Spore rec-assay by dry sheet medium culture plate for bacterial counts

A simple and rapid method for spore rec-assay by utilizing dry sheet medium culture (Compactdry TC, CTC) for determining numbers of bacteria, instead of the spore agar plate, was developed. One mL of spore suspension (2 x 10(6)/mL) of Bacillus subtilis strain M45 Rec- or H17 Rec+ was inoculated in the center of the CTC plate. In the case of metabolic activation, 1 mL of mixed solution (spore suspension of M45 or H17: 9,000 x g supernatant of rat-liver homogenate treated with Aroclor 1254 = 19:1) was used. The spore suspension spreads over the whole sheet in seconds and gels. A paper disk impregnated with 20-40 microL of the sample solution and 20 microL of the cofactor solution was placed on the surface of CTC plate. For the assay of samples that do not require metabolic activation, use of the cofactor solution can be omitted. After 48 hr incubation at 37 degrees C, 0.01% MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] aqueous solution (0.5 mL) was dropped uniformly on the plate. The plate was left for 5 min, and the diameter of the inhibition circle was measured with slide calipers. The samples for which the difference in inhibition zone between M45 and H17 was more than 2 mm were judged positive. Under these conditions, the DNA damaging activities of sodium sulfite, sodium benzoate and citric acid, used as food additives, were investigated by the proposed method. Sodium sulfite and sodium benzoate gave positive results and citric acid gave a negative result with or without metabolic activation, in agreement with the results obtained by the conventional method.     

A new medium for determining the total plate count in food

SimPlate for Total Plate Count-Color Indicator (TPC-CI, IDEXX Laboratories, Inc., Westbrook, Me.) is a new medium that incorporates the redox dye resazurin to detect and quantify bacteria in food. Enumeration is achieved by the most probable number method using a SimPlate device. Viable bacteria are detected in each well of the SimPlate device by the biochemical reduction of resazurin, which is blue, to the pink resorufin or the clear dihydroresorufin indicators. Results after 24 h of incubation for TPC-CI are highly correlated with standard plate count agar after 48 h of incubation. Correlation coefficients from studies conducted at five laboratories ranged from 0.94 to 0.98 in side-by-side comparisons against standard plate count agar. Four additional test sites, using alternative methods for determining the aerobic plate count in food, reported similar results in comparison studies (r = 0.91 to 0.97). The slopes from linear regression analysis at all sites ranged from 0.91 to 0.98, with y intercepts ranging from 0.11 to 0.84. Samples used for the validation of TPC-CI included raw food products (i.e., liver and grains), which may contain natural enzymes that interfere with enzyme-based detection methods. No interference was seen from the foods tested. These results suggest that TPC-CI is a suitable alternative to existing plate count methods and has reduced incubation time.     

应用干制培养基检测食品中金黄色葡萄球菌

目的建立金黄色葡萄球菌X干制培养基(Staphylococcus aureus X medium, XSA)检测食品中金黄色葡萄球菌的分析方法。方法依据GB 4789.10—2016《食品安全国家标准食品微生物学检验金黄色葡萄球菌检验》中前处理的方法制备样品,将样品添加到7.5%NaCl肉汤增菌后接种至干制培养基XSA上进行培养,通过鉴定实验进行定性检测和菌落平板计数进行定量检测,并用国标法同时进行定性及定量检测。结果在定性检测方面,该方法和国标方法假阴性率和假阳性率均为0%,但是检测时间由92 h缩短为48 h,在定量检测方面,该方法与国标法的对数偏差值的绝对值为0.3979<0.45,并且在6次检测同一个样品时的结果相对标准偏差较小,从而说明该方法和国标法的准确度相当,检测时长由78h缩短至24h,大大提高了检验效率。结论该方法操作简便快捷,结果稳定,适用于食品中金黄色葡萄球菌的检测。     

Comparison of the compact dry TC and 3M petrifilm ACP dry sheet media methods with the spiral plate method for the examination of randomly selected foods for obtaining aerobic colony counts

Two hundred thirty-six randomly selected food and milk samples were examined to obtain aerobic colony counts by two dry sheet media methods and a standard Public Health Laboratory Service spiral plate method. Results for 40 samples were outside the limits of detection for one or more of the tested methods and were not considered. (The limits of detection for the spiral plate method were 200 to 1 x 10(8) CFU/ml for the spiral plate method and 100 to 3 x 10(6) CFU/ml for the dry sheet media methods.) The remaining 196 sets of results were analyzed further. When the results from the three methods were compared, correlation coefficients were all >0.80 and slopes and intercepts were close to 1.0 and 0.0, respectively. Mean log values and standard deviations were very similar for all three methods. The results were evaluated according to published UK guidelines for ready-to-eat foods sampled at the point of sale, which include a quality acceptability assessment that is based on aerobic colony counts. Eighty-six percent of the comparable results gave the same verdict with regard to acceptability according to the aerobic colony count guidelines. Both dry sheet media methods were comparable to the spiral plate method and can be recommended for the examination of food.     

Comparison of dry culture medium and conventional plating techniques for enumeration of bacteria in pasteurized fluid milk

Standard plate counts, psychrotrophic bacterial counts, and coliforms were determined by conventional plating techniques and by Petrifilm TM plates, a dry culture medium, for 48 commercially processed milk samples (24 whole milk and 24 skim milk). The Petrifilm SM plate counts were compared with counts on standard methods agar for the standard plate count, psychrotrophic bacterial count, and rapid psychrotrophic bacterial count. The Petrifilm violet red bile plate counts were compared with counts on violet red bile agar for coliform test with a solid medium and the preliminary incubation method for detection of coliforms. Standard plate counts were determined within 24 h of packaging and after 7, 10, and 14 d of storage at 6.1 degrees C. Psychrotrophic bacterial counts and coliform counts were determined with 24 h of packaging and after 7 d storage. There was a strong linear relationship between Petrifilm SM and standard methods agar plates (excluding counts on samples plated within 24 h of packaging) and for the psychrotrophic bacterial count method. Petrifilm SM had a weak linear relationship with Standard Methods Agar plates for the rapid psychrotrophic bacterial count. Coliform counts determined on Petrifilm violet red bile plates were generally within the same range as counts on violet red bile agar plates. The positive predictive values for the Petrifilm violet red bile plates and violet red bile agar plates were essentially the same for samples plated within 24 h of packaging.     

提高培养液中乳酸菌数的方法探讨

乳酸菌在发酵食品中应用非常广泛,发酵乳制品主要依靠添加乳酸菌类发酵剂进行发酵.要得到较高含菌量的发酵剂,首先应该提高培养液中乳酸菌数,以保证后续浓缩干燥等工艺的实施.概述了国内外制备直投式发酵荆工艺中提高培养液中乳酸菌菌数的方法,以及行业发展前景及趋势.     

Bioluminescence-based identification of nisin producers - A rapid and simple screening method for nisinogenic bacteria in food samples

We present a simple and rapid method for screening nisin producers that directly identifies nisinogenic bacteria by induction of bioluminescence within the Lactococcus lactis NZ9800lux biosensor strain (Immonen and Karp, 2007, Biosensors and Bioelectronics 22, 1982-7). An overlay of putative nisinogenic colonies with the biosensor strain gives identification results within 1h. Functionality and specificity of the method were verified by screening nisin producers among 144 raw milk colonies and a panel of 91 lactococcal strains. Studies performed on strains and colonies that did not induce bioluminescence but inhibited growth of the biosensor demonstrated that only nisinogenic bacteria can cause induction. Bacteria known to produce bacteriocins other than nisin failed to induce bioluminescence, further verifying the specificity of the assay. We discovered a non-inducing but inhibitory lactococcal strain harboring a modified nisin Z gene, and demonstrated that the source of the inhibitory action is not a non-inducing variant of nisin, but a bacteriocin of lower molecular weight. The concentration of nisin producers in a raw milk sample was 1.3×10(2)CFU/ml. We identified from raw milk a total of seven nisin Z producing L. lactis subsp. lactis colonies, which were shown by genetic fingerprinting to belong to three different groups. Among the panel of 91 lactococci, four strains were nisin A producers, and one strain harbored the modified nisin Z gene. The method presented here is robust, cost-effective and simple to perform, and avoids the pitfalls of traditional screening methods by directly specifying the identity of the inhibitory substance.     

食品中3M™克罗诺杆菌属分子检测系统评价研究

目的：用不同来源的克罗诺杆菌属（阪崎肠杆菌）、不同来源的非目的菌及不同来源的人工染菌食品样品,考核评估3M? MDS分子检测系统（以下简称3M? MDS）对克罗诺杆菌属的检出限、灵敏度、特异性和准确度,以及与国标法（GB4789.40-2016）检验结果的一致性。方法：用3M? MDS对实验室保存的50株已确认克罗诺杆菌属、40株非目的菌株进行检测,确定该试剂盒的检出限、灵敏度和特异性;对240份人工污染不同浓度目的菌样品,用3M? MDS和国标法同时检验,分析方法的准确度和检验结果的一致性。结果：3M? MDS对50株不同来源的克罗诺杆菌属的检测灵敏度为100%,平均检出限为1.79×103CFU/mL;对40株非目的菌检测结果均为阴性,特异性为100%。用3M? MDS与国标方法对人工污染101 CFU/100g、100 CFU/100g 、10-1 CFU/100g克罗诺杆菌属FC718的240份不同来源乳粉、糊精、乳清和乳糖样品进行检测,结果一致性均大于95%,方法准确度为97.5%。结论：3M? MDS方法具有低检出限、高灵敏和特异性特点,在不同食品样品基质中,检验结果呈现良好的准确度,与国标方法检验结果具有高度一致性,是一项适合基层、企业推广的快速、可靠方法。     

ONPG培养基快速测定食品中大肠菌群的研究

目的探讨邻硝基苯-β-D-吡喃半乳糖苷(o-Nitrophenyl-β-D-Galactopyranoside,ONPG)培养基在快速测定食品中大肠菌群的应用。方法比较ONPG培养基和月桂基硫酸盐胰蛋白胨(lauryl sulfate tryptose,LST)肉汤测定大肠埃希氏菌不同时段的结果,使用χ~2-test分析;比较ONPG培养基和LST肉汤测定食品中大肠菌群的结果,使用Wilcoxon配对法分析。结果测定大肠埃希氏菌时ONPG培养基18 h结果与LST肉汤48 h结果无显著性差异(P0.05);测定食品中大肠菌群时ONPG培养基18 h结果与LST肉汤48 h结果无显著性差异(P0.05)。结论 ONPG培养基可用于快速测定食品中大肠菌群。     

响应面法优化微量热法检测用菌体培养基的实验研究

利用微量热法对酱牛肉中的细菌进行快速定量检测,需要对检测用菌体培养基进行优化从而实现其快速增殖,以期缩短检测时间,同时放大细菌生长热信号。本文采用Plackett-Burman实验设计确定了培养基配方的主要因素后,再用最陡爬坡实验及Box-Behnken实验设计进一步确定了各因素的最优水平。实验结果表明,葡萄糖、pH、胰蛋白胨三个因素是影响酱牛肉中优势细菌生长的主要因素;优化后的最佳培养基配方为:葡萄糖1.42g/L、胰蛋白胨8.45g/L、硫酸镁0.2g/L、氯化钠5g/L、酵母浸膏4.5g/L、pH7.12。用此培养基培养酱牛肉中的细菌,可实现其快速增殖,并显著缩短生长延滞期。     

米线中菌落总数测定结果的不确定度评估

目的对米线中菌落总数测定结果的不确定度进行评估。方法根据GB4789.2-2016《食品安全国家标准食品微生物学检验菌落总数测定》对米线中的菌落总数进行测定,通过建立评估的数学模型,分析不确定度的主要来源,计算出不确定度的各主要分量,得出合成不确定度和扩展不确定度。结果在置信水平为95%时,合成不确定度为0.23856,扩展不确定度为0.49764。结论影响结果不确定度的主要因素为样品的均匀性和重复测定,该评估系统可为今后实验室测定食品中菌落总数的不确定度评估提供参考,为检验检测结果的科学表达提供依据。     

A synthetic culture medium evaluated for detection of coliform bacteria in tomato paprika

A simple selective and differentiating synthetic medium (X broth) that is easy to prepare was developed in liquid form for the selective cultivation of coliform bacteria from foodstuffs. This synthetic medium contains an ammonium salt as nitrogen source, lactose as carbon source, and buffers but is free of inhibitors. Its selectivity is based on the fact that coliform bacteria are able to grow if the minimal medium consists of simple inorganic substances as nitrogen sources and lactose as carbon supply. The selectivity of this medium was tested by the inoculation of pure cultures of different microbes belonging to the genera of Staphylococcus, Bacillus, and Pseudomonas and the family Enterobacteriaceae and was found to be complete in this range. The applicability of the synthetic medium was also tested by a nationwide round of tests, using quick-frozen tomato paprika.The selectivity of X broth proved essentially better than that of the standard brilliant green–bile–lactose broth. The results of nationwide tests of coliform bacteria determination demonstrated that there was no difference in reproducibility, but the comparability was significantly better when X broth was used.The advantages of the new synthetic medium are the standard composition, the absence of inhibitors, the reliability, the long keeping quality and the low costs.     

Comparison of dry sheet media and conventional agar media methods for enumerating yeasts and molds in food

A study was done to compare Nissui Compact Dry Yeast and Mold plates (CDYM), 3M Petrifilm Yeast and Mold count plates (PYM), dichloran-rose bengal chloramphenicol (DRBC) agar, and dichloran 18% glycerol (DG18) agar for enumerating yeasts and molds naturally occurring in 97 foods (grains, legumes, raw fruits and vegetables, nuts, dairy products, meats, and miscellaneous processed foods and dry mixes). Correlation coefficients for plates incubated for 5 days were DG18 versus DRBC (0.93), PYM versus DRBC (0.81), CDYM versus DG18 (0.81), PYM versus DG18 (0.80), CDYM versus DRBC (0.79), and CDYM versus PYM (0.75). The number of yeasts and molds recovered from a group of foods (n = 32) analyzed on a weight basis (CFU per gram) was not significantly different (alpha = 0.05) when samples were plated on DRBC, DG18, PYM, or CDYM. However, the order of recovery from foods (n = 65) in a group analyzed on a unit or piece basis, or a composite of both groups (n = 97), was DRBC > DG18 = CDYM > PYM. Compared with PYM, CDYM recovered equivalent, significantly higher (alpha = 0.05) or significantly lower (alpha = 0.05) numbers of yeasts and molds in 51.5, 27.8, and 20.6%, respectively, of the 97 foods tested; respective values were 68.8, 15.6, and 15.6% in the small group (n = 32) and 43.1, 33.8, and 23.1% in the large group (n = 65) of foods. The two groups contained different types of foods, the latter consisting largely (73.8%) of raw fruits (n = 16) and vegetables (n = 32). Differences in efficacy of the four methods in recovering yeasts and molds from foods in the two groups are attributed in part to differences in genera and predominant mycoflora. While DG18 agar, CDYM, and PYM appear to be acceptable for enumerating yeasts and molds in the foods analyzed in this study, overall, DRBC agar recovered higher numbers from the 97 test foods, thereby supporting its recommended use as a general purpose medium for mycological analysis.     

Evaluation of the petrifilm plate method for the enumeration of aerobic microorganisms and coliforms in retailed meat samples.

This study was designed to compare the effectiveness and applicability of the Petrifilm plate method with the Association of Official Analytical Chemists' (AOAC) standard aerobic count method and violet red bile agar method for meat products. The comparison was carried out using 303 meat samples collected from various retailers: 110 pork samples, 87 chicken samples, and 107 beef samples. In the comparison of the correlation coefficient (R) between the conventional method and the Petrifilm plate method by a linear regression analysis, the correlation coefficient in total microorganisms was 0.99, 0.95, and 0.94 in pork, beef, and chicken samples, respectively. The correlation coefficient in coliform count was 0.83, 0.96, and 0.81 in pork, beef, and chicken samples, respectively. Based on the high correlation in the total microorganism count, it might be possible to replace the conventional methods with the Petrifilm plate method. For coliform counts, the Petrifilm plate method also showed a generally high correlation coefficient, except for pork samples, which are more subject to contamination. The Petrifilm plate method was simpler and less time-consuming in sample preparation and, in procedures, faster than the conventional method. These results suggested that the 3M Petrifilm plate method could replace the conventional methods in the analysis of microorganism contamination measurement in meat products.     

食品感官货架期确定的一般原则与方法

感官品质是描述和判断食品质量最直观的指标,将此品质作为货架寿命的评价指标而确定的货架期为感官货架期,则对应的感官评价方法是确定感官货架寿命的关键技术之一。由此,结合感官评价方法与货架期预测原理,提出了食品感官货架期确定的一般原则与方法,旨在指导构建我国不同类型食品感官品质的货架期确定指南,完善食品货架期检测标准,为我国食品质量与控制的保障提供标准化支撑。     

Kinetic-spectrophotometry method for determination of ultra trace amounts of aluminum in food samples

A simple and sensitive kinetic-spectrophotometry method is developed for the determination of trace amounts of aluminum in food samples based on its catalytic effect on the oxidation of Nile Blue A by potassium bromate in sulfuric acid medium. The absorbance is measured at 595.5 nm with the fixed-time method. The optimization of the operating conditions regarding concentrations of the reagents, temperature and interferences are also investigated. The calibration curve is linear over the concentration range 0.07–0.9 μg ml⁻¹ of aluminum with good precision and accuracy and the detection limit was down to 0.034 μg ml⁻¹. The relative standard deviation for a standard solution of 0.4 μg ml⁻¹ of aluminum is 1.73% (n = 10). The proposed method proved highly sensitive, selective and relatively rapid for the assay of aluminum at ultra trace level without any pre-concentration and separation step. The method was applied to the determination of aluminum in food samples (rice, tea and potato). The analytical results of the real samples were in good agreement with the standard method.