THE INCIDENCE AND THERMAL RESISTANCE OF MESOPHILIC SPORES FOUND IN MILK AND RELATED ENVIRONMENTS

SUMMARY: A spore 'spectrum' is described of aerobic mesophiles capable of resisting different heat treatments. It is shown that B. licheniformis is the most common spore former found in bulk milk but since its spores are rapidly destroyed at 100°, the more heat resistant B. subtilis is the dominant surviving spore former in commercial sterilized milk. The thermal resistance of strains of B. subtilis and B. licheniformis isolated from different sources has been investigated and the strains of B. subtilis typed according to the behaviour of their spores when heated at 100°. All strains of B. licheniformis were destroyed more rapidly by boiling for 2 min than strains of B. subtilis but only those strains of the latter which showed some degree of heat activation were more resistant than B. licheniformis . The 'resistant' and heat activated strains of B. subtilis appear to be sparsely distributed in nature and were only isolated from sterilized milk where the heat treatment applied would tend to eliminate other strains. The spore content of bovine faeces was similar to that in bulk milk and the total spore content varied seasonally, the spore content of faeces being on the average a hundred times greater during indoor feeding than during the period when the cattle were fed outside. A faecal infection of the milk in the ratio of 1:10⁴ would infect the milk with spores at about the same concentration as they are found in bulk raw milk, and it is suggested that bovine faeces could be a primary source of spore formers in milk supplies.     

Characterization of mesophilic bacilli in faeces of feedlot cattle

AIMS: To determine the identity and composition of mesophilic Bacillus spp. in faeces sampled from feedlot cattle. METHODS AND RESULTS: Faecal samples from 10 feedlot cattle were analysed. The total aerobic spore count increased from 4.6 x 10(4) CFU g(-1) (before feedlotting, day 0) to 1.6 x 10(6) CFU g(-1) (feedlot for day 76). A total of 150 randomly selected spore isolates (60 each from days 0 and 76 cattle, 30 from feed) were speciated using a Bacillus group-specific PCR-amplified ribosomal DNA restriction analysis technique (Wu et al. 2006). At day 0, Bacillus subtilis and Bacillus cereus predominated with a prevalence of 58.3% and 26.7%, respectively, whereas three species, B. subtilis (50.0%), Bacillus licheniformis (27.6%) and Bacillus clausii (20.0%) predominated in day 76 faecal samples. Of these, only the first two species were present in feed samples at a frequency of 70% and 30% respectively. All B. cereus isolates on day 0, possessed at least one of three enterotoxin genes (nheA, nheB and nheC) but these were completely eliminated after a period of feedlotting. All isolates of B. licheniformis were genotypically heterogeneous according to pulsed-field gel electrophoresis analysis. CONCLUSIONS: Cattle faeces contain large numbers of Bacillus spores representing different mesophilic species. Stable faecal populations of particular Bacillus spp. mimicking those found in feed, were subsequently established by feedlotting. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained and methods used in this study will help to investigate the indigenous Bacillus composition in the gastrointestinal tract of cattle and will further guide the administration of Bacillus probiotics.     

Incidence and diversity of potentially highly heat-resistant spores isolated at dairy farms

The presence of highly heat-resistant spores of Bacillus sporothermodurans in ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100 degrees C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera. Bacillus licheniformis and Bacillus pallidus were the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems.     

Mesophilic and psychrotrophic aerobe sporulating microorganisms in raw cow’s milk

We studied the occurrence of mesophilic and psychrotrophic aerobic sporulating microorganisms (MPAS) in raw cow’s milk and their relations to microflora in milk. We took 294 samples of raw cow’s milk from 14 farms during one year. Briefly the method for MPAS assessment is to inactivate the milk sample by heating it to 80-82°C for 30 minutes. Mesophilic aerobic sporulates are incubated at 30°C for 3 days-, and psychrotrophic aerobic sporulates at 7°C for 10 days. Results of studied microbiological parameters characterize the sampled milk as complying with requirements of the EU regulation 92/46 and standard STN 57 0529. MPAS count was within the span 2.5–340 CFU/ml. The average value ofMPAS was 59.4 CFU/ml, with variation coefficient 93.1%. Counts up to 50 CFU/ml were in 55.4% samples, the value was not higher than 100 in 85%, and in 3.1% of the samples the MPAS count was higher than 200. MPAS do not show correlation with any of the studied microbiological parameters; marked influences of season were not observed either. On the basis of obtained results, it is possible to support the proposal of an initial limit of maximum 200 CFU/ml for the introduction of a MPAS parameter. MPAS count found in the same dishes at incubation for mesophilic and subsequently strictly psychrophilic microorganisms was 56.9 CFU/ml on average. This represents 95.8% of total CFU sums of individual dishes at two temperatures. The correlation coefficient of these two types of results, r = 0.99,gives evidence of close dependence expressed by the linear regression equation. Use of two incubation temperatures, one after another with an identical set of dishes, enables us to exclude overestimation of results due to sporulates able to grow at both incubation temperatures.     

Studies on the Bacillus flora of milk and milk products

Bacillus licheniformis and B. cereus were the most commonly isolated species of Bacillus found in milk at all stages of processing. Bacillus licheniformis was ubiquitous in the farm environment and counts in raw milks heat-treated in the laboratory were higher during the winter months, whilst B. cereus was associated with cattle feed throughout the year, and tended to be more common in raw milks during the summer months. Although B. licheniformis was usually isolated in larger numbers than B. cereus, this pattern changed after raw and pasteurized milks and reconstituted milk powders were pre-incubated at ambient temperatures, and B. cereus came to dominate the Bacillus population, reaching levels associated with enterotoxin production. Investigation of the growth kinetics of strains of both species showed that B. cereus grew faster than B. licheniformis at ambient temperatures. It is suggested that post-pasteurization contamination, which is commonly blamed for spoilage of milk and milk products by B. cereus, is not necessarily the most important source of this organism.     

Comparative analysis of the diversity of aerobic spore-forming bacteria in raw milk from organic and conventional dairy farms

Bacterial contamination of raw milk can originate from different sources: air, milking equipment, feed, soil, faeces and grass. It is hypothesized that differences in feeding and housing strategies of cows may influence the microbial quality of milk. This assumption was investigated through comparison of the aerobic spore-forming flora in milk from organic and conventional dairy farms. Laboratory pasteurized milk samples from five conventional and five organic dairy farms, sampled in late summer/autumn and in winter, were plated on a standard medium and two differential media, one screening for phospholipolytic and the other for proteolytic activity of bacteria. Almost 930 isolates were obtained of which 898 could be screened via fatty acid methyl ester analysis. Representative isolates were further analysed using 16S rRNA gene sequencing and (GTG)(5)-PCR. The majority of aerobic spore-formers in milk belonged to the genus Bacillus and showed at least 97% 16S rRNA gene sequence similarity with type strains of Bacillus licheniformis, Bacillus pumilus, Bacillus circulans, Bacillus subtilis and with type strains of species belonging to the Bacillus cereus group. About 7% of all isolates may belong to possibly new spore-forming taxa. Although the overall diversity of aerobic spore-forming bacteria in milk from organic vs. conventional dairy farms was highly similar, some differences between both were observed: (i) a relatively higher number of thermotolerant organisms in milk from conventional dairy farms compared to organic farms (41.2% vs. 25.9%), and (ii) a relatively higher number of B. cereus group organisms in milk from organic (81.3%) and Ureibacillus thermosphaericus in milk from conventional (85.7%) dairy farms. One of these differences, the higher occurrence of B. cereus group organisms in milk from organic dairy farms, may be linked to differences in housing strategy between the two types of dairy farming. However, no plausible clarification was found for the relatively higher number of thermotolerant organisms and the higher occurrence of U. thermosphaericus in milk from conventional dairy farms. Possibly this is due to differences in feeding strategy but no decisive indications were found to support this assumption.     

Occurrence of Bacillus sporothermodurans and other aerobic spore-forming species in feed concentrate for dairy cattle

AIMS: To determine the aerobic spore composition and presence of Bacillus sporothermodurans spores in feed concentrate for dairy cattle. METHODS AND RESULTS: Six feed concentrate samples from five different farms were analysed. High levels of spores (up to 10(6) spores g(-1)) were found. Identification of 100 selected isolates was obtained by a combination of fatty acid methyl esters analysis, amplified ribosomal DNA restriction analysis and 16S rDNA sequencing. Ninety-seven isolates could be identified to the species level or assigned to a phylogenetic species group. Most of the isolates obtained after a heat treatment of 10 min at 80 degrees C were identified as members of the B. subtilis group (32 isolates), B. pumilus (25 isolates), B. clausii (eight isolates) and B. licheniformis (eight isolates). The isolates with very heat-resistant spores, obtained after a heat treatment of 30 min at 100 degrees C, were identified as members of the B. subtilis group (five isolates), B. sporothermodurans (three isolates), B. amyloliquefaciens (one isolate), B. oleronius (one isolate) and B. pallidus (one isolate). Bacillus cereus was present in each feed concentrate sample and was isolated using a selective mannitol egg yolk polymyxin agar medium. CONCLUSIONS: Feed concentrate for dairy cattle contains known as well as as yet unknown species of Bacillus and related genera with properties relevant to the dairy sector. SIGNIFICANCE AND IMPACT OF THE STUDY: The results formulate the hypothesis that feed concentrate can be a contamination source of spores, including those of B. sporothermodurans, for raw milk at the farm level.     

Observations on the effect of raw milk quality on the keeping quality of pasteurized milk

Raw milk was stored for up to 14 d at 4°C and pasteurized on days 1, 3, 4, 7, 9 and 14. Precautions were taken to eliminate post-pasteurization contamination. The pasteurized milks were stored at 4°C and analysed at weekly intervals for standard plate counts (SPC), psychrotrophic counts (PC) and aerobic spore counts (ASC). The initial raw milk quality was very good and the keeping quality of all the pasteurized milks tested was greater than 22 d. In some cases the milk still had acceptable SPC after 42 d storage, which shows the keeping quality that can be achieved when the process is well controlled. However, the best keeping quality resulted from milk pasteurized on the third and fourth days. Even milk pasteurized on the seventh and ninth had superior keeping quality to that pasteurized on the first day. The lactoperoxidase anti-microbial system in raw milk may be most active around days 3 and 4.     

Estimating amplification efficiency improves multiplex real-time PCR quantification of Bacillus licheniformis and Bacillus subtilis spores in animal feed

A multiplex real-time PCR assay was developed for absolute quantification in animal feed of Bacillus subtilis CH201 and Bacillus licheniformis CH200 spores, which constitute the viable component of the microbial growth promoter, BioPlus 2B. Spores were lysed using a bead-beating protocol. DNA was extracted and purified from the lysates with the Qiagen DNeasy Plant Kit. Two standard curves for absolute quantification were made and tested. Standard curve-1 was made from feed samples spiked with BioPlus 2B, while standard curve-2 was made from serially diluted DNA extracted from BioPlus 2B powder. Feed samples supplemented with BioPlus 2B were quantified using both standard curves. The detection limit of the assay was 10(4) CFU g(-1) of feed. The amplification efficiency (Eff) of each PCR was determined using the LinRegPCR software and Eff differences between individual samples and standards were corrected for. When compared to plate counts, standard curve-1 slightly under-estimated the number of spores (mean=-2.47% of plate counts). A spore density-dependent Eff was found, and Eff for standard curve-1 could not be determined. Standard curve-2 over-estimated spore numbers when not corrected for individual Eff (mean=+5.46% of plate counts). Standard curve-2 Eff was independent (Eff(mean)=1.96) of spore density. The assay quantified the numbers of spores in feed samples very similar to plate counts (mean=+0.47% of plate counts), when standard curve-2 was used and individual Eff was accounted for.     

Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis.

By using plasmid pMB9, penicillinase genes (penP and penI) from both the wild-type and constitutive strains of Bacillus licheniformis 9945A were cloned in EScherichia coli. When a low-copy-number plasmid was used, both wild-type and constitutive penicillinase genes could be transferred into Bacillus subtilis. However, when a high-copy-number plasmid was used, only the genes of the wild type could be transferred. These recombinant plasmids in B. subtilis could all be transferred by the protoplast transformation procedure into B. licheniformis. Transformants of E. coli were resistant to ampicillin (20 micrograms/ml) in spite of the low penicillinase activities (7 U/mg of cells). However, transformants of B. subtilis and B. licheniformis were sensitive to ampicillin (20 micrograms/ml) even in high penicillinase activities (more than 10,000 U/mg of cells). The secretion of penicillinase was rarely observed in E. coli. In contrast, penicillinases secreted from transformants of B. subtilis and B. licheniformis were around 30 and 60% of the total activities, respectively. We took advantage of the plasmids to permit the construction of hetero- and mero-polyploid structures in host cells, and we discuss a regulatory mechanism of penicillinase synthesis in B. licheniformis.     

Incidence and Diversity of Potentially Highly Heat-Resistant Spores Isolated at Dairy Farms

The presence of highly heat-resistant spores of Bacillus sporothermodurans in ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100°C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera. Bacillus licheniformis and Bacillus pallidus were the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems.     

THE GERMINATION AND ENZYMIC ACTIVITIES OF BACILLUS SPORES AT LOW TEMPERATURES

SUMMARY: Well washed spore preparations of Bacillus cereus and B. subtilis were suspended in various nutrient broths, soil extracts, autoclaved soil of various moisture contents, and in two inorganic solutions, phosphate buffer, pH 7·2 and Ringer's solution. These were incubated at 8°, 5°, 1° and 0° for periods up to 270 days. Periodic total and spore counts on plates indicated a progressive decrease in each, associated with germination taking place in all conditions except in the two inorganic media. Enzymic tests indicated secretion and activity of nitratase and gelatinase and, with spores of B. pasteurii , urease, as a result of germination. B. subtilis germinated to a greater extent than B. cereus in each of the nutrient media. Germination of both organisms at 5° was also observed in L - and D -alanine: in the latter it was probably the result of racemization to the L -form.     

Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products

Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products. After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified. Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B. coagulans , five were B. circulans and four were B. licheniformis . Skim milk powder contributed the maximum number of B. stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%). When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65°C for 30 min. Protease of B. stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70°C for 30 min.     

Enumeration of thermophilic Bacillus species in composts and identification with a Random Amplification Polymorphic DNA (RAPD) protocol

The thermophilic microbial flora of general garden and domestic wastes composts, derived from thermogenic, post-thermogenic and maturation phases, was analysed using spore and total plate counts in combination with an optimised RAPD protocol. A total of 459 isolates were recovered obtained at 55 degrees C, and another 56 at 70 degrees C using tryptic soy-starch agar plates, with near-equal numbers being derived from total plate counts or spore preparations. The isolates were obtained from 11 compost samples and were assigned to eighteen different RAPD fingerprint types, with 76.1% of these ultimately being positively assigned by their RAPD profiles to just 2 species including Bacillus thermodenitrificans and B. licheniformis. Viable cell numbers ranged from 1.4 to 150 x 10(6) colony forming units per gram compost (wet weight), with the highest two counts being from 2 week and 4 week old compost samples with temperatures of 70 degrees C and 55 degrees C, respectively. B. thermodenitrificans was a dominant isolate (representing more than 50% of isolates from total plate counts) in 7 of the 11 individual compost total plate count samples between 30 degrees C to 73 degrees C, and accounted for 68.9% of all isolates overall. Another relatively common Bacillus species that was identified with RAPDs in significant numbers included B. licheniformis (7.2% of all isolates and dominant isolate in 1 sample). Three other relatively common RAPD profiles could not be identified by comparison with known species in a RAPD profile database but were tentatively identified using 16S rDNA sequence comparisons. These were B. sporothermodurans (4.9% of all isolates and dominant in 1 sample), B. thermosphaericus (7.4% and dominant in 1 sample) and Terrabacter tumescens (5.0%). Overall, based on the vegetative and spore count results and the subsequent RAPD-based identification, the data strongly support a significant role for B. thermodenitrificans in the composting process, and casts doubt on the notion that B. stearothermophilus sensu strictu (DSMZ 22) is a prominent member within compost ecology.     

METHODS OF ASSESSING THE SPORICIDAL EFFICIENCY OF AN ULTRA-HIGH-TEMPERATURE MILK STERILIZING PLANT. III. LABORATORY DETERMINATIONS OF THE HEAT RESISTANCE OF SPORES OF BACILLUS SUBTILIS IN WATER AND IN MILK

SUMMARY: Thermal death curves for spores of Bacillus subtilis 786 have been determined in water and in milk. Generally a non-logarithmic order of death was observed. Numbers of survivors were lower in milk than in water, suggesting that there may be inhibitory factors in UHT sterilized milk which affect the germination and/or subsequent growth of heated spores.
The thermal death curves for spores suspended in milk yielded Q₁₀ values of about 30 in the range 110–120°. This is higher than the figures previously reported in the literature for R. subtilis spores. Spores of a number of strains of B. subtilis were compared with strain 786 and all gave high Q₁₀ values.
The results obtained in this work have been used to predict the destruction of spores at higher temperatures in a UHT plant (Burton et al. 1958). The calculated values agree well with the results obtained in the plant by Franklin et al. (1958).     

THE GERMINATION OF SPORES OF BACILLUS SUBTILIS AND BACILLUS LICHENIFORMIS AT TEMPERATURES BELOW THE MINIMUM FOR VEGETATIVE GROWTH

SUMMARY: The germination in nutrient media of the spores of several strains of Bacillus subtilis and B. licheniformis at temperatures too low to permit vegetative growth has been investigated. Germination occurred with only 11 of 54 strains of B. subtilis and with none of 35 strains of B. licheniformis .     

Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products

Proteolytic thermophilic bacterial cultures (171 strains) were isolated from different milk and milk products. After screening these isolates for protease production in a liquid medium, fifty that exhibited enzyme activity in excess of 100 units/ml were selected and identified. Twenty-nine were Bacillus stearothermophilus (constituting 58% of the total), twelve were B. coagulans, five were B. circulans and four were B. licheniformis. Skim milk powder contributed the maximum number of B. stearothermophilus (64.7%) followed by raw milk (63.2%) and pasteurized milk (44.4%). When the culture supernatant liquids from the selected isolates were given heat treatment, five cultures retained 100% protease activity at 65 degrees C for 30 min. Protease of B. stearothermophilus RM-67 had the maximum heat resistance because it retained 87.5% of its activity at 70 degrees C for 30 min.     

长江流域水稻根际芽孢杆菌属固氮菌株的分离与鉴定

生物固氮资源的研究、开发和利用有利于发展持续农业,水稻田的氮素肥力比一般旱地高得多,活跃的生物固氮被认为是主要因素之一.苏宝林、崔宗均等系统地研究了我国北方稻区的固氮菌资源,分离鉴定出8属18种共35株固氮菌.在此基础上,我们对长江流域水稻根际异养固氮菌资源进行了系统研究.本文报道芽孢杆菌属(Bacillus)固氮菌株分离与鉴定的结果.     

'BITTY' CREAM: THE OCCURRENCE AND SIGNIFICANCE OF BACILLUS CEREUS SPORES IN RAW MILK SUPPLIES

SUMMARY: Methods for determining the Bacillus cereus content of milks and of rinses of dairy equipment are described and their limitations discussed. Milk samples from various sources were examined throughout the year for 'bittiness' as well as for B. cereus. The organism was not detected in all samples showing bittiness. There was a marked seasonal variation in the B. cereus index of raw milk supplies; maximum numbers were obtained from July to September and minimum numbers in April and May. Rinses of farm dairy equipment yielded few B. cereus spores but milk cans not uncommonly contained large numbers, especially in the summer months, when 10·5% of cans showed more than 5 × 10³/can. Preliminary observations on other sources of B. cereus are described and some of the problems of the control of this organism in raw milk supplies are discussed.     

Hemicellulases of Bacillus species: preliminary comparative studies on production and properties of mannanases and galactanases

A range of Bacillus subtilis strains and other Bacillus species were screened for mannanase, β-mannosidase and galactanase activities. Maximum mannanase activity, 106.2 units/ml, was produced by B. subtilis NRRL 356. β-Mannosidase and galactanase activities from all strains were relatively low. The effect of carbon and nitrogen source on mannanase and galactanase production by B. brevis ATCC 8186, B. licheniformis ATCC 27811, B. polymyxa NRRL 842 and B. subtilis NRRL 356 was investigated. Highest mannanase production was observed in the four strains tested when the mannan substrate, locust bean gum, was used as carbon source. Induction was most dramatic in the case of B. subtilis NRRL 356 where only basal enzyme levels were produced in the presence of other carbon sources. β-Mannosidase was induced in the four Bacillus cultures by locust bean gum. Results indicated that galactose acted as an inducer for production of galactanase. Organic and inorganic nitrogen sources resulted in induction of high mannanase titres in B. subtilis. Highest galactanase activity was produced by each organism in media containing sodium nitrate as nitrogen source. Mannanases from B. brevis, B. licheniformis, B. polymyxa and B. subtilis retained 100% residual activity after a 3 h incubation at 65°C, 65°C, 60°C and 55°C respectively. Galactanases retained more than 95% activity at 55°C after 3 h. The pH optima of mannanases ranged from 6.5–6.8 whereas galactanases ranged from 5.1 in the case of B. brevis to 7.0 for B. polymyxa.