Rapid and quantitative detection of the microbial spoilage of meat by fourier transform infrared spectroscopy and machine learning

Fourier transform infrared (FT-IR) spectroscopy is a rapid, noninvasive technique with considerable potential for application in the food and related industries. We show here that this technique can be used directly on the surface of food to produce biochemically interpretable "fingerprints." Spoilage in meat is the result of decomposition and the formation of metabolites caused by the growth and enzymatic activity of microorganisms. FT-IR was exploited to measure biochemical changes within the meat substrate, enhancing and accelerating the detection of microbial spoilage. Chicken breasts were purchased from a national retailer, comminuted for 10 s, and left to spoil at room temperature for 24 h. Every hour, FT-IR measurements were taken directly from the meat surface using attenuated total reflectance, and the total viable counts were obtained by classical plating methods. Quantitative interpretation of FT-IR spectra was possible using partial least-squares regression and allowed accurate estimates of bacterial loads to be calculated directly from the meat surface in 60 s. Genetic programming was used to derive rules showing that at levels of 10(7) bacteria.g(-1) the main biochemical indicator of spoilage was the onset of proteolysis. Thus, using FT-IR we were able to acquire a metabolic snapshot and quantify, noninvasively, the microbial loads of food samples accurately and rapidly in 60 s, directly from the sample surface. We believe this approach will aid in the Hazard Analysis Critical Control Point process for the assessment of the microbiological safety of food at the production, processing, manufacturing, packaging, and storage levels.     

改良PCA培养基检测鸡肉胴体中的污染细菌

通过添加鸡肉浸液改良经典PCA培养基获得模拟鸡肉营养条件的近自然的CEA培养基,用于检测鸡肉中对营养要求苛刻的污染细菌。根据添加不同浓度的新鲜鸡肉浸液所获得菌落总数的变化获得最佳添加浓度,并利用生化特性及16SrRNA序列对仅生长于CEA培养基的细菌进行了鉴定。在CEA培养基上获得的细菌数量和多样性都显著高于PCA培养基(P0.05),且分离到3株无法在PCA培养基上生长的细菌,经鉴定其与Enterococcus faecalis、Rothia mucllaginosa,Staphylococcus saprophyticus subsp.saprophyticus的相似性分别为99%、96%、99%。E.faecalis因产生生物胺而被认为是肉品中的腐败菌,后两者则均是条件致病菌株,他们的存在对鸡肉产品的安全可能造成隐患。该方法中的近自然培养法能提高对微生物数量和种类的检测灵敏度,特别适于对营养要求苛刻的污染细菌的检出。     

Estimation of bacteriological spoilage of pork cutlets by electronic nose

The utility of chemosensor array (EN) signals of head-space volatiles of aerobically stored pork cutlets as a non-invasive technique for monitoring their microbiological load was studied during storage at 4, 8 and 12 degrees C, respectively. The bacteriological quality of the meat samples was determined by standard total aerobic plate counts (TAPC) and colony count of selectively estimated Pseudomonas (PS) spp., the predominant aerobic spoilage bacteria. Statistical analysis of the electronic nose measurements were principal component analysis (PCA), and canonical discriminant analysis (CDA). Partial least squares (PLS) regression was used to model correlation between microbial loads and EN signal responses, the degree of bacteriological spoilage, independently of the temperature of the refrigerated storage. Sensor selection techniques were applied to reduce the dimensionality and more robust calibration models were computed by determining few individual sensors having the smallest cross correlations and highest correlations with the reference data. Correlations between the predicted and "real" values were given on cross-validated data from both data reduced models and for full calibrations using the 23 sensor elements. At the same time, sensorial quality of the raw cutlets was noted subjectively on faultiness of the odour and colour, and drip formation of the samples. These preliminary studies indicated that the electronic nose technique has a potential to detect bacteriological spoilage earlier or at the same time as olfactory quality deterioration.     

Early detection of spoilage moulds in bread using volatile production patterns and quantitative enzyme assays

AIMS: Early detection of spoilage fungi (two Eurotium spp., a Penicillium chrysogenum species) in bread analogues over periods of 72 h at 25 degrees C and 0.95 water activity was evaluated using volatile production patterns, hydrolytic enzyme production, and changes in fungal populations. METHODS AND RESULTS: Using an electronic nose system it was possible to differentiate between uninoculated controls and samples contaminated with P. chrysogenum within 28 h. After 40-48 h it was possible to differentiate between the Eurotium spp., P. chrysogenum and the control using Principal Component Analysis (PCA). Cluster analyses could differentiate between the control, P. chrysogenum and the Eurotium spp. after 40 h. Of seven hydrolytic enzymes examined after 48 h, the specific activities of three were significantly different from uninoculated control bread. There were also differences between the mould species in production of three enzymes. Penicillium chrysogenum populations increased fastest, from about 10(3) cfu g(-1) to 10(6)-10(7) cfu g(-1) after 72 h. For the Eurotium spp. this increase was slower. CONCLUSIONS: Overall, this study suggests, for the first time, that an electronic nose system using a surface polymer sensor array is able to detect qualitative changes in volatile production patterns for the early detection of the activity of spoilage moulds in bakery products. SIGNIFICANCE AND IMPACT OF THE STUDY: Potential exists for application of such systems for microbial quality assurance in intermediate moisture food products.     

Determining endotoxin content of ground beef by the Limulus amoebocyte lysate test as a rapid indicator of microbial quality.

Eighty-four samples of ground beef were placed into five half-log cycle groups based upon aerobic plate count (APC) results. Endotoxins were determined by the Limulus amoebocyte lysate test (LAL), and gram-negative viable counts were determined by a violet red bile agar overlay method. Ten samples with a log of APC of less than 5.50 had an APC mean of less than 5.24 and mean endotoxin content by the LAL of 51 ng/g. The 15 samples with APCs between a log of 5.50 and 5.99 had an APC mean of 5.79/g and an endotoxin mean of 103.8 ng/g. Twenty-eight samples had APCs between a log of 6.00 and 6.49 with a mean of 5.28/g and an endotoxin mean of 1106.4 ng/g. The 20 samples with APCs between a log of 6.50 and 7.00 had a mean of 6.77/g and an endotoxin mean of 5067.6 ng/g, while 11 samples had a log of APCs of greater than 7.00 with a mean of 7.53 and an endotoxin mean of 7,472 ng/g. Correlation of half-log cycle mean APC and violet red bile agar counts with mean endotoxin content were both highly significant, indicating that LAL-determined endotoxin content can be used to make a rapid approximation of viable plate counts. Because results can be obtained by LAL in 1 h, the finding of low levels of endotoxins can be taken to indicate low-count meat. The use of additional tests of microbial quality may be necessary when high endotoxin levels are found because the LAL detects both viable and nonviable cells.     

Determining endotoxin content of ground beef by the Limulus amoebocyte lysate test as a rapid indicator of microbial quality.

Eighty-four samples of ground beef were placed into five half-log cycle groups based upon aerobic plate count (APC) results. Endotoxins were determined by the Limulus amoebocyte lysate test (LAL), and gram-negative viable counts were determined by a violet red bile agar overlay method. Ten samples with a log of APC of less than 5.50 had an APC mean of less than 5.24 and mean endotoxin content by the LAL of 51 ng/g. The 15 samples with APCs between a log of 5.50 and 5.99 had an APC mean of 5.79/g and an endotoxin mean of 103.8 ng/g. Twenty-eight samples had APCs between a log of 6.00 and 6.49 with a mean of 5.28/g and an endotoxin mean of 1106.4 ng/g. The 20 samples with APCs between a log of 6.50 and 7.00 had a mean of 6.77/g and an endotoxin mean of 5067.6 ng/g, while 11 samples had a log of APCs of greater than 7.00 with a mean of 7.53 and an endotoxin mean of 7,472 ng/g. Correlation of half-log cycle mean APC and violet red bile agar counts with mean endotoxin content were both highly significant, indicating that LAL-determined endotoxin content can be used to make a rapid approximation of viable plate counts. Because results can be obtained by LAL in 1 h, the finding of low levels of endotoxins can be taken to indicate low-count meat. The use of additional tests of microbial quality may be necessary when high endotoxin levels are found because the LAL detects both viable and nonviable cells.     

Carvacrol and cinnamic acid inhibit microbial growth in fresh-cut melon and kiwifruit at 4 degrees and 8 degrees C

AIM: To establish whether or not carvacrol and cinnamic acid delay microbial spoilage of fresh-cut fruit. METHODS AND RESULTS: Dipping of fresh-cut kiwifruit in carvacrol solutions at 5-15 mM reduced total viable counts from 6.6 to < 2 log cfu g-1 for 21 d at 4 degrees C; however, undesirable colour and odour changes were also observed. Treatment with 1 mM of carvacrol or cinnamic acid reduced viable counts on kiwifruit by 4 and 1.5 log cfu g-1 for 5 d at 4 degrees C and 8 degrees C, respectively. Treatment of fresh-cut honeydew melon with 1 mM of carvacrol or cinnamic acid extended the lag phase of the microbial flora from less than 1 d in the untreated controls to 3 d at 8 degrees C and 5 d at 4 degrees C. Viable counts on the treated melon were 6 log cfu g-1 lower on Day 3 at 8 degrees C and 4 log cfu g-1 lower on Day 5 at 4 degrees C, compared with the untreated controls. IMPACT OF THE STUDY: Treatment with 1 mM of carvacrol or cinnamic acid delays spoilage of fresh-cut kiwifruit and honeydew melon at chill temperatures without adverse sensory consequences.     

A note on microbial spoilage of sheep meat at ambient temperature

Shelf-life and microbial spoilage of sheep carcass meat at ambient temperature under commercial conditions were studied. The initial bacterial count of carcasses ranged 5.6-5.8 log/cm2. Staphylococcus spp. (48%) predominated in the initial microflora of carcasses followed by Micrococcus spp. (19%) and Escherichia spp. (12%). Microbial spoilage of carcasses occurred around 20 h when the bacterial count reached 8.0-9.0 log/cm2. Thus the shelf life of carcasses at ambient temperature was 19 h. The predominant micro-organisms at the time of spoilage were Escherichia and 'Acinetobacter-like' organisms. It was also observed that Enterobacter, Pseudomonas and Staphylococcus spp. could form a major part of the final flora. The presence of Escherichia and Staphylococcus spp. in higher percentages on the surface of carcasses at the time of spoilage presents the scope for health hazards.     

EVALUATION OF THE PETRIFILMTM AND REDIGELTM AS RAPID METHODS TO THE STANDARD PLATE COUNT METHOD FOR THE ENUMERATION OF PROCESSED MEATS AND ENVIRONMENTAL SAMPLES

Aerobic plate counts (APC) are used by the food industry to help determine the sanitary state of equipment and bacterial load of finished product. Conventional aerobic count methods for processed meats require approximately 72 h incubation before results are available. Food processing plants with in-house analytical laboratories require methods that are simple, reliable and rapid. New test methods using Petrifilm^TM (PF) and Redigel^TM (RG) have been developed for determining APC. These methods are faster and easier than culturing in a standard agar medium. A variety of raw, cooked; cured and noncured meat products, cooling brine and environmental surface swabs were collected and analyzed for APC using PF, RG and plate count agar (PCA). Data analyses from over 200 samples indicated that the sensitivity of rapid testing methods for aerobic bacteria can vary depending on sample type.     

Recent findings in pulsed light disinfection

Nonthermal disinfection technologies are gaining increasing interest in the field of minimally processed food in order to improve the microbial safety or to extend the shelf life. Especially fresh‐cut produce or meat and fish products are vulnerable to microbial spoilage, but, due to their sensitivity, they require gentle preservation measures. The application of intense light pulses of a broad spectral range comprising ultraviolet, visible and near infrared irradiation is currently investigated as a potentially suitable technology to reduce microbial loads on different food surfaces or in beverages. Considerable research has been performed within the last two decades, in which the impact of various process parameters or microbial responses as well as the suitability of pulsed light (PL) for food applications has been examined. This review summarizes the outcome of the latest studies dealing with the treatment of various foods including the impact of PL on food properties as well as recent findings about the microbicidal action and relevant process parameters.     

Distribution and sources of microbial contamination on beef carcasses

Three beef dressing lines of different capacity (160, 440 and 800 head d(-1)) were investigated with respect to contamination associated with carcass/hide and carcass/faeces contacts, the distribution of microbial contamination on carcasses and the antimicrobial efficacy of cold water carcass washes. Swab samples were taken from up to 17 sites for determination of Aerobic Plate Counts at 37 degrees C (APC 37 degrees C) and Escherichia coli enumeration using the Petrifilm procedure. The three beef dressing systems produced virtually identical patterns of microbial contamination. High contamination was found at those sites associated with opening cuts and/or subject to hide contact during hide removal. Where contamination is intermittent, the use of mean microbial data tended to obscure evidence of faecal or hide contact. Consequently, worst-case results, as represented by the 95th percentile value, were used to identify probable instances and sources of contact contamination. Sites not subject to faecal contamination or hide contact typically had swab sample APC (37 degrees C) values of less than log 2.00 cfu cm(-2) accompanied by the occasional detection of E. coli at levels below log 1.00 cfu cm(-2). Sites contacted by 'clean' hide typically had APC (37 degrees C) counts of log 3.00 cfu cm(-2) or greater accompanied by occasional E. coli counts not exceeding log 2.00 cfu cm(-2). Sites contaminated by direct faecal contact or contact with faecally contaminated hides typically had APC (37 degrees C) counts equal to, or greater than, log 4.00 cfu cm(-2) accompanied by E. coli counts exceeding log 2.00 cfu cm(-2). Cold water carcass washing was ineffective in removing microbial contamination and tended to bring about a posterior to anterior redistribution, resulting in increased counts at forequarter sites.     

Microbiological survey of prepackaged pâté and ham in New Zealand

AIMS: To gauge the effectiveness of paté and ham manufacturers' management of the microbial safety and quality of their products. METHODS AND RESULTS: A survey of 60 batches of prepackaged paté showed that 41.7% of the batches had aerobic plate counts (APC) exceeding 10(5) CFU g(-1), one of paté sample contained a Bacillus cereus count of >5000 CFU g(-1) and another contained 1700 CFU g(-1) of Listeria monocytogenes. No other pathogens were isolated from any of the samples. The survey of prepackaged ham showed that only 1% (1/104) of the ham samples were positive for L. monocytogenes (50 CFU g(-1)). CONCLUSIONS: The presence of microbial hazards in these foods has generally declined since the early 1990s in New Zealand. Noncomplying APC levels may be due to an over-estimation of product shelf life or poor food handling practices during manufacture. SIGNIFICANCE AND IMPACT OF THE STUDY: Few of the samples tested contained pathogens at significant levels. The prevalences of L. monocytogenes in paté and ham were low. The presence of 1700 CFU g(-1) of L. monocytogenes in a paté sample indicates that occasionally, the population can be exposed to levels of L. monocytogenes above the zero tolerance level set in New Zealand.     

Characterization of bright tobaccos by multivariate analysis of C CPMAS NMR spectra

Univariate and multivariate statistics were applied to characterize cured bright tobacco samples on the basis of their ¹³C CPMAS NMR spectra and leaf constituent analysis. NMR spectra were obtained for 55 samples selected from a set of 134 samples of graded bright tobacco leaves from crop year 1999. Historical leaf constituent analyses were available for total alkaloids, reducing sugars, total nitrogen, and insoluble ash. In addition, we applied HPLC to quantify the two abundant plant polyphenols, chlorogenic acid, and rutin. Principal component analysis (PCA) and partial least squares (PLS) of the NMR spectra revealed systematic relationships between groups of samples related to these substances and afforded predictive quantitative models for the analyzed constituents. Analysis of the PLS significant variables showed that leaf polysaccharides, alkaloids, and minerals are major determinants influencing the grading of cured bright tobacco leaves.     

A rapid method for the evaluation of both extrinsic and intrinsic contamination and resulting spoilage of water-in-oil emulsions

AIMS: To develop a method for studying the microbial spoilage of water-in-oil emulsions and to use this to investigate (i) the intrinsic stability of water-in-oil formulations and (ii) Pseudomonas aeruginosa SP1-induced spoilage of a proprietary emulsion. METHODS AND RESULTS: Aliquots of test emulsion were placed into wells of a microtitre plate and the opacity (492 nm) monitored at 120-min intervals over several hours. Cracking of the emulsion was associated with marked reductions in opacity. Rate and extent of change in O.D. could be used as indicators of spoilage. Spoilage of a laboratory emulsion formulation was investigated where microorganisms with demonstrated spoilage potential were incorporated either into the water phase prior to emulsification or where the proportion of contaminated water droplets was varied by dilution of contaminated emulsion with a sterile formulation. Results suggested that the route of introduction was a critical determinant of the probability of gross spoilage. Ps. aeruginosa SP1-induced spoilage of a proprietary formulation was found to be independent of growth in the formulation; rather it was attributed to the presence of a heat-labile extracellular spoilage-factor that was protease labile and possessed both lipase and polysorbate hydrolytic activity. Such spoilage potential was unique to one Ps. aeruginosa culture filtrate amongst five pseudomonads tested. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is both rapid and reproducible, enables evaluation of the effects of route of contamination upon emulsion spoilage and has potential application in formulation development for cosmetic, pharmaceutical and food products.     

ESTIMATION OF MICROBIOLOGICAL QUALITY OF CHILLED BEEF USING AUTOMATED TURBIDIMETRY

Total bacterial counts on chilled beef samples were estimated by the standard plate count method and by an automated turbidimetric system. The latter method is based on product-specific calibration curves constructed by correlating growth curve parameters calculated for the turbidimeter to the log CFU values obtained by plate counts. A total of 74 beef samples was used to construct the calibration curves. Correlation analysis between turbidimetric parameters and plate count values showed that detection time was the best predictor to estimate microbial loads on fresh (r=0.91) and aged beef (r=0.94). Microbial loads for a different set of aged beef samples (n = 37) refrigerated for 7, 9, 10, 17 and 45 days were compared by turbidimetric measurements and plate counts. Mean total viable counts were log 5.92 ± 1.17 and log 5.54 ± 1.28 CFU/mL, respectively. Results showed that total bacterial counts on chilled beef could be estimated accurately from turbidimetric parameters. Furthermore, setting a cut-off value of log 6 CFU/mL allowed to accepting/rejecting samples according to their microbial condition in shorter periods of time compared to the traditional plate count method.     

Feasibility study on the potential of visible and near infrared reflectance spectroscopy to measure alpaca fibre characteristics

Visible (Vis) and near infrared (NIR) reflectance spectroscopy is a rapid and non-destructive technique that has found many applications in assessing the quality of agricultural commodities, including wool. In this study, Vis and NIR spectroscopy combined with multivariate data analysis was investigated regarding its feasibility in predicting a range of fibre characteristics in raw alpaca wool samples. Mid-side samples (n = 149) were taken from alpacas from a range of colours and ages at shearing time over 4 years (2000 to 2004) and subsequently analysed for fibre characteristics such as mean fibre diameter (MFD) and standard deviation (and coefficient of variation), spin fineness, curvature degree (and standard deviation), comfort factor, medullation percentage (by weight and number in white samples only) using traditional reference laboratory testing methods. Samples were scanned in a large cuvette using a FOSS NIRSystems 6500 monochromator instrument in reflectance mode in the Vis and NIR regions (400 to 2500 nm). Partial least squares (PLS) regression was used to develop a number of calibration models between the spectral and reference data. Mathematical pre-treatment of the spectra (second derivative) as well as various combinations of wavelength range were used in model development. The best calibration model was found when using the NIR region (1100 to 2500 nm) for the prediction of MFD, which had a coefficient of determination in cross-validation (R2) of 0.88 with a root mean square standard error of cross validation (RMSECV) of 2.62 μm. The results show the NIR technique to have promise as a semi-quantitative method for screening purposes. The lack of grease in alpaca wool samples suggests that the technique might find ready application as a rapid measurement technique for preliminary classing of shorn fleeces or, if used directly on the animal, the technology might offer an objective tool to assist in the selection of animals in breeding programmes or shows.     

The survival of three strains of Arcobacter butzleri in the presence of lemon, orange and bergamot essential oils and their components in vitro and on food

AIMS: To test the effect of oils and vapours of lemon, sweet orange and bergamot and their components against three Arcobacter butzleri strains. METHODS AND RESULTS: The disc diffusion method was used to screen the oils and vapours against three strains of A. butzleri. In vitro bergamot was the most inhibitory essential oil (EO) and both citral and linalool were effective. On cabbage leaf, the water isolate was the least susceptible to bergamot EO, citral and linalool (1-2 log reduction), with the chicken isolate being the most susceptible (6-8 log reduction). However, the latter appeared not to be susceptible to vapours over 24 h although type strain and water isolate populations reduced by 8 logs. On chicken skin, the effectiveness of the oils was reduced compared with that on cabbage leaf. CONCLUSIONS: Bergamot was the most effective of the oils tested and linalool the most effective component. All strains tested were less susceptible in food systems than in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Arcobacter isolates vary in their response to EO suggesting that the results of type strain studies should be interpreted with caution. Bergamot EO has the potential for the inhibition of this 'emerging' pathogen.     

Chitosan inactivates spoilage yeasts but enhances survival of Escherichia coli O157:H7 in apple juice

AIMS: To develop new measures for controlling both spoilage and pathogenic micro-organisms in unpasteurized apple juice using chitosan. METHODS AND RESULTS: Micro-organisms were isolated and identified from apple juice treated or untreated with chitosan using enrichment, selective media, microscopy, substrate assimilation patterns and ribosomal DNA profiling. Chitosan (0.05-0.1%) delayed spoilage by yeasts at 25 degrees C for up to 12 days but the effect was species specific: Kloeckera apiculata and Metschnikowia pulcherrima were inactivated but Saccharomyces cerevisiae and Pichia spp. multiplied slowly. In challenge experiments at 25 degrees C, total yeast counts were 3-5 log CFU ml(-1) lower in chitosan-treated juices than in the controls for 4 days but the survival of Escherichia coli O157:H7 was extended from 1 to 2 days; at 4 degrees C, chitosan reduced the yeast counts by 2-3 log CFU ml(-1) for up to 10 days but survival of the pathogen was prolonged from 3 to 5 days. The survival of Salmonella enterica serovar Typhimurium was unaffected by chitosan at either temperature. CONCLUSIONS: The addition of chitosan to apple juice delayed spoilage by yeasts but enhanced the survival of E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the use of chitosan in the treatment of fruit juices may potentially lead to an increased risk of food poisoning from E. coli O157:H7.     

HANDS-FREE, "POP-UP," ADHESIVE TAPE METHOD FOR MICROBIAL SAMPLING OF MEAT SURFACES

Microbial contamination of food is a concern to both food producers and consumers. For the food production industry surface sampling of foods is one of the simplest ways to monitor the microbial load. The objective of this experiment was to investigate the feasibility of using the less expensive and quicker "Pop-up" tape method instead of the conventional swab/rinse method for the microbial sampling of meat surfaces. An analyst can place the unit on the wrist and then use both hands to lay out all the necessary materials and take the sample with one hand. The "Pop-up" tape method was able to measure microbial loads up to 2.2 log CFU/cm² on meat surfaces. The conventional swab/rinse method was able to measure up to 8.3 log CFU/cm² on meat surfaces. The correlation coefficient (R) between the two methods was 0.91 (n=42). These data show that the "Pop-up" tape method is a viable alternative to other methods for estimating microbial surface contamination.