Screening methods for the proteolytic breakdown of gluten by lactic acid bacteria and enzyme preparations

 Gluten is the major protein in wheat and it is largely responsible for the rheological characteristics of bread dough. The sourdough process is a traditional dough fermentation process with lactic acid bacteria. The proteolytic activity of lactic acid bacteria derived from starter cultures used in sourdough or meat fermentation was tested on gluten as a substrate. The activities of commercial enzyme preparations, which were derived from bacterial or fungal sources, were also evaluated. Proteolytic breakdown of gluten protein in an agar medium by either bacterial culture or enzymes was evident by a clear zone around wells after staining with Coomassie blue. The increase in TCA-soluble material due to gluten breakdown was measured spectrophotometrically. A third test evaluated the release of free amino acids due to exoproteolytic activity. The agar based test showed positive results for the commercial enzyme preparations and one Micrococcus strain. All sourdough strains were positive in the enzyme test whereas some starters from the meat fermentation process were not able to break down gluten under test conditions. Variations in the release of free amino acid indicated differences in the enzyme systems of the lactic acid bacteria tested. Received: 25 February 1999     

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。     

Effects of different <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Enterococcus</Emphasis> strains and chemical acidification regarding degradation of gluten proteins during sourdough fermentation

Dough quality and baking performance of wheat dough are significantly affected by the qualitative and quantitative composition of the gluten. Therefore, the degradation was studied of specific fractions of gluten proteins in sourdough as affected by starter cultures. Doughs were fermented for 0, 5, and 24 h at 30 °C after addition of Lactobacillus sakei, L. plantarum, L. sanfranciscensis or Enterococcus faecalis. Chemically acidified doughs were used as controls. All doughs were analyzed quantitatively for their content of albumins, globulins, gliadins, glutenins, and glutenin macropolymer by means of a combined extraction/HPLC procedure. Protein degradation during sourdough fermentation was primarily due to acidic proteases present in flour. While L. sakei, L. plantarum and L. sanfranciscensis were mostly non-proteolytic, E. faecalis clearly contributed to gluten proteolysis. Single gluten protein types were clearly different in their resistance to proteolytic activities of the dough system and E. faecalis, and, in contrast to total glutenins, the amounts of gluten macropolymer were significantly reduced already after 5 h of incubation. When longer fermentation times were applied, gluten was substantially degraded. The strongest decrease was found for the glutenin fraction leading to an increase of alcohol soluble oligomeric proteins in the gliadin fraction. The extent of the decrease of monomeric gliadins was strongest for the γ-type followed by the α- and the ω-types. This indicates that dough properties residing in specific types of gluten fractions can be influenced by the duration of fermentation and the application of proteolytic strains.     

Application of Bifidobacteria as Starter Culture in Whole Wheat Sourdough Breadmaking

This investigation is aimed at developing a new cereal-based product, with increased nutritional quality, by using Bifidobacterium pseudocatenulatum ATCC 27919 as starter in whole wheat sourdough fermentation and evaluating its performance. Four different sourdough levels (5%, 10%, 15%, and 20% on flour basis) in bread dough formulation were analysed. The effects of the use of bifidobacteria in sourdough bread were comparatively evaluated with controls (yeast and/or chemically acidified sourdough with antibiotics). The sourdough and dough fermentative parameters analysed were pH, total titratable acidity, d/l-lactic and acetic acids. Bread performance was evaluated by specific volume, slice shape, crumb structure and firmness, crust and crumb colour, pH, total titratable acidity, and d/l-lactic and acetic acids, phytate, and lower myo-inositol phosphate contents. The sourdough breads showed similar technological quality to the control sample, with the exception of specific bread volume (decreased from 2.46 to 2.22 mL/g) and crumb firmness (increased from 2.61 to 3.18 N). Sourdough inoculated with bifidobacteria significantly increased the levels of organic acids in fermented dough and bread. The Bifidobacterium strain contributed to the fermentation process, increasing phytate hydrolysis during fermentation owing to the activation of endogenous cereal phytase and its own phytase, resulting in bread with significantly lower phytate levels (from 7.62 to 1.45 μmol/g of bread in dry matter). The inclusion of sourdough inoculated with bifidobacteria made possible the formulation of whole wheat bread with positive changes in starch thermal properties and a delay and decrease in amylopectin retrogradation.     

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.     

中国不同地区老酵头对馒头质构和感官品质的影响

目的探讨中国不同地区传统老酵头对馒头品质特征的影响,筛选产品品质较为优良的老酵头。方法收集10个地区的传统面食发酵剂为实验材料制作老酵馒头,测定不同地区老酵面团在发酵过程中微生物的变化及馒头的品质指标,并进行感官评价。结果不同地区老酵馒头的品质指标间具有显著性差异(P0.05),老酵头制作面团发酵过程中不同地区间酵母菌和乳酸菌的生长趋势相似且具有一定的规律性,在发酵前期,酵母菌迅速生长至面团成熟期后,其数量趋于稳定;乳酸菌在发酵前期经过短暂的适应期后进入快速生长期,至面团成熟期后乳酸菌数量显著下降。综合馒头的质构指标、物性指标及感官评价来看,5#老酵头制作出的馒头其综合品质最佳,感官评分最高为89分。结论本研究结果为传统发酵剂在馒头工业化生产中的合理利用提供了理论依据。     

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.     

酸面团发酵过程中面团流变及面筋蛋白降解变化研究

实验选用酿酒酵母、德氏乳杆菌保加利亚亚种和短乳杆菌进行不同菌种的酸面团发酵,研究发酵过程中面团流变及面筋蛋白降解变化,为酸面团制品及其品质与生产提供理论参考。分析了酿酒酵母(Sce)发酵、酿酒酵母和德氏乳杆菌保加利亚亚种混合(Sce-Lb)发酵以及酿酒酵母和短乳杆菌(Sce-Lbr)混合发酵过程中面团流变及面筋蛋白降解变化。研究结果表明:3种发酵方式下面团pH值都随着发酵时间增加而降低,且Sce-Lbr发酵时酸化速度最快;发酵过程中面团的弹性模量和黏性模量都随着发酵时间的增加而降低,发酵过程中弹性模量始终大于黏性模量即面团的损耗因子始终小于1,其中Sce-Lbr发酵过程中黏弹性降低程度最大;发酵过程中面团的湿面筋含量和面筋指数都降低,且降低程度为Sce-Lbr>Sce-Lb>Sce,与酸化程度一致,其中Sce-Lbr发酵至12 h时由于过度酸化导致面筋蛋白结构的完全破坏已经无法洗出湿面筋;发酵过程中面团中的游离巯基含量随着发酵时间的增加而显著增加,其中Sce发酵12 h后游离巯基含量趋于稳定,而Sce-Lb和Sce-Lbr长时发酵后其游离巯基含量持续显著增加。     

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.     

老面发酵程度对馒头比容和白度的影响

针对老面发酵时间对老面馒头比容和白度的影响,采用二次和面时是否添加面粉进行对比试验。发酵面团直接成型时,随着发酵时间的增加,馒头的比容和白度都呈下降的趋势,这种趋势与老面中面筋含量有较大关系,添加面粉时,馒头的比容和白度分别在老面发酵16~18h和18h时达到理想的值,通过对比试验可以看出,老面团的面筋含量、二次和面时添加面粉对馒头的品质有重要的影响。     

Evaluation of Leuconostoc citreum HO12 and Weissella koreensis HO20 isolated from kimchi as a starter culture for whole wheat sourdough

Leuconostoc citreum HO12 and Weissella koreensis HO20 isolated from kimchi were evaluated as starter cultures in the making of whole wheat sourdough bread. After 24 h of fermentation at 25 °C, both lactobacilli grew to the final cell numbers of ca. 10⁹ cfu/g dough, and both doughs had similar pHs and total titratable acidities. In addition, the fermentation quotient of the dough with Lc. citreum HO12 was slightly lower than that of the dough with W. koreensis HO20 (1.6 versus 2.8). Sourdoughs and bread with 50% sourdough produced with the starter cultures exhibited consistent ability to retard the growth of bread spoilage fungi (Penicillium roqueforti and Aspergillus niger) and rope-forming bacterium (Bacillus subtilis). Sourdough breads underwent a significant reduction in bread firming during storage. It seems that both lactobacilli have the potential to improve the shelf-life of wheat bread. The results indicate that the selected lactobacilli have unique fermentation characteristics and produce sourdough breads with overall satisfactory quality.     

Diversity and Stability of Yeast Species During the Fermentation of Tarhana

Tarhana is a traditional cereal-based fermented food produced with a mixture of yoghurt and flour. The main microbiota in the fermentation of tarhana is yeast, together with lactic acid bacteria. In this study, the yeast microbiota of home-made tarhana (HMT) and plant-type tarhana (PTT) dough samples was evaluated and compared during fermentation. Culture-dependent LSU and ITS-5.8S rDNA sequence analysis of yeast isolates collected during the tarhana dough fermentation clarified 45 selected isolates representing different clusters. These yeast isolates displayed high homologywith species Pichia kudriavzevii (11), Candida glabrata (11), Candida humilis (10), Saccharomyces cerevisiae (7), Kluyveromyces marxianus (4), Kazachstania servazzi (1), and Kazachstania unispora (1). Additionally, both culture-dependent and PCR-Denaturated Gradient Gel Electrophoresis (PCR-DGGE) analyses showed that S. cerevisiae, P. kudriavzevii and K. marxianus were abundant in the fermentation of HMT dough samples whereas P. kudriavzevii, C. humilis, and C. glabrata dominated the PTT dough samples. It was concluded that tarhana fermentation was accomplished with the presence of a wide variety of yeast species that mainly included P. kudriavzevii in both HMT and PTT dough samples.     

不同高产胞外多糖乳酸菌发酵荞麦酸面团对面团面筋网络结构和面包烘焙特性的影响

比较研究食窦魏斯氏菌（T5）和融合魏斯氏菌（J28）发酵对荞麦酸面团糖代谢、面包面团面筋网络结构以及面包烘焙特性的影响。结果表明：菌株T5和菌株J28产生的胞外多糖均为高分子葡聚糖;J28发酵荞麦酸面团产糖能力显著高于T5：J28＋组（11.87?g/kg）＞T5＋组（9.36?g/kg）,但J28发酵产生乙酸水平明显低于T5;与添加30%的不产胞外多糖酸面团面包相比,T5和J28产生的胞外多糖都能改善面团面筋网络结构和面包比容以及柔软度,但T5产生的胞外多糖改善作用更加明显;与空白组面包相比,J28＋组面包烘焙品质最佳,并且更受消费者的喜欢。     

复合酶制剂对甜酒酿面包发酵烘焙特性的影响

选用中国特色传统甜酒曲制作的甜酒酿进行面包制作,通过面团流变学分析、生物化学分析、激光共聚焦 显微观察、感官评定等多种手段对甜酒酿面团及面包的品质进行综合评估,与普通小麦面包进行对比,并使用天然 酶制剂来提高含甜酒酿面包的发酵烘焙特性。结果表明：含甜酒酿面团游离巯基含量增加,蛋白酶活力较强,甜酒 酿对面包面团面筋网络结构的形成有破坏作用,会导致面包内聚性、弹性下降,降低口感评分。但甜酒酿能给面包 带来更加诱人的色泽,其特有的酒香使面包更加可口,通过酶制剂的作用,面团面筋网络结构得到强化,含酶甜酒 酿面包品质全面提升,比普通小麦面包更受欢迎。     

乳酸菌发酵剂对杂粮面团及馒头品质的影响

为研究混合乳酸菌发酵对杂粮面团及馒头品质的影响,将黄豌豆粉、莜麦粉、谷朊粉及燕麦β-葡聚糖（oatmeal β-glucan,OBG）复配成杂粮粉基质,利用直投式混合乳酸菌发酵剂发酵面团制作馒头。探究乳酸菌在面团中的酸化能力,分析杂粮面团中OBG、抗性淀粉（resistant starch,RS）和游离氨基酸含量的变化以及面团的微观结构变化,并对发酵前后的杂粮馒头和小麦粉馒头进行品质及营养特性比较。结果表明,在发酵杂粮面团中乳酸菌酸化能力强,发酵24 h后乳酸和乙酸质量浓度分别达到24.85、8.98 mg/mL。与未发酵的杂粮面团相比,乳酸菌发酵杂粮面团中OBG含量下降32.56%,RS含量上升32.88%,游离氨基酸总量提升至1.46倍,氨基酸组成模式更优,部分蛋白质和纤维素发生降解,面筋网络结构更加紧密且连续。在营养方面,乳酸菌发酵前后的杂粮馒头膳食纤维质量分数均高于6%,蛋白质质量分数分别为40.35%和38.38%,属于高纤维高蛋白食品,显著优于小麦粉馒头。乳酸菌发酵杂粮馒头具有更高的体外蛋白消化率和比容,且乳酸菌发酵杂粮馒头感官评价总评分显著高于未发酵杂粮馒头,整体可接受度...     

Studies concerning the use of <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> and <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> for rye sourdough fermentation

The aim of this study was to investigate the fermentation behaviour, antioxidant activity and rheological behaviour of the sourdoughs prepared with Lactobacillus helveticus and Kluyveromyces marxianus. The final acidity of the sourdough after 18 h of fermentation was higher at higher temperatures (40 °C) and for the formulations with whole rye flour. The highest lactic/acetic acid ratios were obtained in case of more fluid sourdoughs fermented at 40 °C. The antioxidant properties of the sourdoughs are correlated with metabolic activity of the microorganisms, the DPPH radical scavenging activity being higher for optimum conditions of Lactobacillus helveticus growth. On the other hand, the rheological properties of the sourdoughs are not significantly influenced by the temperature. Sourdoughs formulations with whole flour are more viscous, as well as the controls fermented by the spontaneous microflora. The results of the bread-making process simulation by means of Mixolab indicate differences between dough formulations mainly depending on flour type.     

Release of free ferulic acid and changes in antioxidant properties during the wheat and rye bread making process

Changes in the free ferulic acid (FFA) contents and antioxidant properties during bread making processes were determined. Experimental breads were produced from whole meal and white wheat and rye flours, and fermented using either baker’s yeast or sourdough starter. Sourdough fermented bread contained the highest content of FFA. Release of occurred mainly during dough fermentation. A further increase in the ferulic acid content in the bread crumb and a decrease in the crust was observed. Total antioxidant properties of sourdough bread, defined as the sum of lipophilic and hydrophilic compound activities, were significantly (p<0.05) higher than for yeast bread. Sourdough bread contained more methanol soluble phenolic compounds, proteins, tocochromanols, and oxidized products of fatty acids than yeast bread. The equilibrium between the anti- and pro-oxidative compound contents resulted in similar antioxidant properties for bread using both types of fermentation, and to results observed for the flour used for baking.     

Overview of Sourdough Technology: from Production to Marketing

Sourdough is a type of dough fermented by yeast and lactic acid bacteria (LAB) used as sponge dough in bread making. Sourdough may have four classifications according to the fermentation type and the technological process used. On sourdough type I, the fermentation of yeasts and LAB present in the flour occurs spontaneously whereas in type II, fermentation occurs after the inoculation of a starter culture. Type III is simply type II sourdough dehydrated and type IV is a mixture of type I and type II sourdough, produced on laboratory scale. With LAB’s growth during fermentation, pH decreases with subsequent hydrolysis of starch and protein, favoring the growth of yeasts. The yeast in turn, releases amino acids during autolysis, contributing to the growth of LAB. Due to this synergistic growth of LAB and yeasts, the products obtained from sourdough show more concentration of flavor, more elastic dough, and a longer shelf life than bread fermented only by yeast. Although, the benefits of this technology, using sourdough, is present just in a few countries, where it is also possible to obtain the dehydrated sourdough starters, for domestic use, in local supermarkets. Due to its fermentative process complexity, this paper presents updated information about the fabrication process of sourdough, main factors affecting this process, benefits of sourdough use for bakery products, and the varieties of dehydrated sourdough commercially available throughout the world.     

类食品乳杆菌412对酸面团发酵的影响

从酸面团中分离得到1株类食品乳杆菌(Lactobacillus parali mentarius)412,添加该菌株的面团在28℃发酵4h后的pH为5.2,发酵24h后pH为3.5;而添加商业化安琪酵母发酵的面团在28℃发酵4h后的pH为5.9,发酵24h后pH为5.2。类食品乳杆菌412与安琪酵母(接种量为107CFU/g)联合发酵测定表明,若菌株412起始接种量为109CFU/g,则面团在28(C发酵24h后的滴定酸度为0.76%(滴定酸度以乳酸计,%为质量分数);若菌株412的起始接种量是108CFU/g或107CFU/g时,则面团在28℃发酵24h后的滴定酸度为0.57%。当向面团添加6.75g/kg的葡萄糖或蔗糖可以促进类食品乳杆菌412的生长。面团在28℃发酵12h后,添加葡萄糖可以使菌株412的细胞数量从起始的2×108CFU/g提高到2.5×1010CFU/g,添加蔗糖则可以使其细胞数量提高到2.6×1010CFU/g;而添加果糖的面团在28℃发酵12h,菌株412的细胞生物量仅3.8×108CFU/g。与25(C或32℃发酵条件相比,28℃下发酵的酸面团中类食品乳杆菌412和安琪酵母菌的活菌数都比较高,且面团酸化速率高于单独采用安琪酵母菌发酵的面团。结果证明,类食品乳杆菌412能够单独或与商业化的安琪酵母联合应用到酸面团发酵中,并对面团的酸化起到积极促进作用。     

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.