering opportunities beyond the conventional use of baker's yeast.

Whole-Genome Sequence Analysis of Bombella intestini LMG 28161T, a Novel Acetic Acid Bacterium Isolated from the Crop of a Red-Tailed Bumble Bee, Bombus lapidarius.

The whole-genome sequence of Bombella intestini LMG 28161T, an endosymbiotic acetic acid bacterium (AAB) occurring in bumble bees, was determined to investigate the molecular mechanisms underlying its metabolic capabilities. The draft genome sequence of B. intestini LMG 28161T was 2.02 Mb. Metabolic carbohydrate pathways were in agreement with the metabolite analyses of fermentation experiments and revealed its oxidative capacity towards sucrose, D-glucose, D-fructose and D-mannitol, but not ethanol and glycerol. The results of the fermentation experiments also demonstrated that the lack of effective aeration in small-scale carbohydrate consumption experiments may be responsible for the lack of reproducibility of such results in taxonomic studies of AAB. Finally, compared to the genome sequences of its nearest phylogenetic neighbor and of three other insect associated AAB strains, the B. intestini LMG 28161T genome lost 69 orthologs and included 89 unique genes. Although many of the latter were hypothetical they also included several type IV secretion system proteins, amino acid transporter/permeases and membrane proteins which might play a role in the interaction with the bumble bee host.

Selected ion flow tube-mass spectrometry for online monitoring of submerged fermentations: a case study of sourdough fermentation.

Selected ion flow tube-mass spectrometry (SIFT-MS) has recently gained interest as an alternative method to traditional GC-MS for the detection of targeted volatile sample compounds, due to its ease of use, its speed and sensitivity, and its potential for real-time quantification. The feasibility of this technique was demonstrated using the case of the production of ethanol during sourdough fermentation. The potential of SIFT-MS as an online monitoring device for food fermentations was further demonstrated by the detection of acetoin in certain sourdough fermentations. This allowed discrimination between sourdough fermentation processes and illustrated the importance of real-time monitoring of food fermentations.