The antimicrobial effect of organic acids, sour dough and nisin against
            <i>Bacillus subtilis</i>
            and
            <i>B. licheniformis</i>
            isolated from wheat bread

130

Two types of white wheat bread (high-and low-type loaves) were investigated for rope spoilage. Thirty of the 56 breads tested developed rope spoilage within 5 days; the high-type loaves were affected by rope spoilage more than the low-type loaves. Sixty-one Bacillus strains were isolated from ropy breads and were characterized on the basis of their phenotypic and genotypic traits. All of the isolates were identified as Bacillus subtilis by biochemical tests, but molecular assays (randomly amplified polymorphic DNA PCR assay, denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of 16S ribosomal DNA) revealed greater Bacillus species variety in ropy breads. In fact, besides strains of B. subtilis, Bacillus licheniformis, Bacillus cereus, and isolates of Bacillus clausii and Bacillus firmus were also identified. All of the ropy Bacillus isolates exhibited amylase activity, whereas only 32.4% of these isolates were able to produce ropiness in bread slices after treatment at 96°C for 10 min. Strains of lactic acid bacteria previously isolated from sourdough were first selected for antirope activity on bread slices and then used as starters for bread-making experiments. Prevention of growth of approximately 10 4 rope-producing B. subtilis G1 spores per cm 2 on bread slices for more than 15 days was observed when heat-treated cultures of Lactobacillus plantarum E5 and Leuconostoc mesenteroides A27 were added. Growth of B. subtilis G1 occurred after 7 days in breads started with Saccharomyces cerevisiae T22, L. plantarum E5, and L. mesenteroides A27.Ropiness is bacterial spoilage of bread that initially occurs as an unpleasant fruity odor, followed by enzymatic degradation of the crumb that becomes soft and sticky because of the production of extracellular slimy polysaccharides (15,43).The species involved in this type of spoilage of bread are primarily Bacillus subtilis and occasionally Bacillus licheniformis, Bacillus pumilus, and Bacillus cereus, even though some rope-producing Bacillus isolates are often not identified at the species level (9,22,41). It is well known that species differentiation within the genus Bacillus is very difficult because of the large number of species and the often incomplete descriptions of a number of newly reported species (19). For these reasons, molecular methods have been used increasingly to simplify characterization procedures in order to provide rapid and reliable identification or to validate phenotypically determined taxa. Among the molecular methods, the randomly amplified polymorphic DNA PCR (RAPD-PCR) assay, 16S ribosomal DNA (rDNA) PCR-denaturing gradient gel electrophoresis (DGGE) analysis, and 16S rDNA sequencing are considered reliable approaches to overcome most of the problems described above. In particular, RAPD-PCR analysis was developed to compare intra-and interspecific differences among Bacillus strains (5, 40), and this method has also been shown to be suitable for identifying a variety of Bacillus species (32). The second approach, DGG...

Section: Discussioncontrasting

confidence: 99%

mentioning

confidence: 99%

Rope-Producing Strains of Bacillus spp. from Wheat Bread and Strategy for Their Control by Lactic Acid Bacteria

Pepe

Blaiotta

Moschetti

et al. 2003

130

“…The ability of the spores to survive during baking (100°C for 15 to 60 min) and high amylase and protease activities have been identified as characteristics of strains of rope-forming bacilli (43,45). The acidification and production of organic acids by heterofermentative lactobacilli in sourdough prevents ropy spoilage of bread (43,44). However, the levels of organic acids in most bread varieties are too low to inhibit B. subtilis.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Reutericyclin Produced by Lactobacillus reuteri LTH2584

Gänzle

Höltzel

Walter

et al. 2000

197

112

Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains of Escherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C 8 chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Lactic acid bacteria (LAB) are the biological basis for the production of a great multitude of fermented foods. Their metabolic activity during these fermentative processes determines and maintains food quality. Food preservation by lactic fermentations relies mainly on the accumulation of organic acids and the acidification of the substrate. Metabolites such as acetaldehyde, diacetyl, hydrogen peroxide, and carbon dioxide contribute to this preservative effect (15). have identified low-molecular-weight compounds from cultures of Lactobacillus plantarum that contribute to the inhibitory effect of lactic acid. Certain strains of Lactobacillus reuteri produce a unique antagonistic activity, reuterin (1). This antimicrobial activity against a broad range of microorganisms was attributed to monomers, hydrated monomers, and cyclic dimers of ␤-hydroxypropionic aldehyde formed during anaerobic catabolism of glycerol. Furthermore, a great number of strains of LAB produce bacteriocins, ribosomally synthesized peptides that exhibit antagonistic activity against closely related species (32, 54). These compounds have received increasing attention since they have the potential to inhibit food pathogens (24, 51). Furthermore, lactobacilli of intestinal origin exhibit antimicrobial activity that could not be attributed to either bacteriocins or organic acids (10, 49). However, to date, no nonbacteriocin antibiotic of lactobacilli has been purified and characterized on the molecular level.The applications of antagonis...

“…Sourdough fermentations, as well as baking agents based on sourdoughs, have retained their importance in contemporary baking technology because of the improved aroma, texture, and shelf life of sourdough breads (7,35,40). The production of a wide variety of traditionally prepared baked goods continues to rely exclusively on the use of sourdough as a leavening agent.…”

mentioning

confidence: 99%

In Situ Production of Exopolysaccharides during Sourdough Fermentation by Cereal and Intestinal Isolates of Lactic Acid Bacteria

Tieking

Korakli

Ehrmann

et al. 2003

204

131

EPS formed by lactobacilli in situ during sourdough fermentation may replace hydrocolloids currently used as texturizing, antistaling, or prebiotic additives in bread production. In this study, a screening of >100 strains of cereal-associated and intestinal lactic acid bacteria was performed for the production of exopolysaccharides (EPS) from sucrose. Fifteen strains produced fructan, and four strains produced glucan. It was remarkable that formation of glucan and fructan was most frequently found in intestinal isolates and strains of the species Lactobacillus reuteri, Lactobacillus pontis, and Lactobacillus frumenti from type II sourdoughs. By the use of PCR primers derived from conserved amino acid sequences of bacterial levansucrase genes, it was shown that 6 of the 15 fructan-producing lactobacilli and none of 20 glucan producers or EPS-negative strains carried a levansucrase gene. In sourdough fermentations, it was determined whether those strains producing EPS in MRS medium modified as described by Stolz et al. (37) and containing 100 g of sucrose liter؊1 as the sole source of carbon also produce the same EPS from sucrose during sourdough fermentation in the presence of 12% sucrose. For all six EPS-producing strains evaluated in sourdough fermentations, in situ production of EPS at levels ranging from 0.5 to 2 g/kg of flour was demonstrated. Production of EPS from sucrose is a metabolic activity that is widespread among sourdough lactic acid bacteria. Thus, the use of these organisms in bread production may allow the replacement of additives.Sourdough has traditionally been used as a leavening agent in bread production. Sourdough fermentations, as well as baking agents based on sourdoughs, have retained their importance in contemporary baking technology because of the improved aroma, texture, and shelf life of sourdough breads (7,35,40). The production of a wide variety of traditionally prepared baked goods continues to rely exclusively on the use of sourdough as a leavening agent. In most industrial applications, sourdough or dried sourdough preparations are added to bread doughs which also contain baker's yeast as a leavening agent (13,14,45). Knowledge of the metabolic activities and corresponding genes of sourdough lactic acid bacteria that are responsible for their positive influence on bread quality is a prerequisite for the deliberate choice of starter cultures for specific applications. The predominant microorganisms isolated from traditional sourdoughs (type I doughs) sustained by continuous propagation are yeasts and lactic acid bacteria, mainly Lactobacillus sanfranciscensis and Lactobacillus pontis. In industrial sourdoughs prepared by using elevated temperatures and/or longer fermentation times (type II sourdough), as well as in cereal fermentations in tropical climates, thermophilic, acid-tolerant lactobacilli, such as L. pontis, Lactobacillus panis, Lactobacillus reuteri, Lactobacillus amylovorus, and Lactobacillus frumenti, are predominant (1,31,45). The microbiotae of type II sourdoughs ar...