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.     

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.     

Key enzymes involved in methionine catabolism by cheese lactic acid bacteria

Cheese microbiota and their enzymatic conversion of l-methionine to volatile sulphur compounds (VSCs) play an important role in aroma formation during cheese ripening. Here, lactic acid bacteria (LAB) strains isolated from raw goats' milk cheeses were screened for the major enzymes critical to the formation of VSCs from l-methionine. A large natural biodiversity in enzyme capabilities and high inter- and intra-species variability was found among the LAB isolates investigated. From those isolates tested, lactococci displayed higher C–S lyase specificities towards the sulphur-containing compounds examined than did Lactobacillus and Leuconostoc, in some cases generating higher levels of VSCs than B. linens, known to be an efficient producer of methanethiol (MTL) and related VSCs. Moreover, these differences in C–S lyase activities (determined spectrophotometrically by measuring the formation of free thiol groups) were shown to correspond with the enzymatic potential of the isolates as determined by visualization of enzymatic activities. This technique could therefore prove valuable for the detection and preliminary characterization of C–S lyase activities among LAB isolates. Lactococci were also found to possess higher aminotransferase activities than lactobacilli and leuconostocs, while glutamate dehydrogenase activities were observed to be highest among Leuconostoc and Lactobacillus spp. Meanwhile, α-keto acid decarboxylase activities were highly variable and were measurable in only a limited number of isolates, mainly lactobacilli. From these data, combining indigenous isolates showing high VSCs-producing capabilities with those that facilitate the completion of the metabolic pathway responsible for degrading l-methionine into volatile compounds may provide an efficient approach to enhance cheese aroma development.     

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     

Volatile sulphur compounds-forming abilities of lactic acid bacteria: C-S lyase activities

Volatile sulphur compounds (VSCs) are of prime importance in the overall aroma of cheese and make a significant contribution to their typical flavours. Thus, the control of VSCs formation offers considerable potential for industrial applications. Here, lactic acid bacteria (LAB) from different ecological origins were screened for their abilities to produce VSCs from L-methionine. From the data presented, VSC-forming abilities were shown to be strain-specific and were correlated with the C-S lyase enzymatic activities determined using different approaches. High VSCs formation were detected for those strains that were also shown to possess high thiol-producing abilities (determined either by agar plate or spectrophotometry assays). Moreover, differences in C-S lyase activities were shown to correspond with the enzymatic potential of the strains as determined by in situ gel visualization. Therefore, the assessment of the C-S lyase enzymatic potential, by means of either of these techniques, could be used as a valuable approach for the selection of LAB strains with high VSC-producing abilities thus, representing an effective way to enhance cheese sulphur aroma compounds synthesis. In this regard, this study highlights the flavour forming potential of the Streptococcus thermophilus STY-31, that therefore could be used as a starter culture in cheese manufacture. Furthermore, although C-S lyases are involved in both biosynthetic and catabolic pathways, an association between methionine and cysteine auxotrophy of the selected strains and their VSCs-producing abilities could not be found.     

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.     

Spanish cheese screening and selection of lactic acid bacteria with high gamma-aminobutyric acid production

Gamma-aminobutyric acid (GABA) is a non-protein four-carbon amino acid which is considered a bioactive component known for its physiological functions, including a regulator of blood pressure, neurotransmitter, diuretic and anti-stress effects. Its use in foods might confer health benefits. Microorganisms such as yeast, fungi or bacteria can produce GABA naturally. Among them, the lactic acid bacteria are being studied for the potential development of fermented foods because their physiological activities and their designation of generally recognized as safe (GRAS). The objective of this study was to evaluate the GABA-production capacity in a whole wheat flour medium of lactic acid bacteria strains that showed a high conversion of glutamic acid to GABA in a screening conducted in 58 Spanish artisanal cheeses. Synthesis of GABA by these strains in a non-optimized whole wheat flour in water solution (1:5) was quantified by High-Performance Liquid Chromatography. The 4 strains showing the highest GABA production were genotypically and phenotypically characterized. Results indicated an interesting fermentative variability between strains. The addition of these isolated lactic acid strains in fermented food products could allow a potentially functional food for regulating hypertension.     

Whey fermentation by thermophilic lactic acid bacteria: evolution of carbohydrates and protein content

Whey, a by-product of the cheese industry usually disposed as waste, is a source of biological and functional valuable proteins. The aim of this work was to evaluate the potentiality of three lactic acid bacteria strains to design a starter culture for developing functional whey-based drinks. Fermentations were performed at 37 and 42 degrees C for 24h in reconstituted whey powder (RW). Carbohydrates, organic acids and amino acids concentrations during fermentation were evaluated by RP-HPLC. Proteolytic activity was measured by the o-phthaldialdehyde test and hydrolysis of whey proteins was analyzed by Tricine SDS-PAGE. The studied strains grew well (2-3log cfu/ml) independently of the temperature used. Streptococcus thermophilus CRL 804 consumed 12% of the initial lactose concentration and produced the highest amount of lactic acid (45 mmol/l) at 24h. Lactobacillus delbrueckii subsp. bulgaricus CRL 454 was the most proteolytic (91 microg Leu/ml) strain and released the branched chain amino acids Leu and Val. In contrast, Lactobacillus acidophilus CRL 636 and S. thermophilus CRL 804 consumed most of the amino acids present in whey. The studied strains were able to degrade the major whey proteins, alpha-lactalbumin being degraded in a greater extent (2.2-3.4-fold) than beta-lactoglobulin. Two starter cultures were evaluated for their metabolic and proteolytic activities in RW. Both cultures acidified and reduced the lactose content in whey in a greater extent than the strains alone. The amino acid release was higher (86 microg/ml) for the starter SLb (strains CRL 804+CRL 454) than for SLa (strains CRL 804+CRL 636, 37 microg/ml). Regarding alpha-lactalbumin and beta-lactoglobulin degradation, no differences were observed as compared to the values obtained with the single cultures. The starter culture SLb showed high potential to be used for developing fermented whey-based beverages.     

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.     

An investigation of the bacteriocinogenic potential of lactic acid bacteria associated with wheat (Triticum durum) kernels and non-conventional flours

One hundred and thirty-seven lactic acid bacteria (LAB), previously isolated from wheat (Triticum durum) grains and non-conventional flour samples, were tested for the production of antibacterial substances. A total of 16 strains (5 Enterococcus faecium, 5 Enterococcus mundtii, 4 Pediococcus pentosaceus, 1 Lactobacillus coryniformis and 1 Lactococcus garvieae) were found to inhibit the growth of Listeria innocua. The antibacterial activities were preliminarily investigated for their general behaviour with proteolytic (proteinase K, protease B and trypsin), amylolytic (α-amylase) and lipolytic (lipase) enzymes, after heat treatment, and exposure to different pHs and ethanol concentrations. Bacteriocin-like inhibitory substances (BLIS) were also characterized for their inhibition spectra against non-pathogenic and pathogenic food-associated and human pathogenic bacteria. LAB showing the best characteristics in terms of inhibition spectrum, inhibition activity and mode of action (bactericidal) belonged to the species Ent. mundtii. The high percentage (11.68%) of BLIS-producing strains detected confirmed previous observations that raw materials may harbour higher numbers of bacteriocinogenic LAB than fermented foods.     

Adaptability of lactic acid bacteria and yeasts to sourdoughs prepared from cereals, pseudocereals and cassava and use of competitive strains as starters

The adaptability of lactic acid bacteria (LAB) and yeasts to sourdoughs prepared from cereals, pseudocereals and cassava was investigated using PCR-DGGE and bacteriological culture combined with rRNA gene sequence analysis. Sourdoughs were prepared either from flours of the cereals wheat, rye, oat, barley, rice, maize, and millet, or from the pseudocereals amaranth, quinoa, and buckwheat, or from cassava, using a starter consisting of various species of LAB and yeasts. Doughs were propagated until a stable microbiota was established. The dominant LAB and yeast species were Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus paralimentarius, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus spicheri, Issatchenkia orientalis and Saccharomyces cerevisiae. The proportion of the species within the microbiota varied. L. paralimentarius dominated in the pseudocereal sourdoughs, L. fermentum, L. plantarum and L. spicheri in the cassava sourdough, and L. fermentum, L. helveticus and L. pontis in the cereal sourdoughs. S. cerevisiae constituted the dominating yeast, except for quinoa sourdough, where I. orientalis also reached similar counts, and buckwheat and oat sourdoughs, where no yeasts could be detected. To assess the usefulness of competitive LAB and yeasts as starters, the fermentations were repeated using flours from rice, maize, millet and the pseudocereals, and by starting the dough fermentation with selected dominant strains. At the end of fermentation, most of starter strains belonged to the dominating microbiota. For the rice, millet and quinoa sourdoughs the species composition was similar to that of the prior fermentation, whereas in the other sourdoughs, the composition differed.     

Populations, types and biochemical activities of aerobic bacteria and lactic acid bacteria from the air of cheese factories

This investigation was carried out in order to identify the airborne bacteria in the processing rooms of two creameries that make, mainly, feta cheese and study some of their biochemical activities. Our results suggest that the numbers of airborne micro-organisms may differ between plants, and the same was observed with the predominant types. The bacteria isolated were primarily Gram-positive, catalase-negative cocci, micrococci and bacilli. The predominant lactic microflora were lactococci and mesophilic lactobacilli, commonly found in feta and other traditional Greek cheeses. A significant number of aerobic bacteria could grow at 4°C and in 6.5% NaCl. In addition, both aerobic and lactic acid bacteria (LAB) exhibited acidifying and proteolytic activities. Slow- and medium-acidification ability of LAB strains were found. There was also a tendency of the strains to preferentially degrade αs-casein. The amounts of amino acids accumulated in the milk were strain dependent. Therefore, there is a possibility that airborne bacteria contaminating cheese milk in the open vats may multiply and contribute to the ripening changes in the cheese.     

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.     

乳酸菌冷冻干燥保护剂的筛选

通过单因素比较和正交试验设计,确定适合保加利亚乳杆菌和嗜热链球菌的最佳冻干保护剂配方。通过对15种冻干保护剂单因素保护效果的比较,以及对选定的4种保护剂的正交试验结果,可以确定嗜热链球菌最佳冻干保护剂配方为脱脂乳粉12%、海藻糖1%、甘油3%、谷氨酸钠1%,保加利亚乳杆菌最佳冻干保护剂配方为脱脂乳粉8%、海藻糖1%、甘油2%、谷氨酸钠1.5%。     

Diversity of lactic acid bacteria population in ripened Parmigiano Reggiano cheese

The diversity of dominant lactic acid bacteria population in 12 months ripened Parmigiano Reggiano cheeses was investigated by a polyphasic approach including culture-dependent and independent methods. Traditional plating, isolation of LAB and identification by 16S rDNA analysis showed that strains belonging to Lactobacillus casei group were the most frequently isolated. Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus parabuchneri, and Lactobacillus buchneri species were detected with lower frequency. PCR-denaturing gradient gel electrophoresis (DGGE) applied to DNA extracted directly from cheese samples and sequencing of rDNA amplicons confirmed the complex microbiological pattern of LAB in ripened Parmigiano Reggiano cheeses, with the significant exception of the Lactobacillus fermentum species, which dominated in several samples, but was not detected by cultivation. The present combination of different approaches can effectively describe the lactic acid bacteria population of Parmigiano Reggiano cheese in advanced stages of ripening, giving useful information for elucidating the role of LAB in determining the final cheese quality.     

Detoxification of cancerogenic compounds by lactic acid bacteria strains

ABSTRACT

Carcinogens in food are an important issue that threat people’s health right now. Lactic acid bacteria (LAB) strains as well-known probiotics have shown numerous perspectives in being used as a good food additive to confront cancerogenic compounds in recent years. Some LAB strains can remove cancerogenic compounds from medium environment via direct physical binding and avoid re-pollution of poisonous secondary metabolites which are generated from degradation of cancerogenic compounds. This article presents a whole overview of the physical-binding of LAB strains to such common cancerogenic compounds existed in food and feed environments as mycotoxins, polycyclic aromatic hydrocarbons (PAHs), heterocyclic amines (HAs) and pthalic acid esters (PAEs).In most cases, summaries of these published researches show that the binding of LAB strains to cancerogenic compounds is a physical process. Binding sites generally take place in cell wall, and peptidoglycan from LAB cells is the chief binding site. The adsorption of lactic acid bacteria to cancerogenic compounds is strain-specific. Specially, the strains from the two genera Lactobacillus and Bifidobacterium show a better potential in binding cancerogenic compounds. Moreover, we firstly used molecular dynamic computer model as a highly potential tool to simulate the binding behavior of peptidoglycan from Lactobacillus acidophilus to DBP, one of pthalic acid esters with genetic toxicity. It was seen that the theoretical data were quite consistent with the experimental results in terms of the ability of this bacterium to bind DBP. Also, the toxicity reduction of cancerogenic compounds by LAB strains could be achieved either in gastrointestinal model or animal tests and clinical researches as well. In conclusion, carefully selected LAB strains should be a good solution as one of safety strategies to reduce potential risk of cancerogenic compounds from food-based products.     

Phenotypic and technological diversity of lactic acid bacteria and staphylococci isolated from traditionally fermented sausages in southern Greece

The physicochemical and microbiological characteristics of spontaneously fermented sausages made by two medium-sized enterprises (MSE) located in southern Greece have been studied. A total of 300 lactic acid bacteria and 300 staphylococcal strains have been isolated and identified by their physiological characteristics. Lactobacillus plantarum strains were found to dominate the lactic acid bacteria microbiota in most of the cases with L. sakei strains prevailing in some of them and L. rhamnosus strains occasionally accompanying the dominant lactic acid bacteria microbiota. On the other hand, S. saprophyticus strains were found to dominate the staphylococcal microbiota in all spontaneously fermented sausages with of S. simulans, S. xylosus, S. gallinarum and S. cohnii cohnii strains being sporadically present. Following the identification, an evaluation of their technological properties, namely proteolytic and lipolytic capacities as well as production of biogenic amines and antimicrobial compounds, took place. None of the lactic acid bacteria and staphylococci was found to possess lipolytic activity whereas a total of 6 lactic acid bacteria and 51 staphylococci strains were found to be able to hydrolyse either the sarcoplasic, myofibrillar or both protein fractions. Furthermore, only one L. sakei strain and 185 staphylococci strains were found to possess decarboxylase activity against lysine, tyrosine, ornithine or histidine. Finally none of the staphylococcal microbiota and 3 lactic acid bacteria strains were found to be able to produce antimicrobial compounds of proteinaceous nature against Listeria monocytogenes.     

Protocols for the isolation and detection of lactic acid bacteria with bacteriocinogenic potential

The objective of this study was to evaluate culture media and methodologies for isolation and detection of lactic acid bacteria (LAB) capable to produce bacteriocin-like substances. Samples of milk and cheese were pour plated on de Mann-Rogosa-Sharpe agar (MRS) and Kang-Fung-Sol agar (KFS) (both at 35 °C/48 h, under anaerobiosis), from which 389 and 256 LAB cultures were selected. The antagonistic activity of them was evaluated using the spot-on-the-lawn and two culture media: brain-heart infusion agar with catalase (BHI + C) and M17 (both at 35 °C/24 h). The proteinaceous nature of the antagonistic cultures was verified using: spot-on-the-lawn (MRS, 25 °C/24 h, under anaerobiosis) and well-diffusion (cultures amplified on modified MRS broth at 25 °C/24 h, and then neutralized using NaOH). Listeria monocytogenes ATCC 7644 was used as indicator. A larger number of antagonist cultures were isolated from MRS (83 by M17 and 65 by BHI + C) in comparison to KFS (24 by M17 and 15 by BHI + C). The spot-on-the-lawn identified a higher frequency of LAB capable of producing bacteriocin-like substances. MRS was considered to be the best culture media for the isolation of LAB capable to produce bacteriocin-like substances, activity that was better identified using the spot-on-the-lawn methodology.