PCR detection of Lactobacillus sanfranciscensis in sourdough and Panettone baked product

Sourdoughs, in which Lactobacillus sanfranciscensis is the predominant bacterial species, are distinctive of some traditional Italian sweet baked products like Panettone. The direct extraction of amplifiable bacterial DNA from products subjected to heat treatment represents a valid tool to identify and trace microbial species originally present in the food matrices. Three types of protocols for the isolation and clean-up of DNA (CTAB, Wizard^® DNA Clean-Up System, NucleoSpin^® Food) were applied on mother, final dough and end-product samples and compared through the determination of the maximum amplifiable dilution by a PCR reaction targeting two fragments (1460 and 153 bp long) of 16S rDNA region of Lb. sanfranciscensis. CTAB extracting protocol was revealed to be the best for isolating DNA. In dough samples the amplification with the 153 bp fragment showed signals at concentration levels that are comparable with the values obtained from the plate counts, and two log cycles higher than those found with the amplification targeting the 1460 bp fragment. In the cooked samples only the 153 bp amplicon was detected, indicating that oven cooking degrades DNA into small fragments.     

传统发面面肥中乳酸菌的分离与鉴定

为改善青贮饲料发酵品质筛选提供乳酸菌资源,实验采用实验室纯培养方法结合生理生化分析,对新疆传统发面面肥样品中乳酸菌的种类、形态学及生理生化特性进行研究,对分离的菌株运用16S r DNA基因序列和系统发育研究进行种属鉴定。结果表明,分离得到三株乳酸菌,其中F5和F28两株被鉴定为Bacillus sp.,F3被鉴定为Paenibacillus sp.。3株菌在p H4~7能生长,在3%和6.5%Na Cl条件下生长状况良好,在10~45℃温度范围能生长。两株芽孢杆菌中F5菌株产酸能力较强,F28菌株生长速率较快,并且这两株菌均能利用多种碳源。所以,实验中筛选出的三株乳酸菌均可选作青贮饲料制作过程中的添加剂。      

乳酸菌素分离纯化和特性及在食品领域的应用研究进展

乳酸菌素是由某些乳酸菌核糖体合成的对相同或相近生境微生物具有拮抗作用的一类多肽、蛋白质类次级代谢产物,作为天然生物防腐剂已用于食品防腐保鲜领域。经过硫酸铵沉淀、凝胶层析和高效液相色谱等分离纯化流程获取乳酸菌素,进而对其热稳定性、p H适应性和分子量等进行分析,其中,乳酸菌素纯化技术是限制其研究和应用的技术瓶颈。本文对近几年乳酸菌素分离纯化如硫酸铵沉淀（ASP）、细胞吸附和解吸（ADPC）、甲醇丙酮萃取（MAE）、凝胶过滤层析（GFC）、阳离子交换色谱（CEC）、琼脂糖凝胶电泳（SP-SFF）和反向液相色谱（RP-HPLC）等技术及其综合运用进行比较分析,并对乳酸菌素性质研究和在食品领域的应用进展进行综述,为开发高效乳酸菌生物保护剂提供借鉴与参考。      

Carbohydrate, peptide and lipid metabolism of lactic acid bacteria in sourdough

The metabolic pathways of lactic acid bacteria that influence bread quality are coupled to the central carbon flux by the availability of cofactors influencing the cellular and environmental redox potential. Homo- and heterofermentative metabolism differ fundamentally with respect to the requirement for regeneration of reduced cofactors, NADH or NADPH. The utilization of co-substrates such as oxygen or fructose as electron acceptors by obligate heterofermentative lactobacilli is coupled to an increased production of acetate in dough. Recently, several oxidoreductases involved in cofactor regeneration were characterized and glutathione and short-chain aldehydes derived from lipid oxidation were identified as substrates for cofactor regeneration by Lactobacillus sanfranciscensis. Based on the different metabolic requirements for cofactor regeneration, homo- and heterofermentative lactobacilli exert divergent effects on redox-reactions in sourdough that influence bread quality beyond the formation of acetate. Proteolysis, followed by peptide or amino acid metabolism by LAB is one of the key routes of flavour formation in bread flavour, and enables the strain-specific formation of antifungal metabolites. Peptide metabolism as well as the metabolism of cysteine, arginine, and phenylalanine in Lactobacillus plantarum, L. sanfranciscensis, and Lactobacillus pontis is increasingly understood and these insights provide new opportunities for the directed application of sourdough LAB for improved bread quality.     

Molecular and functional characterization of Lactobacillus sanfranciscensis strains isolated from sourdoughs

Fifty isolates of Lactobacillus sanfranciscensis from Italian sourdoughs were identified and typed by a polyphasic approach which included genotypic and phenotypic criteria. Genotypic diversity was characterized by Ribosomal Intergenic Spacer Analysis (RISA) of PCR amplified 16S-23S rDNA spacer region, denaturing gradient gel electrophoresis (DGGE) of PCR amplified rpoB (beta subunit of RNA polymerase) gene, and rep-PCR (PCR amplification of repetitive bacterial DNA elements) analyses. The RISA analysis produced a unique electrophoretical profile of four bands (ranging from 300 to 600 bp) for all L. sanfranciscensis isolates. The DGGE analysis of rpoB gene allowed the subdivision of isolates in four clusters. The resolution found by using rep-PCR with primers BOXA1R and REP1R-I/REP2-I allowed the widening of the level of isolates heterogeneity. Phenotypic diversity was evaluated by Biolog System and characterization of several technological traits (e.g., acidification kinetics, proteinase and peptidase activities). L. sanfranciscensis isolates used a large varieties of carbon sources such as dextrin, D-fructose, L-fucose, alpha-D-glucose, maltose, palatinose, L-rhanmose, L- and D,L-lactic acids and L-methionine. The acidification activity and related quotient of fermentation, and the peptidase (PepN, PepV, PepT, PepI, PepX, PepQ and PepR) activities markedly varied among strains. The same was found concerning the capacity to liberate amino acids during sourdough fermentation. This study could be considered as an example of a computerized analysis of the genotypic and phenotypic traits to reliably and rapidly differentiate sourdough isolates. Although some L. sanfranciscensis isolates combined several technological traits, the association of more selected strains seemed to be a requisite to get optimal sourdough characteristics.     

乳酸菌抗真菌活性及其抑制真菌毒素的效果

讨论了乳酸菌抗真菌的活性及其清除真菌毒素的3种可能机制，即①乳酸菌通过对霉菌生长的直接抑制，从而抑制真菌毒素的形成；②通过产生代谢产物抑制真菌菌株或降解真菌毒素到无毒或毒性较低的化合物，以此降低真菌毒素的危害；③借助乳酸菌细胞壁与真菌毒素(黄曲霉毒素B1和玉米赤霉烯酮)的物理结合来清除介质中的毒素危害。乳酸菌的抗真菌活性和清除霉菌毒素的能力显然受到菌株、细胞浓度、细胞处理方式和环境条件的影响。新型发酵乳菌株筛选过程中应该考虑乳酸菌菌株的抗真菌活性及其清除真菌毒素的能力。     

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.     

Added pentosans in breadmaking: fermentations of derived pentoses by sourdough lactic acid bacteria

One hundred and seventy strains of sourdough lactobacilli were screened for their capacity to grow optimally by using pentoses. Lb. alimentarius M106 and M137, Lb. hilgardii S32, Lb. brevis AM11, Lb. fermentum I4 and Lb. brevis AM8 had a higher cell yield, growth rate and acetic acid production on a mix of arabinose, xylose or ribose and maltose than on maltose as carbon sources. In the cofermentation process, pentoses were preferentially consumed instead of maltose. Pentosan extract was treated with α-L-arabinofuranosidase from Aspergillus niger or endo-xylanase from Bacillus subtilis to produce hydrolysates respectively each of arabinose and xylose. Depending on the type of pentose and other unidentified compounds, hydrolysates specifically substantiated the growth and acetic acid production of sourdough lactobacilli. Sourdough fermentation by Lb. hilgardii S32 with addition of pentosans and endo-xylanase enzyme increased the acidification rate, titratable acidity, acetic acid content and cell yield number. In addition to the well known role in retarding bread staling, pentosans may also be used as sources of pentoses which favour the performances of selected sourdough lactobacilli.     

The microflora of sourdough. XVIII. The protein degrading capabilities of lactic acid bacteria in sourdough

Acidification of the dough by the use of sourdough or acidifiers is necessary not only for good baking quality of rye flour but it is also very important for development of the typical sensory characteristics of rye bread. We confirmed that the lactic acid bacteria of sour dough are proteolytic. Proteolytic effects are observed in the increase of the amino acid content during fermentation. A marked increase was found in the content of leucine, alanine, valine, isoleucine, glutamic acid, glutamine, arginine, lysine, methionine, phenylalanine, tyrosine and serine. Lactobacillus plantarum showed a higher proteolytic activity than L. brevis ssp. lindneri or L. fructivorans.     

传统酸面团中抗霉菌乳酸菌的筛选及其在蒸蛋糕中的应用

蒸蛋糕含水量高,不易贮藏。因此,筛选具有抑菌活性的乳酸菌,并将其应用于蒸蛋糕的防腐保鲜具有巨大的应用价值。采用牛津杯法初筛、微量孔板复筛,从传统酵头中筛选得到一株对蒸蛋糕中主要腐败菌具有高抑菌活性的乳酸菌,对该乳酸菌进行分子生物学鉴定,并分析其抑菌活性的有效成分;以该乳酸菌酸面团发酵粉替代低筋粉(替代率分别为0、25%、50%、75%),研究其对蒸蛋糕的质构、感官和微生物的影响。实验结果:经鉴定该乳酸菌为植物乳杆菌(Lactobacillus plantarum),对不同蛋白酶的处理表现出不同的敏感性,发酵液对热稳定,对p H变化最为敏感,推测抑菌作用主要是有机酸。有机酸分析可知,苯基乳酸的含量高达95.91 mg/L。随着替代率的增加,微生物数显著降低;当替代率为25%和50%时,可以显著降低蒸蛋糕的硬度和咀嚼性;当替代率为50%时,蒸蛋糕的感官评价和空白接近,可以被消费者接受。      

Volatile compounds in wheat sourdoughs produced by lactic acid bacteria and sourdough yeasts

The content of volatile compounds in wheat sourdoughs fermented with four different starter cultures of the genusLactobacillus was compared with those in chemically acidified doughs. Sourdoughs as well as chemically acidified doughs were also combined with four different sourdough yeasts to investigate the effect of adding yeasts on the production of volatiles. The volatile compounds in the doughs were isolated by a dynamic headspace technique, analysed by gas chromatography (GC), and identified on the basis of GC retention times for reference compounds and mass spectrometry. Few volatile compounds were identified in the chemically acidified doughs compared to the sourdoughs. The content of volatile compounds in the sourdoughs varied with the starter culture used. Ethanol and ethyl acetate were produced in the highest amounts in sourdoughs fermented with heterofermentative cultures, especiallyL. sanfrancisco. Apart from ethanol,n-hexanol was the dominating alcohol in all the sourdoughs. The content of other compounds was low. When sourdough yeasts were added to the sourdoughs, the number and amount of volatile compounds increased. The content of ethanol, 2-methyl-propanol and 2/3-methyl-1-butanol was highest in the sourdoughs with addedSaccharomyces cerevisiae, whereas the ethyl acetate content was highest in the sourdoughs with added strain A. The content of esters other than ethyl acetate was low. The carbonyls acetoin and diacetyl were produced in small amounts in the sourdoughs with the addition of strain A.

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Flüchtige Aromastoffe in Weizensauerteigen bei der Herstellung unter Zugabe vonLactobacillus und Sauerteighefe

Zusammenfassung Der Gehalt flüchtiger Aromastoffe in Weizensauerteigen, mit 4 verschiedenen Starterkulturen desLactobacillus fermentiert, wurde mit dem Gehalt an flüchtigen Aromastoffen in chemisch gesäuerten Teigen verglichen. Sauerteige sowie chemisch gesäuerte Teige wurden außerdem mit Zugabe von 4 verschiedenen Sauerteighefen hergestellt, um den Einfluß einer Hefezugabe auf die Bildung von flüchtigen Aromastoffen zu untersuchen. Die flüchtigen Aromastoffe der Teige wurden durch eine dynamische Headspace-Technik isoliert, gaschromatographisch analysiert, und auf Grund von Retentionszeiten, Referenzstoffen und Massenspektrometrie identifiziert. Wenige flüchtige Aromastoffe wurden von den chemisch gesäuerten Teigen isoliert im Vergleich zu den Sauerteigen. Der Gehalt an flüchtigen Aromastoffen änderte sich mit der verwendeten Starterkultur. Der höchste Gehalt an Ethanol und Ethylacetat bildete sich in Sauerteigen mit heterofermentativen Kulturen, insbesondere bei der Kultur mitL. sanfrancisco. Neben Ethanol warn-Hexanol der dominierende Alkohol in sämtlichen Sauerteigen. Der Gehalt anderer Aromastoffe war gering. Bei den mit Sauerteighefen angesetzten Sauerteigen erhöhte sich die Anzahl und der Gehalt an Aromastoffen. Ein Höchstgehalt an Ethanol, 2-Methyl-1-Propanol und 2/3-Methyl-1-Butanol erwiesen sich in Sauerteigen mitS. cerevisiae, während der höchste Gehalt an Ethylacetat sich in mit dem Stamm A angesetzten Sauerteigen befand. Der Gehalt an Ester, außer Ethylacetat, war gering. Die Carbonyle Acetoin und Diacetyl bildeten sich in kleinen Mengen in Sauerteigen unter Zugabe von Stamm A.

     

Selective enumeration of Lactobacillus acidophilus,Bifidobacterium spp., starter lactic acid bacteria and non-starter lactic acid bacteria from Cheddar cheese

Twelve media were evaluated for selective and/or differential enumeration of Lactobacillus. acidophilus, Bifidobacterium spp., starter lactic acid bacteria (SLAB) and non-starter lactic acid bacteria (NSLAB) from Cheddar cheese. All media showed variation in counts and selectivity. Some reported selective media failed to inhibit SLAB and NSLAB. The media that were selective and/or differential and also gave better recovery were Reinforced Clostridium Agar with bromocresol green and clindamycin (RCABC), which was selective for L. acidophilus spp. and Reinforced Clostridium Agar with aniline blue and dicloxacillin (RCAAD), which was differential for Bifidobacterium spp. and SLAB. Reinforced Clostridium Agar with bromocresol green and vancomycin (RCABV) was found suitable for NSLAB. Apart from pure cultures, these media were also tested with commercial Cheddar cheese containing L. acidophilus. Additionally, Cheddar cheese containing L. acidophilus and B. lactis was manufactured and the selected media were used to monitor the initial survival of probiotic bacteria, SLAB and NSLAB present.     

Effects of selected lactic acid bacteria on the characteristics of amaranth sourdough

BACKGROUND: As the processing of amaranth in baked goods is challenging, the use of sourdough fermentation is a promising possibility to exploit the advantages of this raw material. In this study the fermentation properties of Lactobacillus plantarum, Lactobacillus paralimentarius and Lactobacillus helveticus in amaranth‐based sourdough were examined in order to validate them as starter cultures. pH, total titratable acidity (TTA) and lactic/acetic acid ratio of the sourdough and sensory properties of the resulting wheat bread were evaluated using fermentation temperatures of 30 and 35 °C. RESULTS: While fermentation pH, TTA and lactic acid concentration showed small variations with the use of L. plantarum and L. paralimentarius, L. helveticus reached the most intensive acidification after initial adaptation to the substrate. Acetic acid production was independent of lactic acid metabolism. Furthermore, the lactic/acetic acid ratio exceeded recommendation by 10–35 times (fermentation quotient 25–82). Sensory evaluation showed no significant differences between the two fermentation temperatures but differences among the three micro‐organisms. CONCLUSION: The results provide relevant information on the fermentation properties required of a customised starter for amaranth flour. Copyright © 2010 Society of Chemical Industry     

Identification of subdominant sourdough lactic acid bacteria and their evolution during laboratory-scale fermentations

Presumptive lactic acid bacterial cocci were found in six sourdoughs (out of 20) from the Abruzzo region (central Italy) and subjected to phenotypic and genotypic characterization. A total of 21 isolates, recognized as seven strains by randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) typing, were identified by a polyphasic approach, consisting of 16S rRNA gene sequencing, multiplex PCR assays and physiological features, as Enterococcus faecium and Pediococcus pentosaceus. Four strains belonging to those species and previously isolated from wheat kernels were inoculated in sterile flour to verify their capacity to grow in sourdough environment. Doughs with several dual bacterial combinations, including Lactobacillus sanfranciscensis, were propagated for 11 days and pH measurements and bacterial counts were carried out.     

Antibacterial activity of sourdough lactic acid bacteria: isolation of a bacteriocin-like inhibitory substance fromLactobacillus sanfranciscoC57

A total of 232 strains from nine species ofLactobacillusisolated from sourdoughs were screened for antagonistic activity against sourdough-related micro-organisms. Seventy-seven strains on agar medium and 52 in culture supernatants, re-adjusted to pH 6.5 and catalase-treated, showed antagonistic activity. The activities were species and strain specific showing different spectra of inhibition against sourdough lactobacilli. All the strains were inhibitory toBacillus subtilisbut not against sourdough yeasts and moulds.Lactobacillus sanfranciscoandLactobacillus plantarumstrains had the largest inhibitory spectrum. All the antimicrobial compounds produced from strains of different species contained a protein moiety and were differently sensitive to different proteinases. A bacteriocin-like inhibitory substance, which was heat-stable (100°C for 20 min), insensitive to lipase and α-amylase, of a protein nature, with an inhibitory spectrum centred about lactic acid bacteria and a bactericidal or bacteriolytic mode of action was isolated fromLb. sanfranciscoC57. The antimicrobial substance also inhibitedListeria monocytogenes, and was mainly produced in the stationary phase of growth and at pH 4.0–5.0.Lb. sanfranciscoC57 variants, which did not contain the nativec. 17 kbp plasmid, maintained their antagonistic activity, therefore, the gene encoding for the bacteriocin-like inhibitory substance fromLb. sanfranciscoC57 is chromosomally located.     

乳酸菌抗突变活性的研究进展

根据近年来国内外报道，对乳酸菌抗突变活性的研究进展进行了综述，并介绍了乳酸菌抗突变活性物质的分离、纯化及其研究方法。     

发酵香肠乳酸菌生理生化特性的研究

采用中国传统发酵香肠作为样品,从中分离纯化出乳酸菌,且对其做生理生化特性鉴定,以对发酵香肠的菌株进一步的筛选,从中选出不产黏液、发酵葡萄糖不产气、不产色素、不产H2S、不具有氨基酸脱羧酶、不产氨、产酸能力强的菌株作为发酵香肠中的优良的乳酸菌,最后测定优势菌株24h的生长曲线和pH值变化曲线.     

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.