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:
BACKGROUND TO THE INVENTION 
       [0001]    The pinking defect in cheese is a worldwide problem. It impacts on a range of ripened cheeses, including Swiss, Cheddar and Italian-type cheese as well as in ripened cheeses coloured with the food colouring, annatto. Its appearance can result in the downgrading or rejection of cheese and a consequential economic loss to producers. Pink discolouration defect can manifest in a number of ways depending on the cheese-type: at the surface of the cheese block either in patches or all over the block surface; as a uniform pink border occurring below the external surfaces of the cheese block conferring a pinked ring appearance; or sporadically distributed within the cheese block. While the cause of this defect is unknown, the topic has been the subject of much debate. It has been suggested that the pink defects are caused by physicochemical factors, though a microbial basis has also been proposed. Indeed, in the latter case, it has been noted that cheeses containing specific starter cultures, and strains of lactobacilli and propionic acid bacteria (PAB) in particular, are more likely to have a pink discolouration 
       STATEMENTS OF INVENTION 
       [0002]    The Applicant has surprisingly discovered that the source of pink discoloration defect in cheese are bacteria that have heretofore not been associated with cheese production,  Thermus thermophilus.  High throughput DNA sequencing of the genome of control cheeses and cheeses having the pink coloration defect revealed the presence of  T. thermophilus  in defect cheeses at a significantly higher level than in control cheeses ( FIG. 3 c   ). In addition, following the production of three experimental continental-type cheeses spiked with  T. thermophilus  and unspiked control cheeses, it was apparent that the pink coloration defect occurred in spiked cheeses only. 
         [0003]    Accordingly, in a first aspect, the invention broadly provides a method of assaying a sample to determine the presence of a source of pink discoloration defect in the sample, the method comprising the steps of assaying the sample to detect the presence of a  Thermus thermophilus  bacteria in the sample, wherein detection of  Thermus thermophilus  bacteria in the sample indicates that the sample contains a source of pink discoloration. 
         [0004]    In particular, the invention relates to a method of determining the presence of a source of pink discolouration of cheese, however the invention may be applied to detecting the source of pink discolouration of other products, for example other dairy products such as, for example, milk, milk products, yoghurt, cream, butter, spreads, milk-derived protein powders and liquids including casein, whey protein, whole milk and skim milk powders. 
         [0005]    The invention also provides a method of determining the presence in a sample of a  Thermus thermophilus  bacteria, comprising the steps of assaying the sample to detect the presence of a  Thermus thermophilus  bacteria, wherein the sample is preferably an ingredient employed in cheese or dairy product manufacture or is obtained from a processing plant, ideally a cheese or dairy processing plant. 
         [0006]    In this specification, the term “pink discoloration defect” should be understood to mean the pink discoloration of products, especially food products, especially dairy products or cheese, that in the case of cheese can occur at the surface of the cheese block in patches or all over the block surface, as a uniform pink border occurring below the external surfaces of the cheese block, or sporadically distributed within the cheese block. 
         [0007]    In this specification, the term “ Thermus thermophilus ” or “ T. thermophilus ” should be understood to mean a bacteria of the genus  Thermus  and the species  thermophilus.  These are gram positive, extremely thermophilic, aerobic microorganisms, and are generally characterised by having a highly conserved region within the polymerase I gene. 
         [0008]    An example of  Thermus thermophilus  is  T. thermophilus  DPC6866 and  T. thermophilus  HB27 (DSMZ Culture Collection, Germany) 
         [0009]    In this specification, the term “detection of  Thermus thermophilus  bacteria in the sample” should typically be understood to mean detection of level of bacteria in the sample in excess of 10 1  cfu g −1 , for example at least 10 2  cfu g −1  or 10 3  cfu g −1  as determined using the qPCR technique described below. 
         [0010]    In the specification, the term “cheese” should be understood to mean any cheese, including hard, soft, semi-soft, or processed cheese products including processed cheese slices or cheese food products. 
         [0011]    In this specification, the term “dairy product” should be understood to mean milk or any product made from milk, including cheese, yoghurt, butter, dairy spreads, and milk-derived protein powders and liquids or concentrates, such as for example casein, whey, whole milk and skim milk powders. 
         [0012]    The sample may be obtained from a food, dairy or cheese processing plant, particularly a plant known to be producing cheese having the pink coloration defect. Generally, the sample is obtained from a surface of piece of machinery employed in the plant. Examples of plant or machinery employed in cheese or milk processing include milk vats, starter culture vats, pasteurisers. Typically, the sample is a swab taken from, for example, a surface of a machine 
         [0013]    The sample may also be an ingredient employed in cheese manufacture, for example, milk, starter culture, hot water, brine or any other ingredient that is used to make or process the cheese. The sample may also be an ingredient employed in dairy product manufacture, for example, milk, starter culture, water, hot water, brine, salt, sugar, or any other ingredient that is used to make or process dairy products. 
         [0014]    Preferably, the sample is hot water. 
         [0015]    In a second aspect, the invention provides a method of testing a dairy product or cheese manufacturing system for a risk of pink discoloration in the dairy product or cheese manufactured by the system, in which the dairy product or cheese manufacturing system comprises a cheese manufacturing plant and cheese manufacturing ingredients processed by the plant, a dairy product manufacturing plant and dairy product manufacturing ingredients processed by the plant, the method comprising the steps of assaying at least one sample obtained from the plant or ingredients to detect the presence of a  Thermus  species of bacteria such as  Thermus thermophilus  bacteria, wherein detection of the  Thermus  species, typically  Thermus thermophilus  bacteria, in at least one sample indicates a risk of pink discoloration in the dairy product or cheese manufactured by the system. 
         [0016]    Suitably, the plurality of samples are obtained from the plant or ingredients and are selected from milk vat, starter culture vat, pasteuriser, steriliser, starter culture, water, hot water, brine, salt, sugar, CIP liquid. Preferably, the plurality of samples includes at least one sample from the cheese manufacturing plant and at least one sample from the cheese ingredients, at least one sample from the dairy product manufacturing plant and at least one sample from the dairy product ingredients. 
         [0017]    In a further aspect, the invention provides a method of identifying an origin of pink coloration defect in a dairy product or cheese manufacturing system of the type comprising a cheese manufacturing plant and cheese manufacturing ingredients processed by the plant, or a dairy product manufacturing plant and dairy product manufacturing ingredients processed by the plant the method comprising the steps of assaying samples obtained from a plurality of different sources selected from locations within the plant and/or ingredients to detect the presence of a  Thermus thermophilus  bacteria in the samples, wherein detection of  Thermus thermophilus  bacteria in one of the samples indicates that the origin of the pink coloration defect is the source of the sample. Thus, if a sample obtained from hot water tests positive for  T. thermophilus,  this indicates that the hot water source is the origin of the pink discoloration defect. This will enable the plant operator to treat the hot water supply lines, storage vessels, and heating vessels, to eliminate the bacteria 
         [0018]    In the further aspect, the invention provides a method of modifying a cheese or dairy product manufacturing system of the type producing cheese or a dairy product having a pink discoloration defect, the method comprising the steps of identifying an origin of the pink coloration or defect according to a method of the invention, and modifying the cheese or diary product manufacturing system to remove or treat the origin of the pink discoloration defect. 
         [0019]    Where the origin of the pink coloration defect is colonisation of a location within the manufacturing plant with  Thermus thermophilus , the surface is generally treated to remove the bacteria. Various methods of treatment to remove the bacteria will be apparent to a person skilled in the art, including treatment of the location with a sterilising liquid or gas. 
         [0020]    Where the origin of the pink coloration defect is the presence of  Thermus thermophilus  in a manufacturing ingredient with, the ingredient is generally replaced with an ingredient not containing  Thermus thermophilus . The presence or otherwise of an ingredient to be used in cheese making may be determined by assaying the ingredient for presence of  Thermus thermophilus  bacteria, preferably quantitatively. 
         [0021]    Preferably, the assay to detect the presence of  Thermus thermophilus  in the sample comprises polymerase chain reaction (PCR), preferably quantitative PCR. The details of PCR and qPCR will be well known to those skilled in the art, and involve amplification of a target sequence that is specific to the target bacteria, and not present in other bacteria known to be present in the sample. One example of a target sequence that is specific to  Thermus thermophilus  is a region of the polymerase I gene that can be amplified with PCR using the following primers: 
         [0000]    
       
         
               
             
           
               
                 [SEQ ID NO: 1] 
               
               
                 Forward Primer (TpolFor): AGCCTCCTCCACGAGTTC 
               
               
                   
               
               
                 [SEQUENCE ID NO: 2] 
               
               
                 Reverse Primer: (TpolRev): GTAGGCGAGGAGCATGGGGT 
               
             
          
         
       
     
         [0022]    Thus, in one preferred embodiment of the invention, the method employs PCR that is adapted to detect the presence of an amplicon that is unique to  Thermus thermophilus , for example a target sequence that can be amplified using the primers of SEQUENCE ID NO&#39;S 1 and 2, or a variant thereof that is specific to  Thermus thermophilus.    
         [0023]    Thus, in another aspect, the invention provides a PCR kit, preferably a qPCR kit, for detecting the presence of  Thermus thermophilus  in a sample. Preferably, the kit comprises a forward primer of SEQUENCE ID NO: 1 and a reverse primer of SEQUENCE ID NO: 2. Suitably, the kit comprises a control primer pair. 
         [0024]    In another aspect, the invention provides a quantitative PCR kit for specific quantitative detection of  Thermus thermophilus , and comprising:
       a forward primer of SEQUENCE ID NO: 1 and a reverse primer of SEQUENCE ID NO:2; and optionally including one or more further reagents selected from buffer, dNTPs, thermostable hot-start DNA polymerase, and an appropriate dye (such as SYBR® Green).       
 
         [0026]    Other methods for detecting the presence of  Thermus thermophilus  in a sample will be apparent to person skilled in the art, including use of antibodies that are specific to  Thermus thermophilus,  (doi:10.3390/antib2030501), for example a suitable ELISA or quantitative ELISA kit, or use of mass spectrophotometry. 
         [0027]    It will be appreciated that other species of  Thermus  bacteria may be employed to detect the presence of a source of pink discolouration defect in a sample. Other  Thermus  species include  T. antranikianii, T. aquaticus, T. brockianus, T. caldophilus, T. filiformis, T. igniterrae, T. kawarayuensis, T. nonproteolyticus, T. oshimai, T. rehai, T. scotoductus, T. thermophilus, T. yunnanensi, T.  sp.  Manikaranii.  Methods for detecting these species of  Thermus  bacteria will be known by a person skilled in the art and from the literature in the field. (http://rd.springer.com/referenceworkentry/10.1007%2F0-387-30747-8_32. The Prokaryotes 2006, pp 797-812 The Genus  Thermus  and Relatives Milton S. Da Costa, Fred A. Rainey, M. Fernanda Nobre) 
         [0028]    The invention also relates to a kit for assaying a sample for the presence of a cause of pink discoloration defect, the kit comprising means for detecting of  Thermus thermophilus  in a sample. Typically, the kit comprises means for quantitative detection of at least 10 2  or 10 3  cfu.g −1 . Suitably the kit is a PCR, ideally a quantitative PCR kit. Typically, the kit comprises primer pairs adapted to quantitatively detect a target sequence in the  T. Thermophilus  DNA polymerase I gene that is unique to  T. Thermophilus,  typically a target sequence that can be amplified using a primer pair of SEQ ID NO&#39;s 1 and 2. Preferably, the PCR kit comprises a primer pair of SEQ ID NO: 1 and 2. In another embodiment, the kit comprises a probe adapted to specifically bind to a target sequence that is unique to  T. thermophilus.    
         [0029]    The invention also relates to kit of the invention, for use in a method of the invention, for example for use in a method of determination of the presence of a source of pink discoloration in a sample. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0030]      FIG. 1 . Rarefaction curve of Shannon diversity indicating satisfactory coverage of all samples sequenced. 
           [0031]      FIG. 2 . Principal Co-ordinate Analysis (PCoA) plot based on Weighted Unifrac, highlighting a split of the bacterial population into two clusters. Two different cheese types manufactured using thermophillic cultures with cheese type 1 represented as dark red (control) and bright red (defect) and cheese type 2 represented in dark blue (control) and light blue (defect). 
           [0032]      FIG. 3 . Bacterial composition of defect and control cheeses as determined by high throughput sequencing. 16S rRNA sequences assigned according to MEGAN using the Silva database at the (a) phylum, (b) family and (c) genus levels with the three cheese types affected by the pink discolouration defect and controls populations. 
           [0033]      FIG. 4 . Counts of ripening bacteria,  Lactobacillus helveticus  (Lh),  Streptococcus thermophilus  (St), propionic acid bacteria (PAB) and non-starter lactic acid bacteria (NSLAB) throughout ripening, 1 d, 11 d, 46 d, 60 d, 88 d, 116 d. 
           [0034]      FIG. 5 .  Thermus thermophilus  levels, as determined by qPCR, throughout manufacture. M-inoculated milk, W-whey          , C-curd. Experimental cheese           1, experimental cheese 2, experimental cheese 3          . 
           [0035]      FIG. 6 . The effect of different treatments on cheese pH over ripening. Control cheese          , experiment 1 cheese          , experiment 2 cheese           and experiment 3 cheese          . 
           [0036]      FIG. 7 . The effect of different experimental set-up on cheese % pH 4.6 soluble nitrogen over ripening time. Control cheese          , experiment 1 cheese          , experiment 2 cheese           and experiment 3 cheese          . 
           [0037]      FIG. 8 . The effect of different experimental set-up on free amino acid levels after 116 days ripening. Control cheese, experiment cheese 1, experiment cheese 2, experiment cheese 3. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Materials and Methods 
     DNA Extraction from Cheeses 
       [0038]    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. 
       Generation of 16S rRNA Amplicons for High Throughput Sequencing 
       [0039]    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. 
       High-Throughput Sequencing and Bioinformatic Analysis 
       [0040]    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. 
       Culturing of  Thermus    
     Culture-Based Method 
       [0041]    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. 
       PCR and qPCR-Based Detection of  Thermus    
       [0042]    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. 
         [0043]    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. 
       Cheese Spiking Studies 
     Cheese Manufacture and Analysis 
       [0044]    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). 
       Visual Detection of Pinking 
       [0045]    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. 
       Statistical Analysis 
       [0046]    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. 
       Results 
     Compositional Sequencing Reveals Higher Proportions of the Genus  Thermus  in Cheeses with a Pink Defect 
       [0047]    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 ( FIG. 1 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 ( FIG. 1 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 ( FIG. 1 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). 
       Detection of  Thermus  in Cheese 
       [0048]    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. 
         [0049]    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. 
       Formation of “Pinking” in Spiked Cheese 
       [0050]    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. 
       Starter, PAB and NSLAB Viability During Cheese Ripening 
       [0051]    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 ( FIG. 4 ) 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 ( FIG. 4 ). 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) ( FIG. 4 ), however they did not differ significantly between treatments. Viable NSLAB numbers increased significantly until the end of warm room ripening ( FIG. 4 ) (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). 
       Survival of  Thermus thermophilus  Throughout Cheese Manufacture and Ripening 
       [0052]      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 ( FIG. 5 ).  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 ( FIG. 5 ). 
       Composition of Cheeses 
       [0053]    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 ( FIG. 6 ), pH 4.6 SN (soluble nitrogen) ( FIG. 7 ) 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  FIG. 8 . 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). 
       Visual Examination for “Pinking” Formation in Cheese 
       [0054]    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). 
       Location of  Thermus    
       [0055]      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). 
         [0056]    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. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Details and differences between manufacture of continental-type spiked cheese trials. 
               
             
          
           
               
                   
                 Control 
                 Experiment 1 
                 Experiment 2 
                 Experiment 3 
               
               
                 Treatment 
                 Cheese 
                 Cheese 
                 Cheese 
                 Cheese 
               
               
                   
               
               
                 Milk Volume 
                  454 kg 
                  454 kg 
                  454 kg 
                  454 kg 
               
               
                 Starter Culture (w/v) 
               
               
                 
                   Streptococcus thermophilus 
                 
                 500 g 
                 500 g 
                 500 g 
                 250 g 
               
               
                 
                   Lactobacillus helveticus 
                 
                 250 g 
                 250 g 
                 500 g 
                 500 g 
               
               
                 
                   Propionibacterium freudenreichii 
                 
                  4 g 
                  4 g 
                  4 g 
                  4 g 
               
               
                 Test Bacterium cfu ml −1   
               
               
                 
                   Thermus thermophilus 
                 
                 0 
                 10 6   
                 10 6   
                 10 6   
               
             
          
           
               
                 Curd Formation 
                 As Standard 
               
               
                 Cook 
                 0.5° C. min to 45° C. 
               
               
                   
                   1° C. min to 53° C. 
               
               
                 Drain pH 
                 pH 6.30 
               
               
                 Curd Handling 
                 Pre-press and mould 
               
               
                 Salting Method 
                 Brine 
               
               
                 Cheese Size 
                 10 kg 
               
               
                 Cool Room Ripening 
                 8.5° C. x 10 days 
               
               
                 Hot Room Ripening 
                 22° C. x 7 weeks 
               
               
                 Ripening Regime 
                 4.5° C. after hot room step 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Assessment carried out at different stages of manufacture and ripening. 
               
             
          
           
               
                 Ripening 
                 Stages of 
                   
                 Microbiological 
                 Compositional 
               
               
                 Time (days) 
                 Ripening 
                 Sample Type 
                 Analysis 
                 Analysis 
               
               
                   
               
             
          
           
               
                 0 
                 Day of 
                 Milk, Wey, 
                 Tt 
                 PH 
               
               
                   
                 manufacture 
                 Curd 
               
               
                 1 
                 After Brining 
                 Cheese 
                 Tt, St, 
                 pH, Moisture, Salt, 
               
               
                   
                   
                   
                 Lh, PAB 
                 Proteins, pH4.6SN, 
               
               
                   
                   
                   
                   
                 FAA 
               
               
                 11 
                 After 10 days at 
                 Cheese 
                 Tt, St, 
                 pH, Moisture, Salt, 
               
               
                   
                 cool room 
                   
                 Lh, PAB, 
                 Proteins, pH4.6SN, 
               
               
                   
                 ripening (8.5° C.) 
                   
                 NSLAB 
                 FAA 
               
               
                 46 
                 After 5 weeks at 
                 Cheese 
                 Tt, St, 
                 pH, pH4.6SN, FAA, 
               
               
                   
                 warm room 
                   
                 Lh, PAB, 
                 visual examination 
               
               
                   
                 ripening (22° C.) 
                   
                 NSLAB 
               
               
                 60 
                 End of warm room 
                 Cheese 
                 Tt, PAB, 
                 pH, pH4.6SN, FAA, 
               
               
                   
                 ripening (22° C.) 
                   
                 NSLAB 
                 visual examination 
               
               
                 88 
                 After 1 month in 
                 Cheese 
                 Tt, NSLAB 
                 pH, pH4.6SN, FAA, 
               
               
                   
                 cold room (4.5° C.) 
                   
                   
                 visual examination 
               
               
                 116 
                 After 2 months in 
                 Cheese 
                 Tt, NSLAB 
                 pH, pH4.6SN, FAA, 
               
               
                   
                 cold room (4.5° C.) 
                   
                   
                 visual examination 
               
               
                 144 
                 After 3 months in 
                 Cheese 
                 Tt 
                 pH, pH4.6SN, FAA, 
               
               
                   
                 cold room (4.5° C.) 
                   
                   
                 visual examination 
               
               
                   
               
               
                 Tt— Thermus thermophilus ; 
               
               
                 St— Streptococcus thermophilus ; 
               
               
                 Lh— Lactobacillus helveticus ; 
               
               
                 PAB—Propionic Acid Bacteria; 
               
               
                 NSLAB—Non-starter lactic acid bacteria; 
               
               
                 pH 4.6SN - pH 4.6 s 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Composition of cheeses at 11 days post manufacture. 
               
             
          
           
               
                   
                 pH 
                 % Moisture 
                 % Salt 
                 % Protein 
               
               
                   
                   
               
             
          
           
               
                   
                 Control 
                 5.21 
                 41.10 
                 1.36 
                 24.931 
               
               
                   
                 Exp 1 
                 5.24 
                 40.80 
                 1.25 
                 25.271 
               
               
                   
                 Exp 2 
                 5.21 
                 41.50 
                 1.22 
                 25.723 
               
               
                   
                 Exp 3 
                 5.23 
                 40.94 
                 1.28 
                 24.804 
               
               
                   
                   
               
               
                   
                 Data presented in this table are means for three replicate trials. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Effect of treatment on colour properties as 
               
               
                 determined by Hunter L, a, b, dimensions. 
               
             
          
           
               
                   
                 Cheese 
                 Area 
                 a* value 
                   
               
             
          
           
               
                   
                 Sample 
                 Assessed 
                 116 d 
                 144 d 
               
               
                   
                   
               
             
          
           
               
                   
                 Control 
                 Top 
                 −2.22 
                 −2.22 
               
               
                   
                   
                 Side 
                 −2.20 
                 −2.17 
               
               
                   
                   
                 Base 
                 −2.01 
                 −2.32 
               
               
                   
                   
                 Centre 
                 −2.08 
                 −2.38 
               
               
                   
                 Exp 1 
                 Top 
                 −2.20 
                 −2.21 
               
               
                   
                   
                 Side 
                 −2.05 
                 −2.28 
               
               
                   
                   
                 Base 
                 −2.23 
                 −2.21 
               
               
                   
                   
                 Centre 
                 −1.91 
                 −1.95 
               
               
                   
                 Exp 2 
                 Top 
                 −2.57 
                 −2.18 
               
               
                   
                   
                 Side 
                 −2.20 
                 −2.16 
               
               
                   
                   
                 Base 
                 −2.52 
                 −2.10 
               
               
                   
                   
                 Centre 
                 −1.75 
                 −1.34* 
               
               
                   
                 Exp 3 
                 Top 
                 −2.08 
                 −2.14 
               
               
                   
                   
                 Side 
                 −1.91 
                 −2.35 
               
               
                   
                   
                 Base 
                 −1.75 
                 −2.13 
               
               
                   
                   
                 Centre 
                 −1.73 
                 −1.82 
               
               
                   
                   
               
               
                   
                 Here we represent a values which indicate formation of redness colour. The results are those taken from 144 d old cheeses 
               
               
                   
                 *Statistically significant difference compared to control cheese p = 0.0009. 
               
               
                   
                 Data presented in this table are means for three replicate trials. 
               
             
          
         
       
     
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