Novel metabolites from cereal-associated lactobacilli – Novel functionalities for cereal products?

Predictions from genome sequence data of sourdough lactobacilli, novel applications of known metabolic traits such as glycansucrases, as well as the exploitation of biodiversity of lactobacilli from traditional fermentations remain an important resource for identification of novel metabolic traits of lactobacilli for use in bread production and the production of value-added food ingredients. Cornerstones of heterofermentative lactic metabolism in cereal fermentations are the rapid utilization of maltose as preferred carbon source, and the production of lactate, CO₂, and the alternative products ethanol and acetate. This review will highlight selected novel aspects of carbohydrate metabolism that are related to the production of maltose and the utilisation of lactate by lactobacilli in cereal fermentations. Several species of lactobacilli convert glycerol and lactate to 1,3 and 1,2 propanediol, respectively. Both metabolic pathways are relevant for food preservation as reuterin is an intermediate of 1,3 propanediol formation, and 1,2 propanediol is further converted to propionate. Glycansucrases, disaccharide hydrolases and disaccharide phosphorylases catalyse oligosaccharide formation from sucrose, maltose, or lactose. Lactobacilli in sourdough generally harbour several enzymes capable of oligosaccharide formation from disaccharides. Oligosaccharide formation by sourdough lactobacilli can be exploited for fermentative production of novel oligosaccharides in bread and a wide spectrum of other food applications.     

传统食品源乳酸菌对酸面团面包风味的影响

乳酸菌在酸面团制作中扮演着重要角色,对其风味等品质有显著影响。不同种类的乳酸菌在性能上存在差异,为了选育特色酸面团用乳酸菌发酵剂,开发差异化酸面团面包,本研究对来自不同种类传统发酵食品中的乳酸菌,进行糖代谢能力、产有机酸能力及风味特征评估,并验证其对酸面团和面包风味的影响。结果显示,有6株乳酸菌具有较强的糖代谢能力和产酸性能,风味特征优良。其产生的挥发性风味物质主要包括醇类、酯类、酮类、醛类、酸类和其他类化合物。其中有2株植物乳杆菌可为酸面团面包贡献酸味、果味和酒酿味等特殊风味。这些筛选出的菌株为开发具有特征性香气成分的酸面团发酵剂提供了资源,具有工业应用潜力。     

Microbial re-inoculation reveals differences in the leavening power of sourdough yeast strains

A method based on microbial re-inoculation, or the so-called backslopping and subsequent proofing of rye bread dough simulating commercial one-stage sourdough process, was used for the screening of the leavening capacity of sourdough yeast strains. Two yeast strains were initially tested with seven Lactobacillus strains. Thereafter, 17 yeast strains, mostly of sourdough origin, were tested with a backslopping procedure with heterofermentative Lactobacillus brevis as an acidifying lactic acid bacteria (LAB). The highest leavening capacity was found in sourdoughs containing Candida milleri, in particular when it was accompanied by obligately homofermentative Lactobacillus acidophilus or facultatively heterofermentative Lactobacillus plantarum when it acted homofermentatively. The leavening capacity of the reference strain Saccharomyces cerevisiae was about half that of C. milleri in all sourdoughs tested. The re-inoculation procedure increased the differences found in the leavening capacity of the tested yeast strains during final proofing of rye bread dough. The backslopped sourdoughs containing a heterofermentative Lactobacillus strain were more suppressive than those containing a homofermentative strain. The highest leavening capacity was found in C. milleri strains. The use of one backslopping cycle before assaying the leavening capacity of a laboratory sourdough is recommended since it helps to differentiate between yeast strains to be tested for their leavening power in the final bread dough.     

不同发酵基质的酸面团对酵母面团体系面包烘焙及老化特性的影响

应用分离自我国传统酸面团的区域特色乳酸菌--旧金山乳杆菌分别发酵小麦粉和小麦麸皮基质制成（小麦/麦麸）酸面团,研究了两种不同发酵基质的酸面团及其添加量对酵母面团体系面包烘焙及老化特性的影响。结果表明：与小麦粉制作的空白组面包相比,小麦酸面团可以明显改善面包的比容和感官品质;添加未发酵麦麸制作的非酸面团麦麸面包品质低于空白组,但引入麦麸酸面团（10%、20%、30%）后面包比容和感官评定得分均高于相对应的非酸面团麦麸面包。小麦酸面团和麦麸酸面团以及小麦麸皮均可以改善面包的老化特性,在相同贮藏期内,酸面团面包和麦麸面包的硬度增加量、水分迁移量和老化焓值都低于空白组,并且添加麦麸酸面团的面包其硬度和老化焓值都低于相对应的非酸面团麦麸面包。     

Utilization of electron acceptors by lactobacilli isolated from sourdough

The metabolism of maltose and the use of electron acceptors has been investigated in strains of lactobacilli which are known to be stable elements in sourdoughs, which, traditionally, have been used for a long time. The metabolic features ofLactobacillus sanfrancisco have been described by us in a previous communication. Similar principles have been detected for the competitiveness ofL. pontis, L. reuteri, L. fermentum andL. amylovorus, as well as species-specific characteristics. Based on these findings the metabolic key reactions have been identified and the use of electron acceptors present in sourdough are presented in a schematic overview. In contrast toL. sanfrancisco, these species can not use oxygen as an electron acceptor, and the length of their lag phase was not affected by agitation. Malate and fumarate were reduced to succinate, and fructose was used, depending on the species, as an electron acceptor, carbon source or both. All heterofermentative sourdough lactobacilli efficiently split maltose using maltose phosphorylase. Glucose was excreted, which induced glucose repression in competing indigenous micro-organisms, without affecting the maltose metabolism of sourdough lactobacilli. Lactobacilli generate additional adenosine 5-triphosphate (ATP) from acetyl phosphate in the presence of electron acceptors. These special features are suggested to represent a general principle which accounts for the prevalence of specific heterofermentative lactobacilli which are propagated over long periods present in sourdough fermentations.     

The potential of dairy lactic acid bacteria to metabolise amino acids via non-transaminating reactions and endogenous transamination

The metabolism of amino acids by 22 starter and 49 non-starter lactic acid bacteria (LAB) was studied in a system consisting of amino acids and non-growing cells without added amino acceptors such as alpha-ketoglutarate. There were significant inter- and intra-species differences in the metabolism of amino acids. Some amino acids such as alanine, arginine, aspartate, serine and branched-chain amino acids (leucine, isoleucine and valine) were utilised, whereas other amino acids such as glycine, ornithine and citrulline were produced. Alanine and aspartate were utilised by some LAB and accumulated during the incubation of other LAB. Arginine was degraded not only by Lactococcus lactis subsp. lactis (the lactococcal subspecies known to catabolise arginine), but also by pediococci, heterofermentative lactobacilli (Lactobacillus brevis and Lb. fermentum) and some unidentified homofermentative lactobacilli. Serine was utilised predominantly by homofermentative Lb. paracasei subsp. paracasei, Lb. rhamnosus and Lb. plantarum. Of the LAB studied, Lb. brevis and Lb. fermentum were the most metabolically active, utilising alanine, arginine, aspartate, glutamate and branched-chain amino acids. Leuconostocs were the least metabolically active, showing little potential to metabolise amino acids. The formation of ammonia and acetate from amino acid metabolism varied both between species and between strains within species. These findings suggest that the potential of LAB for amino acid metabolism via non-transaminating reactions and endogenous transamination will impact both on the physiology of LAB and on cheese ripening, especially when transamination is rate-limiting in the absence of an exogenous amino acceptor such as alpha-ketoglutarate.     

产乳化活性多糖乳酸菌的筛选及其发酵荞麦酸面团、面包的特性

本研究从不同来源的原料中分离得到18株具有产胞外多糖(EPS)能力的乳酸菌,通过对比发酵液及乳酸菌荞麦酸面团的乳化活性,获得一株产EPS且乳化活性较高的乳酸菌,并应用于荞麦酸面团的制作。采用动态流变测定、激光共聚焦观察、质构分析等手段,研究其对荞麦酸面团面包烘焙学特性的影响。结果表明:优选产乳化性多糖的乳酸菌YC'-22经鉴定为发酵乳杆菌(Lactobacillus fermentum strain HBUAS54017),且同源性为100%,EPS产量为1.63 g/L。通过添加乳酸菌荞麦酸面团,使面包面团的面筋网络结构得到强化。相比空白面包(CB),添加1.0%阿拉伯胶的荞麦面包(AGB)、荞麦酸面团面包(SDB)的比容分别增加了12.42%和14.08%,硬度分别降低了13.90%和22.87%;其中SDB面包整体可接受度最高,改善效果最明显。因此,产乳化性多糖的乳酸菌发酵的荞麦酸面团可以作为一种天然的面包功能配料。     

Use of selected sourdough strains of Lactobacillus for removing gluten and enhancing the nutritional properties of gluten-free bread

Forty-six strains of sourdough lactic acid bacteria were screened for proteolytic activity and acidification rate in gluten-free (GF) flours. The sourdough cultures consisted of Lactobacillus sanfranciscensis LS40 and LS41 and Lactobacillus plantarum CF1 and were selected and used for the manufacture of GF bread. Fermentation occurred in two steps: (i) long-time fermentation (16 h) and (ii) fast fermentation (1.5 h) using the previous fermented sourdough as inoculum (ca. 43%, wt/wt) with Saccharomyces cerevisiae (baker's yeast). GF bread started with baker's yeast alone was used as the control. Gluten was added to ingredients before fermentation to simulate contamination. Initial gluten concentration of 400 ppm was degraded to below 20 ppm only in the sourdough GF bread. Before baking, sourdough GF bread showed phytase activity ca. sixfold higher than that of GF bread started with baker's yeast alone. Atomic absorption spectrophotometric analysis revealed that the higher phytase activity resulted in an increased availability of free Ca2+, Zn2+, and Mg2+. The concentration of free amino acids also was the highest in sourdough GF bread. Sourdough GF bread had a higher specific volume and was less firm than GF bread started with baker's yeast alone. This study highlighted the use of selected sourdough cultures to eliminate risks of contamination by gluten and to enhance the nutritional properties of GF bread.     

Exopolysaccharides from Sourdough Lactic Acid Bacteria

The use of sourdough improves the quality and increases the shelf life of bread. The positive effects are associated with metabolites produced by lactic acid bacteria (LAB) during sourdough fermentation, including organic acids, exopolysaccharides (EPS), and enzymes. EPS formed during sourdough fermentation by glycansucrase activity from sucrose influence the viscoelastic properties of the dough and beneficially affect the texture and shelf life (in particular, starch retrogradation) of bread. Accordingly, EPS have the potential to replace hydrocolloids currently used as bread improvers and meet so the consumer demands for a reduced use of food additives. In this review, the current knowledge about the functional aspects of EPS formation by sourdough LAB especially in baking applications is summarized.     

传统酸面团中细菌与酵母菌的分离与鉴定

为进一步描述我国传统面食发酵剂的理化性质和菌落组成,收集了北方地区6份发酵剂样品,测定了其酸度和菌落总数,并对分离、纯化、初筛后得到的75株细菌和60株酵母菌进行了测序鉴定。结果显示,样品pH值范围为3.73~5.46,总滴定酸度为8.3~19.8 mL;乳酸菌和酵母菌的计数结果分别为8.35±0.07~9.75±0.12 Log cfu/g,6.31±0.22~8.68±0.04 Log cfu/g。鉴定出包括短乳杆菌(Lactobacillus brevis)、植物乳杆菌(Lactobacillus plantarum)和旧金山乳杆菌(Lactobacillus sanfranciscensis)在内的乳酸菌8种;酵母菌4种,其中优势菌为酿酒酵母(Saccharomyces cerevisiae);其他细菌6种,主要为解淀粉芽孢杆菌(B.amyloliquefaciens)、地衣芽孢杆菌(B.licheniformis)和醋杆菌。结果表明,我国传统面食发酵剂菌相复杂,以酵母菌和乳酸菌为主,还包括芽孢杆菌、醋酸杆菌在内的多种其他细菌,甚至可能含有致病菌。通过比较细菌和酵母在不同酸面团样品中的分布,发现不同来源样品的微生物种类组成存在差异。     

Quality attributes and firming kinetics of partially baked frozen wholewheat bread with sourdough

Partially baked frozen (PBF) process prolongs bread shelf life, but diminishes its volume and crumb texture. Therefore, we investigated the possibility of using sourdough for the quality improvement in PBF wholewheat bread. Sourdough was fermented with either Lactobacillus plantarum, Lb. brevis or Leuconostoc mesenteroides mixed with yeast Candida humilis and added at 7.5, 15, 22.5 or 30% on bread dough basis. The choice of sourdough starter significantly affected bread acidity characteristics, flavour, specific volume, shape and crumb firmness. The sourdough amount and acetic acid content of bread inversely correlated to flavour score, specific volume, shape and crumb softness. The overall quality of PBF wholewheat bread was most efficiently improved after adding Lb. plantarum sourdough at 15–22.5%, resulting in retarded firming rate (74–94%) and improved specific volume (25–28%) in comparison with PBF bread without sourdough. Such sourdough has lactic to acetic acid higher than previously recommended for traditional sourbreads.     

Identification of lactic acid bacteria isolated from oat sourdoughs and investigation into their potential for the improvement of oat bread quality

The use of sourdough in wheat and rye breads has been extensively studied; however, little is known about its potential effect when baking oat bread. Consequently, the impact of sourdough on oat bread quality was investigated. Two different sourdoughs were prepared from wholegrain oat flour without the addition of starter cultures, by continuous propagation at 28 (SD 28) or 37 °C (SD 37) until the composition of the lactic acid bacteria remained stable. The dominant LAB were identified by sequence analysis of the 16S rDNA isolated from pure cultures. LAB from SD 28 belonged to the species Leuconostoc argentinum, Pedicoccus pentosaceus and Weissella cibaria, while Lactobacillus coryniformis dominated SD 37. The isolated LAB were further used as starter cultures for the production of oat sourdoughs. Fundamental rheology revealed softening of the sourdoughs compared to non-acidified and chemically acidified controls, which could not be attributed to proteolytic activity. Incorporation of oat sourdough into an oat bread recipe resulted in significantly increased loaf-specific volume as well as improved texture, independent of addition level or sourdough type. Overall, the results of this study show that sourdoughs containing lactic acid bacteria isolated from oats have the potential to enhance oat bread quality.     

Fundamental study on the effect of hydrostatic pressure treatment on the bread-making performance of oat flour

The use of sourdough in wheat and rye breads has been extensively studied; however, little is known about its potential effect when baking oat bread. Consequently, the impact of sourdough on oat bread quality was investigated. Two different sourdoughs were prepared from wholegrain oat flour without the addition of starter cultures, by continuous propagation at 28 (SD 28) or 37 °C (SD 37) until the composition of the lactic acid bacteria remained stable. The dominant LAB were identified by sequence analysis of the 16S rDNA isolated from pure cultures. LAB from SD 28 belonged to the species Leuconostoc argentinum, Pedicoccus pentosaceus and Weissella cibaria, while Lactobacillus coryniformis dominated SD 37. The isolated LAB were further used as starter cultures for the production of oat sourdoughs. Fundamental rheology revealed softening of the sourdoughs compared to non-acidified and chemically acidified controls, which could not be attributed to proteolytic activity. Incorporation of oat sourdough into an oat bread recipe resulted in significantly increased loaf-specific volume as well as improved texture, independent of addition level or sourdough type. Overall, the results of this study show that sourdoughs containing lactic acid bacteria isolated from oats have the potential to enhance oat bread quality.     

Propionic acid production by cofermentation of Lactobacillus buchneri and Lactobacillus diolivorans in sourdough

Cooperative metabolism of lactobacilli in silage fermentation converts lactate to propionate. This study aimed to determine whether propionate production by Lactobacillus buchneri and Lactobacillus diolivorans can be applied for bread preservation. Propionate formation was observed in cofermentation with L. buchneri and L. diolivorans in modified MRS broth as well as sourdough with low, medium and high ash contents. 48 mM of propionate was formed in sourdough with medium ash content, but only 9 and 28 mM propionate were formed in sourdoughs prepared from white wheat flour or whole wheat flour, respectively. Acetate levels were comparable in all three sourdoughs and ranged from 160 to 175 mM. Sourdough fermented with L. buchneri and L. diolivorans was used in breadmaking and its effect on fungal spoilage was compared to traditional sourdough or propionate addition to straight doughs. Bread slices were inoculated with Aspergillus clavatus, Cladosporium spp., Mortierella spp. or Penicillium roquefortii. The use of 20% experimental sourdough inhibited growth of three of the four moulds for more than 12 days. The use of 10% experimental sourdough deferred growth of two moulds by one day. Bread from traditional sourdough with added acetate had less effect in inhibiting mould growth.     

Biodiversity of lactic acid bacteria and yeasts in spontaneously-fermented buckwheat and teff sourdoughs

In this study, four different laboratory scale gluten-free (GF) sourdoughs were developed from buckwheat or teff flours. The fermentations were initiated by the spontaneous biota of the flours and developed under two technological conditions (A and B). Sourdoughs were propagated by continuous back-slopping until the stability was reached. The composition of the stable biota occurring in each sourdough was assessed using both culture-dependent and -independent techniques. Overall, a broad spectrum of lactic acid bacteria (LAB) and yeasts species, belonging mainly to the genera Lactobacillus, Pediococcus, Leuconostoc, Kazachstania and Candida, were identified in the stable sourdoughs. Buckwheat and teff sourdoughs were dominated mainly by obligate or facultative heterofermentative LAB, which are commonly associated with traditional wheat or rye sourdoughs. However, the spontaneous fermentation of the GF flours resulted also in the selection of species which are not consider endemic to traditional sourdoughs, i.e. Pediococcus pentosaceus, Leuconostoc holzapfelii, Lactobacillus gallinarum, Lactobacillus vaginalis, Lactobacillus sakei, Lactobacillus graminis and Weissella cibaria. In general, the composition of the stable biota was strongly affected by the fermentation conditions, whilst Lactobacillus plantarum dominated in all buckwheat sourdoughs. Lactobacillus pontis is described for the first time as dominant species in teff sourdough. Among yeasts, Saccharomyces cerevisiae and Candida glabrata dominated teff sourdoughs, whereas the solely Kazachstania barnetti was isolated in buckwheat sourdough developed under condition A. This study allowed the identification and isolation of LAB and yeasts species which are highly competitive during fermentation of buckwheat or teff flours. Representatives of these species can be selected as starters for the production of sourdough destined to GF bread production.     

Optimization of sourdough process for improved sensory profile and texture of wheat bread

The aim of the study was to determine optimum sourdough process conditions for improved flavour and texture of wheat bread. The influence of process conditions and the starter culture on the characteristics of wheat sourdough bread was established by using response surface methodology. Influence of fermentation temperature (16-32 °C), ash content of flour (0.6-1.8 g/100 g), and fermentation time (6-20 h) were considered as independent factors and their effects were studied in sourdough bread fermented with Lactobacillus plantarum, Lactobacillus brevis, Saccharomyces cerevisiae or with a combination of yeast and lactic acid bacteria. Intensity of sensory attributes, specific volume and bread hardness were considered as the main responses. Ash content of flour and fermentation time were the main factors determining the intensity of sensory attributes. The possibility to enhance intensity of overall flavour, aftertaste and roasted flavour without excessive pungent flavour and without reduced fresh flavour in wheat bread containing 20 g sourdough/100 g of wheat dough was demonstrated by choosing e.g. Lb. brevis for a starter and by utilization of high ash content of flour, long fermentation time and reduced temperature. Bread specific volume was improved 0.2-0.5 ml/g and hardness was reduced (after 4 days of storage) up to 260 g by using low ash content of flour and by optimizing fermentation time according to the microbial strain. Lactic acid fermentation had more profound influence on both desired and undesired flavour attributes, as well as textural features of bread in comparison with yeast fermentation.     

On the microbiological profile of traditional Portuguese sourdough

Traditional manufacture of bread from maize has been noted to play important roles from both economic and social standpoints; however, enforcement of increasingly strict hygiene standards requires thorough knowledge of the adventitious microbiota of the departing dough. To this goal, sourdough as well as maize and rye flours from several geographic locations and in two different periods within the agricultural year were assayed for their microbiota in sequential steps of quantification and identification. More than 400 strains were isolated and taxonomic differentiation between them was via Biomerieux API galleries (375 of which were successfully identified) following preliminary biochemical and morphological screening. The dominant groups were yeasts and lactic acid bacteria (LAB). The most frequently isolated yeasts were Saccharomyces cerevisiae and Candida pelliculosa. The most frequently isolated LAB were (heterofermentative) Leuconostoc spp. and (homofermentative) Lactobacillus spp.; L. brevis, L. curvatus, and L. lactis ssp. lactis were the dominant species for the Lactobacillus genera; Lactococcus lactis ssp. lactis for lactococci; Enterococcus casseliflavus, E. durans, and E. faecium for enterococci; and Streptococcus constellantus and S. equinus for streptococci.     

Prevalence and impact of single-strain starter cultures of lactic acid bacteria on metabolite formation in sourdough

Flavour of type II sourdoughs is influenced by the ingredients, processing conditions, and starter culture composition. It is, however, not fully clear to what extent different sourdough lactic acid bacteria (LAB) contribute to flavour. Therefore, two types of flour (rye and wheat) and different LAB starter culture strains were used to prepare sourdoughs, thereby leaving the yeast microbiota uncontrolled. All LAB starter culture strains tested were shown to be prevalent and to acidify the flour/water mixture to pH values between 3.1 and 3.9 after 24 h of fermentation. Multiple aldehydes, alcohols, ketones, and carboxylic acids were produced by the sourdough-associated microbiota throughout the fermentation period. Based on the organoleptic evaluation of breads produced with these sourdoughs, five LAB strains were selected to perform prolonged wheat and rye fermentations as to their capacity to result in an acidic (Lactobacillus fermentum IMDO 130101, Lactobacillus plantarum IMDO 130201, and Lactobacillus crustorum LMG 23699), buttermilk-like (Lactobacillus amylovorus DCE 471), or fruity flavour (Lactobacillus sakei CG1). Upon prolonged fermentation, higher metabolite concentrations were produced. For instance, L. sakei CG1 produced the highest amounts of 3-methyl-1-butanol, which was further converted into 3-methylbutyl acetate. The latter compound resulted in a fruity banana flavour after 48 h of fermentation, probably due to yeast interference. Rye fermentations resulted in sourdoughs richer in volatiles than wheat, including 3-methyl-1-butanol, 2-phenylethanol, and ethyl acetate.     

Effects of wheat sourdough process on the quality of mixed oat-wheat bread

The aim of this work was to study the effects of sourdough fermentation of wheat flour with Lactobacillus plantarum, on the quality attributes of mixed oat-wheat bread (51 g whole grain oat flour and 49 g/100 g white wheat flour). Emphasis was laid both on β-glucan stability as well as bread structure and sensory quality. The variables of the sourdough process were: dough yield (DY), fermentation time, fermentation temperature, and amount of sourdough added to the bread dough. The sourdough process was shown to be a feasible method for mixed oat-wheat bread, and, when optimized, provided bread quality equal to straight dough baking. A small amount (10g/100 g dough) of slack sourdough fermented at high temperature for a long time resulted in the most optimal sourdough bread with the highest specific volume (3.5 cm³/g), the lowest firmness after 3 days storage (0.31 kg), and low sensory sourness with high intensity of the crumb flavour. Wheat sourdough parameters did not affect the content of oat β-glucan in the bread. Additionally, both straight dough and sourdough bread contained 1.4-1.6 g β-glucan/100 g fresh bread. The average molecular weight of β-glucan was 5.5 × 10⁵ in both types of bread, while that of oat flour was 10 × 10⁵. This indicates that a slight degradation of β-glucan occurred during proofing and baking, and it was not affected by variation in the acidity of the bread between pH 4.9-5.8.