A RAPD-based survey of thermophilic bacilli in milk powders from different countries

Twenty-eight milk powders from 18 different countries were examined for the number and type of contaminating thermophilic bacilli. Of 742 isolates examined, 96.8% were assigned to the same strains of bacilli as previously found in New Zealand powders. The dominant isolate was Anoxybacillus flavithermus strain C followed by Bacillus licheniformis strain F. The former was also prevalent in New Zealand powders and the results demonstrate that A. flavithermus represents a widespread contaminant, seemingly ubiquitous in factories producing milk powder. The presence of thermophilic strains of Geobacillus stearothermophilus and to a lesser extent of Bacillus subtilis in milk powders was reconfirmed.     

Survival of thermophilic spore-forming bacteria in a 90+ year old milk powder from Ernest Shackelton's Cape Royds Hut in Antarctica

Milk powder taken to Antarctica on Shackelton's British Antarctic Expedition in 1907 was produced in New Zealand by a roller drying process in the first factory in the world dedicated to this process. Thermophilic bacilli are the dominant contaminants of modern spray-dried milk powders and the 1907 milk powder allows a comparison to be made of contaminating strains in roller-dried and spray-dried powders. Samples of milk powder obtained from Shackelton's Hut at Cape Royds had low levels of thermophilic contamination (< 500 cfu ml-1) but the two dominant strains (Bacillus licheniformis strain F and Bacillus subtilis) were typical of those found in spray-dried powders. Soil samples from the floor of the hut also contained these strains, whereas soils distant from the hut did not. Differences in the RAPD profiles of isolates from the milk powder and the soils suggest that contamination of the milk from the soil was unlikely. It is significant that the most commonly encountered contaminant strain in modern spray-dried milk (Anoxybacillus flavithermus strain C) was not detected in the 1907 sample.     

A RAPD-based comparison of thermophilic bacilli from milk powders

The similarity of strains of thermophilic Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), Anoxybacillus flavithermus (formerly Bacillus flavothermus), Bacillus licheniformis and Bacillus subtilis isolated from separate milk powder production runs from multiple factories was examined using a random amplified polymorphic DNA (RAPD) protocol. As a result of the analysis of the RAPD fingerprints and data relating to general growth and biochemical tests, over 98% of the 1470 isolates examined (grown at 55 degrees C) were assigned to the species G. stearothermophilus, A. flavithermus, B. licheniformis and B. subtilis. The G. stearothermophilus isolates were identified as being nearly identical to G. stearothermophilus (DSMZ 22; equivalent to ATCC 12980), or G. stearothermophilus var. calidolactis (DSMZ 1550). Three groups of isolates were found to be related to A. flavithermus (DSMZ 2641) by partial small ribosomal subunit (16S) sequence comparisons and shown to be interrelated by RAPD analyses with multiple primer sets. The thermophilic isolates of B. licheniformis were positively identified by comparison with type strains of B. licheniformis DSMZ 13 and DSMZ 8785. All of the B. subtilis strains shared bands in their RAPD profiles and were similar to a common B. subtilis type strain (DSMZ 10 and DSMZ 347). Overall, the most common and prevalent group of strains (group A) was demonstrated to be closely related to G. stearothermophilus (DSMZ 22).     

Concurrence of spores of Clostridium tyrobutyricum,Clostridium beijerinckii and Paenibacillus polymyxa in silage,dairy cow faeces and raw milk

Spores of lactate-fermenting clostridia, known as butyric acid bacteria (BAB), can cause severe quality defects in semi-hard cheeses, called late-blowing. The routine detection method of BAB spores involves most probable number quantification of spores of anaerobic, gas-forming bacteria in a medium containing lactic acid. In this study, BAB spores were detected in 296 samples of soil, maize and grass silage, dairy cow faeces and farm tank milk collected from dairy farms in The Netherlands and the most abundant populations identified. Three major populations were detected, namely Clostridium tyrobutyricum, Clostridium beijerinckii and Paenibacillus spp. Paenibacillus polymyxa was the most abundant species of the latter group. The results indicate that the three spore populations share the same sources and the same contamination route of milk. In contrast to the Clostridium species, P. polymyxa isolates were unable to ferment lactate into butyrate. P. polymyxa spores are presumably unable to cause cheese defects.     

Stability of cyclopiazonic acid during storage and processing of milk

The effect of refrigerated storage (4°C) of raw or processed milk on the stability of cyclopiazonic acid (CPA) in milk was investigated. CPA decreased by 1.4% following 4 days of simulating collecting, storing and transporting of contaminated (1 μg CPA ml⁻¹) raw milk. Storage at 4°C for 21 days, simulating retail milk, moderately reduced the CPA level by 5.8%. A similar trend of CPA decrease was observed in frozen and freeze dried milk stored at −18°C. However, in both products, less than 12% of CPA decreased in spite of a storage period of 140 days. The effect of processing milk on component separation of CPA was also studied. Simulating unsweetened condensed milk production by preheating 4 l contaminated milk to 100°C and concentrating under steam injection to 1.5 l led to a decrease of CPA by 39.7%. In contrast, very little CPA (0.7%) was lost from the production of evaporated milk using low temperature (60°C) heating under vacuum to remove water. CPA was stable in both concentrated and evaporated milks throughout an 8 weeks storage period at 4°C. There was no decrease of CPA during the manufacturing of milk powder by spray drying. Persistence of CPA during the above milk storage and processing methods confirmed the potential of CPA to reach consumers of dairy products when the mycotoxin was carried over into processed milks.     

A standard set of testing methods reliably enumerates spores across commercial milk powders

Contamination of dairy powders with sporeforming bacteria is a concern for dairy processors who wish to penetrate markets with stringent spore count specifications (e.g., infant powders). Despite instituted specifications, no standard methodology is used for spore testing across the dairy industry. Instead, a variety of spore enumeration methods are in use, varying primarily by heat-shock treatments, plating method, recovery medium, and incubation temperature. Importantly, testing the same product using different methodologies leads to differences in spore count outcomes, which is a major issue for those required to meet specifications. As such, we set out to identify method(s) to recommend for standardized milk powder spore testing. To this end, 10 commercial milk powders were evaluated using methods varying by (1) heat treatment (e.g., 80°C/12 min), (2) plating method (e.g., spread plating), (3) medium type (e.g., plate count milk agar), and (4) incubation time and temperature combinations (e.g., 32°C for 48 h). The resulting data set included a total of 48 methods. With this data set, we used a stepwise process to identify optimal method(s) that would explain a high proportion of variance in spore count outcomes and would be practical to implement across the dairy industry. Ultimately, spore pasteurized mesophilic spore count (80°C/12 min, incubated at 32°C for 48 h), highly heat resistant thermophilic spore count (100°C/30 min, incubated at 55°C for 48 h), and specially thermoresistant spore enumeration (106°C/30 min, incubated at 55°C for 48 h) spread plating on plate count milk agar were identified as the optimal method set for reliable enumeration of spores in milk powders. Subsequently, we assessed different powder sampling strategies as a way to reduce variation in powder spore testing outcomes using our recommended method set. Results indicated that 33-g composite sampling may reduce variation in spore testing outcomes for highly heat resistant thermophilic spore count over 11-g and 33-g discrete sampling, whereas there was no significant difference across sampling strategies for specially thermoresistant spore enumeration or spore pasteurized mesophilic spore count. Finally, an interlaboratory study using our recommended method set and a modified method set (using tryptic soy agar with 1% starch) among both university and industry laboratories showed increased variation in spore count outcomes within milk powders, which not only was due to natural variation in powders but also was hypothesized to be due to technical errors, highlighting the need for specialized training for technicians who perform spore testing on milk powders. Overall, this study addresses challenges to milk powder spore testing and recommends a method set for standardized spore testing for implementation across the dairy industry.     

奶粉厂节能措施与设计

奶粉是一种耗能很高的加工制品，在制造过程中又以浓缩和干燥两个环节耗能最多。针对我国，小型奶粉厂现状，在这两个方面提出了一些切实可行的节能措施与设计，以降低成本，提高效益。     

The characterisation of Bacillus spores occurring in the manufacturing of (low acid) canned products

Spore-forming bacteria can be a problem in the food industry, especially in the canning industry. Spores present in ingredients or present in the processing environment severely challenge the preservation process since their thermal resistance may be very high. We therefore asked the question which bacterial spore formers are found in a typical soup manufacturing plant, where they originate from and what the thermal resistance of their spores is. To answer these questions molecular techniques for bacterial species and strain identification were used as well as a protocol for the assessment of spore heat stress resistance based on the Kooiman method. The data indicate the existence and physiological cause of the high thermal resistance of spores of many of the occurring species. In particular it shows that ingredients used in soup manufacturing are a rich source of high thermal resistant spores and that sporulation in the presence of ingredients rich in divalent metal ions exerts a strong influence on spore heat resistance. It was also indicated that Bacillus spores may well be able to germinate and resporulate during manufacturing i.e. through growth and sporulation in line. Both these spores and those originating from the ingredients were able to survive certain thermal processing settings. Species identity was confirmed using fatty acid analysis, 16SrRNA gene sequencing and DNA-DNA hybridisation. Finally, molecular typing experiments using Ribotyping and AFLP analysis show that strains within the various Bacillus species can be clustered according to the thermal resistance properties of their spores. AFLP performed slightly better than Ribotyping. The data proofed to be useful for the generation of strain specific probes. Protocols to validate these probes in routine identification and innovation aimed at tailor made heat processing in soup manufacturing have been formulated.     

An alternative process for the manufacture of whole milk powder

The traditional process of manufacturing whole milk powder has some negative aspects: high heat treatment of the milk and, owing to fouling during evaporation, loss of product. To reduce these negative aspects an alternative way of producing whole milk powder was investigated in pilot-plant experiments. Milk was first separated into skim milk and cream and then treated further. Skim milk was subjected to a low heat treatment and concentrated by evaporation. The cream was subjected to a high heat treatment and mixed with the concentrated skim milk. The standardized whole milk concentrate was then spray dried. This process of manufacturing whole milk powder compared favourably with the traditional process with respect to product losses, the physical properties of the whole milk powder and the flavour of the reconstituted milk.     

Reservoir and routes of transmission of Enterobacter sakazakii (Cronobacter spp.) in a milk powder-producing plant

Several outbreaks of Cronobacter spp. (Enterobacter sakazakii) have been described as food-borne illness in neonates and infants. Powdered infant formula has been identified as a source of infection, especially in hospital nurseries, where a bulk of formula nutrient is prepared for the whole day and instructions for preparation are not always followed correctly. Neonates who are underweight or immunosuppressed are especially at risk for an E. sakazakii infection. Considering that milk powder is the main ingredient of powdered infant formula, we analyzed the incidence and distribution of E. sakazakii in a milk powder-producing plant. We looked specifically at the spray-drying towers and roller dryers. Selected isolates from samples taken from the environment and final product were typed by pulsed-field gel electrophoresis to investigate the epidemiology of the organism within the production area of the plant. Seven pulsed-field gel electrophoresis types were detected in the spray-drying area, which presumably entered the plant through an aperture for process air and an improperly controlled roller shutter. Furthermore, textile filters for exhaust air of both the spray-drying towers were identified as internal reservoirs of the pathogen. For economic reasons, powder from the textile filters is reintroduced into the product flow; this can contaminate the final product. For the production of milk powder to be used as an ingredient of powdered infant formula, it was suggested to terminate the process of reintroducing the filtered powder into the product flow. A second transmission route was identified in the roller dryer section of the factory. It could be shown that contaminated milk concentrate could pass the process unheated, thus leading to a contamination of the product with E. sakazakii.     

Aerobic spore-forming bacteria in bulk raw milk: factors influencing the numbers of psychrotrophic, mesophilic and thermophilic Bacillus spores

Psychrotrophic, mesophilic and thermophilic spore concentrations of Bacillus spp. were determined on a weekly basis in bulk raw milk samples obtained from a central processing facility, over one calendar year. These data were correlated with concentrations of metal ions, free amino acids and somatic cell counts obtained from the same samples, as well as local meteorological mean temperature and relative humidity measurements relating to the same test period. A heat treatment of 80°C for 20 min followed by the addition of L-alanine to the milk at 0.1% w/v and incubation at 55°C for 7 days gave optimal recovery conditions for thermophilic spores. Free amino acids, metal ions, somatic cell counts, temperature, and relative humidity measurements were each significantly correlated with the recovery of spores of Bacillus spp. from bulk raw milk, although no single factor was shown to demonstrate a consistent effect with the psychrotrophic, mesophilic and thermophilic spore groups studied.     

Evaluation of dairy powder products implicates thermophilic sporeformers as the primary organisms of interest

Dairy powder products (e.g., sweet whey, nonfat dry milk, acid whey, and whey protein concentrate-80) are of economic interest to the dairy industry. According to the US Dairy Export Council, customers have set strict tolerances (<500 to <1,000/g) for thermophilic and mesophilic spores in dairy powders; therefore, understanding proliferation and survival of sporeforming organisms within dairy powder processing plants is necessary to control and reduce sporeformer counts. Raw, work-in-process, and finished product samples were collected from 4 dairy powder processing facilities in the northeastern United States over a 1-yr period. Two separate spore treatments: (1) 80°C for 12 min (to detect sporeformers) and (2) 100°C for 30 min (to detect highly heat resistant sporeformers) were applied to samples before microbiological analyses. Raw material, work-in-process, and finished product samples were analyzed for thermophilic, mesophilic, and psychrotolerant sporeformers, with 77.5, 71.0, and 4.6% of samples being positive for those organisms, respectively. Work-in-process and finished product samples were also analyzed for highly heat resistant thermophilic and mesophilic sporeformers, with 63.7 and 42.6% of samples being positive, respectively. Sporeformer prevalence and counts varied considerably by product and plant; sweet whey and nonfat dry milk showed a higher prevalence of thermophilic and mesophilic sporeformers compared with acid whey and whey protein concentrate-80. Unlike previous reports, we found limited evidence for increased spore counts toward the end of processing runs. Our data provide important insight into spore contamination patterns associated with production of different types of dairy powders and support that thermophilic sporeformers are the primary organism of concern in dairy powders.     

Enumeration and confirmation of Clostridium tyrobutyricum in silages using neutral red, D-cycloserine, and lactate dehydrogenase activity

Spores of clostridia in big bale silages, manure, and dairy products were enumerated and distinguished from other spore formers by using Reinforced Clostridium Agar containing .005% neutral red. Spores of Clostridium tyrobutyricum predominated, but spores of Clostridium butyricum, Clostridium sporogenes, Clostridium bifermentans, Clostridium putrificum, and Clostridium sphenoides occurred to a lesser extent. In samples with high bacterial spore counts, growth of Bacillus spp., but not C. tyrobutyricum, was retarded by the addition of 200 ppm D-cycloserine. Clostridia isolated from silages and milk products were identified and tested on lactate dehydrogenase activity. Of 275 investigated strains, only strains identified as C. tyrobutyricum tested positively. Only 65% of the tested strains of C. tyrobutyricum grew in the confirmatory substrate containing minerals, lactic acid, and acetic acid. Tyrobutyricum Broth was not selective for C. tyrobutyricum, since C. butyricum and C. sporogenes also grew in this medium.     

Nitrite production by thermophilic aerobic bacteria isolated during the manufacture of milk powder

Nitrite contamination in milk powder is hypothesised to be caused by thermophilic bacteria that form biofilms in milk powder manufacturing plants. Regulatory limits in some countries have made nitrite contamination in milk powder an issue influencing product sales. In this study, thermophilic bacteria were isolated from milk samples obtained from a milk processing plant to investigate the potential of different isolates to convert nitrate to nitrite. Eight bacteria species were identified through 16s rDNA gene sequencing (Bacillus licheniformis, Bacillus aerius, Bacillus subtilis, Bacillus sonorensis, Bacillus firmus, Enterococcus faecium, Geobacillus stearothermophilus and Macrococcus caseolyticus). Most bacteria isolated were able to convert nitrate to nitrite, and surprisingly in aerobic conditions. The most interesting species was G. stearothermophilus as its nitrate reducing capability was highly variable between different isolates. This could explain why there was no relationship reported between nitrite level in milk powder and high thermophile count.     

Incidence, source and some properties of psychrotrophic Bacillus spp found in raw and pasteurized milk

Spores of psychrotrophic Bacillus spp were isolated from 58% of farm bulk tank milks and about 69% of pasteurized milks. Counts of Bacillus spp in about 10% of raw milk samples reached 1 × 10⁵ cfu/ml and above within seven days at 6°C. Psychrotrophic spore counts in pasteurized milks ranged from <0.5 to 170 spores/litre with an average of about 17/1. There was little correlation between the total bacterial count of the raw milk and presence of psychrotrophic Bacillus spores. There was some evidence that the bulk tank itself may be a source of contamination. The spores in pasteurized milk probably were not the result of postpasteurization contamination. The optimum germination temperature for psychrotrophic Bacillus spores was lower than that for spores of mesophilic strains. About 50% of the psychrotrophic Bacillus strains isolated from milk were capable of growth at 2°C.     

黑曲霉孢子粉真空冷冻干燥保护剂配方优化

采用单因素试验和混合均匀试验设计,对黑曲霉孢子粉真空冷冻干燥保护剂配方进行筛选和优化。通过Design-Expert软件对实验数据进行回归分析,得到保护剂最佳配方为脱脂奶粉6.43 g/100 mL、蔗糖13.03 g/100 mL、甘油6.88 g/100 mL,孢子存活率预测值为88.82%,实际值为87.03%。     

Investigation of the migration of triclabendazole residues to milk products manufactured from bovine milk,and stability therein,following lactating cow treatment

Triclabendazole (TCB) is a flukicide used in the treatment of liver fluke in cattle; however, its use is currently prohibited in lactating dairy cows. In this study, following administration of 10% Fasinex (triclabendazole, Novartis Animal Health UK Ltd., Camberley, UK) the milk of 6 animals was used to manufacture dairy products, to ascertain if TCB residues in milk migrate into dairy products. The detection limit of the ultra-high-performance liquid chromatography-tandem mass spectrometry method used was 0.67 μg/kg. The highest concentrations of TCB residue measured, within the individual cow milk yield, was 1,529 ± 244 µg/kg (n = 6), on d 2 posttreatment. Days 2 and 23 posttreatment represented high and low residue concentrations, respectively. At each of these 2 time points, the milk was pooled into 2 independent aliquots and refrigerated. Milk products, including cheese, butter, and skim milk powder were manufactured using pasteurized and unpasteurized milk from each aliquot. The results for high residue milks demonstrated that TCB residues concentrated in the cheese by a factor of 5 (5,372 vs. 918 µg/kg for cheese vs. milk) compared with the starting milk. Residue concentrations are the sum of TCB and its metabolites, expressed as keto-TCB. Residues were concentrated in the butter by a factor of 9 (9,177 vs. 1,082 μg/kg for butter vs. milk) compared with the starting milk. For milk, which was separated to skim milk and cream fractions, the residues were concentrated in the cream. Once skim milk powder was manufactured from the skim milk fraction, the residue in powder was concentrated 15-fold compared with the starting skim milk (7,252 vs. 423 µg/kg for powder vs. skim milk), despite the high temperature (185°C) required during powder manufacture. For products manufactured from milk with low residue concentrations at d 23 posttreatment, TCB residues were detected in butter, cheese, and skim milk powder, even though there was no detectable residue in the milk used to manufacture these products. Triclabendazole residues were concentrated in some milk products (despite manufacturing treatments), exceeding residue levels in the starting milk and, depending on the storage conditions, may be relatively stable over time.     

Farmers' preferences for adopting on-farm concentration of raw milk: Results from a discrete choice experiment in Germany

Milk concentrates are used in the manufacturing of dairy products such as yogurt and cheese or are processed into milk powder. Processes for the nonthermal separation of water and valuable milk ingredients are becoming increasingly widespread at farm level. The technical barriers to using farm-manufactured milk concentrate in dairies are minimal, hence the suspicion that the practice of on-farm raw milk concentration is still fairly uncommon for economic reasons. This study, therefore, set out to investigate farmers' potential willingness to adopt a raw milk concentration plant. The empirical analysis was based on discrete choice experiments with 75 German dairy farmers to identify preferences and the possible adoption of on-farm raw milk concentration. The results showed that, in particular, farmers who deemed the current milk price to be insufficient viewed on-farm concentration using membrane technology as an option for diversifying their milk sales. We found no indication that adoption would be impeded by a lack of trustworthy information on milk processing technologies or capital.     

Germination, Growth, and Toxin Production of Nonproteolytic Clostridium botulinum as Affected by Multiple Barriers

ABSTRACT The effect of combinations of pH (6.5, 5.75), NaCl (0.25,1.75%), and incubation temperatures (7,13 °C) on spore germination, outgrowth, and time to toxicity of nonproteolytic Clostridium botulinum was examined in a broth system. Spores of four toxin type E and nonproteolytic type B were inoculated (10⁴/ml) into Tryptone Peptone Glucose Yeast Extract (TPGY) broth. Cultures were monitored for three weeks, or until toxin was detected. A modified FSIS‐amplified ELISA (comparable in sensitivity to the mouse bioassay) was used to screen cultures for neurotoxin. Combinations of the most inhibitory level for each barrier reduced the degree and rate of germination, the lag and growth rate of vegetative cells, and the time to toxicity.     

Spore inactivation on solid surfaces by vaporized hydrogen peroxide—Influence of carrier material surface properties

The aim of this study was to investigate the influence of surface hydrophobicity and roughness of carrier materials on the inactivation of bacterial spores with gaseous hydrogen peroxide whereas condensate formation is prevented. Spores of Bacillus subtilis and Bacillus atrophaeus were applied either as single spore culture or as a mixed spore population to simulate natural contamination with microorganisms of different characteristics. Inactivation with gaseous hydrogen peroxide was carried out at 5200 ppm hydrogen peroxide without condensate formation. The inactivation results of B. subtilis and B. atrophaeus spores on carrier materials with varying surface hydrophobicity differed significantly. However, inactivation of the mixed spore populations resulted in similar resistance compared to the single spore batches. The results of this study indicate that surface hydrophobicity most probably has an impact on the inactivation with gaseous hydrogen peroxide whereas surface roughness only plays a minor role.