Adjunct Cultures: Recent Developments and Potential Significance to the Cheese Industry 1 M. El Soda, S.A. Madkor* and P.S. Tong*, , * Dairy Products Technology Center, California Polytechnic State University, San Luis Obispo, California 93407 Department of Dairy Science and Technology, Faculty of Agriculture, Alexandria University Alexandria, EgyptReceived 6 July 1999; accepted 16 November 1999. Available online 3 April 2010. AbstractSeveral previous reviews have described different ways to enhance the flavor and texture of cheese, including use of live cells and nonviable attenuated cells as adjunct cultures. However, comparisons between viable and nonviable cultures were never discussed in these reviews. In addition, recent publications on adjunct cultures have not been covered in previous reviews. This article will survey the more recent work on adjunct cultures with particular attention to whether the adjuncts contained viable or nonviable cellsand propose areas where additional research is needed.Key words: adjunct lactobacilli; attenuation; autolysis; cheese ripeningAbbreviations: LAB, lactic acid bacteria; NSLAB, nonstarter lactic acid bacteriaCorresponding author.1 Part of this review was presented by Morsi El Soda, recipient of the 1998 Marschall Rhodia International Dairy Science Award at the 93rd ADSA meeting, Denver, Colorado. Journal of Dairy ScienceVolume 83, Issue 4, April 2000, Pages 609-619 A novel real-time polymerase chain reaction-based method for the detection and quantification of lactose-fermenting Enterobacteriaceae in the dairy and other food industries M.C. Martn1, a, N. Martnez1, a, B. del Rioa, V. Laderoa, M. Fernndeza and M.A. Alvarez , a, a Instituto de Productos Lcteos de Asturias, CSIC, 33300 Villaviciosa, Asturias, SpainReceived 12 June 2009; accepted 25 November 2009. Available online 19 February 2010. AbstractThe presence of lactose-fermenting Enterobacteriaceae and coliforms is routinely assessed to determine the hygienic quality of water and foods, particularly dairy products. This paper reports the use of lacZ-specific primers in an SYBR green I-based real-time PCR method for the easy and rapid detection of coliforms in dairy products. A large number of bacterial species were assayed to establish the specificity of the method. The sensitivity of the method was assessed using artificially contaminated cheeses. The limit of detection was 1 coliform cell in cheese samples enriched for 8 h in a culture medium. The entire procedure, including sample processing, enrichment, DNA extraction, and real-time PCR amplification, can be completed within 10 to 12 h, making it a single-day assay.Key words: Enterobacteriaceae; coliform; real-time polymerase chain reaction detection; cheeseArticle OutlineIntroductionMaterials and MethodsBacterial Strains Extraction of DNA for the RT-qPCR Assay lacZ Sequencing and Nucleotide Sequence Analysis RT-qPCR Conditions Data Analysis Artificial Contamination and Enrichment of Cheese Nucleotide Sequence Accession NumbersResultsSequencing of the lacZ Genes and Primer Design Optimization of RT-qPCR Specificity and Sensitivity Melting Curve Analysis of the Amplified DNA RT-qPCR Detection LimitsDiscussionAcknowledgementsReferencesFigure 1. Alignment of a 63-bp region of the -galactosidase (lacZ) gene from 22 representative coliform strains (Citrobacter freundii, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Enterobacter sakazakii, Citrobacter koseri, Citrobacter amalonaticus, and Raoultella planticola) using Clustal W software (Altschul et al., 1997). The locations of the primers are shown by arrows, an asterisk denotes identical sequences, and a dash indicates a mismatch with the consensus sequence. Figure 2. Standard curves for the log number of coliform cells per reaction versus the cycle threshold (Ct) value for the fluorescent signal for A) Escherichia coli CECT 515, B) Enterobacter cloacae CECT 194, C) Citrobacter freundii CECT 401, and D) Klebsiella pneumoniae ssp. pneumoniae CECT 143. The error bars indicate standard deviations for 3 independent experiments. CECT = Coleccin Espaola de Cultivos Tipo, Burjasot, Valencia, Spain. Table 1. Strains used in this work1 CECT = Coleccin Espaola de Cultivos Tipo, Burjasot, Valencia, Spain; LSP = Laboratorio de Salud Pblica, Principado de Asturias, Spain; CNRZ = Centre National de Recherch Zootechniques, Jouyen-Josas, France; LMD = Laboratory of Microbiology, Technical University, Delft, the Netherlands; ATCC = American Type Culture Collection, Rockville, Maryland.2 RT-qPCR = real-time quantitative PCR; + indicates a positive RT-PCR result, indicates a negative result. Table 2. Detection of coliforms in cheese by culture enrichment and real-time quantitative PCR11 CECT = Coleccin Espaola de Cultivos Tipo, Burjasot, Valencia, Spain; 1, 10, and 100 indicate the contamination level of coliforms (cfu/mL) before enrichment; + indicates a positive RT-PCR r