Development of Two Quantitative Real-Time PCR Methods Based on SYBR Green and TaqMan to Quantify Sterigmatocystin-Producing Molds in Foods

Sterigmatocystin (ST) is a mycotoxin produced by different species of molds that can grow and contaminate some foods such as dry-ripened foods, cereals, and spices. To improve food safety, the presence of ST-producing molds in foods should be quantified. In the present work, two real-time quantitative PCR (qPCR) protocols based on SYBR Green and TaqMan were developed. Twenty-eight ST-producing and non-producing strains belonging to different species usually reported in food products were used. The ST production of the reference strains was checked by micellar electrokinetic capillary electrophoresis and high-performance liquid chromatography–mass spectrometry. For development of the qPCR methods, a primer pair (FluGF1/FluGR1) and a TaqMan probe (FluGp) were designed on the basis of fluG gene, involved in the ST biosynthesis. The presence of fungal DNA was tested by using a qPCR method based on the universal fungal β-tubulin gene. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the fluG gene copies number and Ct values for the different ST producers tested. The ability to quantify ST producers of the developed SYBR Green and TaqMan assays in different artificially inoculated food samples and naturally infected samples was successful, with a minimum detection limit of 1 log cfu/g. In conclusion, both qPCR methods developed allow quantifying ST-producing molds, in raw materials/ingredients and pre-processed foods, which they would be very useful for monitoring these toxigenic molds in HACCP programs, to prevent ST accumulation in processed foods.     

Development of real-time PCR methods to quantify patulin-producing molds in food products

Patulin is a mycotoxin produced by different Penicillium and Aspergillus strains isolated from food products. To improve food safety, the presence of patulin-producing molds in foods should be quantified. In the present work, two real-time (RTi) PCR protocols based on SYBR Green and TaqMan were developed. Thirty four patulin producers and 28 non-producers strains belonging to different species usually reported in food products were used. The patulin production was tested by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair F-idhtrb/R-idhtrb and the probe IDHprobe were designed from the isoepoxydon dehydrogenase (idh) gene, involved in patulin biosynthesis. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the idh gene copy number and Ct values for the different patulin producers tested. The ability to quantify patulin producers of the developed SYBR Green and TaqMan assays in artificially inoculated food samples was successful, with a minimum threshold of 10 conidia g⁻¹ per reaction. The developed methods quantified with high efficiency fungal load in foods. These RTi-PCR protocols, are proposed to be used to quantify patulin-producing molds in food products and to prevent patulin from entering the food chain.     

Quantification of ochratoxin A-producing molds in food products by SYBR Green and TaqMan real-time PCR methods

Ochratoxin A (OTA) is a mycotoxin synthesized by a variety of different fungi, most of them from the genera Penicillium and Aspergillus. Early detection and quantification of OTA producing species is crucial to improve food safety. In the present work, two protocols of real-time qPCR based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the non-ribosomal peptide synthetase (otanpsPN) gene involved in OTA biosynthesis. Seventy five mold strains representing OTA producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for OTA production by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). The ability of the optimized qPCR protocols to quantify OTA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1 x 10⁴ to 10 conidia/g per reaction for all qPCR assays in the different food matrices (cooked and cured products and fruits). The detection limit in all inoculated foods ranged between 1 and 10 conidia/g for SYBR Green assay and TaqMan. No significant differences were found between the Ct values obtained from pure mold DNA and pure mold DNA mixed with food DNA. The ability of the designed qPCR methods to quantify two known conidial suspensions inoculated on several foods was evaluated. The amount of conidia assessed by both qPCR methods was close to the inoculated amount for most foods and indicates that the described procedure holds potential for use for the detection and quantification of OTA producing molds in foods.     

Duplex real-time PCR method with internal amplification control for quantification of verrucosidin producing molds in dry-ripened foods

Verrucosidin, which is a tremorgenic mycotoxin responsible for neurological diseases, has been detected in different dry-ripened foods as consequence of the growth of toxigenic molds. To improve food safety, the presence of verrucosidin producing molds in these kind foods should be quantified. The aim of this study was to design a duplex real-time PCR (qPCR) protocol based on TaqMan methodology with an internal amplification control (IAC). Eleven verrucosidin producing and 11 non producing strains belonging to different species often reported in food products were used. Verrucosidin production was tested by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair (VerF1/VerR1) and a TaqMan probe (Verprobe) were designed from the SVr1 probe sequence of a verrucosidin producing Penicillium polonicum. The conserved regions of the β-tubulin gene were used to design primers (TubF1/TubR1) and probe (Tubprobe) of the non-competitive IAC. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves which relating Ct values and DNA template of the tested verrucosidin producers using the verrucosidin and IAC primers. The ability to quantify verrucosidin producers of the developed TaqMan assay in all artificially inoculated food samples was successful, with a minimum detection limit of 1 log cfu per gram of food. This qPCR protocol including an IAC could be very useful to quantify verrucosidin producing molds in dry-ripened foods avoiding false negative results. This method should be proposed to monitor the target molds in HACCP programs to prevent the risk of verrucosidin formation and consequently avoid its presence in the food chain.     

Rapid and reliable identification of Staphylococcus aureus harbouring the enterotoxin gene cluster (egc) and quantitative detection in raw milk by real time PCR

A TaqMan and a SYBR Green real time PCR (rt-PCR) were developed for the reliable identification and quantitative detection of Staphylococcus (S.) aureus strains harbouring the enterotoxin gene cluster (egc) regardless of its variants. Both approaches revealed 100% specificity against a panel of 70 reference strains, including 29 clinical and foodborne S. aureus strains harbouring all the egc variants to date known, 4 egc⁻S. aureus strains and 37 strains of phylogenetically closely and distantly related species. Standard curves made by 10 fold dilutions of either genomic DNA or cells from an egc⁺S. aureus log-phase broth culture showed a good linearity of response (R² ≥ 0.993) for six orders of magnitude, with about 100% relative accuracy and a low inter-assay variability (CV ≤ 3.02). The overall limit of quantification (LOQ) for both rt-PCR assays (about 100% PCR efficiency; running time 30 min) was 10 cfu or 10 genome equivalents per reaction mixture although 1 cfu or 1 genome equivalent was detected with a 33.33% probability. These performances were confirmed in raw milk artificially contaminated with log-phase broth cultures of either a single egc⁺S. aureus strain or a mixture of S. aureus strains harbouring all the egc variants to date known. Similar results were also obtained with a raw milk based standard curve of the S. aureus egc⁺ mixture in the presence of 10⁶ cfu/mL of egc⁻S. aureus strains harbouring some of the commonest enterotoxin genes associated to the staphylococcal food poisoning. Nonetheless, the TaqMan based approach resulted in a lower sensitivity (LOQ = 100 cfu equivalents per reaction mixture) than the SYBR Green based assay (LOQ = 10 cfu equivalents per reaction mixture). When applied to real milk samples, both PCR assays provided a good response with 100% diagnostic specificity and 96-107% relative accuracy, as compared to conventional culture-based PCR approaches. Due to the high specificity, the wide dynamic range of detection and the high sensitivity demonstrated even in a complex and potentially highly contaminated raw milk matrix, the SYBR Green rt-PCR assay is a useful diagnostic tool for quick, high throughput and reliable routine screening of egc⁺S. aureus isolates. Moreover, the SYBR Green based quantitative detection of these pathogens in raw milk could remarkably contribute to clarify their actual role in staphylococcal food poisoning and other clinical syndromes associated with the consumption of milk and milk-based products.     

Real-time polymerase chain reaction for the quantitative detection of tetA and tetB bacterial tetracycline resistance genes in food

A new, rapid, sensitive and specific method was developed to directly detect and quantify tetA and tetB in food. Both tet genes are two of the most frequently present tetracycline resistance genes in Gram-negative bacteria. A set of primers and Taqman probes was designed for each gene. The standard curves were performed using Escherichia coli BM13 (C600 RifR)/RP4 and E. coli NCTC 50365, which carry tetA and tetB, respectively. Meat and fish samples inoculated with these reference strains were used as a matrix to construct the standard curves for the analysis of 20 samples of chicken meat and 10 samples of hake (Merlucius merlucius). The limits of detection in pure culture were 5 cfu/mL (0.7 log cfu/mL) in the case of tetA, 50 cfu/mL (1.7 log cfu/mL) for tetB and 5 × 10² cfu/g (2.7 log cfu/g) for both genes in food samples. The results obtained by real-time quantitative polymerase chain reaction (qPCR) were compared to counts of tetracycline-resistant bacteria obtained by plating extracts of poultry and hake samples in culture media supplemented with 16 mg/L of tetracycline. Counts of tetracycline-resistant bacteria obtained by qPCR showed a positive correlation, especially interesting when compared with microbiological counts of tetracycline-resistant Enterobacteriaceae in poultry meat (r = 0.5509) and with tetracycline-resistant mesophilic aerobic bacteria in hake samples (r = 0.7146). The obtained results demonstrate that this method could be a useful tool for the direct quantification of the amount of bacterial strains that carry tetA and/or tetB genes in food samples.     

Development of a Multiplex PCR Method for the Detection of Patulin-, Ochratoxin A- and Aflatoxin-Producing Moulds in Foods

A multiplex PCR was developed for the simultaneous detection of patulin-, ochratoxin A-, and aflatoxin-producing moulds. The idh, otanpsPN and omt-1 genes involved in the patulin, ochratoxin A and aflatoxin biosynthesis, respectively, were used as target sequences in the multiplex PCR. The expected amplicons of 496, 373, and 289 bp for the corresponding primer pairs were detected in all tested toxigenic mould strains. These results were closely related to the mycotoxin detection by micellar electrokinetic capillary electrophoresis and high-performance liquid chromatography–mass spectrometry. The sensitivity of the method was tested on DNA from mould pure cultures and DNA from artificially contaminated food products (dry-fermented sausage “salchichón”, paprika, apple, wheat and peanut). The method was able to detect down to 1 ng of pure DNA from producing strains and from 10³ to 10⁴?cfu/g in inoculated foods. Inhibition from the food matrices was no detected in the PCR assay. The developed multiplex PCR protocol is a good alternative to traditional diagnostic methods for the simultaneous and early detection of different types of toxigenic moulds in foods.     

Development of a PCR protocol to detect aflatoxigenic molds in food products

Aflatoxins are secondary metabolites produced mainly by Aspergillus species growing in foodstuffs. Because aflatoxins have important health effects, the detection of early contamination of foods by aflatoxigenic molds should be useful. In the present work, a reliable conventional PCR method for detecting aflatoxigenic molds of various species was developed. Fifty-six aflatoxigenic and nonaflatoxigenic strains commonly reported in foodstuffs were tested. Aflatoxin production was first confirmed by micellar electrokinetic capillary electrophoresis or/and high-pressure liquid chromatography-mass spectrometry. Based on the conserved regions of the O-methyltransferase gene (omt-1) involved in the aflatoxin biosynthetic pathway, six primer pairs were designed. With only the designed primer pair AFF1-AFR3, the expected PCR product (381 bp) was obtained in all of the tested aflatoxigenic strains of various species and genera. Amplification products were not obtained with this primer pair for any of the nonaflatoxigenic reference molds. However, an amplicon of 453 bp was obtained for all aflatoxigenic and nonaflatoxigenic mold reference strains with a PCR protocol based on the constitutive fungal β-tubulin gene, which was used as a positive fungal control. The PCR protocol based on omt-1 detected as little as 15 pg of DNA from aflatoxigenic molds and 10(2) to 10(3) CFU/g in contaminated food samples. This PCR protocol should be used as a routine technique to detect aflatoxigenic molds in foods.     

Detection of aflatoxin-producing molds in Korean fermented foods and grains by multiplex PCR

An assay based on multiplex PCR was applied for the detection of potential aflatoxin-producing molds in Korean fermented foods and grains. Three genes, avfA, omtA, and ver-1, coding for key enzymes in aflatoxin biosynthesis, were used as aflatoxin-detecting target genes in multiplex PCR. DNA extracted from Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, Aspergillus niger, Aspergillus terreus, Penicillium expansum, and Fusarium verticillioides was used as PCR template to test specificity of the multiplex PCR assay. Positive results were achieved only with DNA that was extracted from the aflatoxigenic molds A. flavus and A. parasiticus in all three primer pairs. This result was supported by aflatoxin detection with direct competitive enzyme-linked immunosorbent assay (DC-ELISA). The PCR assay required just a few hours, enabling rapid and simultaneous detection of many samples at a low cost. A total of 22 Meju samples, 24 Doenjang samples, and 10 barley samples commercially obtained in Korea were analyzed. The DC-ELISA assay for aflatoxin detection gave negative results for all samples, whereas the PCR-based method gave positive results for 1 of 22 Meju samples and 2 of 10 barley samples. After incubation of the positive samples with malt extract agar, DC-ELISA also gave positive results for aflatoxin detection. All Doenjang samples were negative by multiplex PCR and DC-ELISA assay, suggesting that aflatoxin contamination and the presence of aflatoxin-producing molds in Doenjang are probably low.     

小麦粉污染霉菌的分离鉴定及产黄曲霉毒素 能力的研究

目的对污染小麦粉中所含霉菌进行分离和菌株鉴定,并对所分离菌株的产黄曲霉毒素能力进行评价。方法使用马铃薯-葡萄糖琼脂培养基和麦汁琼脂培养基对小麦粉污染的霉菌进行分离和纯化,根据菌落形态、显微形态观察和ITS序列分析结果对分离菌株进行鉴定,采用PCR技术检测黄曲毒霉合成路径的关键基因来判断菌株的潜在产毒能力,最后用高效液相色谱法确认菌株是否产毒。结果共分离出5株菌株,分别鉴定为链格孢霉(NHF1)、橘灰青霉(NHF2)、黑曲霉(NHF3)和米曲霉(NHF4、NHF5),其中2株米曲霉具有潜在的产黄曲霉素的能力,在一定条件下会产生黄曲霉毒素。结论需要加强小麦粉微生物检测,尤其是霉菌污染的检测、管理和控制,全面制定小麦粉中污染微生物的限量标准,尤其是霉菌的限量值。     

Detection and enumeration of Staphylococcus aureus from bovine milk samples by real-time polymerase chain reaction

The aim of this study was to develop and evaluate a real-time quantitative PCR (qPCR)-based method to detect and quantify Staphylococcus aureus in bronopol-preserved milk samples from subclinical intramammary infections (IMI). Serial dilutions of milk artificially inoculated with Staph. aureus ATCC 29213 were used to establish a standard curve (cfu/mL) of the qPCR assay targeting the Staph. aureus thermonuclease-encoding gene nuc according to the strain plate count. The analytical sensitivity, specificity, and repeatability of the qPCR assay were determined. A total of 60 milk samples, collected from mammary quarters without abnormal appearance and with positive isolation of Staph. aureus, were submitted to both the qPCR protocol and Staph. aureus plate counting and results from both methods were compared. Staphylococcus aureus from bronopol-preserved, subclinical IMI milk samples were not accurately enumerated by qPCR compared with plate counting of the nonpreserved, raw milk sample. The detection limit of the qPCR protocol of inoculated Staph. aureus ATCC 29213 in bronopol-preserved milk samples was 1.04 × 10¹ cfu/mL. The qPCR protocol can be a high-throughput and rapid diagnostic assay to accurately detect Staph. aureus IMI from bronopol-preserved milk samples compared with a traditional culturing method. However, the proposed qPCR protocol is not accurate for counting of Staph. aureus in bronopol-preserved milk samples from naturally infected mammary glands.     

QUALITY CHANGES OF SARDINE (SARDINA PILCHARDUS) PATTIES DURING REFRIGERATED STORAGE

In this study, quality changes in sardine patties stored at 4C were investigated. Total viable bacteria, psychrotrophic and coliform bacteria counts of sardine patties increased from 2.50 log cfu/g, 2.60 log cfu/g, 19 MPN/g to 6.15 log cfu/g, 6.45 log cfu/g, 70 MPN/g on day 6, respectively. Yeast–molds, Staphylococcus aureus and Escherichia coli were not detected during the storage period. Total volatile basic nitrogen, thiobarbituric acid and trimethylamine nitrogen values of sardine patties were 13.66 mg/100 g, 1.50 mg MA/kg and 1.20 mg/100 g at the beginning and 29.55 mg/100 g, 3.60 mg MA/kg and 5.01 mg/100 g at the end of the storage period (at day 6), respectively. Based on sensory evaluation, the shelf life of sardine patties was determined to be 4 days during refrigerated storage. When chemical, microbiological and sensory analyses were compared, sensory analysis was the found to be most effective for determining the shelf life of sardine patties .

PRACTICAL APPLICATIONS

There is a possibility of consuming sardines as fish patties when catches are large. Sardines are very suitable for patties because of their flavor and taste, and their consumption is very large as fresh fish in Turkey. In recent years, the increase in civilization or socioeconomic factors such as the increasing numbers of working women have led to direct consumers' preference for ready-to-eat foods. Thus, sardine patties were made and quality changes in sardine patties during refrigerated storage were investigated.     

A multiplex RTi-PCR reaction for simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus on fresh, minimally processed vegetables

In this work, a new multiplex single-tube real-time PCR approach is presented for the detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus, three of the more frequent food-borne bacterial pathogens that are usually investigated in a variety of food matrices. The study includes the design and specificity testing, of a new primer and probe specific for Salmonella spp. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the beta-glucuronidase (uidA, E. coli) and Thermonulease (nuc, Sta. aureus) genes, and in the replication origin sequence (oriC, Salmonella spp.). Melting-curve analysis using a SYBR Green I RTi-PCR approach showed characteristic T(m) values demonstrating the specific and efficient amplification of the three fragments. Subsequently, a TaqMan RTi-PCR approach was settled, using FAM, NED and VIC fluorescently labelled specific probes for an automated detection. It was equally sensitive than uniplex RTi-PCR reactions in Sta. aureus and E. coli O157:H7, using same amounts of purified DNA, and allowed detection of 10 genome equivalents in the presence of 10(2) or 10(4) genome equivalents of the other two pathogens. Finally, it was tested in artificially inoculated fresh, minimally processed vegetables, revealing a sensitivity of 10(3)CFUg(-1) each of these pathogens in direct detection, following DNA extraction with DNeasy Tissue Kit (Qiagen). The multiplex RTi-PCR developed scored the sensitivity recognised for PCR in food and it allows a high-throughput and automation, thus it is promising as a rapid and cost-effective test for the food industry.     

Worldwide aflatoxin contamination of agricultural products and foods: From occurrence to control

Aflatoxins represent a global public health and economic concern as they are responsible for significant adverse health and economic issues affecting consumers and farmers worldwide. Produced by fungal species from the Aspergillus genus, aflatoxins are a toxic, mutagenic, and carcinogenic group of fungal metabolites that routinely contaminate food and agricultural products. Climate and diet are essential factors in the aflatoxin contamination of food and subsequent human exposure process. Countri es with warmer climates and staple foods that are aflatoxin-susceptible shoulder a substantial portion of the global aflatoxins burden. Enactment of regulations, prevention of pre- and postharvest contamination, decontamination, and detoxification have been used to prevent human dietary exposure to aflatoxin. Exploiting their chemical and structural properties, means are devised to detect and quantify aflatoxin presence in foods. Herein, recent developments in several important aspects impacting aflatoxin contamination of the food supply, including: fungal producers of the toxin, occurrence in food, worldwide regulations, detection methods, preventive strategies, and removal and degradation methods were reviewed and presented. In conclusion, aflatoxin continues to be a major food safety problem, especially in developing countries where regulatory limits do not exist or are not adequately enforced. Finally, knowledge gaps and current challenges in each discussed aspect were identified, and new solutions were proposed.     

Multiplex PCR assay for the detection of aflatoxigenic and non-aflatoxigenic fungi in meju, a Korean fermented soybean food starter

Aflatoxins are toxic secondary metabolites produced commonly by Aspergillus flavus and Aspergillus parasiticus. In this study, the possibility of using multiplex PCR was investigated to speed up and specify the detection of aflatoxigenic Aspergillus species in meju, a traditional Korean fermented soybean food starter. Two different sets of three primers were designed specifically for the omtB, ver-1, aflR, and omtA genes present in the aflatoxin biosynthesis cluster. The optimized multiplex PCR showed that only aflatoxigenic Aspergillus species gave three band patterns in both primer sets. The detection limits were determined as 125 pg/μl for genomic DNA from aflatoxigenic A. parasiticus KCCM 35078, and 10⁵ spores/g of meju sample for DNA extracted directly from meju. A total of 65 Aspergillus isolates from meju were tested for the presence of aflatoxigenic fungi by the application of multiplex PCR, and were analyzed by TLC and HPLC for the aflatoxin production in the culture filtrates. Results showed a good correlation between the presence of the aflatoxin biosynthesis genes analyzed by multiplex PCR and aflatoxin production by TLC and HPLC. This suggests that this multiplex PCR method may provide an accurate and specific detection of aflatoxigenic Aspergillus species in fermented soybean foods.     

Optimization of pressure-induced germination of Bacillus sporothermodurans spores in water and milk

Bacillus sporothermodurans produces highly resistant endospores that can survive ultra-high-temperature treatment in milk. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk sterility. In this work, the rate of spore germination of B. sporothermodurans LTIS27 was measured in distilled water after high-pressure treatments with varying pressure (50–600 MPa), treatment temperature (20–50 °C), pressure-holding time (5–30 min) and post-pressurization incubation time (30–120 min) at 37 °C or 4 °C. The results showed that pressure-induced germination was maximal (62%) after a treatment at 200 MPa and 20 °C and increased with pressure-holding time and post-pressurization incubation time. Treatment temperature had no significant effect on germination. A central composite experimental design with three factors (pressure, pressure-holding time, and post-pressurization incubation time) using response surface methodology was used to optimize the germination rate in distilled water and in skim milk. No factor interaction was observed. Germination was induced at lower pressure and was faster in milk than in distilled water, but complete germination was not reached. The optimum germination obtained with experimental data was 5.0 log cfu/mL in distilled water and 5.2 log cfu/mL in milk from 5.7 log cfu/mL of spores initially present in the suspension. This study shows the potential of using high hydrostatic pressure to induce the germination of B. sporothermodurans spores in milk before a heat treatment.     

Cold atmospheric pressure plasma treatment of ready-to-eat meat: inactivation of Listeria innocua and changes in product quality

The application of cold atmospheric pressure plasma for decontamination of a sliced ready-to-eat (RTE) meat product (bresaola) inoculated with Listeria innocua was investigated. Inoculated samples were treated at 15.5, 31, and 62 W for 2–60 s inside sealed linear-low-density-polyethylene bags containing 30% oxygen and 70% argon. Treatments resulted in a reduction of L. innocua ranging from 0.8 ± 0.4 to 1.6 ± 0.5 log cfu/g with no significant effects of time and intensity while multiple treatments at 15.5 and 62 W of 20 s with a 10 min interval increased reduction of L. innocua with increasing number of treatments. Concentrations of thiobarbituric acid reactive substances (TBARS) increased with power, treatments and storage time and were significantly higher than those of control samples after 1 and 14 days of storage at 5 °C. However, the levels were low (from 0.1 to 0.4 mg/kg) and beneath the sensory threshold level. Surface colour changes included loss of redness of ∼40% and 70% after 1 and 14 days of storage, respectively, regardless of plasma treatment. The results indicate that plasma may be applicable in surface decontamination of pre-packed RTE food products. However, oxidation may constitute an issue in some products.     

A real-time PCR method for the detection and quantification of lupin flour in wheat flour-based matrices

Lupin flour is growingly being used in bakery products, mainly as a soybean protein substitute. The aim of the present work was to detect and quantify the presence of lupin flour in wheat-based foods using a newly set up qPCR system based on SYBR green. Although DNA sequence information for lupin is scarce, it has been possible to design a primer pair highly specific for the target gene and devoid of any primer-dimers amplification capacity. Lupin flour revealed to be a difficult matrix, since large amounts of compounds tend to co-purify with DNA, even adopting well established extraction protocols. Nonetheless, the primers used allowed to reach high PCR efficiencies and did not show any cross-reactivity with DNAs extracted from various plant and animal foods. The sensitivity achieved was 7 pg of lupin DNA, corresponding to a percentage of less than 0.1% of lupin flour in the foods.     

Development of a locally sustainable functional food based on mutandabota,a traditional food in southern Africa

A probiotic dairy product was developed on the basis of a traditional dish called mutandabota to enable resource-poor populations in southern Africa to benefit from a functional food. Mutandabota is widely consumed in rural southern Africa, making it an ideal food matrix to carry probiotics. First, a process to produce probiotic mutandabota was designed. Raw cow milk was boiled and subsequently cooled to ambient temperature (25°C). Next, dry pulp from the fruit of the baobab tree (Adansonia digitata L.) was added to the milk at a concentration of 4% (wt/vol). This mixture was inoculated with the probiotic Lactobacillus rhamnosus yoba and left to ferment for 24 h, while the growth of the bacterial culture was monitored. Final ingredients were then added to produce probiotic mutandabota that had 14% (wt/vol) baobab fruit pulp and 7% (wt/vol) sugar in cow milk. The pH of probiotic mutandabota was pH 3.5, which ensures that the product is microbiologically safe. The viable plate count of L. rhamnosus yoba increased from 5.8 ± 0.3 log cfu/mL at the point of inoculation to 8.8 ± 0.4 log cfu/mL at the moment of consumption, thereby meeting the criterion to have a viable count of the probiotic bacterium in excess of 6 log cfu/mL of a product. Baobab fruit pulp at 4% promoted growth of L. rhamnosus yoba with a maximal specific growth rate (μ_max) of 0.6 ± 0.2/h at 30°C. The developed technology, though specific for this particular product, has potential to be applied for the delivery of probiotics through a variety of indigenous foods in different regions of the world. Upon consumption, probiotic mutandabota is expected to improve the population's intestinal health, which is especially relevant for vulnerable target groups such as children and elderly people.