Performance of <Emphasis Type="Italic">Lactobacillus sanfranciscensis</Emphasis> TMW 1.392 and its levansucrase deletion mutant in wheat dough and comparison of their impact on bread quality

The performance of Lactobacillus sanfranciscensis TMW 1.392 and its levansucrase deletion mutant TMW 1.392 Δlev in wheat dough was compared. The effects of both strains on dough and bread characteristics were determined in order to find benchmarks for in situ production of exopolysaccharides (EPS). Growth and acidification were lower in doughs prepared with the Δlev mutant than in those employing the wild type. Extensogram resistance of the dough was reduced and extensibility increased with the addition of L. sanfranciscensis levan. Added EPS positively influenced water absorption, bread volume and firming of the crumb. In situ production of EPS was not sufficient to achieve the same positive effects of EPS, as they partially overlapped with effects resulting from enhanced acidification. Control doughs were made to separate effects of predough, EPS and different metabolism/acidification. High acetic acid levels decreased extensibility and volume. High lactic acid levels negatively influenced crumb hardness and firming kinetics. The use of knock out mutants proved helpful to judge overall performance of a strain, although the interpretation of specific effects must consider all changes in its metabolism.     

Influence of pH on the Formation of Glucan by Lactobacillus reuteri TMW 1.106 Exerting a Protective Function Against Extreme pH Values

Lactobacillus reuteri TMW 1.106, a dominant type II sourdough bacterium, produces glucan from sucrose in vitro and in situ. Exopolysaccharides positively affect the texture and mouth feel of foods and their in situ production in fermented foods could be an alternative to the addition of hydrocolloids from plants or non-GRAS microorganisms. The aim of this study was to elucidate a probable function of the EPS for the bacterium. Lb. reuteri TMW 1.106 harbors two glucosyltransferases, Gtf106A and Gtf106B and produces a dextran. Gtf106B exhibited hydrolysis but no transferase activity. Enzymatic production of dextran with the heterologously expressed, N-terminally truncated ΔN Gtf106A was highest at a pH of 4.0, whereas dextran formation in pH static fermentations was optimal between pH 4.7 and 5.4. The dextran synthesised at these pH values had the highest molecular mass (1.2 × 10⁷) and 15% α–(1–4) linkages. A protective effect of this EPS on Lb. reuteri TMW 1.106 against low pH, explaining the low pH-production maximum, could be demonstrated through the delay of cell death.     

Impact of ecological factors on the stability of microbial associations in sourdough fermentation

The limits for the stability of the microbial association 1 (Lactobacillus sanfranciscensis and Candida humilis) and association 2 (Lactobacillus reuteri, Lactobacillus johnsonii and Issatchenkia orientalis) during sourdough fermentation were evaluated by investigating the effects of the ecological factors substrate, refreshment time, temperature, amount of backslopping and competing species in different combinations on their growth. Sourdoughs were fermented in 28 batches under different conditions using the associations and possible competing strains as starters. The dominating microbiota was characterized by bacteriological culture, rRNA gene sequence analysis and RAPD-PCR. Association 1 was found to be competitive in doughs with rye and wheat flour at temperatures between 20 and 30 °C, refreshment times of 12 and 24 h, amounts of backslopping dough from 5 to 20% and against all competing lactic acid bacteria and yeasts. The processing parameters for the competitiveness of the association 2 were temperatures of 35-40 °C, refreshment times of 12-24 h and the substrates rye bran, wheat and rye flour, but not in every case. Issatchenkia orientalis could only grow when enough oxygen was available. Its cell counts fell rapidly under the limit of detection when using high amounts of doughs (small ratio of surface to volume) and refreshment times of 12 h. In conclusion, the results demonstrated that the two associations were remarkably stable under most of the investigated process conditions.     

The bacteriocin producer Lactobacillus amylovorus DCE 471 is a competitive starter culture for type II sourdough fermentations

Type II sourdoughs were prepared using Lactobacillus amylovorus DCE 471, a producer of the bacteriocin amylovorin L. The strain was used as a starter culture for rye and wheat sourdoughs on laboratory scale (10 L), and in rye sourdough on pilot scale (100 L). The sourdoughs were acidified to a pH of around 3.5 within 15 h (laboratory dough) to 25 h (pilot‐scale dough). Final amylovorin L titres of 0.3–0.4 (laboratory scale) and 0.2 (pilot scale) MAU kg^?1 of sourdough were detected. After baking of wheat dough that was supplemented with the pilot‐scale sourdough, no amylovorin L activity was recovered from the breadcrumbs. On laboratory scale, aeration or the addition of complex carbohydrates hardly affected growth or amylovorin L production. Rye and wheat sourdough fermentation were rather similar despite differences in sugar concentrations. The persistence of L. amylovorus DCE 471 during rye sourdough fermentation, both on laboratory and pilot scale, was confirmed by repetitive sequence‐based polymerase chain reaction (rep‐PCR) and by testing isolates towards an amylovorin L‐sensitive organism. Further, rep‐PCR indicated that the background microbiota of the flour—probably responsible for the production of low amounts of acetic acid—grew poorly and were overgrown by L. amylovorus DCE 471 during the pilot‐scale fermentation. Copyright © 2007 Society of Chemical Industry     

Effects of enzymes in fibre-enriched baking

The aim of the present study was to improve the quality of fibre-enriched wheat breads by enzymic treatment of the fibre fraction. The suitability of different enzymes in fibre-enriched baking and their effects on the dietary fibre content and the ratio of insoluble: soluble fibre content of the breads were studied. The enzyme preparations used were a hemicellulolytic culture filtrate of Trichoderma reesei, a specific (pI 9) xylanase of T reesei and Fermizyme, an α-amylase preparation containing a standardised level of hemicellulase activity. Rye bran was extracted in water (10% (w/w) suspension) to determine the solubilities of the β-glucans and pentosans. Addition of T reesei culture filtrate significantly increased the amount of extractable pentosan obtained from nonautoclaved rye bran. Rye bran supplementation (5%) of wheat flour increased the farinograph absorption and dough development time, but had little or no effect on stability and softening of the dough. The added enzymes decreased dough stability and increased softening. Addition of enzymes caused significant differences in the stickiness of the wheat doughs both with (P<0·003) and without (P<0·001) rye bran. Fermizyme significantly increased the stickiness of wheat doughs both with and without rye bran. The baking results of the fibre-enriched breads were improved by the added enzymes. Addition of T reesei culture filtrate increased the specific volume of the wheat breads both with and without rye bran by almost 20%. Enzyme mixtures were more efficient than individual xylanase in softening the bread crumb and reducing the staling rate of wheat breads both with and without rye bran. Incorporation of enzymes reduced the total dietary fibre content of the breads, but at least doubled the amount of soluble pentosan. The proportions of fluorescent cell walls in the breads were detected by microscopical image analysis. Enzyme addition caused the surface area of insoluble cell walls originating from wheat flours to decrease, suggesting that the enzymes exert more effects on wheat endorsperm cell walls than on bran particles. © 1998 SCI.     

Phytate degradation by Bifidobacterium on whole wheat fermentation

Whole cereal-based products have a beneficial effect on health, but they also contain high levels of phytate, defined as anti-nutrient. The possible use of different strains of bifidobacterial species (B. catenulatum, B. longum and B. breve) with phytate degrading activity as starters in the fermentation process was investigated. Fermentative parameters of doughs (pH, volume, total titrable acidity [TTA] and acetic and lactic acids production) and the hydrolysis profile of the phytic acid were determined during whole wheat dough fermentation. During fermentation in the presence of different bifidobacterial strains, the concentration of phytic acid showed a progressive decrease, leading to a higher release of hydrolysis products than in the control, within short fermentation time. According to the fermentative parameters bifidobacterial strains showed a good adaptation to the dough ecosystem. The range of TTA and lactic acid production was dependent on the strain. Among the tested bifidobacterial strains, those belonging to the species B. breve and B. longum induced a higher hydrolysis of phytic acid with simultaneous production of lower inositol phosphates. In addition, dough containing those strains had high pH and minor acidity than those containing a commercial starter (L. plantarum). Therefore, the tested bifidobacterial strains could be used as breadmaking starters contributing to different acidification degrees and promoting simultaneously the degradation of phytic acid in the whole wheat dough.     

Identification of Lactobacillus curvatus TMW 1.624 dextransucrase and comparative characterization with Lactobacillus reuteri TMW 1.106 and Lactobacillus animalis TMW 1.971 dextransucrases

Recently, it was affirmed that the exopolysaccharides (EPSs) of Lactobacillus curvatus TMW 1.624, Lactobacillus reuteri TMW 1.106 and Lactobacillus animalis TMW 1.971 improve the quality of gluten-free breads and that they can be produced in situ to levels enabling baking applications. In this study we provide insight into the molecular and biochemical background of EPS production of these three strains. EPS formation strongly correlated with growth and took place during the exponential phase. Gtf genes were heterologously expressed, purified and their enzymatic properties as well as the structures of the EPSs formed were compared. Structural comparison of EPS formed by heterologously expressed glucosyltransferases (Gtfs) and of those formed by the wildtype lactobacilli confirmed that the respective genes/enzymes were identified and examined. The glucan formed by L. animalis Gtf was identified as a linear low molecular weight dextran. Optimal enzymatic conditions were pH 4.4 and 45 °C for the L. reuteri Gtf and pH 4.4 and 31 °C for L. curvatus Gtf. The Gtf from L. animalis had an optimal pH of 5.8 and displayed more than 50% of activity over a broad temperature profile (22–59 °C). The three Gtfs were stimulated by various mono- and divalent metal ions, dextran, as well as levan to different extents.     

Lactobacillus reuteri CRL 1100 as starter culture for wheat dough fermentation

The effect of sucrose on the fermentation balance of Lactobacillus reuteri CRL 1100 and the invertase activity of this strain in wheat dough and culture medium (MRSs) was evaluated. The enzyme activity was dependent on the environmental pH releasing glucose and fructose from sucrose hydrolysis. Glucose was used as carbon source, while fructose was mainly used as electron acceptor to produce mannitol up to 10h of fermentation. Thereafter, fructose seemed to be metabolized by the heterofermentative pathway, which determined an increase in the concentration of acetate (6 mmol l(-1)), lactate (2 mmol l(-1)) and ethanol (1 mmol l(-1)) and the lack of mannitol formation after glucose depletion. The fermentation balance of Lb. reuteri CRL 1100 during the dough fermentation resulted in lower (63%) ethanol, higher (75%) acetate production and soluble carbohydrates concentrations, like MRSs cultures. This fermentation profile would be important to obtain an optimal growth of yeast and the optimal bread flavor and taste.     

乳酸菌固态发酵过程中麦麸组分的动态变化

麦麸是小麦加工的主要副产物,营养丰富且产量大,采用乳酸菌处理麦麸可提高其附加值。为明确乳酸菌发酵对麦麸各组分的影响,作者采用植物乳杆菌、鼠李糖乳杆菌、戊糖片球菌和布氏乳杆菌分别对麦麸进行固态发酵,在48 h内每隔8 h取样,分析可溶性膳食纤维、粗蛋白质、淀粉、总酚、植酸等成分的质量分数及DPPH自由基清除能力的动态变化。结果表明,在麦麸基质中,4株乳酸菌在24 h内生长较为迅速;麦麸经乳酸菌发酵后可溶性膳食纤维质量分数显著提高,其中布氏乳杆菌发酵48 h后可溶性膳食纤维质量分数由4.72%增加至6.58%;随着发酵时间的增加,麦麸中淀粉质量分数逐渐降低,粗蛋白质量分数先增加后降低最后趋于稳定;植物乳杆菌在提高麦麸多酚质量分数方面有更好的效果,多酚质量分数由1.34 mg/g增加至3.86 mg/g,麦麸抗氧化活性显著增加;此外,乳酸菌发酵麦麸可显著降低其植酸质量分数。综合而言,植物乳杆菌和布氏乳杆菌在提高麦麸的营养特性方面具有较好的效果,可有效改善麦麸的综合利用价值。     

Identification and characterization of a glucan-producing enzyme from Lactobacillus hilgardii TMW 1.828 involved in granule formation of water kefir

Water kefir is a home made fermented beverage based on a sucrose solution with fruit extracts. The inoculum of such fermentations consists of macroscopic granula containing lactic and acetic acid bacteria, and yeasts, which are embedded in an exopolysaccharide (EPS) matrix. In this work, a strain of Lactobacillus hilgardii producing large amounts of the granule-forming dextran could be isolated. The glycosyltransferans (Gtf) commonly called glucansucrase responsible for the production of this dextran was purified from L. hilgardii. Characteristic enzyme kinetic data were obtained. Optimum activity was observed between pH 4.3 and 4.6 and temperatures between 40 °C and 45 °C. A Michaelis–Menten kinetic could be fit to the experimental data and a K_M of 0.0385 M was calculated. The corresponding gtf gene was identified and characterized. It encodes a 1448 amino acid protein with higher homologies to Gtfs of Lactobacillus parabuchneri, Lactobacillus sakei and Lactobacillus fermentum followed by lower homologies to Lactobacillus reuteri Gtfs. By knockout experiments the role of this gene in granule dextran production was demonstrated.     

Structural and rheological characterisation of heteropolysaccharides produced by lactic acid bacteria in wheat and sorghum sourdough

Hydrocolloids improve the volume, texture, and shelf life of bread. Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) during sourdough fermentation can replace hydrocolloids. It was the aim of this study to determine whether heteropolysaccharides (HePS) synthesized intracellularly from sugar nucleotides by glycosyltransferases are produced in wheat and gluten-free sorghum sourdough at effective levels. The HePS-producing strains Lactobacillus casei FUA3185, L. casei FUA3186, and Lactobacillus buchneri FUA3154 were used; Weissella cibaria 10M producing no EPS in the absence of sucrose served as control strain. Cell suspensions of L. buchneri in MRS showed the highest viscosity at low shear rate. Glycosyltransferase genes responsible of HePS formation in LAB were expressed in sorghum and wheat sourdough. However, only HePS produced by L. buchneri influenced the rheological properties of sorghum sourdoughs but not of wheat sourdoughs. Sorghum sourdough fermented with L. buchneri exhibited a low |G^∗| compared to the control, indicating a decrease in resistance to deformation. An increase in tan δ indicated decreased elasticity.The use of LAB producing HePS expands the diversity of EPS and increases the variety of cultures for use in baking.     

Headspace flavour compounds produced by yeasts and lactobacilli during fermentation of preferments and bread doughs.

Production of volatile flavour compounds during fermentation with pure cultures of Saccharomyces cerevisiae and Candida guilliermondii, Lactobacillus brevis and Lactobacillus plantarum have been investigated, using wheat doughs and several preferements as substrates. For yeast, preferments consisted of 10% (w/v) glucose, maltose and sucrose solutions, whereas for lactobacilli they consisted of supplemented and unsupplemented (3% and 10% (w/v)) glucose solutions, and a 10% (w/v) wheat flour slurry. Seven volatile compounds (acetaldehyde, acetone, ethyl acetate, ethanol, hexanal+isobutyl alcohol, and propanol) were detected when using yeasts. All these compounds, except propanol, appeared for all the substrates assayed, with ethanol as the predominant component. Generally, S. cerevisiae produced higher amounts of the different components than C. guilliermondii. Both yeasts produced larger amounts of volatile flavour compounds during fermentation in glucose and sucrose solutions than in maltose or wheat dough. In general the yeasts examined produced more flavour components than the lactobacilli. For the lactobacilli the highest number of volatile flavour compounds were observed for substrates containing flour.     

Amino Acid Metabolism by Yeasts and Lactic Acid Bacteria during Bread Dough Fermentation

Metabolism of 22 free amino acids (AA) during fermentation(early and later steps) of wheat dough samples started with pure and associated cultures of yeast and lactic acid bacteria, and commercial compressed yeast (CY) were investigated. Unfermented and fermented straight doughs were studied byReversed-Phase HPLC of their dansyl derivatives. Used as starters were strains of Saccharomyces cervisiae, Lactobacillus brevis, Lactobacillus plantarum and Enterococcus faecium. Statistical data analysis indicated clustering of samples with yeast and samples without yeast (including uninoculated doughs) respectively, based on rate of metabolism of acidiC., basiC., aliphatic and aromatic A A. Differences in AA metabolism during the later fermentation step (DF2) categorized bacterial starters into groups according to balance between degree of assimilation(D F2<0) and exoproteolytic release(DF2>0) of amino acids.     

Comparison of the effect of exopolysaccharide-producing lactic acid bacteria from sourdough on dough characteristics and steamed bread quality

The use of exopolysaccharide (EPS)-producing lactic acid bacteria (LAB) is promising in sourdough fermentation. However, the knowledge of the effects of various species of LAB on steamed bread making remains limited. In this study, the effects of two LAB with high EPS-producing capacity, namely Weissella cibaria L32 and Lactobacillus brevis L17 on dough fermentation and steamed bread quality were estimated. The addition of these two LAB strains significantly increased the titratable acidity and protease activity during the dough fermentation, especially L. brevis L17. Although the in situ EPS synthesised by LAB could improve the steamed bread quality, excessive acidification of L. brevis L17 would still increase the protease activity and thus destroy its gluten network structure. As a result, the steamed bread fermented with L. brevis L17 had the lowest specific volume and hardest texture in comparison with the steamed bread fermented with W. cibaria L32 and with added EPS produced by W. cibaria L32 and L. brevis L17. These results indicated that different EPS-producing LAB exhibited distinctive dough fermentation characteristics, and the in situ EPS-producing W. cibaria L32 could improve steamed bread quality, which confirmed its potential application in steamed bread making.     

Development of fermented instant Chinese noodle using Lactobacillus plantarum

A new-type of instant Chinese noodle was developed with the application of lactic acid fermentation by lactobacilli. Since the pH value of the noodle sheets is alkaline with kansui (around 8.5), alkaline tolerance is required for the lactobacilli to ferment noodle sheets. The screening of the lactobacilli strains suitable for the fermentation was conducted using 46 strains from 12 species (including subspecies) of lactobacilli. Several strains of Lactobacillus pentosus and Lactobacillus plantarum were found to be fermenters. Among these, L. plantarum NRIC 0380, that showed the highest fermentation rate and favorable modification of noodle, was selected as the best strain, and was employed for the pilot scale manufacture of instant Chinese noodle. During fermentation, L. plantarum NRIC 0380 produced lactic acid to about 11 g/kg noodle sheet after 24 h with a concomitant pH decrease from an initial of about 7.9 down to 3.9. Sensory test after rehydration with boiled water revealed that the fermented instant Chinese noodle sheets at pH 7.5 had increased hardness, elasticity and light sour taste.     

The sourdough microflora Microbiological, biochemical and breadmaking characteristics of doughs fermented with freeze-dried mixed starters, freeze-dried wheat sourdough and mixed fresh-cell starters

Freeze-dried mixed starters, freeze-dried wheat sourdough and mixed fresh-cell starters made withLactobacillus sanfrancisco CBI,L. plantarum DC400 andSaccharomyces cerevisiae 141 and/orS. exiguus M14 were used for leavening wheat doughs, and their microbiological, biochemical and breadmaking characteristics were compared with those of Italian traditional doughs produced by baker's yeast. All the doughs fermented with starters had more balanced microbiological and biochemical characteristics than dough started with baker's yeast in which alcoholic fermentation end-products largely predominated. By using starters, the greatest lactic acid bacteria cell number and acetic acid production, were achieved, along with more complete profiles of volatile compounds and greater structural stability of fermented doughs. Fresh-cell starters showed higher microbial functionality and represented the only way to enrich the doughs withS. exiguus M14, some of which survived the freeze-drying process. No differences were detected between the two different types of freeze-dried starters and the subsequent use (10 times) of doughs initially produced with freezedried starters eliminated initial differences in the microbial functionality with respect to fresh-cell starters.     

Co-metabolism of citrate and maltose byLactobacillus brevis subsp.lindneri CB1 citrate-negative strain: effect on growth, end-products and sourdough fermentation

Growth, substrates and end-product formation of the maltose and citrate co-metabolization byLactobacillus brevis subsp.lindneri CB1 citrate-negative strain were initially studied in synthetic medium. Compared to maltose (19 g/l) fermentation, the co-metabolization of maltose (10 g/l) plus citrate (9 g/l) caused faster cell growth, increased the concentrations of lactic acid and especially of acetic acid (from 0.7 g/l to 2.9 g/l), produced succinic acid (0.5 g/l) and reduced ethanol synthesis. Highest activities of acetate kinase, the same of lactate dehydrogenase and a reduced alcohol dehydrogenase activity were detected in cytoplasmic extracts of cells growing on maltose plus citrate. The breakdown of citrate depended upon the continuous presence of maltose in the growth medium. Upon depletion of citrate, the cells continued through the normal maltose fermentation, having a diauxic metabolic curve as shown by impedance measurements. Concentrations of citrate from 3 g/l to 15 g/l led to increases of acetic acid from 1.25 g/l to 5.55 g/l. Since maltose was naturally present during sourdough fermentation, the addition of 9 g citrate per kg wheat dough enabled the co-metabolization of maltose and citrate byL. brevis subsp.lindneri CB1. Compared with traditional sourdough fermentation, faster cell growth, a higher acetic acid concentration and a reduced quotient of fermentation were obtained by co-metabolism.     

Study of the behaviour of Lactobacillus plantarum and Leuconostoc starters during a complete wheat sourdough breadmaking process

The acidification properties, metabolic activity and technological performance of four individual Lactobacillus plantarum or Leuconostoc freeze-dried starters were investigated during a complete wheat sourdough breadmaking process including 0.2 g/100 g baker's yeast. Microbiological contents (lactic acid bacteria and yeasts), acidification characteristics (pH and total titratable acidity), soluble carbohydrates (maltose, glucose and fructose) and fermentative end-products (lactic and acetic acids, ethanol) contents were evaluated during both sourdough and corresponding bread dough fermentation. Biochemical and technological analysis of the resulting bread products are also presented. Some differences among strains in acidification properties and soluble carbohydrates availability were outlined both in sourdough and bread dough. Each individual Leuconostoc or Lb. plantarum starter was able to produce a characteristic fermentation and was found to ensure the production of breads with overall satisfactory acceptance.