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

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.     

Detection of rope spoilage in bread caused by Bacillus species

Rope spoilage of bread by eight Bacillus isolates obtained from a bakery environment was examined via direct inoculation of slices of bread with bacterial culture. The three types of loaf examined were two soft grain varieties, one containing vinegar and the other containing calcium propionate as the preservative agent, and a white variety containing calcium propionate. Differences in rope production caused by batch variation were studied by comparing seven loaves of each type of bread. Not all isolates were capable of causing extensive rope, but isolates of Bacillus subtilis , B. licheniformis , B. megaterium and B. pumilus were able to produce such spoilage. Limited rope was also caused by pre-existing Bacillus species within the loaves. The amount of rope production by an isolate was not constant on all loaf types or even between different batches of the same variety, indicating that approaches that rely on direct inoculation of loaves with culture are not applicable for assessing the rope-inducing potential of Bacillus isolates. However, it was clear from this study that vinegar in soft grain loaves was more effective than calcium propionate at inhibiting rope.     

Genetic diversity and involvement in bread spoilage of Bacillus strains isolated from flour and ropy bread

AIMS: To study Bacillus contamination of wheat flour and ropy bread, to analyse genetic diversity of isolated strains and to evaluate the ability of these strains to produce ropy bread. METHODS AND RESULTS: Classical and molecular methods [16S rDNA sequencing and random amplified polymorphic DNA (RAPD)-PCR] were used to identify and type-isolated strains. The predominant species isolated were Bacillus subtilis and B. licheniformis. RAPD analysis demonstrated that the same sample may harbor different strains. Ten of 15 strains of B. subtilis and four of six strains of B. licheniformis were able to cause rope spoilage of the laboratory-baked bread. CONCLUSION: RAPD typing can be useful in the tracking of Bacillus strains during bakery processing and in the understanding of the role of different Bacillus strains in the rope spoilage of bread. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate the variability of Bacillus strains isolated from flour and responsible for rope spoilage of bread.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27·5°C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10⁴ spores/g whereas with 800 g loaves survival occurred with doughs containing 5·0 times 10³ spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0·2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

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

Prevention of growth in wheat bread for more than 6 d of approximately 10⁶ rope-producing Bacillus subtilis spores per gram of dough was achieved by addition of propionic or acetic acids at levels of 0·10% v/w (based on flour weight), or by addition of 15% sour dough fermented with Lactobacillus plantarum C11, Lact. brevis L62, Lact. plantarum ('vege-start 60'), Lact. plantarum (ch 20), Lact. maltaromicus (ch 15), or the commercial sour dough starter culture, Lact. sanfrancisco L99. These cultures resulted in an amount of total titratable acids above 10 in the sour dough and a pH value below 4·8 in the final bread. Bacteriocin-producing lactic acid bacteria added as starter cultures in wheat dough and nisin (Nisaplin) at levels up to 100 p.p.m. g⁻¹ flour had no effect against B. subtilis and B. licheniformis strains, despite the fact that nisin-producing strains of Lactococcus lactis ssp. lactis among 186 strains of lactic acid bacteria had demonstrated inhibitory activity against B. subtilis and B. licheniformis in an agar spot assay.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27.5 degrees C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10(4) spores/g whereas with 800 g loaves survival occurred with doughs containing 5.0 X 10(3) spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0.2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa

Twenty-five Bacillus strains capable of producing gamma-polyglutamic acid (PGA) were isolated from fermented locust bean products manufactured in the savanna area of Ghana. To clarify the phylogeny of these PGA-producing strains, phylogenetic analyses based on sequences of 16S rDNA, rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) genes were performed. A phylogenetic tree based on 16S rDNA indicated that ten isolates were clustered in the same group of Bacillus subtilis. Another ten isolates were located in the cluster of B. amyloliquefaciens, and the remaining isolates were identified as B. pumilus (three isolates) and B. licheniformis (two isolates), respectively. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. Thirty-four strains in 27 species belonging to the genus Bacillus and its neighbors were also investigated for PGA production. It was found that PGA was produced by B. amyloliquefaciens NBRC 14141 and NBRC 15535(T), B. atrophaeus NBRC 15539(T), B. licheniformis NBRC 12107, B. mojavensis NBRC 15718(T), B. pumilus NBRC 12094, B. subtilis NBRC 16449, and Lysinibacillus sphaericus NBRC 3525. Except for L. sphaericus, the above Bacillus species are very closely related in phylogeny, indicating that PGA-producing Bacillus strains constitute a cluster.     

Characterization and distribution of lactic acid bacteria in cassava fermentation during fufu production

One hundred and thirty four lactic acid bacterial strains isolated during the 96-h period of cassava fermentation for fufu production were identified. The spectrum and proportion of the strains include Lactobacillus plantarum , 81%; Leuconostoc mesenteroides , 16%; Lact. cellobiosus , 15%; Lact. brevis , 9%; Lact, coprophilus , 5%; Lact. lactis , 4%; Leuc. lactis , 3% and Lact. bulgaricus , 1%. The isolates were characterized into strains. The succession among the lactic isolates was established. Lactobacillus plantarum was identified as the most dominant lactic acid bacterial strain involved in the fermentation.     

Degradation of African locust bean oil by Bacillus subtilis and Bacillus pumilus isolated from soumbala,a fermented African locust bean condiment

AIMS: To investigate predominant isolates of Bacillus subtilis and B. pumilus in soumbala, a fermented African locust bean condiment, for their ability to degrade African locust bean oil (ALBO). METHODS AND RESULTS: Agar diffusion test in tributyrin and ALBO agar was used for screening of the isolates for esterase and lipase activity, respectively. The quantity and the profile of free fatty acids (FFA) during 72 h of degradation of ALBO by the Bacillus isolates were studied by titration and gas chromatography. The degradation of tributyrin and ALBO was variable among the isolates. Two strains of B. subtilis and two strains of B. pumilus showed significantly higher esterase and lipolytic activities than the others. The degradation ALBO was most pronounced in enriched nutrient agar except for one isolate of B. pumilus degrading ALBO to the same extent regardless of the enrichment. The quantity of FFA released from ALBO by the most lipolytic strains of Bacillus increased mainly between 0 and 24 h and differed among the isolates. The profile of FFA was similar for the Bacillus isolates with oleic acid (C18:2) occurring as the major FFA in all the samples except in samples incubated with B. subtilis B9 where stearic acid (C18) was dominant. CONCLUSION: Bacillus isolates from soumbala showed high strain dependent lipolytic activity against ALBO. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus strains to be used as starter cultures for controlled production of soumbala.     

Microbiology of controlled rotting of egusi (Colocynthis citrullus L.) fruits for the harvesting of the seeds

Microorganisms isolated from naturally rotting egusi fruits which are used as a source of dietary seeds, were mainly bacteria: Bacillus subtilis, B. licheniformis, B. polymyxa, B. megaterium, B. pumilus, Lactobacillus plantarum, La. brevis, Leuconostoc mesenteroides, Enterobacter aerogenes, E. cloacae, Klebsiella aerogenes, K. pneumoniae, Staphylococcus epidermidis, S. aureus, Micrococcus and Candida spp. The rotting period of egusi fruits could be decreased from 120 to 72 h by using a mixture of B. subtilis and B. licheniformis.     

Specific antibody-mediated detection of Brochothrix thermosphacta in situ in British fresh sausage

S.C. STRINGER, B.J. CHAFFEY, C.E.R. DODD, M.R.A. MORGAN AND W.M. WAITES. 1995. A rabbit polyclonal antibody-linked probe was developed which detected 76% of 800 food isolates of the spoilage bacterium Brochothrix thermosphacta when cells were bound to nitrocellulose. In slide cross-reaction tests all six environmental isolates tested were stained but the type strain was not. The antibody did not cross-react with Listeria grayi, L. monocytogenes, Lactobacillus plantarum, Lactococcus lactis, Streptococcus mutans, Bacillus cereus or B. subtilis.
The antibody-linked probe detected Br. thermosphacta in thin sections of British fresh sausage when the viable count was greater than 10⁶ g⁻¹ Cells were detected mainly within 1 or 2 mm of the surface on the loose starchy material. They were not detected within muscle blocks or in the centre of the sausage. Such results suggest that growth of this organism occurs close to the surface of the sausage.     

Microbial assessment and quality evaluation of ogi during spoilage

The changes during the fermentation of ogi, a cereal-based traditional lactic acid-fermented weaning food were studied up to the spoilage stage. Ogi off-odour was first noticed at the fourth day of fermentation (including 24 h steeping). Yeast isolates such as Candida valida, C. krusei, Geotrichum candidum and bacteria like Lactobacillus brevis, L. plantarum, Pediococcus pentosaceus, Bacillus subtilis, Brevibacterium linens, Br. oxydans and other two Brevibacterium spp. dominated the fermenting mash at the spoilage stage. The brevibacteria contributed most significantly to ogi off-odour. Ogi samples inoculated with lactic acid bacteria increased acidity and product acceptability over the time of fermentation. The pH, titratable acidity, dissolved hydrogen sulphide and the presence of brevibacteria appear as good indices for monitoring spoilage. Hydrogen sulphide (H₂S) levels appear to be the most useful of the parameters studied. No coliforms and clostridia were identified during spoilage.     

In vitro and in situ growth characteristics and behaviour of spoilage organisms associated with anaerobically stored cooked meat products

AIMS: Understanding spoilage caused by different types of spoilage organisms, associated with vacuum-packaged sliced cooked meat products (CMP). METHODS AND RESULTS: First, strains were characterized in a broth at 7 degrees C under anaerobic conditions to compare their growth rate, acidifying character and metabolite production under conditions simulating refrigerated vacuum-packaged conditions. Brochotrix thermosphacta grew faster than the lactic acid bacteria (LAB). Within the group of the LAB, all strains grew fast except Leuconostoc mesenteroides subsp. dextranicum and Leuconostoc carnosum. Secondly, the organisms were inoculated on a model cooked ham to better understand the relationship between spoilage, microbial growth, pH, metabolite production and accompanying sensory changes. Most rapidly growing strains were Leuc. mesenteroides subsp. mesenteroides followed by B. thermosphacta, while Leuc. mesenteroides subsp. dextranicum and Leuc. carnosum grew very slowly compared with the other LAB. Brochotrix thermosphacta caused sensory deviations at a lower cell number compared with the LAB. The related pH changes, metabolite production and sensory perception are presented. CONCLUSIONS: In this pure culture study, B. thermosphacta and Leuc. mesenteroides subsp. mesenteroides had the highest potential to cause rapid spoilage on CMP. SIGNIFICANCE AND IMPACT OF THE STUDY: A systematic study on the behaviour of spoilage organisms on a model cooked ham to establish the relationship between microbial growth, pH, metabolite formation and organoleptic deviations.     

Identification and characterization of Lactic Acid Bacteria isolated from Tibetan Qula cheese

Fourteen strains of Lactic Acid Bacteria (LAB) isolated from Qula, a Tibetan traditional yak cheese, were divided into four groups (A-D) according to morphological and biochemical characteristics. On the basis of the 16S rRNA gene sequence analysis, group A and group B strains were placed in the cluster making up the genus Leuconostoc, which together with Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, formed a distinct cluster. The group C strain was clearly identified as Enterococcus faecium by forming a very well defined cluster with this species. The group D strains were placed in the lactobacilli cluster with Lactobacillus plantarum and Lactobacillus pentosus being the closely related species. On the basis of DNA-DNA hybridization, strains in the groups A, B, C and D were identified as Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc pseudomesenteroides, Enterococcus faecium and Lactobacillus plantarum, respectively. Leuconostoc mesenteroides subsp. dextranicum was the dominate member of the population.     

Directed evolution of a maltogenic alpha-amylase from Bacillus sp. TS-25

Directed evolution coupled with a high-throughput robotic screen was employed to broaden the industrial use of the maltogenic alpha-amylase Novamyl from Bacillus sp. TS-25. Wild-type Novamyl is currently used in the baking industry as an anti-staling agent in breads baked at neutral or near neutral pH. However, the enzyme is rapidly inactivated during the baking process of bread made with low pH recipes and Novamyl thus has very limited beneficial effect for this particular application. In an effort to improve the performance of Novamyl for low pH bread applications such as sourdough and rye, two error-prone PCR libraries were generated, expressed in Bacillus subtilis and screened for variants with improved thermal stability and activity under low pH conditions. Variants exhibiting improved performance were iteratively recombined using DNA shuffling to create two generations of libraries. Relative to wild-type Novamyl, a number of the resulting variants exhibited more than 10 degrees C increase in thermal stability at pH 4.5, one of which demonstrated substantial anti-staling properties in low pH breads.     

Biosynthesis of linkage units for teichoic acids in gram-positive bacteria: distribution of related enzymes and their specificities for UDP-sugars and lipid-linked intermediates.

The distribution and substrate specificities of enzymes involved in the formation of linkage units which contain N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) or glucose and join teichoic acid chains to peptidoglycan were studied among membrane systems obtained from the following two groups of gram-positive bacteria: group A, including Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Staphylococcus aureus, and Lactobacillus plantarum; group B, Bacillus coagulans. All the membrane preparations tested catalyzed the synthesis of N-acetylglucosaminyl pyrophosphorylpolyprenol (GlcNAc-PP-polyprenol). The enzymes transferring glycosyl residues to GlcNAc-PP-polyprenol were specific to either UDP-ManNAc (group A strains) or UDP-glucose (group B strains). In the synthesis of the disaccharide-bound lipids, GlcNAc-PP-dolichol could substitute for GlcNAc-PP-undecaprenol. ManNAc-GlcNAc-PP-undecaprenol, ManNAc-GlcNAc-PP-dolichol, Glc-GlcNAc-PP-undecaprenol, Glc-GlcNAc-PP-dolichol, and GlcNAc-GlcNAc-PP-undecaprenol were more or less efficiently converted to glycerol phosphate-containing lipid intermediates and polymers in the membrane systems of B. subtilis W23 and B. coagulans AHU 1366. However, GlcNAc-GlcNAc-PP-dolichol could not serve as an intermediate in either of these membrane systems. Further studies on the exchangeability of ManNAc-GlcNAc-PP-undecaprenol and Glc-GlcNAc-PP-undecaprenol revealed that in the membrane systems of S. aureus strains and other B. coagulans strains both disaccharide-inked lipids served almost equally as intermediates in the synthesis of polymers. In the membrane systems of other B. subtilis strains as well as B. licheniformis and B. pumilus strains, however, the replacement of ManNAc-GlcNAc-PP-undecaprenol by Glc-GlcNAc-PP-undecaprenol led to a great accumulation of (glycerol phosphate)-Glc-GlcNAc-PP-undecaprenol accompanied by a decrease in the formation of polymers.     

Genotyping and toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages

Artisanal and industrial sausages were analyzed for their aerobic, heat-resistant microflora to assess whether new emerging pathogens could be present among Bacillus strains naturally contaminating cured meat products. Sixty-four isolates were characterized by randomly amplified polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (fAFLP). The biotypes, identified by partial 16S rRNA gene sequence analysis, belonged to Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens species. Both RAPD-PCR and fAFLP analyses demonstrated that a high genetic heterogeneity is present in the B. subtilis group even in strains harvested from the same source, making it possible to isolate 56 different biotypes. Moreover, fAFLP analysis made it possible to distinguish B. subtilis from B. pumilus strains. The strains were characterized for their toxigenic potential by molecular, physiological, and immunological techniques. Specific PCR analyses revealed the absence of DNA sequences related to HBL, BcET, NHE, and entFM Bacillus cereus enterotoxins and the enzymes sphingomyelinase Sph and phospholipase PI-PLC in all strains; also, the immunological analyses showed that Bacillus strains did not react with NHE- and HBL-specific antibodies. However, some isolates were found to be positive for hemolytic and lecithinase activity. The absence of toxigenic potential in Bacillus strains from the sausages analyzed indicates that these products can be considered safe under the processing conditions they were produced; however, great care should be taken when the ripening time is shortened, particularly in the case of traditional sausages, which could contain high amounts of Bacillus strains and possibly some B. cereus cells.     

Cytotoxic Bacillus spp. belonging to the B. cereus and B. subtilis groups in Norwegian surface waters

AIMS: To investigate the presence and numbers of Bacillus spp. spores in surface waters and examine isolates belonging to the B. cereus and B. subtilis groups for cytotoxicity, and to discuss the presence of cytotoxic Bacillus spp. in surface water as hazard identification in a risk assessment approach in the food industry. METHODS AND RESULTS: Samples from eight different rivers with variable degree of faecal pollution, and two drinking water sources, were heat shocked and examined for the presence of Bacillus spp. spores using membrane filtration followed by cultivation on bovine blood agar plates. Bacillus spp. was present in all samples. The numbers varied from 15 to 1400 CFU 100 ml(-1). Pure cultures of 86 Bacillus spp. isolates representing all sampling sites were characterized using colony morphology, atmospheric requirements, spore and sporangium morphology, and API 50 CHB and API 20E. Bacillus spp. representing the B. cereus and B. subtilis groups were isolated from all samples. Twenty-one isolates belonging to the B. cereus and B. subtilis groups, representing eight samples, were screened for cytotoxicity. Nine strains of B. cereus and five strains belonging to the B. subtilis group were cytotoxic. CONCLUSIONS: The presence of cytotoxic Bacillus spp. in surface water represents a possible source for food contamination. Filtration and chlorination of surface water, the most common drinking water treatment in Norway, do not remove Bacillus spores efficiently. This was confirmed by isolation of spores from tap water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of food with water containing low numbers of Bacillus spores implies a risk for bacterial growth in foods. Consequently, high numbers of Bacillus spp. may occur after growth in some products. High numbers of cytotoxic Bacillus spp. in foods may represent a risk for food poisoning.     

Growth of Bacillus cereus in fermenting tempeh made from various beans and its inhibition by Lactobacillus plantarum

Counts of Bacillus cereus reached ca 10⁸ cfu/g within 40 h in fermenting unacidified horsebean tempeh and resulted in complete spoilage of the product. In fermenting unacidified pea, chickpea and soybean tempeh, B. cereus counts reached 10⁶–10⁷ cfu/g, although the products were not spoiled. Inoculation of these unacidified beans with Lactobacillus plantarum decreased the final count of B. cereus by 2 log units, but had no effect on its growth in unacidified horsebean tempeh and its subsequent spoilage. Acidification of the beans during soaking resulted in a lower rate of B. cereus growth during fermentation. Inoculation of acidified beans with Lact. plantarum resulted in a markedly lower growth rate of B. cereus . In an associative broth culture study, B. cereus was completely inhibited by Lact. plantarum at pH values of about 5·5. Lactobacillus plantarum may be used to control the growth of B. cereus during tempeh production.     

Isolation of plant-growth-promoting Bacillus strains from soybean root nodules

Endophytic bacteria reside within plant tissues and have often been found to promote plant growth. Fourteen strains of putative endophytic bacteria, not including endosymbiotic Bradyrhizobium strains, were isolated from surface-sterilized soybean (Glycine max. (L.) Merr.) root nodules. These isolates were designated as non-Bradyrhizobium endophytic bacteria (NEB). Three isolates (NEB4, NEB5, and NEB17) were found to increase soybean weight when plants were co-inoculated with one of the isolates and Bradyrhizobium japonicum under nitrogen-free conditions, compared with plants inoculated with B. japonicum alone. In the absence of B. japonicum, these isolates neither nodulated soybean, nor did they affect soybean growth. All three isolates were Gram-positive spore-forming rods. While Biolog tests indicated that the three isolates belonged to the genus Bacillus, it was not possible to determine the species. Phylogenetic analysis of 16S rRNA gene hypervariant region sequences demonstrated that both NEB4 and NEB5 are Bacillus subtilis strains, and that NEB17 is a Bacillus thuringiensis strain.