• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. Apr 2003; 69(4): 2321–2329.
PMCID: PMC154770

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

Abstract

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 104 rope-producing B. subtilis G1 spores per cm2 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, DGGE analysis of the V3 region of 16S rDNA, is a powerful method for differentiating microorganisms at the species level (8, 14), for inferring the phylogenetic affiliations of community members (25), for evaluating the microbial diversity of several environments (3, 16), and for profiling complex microbial communities (24). However, as far as we know, no previous studies have exploited these techniques to characterize rope-producing Bacillus strains from ropy bread.

Spores of B. subtilis have been isolated from ropy bread, raw materials, and bakery environments and also from additives, including yeast, bread improvers, and gluten (4, 9, 33, 43). B. subtilis spores are heat resistant and can survive during baking in the core of the crumb, where the maximum temperature is 97 to 101°C for a few minutes (31, 33). Ropiness occurs particularly when warm (25 to 30°C) and humid (water activity, ≥0.95) environmental conditions allow germination of Bacillus spores (46). The water activity, pH, and temperature during storage may also play important roles in spore germination and growth of vegetative cells of Bacillus spp. (10, 28, 29). Moreover, rope-causing strains are also characterized by faster development and enhanced protease and amylase production during growth in the bread crumb (31). Current international trends towards elimination of chemical preservatives, including calcium propionate (34, 35), which may cause cancer tumors (34), from bread and other foodstuffs is expected to increase the risk of bread spoilage by rope-producing Bacillus strains (4). The traditional sourdough fermentation in bread making allows natural acidification that is able to control rope spoilage. Studies have shown that starting wheat bread with increasing sourdough contents enhances thermal inactivation of B. subtilis spores (30). However, although there are many advantages associated with sourdough fermentation in bread making, this process is quite complex and time-consuming compared to the simpler straight dough process.

The aims of the present study were to investigate the occurrence of rope-producing Bacillus spp. in different breads, to characterize the isolates at the phenotypic and genotypic levels, and to determine the antirope activities of selected starter cultures of lactic acid bacteria (LAB) employed in bread making.

MATERIALS AND METHODS

Bread samples.

Fifty-six white wheat loaves were obtained from different retail bakers and supermarket stores in southern Italy during a 1-year period. Thirty-two samples of artisanal and industrial high-type breads (height, ≥10 cm) and 24 samples of artisanal low-type breads (height, ≤10 cm) were studied. The samples were refrigerated at 4°C, transported to the laboratory, and immediately sliced aseptically.

Detection of rope spoilage in bread.

Two slices from each loaf were placed into petri dishes (diameter, 150 mm), uniformly soaked with 5 ml of sterile 0.25× Ringer's solution (Oxoid), and incubated at 23 and 30°C. At daily intervals, the slices were examined for rope appearance (sweet rope odor, discoloration of the crumb, and sticky threads).

Isolation and phenotypic characterization of Bacillus strains.

In order to obtain isolated colonies from ropy breads, the same portions of slimy crumb were streaked on three starch agar (SA) plates (20). After overnight incubation at 30°C, 3 to 10 isolated colonies were picked from the agar plates and purified on SA. Isolates were recognized as Bacillus spp. by assessing the presence of spores by phase-contrast microscopy and the Gram reaction as described by Gregersen (18). Identification was performed by using the dichotomous key of Norris et al. (27). Amylase activity was detected by adding drops of an iodine solution (20) to the purified colonies grown on SA. Unhydrolyzed starch formed a blue color with the iodine, while areas of hydrolysis appeared as clear zones resulting from β-amylase activity of the Bacillus isolates (20). The isolates were assayed for rope production on sterile bread slices, as described below.

Molecular characterization of Bacillus strains. (i) DNA extraction.

Crude cell extracts were prepared as previously described (23). One microliter of a mixture was used directly as a template for PCR amplification as described below.

(ii) RAPD-PCR assay and statistical analysis of RAPD profiles.

The RAPD-PCR conditions used with primer XD9 (5′ GAAGTCGTCC 3′) were the conditions described previously (23). The amplified products (25 μl) were resolved by electrophoresis on a 1.5% (wt/vol) agarose-Tris-borate gel at 5 V cm−1 for 4 h. A 1-kb DNA ladder (Gibco BRL) was used as the molecular weight marker. B. subtilis strain ATCC 3366 was used as the reference strain. RAPD fingerprints were normalized with molecular weight markers, and the bands in each RAPD-PCR profile were automatically detected and matched by using the software Phoretic 1, advanced version 3.0.1 (Phoretix International Ltd., Newcastle upon Tyne, England). A correlation matrix for the RAPD patterns of the strains was obtained by using the formula described by Nei and Li (26): Fxy = 2nxy/(nx + ny), where Fxy is the proportion of the reproducible bands that are common to strains x and y, nxy is the number of the reproducible bands shared by strains x and y, and nx and ny are the total numbers of reproducible bands obtained for strains x and y, respectively. A similarity matrix was created and used with the average linkage method by using the Cluster procedure of Systat 5.2.1 (42) in order to estimate the percentages of similarity of the RAPD patterns of strains.

(iii) Amplification of the V3 region of 16S rDNA and DGGE analysis.

Primers V3f (5′ CCTACGGGAGGCAGCAG 3′) and V3r (5′ ATTACCGCGGCTGCTGG 3′) spanning the V3 region of the 16S rDNA of Escherichia coli (positions 341 to 534) (24) were used for amplification. A GC clamp was added to the forward primer, as described by Muyzer et al. (24). Amplification was performed in a programmable heating incubator (MJ Research Inc., Madison, Wis.). Each mixture (final volume, 25 μl) contained 1 μl (about 25 ng) of template DNA, each primer at a concentration of 0.2 μM, each deoxynucleoside triphosphate at a concentration of 0.25 mM, 2.5 mM MgCl2, 2.5 μl of 10× PCR buffer, and 2.5 U of Taq polymerase (Gibco BRL). The template DNA was denatured for 5 min at 94°C. To increase the specificity of amplification and to reduce the formation of spurious by-products, a touchdown PCR was performed (24). The initial annealing temperature used was 10°C above the expected annealing temperature (65°C), and the temperature was decreased by 1°C every second cycle until the touchdown temperature (55°C) was reached; then additional 10 cycles were carried out at 55°C. Primer extension was carried out at 72°C for 3 min. Finally, the samples were incubated for 10 min at 72°C (final extension). The PCR products were analyzed by DGGE by using a Dcode apparatus (Bio-Rad Laboratories, Hercules Calif.). Samples were applied to 8% (wt/vol) polyacrylamide gels in 1× Tris acetate buffer. Parallel electrophoresis experiments were performed at 60°C by using gels containing a 15 to 55% urea-formamide denaturing gradient (100% corresponded to 7 M urea and 40% [wt/vol] formamide) increasing in the direction of electrophoresis. The gels were electrophoresed for 10 min at 50 V and for 3.5 h at 200 V, stained with ethidium bromide for 5 min, and rinsed for 20 min in distilled water.

(iv) Sequencing of DGGE fragments.

DGGE fragments to be sequenced were excised from the gel with a sterile scalpel and processed with a Qiaex II DNA extraction kit (Qiagen S.p.a., Milan, Italy) according to the suggestions of the manufacturer. Twenty microliters of the eluted DNA from each DGGE band was reamplified by using the conditions described above. Sequences were determined by the dideoxy chain termination method (36) by using a DNA sequencing kit (Perkin-Elmer Cetus, Emeryville, Calif.) according to the manufacturer's instructions and primer V3r.

(v) Analysis of the sequence data.

To determine the closest known relatives of the partial 16S rDNA sequences obtained, searches were performed by using public data libraries (GenBank [http://www.ncbi.nlm.nih.gov/Blast.cgi]) and the Blast program (1). Similarities were calculated by considering the same length of the sequenced fragments and by using the DNasis pro. v. 3.0.7 program (Hitachi).

Rope production on bread slices by Bacillus strains.

Rope-positive Bacillus strains isolated from breads were grown overnight at 30°C in 15 ml of bread extract broth (BEB). This medium was prepared by stomaching 350 ml of distilled water and 100 g of commercial sliced white bread for 2 min; the suspension was then filtered through filter paper (Whatman no. 1), and the pH was adjusted to 6.8 with 1 mol of NaOH per liter. The medium was sterilized in tubes at 121°C for 15 min.

The BEB cultures were divided into three 5-ml aliquots. One of the aliquots was heat treated for 10 min at 96°C, one was heat treated for 10 min at 100°C, and the third was used untreated. Each aliquot was homogeneously distributed on slices of autoclaved (121°C, 15 min) commercial slices of carrè white bread to obtain a final concentration of about 104 spores cm−2. The slices were incubated at 30°C and examined daily for rope spoilage.

Screening for antagonistic activity of LAB against B. subtilis G1.

On the basis of slime production, heat resistance, and amylase activity, strain G1 of B. subtilis was selected as an indicator to determine the antagonistic activities of 118 homo- and heterofermentative LAB strains. These strains were isolated previously from pizza doughs and baker's yeasts (11, 12), and the numbers of strains belonging to the different taxa are shown in Table Table1.1. Overnight cultures of LAB were assayed by an agar-deferred spot method (45) by using plate count agar (Oxoid SPA, Garbagnate Milanese, Italy) as the culture medium. Inhibition due to H2O2 was ascertained by adding catalase (1,000 U/ml) on one side of the spotted cultures. After incubation for 24 h at 30°C, the plates were overlaid with soft SA seeded with about 104 CFU of spores of the indicator strain B. subtilis G1 per ml. After incubation for an additional 24 h at 30°C, the plates were examined to determine the presence, diameters, and appearance of inhibition zones around the spots.

TABLE 1.
Antagonistic activities of LAB against B. subtilis G1 determined by the agar deferred spot method

To ascertain the inhibition due to acidification, a well diffusion assay (37, 45) was used. Overnight cultures of LAB strains grown in appropriate broth media were centrifuged for 10 min at 8,000 × g. The pH values of the cell-free supernatants were adjusted to 7 with 2 mol of NaOH per liter, and the supernatants were filter sterilized (pore size, 0.45 μm; Sterile Acrodisc; Gelman Science, Ann Arbor, Mich.). Portions (50 μl) of the supernatant fluids were added to wells (diameter, 6 mm) bored into agar plates containing the indicator strain (about 104 CFU g−1). After incubation at 30°C for 24 h, the antimicrobial activity was determined by examining the inhibition zones.

Inhibition of B. subtilis G1 on sterile bread slices by heat-treated selected LAB cultures.

The LAB strains Lactobacillus plantarum E5, Lactobacillus sanfranciscensis M207, Lactobacillus sakei T56, Leuconostoc mesenteroides A27, Weissella paramesenteroides A51, and Enterococcus faecium A86, which showed the best inhibitory activity against B. subtilis G1, were further tested to determine their antirope activities on bread slices. LAB broth cultures that were grown overnight in BEB at 30°C were centrifuged at 2,500 × g for 10 min, and 5 ml of each supernatant fluid was heat treated at 96°C for 10 min, mixed with a heat-treated suspension of B. subtilis G1, and distributed onto sterile bread slices to obtain about 5 × 104 spores/cm2. During incubation at 30°C the slices were examined daily to detect rope slime production and to determine the pH values (2). Slices of bread soaked with a B. subtilis G1 spore suspension (5 × 103 spores/cm2) were used as a control. The experiment was performed in triplicate.

Bread-making and storage tests.

L. plantarum E5 and L. mesenteroides A27, along with Saccharomyces cerevisiae T22, previously isolated from pizza doughs (12), were used as an antirope starter in bread-making and storage tests. All bacterial cultures were propagated in BEB at 30°C for 16 h. The bread doughs were obtained by kneading in a mixer (model KPM50 Professional; KitchenAid, St. Joseph, Mich.) 1,000 g of wheat flour (labeled OO according to the Italian classification) and 560 g of tap water for 5 min at room temperature. The starter suspension was also added at this stage at a concentration that resulted in viable counts of about 5 × 107 CFU g−1 for both the yeast and LAB in the final doughs. The doughs were artificially contaminated by adding 5 × 104 spores of B. subtilis G1 g−1 to the mixing bowl. A preparation inoculated with 5 × 107 CFU of S. cerevisiae g−1 and 5 × 104 CFU of B. subtilis G1 g−1 was used as a control. After mixing, the doughs were shaped into ca. 400-g loaves, placed in aluminum pans, and leavened at 30°C until the volume was twice the initial volume. The leavened doughs were cooked in an oven at 250°C for 20 min. After cooling, the treated breads and the control were stored at 23 and 30°C. For 7 days the loaves were examined externally for the presence of the typical sweet fruity odor which characterizes the first stage of ropiness. The breads that gave off an intense smell were cut into two parts to examine the internal loaf crumb. The pH and total titratable acidity (2) were measured at the end of the incubation period.

Nucleotide sequence accession numbers.

The GenBank accession numbers for 16S rDNA partial sequences retrieved from DGGE bands are shown in Table Table22.

TABLE 2.
RAPD-PCR and DGGE patterns and molecular identification of some Bacillus spp. strains

RESULTS

Detection of ropy breads.

A total of 53.6% of the loaves examined showed typical rope production; 63% of the rope-positive samples were high-type breads, whereas 37% were low-type breads. As shown in Fig. Fig.11 the majority of the bread samples exhibited rope symptoms after 2 or 3 days of storage at 23 and 30°C, although the higher temperature (30°C) resulted in faster development of ropiness (1 to 2 days). No other ropy breads were detected between days 5 and 15 of storage at either 23 or 30°C.

FIG. 1.
Cumulative total numbers of rope-spoiled breads during storage at 23 and 30°C. A total of 56 samples were analyzed.

Characterization of Bacillus isolates. (i) Isolation and phenotypic characterization.

A total of 61 cultures of gram-positive spore-forming rods were isolated from the 30 ropy bread samples. All the isolates were identified as B. subtilis by using the dichotomous key of Norris et al. (27).

The B. subtilis isolates were characterized to determine their rope activity, resistance to heating, and amylase activity. As shown in Table Table3,3, 37 (66%) of the 61 strains assayed without heat treatment were able to cause ropiness in sterile bread slices. When the Bacillus strains were heat treated for 10 min at 96°C, only 12 (32.4%) of them still produced ropiness. No ropiness was observed when the strains were treated at 100°C for 10 min. Moreover, all rope-positive Bacillus isolates were able to hydrolyze the SA to dextrin and maltose (Table (Table3).3). Among the heat-resistant rope-producing Bacillus isolates, strain G1 produced the largest zone of amylase activity (Table (Table3)3) and hence was chosen as the indicator strain for the subsequent experiments.

TABLE 3.
Rope formation and amylase activities of the B. subtilis strains isolated from ropy bread

(ii) Molecular characterization.

Thirty-four strains of B. subtilis were chosen on the basis of their origins (isolation samples) and their biochemical activities and were characterized by molecular methods. These strains showed 11 different patterns after RAPD-PCR analysis (Fig. (Fig.2).2). As shown in Table Table2,2, most RAPD-PCR patterns were obtained for more than one strain; the only exceptions were patterns B and H, which were obtained only for strain R1 and B. subtilis ATCC 9799, respectively. In most cases, a RAPD-PCR pattern was produced by strains isolated from different samples. Nevertheless, the levels of similarity among RAPD-PCR patterns were very low, ranging from 20 to 60% (data not shown).

FIG. 2.
Ethidium bromide-stained 1.5% (wt/vol) agarose gel of RAPD-PCR products of 13 Bacillus spp. isolates generated by using primer XD9. Lane 1, strain G3 (pattern D); lane 2, strain F1 (pattern J); lane 3, strain G1 (pattern C); lane 4, strain M1p (pattern ...

Sequence-specific separation of the V3 region of 16S rDNA by DGGE resulted in grouping of the 34 strains in four profiles (Fig. (Fig.3).3). As shown in Table Table2,2, the DGGE profile a group contained seven strains that produced three different RAPD-PCR patterns (patterns A, D, and K); the DGGE profile b group contained eight strains that produced two RAPD-PCR patterns (patterns B and G); the DGGE profile c group contained 17 strains, including B. subtilis ATCC 9799, that produced RAPD-PCR patterns C, E, H, I, and J; and the DGGE profile d group contained two strains that produced RAPD-PCR pattern F.

FIG. 3.
Sequence-specific DGGE separation of the V3 regions of 16S rDNA of 14 Bacillus spp. strains. Lane 1, strain R1 (pattern b); lane 2, strain G1 (pattern c); lane 3, strain A3 (pattern a); lane 4, strain K2 (pattern b); lane 5, strain G3 (pattern a); lane ...

Molecular identification of 10 strains, representing both DGGE and RAPD-PCR patterns, was performed by sequencing the V3 region of 16S rDNA (E. coli positions 341 to 534). The accession numbers for the sequences retrieved are shown in Table Table2.2. Strains that produced DGGE pattern a were most closely related (level of similarity, 99%) to B. cereus, Bacillus thuringiensis, and Bacillus anthracis; strains that produced DGGE pattern b were most closely related (95 to 98%) to Bacillus clausii; strains that produced DGGE pattern c were most closely related (98 to 99%) to B. subtilis, B. licheniformis, and Bacillus vallismortis; and finally, strains that produced DGGE pattern d were most closely related (97%) to Bacillus firmus.

Activity of LAB against B. subtilis G1 as determined by the agar well diffusion method.

The antirope activities of LAB, as shown in Table Table4,4, were demonstrated by an agar spot deferred antagonism method and were confirmed by an agar well diffusion assay performed with neutralized and filtered culture supernatants. B. subtilis G1, selected on the basis of its heat resistance and amylase activity as described above, was used as the indicator strain. The possibility of interference of hydrogen peroxide production was eliminated by adding catalase (1,000 U/ml) on one side of the spotted cultures. As shown in Table Table1,1, 15 (12.7%) of 118 LAB strains assayed produced zones of inhibition of B. subtilis G1. The largest inhibition haloes (diameters, 5 to 7 mm) were produced by L. sakei T56, L. plantarum E5, L. sanfranciscensis M207, W. paramesenteroides A51, L. mesenteroides A27, and E. faecium A86. The inhibition zones disappeared when neutralized cell-free supernatants were assayed; therefore, the antimicrobial activity was due to acidification.

TABLE 4.
Antirope activities of LAB with B. subtilis G1, detected on sterile bread slices during storage at 30°C for 15 daysa

Inhibition of B. subtilis G1 by LAB on bread slices.

The inhibitory activities of heat-treated supernatants of the LAB strains selected as described above were tested by using sterile bread slices and B. subtilis G1 (Table (Table4).4). L. plantarum E5 and L. mesenteroides A27 inhibited ropiness in the bread for more than 15 days, whereas the other strains were able to inhibit the growth of B. subtilis G1 for a minimum of 2 days and a maximum of 5 days during incubation at 30°C. The pH decreased from 4.9 at the beginning of storage (data not shown) to values ranging from 4.3 to 3.7 during the inhibition period (Table (Table4).4). The development of rope in the bread slices caused a rapid increase in the pH to 5. The control slices inoculated with B. subtilis G1 spores without LAB were ropy after overnight incubation (Table (Table44).

Antirope activity in baked bread.

L. plantarum E5 and L. mesenteroides A27 were used as components of the antirope starter in the bread-making experiments. In order to obtain leavening, the starter was complemented with S. cerevisiae T22. The preservative starter delayed rope spoilage of bread for 1 week when the bread was stored at 23°C (Fig. (Fig.4A).4A). Incubation of the bread at 30°C resulted in development of rope after 4 days of storage (data not shown). Bread started with S. cerevisiae alone exhibited ropiness after overnight incubation at both 23 and 30°C (Fig. (Fig.4B).4B). The pH values of the loaves of bread prepared with and without the antirope starter were 4.6 and 5.5, respectively, whereas the total titratable acidity values were 1.8 and 0.7 ml of 0.1 N NaOH 10 g−1, respectively (data not shown).

FIG. 4.
Rope production in bread started with S. cerevisiae T22 after 1 day of storage at 23°C (A) and rope production in bread started with L. plantarum E5, L. mesenteroides A27, and S. cerevisiae T22 after 7 days of storage at 23°C (B). The ...

DISCUSSION

Detection of rope spoilage in bread.

According to previous studies (4, 15, 44), ropy samples develop a typical fruity odor after 12 to 24 h of incubation at 23 and 30°C, whereas the crumb becomes stickier and softer during the following days. More than one-half of the breads tested developed rope in 5 days; however, high-type breads were more frequently affected by ropiness than low-type breads. These results demonstrate that failure of the baking process is expected especially in high-type bread, in which heat-resistant Bacillus spores can survive in the center of the crumb, where the temperature often is not 96 to 101°C for 10 min (33, 38).

Identification and characterization of Bacillus strains.

Detection of rope spoilage, performed directly with bread samples, allowed isolation of different strains of spoilage-causing bacteria. Identification and differentiation of Bacillus spp. isolates from bakery environments on the basis of their phenotypic traits are very difficult (9). Collins et al. (9) suggested that additional methods, such as numerical taxonomy with combined results, can be used to clarify discrepancies in the identification of isolates. In this work a polyphasic approach, in which both biochemical and molecular methods were used, was used to characterize the Bacillus strains isolated from ropy bread. In some cases, the identities obtained by using biochemical and molecular assays differed considerably. Using the key of Norris et al. (27) resulted in identification of all of the isolates as B. subtilis, which, according to Rosenkvist and Hansen (33), could be considered the only species associated with ropiness in bread. In this study, molecular identification resulted in a greater variety of species than phenotypic characterization. In fact, strains were grouped into 11 biotypes with low degrees of similarity (<62%) as a result of using the RAPD-PCR technique. On the basis of the electrophoretic mobility of the 16S rDNA V3 region in DGGE gels, these biotypes were further clustered into four groups, suggesting the presence of at least four different species according to Muyzer et al. (24). Furthermore, on the basis of a 16S rDNA V3 region sequence analysis of representative strains of the two groups obtained by the molecular techniques mentioned above, it was possible to identify the following four taxonomic groups: (i) a B. cereus group (including B. cereus, B. anthracis, and B. thuringiensis) (group a); (ii) B. clausii (group b); (iii) a B. subtilis group (including B. subtilis, B. licheniformis, and B. vallismortis) (group c); and (iv) B. firmus (group d). Unfortunately, due to the high levels of sequence similarity of the 16S rDNA V3 regions of B. cereus, B. anthracis, and B. thuringiensis and the high levels of sequence similarity of the 16S rDNA V3 regions of B. subtilis, B. licheniformis, and B. vallismortis (data not shown), it was impossible to identify the strains of taxonomic groups a and c at the species level.

The molecular approach used in this study can therefore be considered valid for detecting the occurrence of species with unequivocal DGGE profiles and useful for confirming or facilitating taxonomic identification of Bacillus cultures. The latter use is enhanced by knowledge of additional cultural or biochemical traits. In the case of species belonging to groups whose members have similar or identical DGGE profiles, identification is facilitated by the possibility of selecting a specific biochemical assay or species-specific PCR primers, as suggested by Walters et al. (48), which distinguish a smaller number of candidate taxa.

Hence, further investigations involving other 16S rDNA regions or other target genes are necessary in order to find fragments that result in specific DGGE profiles. Indeed, recently, the 16S rDNA V1 region and the gyrA (gyrase A) gene have been evaluated as potential species-specific markers for the B. subtilis and B. cereus groups (6, 7).

The taxonomic profile obtained by the molecular methods used in this study appeared to be similar to the profiles detected by Collins et al. (9), Bailey and von Holy (4), and Thompson et al. (43), even if some differences were found. Strains of B. subtilis, B. licheniformis, B. cereus, and B. firmus are generally recognized as contaminants in bread manufacturing (4, 9, 43). However, we also identified other isolates obtained from spoiled bread, such as B. clausii. Strains of B. firmus were previously isolated only from raw materials and wheat grains (9, 33) and, along with B. clausii, were able to cause ropiness in bread. Some authors (4, 9, 21, 33) consider the possibility of high levels of contamination by B. cereus in bread remote, while other studies have documented cases of food poisoning involving nausea, vomiting, diarrhea, headaches, and chills associated with the consumption of ropy bread (22, 44, 47). However, the presence of rope-producing B. cereus in bread has to be considered a factor that could result in food poisoning and suggests that contamination should be prevented.

All of the strains isolated were also characterized to determine their amylase activities and heat resistance. The relationship between ropiness and hydrolysis of starch by microbial amylases, as reported previously by Fisher and Halton (15), Kirschner and von Holy (22), and Rosenkvist and Hansen (33), was confirmed by our findings, since all rope-positive isolates of Bacillus showed amylase activity on SA.

Rope production by heat-treated broth cultures of Bacillus strains was determined by using sterilized bread slices. Heat treatments greatly decreased the number of strains able to cause rope in bread slices. In agreement with other studies (4, 33), in which isolated B. subtilis strains survived the baking process, most of the Bacillus strains detected in this study were resistant to heating at 96°C for 10 min. Moreover, our experiment also revealed rope activity by heat-resistant strains of B. clausii, a new species related to rope spoilage in bread. These findings highlight the danger of underbaking bread, which might result in increasing the number of Bacillus species that survive the baking process, thus reducing the product shelf life.

Inhibitory activity of LAB against B. subtilis G1.

Antirope activity has to be considered an important characteristic for selecting LAB able to extend the shelf life of baked products. Only 12.7% of the LAB strains assayed for antagonistic activity showed inhibitory activity against B. subtilis G1. As shown by a well diffusion assay, the inhibition of B. subtilis G1 was due to the acidity of the broth supernatants obtained from LAB cultures. Bacteriocins have been found to confer antagonistic potential to LAB isolates from dough (13, 34). However, we did not detect any bacteriocin-like substance produced by the LAB strains used.

The inhibitory effects of LAB strains (L. sakei T56, L. plantarum E5, L. sanfranciscensis M207, W. paramesenteroides A51, L. mesenteroides A27, and E. faecium A86) on B. subtilis G1 were evaluated by testing supernatant fluids heated at 96°C for 10 min on bread slices. With this treatment we aimed to select LAB strains under conditions resembling those during the baking process. L. plantarum E5 and L. mesenteroides A27 showed the most effective antirope activity, inhibiting the development of ropiness for more than 15 days. Many authors (28, 29, 41) have described the importance of pH as a controlling factor in the development of ropiness. According to our observations, the time of inhibition of rope symptoms increases at a low pH (range, pH 3.7 to 4.3). This finding differs from the observations of other authors that indicated that the rope-inhibiting pH values were 4.69 (39) and 4.8 (34). The differences may have been due to the different experimental conditions and the different strains used as indicators.

The screening steps used in this work were useful for selecting the most effective antirope LAB strains for use in bread-making and storage tests. This approach was chosen based on the results obtained by authors who studied the activities of LAB strains against Bacillus in agar media or in bread. As reported by Rosenquist and Hansen (34), cultures of bacteriocin-producing LAB added as starters in wheat dough had no effect on B. subtilis and B. licheniformis strains, despite the fact that the same nisin-producing LAB strains had shown inhibitory activity against B. subtilis and B. licheniformis in an agar spot assay. This suggested that screening LAB by using conventional laboratory media might provide results that differ from the real activities of the strains selected. It might be advisable to select antirope starter cultures by using a medium with a composition very similar to that of bread. In our study this objective was achieved by growing the antagonistic LAB in BEB, a natural medium obtained by bread extraction, followed by an antagonistic assay with B. subtilis G1 directly on bread slices.

Antirope activity in baked bread.

The possible use of microbial cultures to increase the shelf life and safety of food products requires development of techniques which demonstrate specific inhibitory activities by starters directly in food manufacturing. Compared to bread started with S. cerevisiae T22 alone, bread manufactured with L. plantarum E5 and L. mesenteroides A27 associated with S. cerevisiae T22 delayed rope symptoms for a time that depended on the storage temperature used. A lower storage temperature (23°C) extended the bread shelf life for 7 days, whereas storage at 30°C controlled ropiness for 4 days. Storage of bread at temperatures below 25°C, which has been indicated by some authors to be an important controlling factor (28), has to be associated with the antirope activity of the starter in order to prevent the growth of Bacillus and prolong the shelf life of the bread.

The high percentages of ropy breads detected in this study and the climatic conditions of southern Italy, which favor growth of the bacteria, are expected to increase the risk of spoilage caused by Bacillus in artisanal and industrially baked products. Phenotypic identification and molecular identification highlight the fact that together with B. subtilis and B. licheniformis, which are always related to rope activity, other Bacillus species might be able to cause ropiness in bread. Rope spoilage cannot be completely prevented by using only the recommended control procedures, such as analysis of the raw material, controlling the hygiene of the bakery equipment, and strict temperature control during baking. The use of an antirope starter consisting of selected LAB strains may be considered a natural and effective method to prevent rope spoilage in bread.

Acknowledgments

We are grateful to S. Coppola, D. Ercolini, and Mark Walters for reading the manuscript and providing helpful suggestions.

REFERENCES

1. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. [PMC free article] [PubMed]
2. American Association of Cereal Chemists, Inc. 1975. Cereal laboratory methods, method 44-15A. American Association of Cereal Chemists, Inc., St. Paul, Minn.
3. Ampe, F., N. ben Omar, C. Moizan, C. Wacher, and J. P. Guyot. 1999. Polyphasic study of the spatial distribution of microorganisms in Mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations. Appl. Environ. Microbiol. 65:5464-5473. [PMC free article] [PubMed]
4. Bailey, C. P., and A. von Holy. 1993. Bacillus spore contamination associated with commercial bread manufacture. Food Microbiol. 10:287-294.
5. Brousseau, R., A. Saint-Onge, G. Préfontaine, L. Masson, and J. Cabana. 1993. Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains. Appl. Environ. Microbiol. 59:114-119. [PMC free article] [PubMed]
6. Chen, M. L., and H. Y. Tsen. 2002. Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrA gene based PCR primers and sequencing of their annealing sites. J. Appl. Microbiol. 92:912-919. [PubMed]
7. Chun, J., and K. S. Bae. 2000. Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequence. Antonie Leeuwenhoek 78:123-127. [PubMed]
8. Cocolin, L., M. Manzano, C. Cantoni, and G. Comi. 2000. Development of a rapid method for the identification of Lactobacillus spp. isolated from fermented Italian sausages using a polymerase chain reaction-temperature gradient gel electrophoresis. Lett. Appl. Microbiol. 30:126-129. [PubMed]
9. Collins, N. F., L. A. M. Kirshner, and A. von Holy. 1991. A characterisation of Bacillus isolates from ropy bread, bakery equipment and raw materials. S. Afr. J. Sci. 87:62-66.
10. Condon, S., A. Palop, J. Raso, and F. J. Sala. 1996. Influence of the incubation temperature after heat treatment upon the estimated heat resistance values of spores of Bacillus subtilis. Lett. Appl. Bacteriol. 22:149-152.
11. Coppola, S., O. Pepe, and G. Mauriello. 1998. Effect of leavening microflora on pizza dough properties. J. Appl. Microbiol. 85:891-897. [PubMed]
12. Coppola, S., O. Pepe, P. Masi, and M. Sepe. 1996. Characterisation of leavened doughs for pizza in Naples. Adv. Food Sci. 18:160-162.
13. Corsetti, A., M. Gobbetti, and E. Smacchi. 1996. Antibacterial activity of sourdough lactic acid bacteria: isolation of a bacteriocin-like inhibitory substance from Lactobacillus sanfrancisco C57. Food Microbiol. 13:447-456.
14. Ercolini, D., G. Moschetti, G. Blaiotta, and S. Coppola. 2001. Behaviour of variable V3 region from 16S rDNA of lactic acid bacteria in denaturing gradient gel electrophoresis. Curr. Microbiol. 42:199-202. [PubMed]
15. Fisher, E. A., and P. Halton. 1928. A study of rope in bread. Cereal Chem. 5:192-208.
16. Gelsomino, A., A. C. Keijzer-Wolters, G. Cacco, and J. D. van Elsas. 1999. Assessment of bacterial community structure in soil by polymerase chain reaction and denaturing gradient gel electrophoresis. J. Microbiol. Methods 38:1-15. [PubMed]
17. Green, D. H., P. R. Wakeley, A. Page, A. Barnes, L. Baccigalupi, E. Ricca, and S. M. Cutting. 1999. Characterization of two Bacillus probiotics. Appl. Environ. Microbiol. 65:4288-4291. [PMC free article] [PubMed]
18. Gregersen, T. 1978. Rapid method for distinction of Gram negative from Gram positive bacteria. Eur. J. Appl. Microbiol. Biotechnol. 5:123-127.
19. Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, and S. T. Williams. 1994. Bergey's manual of determinative bacteriology, 9th ed. Williams & Wilkins, Baltimore, Md.
20. Hurrigan, W. F., and M. E. McCance. 1976. Laboratory methods in food and dairy microbiology. Academic Press, London, United Kingdom.
21. Kaur, P. 1986. Survival and growth of Bacillus cereus in bread. J. Appl. Bacteriol. 60:513-516. [PubMed]
22. Kirschner, L. M., and A. von Holy. 1989. Rope spoilage of bread. S. Afr. J. Sci. 85:425-427.
23. Moschetti, G., G. Blaiotta, M. Aponte, P. Catzeddu, F. Villani, P. Deiana, and S. Coppola. 1998. Random amplified polymorphic DNA (RAPD) and amplified ribosomal DNA spacer polymorphism: powerful methods to differentiate Streptococcus thermophilus strains. J. Appl. Microbiol. 85:25-36. [PubMed]
24. Muyzer, G., E. C. De Waal, and A. G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695-700. [PMC free article] [PubMed]
25. Muyzer, G., A. Teske, C. O. Wirsen, and H. W. Jannasch. 1995. Phylogenetic relationship of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch. Microbiol. 164:165-172. [PubMed]
26. Nei, M., and W. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76:5269-5273. [PMC free article] [PubMed]
27. Norris, J. R., R. C. W. Berkeley, N. A. Logan, and A. G. O'Donnell. 1982. The genera Bacillus and Sporolactobacillus, p. 1711-1742. In M. P. Starr, H. Stolp, H. G. Trüper, A. Balows, and H. G. Schlegel (ed.), The prokaryotes. Springer-Verlag, New York, N.Y.
28. Quintavalla, S., and G. Parolari. 1993. Effects of temperature, aw and pH on the growth of Bacillus cells and spores: a response surface methodology study. Int. J. Food Microbiol. 19:207-216. [PubMed]
29. Quintavalla, S., and S. Gola. 1987. Effetti del pH e dell'aw sull'accrescimento di Bacillus in substrato colturale. Ind. Conserve 62:131-134.
30. Röcken, W. 1996. Applied aspects of sourdough fermentation. Adv. Food Sci. 18:212-216.
31. Röcken, W., and P. A. Voysey. 1995. Sourdough fermentation in bread making. J. Appl. Bacteriol. Symp. Suppl. 79:38-48.
32. Ronimus, R. S., L. E. Parker, and H. W. Morgan. 1997. The utilization of RAPD-PCR to identifying thermophilic and mesophilic Bacillus species. FEMS Microbiol. Lett. 147:75-79. [PubMed]
33. Rosenkvist, H., and A. Hansen. 1995. Contamination profiles and characterisation of Bacillus species in white bread and raw materials of bread production. Int. J. Food Microbiol. 26:353-363. [PubMed]
34. Rosenkvist, H., and A. Hansen. 1998. The antimicrobial effect of organic acid sourdough and nisin against Bacillus subtilis and Bacillus licheniformis isolated from wheat bread. J. Appl. Microbiol. 85:621-631.
35. Russ, J. J., W. Reeder, and D. W. Hatch. 1961. A rapid bacteriological method for predicting ropiness in bread. Cereal Sci. Today 3:89-91.
36. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. [PMC free article] [PubMed]
37. Schillinger, U., and F. K. Lücke. 1989. Antibacterial activity of Lactobacillus sake isolated from meat. Appl. Environ. Microbiol. 55:1901-1906. [PMC free article] [PubMed]
38. Spicher, G. 1983. Baked goods, p. 1-80. In H. J. Rehm and G. Reed (ed.), Bio/technology. Verlag Chemie, Deerfield Beach, Fla.
39. Spicher, G., and G. Mastik. 1988. Uber die Wechselwirkungen zwischen den Lactobacillen des Sauerteiges und Dermikroflora des Mehles. Getreide Mehl Brot 42:338-342.
40. Stephan, R., H. Schraft, and F. Untermann. 1994. Characterization of Bacillus licheniformis with the RAPD technique (randomly amplified polymorphic DNA). Lett. Appl Microbiol. 18:260-263. [PubMed]
41. Streuli, H., and M. Staub. 1955. Über die Erreger der Brotenkrankheit. Mitt. Lebensmittelunters. Hyg. 46:312-339. [PubMed]
42. Systat, Inc. 1992. Statistics, version 5.2. Systat Inc., Evanston, Ill.
43. Thompson, J. M., C. E. R. Dodd, and W. M. Waites. 1993. Spoilage of bread by Bacillus. Int. Biodeterior. Biodegrad. 32:55-66.
44. Thompson, J. M., W. M. Waites, and C. E. R. Dodd. 1998. Detection of rope spoilage in bread caused by Bacillus species. J. Appl. Microbiol. 85:481-486.
45. Villani, F., G. Salzano, E. Sorrentino, O. Pepe, P. Marino, and S. Coppola. 1993. Enterocin 226NWC, a bacteriocin produced by Enterococcus faecalis 226, active against Listeria monocytogenes. J. Appl. Bacteriol. 74:380-387. [PubMed]
46. Volavsek, P. J. A., L. A. M. Kirshner, and A. von Holy. 1992. Accelerated methods to predict the rope-inducing potential of bread raw materials. S. Afr. J. Sci. 87:99-102.
47. Voysey, P. A. 1989. Rope: a problem for bakers. J. Appl. Bacteriol. 67:xxv-xxvi.
48. Walters, J., G. W. Tannock, A. Tilsal-Timisiarvi, S. Rodtong, D. M. Loach, K. Munro, and T. Alatossava. 2000. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl. Environ. Microbiol. 66:297-303. [PMC free article] [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...