Patent Application: US-201515319422-A

Abstract:
provided herein are methods and kits directed to a method of assaying a sample to determine the presence of a source of pink discoloration defect of a dairy product , such as cheese . in some embodiments , the method comprises the steps of assaying the sample to detect the presence of a thermus species of bacteria , such as thermus thermophilus bacteria , wherein detection of thermus thermophilus bacteria in the sample indicates that the sample contains a source of pink discoloration of a dairy product .

Description:
for nucleic acid extraction , 1 g of cheese from the defect or control cheese was combined with 9 ml 2 % tri - sodium citrate and homogenised before dna was extracted using the powerfood ™ microbial dna isolation kit ( mobio laboratories inc ., usa ) ( quigley et al ., 2012a ). as described previously ( quigley et al ., 2012a ), additional steps , whereby the homogenate was treated with 50 μg ml − 1 lysozyme and 100 u mutanolysin at 37 ° c . for 1 hour followed with protein digestion by adding 250 μg ml − 1 proteinase k and incubating at 55 ° c . for 1 hour , were added to the standard manufacturer &# 39 ; s instructions . dna extracts were used as a template for pcr amplification of 16s rrna tags ( v4 region ; 408 nt long ) using universal 16s primers predicted to bind to 94 . 6 % of all 16s genes i . e . the forward primer f1 , 5 ′- aytgggydtaaagng , seq id 3 (( rdp &# 39 ; s pyrosequencing pipeline : http :// pyro . cme . msu . edu / pyro / help . jsp ) and reverse primer v5 , 5 - ccgtcaattyytttragttt - 3 ′ seq id 4 ( claesson et al ., 2010 ). the primers incorporated the proprietary 19 - mer sequences at the 5 ′ end to allow emulsion - based clonal amplification for the 454 - pyrosequencing system . unique molecular identifier ( mid ) tags were incorporated between the adaptamer and the target - specific primer sequence , to allow identification of individual sequences from pooled amplicons . the pcr reaction contained 25 μl biomix red ™ ( bioline reagents ltd ., london , uk ), 1 μl of each primer ( 10 pmol ), 5 μl dna template and nuclease free h 2 o to give a final reaction volume of 50 μl . pcr amplification was performed using a g - storm thermal cycler ( gene technologies , uk ). the amplification programme consisted of an initial denaturation step at 94 ° c . for 2 min , followed by 40 cycles ; denaturation at 94 ° c . for 1 min , annealing at 52 ° c . for 1 min and extension at 72 ° c . for 1 min . a final elongation step at 72 ° c . for 2 mins was also included . amplicons were cleaned using the ampure xp purification system ( beckman coulter , takeley , united kingdom ). the quantity of dna extracted by the different methods was assessed using the quant - it ™ picogreen ® dsdna reagent ( invitrogen , usa ) used in accordance with the manufacturer &# 39 ; s instructions and a nanodrop ™ 3300 fluorospectrometer ( thermo fisher scientific inc , usa ). the nd3300 excites in the presence of dsdna bound with picogreen ® at 470 nm and monitors emission at 525 nm . the 16s rrna v4 amplicons were sequenced on a 454 genome sequencer flx platform ( roche diagnostics ltd , burgess hill , west sussex , uk ) according to 454 protocols . read processing was performed using techniques implemented in the rdp pyrosequencing pipeline sequences not passing the flx quality controls were discarded , the 454 specific portion of the primer were trimmed , the raw sequences were sorted according to tag sequences and reads with low quality scores ( quality scores below 40 ) and short length ( less than 150 bp for the 16s rrna v4 region ) were removed as well as reads that did not have exact matches with the primer sequence . the qiime suite of programs was used to align , chimera check , cluster and carry out phylogenetics on sequence reads , as well as , to measure microbial α - diversities and to plot rarefaction curves to determine if sequencing was carried out to sufficient depth β - diversities were calculated on the sequence reads based on weighted unifrac and principal coordinate analysis ( pcoa ) performed . kingviewer was used to visualise pcoa plots . trimmed fasta sequences were assessed using blast against the silva version 100 database . the resulting blast output was parsed using megan version 62 . 3 . 0 . megan assigns reads to ncbi taxonomies by employing the lowest common ancestor algorithm which assigns each rna - tag to the lowest common ancestor in the taxonomy from a subset of the best scoring matches in the blast result . bit scores were used from within megan for filtering the results prior to tree construction and summarisation ( absolute cut - off : blast bit - score 86 , relative cut - off : 10 % of the top hit ). the statistical significance of differences in proportions of microbial taxa was determined by the non - parametric kruskal - wallis test ( kruskal and wallis , 1952 ) using minitab ® statistical package . castenholz tye medium was chosen to selectively support the growth of strains from the genus thermus . castenholz tye medium was prepared by mixing 5 parts 2 × castenholz salts with one part 1 % tye and 4 parts distilled water . castenholz salts , 2 × contained 0 . 2 g nitrilotriacetic acid , 0 . 12 g caso 4 . 2h 2 o , 0 . 2 g mgso 4 . h 2 o , 0 . 016 g nacl , 0 . 21 g kno 3 , 1 . 4 g nano 3 , 0 . 22 g na 2 hpo 4 , 2 . 0 ml fecl 3 solution ( 0 . 03 %) and 2 . 0 ml nitsch &# 39 ; s trace elements { 0 . 5 ml h 2 so 4 , 2 . 2 g mnso 4 , 0 . 5 g znso 4 . 7h 2 o , 0 . 5 g h 3 bo 3 , 0 . 016 g cuso 4 . 5h 2 o , 0 . 025 g na 2 moo 4 . 2h 2 o , 0 . 046 g cocl 2 . 6h 2 o distilled water 1 l }, adjusted to a final volume of 1 l and final ph of 8 . 2 . 1 % tye solution consisted of 10 . 0 g tryptone , 10 . 0 g yeast extract dissolved in 1 l distilled water . the final ph of castenholz tye medium should be 7 . 6 . for preparation of the corresponding agar , 3 % ( w / v ) bacteriological agar was added to the final solution . thermus was isolated by enriching for 3 days at 70 ° c . in castenholz medium followed by isolation on castenholz agar at 55 ° c . for a further 3 days . a set of primers ( tpolfor ; 5 ′- agcctcctccacgagttc - 3 ′ and tpolrev ; 5 ′- gtaggcgaggagcatggggt - 3 ′) ( seq id 1 and 2 ) targeting a region specifically conserved within the polymerase 1 gene of thermus were designed to facilitate pcr and qpcr - based detection of the genus . the theoretical specificity of these primers was tested using the oligo probe search tools in the blast classifier database . pcr amplification of the polymerase 1 gene using these primers was carried out under the following parameters : 95 ° c . for 2 min initial denaturation , followed by 40 cycles of 94 ° c .× 30 s , 63 ° c .× 30 s , 72 ° c .× 45 s , and a final elongation of 72 ° c . for 2 min . the resultant products were visualised by agarose gel electrophoresis . amplicons generated were cleaned using the roche high pure pcr clean - up kit and sequenced ( source bioscience ; dublin , ireland ). the specificity of the primer pair was tested using dna from a selection of cheese - associated gram - positive and gram - negative cultures i . e . streptococcus thermophilus ( defined starter mix , tfp , france ), lactobacillus helveticus dpc6865 , propionibacterium freudenreichii dpc6451 and lactococcus lactis hp as well as escherchia coli , listeria monocytogenes egde , salmonella typhimurium lt2 and bifidobacterium longum dpc15697 . to facilitate the quantification of thermus by molecular means , a quantitative real - time ( qpcr ) protocol was designed . genomic dna was extracted from thermus thermophilus hb27 ( dsmz culture collection , germany ) using the powerfood microbial dna extraction kit ( cambio ). a pcr product from within the polymerase1 gene was generated using the genus - specific primers , as described above . purified amplicons were cloned into the pcr ® 2 . 1 - topo vector using the topo - ta cloning system ( invitrogen , life technologies , carlsbad , calif .) in accordance with manufacturer &# 39 ; s instructions . following cloning , the complete vector was transformed into chemically competent top - 10 e . coli cells ( invitrogen ) and harvested on lb media containing 100 μg ml − 1 ampicillin . the accuracy of the cloned amplicon was confirmed by restriction analysis and dna sequencing . qpcr standards were prepared following the linearization of plasmid dna with pst restriction enzyme and quantification with the nanodrop nd - 1000 ( thermo fisher scientific inc ). a standard curve was then generated via a series of dilutions from 10 2 to 10 8 copies μl − 1 dna . the lightcycler 480 sybr green i master kit ( roche diagnostics gmbh , mannheim , germany ) was used for quantification according to the manufacturer &# 39 ; s instructions . each pcr reaction contained 5 μl sybr green master mix ( roche ), 1 μl of both forward and reverse primer ( 7 . 5 pmol ), 2 μl of dna and was made up to a final volume of 10 μl with nuclease free dsh 2 o . the pcr conditions were as follows : an initial denaturation at 95 ° c . for 10 min , followed by 45 cycles of denaturation at 95 ° c . for 20 sec , annealing at 61 ° c . for 15 sec and elongation 72 ° c . for 20 sec . assays were performed in triplicate . to facilitate quantification by qpcr , we applied the formula of quigley et al ., 2013 , to convert from copies μl − 1 to cfu g − 1 of cheese . the starter cultures s . thermophilus ( defined starter mix , tpf , france ) and l . helveticus dpc6865 were each grown overnight at 37 ° c . in reconstituted low heat - skim milk powder , which had first been heat - treated at 90 ° c . for 30 min . propionibacterium freudenreichii dpc6451 was grown for 3 days at 30 ° c . in sodium lactate broth . t . thermophilus dpc6866 , obtained from a cheese with a pink defect , was grown in castenholz broth at 60 ° c . with shaking for 36 hours . cells were collected by centrifugation at 14 , 000 g for 20 min , washed once to remove trace media and resuspended in sterile water . raw milk was obtained from teagasc , moorepark dairy herd , standardised , pasteurised at 72 ° c . for 15 sec and pumped at 32 ° c . into four individual cylindrical stainless steel vats with automated variable speed cutters and stirrers . this milk was employed to manufacture continental - type cheese at pilot - scale level in moorepark technology ltd ( fermoy , cork , ireland ). details with respect to the manufacture of control and test cheeses can be found in table 1 . enumeration of microbiological content , composition of cheeses and proteolysis were measured at various stages of ripening ( table 2 ). to enumerate specific bacterial components , cheese samples were aseptically removed , placed in a stomacher bag , diluted 1 : 10 with sterile tri - sodium citrate ( 2 % w / v , sigma ) and homogenised in a seward stomacher ® 400 lab system ( seward ltd ., west sussex , uk ) for 2 min . further dilutions were prepared as required . viable s . thermophilus were enumerated on m17 agar ( oxoid ) with 0 . 5 % lactose ( oxoid ) at 42 ° c . for 3 days . l . helveticus were enumerated on mrs agar ( oxoid ) adjusted to ph 5 . 4 at 37 ° c . for 3 days under anaerobic conditions . pab levels were enumerated on sodium lactate agar containing 40 μg ml − 1 kanamycin ( sigma ) at 30 ° c . for 7 days under anaerobic conditions . non - starter lactic acid bacteria ( nslab ) were enumerated on lactobacillus selective agar ( lbs ; difco ) at 30 ° c . for 5 days aerobically . t . thermophilus was monitored using qpcr methods . to facilitate this , dna was extracted from milk , whey or 10 ml cheese homogenate using the powerfood dna isolation kit as described above . grated samples from cheeses were analysed for salt ( idf , 1988 ), moisture ( idf , 1982 ) and protein ( idf , 1993 ) after 11 days of manufacture , ph ( standards , 1976 ) was measured throughout ripening . the levels of nitrogen soluble at ph 4 . 6 ( ph 4 . 6 sn ) were measured as described by sheehan et al . ( 2007 ). free amino acid analysis was carried out on ph 4 . 6 sn extract as described by fenelon et al . ( 2000 ). cheese rounds were examined visually throughout ripening for the formation of pink discolouration defect . pink colour formation was quantified with a chroma meter using hunter , l , a , b colour scale . the colour was measured using fresh sliced exposed cheese surface . the colour meter was standardised with a white standard plate ( y = 88 . 31 , x = 0 . 3160 , y = 0 . 3226 ). hunter a ( redness ) values were recorded . a randomised complete block design that incorporated the four treatments and 3 blocks ( replicate trials ) was used for the analysis of response variables relating to the composition of cheeses , moisture , salt and protein , as well as starter bacteria , pab , nslab , t . thermophilus , ph , ph 4 . 6 sn , faa and apparent colour differences . analysis of variance was carried out on data using the general linear model procedure of sas ( sas institute ). the tukey honestly significant difference test was used to determine the significance of difference between the means . the level of significance was determined at p & lt ; 0 . 05 . compositional sequencing reveals higher proportions of the genus thermus in cheeses with a pink defect compositional ( 16s rdna ) sequencing was performed on dna extracted from control ( n = 9 ) and pink defect ( n = 9 ) samples of a commercially produced continental - type cheese . sequencing coverage was satisfactory for all samples ( si figure s1 ). phylogenetic analysis established that the sequence reads corresponded to five different bacterial phyla ( fig1 a ), i . e . firmicutes , proteobacteria , bacteroides , actinobacteria and deinococcus - thermus . firmicutes and deinococcus - thermus dominated with less than 1 % of assigned reads corresponding to other phyla . the proportions of firmicutes present did not differ between control and defect samples . reads corresponding to the phylum deinococcus - thermus were detected in defect - associated samples only ( 6 %). when reads were assigned at the family level , eleven families were identified ( fig1 b ). all reads from the phylum deinococcus - thermus were assigned to the family thermaceae and , again , this was the only taxon for which significant differences were observed , i . e . 6 % and 0 % in defect and control , respectively . when these reads were assigned at genus level , 15 genera were identified ( fig1 c / si figure s2 ). reads corresponding to deinococcus - thermus and thermaceae were assigned to the genus thermus and , again , this was the only taxonomic group for which there were significant differences ( p = 0 . 002 ). following the identification of reads assigned to the genus thermus in samples of cheeses containing the pink discolouration defect , attempts were made to isolate this bacterium , which is not regarded as being a typical cheese - associated genus , from the defect cheeses . castenholz medium was employed as it has previously been shown to support the growth of thermus ( brock and freeze , 1969 ) but , due to its minimal nutrient content , was unlikely to support the growth of other genera . an enrichment step , whereby cheese was homogenised in castenholz medium and incubated at 70 ° c . for 3 days , was employed to encourage the growth of thermus , which are characterised by their highly thermophilic nature , and to prevent the growth of more moderately thermophilic cultures such as those within the starter culture population . a 3 % agar was employed to allow incubation at high temperature ( 55 ° c .) without rapid dehydration of the media . use of this approach resulted in the successful isolation of thermus from defect cheese . rapid , culture - independent pcr - based methods to detect thermus were also developed . a primer pair was designed to selectively amplify the polymerase i gene of thermus and assays with a broad variety of controls established the primers to be specific . to take full advantage of the specificity of these primers , a corresponding qpcr - based protocol was developed . quantitative pcr analysis ( of the cheeses used for 16s rdna analysis ) confirmed that thermus was absent from the control cheeses and that defect cheeses contained on average 1 . 77 × 103 cfu g − 1 . sequencing of pcr amplicons from defect cheeses and from thermus strains isolated from these cheeses revealed that the species in question was t . thermophilus . a representative defect cheese isolate , t . thermophilus dpc6866 , was employed in subsequent studies . to establish definitively that thermus is responsible for the formation of pink defects in cheese , a trial was carried out whereby cheese containing t . thermophilus was produced and the degree of pink development compared to that of a control cheese . for this , a continental - type cheese was manufactured . trials were carried out in triplicate and in each instance four cheeses were produced . these included the control cheese , which contained no t . thermophilus , and three experimental cheeses , all of which contained t . thermophilus at 10 6 cfu g − 1 . experiment 1 ( exp 1 ) cheese contained t . thermophilus with starter cultures at normal levels ( 500 g l . helveticus , 250 g s . thermophilus , 4 g pab ). experiment 2 ( exp 2 ) cheese contained t . thermophilus with higher levels of l . helveticus ( 500 g ). finally , experiment 3 ( exp 3 ) cheese contained t . thermophilus with higher levels of l . helveticus ( 500 g ) and lower levels of s . thermophilus ( 250 g ) ( table 1 ). the reasoning behind the varying levels of l . helveticus and s . thermophilus was due to the increased and decreased levels of these bacteria ( respectively ), as detected by the pyrosequencing data where thermus was present . mean viable cell numbers of s . thermophilus were determined to be 10 7 cfu g − 1 at day 1 of ripening in control , exp 1 and exp 2 cheeses and at 10 6 cfu g − 1 in exp 3 cheese , which correlates with levels of starter s . thermophilus inoculated into the cheese milk . there were significant increases ( p = 0 . 0063 ) in the numbers of s . thermophilus over time ( fig4 ) but there were no significant differences between treatments . l . helveticus numbers were 1 × 10 6 cfu g − 1 at 1 d ripening , in control and exp 1 cheese , while exp 2 and exp 3 cheese contained 5 × 10 6 cfu g − 1 , again reflecting the different levels of l . helveticus starter added ( fig4 ). the changes observed in levels of l . helveticus during cheese production were not significant . counts of pab increased significantly until 46 d ripening ( p & lt ; 0 . 0001 ) ( fig4 ), however they did not differ significantly between treatments . viable nslab numbers increased significantly until the end of warm room ripening ( fig4 ) ( p & lt ; 0 . 0001 ). we observed a significant difference in the levels of nslab between control cheese and exp 2 cheese ( p = 0 . 0438 ) and control cheese and exp 3 cheese at 60 d ripening ( p = 0 . 0225 ). thermus thermophilus was inoculated with a view to obtaining & gt ; 10 4 cfu g − 1 in the three experimental cheeses . using culture - independent qpcr , the levels of t . thermophilus present in the inoculated milk , lost in whey , and retained in curd , as well as throughout ripening were determined ( fig5 ). thermus was present at 10 6 cfu ml − 1 in milk after 1 h inoculation ( sampled prior to rennet addition ). there was some loss of t . thermophilus in whey , i . e . 10 2 cfu ml − 1 , however , considerable levels were retained within the curd ( 10 5 cfu g − 1 ). control cheeses , which were not spiked with t . thermophilus , were also assessed and were found not to contain thermus ( data not shown ), establishing that no natural contamination , or cross - contamination , occurred during production . slight numerical increases in the levels of t . thermophilus were noted during hot room ripening , however these were not significant . following transfer to the cold room for continued ripening , we observed a slight decrease in the levels of t . thermophilus to 10 4 cfu g − 1 . this was consistent across all three experimental cheeses ( fig5 ). the gross composition of cheeses at 11 d ripening was assessed and is summarised in table 3 . all cheeses had statistically similar ph values , levels of moisture , salt and protein . the consistency of these results between cheeses and cheese trials indicate good repetition across each day of manufacture i . e . no significant differences were detected between these variables . significant increases in ph ( fig6 ), ph 4 . 6 sn ( soluble nitrogen ) ( fig7 ) and total faa ( p & lt ; 0 . 0001 for all three parameters assessed ) were observed throughout ripening . the concentrations of individual faas ( mg kg − 1 of cheese ) in all cheeses at 116 d of ripening are shown in fig8 . the faas present at greatest concentrations in the cheeses at most ripening times were glutamic acid , valine , leucine , lysine and proline , and were in line with that expected in swiss - type cheeses ( sheehan et al ., 2008 ). to quantify the formation of “ pinking ” in the cheese samples we applied a chroma meter using hunter l , a , b colour scale throughout ripening . hunter a values determine the level of redness (+) to greenness (−) ( wadhwani and mcmahon , 2012 ). changes in the a values are summarised in table 4 . the a reading is a negative value , establishing that the overall colour is in the green spectrum . however , throughout the centre of the experimental cheeses there is a shift towards a more positive value . these differences were first noted after 116 d ripening ( a =− 2 . 08 , − 1 . 91 , − 1 . 75 , − 1 . 73 , for control , exp 1 , exp 2 and exp 3 cheeses , respectively ) and the intensity of this value and the formation of a pink hue developed further in exp 2 cheese at 144 d , ( a =− 2 . 38 − 1 . 95 , − 1 . 34 and − 1 . 82 for control , exp 1 , exp 2 and exp 3 cheeses , respectively ). this further pinking of exp 2 cheese between 116 d and 144 d was statistically significant ( p = 0 . 0108 ). the exp 2 144 d values were also significantly less negative than those of the control ( p = 0 . 0009 ) and exp 1 cheeses ( p = 0 . 0235 ). thermus was consistently detected in hot water sources . notably , thermus is a known thermophillic water bacterium and has been isolated previously from hot tap water ( pask - hughes and williams , 1975 ). the invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention . masoud , w ., takamiya , m ., vogensen , f . k ., lillevang , s ., abu al - soud , w ., sorensen , s . j . & amp ; jakobsen , m . 2011 . characterization of bacterial populations in danish raw milk cheeses made with different starter cultures by denaturating gradient gel electrophoresis and pyrosequencing . international dairy journal , 21 , 142 - 148 . mortensen , g ., bertelsen , g ., mortensen , b . k . & amp ; stapelfeldt , h . 2004 . light - induced changes in packaged cheeses — a review . international dairy journal , 14 , 85 - 102 . mortensen , g ., sørensen , j . & amp ; stapelfeldt , h . 2002 . light - induced oxidation in semihard cheeses . evaluation of methods used to determine levels of oxidation . journal of agricultural and food chemistry , 50 , 4364 - 4370 . paramita , a . & amp ; broome , m . pink discolouration in romano style cheese — role of a - 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7196 . quigley , l ., mccarthy , r ., o &# 39 ; sullivan , o ., beresford , t . p ., fitzgerald , g . f ., ross , r . p ., stanton , c . & amp ; cotter , p . d . 2013 . the microbial content of raw and pasteurized cow milk as determined by molecular approaches . journal of dairy science , 96 , 4928 - 4937 . quigley , l ., o &# 39 ; sullivan , o ., beresford , t ., paul ross , r ., fitzgerald , g . & amp ; cotter , p . 2012a . a comparison of methods used to extract bacterial dna from raw milk and raw milk cheese . journal of applied microbiology , 113 , 96 - 105 . quigley , l ., o &# 39 ; sullivan , o ., beresford , t . p ., ross , r . p ., fitzgerald , g . f . & amp ; cotter , p . d . 2011 . molecular approaches to analysing the microbial composition of raw milk and raw milk cheese . international journal of food microbiology , 150 , 81 - 94 . quigley , l ., o &# 39 ; sullivan , o ., beresford , t . p ., ross , r . p ., fitzgerald , g . f . & amp ; cotter , p . d . 2012b . high - throughput sequencing for detection of subpopulations of bacteria not previously associated with artisanal cheeses . applied and environmental microbiology , 78 , 5717 - 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