High-Pressure Processing of Human Milk: A Balance between Microbial Inactivation and Bioactive Protein Preservation.

BACKGROUND: Donor human milk banks use Holder pasteurization (HoP; 62.5°C, 30 min) to reduce pathogens in donor human milk, but this process damages some bioactive milk proteins. OBJECTIVES: We aimed to determine minimal parameters for high-pressure processing (HPP) to achieve >5-log reductions of relevant bacteria in human milk and how these parameters affect an array of bioactive proteins. METHODS: Pooled raw human milk inoculated with relevant pathogens (Enterococcus faecium, Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii) or microbial quality indicators (Bacillus subtilis and Paenibacillus spp. spores) at 7 log CFU/mL was processed at 300-500 MPa at 16-19°C (due to adiabatic heating) for 1-9 min. Surviving microbes were enumerated using standard plate counting methods. For raw milk, and HPP-treated and HoP-treated milk, the immunoreactivity of an array of bioactive proteins was assessed via ELISA and the activity of bile salt-stimulated lipase (BSSL) was determined via a colorimetric substrate assay. RESULTS: Treatment at 500 MPa for 9 min resulted in >5-log reductions of all vegetative bacteria, but <1-log reduction in B. subtilis and Paenibacillus spores. HoP decreased immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G, lactoferrin, elastase and polymeric immunoglobulin receptor (PIGR) concentrations, and BSSL activity. The treatment at 500 MPa for 9 min preserved more IgA, IgM, elastase, lactoferrin, PIGR, and BSSL than HoP. HoP and HPP treatments up to 500 MPa for 9 min caused no losses in osteopontin, lysozyme, α-lactalbumin and vascular endothelial growth factor. CONCLUSION: Compared with HoP, HPP at 500 MPa for 9 min provides >5-log reduction of tested vegetative neonatal pathogens with improved retention of IgA, IgM, lactoferrin, elastase, PIGR, and BSSL in human milk.

Bioactive milk peptides: an updated comprehensive overview and database.

Partial digestion of milk proteins leads to the formation of numerous bioactive peptides. Previously, our research team thoroughly examined the decades of existing literature on milk bioactive peptides across species to construct the milk bioactive peptide database (MBPDB). Herein, we provide a comprehensive update to the data within the MBPDB and a review of the current state of research for each functional category from in vitro to animal and clinical studies, including angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, antioxidant, dipeptidyl peptidase (DPP)-IV inhibitory, opioid, anti-inflammatory, immunomodulatory, calcium absorption and bone health and anticancer activity. This information will help drive future research on the bioactivities of milk peptides.

Peptides Derived from In Vitro and In Vivo Digestion of Human Milk Are Immunomodulatory in THP-1 Human Macrophages.

BACKGROUND: Milk proteins contain many encrypted bioactive peptides. Whether these bioactive peptides are released in the infant intestine and exert immunomodulatory activity remains unknown. OBJECTIVE: This study examined in vitro immunomodulatory activities of peptides from in vitro- and in vivo-digested human milk. METHODS: Peptides were extracted from in vitro-digested human milk and pooled intestinal samples from 8 infants fed human milk. Peptides extracted from in vitro-digested samples were fractionated. The in vitro effects of these peptides and fractions on the secretion of TNF-α and IL-8 in LPS-treated human immune THP-1 macrophages were evaluated. The significance of differences between in vitro peptide fraction treatment and control on cytokine production was analyzed by t test. LC-MS/MS-based peptidomics was conducted to identify the peptides. The peptides were screened for potential bioactivity using a sequence homology search using the Milk Bioactive Peptide Database (MBPDB). RESULTS: Six fractions of the peptide mixture extracted from the in vitro-digested human milk significantly inhibited TNF-α production by LPS-challenged THP-1 macrophages. Fractions F4, F8, F11, F14, and F17 attenuated IL-8 secretion, and F6/7 and F18 increased IL-8 secretion. Peptides extracted from the pooled in vivo intestinal samples attenuated both TNF-α and IL-8 secretion. There were 266 and 418 peptides identified in the in vitro and in vivo samples, respectively. Among the peptides, 34 and 50 in the in vitro and in vivo samples, respectively, had >80% sequence similarity to bioactive peptides in the MBPDB. CONCLUSIONS: Peptides released by in vitro and in vivo infant digestion of human milk were immunomodulatory in human immune cells; fractions F4, F8, and F11 were anti-inflammatory; and F6/7 and F18 were proinflammatory. Thirteen peptides were present in all fractions with anti-inflammatory activity, and 38 peptides were present in all fractions with proinflammatory activity. These peptides potentially contributed to the observed immunomodulatory activity of the peptide mixtures.

Chlorogenic acid isomers directly interact with Keap 1-Nrf2 signaling in Caco-2 cells.

Chlorogenic acid (CGA) exists as multiple isomers (e.g., 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) in foods such as coffee beverages, fruits and vegetables. This study aimed to investigate relative activities of these six different CGA isomers to modify redox biology in inflamed Caco-2 cells that involved Nrf2 signaling. Caco-2 cells were pre-treated with individual CGA isomers to assess the relative effectiveness to mitigate oxidative stress. Isomer-specific capacity of different CGA isomers for direct free radical scavenging activity and potential endogenous control of oxidative stress were determined using chemical assays and cell-based experiments, respectively. Molecular dynamics simulations of the CGA and Keap1-Nrf2 complex were performed to predict CGA structure-specific interactions. Results demonstrated that dicaffeoylquinic acid (diCQA including 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) isomers had greater (p < 0.05) affinity to ameliorate oxidative stress through direct free radical scavenging activity. This observation corresponded to greater (p < 0.05) capacity to activate Nrf2 signaling compared to caffeoylquinic acid (CQA including 3-CQA, 4-CQA, and 5-CQA) isomers in inflamed differentiated Caco-2 cells. Simulations revealed that differences between the ability of CQA and diCQA to interact with the Keap1-Nrf2 complex may be due to differences in relative orientation within this complex. The observed CGA isomer-specific affinity for CQA to activate Nrf2 signaling was confirmed by nuclear translocation of Nrf2 induced by CGA and greater (p < 0.05) upregulation of genes related to Nrf2 expression.

Chlorogenic Acid (CGA) Isomers Alleviate Interleukin 8 (IL-8) Production in Caco-2 Cells by Decreasing Phosphorylation of p38 and Increasing Cell Integrity.

The objective of this study was to determine the effect of six chlorogenic acid (CGA) isomers known to be present in coffee and other plant foods on modulating the inflammatory response induced by pro-inflammatory cytokines in the Caco-2 human intestinal epithelial cell line. Compared to caffeoylquinic acids (CQA), dicaffeoylquinic acids (DiCQA) had significantly stronger (p < 0.05) capacities to reduce phosphorylation of one of mitogen-activated protein kinases (MAPK) cascades, namely p38. Compared to the control, CQA isomers treatment resulted in around 50% reduction in an interleukin-8 (IL-8) secretion, whereas DiCQA, at the same concentration, resulted in a 90% reduction in IL-8 secretion, compared to the control cells. CGA isomer treatment also showed a significant effect (p < 0.05) on the up-regulation of NFκB subunit p65 nuclear translocation by more than 1.5 times, compared to the control. We concluded that CGA isomers exert anti-inflammatory activity in a mixture of interferon gamma (IFNγ) and phorbol myristate acetate (PMA)-challenged Caco-2 cells, by decreasing the phosphorylation of p38 cascade and up-regulating NFκB signaling.

Antioxidant property of coffee components: assessment of methods that define mechanisms of action.

Coffee is a rich source of dietary antioxidants, and this property, coupled with the fact that coffee is one of the world's most popular beverages, has led to the understanding that coffee is a major contributor to dietary antioxidant intake. Brewed coffee is a complex food matrix with numerous phytochemical components that have antioxidant activity capable of scavenging free radicals, donating hydrogen and electrons, providing reducing activity and also acting as metal ion pro-oxidant chelators. More recent studies have shown that coffee components can trigger tissue antioxidant gene expression and protect against gastrointestinal oxidative stress. This paper will describe different in vitro, cell-free and cell-based assays that both characterize and compare the antioxidant capacity and mechanism of action of coffee and its bioactive constituents. Moreover, evidence of cellular antioxidant activity and correlated specific genomic events induced by coffee components, which are relevant to antioxidant function in both animal and human studies, will be discussed.

Optimized Ultraviolet-C Processing Inactivates Pathogenic and Spoilage-Associated Bacteria while Preserving Bioactive Proteins, Vitamins, and Lipids in Human Milk.

Holder pasteurization (HoP) enhances donor human milk microbiological safety but damages many bioactive milk proteins. Though ultraviolet-C irradiation (UV-C) can enhance safety while better preserving some milk proteins, it has not been optimized for dose or effect on a larger array of bioactive proteins. We determined the minimal UV-C parameters that provide >5-log reductions of relevant bacteria in human milk and how these treatments affect an array of bioactive proteins, vitamin E, and lipid oxidation. Treatment at 6000 and 12 000 J/L of UV-C resulted in >5-log reductions of all vegetative bacteria and bacterial spores, respectively. Both dosages improved retention of immunoglobulin A (IgA), IgG, IgM, lactoferrin, cathepsin D, and elastase and activities of bile-salt-stimulated lipase and lysozyme compared with HoP. These UV-C doses caused minor reductions in α-tocopherol but not γ-tocopherol and no increases in lipid oxidation products. UV-C treatment is a promising approach for donor human milk processing.

Corrigendum: Structural and functional changes of bioactive proteins in donor human milk treated by vat-pasteurization, retort sterilization, ultra-high-temperature sterilization, freeze-thawing and homogenization.

[This corrects the article DOI: 10.3389/fnut.2022.926814.].

Macrophage-Immunomodulatory Actions of Bovine Whey Protein Isolate, Glycomacropeptide, and Their In Vitro and In Vivo Digests.

Whey protein isolate (WPI) consists of an array of proteins and peptides obtained as a byproduct of the cheesemaking process. Research suggests that WPI, along with its peptides such as glycomacropeptide (GMP), possesses immunomodulatory properties. These properties hold potential for alleviating the adverse effects of inflammatory conditions such as inflammatory bowel disease. Although promising, the immunoregulatory properties of the digested forms of WPI and GMP-those most likely to interact with the gut immune system-remain under-investigated. To address this knowledge gap, the current study examined the effects of in vitro-digested WPI and GMP, in vivo-digested WPI, and undigested WPI and GMP on the secretion of pro-inflammatory cytokines (TNF-α and IL-1ß) in lipopolysaccharide-stimulated macrophage-like cells. Our results indicate that digested WPI and GMP reduced the expression of TNF-α and IL-1ß, two pro-inflammatory cytokines. Whole WPI had no effect on TNF-α but reduced IL-1ß levels. In contrast, in vivo-digested WPI reduced TNF-α but increased IL-1ß. Undigested GMP, on the other hand, increased the secretion of both cytokines. These results demonstrate that digestion greatly modifies the effects of WPI and GMP on macrophages and suggest that digested WPI and GMP could help mitigate gastrointestinal inflammation. Further clinical studies are necessary to determine the biological relevance of WPI and GMP digestion products within the gut and their capacity to influence gut inflammation.

Bioavailability of Peptides Derived from the In Vitro Digestion of Human Milk Assessed by Caco-2 Cell Monolayers.

Human milk-protein-derived peptides exhibit an array of bioactivities. Certain bioactivities cannot be exerted unless the peptides are absorbed across the gastrointestinal lumen into the bloodstream. The purpose of study was to determine which peptides derived from in vitro digestion of human milk could cross human intestinal Caco-2 cell monolayers. Our results showed that the numbers of peptides absorbed by the Caco-2 cell monolayer were different at different concentrations (44 peptides out of 169 peptides detected at 10 µg/mL, 124 peptides out of 204 peptides detected at 100 µg/mL, and 175 peptides out of 236 peptides detected at 1000 µg/mL). Four peptides (NLHLPLP (ß-casein [138-144]), PLAPVHNPI (ß-casein [216-224]), PLMQQVPQPIPQ (ß-casein [148-159]), and FDPQIPK (ß-casein [126-132])) crossed to the basolateral chamber of the Caco-2 monolayer incubated with peptides at all three concentrations. Among the peptides identified in the basolateral chambers, three peptides (NLHLPLP (ß-casein [138-144]), LENLHLPLP (ß-casein [136-144]), and QVVPYPQ (ß-casein [182-188])) are known ACE-inhibitors; one peptide (LLNQELLLNPTHQIYPV (ß-casein [197-213])) is antimicrobial, and another peptide (QVVPYPQ (ß-casein [182-188])) has antioxidant activity. These findings indicate that specific milk peptides may be able to reach the bloodstream and exert bioactivity.

Structural and functional changes of bioactive proteins in donor human milk treated by vat-pasteurization, retort sterilization, ultra-high-temperature sterilization, freeze-thawing and homogenization.

Background: Donor human milk should be processed to guarantee microbiological safety prior to infant feeding, but this process can influence the structure and quantity of functional proteins. Objective: The aim of this study was to determine the effect of thawing, homogenization, vat-pasteurization (Vat-PT), retort sterilization (RTR) and ultra-high-temperature (UHT) processing on the structure of bioactive proteins in donor milk. Methods: Pooled donor milk was either not treated (Raw) or treated with an additional freeze-thaw cycle with and without homogenization, Vat-PT, RTR with and without homogenization, and UHT processing with and without homogenization. Overall protein retention was assessed via sodium-dodecyl sulfate (SDS-PAGE), and the immunoreactivity of 13 bioactive proteins were assessed via enzyme-linked immunosorbent assay (ELISA). Results: Freeze-thawing, freeze-thawing plus homogenization and Vat-PT preserved all the immunoglobulins (sIgA/IgA, IgG, IgM) in donor milk, whereas RTR and UHT degraded almost all immunoglobulins. UHT did not alter osteopontin immunoreactivity, but Vat-PT and retort decreased it by ~50 and 70%, respectively. Freeze-thawing with homogenization, Vat-PT and UHT reduced lactoferrin's immunoreactivity by 35, 65, and 84%, respectively. Lysozyme survived unaltered throughout all processing conditions. In contrast, elastase immunoreactivity was decreased by all methods except freeze-thawing. Freeze-thawing, freeze-thawing plus homogenization and Vat-PT did not alter polymeric immunoglobulin receptor (PIGR) immunoreactivity, but RTR, RTR plus homogenization and UHT increased detection. All heat processing methods increased α-lactalbumin immunoreactivity. Vat-PT preserved all the growth factors (vascular/endothelial growth factor, and transforming growth factors ß1 and ß2), and UHT treatments preserved the majority of these factors. Conclusion: Different bioactive proteins have different sensitivity to the treatments tested. Overall, Vat-PT preserved more of the bioactive proteins compared with UHT or RTR. Therefore, human milk processors should consider the impact of processing methods on key bioactive proteins in human milk.

Amelioration of Oxidative Stress in Caco-2 Cells Treated with Pro-inflammatory Proteins by Chlorogenic Acid Isomers via Activation of the Nrf2-Keap1-ARE-Signaling Pathway.

Understanding the potential effects of chlorogenic acid (CGA) isomers on the intestinal epithelium is important because coffee intake exposes consumers to the six major CGA isomers: 3-caffeoylquinic acid (3-CQA), 4-caffeoylquinic acid (4-CQA), 5-caffeoylquinic acid (5-CQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), 3,5-dicaffeoylquinic acid (3,5-diCQA), and 4,5-dicaffeoylquinic acid (4,5-diCQA). The present study determined the relative effects of CGA isomers on the antioxidant status of inflamed Caco-2 intestinal cells by investigating the oxidative-stress-responsive pathway and nuclear-factor-erythroid-derived-2-like 2 (Nrf2) signaling. Differentiated Caco-2 cells were challenged with the inflammatory mediators PMA and IFNγ, as a model of intestinal inflammation in vitro. Significant redox ( p < 0.05) responses to these mediators were assessed by indirect measurement of induced generation of reactive oxygen species (ROS), as well as the expression of reduced (GSH) and oxidized (GSSG) glutathione. This translated to a 40% reduction in the GSH/GSSG ratio. We found that responses in these parameters were associated with increased Nrf2 activation ( p < 0.05). ROS generation and increased IL-8 secretion were found in challenged cells, indicating an association between induced oxidation and inflammatory status. Oxidative stress was ameliorated by CGA isomers, which scavenged intracellular ROS, increased GSH, and activated Nrf2 signaling. diCQA isomers were relatively more effective at reducing IL-8 ( p < 0.05). The observed increase in Nrf2 signaling led to upregulated expression of some Nrf2 target genes ( GPX2, KEAP1, and NFE2L2) in Caco-2 cells and activated the Nrf2-Keap1-ARE-signaling pathway. These findings indicate that CGA isomers present in coffee have bioactivity toward alleviating oxidative stress associated with intestinal inflammation.

Interactions between major chlorogenic acid isomers and chemical changes in coffee brew that affect antioxidant activities.

Coffee bean source and roasting conditions significantly (p<0.05) affected the content of chlorogenic acid (CGA) isomers, several indices of browning and subsequent antioxidant values. Principal component analysis was used to interpret the correlations between physiochemical and antioxidant parameters of coffee. CGA isomer content was positively correlated (p<0.001) to capacity of coffee to reduce nitric oxide and scavenge Frémy's salt. Indices of browning in roasted coffee were positively correlated (p<0.001) to ABTS and TEMPO radical scavenging capacity, respectively. Only the CGA content of coffee corresponded to intracellular antioxidant capacity measured in Caco-2 intestinal cells. This study concluded that the intracellular antioxidant capacity that best describes potential health benefits of coffee positively corresponds best with CGA content.

Application of Attenuated Total Reflectance-Fourier Transformed Infrared (ATR-FTIR) Spectroscopy To Determine the Chlorogenic Acid Isomer Profile and Antioxidant Capacity of Coffee Beans.

The chlorogenic acid isomer profile and antioxidant activity of both green and roasted coffee beans are reported herein using ATR-FTIR spectroscopy combined with chemometric analyses. High-performance liquid chromatography (HPLC) quantified different chlorogenic acid isomer contents for reference, whereas ORAC, ABTS, and DPPH were used to determine the antioxidant activity of the same coffee bean extracts. FTIR spectral data and reference data of 42 coffee bean samples were processed to build optimized PLSR models, and 18 samples were used for external validation of constructed PLSR models. In total, six PLSR models were constructed for six chlorogenic acid isomers to predict content, with three PLSR models constructed to forecast the free radical scavenging activities, obtained using different chemical assays. In conclusion, FTIR spectroscopy, coupled with PLSR, serves as a reliable, nondestructive, and rapid analytical method to quantify chlorogenic acids and to assess different free radical-scavenging capacities in coffee beans.

Performance review of a fast HPLC-UV method for the quantification of chlorogenic acids in green coffee bean extracts.

The aim of this study was to test the performance of a HPLC method, designated for rapid quantification of chlorogenic acids (CGA) in green coffee extract (GCE). The precision statistics associated with the method were assessed using three independent laboratories with five samples analyzed in triplicate. Seven main CGA isomers (3-CQA, 5-CQA, 4-CQA, 5-FQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA) were quantified. The concentration of total CGA in the samples varied from 32.24% to 52.65% w/w. The repeatability and reproducibility standard deviations for the determination of individual isomers varied, respectively, from 0.01 to 0.28 and 0.05-1.59. The repeatability and reproducibility standard deviations of the calculated total CGA, corresponding to the sum of the seven main CGA isomers, varied respectively, from 0.17 to 0.58 and 0.55-2.01. The fast HPLC method evaluated in this study was considered precise and appropriate for the determination of CGA in GCE.

Role of Chlorogenic Acids in Controlling Oxidative and Inflammatory Stress Conditions.

Chlorogenic acids (CGAs) are esters formed between caffeic and quinic acids, and represent an abundant group of plant polyphenols present in the human diet. CGAs have different subgroups that include caffeoylquinic, p-coumaroylquinic, and feruloyquinic acids. Results of epidemiological studies suggest that the consumption of beverages such as coffee, tea, wine, different herbal infusions, and also some fruit juices is linked to reduced risks of developing different chronic diseases. These beverages contain CGAs present in different concentrations and isomeric mixtures. The underlying mechanism(s) for specific health benefits attributed to CGAs involves mitigating oxidative stress, and hence the related adverse effects associated with an unbalanced intracellular redox state. There is also evidence to show that CGAs exhibit anti-inflammatory activities by modulating a number of important metabolic pathways. This review will focus on three specific aspects of the relevance of CGAs in coffee beverages; namely: (1) the relative composition of different CGA isomers present in coffee beverages; (2) analysis of in vitro and in vivo evidence that CGAs and individual isomers can mitigate oxidative and inflammatory stresses; and (3) description of the molecular mechanisms that have a key role in the cell signaling activity that underlines important functions.

Chemical properties and reactive oxygen and nitrogen species quenching activities of dry sugar-amino acid maillard reaction mixtures exposed to baking temperatures.

Maillard reaction products (MRPs) derived from 10 different, dry sugar-amino acid reaction model systems were examined for changes in color index (E), sugar loss, and formation of α-dicarbonyl compounds; the changes were correlated with relative activities to quench both reactive oxygen (ROS) and reactive nitrogen (RNS) species. Reducing sugars, xylose, ribose, fructose, glucose, and non-reducing sucrose were reacted with glycine (Xyl-Gly, Rib-Gly, Fru-Gly, Glc-Gly, and Suc-Gly), or lysine (Xyl-Lys, Rib-Lys, Fru-Lys, Glc-Lys, and Suc-Lys), respectively, at temperatures of 150°C and 180°C for time periods ranging from 5 to 60min. ROS quenching capacity was negatively correlated with color index (E) (r=-0.604, P<0.001), and positively correlated with sugar loss (r=0.567, P<0.001). MRPs also exhibited activity to quench RNS as assessed by nitric oxide (NO) inhibition in differentiated Caco-2 cells that were induced with interferon-γ (IFN-γ) and phorbol ester (PMA) cocktail. We also showed a correlation between RNS and color index, sugar loss, and ROS quenching activities for MR mixtures that were heated for a short time (e.g. 10min) at 150°C. MRP quenching of ROS was largely influenced by sugar type, whereas, RNS quenching was dependent more so on the interaction between reactants and reaction conditions used to generate MRPs.

Emotional conflict in facial expression processing during scene viewing: an ERP study.

Facial expressions are fundamental emotional stimuli as they convey important information in social interaction. In everyday life a face always appears in complex context. Scenes which faces are embedded in provided typical visual context. The aim of the present study was to investigate the processing of emotional conflict between facial expressions and emotional scenes by recording event-related potentials (ERPs). We found that when the scene was presented before the face-scene compound stimulus, the scene had an influence on facial expression processing. Specifically, emotionally incongruent (in conflict) face-scene compound stimuli elicited larger fronto-central N2 amplitude relative to the emotionally congruent face-scene compound stimuli. The effect occurred in the post-perceptual stage of facial expression processing and reflected emotional conflict monitoring between emotional scenes and facial expressions. The present findings emphasized the importance of emotional scenes as a context factor in the study of the processing of facial expressions.

Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR.

Contamination of lettuce by Salmonella has caused serious public health problems. Polymerase chain reaction (PCR) allows rapid detection of pathogenic bacteria in food, but it is inaccurate as it might amplify DNA from dead target cells as well. This study aimed to investigate the stability of DNA of dead Salmonella cells in lettuce and to develop an approach to detecting viable Salmonella in lettuce. Salmonella-free lettuce was inoculated with heat-killed Salmonella Typhimurium cells and stored at 4 °C. Bacterial DNA extracted from the sample was amplified by real-time PCR targeting the invA gene. Our results indicate that DNA from the dead cells remained stable in lettuce for at least 8 d. To overcome this limitation, propidium monoazide (PMA), a dye that can selectively penetrate dead bacterial cells and cross-link their DNA upon light exposure, was combined with real-time PCR. Lettuce samples inoculated with different levels of dead or viable S. Typhimurium cells were treated or untreated with PMA before DNA extraction. Real-time PCR suggests that PMA treatment effectively prevented PCR amplification from as high as 10(8) CFU/g dead S. Typhimurium cells in lettuce. The PMA real-time PCR assay could detect viable Salmonella at as low as 10(2) CFU/mL in pure culture and 10(3) CFU/g in lettuce. With 12-h enrichment, S. Typhimurium of 10(1) CFU/g in lettuce was detectable. In conclusion, the PMA real-time PCR assay provides an alternative to real-time PCR assay for accurate detection of Salmonella in food.