Real-time PCR detection of Paenibacillus spp. in raw milk to predict shelf life performance of pasteurized fluid milk products

Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (C(T)) of 19.14 ± 0.54. While 14/17 Bacillus isolates showed no signal (C(T) > 40), 3 Bacillus isolates showed very weak positive signals (C(T) = 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 10(1) CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.     

Detection of Helicobacter pylori in cow's milk

AIMS: To investigate the existence of Helicobacter pylori in cow's milk as one of the foods which most Japanese children eat. METHODS AND RESULTS: Detection of H. pylori was demonstrated by the semi-nested polymerase chain reaction (PCR), a culture method and electron microscopy. Semi-nested PCR demonstrated the ureA gene of H. pylori in 13 of 18 (72.2%) raw milk samples and in 11 of 20 (55%) commercial pasteurized milk samples. Helicobacter pylori binding immunomagnetic beads with H. pylori-specific goat anti-H. pylori antibody was shown by electron microscopy in both raw and pasteurized milk positive for the ureA gene. Helicobacter pylori was cultured in one raw milk sample, whereas it was not cultured in pasteurized milk samples. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: There is a possibility that cow's milk is a transmission vehicle in childhood H. pylori infection, although we failed to confirm the survival of H. pylori in pasteurized milk.     

The cytochrome c oxidase test for the rapid detection of psychrotrophic bacteria in milk

A new, rapid, simple and cheap method for the detection of the predominant psychrotrophic bacteria in raw and pasteurized milks is described, using tetramethyl-p-phenylene-diamine dihydrochloride to detect cytochrome c oxidase which in general is not present in the non-psychrotrophic bacterial milk flora. The test is sensitive to samples containing over 10(4) organisms/ml. Correlation coefficients of 0.92 and 0.84 between dye oxidation and viable counts for pasteurized and raw milk samples, respectively, were found.     

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.     

Incidence of Mycobacterium paratuberculosis in bulk raw and commercially pasteurized cows' milk from approved dairy processing establishments in the United Kingdom

Over a 17-month period (March 1999 to July 2000), a total of 814 cows' milk samples, 244 bulk raw and 567 commercially pasteurized (228 whole, 179 semi-skim, and 160 skim), from 241 approved dairy processing establishments throughout the United Kingdom were tested for the presence of Mycobacterium paratuberculosis by immunomagnetic PCR (to detect all cells living and dead) and culture (to detect viable cells). Overall, M. paratuberculosis DNA was detected by immunomagnetic PCR in 19 (7.8%; 95% confidence interval, 4.3 to 10.8%) and 67 (11.8%; 95% confidence interval, 9.0 to 14.2%) of the raw and pasteurized milk samples, respectively. Confirmed M. paratuberculosis isolates were cultured from 4 (1.6%; 95% confidence interval, 0.04 to 3.1%) and 10 (1.8%; 95% confidence interval, 0.7 to 2.8%) of the raw and pasteurized milk samples, respectively, following chemical decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h. The 10 culture-positive pasteurized milk samples were from just 8 (3.3%) of the 241 dairy processing establishments that participated in the survey. Seven of the culture-positive pasteurized milk samples had been heat treated at 72 to 74 degrees C for 15 s; the remainder had been treated at 72 to 75 degrees C for the extended holding time of 25 s. When typed by restriction fragment length polymorphism and pulsed-field gel electrophoresis methods, some of the milk isolates were shown to be types distinct from those of laboratory strains in regular use within the testing laboratory. From information gathered at the time of milk sample collection, all indications were that pasteurization had been carried out effectively at all of the culture-positive dairies. That is, pasteurization time and temperature conditions complied with the legal minimum high-temperature, short-time process; all pasteurized milk samples tested phosphatase negative; and post-process contamination was considered unlikely to have occurred. It was concluded that viable M. paratuberculosis is occasionally present at low levels in commercially pasteurized cows' milk in the United Kingdom.     

Determining the source of Bacillus cereus and Bacillus licheniformis isolated from raw milk, pasteurized milk and yoghurt

Aims:  Strain-specific detection of Bacillus cereus and Bacillus licheniformi s in raw and pasteurized milk, and yoghurt during processing.
Methods and Results:  Randomly selected isolates of Bacillus spp. were subjected to PCR analysis, where single primer targeting to the repetitive sequence Box elements was used to fingerprint the species. The isolates were separated into six different fingerprint patterns. The results show that isolates clustered together at about the 57% similarity level with two main groups at the 82% and 83% similarity levels, respectively. Contamination with identical strains both of B. cereus and B . licheniformis in raw and pasteurized milk was found as well as contaminated with different strains (in the case of raw milk and yoghurt/pasteurized milk and yoghurt). Several BOX types traced in processed milk samples were not discovered in the original raw milk.
Conclusions:  BOX-PCR fingerprinting is useful for characterizing Bacillus populations in a dairy environment. It can be used to confirm environmental contamination, eventually clonal transfer of Bacillus strains during the technological processing of milk.
Significance and Impact of the Study:  Despite the limited number of strains analysed, the two Bacillus species yielded adequately detectable banding profiles, permitting differentiation of bacteria at the strain level and showing their diversity throughout dairy processing.     

Selenium concentration and glutathione peroxidase activity in cow’s milk

Samples of raw or pasteurized cow’s milk and infant formula were assayed for glutathione peroxidase (GSH-Px) activity, selenium (Se), and total protein. GSH-Px activity was detected in raw milk, but not in market-pasteurized milk or infant formula. The correlation between GSH-Px activity and Se levels was significant, even when the influence of protein level was removed. This result implies a role of GSH-Px as one of biologically active forms of Se in raw milk. Absence of GSH-Px activity in pasteurized milk and infant formula result from the heating process in these productions, because the heating of raw milk gave an irreversible inactivation of GSH-Px. Both GSH-Px activity and Se levels had significant correlation with protein level, but not so when the respective influences of Se and GSH-Px levels were removed. In raw cow’s milk, Se content was 23 ng/mL and GSH-Px activity was 20 U/mL. About 12% of Se was bound to GSH-Px, and 0.003% of protein was GSH-Px. Raw milk obtained in July contained higher levels of Se, GSH-Px, and protein than that in November. Data for cow’s milk were discussed in relation to those for human milk and those in New Zealand.     

Incidence of Mycobacterium paratuberculosis in Bulk Raw and Commercially Pasteurized Cows' Milk from Approved Dairy Processing Establishments in the United Kingdom

Over a 17-month period (March 1999 to July 2000), a total of 814 cows' milk samples, 244 bulk raw and 567 commercially pasteurized (228 whole, 179 semiskim, and 160 skim), from 241 approved dairy processing establishments throughout the United Kingdom were tested for the presence of Mycobacterium paratuberculosis by immunomagnetic PCR (to detect all cells living and dead) and culture (to detect viable cells). Overall, M. paratuberculosis DNA was detected by immunomagnetic PCR in 19 (7.8%; 95% confidence interval, 4.3 to 10.8%) and 67 (11.8%; 95% confidence interval, 9.0 to 14.2%) of the raw and pasteurized milk samples, respectively. Confirmed M. paratuberculosis isolates were cultured from 4 (1.6%; 95% confidence interval, 0.04 to 3.1%) and 10 (1.8%; 95% confidence interval, 0.7 to 2.8%) of the raw and pasteurized milk samples, respectively, following chemical decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h. The 10 culture-positive pasteurized milk samples were from just 8 (3.3%) of the 241 dairy processing establishments that participated in the survey. Seven of the culture-positive pasteurized milk samples had been heat treated at 72 to 74°C for 15 s; the remainder had been treated at 72 to 75°C for the extended holding time of 25 s. When typed by restriction fragment length polymorphism and pulsed-field gel electrophoresis methods, some of the milk isolates were shown to be types distinct from those of laboratory strains in regular use within the testing laboratory. From information gathered at the time of milk sample collection, all indications were that pasteurization had been carried out effectively at all of the culture-positive dairies. That is, pasteurization time and temperature conditions complied with the legal minimum high-temperature, short-time process; all pasteurized milk samples tested phosphatase negative; and postprocess contamination was considered unlikely to have occurred. It was concluded that viable M. paratuberculosis is occasionally present at low levels in commercially pasteurized cows' milk in the United Kingdom.     

Effect of commercial-scale high-temperature, short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows' milk

Raw cows' milk naturally infected with Mycobacterium paratuberculosis was pasteurized with an APV HXP commercial-scale pasteurizer (capacity 2,000 liters/h) on 12 separate occasions. On each processing occasion, milk was subjected to four different pasteurization treatments, viz., 73 degrees C for 15 s or 25 s with and without prior homogenization (2,500 lb/in(2) in two stages), in an APV Manton Gaulin KF6 homogenizer. Raw and pasteurized milk samples were tested for M. paratuberculosis by immunomagnetic separation (IMS)-PCR (to detect the presence of bacteria) and culture after decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h (to confirm bacterial viability). On 10 of the 12 processing occasions, M. paratuberculosis was detectable by IMS-PCR, culture, or both in either raw or pasteurized milk. Overall, viable M. paratuberculosis was cultured from 4 (6.7%) of 60 raw and 10 (6.9%) of 144 pasteurized milk samples. On one processing day, in particular, M. paratuberculosis appeared to have been present in greater abundance in the source raw milk (evidenced by more culture positives and stronger PCR signals), and on this occasion, surviving M. paratuberculosis bacteria were isolated from milk processed by all four heat treatments, i.e., 73 degrees C for 15 and 25 s with and without prior homogenization. On one other occasion, surviving M. paratuberculosis bacteria were isolated from an unhomogenized milk sample that had been heat treated at 73 degrees C for 25 s. Results suggested that homogenization increases the lethality of subsequent heat treatment to some extent with respect to M. paratuberculosis, but the extended 25-s holding time at 73 degrees C was found to be no more effective at killing M. paratuberculosis than the standard 15-s holding time. This study provides clear evidence that M. paratuberculosis bacteria in naturally infected milk are capable of surviving commercial high-temperature, short-time pasteurization if they are present in raw milk in sufficient numbers.     

Isolation of Yersinia enterocolitica from Milk and a Dairy Farm in Australia

Forty-eight isolates obtained from pasteurized milk and cream, raw milk, and various sites on a dairy farm were identified as Yersinia enterocolitica biotype 1 but were unusual in their ability to ferment lactose. The majority belonged to serotype 5a. Serotypes 5b, 6, 13/15 and 23/15 were also found. A few isolates were not typable. There is no evidence that strains isolated in this study are pathogenic for humans. Their presence in pasteurized products and growth at refrigeration temperatures are, however, cause for concern.     

A note on the use of the Catalasemetre in assessing the quality of milk

The Catalasemetre, for assessing the quality of raw and pasteurized milk, has been studied. No correlation was found between catalase activity and bacterial counts for farm bulk tank milks within the range 5.2 ± 10²-5.4 ± 10⁵ cfu/ml. Similarly, no relation was observed between catalase activity and somatic cell counts of milk (range of counts from 0.08 to 3.5). However, the catalase activity and bacterial count of pasteurized milks which had been pre-incubated at 21.C for 25 h in the presence of crystal violet-penicillin-nisin to inhibit Gram-positive bacterial growth were significantly related. Thus, the use of this pre-incubation procedure coupled with the Catalasemetre to estimate bacterial growth, has potential in assessing the keeping quality of pasteurized milk samples within 25.5 h of production. Results on the thermostability of native milk catalase are also presented.     

Germination of Spores of Bacillus cereus in Milk and Milk Dialysates: Effect of Heat Treatment

S ummary . The germination of spores of Bacillus cereus was studied in milk and in media consisting of the low M.W. fraction of milk. Dialysates, centrifugates, filtrates and acid whey supported germination to an extent similar to that in the milk from which they were derived. HTST (72° for 15 sec) pasteurized milk or derived media supported appreciable germination whereas raw milk or media derived from it supported little or none. Whey produced by the action of rennet was an exception in that it was equally stimulatory for germination whether derived from raw or pasteurized milk. Heat treatments for 15 see using temperatures between 65–75° rendered the milk most suitable as a germination medium but temperatures > 80° were necessary for spore activation. Of the 2 effects, activation was the more important; at treatment temperatures > 80° germination was increased despite the less favourable medium which resulted. The extent of germination in pasteurized milk varied with different isolates and could be related to their source, those from pasteurized milk germinating the most readily. The practical implications of these findings are discussed together with the preliminary work to examine the nature of the germination factor(s) produced during HTST pasteurization.     

Quantitative isolation efficiency of O26, O103, O111, O145 and O157 STEC serotypes from artificially contaminated food and cattle faeces samples using a new isolation protocol

Aims:  A range of new differential and confirmation plating media for some non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes (O26, O103, O111, O145) and both sorbitol-positive and -negative O157 were evaluated using artificially contaminated samples.
Methods and Results:  Dairy products (raw milk, cheese made from pasteurized milk and raw milk), meat (ground beef, fermented meat) and cattle faeces were artificially contaminated using clinical STEC strains. Isolation efficiency was 100%, 82·3%, 88·5%, 65·9%, 64·3% and 15·8%, respectively, for an inoculum size of ≤100 CFU 25 g⁻¹. The consecutive use of differential and confirmation media limited the incidence of false positive isolates from 0% for raw milk samples, cheese made from pasteurized milk and for fermented meat to 2·1% for cheese made from raw milk, and to 8·9% for ground beef.
Conclusions:  Data presented in this paper indicated that the efficiency of the applied isolation method was dependent on sample-to-sample variation but not on the inoculum size.
Significance and Impact of Study:  Data in this paper indicated that isolation of low levels of non-O157 and sorbitol-positive O157 STEC from food samples is possible.     

Effect of Commercial-Scale High-Temperature, Short-Time Pasteurization on the Viability of Mycobacterium paratuberculosis in Naturally Infected Cows' Milk

Raw cows' milk naturally infected with Mycobacterium paratuberculosis was pasteurized with an APV HXP commercial-scale pasteurizer (capacity 2,000 liters/h) on 12 separate occasions. On each processing occasion, milk was subjected to four different pasteurization treatments, viz., 73°C for 15 s or 25 s with and without prior homogenization (2,500 lb/in² in two stages), in an APV Manton Gaulin KF6 homogenizer. Raw and pasteurized milk samples were tested for M. paratuberculosis by immunomagnetic separation (IMS)-PCR (to detect the presence of bacteria) and culture after decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h (to confirm bacterial viability). On 10 of the 12 processing occasions, M. paratuberculosis was detectable by IMS-PCR, culture, or both in either raw or pasteurized milk. Overall, viable M. paratuberculosis was cultured from 4 (6.7%) of 60 raw and 10 (6.9%) of 144 pasteurized milk samples. On one processing day, in particular, M. paratuberculosis appeared to have been present in greater abundance in the source raw milk (evidenced by more culture positives and stronger PCR signals), and on this occasion, surviving M. paratuberculosis bacteria were isolated from milk processed by all four heat treatments, i.e., 73°C for 15 and 25 s with and without prior homogenization. On one other occasion, surviving M. paratuberculosis bacteria were isolated from an unhomogenized milk sample that had been heat treated at 73°C for 25 s. Results suggested that homogenization increases the lethality of subsequent heat treatment to some extent with respect to M. paratuberculosis, but the extended 25-s holding time at 73°C was found to be no more effective at killing M. paratuberculosis than the standard 15-s holding time. This study provides clear evidence that M. paratuberculosis bacteria in naturally infected milk are capable of surviving commercial high-temperature, short-time pasteurization if they are present in raw milk in sufficient numbers.     

Survival of Campylobacter jejuni in raw, pasteurized and ultra-heat-treated goat's milk stored at different temperatures

The survival of a human strain of Campylobacter jejuni in raw, pasteurized and ultra-heat-treated goat's milk stored at 5°, 10°, 15° and 20°C was studied. No viable units were detected in raw milk after 24 h at 20°C and 48 h at 15°C. None were detected in pasteurized milk after 48 h at 20°C. In all other samples, there was a decline in viable units in the first 24 h but very little decline in the next 24 h period. The organism survived best at 5° and 10° C.     

An attempt to improve the flavour of 'nono' by the use of starter cultres

Three close-fitting lid fermenters were incubated at 27 ± 2°C for 120 h. 'A' served as a control and contained 100 ml raw goats' milk; 'B' contained 50 ml 48-h-old nono as starter culture plus 50 ml pasteurized milk; and 'C' contained 100 ml pasteurized milk only. The pH and lactic acid contents were determined at intervals. Fermenter 'B' gave the best results in that the milk reached the lowest pH values and the highest lactic acid contents, and this resulted in a more appetizing and bitter product.     

Challenge testing of the lactoperoxidase system in pasteurized milk

AIMS: To determine the role of lactoperoxidase (LP) in inhibiting the growth of micro-organisms in pasteurised milk. METHODS AND RESULTS: Four micro-organisms of importance in the spoilage of pasteurized milk were challenged in lactoperoxidase (LP)-enriched ultra-heat treated (UHT) milk after subsequent pasteurization. Milk samples were stored at the optimum temperatures for growth of the individual bacteria. Pasteurization was carried out at 72 degrees C/15 s and 80 degrees C/15 s to determine the effect of the LP system on the micro-organisms. An active LP system was found to greatly increase the keeping quality (KQ) of milks inoculated with Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus thermophilus and pasteurized at 72 degrees C, but had little or no effect in milks heated at 80 degrees C, presumably due to virtual inactivation of LP at 80 degrees C. However, pasteurization temperature had no effect on the KQ of milks challenged with Bacillus cereus spores. CONCLUSIONS: This study suggests that the LP system, rather than heat-shocking of spores, is responsible for the greater KQ of milk pasteurized at 72 degrees C/15 s compared with 80 degrees C/15 s. SIGNIFICANCE AND IMPACT OF THE STUDY: The study emphasizes the care required in selecting pasteurization temperatures in commercial practice and to avoid the temptation to compensate for inferior quality of raw milk by increasing pasteurization temperature.     

Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow's milk in the Czech Republic

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7 degrees C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.     

Surveillance of bulk raw and commercially pasteurized cows' milk from approved Irish liquid-milk pasteurization plants to determine the incidence of Mycobacterium paratuberculosis

Over the 13-month period from October 2000 to November 2001 (inclusive), the Food Safety Authority of Ireland (FSAI) carried out surveillance of Irish bulk raw (n = 389) and commercially pasteurized (n = 357) liquid-milk supplies to determine the incidence of Mycobacterium paratuberculosis. The pasteurization time-temperature conditions were recorded for all pasteurized samples. Overall, 56% of whole-milk pasteurized samples had been heat treated at or above a time-temperature combination of 75 degrees C for 25 s. All analyses were undertaken at the Department of Food Science (Food Microbiology) laboratory at Queen's University Belfast. Each milk sample was subjected to two tests for M. paratuberculosis: immunomagnetic separation-PCR (IMS-PCR; to detect the presence of M. paratuberculosis cells, live or dead) and chemical decontamination and culture (to confirm the presence of viable M. paratuberculosis). Overall, M. paratuberculosis DNA was detected by IMS-PCR in 50 (12.9%; 95% confidence interval, 9.9 to 16.5%) raw-milk samples and 35 (9.8%; 95% confidence interval, 7.1 to 13.3%) pasteurized-milk samples. Confirmed M. paratuberculosis was cultured from one raw-milk sample and no pasteurized-milk samples. It is concluded that M. paratuberculosis DNA is occasionally present at low levels in both raw and commercially pasteurized cows' milk. However, since no viable M. paratuberculosis was isolated from commercially pasteurized cows' milk on retail sale in the Republic of Ireland, current pasteurization procedures are considered to be effective.     

Enhancing Milk Preservation with Esterified Legume Proteins

Three methylated legume proteins; soybean protein, broad bean protein and chickpea protein as well as their respective native proteins were applied at two different concentrations (0.1 and 1%) to either raw or pasteurized milk before preservation at 4 °C for 7–14 days. Supplementation of raw milk with esterified legume proteins could ameliorate its preservation quality at 4 °C for 5 days, based on the total bacterial count (TBC) or the titratable acidity levels. Supplementing pasteurized milk with esterified legume proteins (0.1%) has significantly improved its keeping quality as it significantly reduced the total bacterial count by 3.33 and 1.80 log when preserved at 4 °C for 7 and 14 days, respectively. Esterified legume proteins (0.1%) could maintain the level of bacterial load of the pasteurized milk at its initial level of pasteurization (zero time) after 14 days of preservation at 4 °C under closed conditions.