Use of selected sourdough lactic acid bacteria to hydrolyze wheat and rye proteins responsible for cereal allergy

This work was aimed at showing the capacity of selected sourdough lactic acid bacteria to hydrolyze wheat and rye allergens. Hydrolysis was investigated after wheat sourdough fermentation and after treatment of wheat and rye sourdough breads with pepsin, trypsin and pancreatin, which mimicked the digestive process. As shown by immunoblotting with sera from allergic patients, wheat sourdough fermentation caused the disappearance of some IgE-binding proteins (albumins/globulins and gliadins mainly) with respect to the chemically acidified dough used as the control. The IgE-binding protein profile of wheat and rye sourdough breads differed from those of baker's yeast breads. The signals of the IgE-binding proteins contained in the sourdough breads disappeared after in vitro digestion with pepsin, trypsin and pancreatin. The same effect by digestive enzymes was not found for baker's yeast breads which showed persistent IgE-binding proteins. As shown by ELISA inhibition assays, the presence of IgE-binding low molecular weight proteins/peptides in sourdough breads significantly decreased with respect to baker's yeast breads. Proteolytic activity by selected sourdough lactic acid bacteria may have an importance during food processing to produce pre-digested wheat and rye dough which contains IgE-binding proteins degradable by digestive enzymes.     

Defined multi-species semi-liquid ready-to-use sourdough starter

This paper aimed at setting up a protocol for the manufacture of a defined multi-species semi-liquid ready-to-use sourdough starter consisting of lactic acid bacteria (LAB) selected on the basis of their capacity of rapid acidification and adaptation to several technological factors. Preliminarily, 56 strains of sourdough LAB were screened based on the acidification kinetics during sourdough fermentation. The influence of temperature (25-37 degrees C), NaCl (2%, w/w, of wheat flour), initial cell number (10(7)-10(9) cfu g(-1)), dough yield (150-180), substrate for cell cultivation (mMRS or SDB media) and phase of cell growth (exponential or stationary phases) on the kinetics of acidification was also determined. Lactobacillus casei DPPMA27, Weissella confusa DPPMA20 and Lactobacillus fructivorans DPPMA8 were selected and used to produce a defined multi-species semi-liquid sourdough starter. Fermentation was carried out in a 2l batch fermentation bioreactor (12 h at 37 degrees C), under controlled pH, by cultivating cells in a medium made of water (60%), flour and other nutrients (40%). The semi-liquid sourdough starter was stored for 30 days under different conditions and used for dough fermentation (dough yield 160). The microbial composition, acidifying activity and fermentation end-products were kept constant during 21 days of storage. The findings of this study may be useful to increase the use of sourdough in bakery industries.     

The use of sourdough fermented by antifungal LAB to reduce the amount of calcium propionate in bread

Addition of sourdough is a common practice in the bakery industry to improve, among other quality parameters, the shelf life of bread. In this study, sourdough fermented by antifungal Lactobacillus plantarum strains was investigated for the ability to inhibit growth of common bread spoilage fungi. In both in vitro and sourdough wheat bread system, the antifungal sourdoughs significantly affected the outgrowth of Aspergillus niger, Fusarium culmorum, or Penicillium expansum spores, however on wheat bread outgrowth of Penicillium roqueforti spores was not affected. In an attempt to reduce the amounts of chemical additives in bread, the antifungal sourdoughs were used in combination with calcium propionate (CAP) and possible synergistic effects were evaluated. Presence of 3000 ppm CAP in the bread did not affect the outgrowth of P. roqueforti, whereas outgrowth of the other fungi was retarded. A strong synergistic effect was observed when CAP and antifungal sourdoughs were combined into the bread formulation, and outgrowth of P. roqueforti was affected. The use of reduced CAP amount (1000 ppm) showed significant inhibition only when antifungal sourdough was added. Remarkably, the increase in shelf life achieved was higher than that obtained using 3000 ppm of CAP alone. In conclusion, the results of this study clearly show that the addition of antifungal sourdough has the potential to reduce the levels of chemical additives needed in the bakery industry to ensure the microbiological safety of bread.     

酸面团发酵技术应用研究进展

酸面团发酵技术是一种传统的生物加工技术,但目前又拓展了新的内容。本文介绍了酸面团发酵技术在提高发酵产品的营养价值、改善焙烤产品的流变学特性与感官品质、延长产品的货架期、制备低血糖生成指数的面包及研制可供乳糜泻患者安全食用的低致敏焙烤产品等方面的应用,并对目前酸面团发酵技术所具有的优势和存在的问题进行了总结和分析。总之,酸面团发酵技术在食品工业中的应用前景广阔,值得深入研究。      

Effect of sourdough fermentation on stabilisation,and chemical and nutritional characteristics of wheat germ

Lactic acid bacteria strains were identified from wheat germ by 16S rRNA partial sequencing, subjected to RAPD-PCR typing and screened. Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5 were used as starters to produce sourdough fermented wheat germ (SFWG). The chemical and nutritional characteristics of SFWG were compared to those of the raw wheat germ (RWG). Lipase activity in SFWG was ca. 2.6-fold lower than that found in RWG. As shown by SPME/GC/MS analysis, most of the volatile compounds derived from lipid oxidation during storage (40 days) were at markedly lower levels in SFWG compared to RWG. Fermentation of wheat germ increased of ca. 50% the concentration of free amino acids. Glu markedly decreased in SFWG, due to its conversion in GABA. The concentration of the anti-nutritional factor raffinose also decreased in SFWG. The in vitro protein digestibility, the concentration of total phenols, phytase and antioxidant activities were increased by fermentation.     

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.     

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.     

Generation of aroma compounds in sourdough: effects of stress exposure and lactobacilli-yeasts interactions

The effects of the interaction between Saccharomyces cerevisiae LBS and Lactobacillus sanfranciscensis LSCE1 and of their responses to acid, oxidative or osmotic stress on alcohol and aroma production were assessed. The exposure of S. cerevisiae LBS and L. sanfranciscensis LSCE1 cells to oxidative, acid or osmotic sub-lethal stress gave rise to a common or specific responses. Gamma-decalactone, 2(5H)-furanones and aldehydes were overproduced by LAB following oxidative stress. The acid stress induced both in yeasts and LAB, as well as in their co-cultures, a relevant accumulation of isovaleric and acetic acids and higher alcohols. A cross-exposure of yeasts and LAB to their preconditioned media, generated in S. cerevisiae a release of esters including esters of long-chain unsaturated fatty acids coming from membrane phospholipids. These esters were excreted also by yeasts following a pressure stress.     

Characteristics of bread making doughs: influence of sourdough fermentation on the fundamental rheological properties

The proofing process is an essential step in the bread making technology in providing a link between the bubble structure created in the mixer and the final baked loaf structure. This research was carried out to test the effect of two different types of fermentation on bread making dough properties. Fundamental rheological tests were used to evaluate and compare doughs prepared by using compressed yeast and only natural yeast. Dynamic oscillation tests were performed using a controlled stress--strain rheometer; the phase angle (δ) and the value of the storage modulus (G′) were measured for all samples. Significant differences were found between the doughs which were made using commercial compressed yeast (baker's yeast (BY)) and those prepared using sourdough (SD). In particular SD was less elastic, less firm and easily extensible having higher phase angle and lower G′.     

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.     

Emerging fermentation technologies: development of novel sourdoughs

The increasing knowledge of sourdough fermentation generates new opportunities for its use in the bakery field. New fermentation technologies emerged through in depth sourdough research. Dextrans are extracellular bacterial polysaccharides produced mainly by lactic acid bacteria (LAB). These bacteria convert sucrose thanks to an inducible enzyme called dextransucrase into dextran and fructose. The structure of dextran depends on the producing micro-organism and on culture conditions. Depending on its structure, dextran has specific properties which lead to several industrial applications in different domains. The use of dextran is not widely spread in the bakery field even if its impact on bread volume and texture was shown. A new process has been developed to obtain a sourdough rich in dextran using a specific LAB strain able to produce a sufficient amount of HMW dextran assuring a significant impact on bread volume. The sourdough obtained permits to improve freshness, crumb structure, mouthfeel and softness of all kinds of baked good from wheat rich dough products to rye sourdough breads. From fundamental research on dextran technology, a new fermentation process has been developed to produce an innovative functional ingredient for bakery industry.     

Biodiversity of lactic acid bacteria in French wheat sourdough as determined by molecular characterization using species-specific PCR

The lactic acid microflora of nine traditional wheat sourdoughs from the Midi-Pyrénées area (South western France) was previously isolated and preliminary characterized using conventional morphological and biochemical analysis. However, such phenotypic methods alone are not always reliable and have a low taxonomic resolution for identification of lactic acid bacteria species. In the present study, a total of 290 LAB isolates were identified by PCR amplification using different sets of specific primers in order to provide a thorough characterization of the lactic flora from these traditional French sourdoughs. Overall, the LAB isolates belonged to 6 genera: Lactobacillus (39%, 8 species), Pediococcus (38%, 1 species), Leuconostoc (17%, 2 species), Weissella (4%, 2 species), Lactococcus (1%, 1 species) and Enterococcus (< 1%, 1 species) and 15 different species were detected: L. plantarum, L. curvatus, L. paracasei, L. sanfranciscensis, L. pentosus, L. paraplantarum, L. sakei, L. brevis, P. pentosaceus, L. mesenteroides, L. citreum, W. cibaria, W. confusa, L. lactis and E. hirae. Facultative heterofermentative LAB represent more than 76% of the total isolates, the main species isolated herein correspond to L. plantarum and P. pentosaceus. Obligate heterofermentative lactobacilli (L. sanfranciscencis, L. brevis) represent less than 3% of the total isolates whereas Leuconostoc and Weissella species represent 21% of the total isolates and have been detected in eight of the nine samples. Detection of some LAB species was preferentially observed depending on the isolation culture medium. The number of different species within a sourdough varies from 3 to 7 and original associations of hetero- and homofermentative LAB species have been revealed. Results from this study clearly confirm the diversity encountered in the microbial community of traditional sourdough and highlight the importance of LAB cocci in the sourdough ecosystem, along with lactobacilli.     

乳酸菌细胞固定化发酵的研究进展

乳酸菌足能利用糖发酵产生乳酸的一类细菌,在食品、医药、工业、农业和科研等领域均有广泛的应用。本文综述了利用乳酸菌进行细胞固定化发酵生产乳酸、乳制品以及Nisin的国内外研究进展情况。并对乳酸菌固定发酵技术的应用前景进行了简要论述。     

Antifungal activity of sourdough fermented wheat germ used as an ingredient for bread making

This study aimed at investigating the antifungal activity of sourdough fermented (Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5) wheat germ (SFWG). Preliminarily, methanol and water/salt-soluble extracts from SFWG were assayed by agar diffusion towards Penicillium roqueforti DPPMAF1. As shown by hyphal radial growth rate, the water/salt-soluble extract showed the inhibition of various fungi isolated from bakeries. The antifungal activity was attributed to a mixture of organic acids and peptides which were synthesized during fermentation. Formic (24.7 mM) acid showed the highest antifungal activity. Four peptides, having similarities with well known antifungal sequences, were identified and chemically synthesized. The minimal inhibitory concentration was 2.5–15.2 mg/ml. Slices of bread made by addition of 4% (wt/wt) of freeze dried SFWG were packed in polyethylene bags and stored at room temperature. Slices did not show contamination by fungi until at least 28 days of storage and behaved as the calcium propionate (0.3%, wt/wt).     

高浓度培养乳酸菌发酵培养基的优化

对一株已经证实具有耐酸、耐胆汁的乳杆菌R8菌株进行培养基的优化研究,以菌体密度为检测指标,采用单因素实验和正交设计实验优化发酵培养基组分,为该益生菌应用于改善胃肠道的健康食品奠定基础。结果显示,该菌最佳发酵配方为:葡萄糖20.0 g,酵母粉30.0 g,MnSO₄·4H₂O 0.075 g,MgSO₄·7H₂O 2.0 g,KH₂PO₄ 2.5 g,柠檬酸铵2.5 g,CH₃COONa·3H₂O 6.25 g,Tween80 1.0 mL,pH6.2。培养基配方经过系统筛选和优化后,菌体浓度(OD_{600 nm})达到2.38。     

Production of microbial exopolysaccharides in the sourdough and its effects on the rheological properties of dough

Exopolysaccharides (EPS) are exogenous microbial metabolites which are secreted mainly by bacteria and microalgae during growth. In addition to natural polysaccharides present in cereal grains flour and dough, microbial flora is usually involved in production of polysaccharide on sourdough fermentation. Total polysaccharides (microbial and flour) were extracted from sourdough and dough samples dehydrated and were added at the rate of 0%, 0.25%, 0.5%, 1%, 1.5%, 2% and 2.5% (w/w flour based) on the dough to investigate its effects on the rheological properties of the dough. Addition of polysaccharides to the dough increased the water absorption and decreased the dough softening after 20 min. Resistance to extension after 45, 90 and 135 min resting time was decreased by increasing the percentage of the added polysaccharides. Longer fermentation time for each level of polysaccharides led to greater stability. No significant differences were observed in the extensibility of dough. The overall effects of different levels of added polysaccharides resulted in a decrease in resistance to extension ratio of the samples. Energy input decreased in all cases. It seems therefore that addition of polysaccharides may be useful when bread is to be made with stronger flour and longer fermentation time is needed.     

Enrichment of isoflavone aglycones in soymilk by fermentation with single and mixed cultures of Streptococcus infantarius 12 and Weissella sp. 4

Soymilk was fermented with either Streptococcus infantarius 12 (Si 12), Weissella sp. 4 (Ws 4), or their mixed cultures with different mixing ratios (Si 12:Ws 4 = 1:1, 1:3, 1:5, and 1:10, v/v) for 12 h at 37 °C. All cultures in soymilk readily proliferated and reached about 10^8–9 CFU/mL. After 12 h, pH and titratable acidity of soymilk ranged 4.19–4.47 and 0.57%–0.64%, respectively. The pH of soymilk fermented with Si 12 was the lowest while that obtained with Ws 4 the highest. A sharp increase in β-glucosidase (β-glu) activity corresponded well with a rapid decrease in isoflavone glucosides and an increase in aglycone contents. The rate of hydrolysis of isoflavone glucosides was the least with Si 12 while the highest with Ws 4, resulting in about 23%–33% and 98%–99% hydrolysis of the glucosides with Si 12 and Ws 4, respectively, after 12 h. Mixed cultures with 1:3, 1:5, and 1:10 ratios seem to be more effective starters for bioactive fermented soymilk with more aglycones and appropriate acidity in a short time than single cultures.     

Microbiological profile of maize and rye flours, and sourdough used for the manufacture of traditional Portuguese bread

A thorough microbiological study of maize and rye flours, and sourdoughs obtained therefrom for eventual manufacture of broa – a dark sour bread typical in Northern Portugal, following artisanal practices, was carried out. Towards this purpose, samples were supplied by 14 artisanal producers, selected from 4 sub-regions, during two periods of the year. Total viable counts, as well as viable mesophilic and thermophilic microorganisms, yeasts and molds, Gram⁻ rods, endospore-forming and nonsporing Gram⁺ rods, and catalase⁺ and catalase⁻ Gram⁺ cocci were assayed for. The comprehensive experimental dataset unfolded a unique and rather complex wild microflora in flours and sourdoughs throughout the whole region, which did not discriminate among sub-regions or seasons, or flour source for that matter. However, fermentation played a major role upon the numbers of the various microbial groups: the viable counts of yeasts, lactobacilli, streptococci, lactococci, enterococci and leuconostocs increased, whereas those of molds, Enterobacteriaceae, Pseudomonadaceae, staphylococci and micrococci decreased.     

Aglycone production by Lactobacillus rhamnosus CRL981 during soymilk fermentation

Lactobacillus rhamnosus CRL981 showed the highest levels of β-glucosidase and was selected to characterize this enzyme system, among 63 strains of different Lactobacillus species. The maximum activity was obtained at pH 6.4 and 42 °C. The enzyme showed weak resistance to thermal inactivation maintaining only 20% of the initial activity when it was exposed at 50 °C for 5 min. It also, showed stability when stored at 4 °C for 60 days. Afterwards, L. rhamnosus was evaluated for hydrolysis of isoflavones to aglycones, cell population, residual sugars and organic acid produced during fermentation on soymilk (37 °C for 24 h). Higher viable counts were obtained after 12 h of fermentation (8.85 log CFU ml⁻¹) followed by a drop of pH and an increase of acidity during fermentation due the production of organic acids. L. rhamnosus CRL981 was able to proliferate in soymilk and produce a high β-glucosidase activity achieving a complete hydrolysis of glucoside isoflavones after 12 h of fermentation. The present study indicates that L. rhamnosus CRL981 could be used in the development of different aglycone-rich functional soy beverages.