Effect of heat-treated <Emphasis Type="Italic">Nuruk</Emphasis> on the quality characteristics of aged <Emphasis Type="Italic">Yakju</Emphasis>

Long-term aging of Yakju, a traditional Korean liquor made of rice and Nuruk (a fermentation agent), causes browning and odor and flavor development. This study investigated the effects of heat-treated Nuruk (50–80 °C, 30 min) on Yakju quality. The saccharogenic powers and glucoamylase, α-amylase, and carboxypeptidase activities were similar in non-heat-treated Nuruk and that treated at 50 °C. However, acidic protease and alcohol dehydrogenase decreased above 50 °C. The content of nitrogen-containing compounds was inversely proportional to the heat-treatment temperature. Compounds that cause off-flavors decreased at 50–60 °C, but increased at 70–80 °C, whereas compounds that provide fragrance increased at 50–60 °C. Sensory evaluation indicated that bad taste attributes were higher in Yakju produced using non-heat-treated Nuruk. Therefore, heat treatment of Nuruk at 50 °C can be adopted as a method for improving Yakju quality, as enzymatic activities that affect color, aroma, and taste are regulated.     

Development and characterization of reconstituted hydrogel from <Emphasis Type="Italic">Aloe vera</Emphasis> (<Emphasis Type="Italic">Aloe barbadensis</Emphasis> Miller) powder

Structural and rheological characterization of reconstituted hydrogels developed from A. vera non-fibrous alcohol insoluble residue (NFAIR) powder using different methods [viz., shaking (S), heating-shaking (HS), and heating (H)] and concentrations (viz., 0.2–1.6 %, w/v) was carried out. Functional group distribution by FTIR spectroscopy and Congo red (CR) method revealed the presence of acetylated acemannan in A. vera powder. Dynamic oscillation studies of A. vera (NFAIR) fluids at all concentrations of 0.2–1.6 %, w/v, showed gel strength in the order of H > HS > S method. However, in H method, increase in concentration from 0.2 to 1.6 %, w/v showed the conformational transition from semi-diluted solution to weak gel nature. Rheological models described the effect of heating temperatures (HT); 30–90 °C, and times (Ht); 15–60 min on viscoelastic behavior in reconstituted A. vera fluids. The reconstituted A. vera hydrogel prepared with a concentration of 1.6 %, w/v using 50 °C (HT) and 30 min (Ht) condition showed a good agreement with the Power law (storage modulus, G′) and Weak gel model (complex modulus, G*) fitted data (R² > 0.94) resulting higher viscoelastic moduli intercepts; G′₀ (71.5 Pa s ^n′), G″₀ (33.5 Pa s ^n″), lower slopes; n′ (0.22), n″ (0.06), higher network strength (A _F , 121.3 Pa s^1/z ) and number of network (z, 5.3) values. The obtained results suggested that heating at 50 °C/30 min can develop aqueous weak gel networks of A. vera with enhanced gel strength which may be utilized as a novel gelling agent for wide variety of targeted applications in food and pharmaceutical sectors.     

A Rapid and Visual Single Primer Isothermal Amplification-Based Method for the Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Raw Pork Products

Staphylococcus aureus (S. aureus) is an important food-borne pathogen which poses a severe threat to public health worldwide. Rapid detection of S. aureus with high sensitivity is of particular importance for food safety. In this study, a novel single primer isothermal amplification (SPIA) method was established to detect S. aureus in food, targeting the accessory gene regulator (agr) gene with a DNA/RNA primer. The developed SPIA method has the advantages of visualization and avoiding tedious electrophoresis. In order to confirm the specificity of this method, 7 S. aureus strains and 26 non-S. aureus strains were detected with their pure cultures. The sensitivity and detection limit of S. aureus with artificially inoculated raw pork products by SPIA were evaluated through fluorescence and turbidity by naked eye and the amplification curve, which were 4.3?×?10⁰ CFU/mL and 5.6?×?10⁰ CFU/g, respectively. Compared with the conventional PCR method, the SPIA has 100-fold higher sensitivity and 100-fold lower detection limit. Therefore, the developed SPIA method is a potentially reliable tool for rapid and visual detection of S. aureus in food.     

Probe-Based Fluorescence Melting Curve Analysis for Differentiating <Emphasis Type="Italic">Larimichthys polyactis</Emphasis> and <Emphasis Type="Italic">Larimichthys crocea</Emphasis>

Larimichthys polyactis (redlip yellow croaker) and Larimichthys crocea (large yellow croaker) are commercially important fish species in East Asia with high differences in their market values. In Korea, consumers prefer L. polyactis to L. crocea, although it is difficult to distinguish them based on their morphological traits. The objective of this study was to develop an assay for differentiating L. polyactis and L. crocea using fluorescence melting curve analysis (FMCA) with a single locked nucleic acid (LNA) probe. Species-specific regions of the mitochondrial 16S rDNA were selected as LNA probes. The target sequences of L. polyactis and L. crocea had a 2-bp difference, and a single LNA probe was identified using melting temperature (Tm) shift. LNA probe was 100 % complementary to the target sequence of ten L. polyactis samples, giving a significantly higher Tm value (66 °C) than that of five L. crocea samples (42 °C). Overall, the developed LNA-based FMCA system had high efficiency, multiplexity, and simplicity, and could be effectively used for differentiating L. polyactis and L. crocea, and as a food analyzing method based on DNA sequence.     

Rapid Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Food Using a Recombinase Polymerase Amplification-Based Assay

Staphylococcus aureus (S. aureus) is of great importance and is a leading cause of food poisoning, which is a public health concern in terms of the frequency and seriousness of the disease. In the present study, RPA and real-time RPA assays were developed and validated to detect S. aureus with high sensitivity and specificity by targeting the nuc gene for the first time. The analytical sensitivity of real-time RPA was 10² copies/reaction, which was higher than the sensitivity of the real-time PCR method. The analysis time was reduced to 10 min, but this method was as reliable as real-time PCR. Furthermore, the potential use of RPA to detect S. aureus was validated with five different artificially contaminated foods. In conclusion, the RPA and real-time RPA assays developed here, similar to real-time PCR, are rapid and simple and exhibit with high sensitivity and specificity. These assays serve as efficient tools for the detection of S. aureus in less advanced laboratories and are suitable for point-of-care detection.     

Modeling the Inactivation of <Emphasis Type="Italic">Listeria innocua</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> in Fresh-Cut Tomato Treated with Pulsed Light

The effectiveness of pulsed light (PL) treatments to inhibit microorganisms on fresh-cut tomatoes (Lycopersicon esculentum Mill., cv. Daniela) was investigated. Tomato slices inoculated with Escherichia coli or Listeria innocua were exposed to PL treatments (4, 6, or 8 J cm^?2 fluence) and kept cold at 4 °C for 20 days. L. innocua and E. coli counts, gases in the headspace of the containers (O₂ and CO₂), pH, titratable acidity, and soluble solid content were monitored throughout the cold storage. The PL treatments reduced significantly (p < 0.05) initial loads of both microbes. The effect of the PL fluence on the survival number of microoganisms was described by a log-linear model (R ² = 0.849–0.999). At any fixed time within the cold storing, the microbial counts for untreated samples were always higher than those cut tomatoes that had been previously PL-treated. The behavior of L. innocua and E. coli during the storage were well adjusted (R ² > 0.930) by Gompertzian models; the studied microorganisms exhibited different patterns during the storage period. On the other hand, O₂ and CO₂ partial pressures in containers with fresh-cut tomatoes were also significantly affected by PL treatments (p < 0.05). The highest PL fluence caused the greatest changes of O₂ and CO₂ contents. In addition, the application of PL triggered an acceleration of the O₂ consumption during the cold stage. PL treatments might be used to effectively extend the safety of fresh-cut tomatoes over 12 days of storage against E. coli and L. innocua growth.     

Comparative inactivation studies of <Emphasis Type="Italic">Aegle marmelos</Emphasis> (<Emphasis Type="Italic">bael</Emphasis>) peroxidase in crude extract of fruit by heat processing and ultrasonication treatment

Bael (Aegle marmelos) is considered as a holy fruit comprised of vast number of phytonutrients. Whole bael tree including all its parts has medicinal significance. Lack of awareness and seasonal nature makes its processing rather challenging. Conventional heat processing may lead to inactivation of quality hampering enzymes such as peroxidase, but at the cost of loss in essential phytonutrients. In the present work it was observed that thermal inactivation of bael peroxidase obeyed first order kinetics with enzyme activation energy of 7.7 kJ mol^?1. Complete inactivation of bael peroxidase was achieved within 11 min at 85?°C while ultrasound treatment attained in lesser time of 4 min at 64.07 W cm^?2 ultrasonic intensity. Loss of marmelosin a well-known phytonutrient in bael fruit was found to be 83.29?% by heat (11 min, 85?°C) and only 50.20?% by ultrasonication (4 min, 64.07 W cm^?2 ultrasonic intensity). Ultrasonication has potential to overcome harmful effects of heat processing with retention of phyto-constituents and hence has promising future in various food processing applications.     

Effect of Solids Concentration on the Physicochemical and Flow Properties of Cactus Pear Juices of Two Varieties (<Emphasis Type="Italic">Opuntia ficus-indica</Emphasis> and <Emphasis Type="Italic">Opuntia streptacantha</Emphasis>)

Juices from two varieties of cactus pear, a green (Opuntia ficus-indica) and a red (Opuntia streptacantha), were obtained and concentrated by evaporation. Both fruit varieties and their juices at different concentrations were characterized. Green cactus pears had significantly higher amount of pulp than red cactus pears; the peel of O. ficus-indica represented only 38 versus 52 % of the fruit for the O. streptacantha. Both varieties had no significant differences on moisture, density, pH, and titratable acidity, in contrary to soluble solids. Juice was concentrated under vacuum conditions to reach a final concentration of 42, 53–55, and 58–60 °Brix, respectively, and stored under refrigeration (10 °C) during 4 weeks. Physicochemical properties of the pears and juices were determined as fresh items (time zero) and every week for the concentrate juices through storage; similarly, flow parameters were measured at 10 and 25 °C. Concentrate density (1160–1283 kg/m³) was mainly affected by final soluble solids, while pH and acidity were affected differently depending on the variety. Concentrated juices at 42 °Brix were considered with Newtonian behavior with a viscosity of 2–22 mPa s, while those at higher concentrations were of pseudoplastic nature (n < 1.0 and K > 69 mPa sⁿ). Power Law model fitted better the flow behavior than Herschel-Bulkley model of concentrates of both varieties. Temperature, solid concentration, and/or storage time affected the consistency coefficient (K) and flow index (n) depending on the cactus pear variety. Overall, those concentrated juices from O. streptacantha were more stable and exhibited lower apparent viscosity.     

LAMP,PCR, and real-time PCR detection of <Emphasis Type="Italic">Acetobacter aceti</Emphasis> in yogurt

Acetic acid bacteria (AAB) can spoil food. Acetobacter aceti as a core subgroup of AAB is usually isolated from yogurt. A. aceti should be timely and effectively detected to prevent yogurt contamination. The present study focused on A. aceti to establish an assay that can be performed to detect AAB in yogurt. LAMP, PCR, and real-time PCR were applied and compared for detecting A. aceti from pure culture and artificially contaminated yogurt samples. In pure culture, LAMP showed the highest detection sensitivity with 10^?1 CFU/mL. For yogurt samples, the sensitivity limit of LAMP was 10² CFU/mL, which was lower than that of real-time PCR (10¹ CFU/mL). The results indicated that these methods could be quickly and efficiently applied to detect A. aceti. As LAMP technology has low cost and high detection efficiency, it can potentially be applied for detecting A. aceti in production and quality control programs of yogurt.     

Thermal transition and thermo-physical properties of potato (<Emphasis Type="Italic">Solanum tuberosum L</Emphasis>.) var. Russet brown

Potatoes are an important food in many regions of the world and are commonly used in a variety of food products. Thermal transition and thermo-physical properties of potatoes are important in order to design efficient food processes and select appropriate storage conditions. In this study, we determined the thermal transitions and thermophysical properties of raw and blanched/par-fried potato for a temperature range of ??32 to 21.1 °C. Using differential scanning calorimetry, we found an initial freezing point (T_f) at ??1.8?±?0.1 °C, an onset of melting (T_m^′) at ??9.9?±?0.2 °C and an unfreezable water content (X^′_w) for maximally freeze-concentrated raw potato at 0.21 kg water/kg potato. Corresponding values for blanched/par-fried potatoes were ??0.9?±?0.1 °C, ??11.0?±?0.2 °C and 0.18 kg water/kg potato. Results show that an increase in solids content decreased T_f of both raw and blanched potatoes. We modelled the relationship between them using the Chen model. The apparent specific heat (C_app) increased around T_f to 31.7?±?1.13 kJ/kg K for raw potato and 26.7?±?0.62 kJ/kg K for blanched/par-fried potato. For frozen raw potato at ??32 °C, thermal diffusivity (α) was 0.89?±?0.01?×?10?^?6 m²/s and thermal conductivity (k), 1.82?±?0.14 W/m K, respectively. These values were higher for frozen raw potato than for the unfrozen raw potato (0.15?±?0.01?×?10?^?6 m²/s and 0.56?±?0.08 W/m K, respectively at 21.1 °C). The apparent density (ρ) of frozen raw potato (992?±?4.00 kg/m³ at ??32 °C) was less than that for unfrozen raw potato (1053?±?4.00 kg/m³ at 21.1 °C), and a similar trend was obtained for blanched/par-fried potato (993?±?2.00 kg/m³ at ??32 °C and 1188?±?7.00 kg/m³ at 21.1 °C, respectively). This study established a correlation between thermo-physical properties and temperature. Findings may be used to inform the design and optimization of freezing processes and frozen storage for potato products.     

Isolation of lactic acid bacteria starters from <Emphasis Type="Italic">Jeung</Emphasis>-<Emphasis Type="Italic">pyun</Emphasis> for sourdough fermentation

Lactic acid bacteria (LAB) are key for the fermentation of sourdoughs to improve the quality and nutritive value of bread. The aim of this study was to isolate the LAB starter for sourdough fermentation from Jeung-pyun, a Korean traditional rice cake. Among the twenty two LAB screened, five isolates were selected based on exo-polysaccharide production. Among them, three isolates showed cell growth greater than 8.5 Log CFU/g, maximum increase in the volume of dough, and dextran concentration up to 0.16%. During the sourdough fermentation, pH and total titratable acidity (TTA) were changed, as the three isolates synthesized lactic acid and acetic acid with fermentation quotients less than 2.0. They were identified as Leuconostoc lactis EFEL005, Lactobacillus brevis EFEL004, and Le. citreum EFEL006. They displayed good fermentation properties (growth, dextran production, pH, and TTA) in dough and they are regarded as potential starters to be used in sourdough fermentation.     

A mathematical model for moisture movement during continous and intermittent drying of <Emphasis Type="Italic">Eucalyptus saligna</Emphasis>

A modified diffusion-based mathematical model is proposed to describe the moisture movement during continuous and intermittent drying of Eucalyptus saligna. This model includes the temperature change, the surface drying coefficient (β _n ) and 2 diffusion coefficients [from green to FSP (D _f ) and from FSP to dry condition (D _o )] as important parameters. The final model expression obtained was M?=?exp (??25 β _n ² D _t /l²) with the β _n used was 1.5807 kg m^?2 s^?1, the D _f was 2.26?×?10^?11 m² s^?1, and the D _o was 5.85?×?10^?12 m² s^?1. The range of temperature change between heating and non-heating phases in the intermittent drying regimes was from 24.9 to 31.8 °C. The R² values obtained when the model was fitted into the drying data of different intermittent regimes ranged from 71.5 to 85.9%. The R² value was 87.4% when the model was fitted into continuous trial data. The high values of R² indicate that the model can be used to understand the moisture reduction both in intermittent and continuous regimes.     

Biodegradable Polymeric Films Incorporated with Nisin: Characterization and Efficiency against <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>

Nisin (0.2 IU per cm² films) containing biodegradable films were produced from pea protein isolate (PPI), whey protein isolate (WPI), and polylactic acid (PLA). Nisin was released over 4 h at 22 °C and 8 h at 4 °C. PPI released more nisin compared to other films suppressing the growth of Listeria monocytogenes (P?<?0.05) based upon diffusion into agar and liquid culture media. The population of bacteria after 48 h in liquid media was 6 CFU/mL (1 log₁₀ increase) in PPI, 8.47 CFU/mL (3.47 log₁₀ increase) in WPI and 9 CFU/mL (4 log₁₀ increase) in PLA, which was significantly lower in protein based films compared to PLA (P?<?0.05). The inhibition zone in agar test was significantly higher (P?<?0.05) in PPI and WPI, compared to PLA film, which might be due to the higher hydration in protein based films. Fourier transform infrared spectroscopy (FTIR) showed that nisin altered the intensity of amide I peaks in protein based films suggesting that nisin can bind to the protein functional groups in PPI and WPI. Thermogram showed that nisin did not influence the glass transition and melting temperatures of the films. Nisin containing films exhibited significantly lower enthalpy compared to control films (P?<?0.05). PeakForce Quantitative Nano Mechanical Property Mapping (PeakForce QNM) was applied to extract material and mechanical properties in PPI, WPI and PLA films with and without nisin. Results showed significant reductions in material and mechanical properties of protein based films containing nisin compared to PLA films.     

Characterization of brewing microorganisms isolated from Korean traditional <Emphasis Type="Italic">nuruk</Emphasis> for <Emphasis Type="Italic">Cheongju</Emphasis> production

The objective of this study was to select and identify yeasts and molds isolated from traditional nuruk and to investigate their brewing characteristics for Cheongju production. The yeast strains Y190 (Accession ID-KACC 93251P), Y263 (Accession ID-KACC 93252P), and Y270 (Accession ID-KACC 93253P) showing high alcohol and flavor productivity were isolated and identified by phylogenetic inference based on an internal transcribed spacer 2 region sequence analysis. In addition, Aspergillus oryzae 83-10 (Accession ID-KACC 93254P) showing the highest enzyme activity was isolated. This study provides basic data for the production of Korean Cheongju and assesses the applicability of these three yeast strains and A. oryzae 83-10 isolated from traditional nuruk.     

Effect of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> Bauer and <Emphasis Type="Italic">Bifidobacterium animalis</Emphasis> ssp. <Emphasis Type="Italic">lactis</Emphasis> BB12 on proteolytic changes in dry-cured loins

The effect of the potentially probiotic bacteria strain of Lactobacillus acidophilus Bauer and probiotic bacteria Bifidobacterium animalis ssp. lactis BB12 on proteolytic changes of proteins in dry-cured loins during fermentation and cold storage was studied. Results of the conducted tests demonstrated that the use of probiotic bacteria for the production of dry-cured meats impacts the generation of products of protein proteolysis with high antioxidant activity. The highest antioxidant activity of peptides after fermentation and cold storage was observed in the loin with the strain B. animalis ssp. lactis BB12 and the loin with the mixture of strains L. acidophilus Bauer and B. animalis ssp. lactis BB12. Qualitative analysis of peptides demonstrated that peptides with weight below 3.5 kDa are characterized by the highest capacity of quenching the ABTS cation radical, including the peptides in loins with the strain B. animalis ssp. lactis BB12.     

Properties of a fibrinolytic enzyme secreted by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> JS2 isolated from saeu (small shrimp) <Emphasis Type="Italic">jeotgal</Emphasis>

Bacillus species were screened to be used as starters for jeotgals, salted and fermented Korean sea foods. A strain, JS2, showing strong fibrinolytic activity was isolated from saeu (small shrimp) jeotgal, and identified as Bacillus subtilis. Bacillus subtilis JS2 grew well at 20% (w/v) NaCl concentration. SDS-PAGE of culture supernatant from JS2 showed 3 major bands of 27, 29, and 60 kDa in size. Fibrin zymography showed that the 27 kDa band was the major fibrinolytic protein. The gene, aprEJS2, was cloned and introduced into B. subtilis WB600 using pHY300PLK. A B. subtilis transformant harboring pHYJS2 showed higher fibrinolytic activity than B. subtilis JS2. aprEJS2 was overexpressed in Escherichia coli BL21 (DE3). The optimum pH and temperature for AprEJS2 were pH 8.0 and 40 °C, respectively. Km and V_max values were determined. AprEJS2 has strong α-fibrinogenase activity and moderate β-fibrinogenase activity.     

Nutritional,functional, thermal and structural characteristics of <Emphasis Type="Italic">Citrullus lanatus</Emphasis> and <Emphasis Type="Italic">Limonia acidissima</Emphasis> seed flours

Seeds from fruits such as Citrullus (C.) lanatus (watermelon) and Limonia (L.) acidissima (wood apple) are not commonly utilized but could be suitable in numerous food formulations. It was shown that the protein content of defatted seed flours was 71.38 and 49.51 % and that these contained considerable amounts of minerals such as Na, Mn, Mg, K, Cu, Fe and Zn. The defatted L. acidissima seed flour was superior to C. lanatus in essential amino acids. The flours obtained from both seeds were also evaluated for functional properties and characterized by X-ray diffraction, differential scanning calorimeter and scanning electron microscope (SEM). Amorphous nature was observed in defatted C. lanatus and L. acidissima flours due to the low percentage of degree of crystallinity. Spherical morphologies were observed through SEM. The exothermic peak was recorded in defatted C. lanatus and L. acidissima flour.     

Dual-Labeled PCR-Based Immunofluorescent Assay for the Rapid and Sensitive Detection of Enterotoxic <Emphasis Type="BoldItalic">Staphylococcus aureus</Emphasis> Using Cocktail-Sized Liposomal Nanovesicles as Signal Enhancer

Staphylococcus aureus is a major foodborne pathogen worldwide, and as little as 1 μg of staphylococcal enterotoxins (SEs) can cause food poisoning. Among SEs, enterotoxin A is the most common toxin that causes staphylococcal food poisoning. Hence, this work has developed a dual-labeled PCR-based immunofluorescent assay using anti-digoxigenin (DIG) antibody-tagged immunomagnetic beads (IMBs) as the capture reagent and cocktail-sized NeutrAvidin-tagged liposomal nanovesicles (NA-LNs) that encapsulate fluorescent dyes as the detection reagent. In this approach, the amplicon of sea gene was doubly labeled with DIG and biotin using modified primers and biotin-11-dUTP. The system depends on the immunocapture of IMB to pre-concentrate the labeled amplicons, which were further quantified using cocktail-sized NA-LNs, based on the release and subsequent measurement of encapsulated fluorescent dyes following the lysis of NA-LNs. After optimization, the developed assay could detect S. aureus and differentiate it from other common foodborne bacteria, such as Salmonella enterica and Escherichia coli, with a limit of detection (LOD) of 10¹ CFU mL^?1 without pre-enrichment. With a 2-h pre-enrichment, this developed assay could detect as little as 1 CFU in 25 mL of milk within a workday. Hence, this work established a rapid and sensitive PCR-based immunofluorescent assay using liposomal nanovesicles as an instant signal enhancer to detect the contamination of enterotoxic S. aureus in milk.     

Improvement of the Detection Sensitivity for <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Spices and Herbs

Various variants of ISO 6888-1:1999 and ISO 6888-3:2003 methods for the detection of Staphylococcus aureus were evaluated and improved for their application to conduct analysis in spices and herbs. Improvement substantiated in washing of the sample to remove compounds interfering with the analysis and in the use of PCR for final detection, instead of plating on Baird-Parker agar, to improve sensitivity at high backgrounds and to save time. The evaluation of the method variants was based on determination of the detection limit (LOD) using series of artificially contaminated spices (allspice, black pepper, cinnamon, nutmeg, paprika, vanilla) and herbs (basil, oregano, parsley, thyme). The method without enrichment, ISO 6888-1:1999, produced LODs of 10³–10⁵ CFU/g with no positive effect of washing the sample or use of PCR for final detection. The method with enrichment, ISO 6888-3:2003, had LOD of 10⁰ CFU/g for basil, black pepper, paprika and parsley. If the washing step was added and PCR was used for final detection, LOD of 10⁰ CFU/g was determined also for cinnamon, nutmeg and vanilla, and LOD of 10¹ CFU/g was determined for allspice. For oregano and thyme, which strongly inhibit the growth of S. aureus, an alternative enrichment-independent method based on direct DNA extraction coupled to real-time PCR may be advantageous.     

Evaluation of <Emphasis Type="Italic">eryC</Emphasis> as a Molecular Marker for the Quantitative Detection of <Emphasis Type="Italic">Brucella</Emphasis> Spp. by Real-Time PCR in Food Samples

We evaluated the capacity of the Brucella sp. eryC gene as a diagnostic marker for brucellosis by quantitative real-time PCR. eryC gene encodes the enzyme d-erythrulose-1-phosphate dehydrogenase that plays an important role in the erythritol metabolism and is related with the Brucella survival in the intracellular environment of the macrophage. The assay includes an internal amplification control (IAC) in order to avoid false negative results. It was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE) in 43% of the reactions, being the quantification highly linear (R ² > 0.9953) and efficient (PCR efficiency >0.8820) over a 6-log dynamic range, down to 10 GE. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk and pig blood. The eryC-IAC real-time PCR assay allowed detection of as few as ten Brucella cells per 25 ml of sheep raw milk or per 1 ml of pig blood. In conclusion, we present an alternative for the detection of Brucella genus and therefore facilitate the establishment of preventive and prophylactic measures in food and farm environments.