Limits of a PCR-based detection method for genetically modified soya beans in wheat bread production

 The official PCR-based method for the detection of recombinant DNA from glyphosate-tolerant soya beans (GTS), laid down in the collection of methods according to Sect. 35 of the German Food Law, was investigated for applicability. As a model, wheat bread was produced with a 1% addition of baking aid consisting of 45% GTS flour. DNA extraction of samples drawn at various stages of the production process revealed that during the process a degradation of DNA took place, resulting in fragment sizes in bread of <500 bp. GTS DNA was detectable at all stages, although the content of GTS flour in the dough and bread had dropped to only 0.4%. In 2 out of 15 commercial baking aids, GTS DNA was detected, reflecting the status of the use of GTS in that area. A model was also developed to study the effect on the detection of GTS under conditions when the target gene sequence of one primer is present in food originating from natural contamination or genetically modified organisms other than GTS. It was observed that high concentrations of competing DNA inhibited the PCR. This inhibition was overcome by increasing the concentration of Taq polymerase in the reaction mixture. Received: 30 April 1998 / Revised version: 17 June 1998     

Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction

Pork identification in raw meat using real-time polymerase chain reaction (PCR) was developed. Total DNA from meat samples were successfully extracted and found to be of high quality and produced clear PCR products. Porcine-specific molecular beacon probe and primers that amplifies 119 bp of the cytochrome b gene fragment of swine (Sus scrofa domestica) was used. Analysis of data showed that the C _q (quantification cycle) from 10 ng/μl porcine DNA is (18.70 ± 0.12 to 19.08 ± 0.06). Meanwhile, the other samples exhibited negative result, which confirmed the specificity of the primers. The method also showed that the limit of detection of pork was 0.0001 ng. Based on the regression analysis of the standard curve, the 96% efficiency of real-time PCR was achieved with high correlation coefficient (r ² = 0.9989). Sensitivity of the assay in discriminating pork as low as 0.1% (w/w) pork in pork–beef mixtures was also obtained. Reproducibility of the assay was successfully validated by applying sample and experimental replicates in every assay being conducted. Thus, this methodology could serve as a fast and sensitive method for detection of pork for meat species verification.     

Effect of selected strains of Debaryomyces hansenii on the volatile compound production of dry fermented sausage “salchichón”

Different biotypes of Debaryomyces hansenii, characterized by mitochondrial DNA (mtDNA) restriction analysis, were inoculated in dry fermented sausages to evaluate their influence as single starter culture on volatile compound generation throughout the ripening process. Similar evolution of physicochemical parameters and microbial population was observed in both uninoculated and inoculated sausages. The tested biotypes modified the volatile compound profile of sausages specially in esters, branched alcohols and aldehydes. The biotype of D. hansenii with the E mtDNA restriction pattern is the most suitable to be used as starter culture since it produced volatile compounds involved in flavour development of dry-cured meat products such as 3-methylbutanol, 3-methylbutanal and 2-propanone. Moreover, the use of D. hansenii strains with the B, C2 and E mtDNA restriction patterns, as a mixed starter culture, should be also considered to generate low amount of sulphur compounds in dry-cured meat products.     

The use of commercial starter cultures in the production of Turkish semi-dry fermented sausages

 The effects of three different starter cultures (Pediococcus acidilactici, Staphylococcus xylosus plus P. pentosaceus, S. carnosus plus Lactobacillus pentosus) were evaluated during the production of Turkish semi-dry fermented sausages. Sausages were studied during the fermentation phase, after heat processing and after drying for 24 h and 72 h. Chemical and organoleptical results indicated that in the processing of these semi-dry sausages a starter culture of P. acidilactici should be used. The use of this culture significantly reduced the pH, increased the lactic acid content and percentage of total heme pigments converted to the cured pigment and improved the development of the sausages' characteristics, i.e. color, appearance, flavor and general acceptability. Received: 20 February 1998 / Revised version: 7 May 1998     

The effect of starter cultures on proteolytic changes and amino acid content in fermented sausages

The main objective of this work was to examine the effects of using five types of commercial starter cultures in fermented sausages. During the fermentation stage, changes in proteolytic characteristics were observed in fermented sausages. Proteolytic activity was high in Ls_b + Sc:(Lactobacillus sakei + Staphylococcus carnosus) and Pp + Sx:(Pediococcus pentosaceus + Staphylococcus xylosus) starter-inoculated sausages during processing. Moreover, a slight increase in proteolytic activity was detected during storage in both these sausages. Sarcoplasmic and myofibrillar proteins were also affected by this starter culture addition, during the fermentation, ripening and intense proteolysis were observed in both the fermented sausages. The content of free amino acids was similar at the beginning of the fermentation stage for all the studied batches. However, the high differences in the content of free amino acids at the end of the process could be attributed to the starter culture activity.     

Oxidative stability of frozen-stored raw pork chops, chill-stored pre-frozen raw pork chops, and frozen-stored pre-cooked sausages in relation to dietary CuSO4, rapeseed oil and vitamin E

 Danish Landrace× Danish Yorkshire female pigs were fed either a standard diet or a standard diet enriched with 6% rapeseed oil and supplemented with increasing amounts of vitamin E (0, 100 or 200 mg all-rac-α-tocopheryl acetate/kg feed) and copper (0, 35 or 175 mg CuSO₄/kg feed), and the effect of dietary regimen on the oxidative stability of (1) frozen-stored raw pork chops packed in atmospheric air, (2) chill-stored pre-frozen pork chops packed in atmospheric air, and (3) freezer-stored, vacuum-packed pre-cooked sausages was investigated. The addition of 6% rapeseed oil did not influence the oxidative stability of the pork chops negatively, whereas the oxidative stability of a product such as the pre-cooked sausages (Danish dinner sausages) with a higher fat content (15%) decreased as a result of rapeseed oil feeding. Inclusion of rapeseed oil in the diets increased the amount of monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs) in the meat and fat for the production of sausages at the expense of the content of the saturated fatty acids, and the higher content of PUFAs readily explains the decreased oxidative stability of the pre-cooked sausages. Feeding pigs 100 mg or 200 mg all-rac-α-tocopheryl acetate/kg feed significantly increased the oxidative stability of the pork chops and the detrimental effect of rapeseed oil observed in the pre-cooked sausages was effectively neutralised by both levels of vitamin E supplementation. Supplementation with copper did not affect the oxidative stability of any of the products. The presented results show that it is possible to produce pork products with a nutritionally improved fatty acid profile by inclusion of 6% rapeseed oil, without affecting the oxidative stability of the products negatively, through the protection provided by dietary vitamin E. Received: 23 March 1998 / Revised version: 28 May 1998     

Effect of processing methods and starter culture (Staphylococcus xylosus and Pediococcus pentosaceus) on proteolytic changes in Turkish sausages (sucuk) during ripening and storage

Proteolytic changes in Turkish sausages (sucuk) produced by two methods (heat processing and traditional) were determined during processing and storage for 90 days. The sausages were produced with or without starter culture in both methods. A mixture of Staphylococcus xylosus and Pediococcus pentosaceus was used as starter culture for their acidic and proteolytic characteristics.The major changes in proteolytic characteristics of sucuk took place during the fermentation stage, with an increase in non-protein nitrogen (NPN) and a decrease in protein solubility. Proteolytic activity was observed in both starter-inoculated and non-inoculated (control) sausages during processing. Moreover, a slight increase in proteolytic activity was detected during storage in both starter-inoculated and control traditional sausages, and also in heat-processed sausages due to some heat-resistant proteolytic enzymes. Protein solubility was significantly affected by processing time and heat treatment. Sarcoplasmic and myofibrillar proteins were also affected by starter addition, fermentation, drying and heat processing. During fermentation, starter-inoculated and control sausages showed intense proteolysis in both the traditional and heat processing methods. After heating, intensive degradation of both sarcoplasmic and myofibrillar proteins due to denaturation was observed in heat-processed samples.     

Metabolism of nitrate in fermented meats: the characteristic feature of a specific group of fermented foods

Within the universe of food fermentation processes the multi-purpose use of nitrate and/or nitrite is a unique characteristic of meat fermentations. These curing agents play a decisive role in obtaining the specific sensory properties, stability and hygienic safety of products such as fermented sausages, ham and, more recently, emulsion type of sausages. The use of nitrate is the traditional method in curing processes and requires its reduction to reactive nitrite. Thus, nitrate reduction is the key event that is exclusively performed by microorganisms. Under controlled fermentation conditions starter cultures are used that contain staphylococci and/or Kocuria varians, which in addition to strongly affecting sensory properties exhibit efficient nitrate reductase activity. To obtain clean label products some plant sources of nitrate have been in use. When producing thermally treated sausages (e.g. of emulsion type), starter cultures are used that form nitrite before cooking takes place. Staphylococci reduce nitrite to ammonia after nitrate has been consumed. K. varians is devoid of nitrite reductase activity. Nitrate and nitrite reductases are also present in certain strains of lactobacilli. It was shown that their application as starter cultures warrants efficient activity in sausages made with either nitrate or nitrite. NO is formed from nitrite in numerous chemical reactions among which disproportionation and reaction with reductants either added or endogenous in meat are of practical importance. Numerous nitrosation and nitrosylation reactions take place in the meat matrix among which the formation of nitrosomyoglobin is of major sensory importance.Safety considerations in meat fermentation relate to the safe nature of the starter organisms and to the use of nitrate/nitrite. Staphylococci (“micrococci”) in fermented meat have a long tradition in food use but have not received the QPS status from the EFSA. They require, therefore, thorough assessment with regard to toxigenicity and pathogenicity determinants as well as presence of transferable antibiotic resistance. Nitrate and nitrite are still considered basically undesired in food. The main objections are based on their potential to form nitrosamines with carcinogenic potential. In view of new results from intensive research of NO, potential risks are opposed by positive effects on human health.     

复合发酵剂和天然香辛料对羊肉发酵香肠品质特性的影响

通过接种复合发酵剂和添加天然香辛料,采用相同的工艺条件生产4组羊肉发酵香肠,1组为对照组,2组为接种发酵剂组,3组为发酵剂+黑胡椒组,4组为发酵剂+黑胡椒+孜然组。分别测定4个组羊肉发酵香肠在发酵成熟和贮藏过程中pH值、Aw值、水分含量及微生物的变化。结果表明:在发酵结束(即第1.5天)时,试验组和对照组的pH值、Aw值和水分含量均呈下降趋势,试验组的pH值迅速降到最低值(即5.0以下),与对照组差异显著(P<0.05);在干燥成熟过程中,试验组的pH值、Aw值和水分含量迅速下降,添加香辛料组的pH值迅速回升,同时乳酸菌数维持在108cfu/g以上水平,高于对照组,细菌总数却低于对照组;在贮藏过程中,试验组的pH值和水分含量基本保持不变,且均低于对照组,而Aw值始终呈下降趋势,细菌总数小于对照组。上述结果表明,接种复合发酵剂和添加天然香辛料能迅速降低pH值、Aw值和水分含量,有效抑制腐败菌和致病菌的生长,提高羊肉发酵香肠的品质和保藏性。     

Effect of reuterin,produced by Lactobacillus reuteri on the surface of sausages to inhibit the growth of Listeria monocytogenes and Salmonella spp.

Reuterin is a bacteriocin produced by some strains of Lactobacillus reuteri. The strain used in this study was isolated from raw milk from a dairy farm nearby Ankara. Beef sausage is a long years produced bratwurst style meat product in Turkey, as well as in some other countries in the Mediterranean region. Sausages are produced by raw meat; sometimes lactic starter cultures are added or spontaneous fermentation is employed. The production and storage conditions of the product promotes the growth of Listeria monocytogenes and Salmonella spp. Although nitrate is added as an antimicrobial substance against many pathogens, sometimes however nitrate application is not preventive enough on the surface because of the natural film around the sausages. Since most of the contaminations take place at post production steps, pathogenic growth is more effective on the surface of the sausages in refrigerated conditions. In this study, reuterin was applied to the surface of the sausages in order to prevent the growth of these two pathogens along with nitrate used as an additive in the product. Reuterin has inhibited the growth of L. monocytogenes considerably but not of Salmonella spp. on the surface of the sausages.     

Microbiological Profile of Goat Meat Inoculated with Lactic Acid Bacteria Cultures and Stored at 30��C for 7?days

The ability of lactic acid bacteria (LAB) cultures to preserve goat meat at 30°C was evaluated in the present study. Strains of Pediococcus pentosaceus GOAT 01 and Lactobacillus plantarum GOAT 012, individually and in combination, were applied as starters on sliced meat samples at 6 log cfu/g and stored for 7 days at 30°C to simulate ambient temperature in Nigeria. They were evaluated for microbiological profile during storage. Reduction in bacterial counts was recorded for enterobacteriaceae, Staphylococcus, yeasts and moulds in starter culture inoculated samples (SCIS), whereas an increase occurred in uninoculated control samples (UCS). In challenge experimental trials, two different sets of meat were inoculated individually with 6 log cfu/g each of pathogenic organisms, Listeria monocytogenes and Salmonella Typhimurium. The inoculated pathogens were monitored during storage to assess the influence of starter cultures on them. Approximately 1 log reduction was recorded in the viable count of L. monocytogenes on day 1, while counts were below detection limit (<2 log) on day 2 in meat samples inoculated with P. pentosaceus alone and in combination with L. plantarum. Counts of Salmonella Typhimurium showed about 2 log reduction in SCIS, inoculated with P. pentosaceus alone and in combination with L. plantarum, on day 2 while an increase by 4 logs was observed in UCS. Our findings suggest that the protective effect of the LAB strains could be exploited in shelf life extension and control of foodborne pathogens in goat meat. If the starter strains could be improved upon, their potential as biopreservatives may be engaged in the preservation of the meat in Nigeria, where storage systems have been very inadequate.     

The fate of forage plant DNA in farm animals: a collaborative case-study investigating cattle and chicken fed recombinant plant material

 The fate of ingested recombinant plant DNA in farm animals (cattle and chicken) being fed a diet containing conventional maize or recombinant Bacillus thuringiensis toxin-maize (Bt-maize) is described. The probability of the detection by polymerase chain reaction of chloroplast-specific gene fragments of different lengths (199 bp and 532 bp) and a Bt-maize-specific fragment [truncated version of CryIA(b)] is shown. First data indicated that only short DNA fragments (<200 bp) derived from plant chloroplasts could be detected in the blood lymphocytes of cows. In all other cattle organs investigated (muscle, liver, spleen, kidney) plant DNAs were not found, except for faint signals in milk. Furthermore, Bt-gene fragments possibly recording the uptake of recombinant maize, were not detected in any sample from cattle. However, in all chicken tissues (muscle, liver, spleen, kidney) the short maize chloroplast gene fragment was amplified. In contrast to this, no foreign plant DNA fragments were found in eggs. Bt-gene specific constructs originating from recombinant Bt-maize were not detectable in any of these poultry samples either. Received: 23 February 2000 / Revised version: 20 March 2000     

Quantification of beef,pork, chicken and turkey proportions in sausages: use of matrix-adapted standards and comparison of single versus multiplex PCR in an interlaboratory trial

Quantitative PCR methods for the determination of beef, pork, chicken and turkey proportions in sausage were tested in an interlaboratory trial. Twelve different laboratories analysed six meat products each made of different compositions of beef, pork, chicken and turkey. Two kinds of calibrators were used: sausages of known proportions of meat and DNA from muscle tissue. Results generated using calibration sausages were more accurate than those resulting from the use of muscle tissue DNA. Regardless of the method used (either multiplex or single PCR), when using calibration sausages, it was always possible to quantify the proportions of meats in the unknown samples (in the range of 0.5–80%) with high precision and accuracy.     

Accelerated ripening of dry fermented sausages

Research on accelerated ripening of dry fermented sausages started in the early 1990s. Fermented sausages manufacture is a very important part of meat industry in many countries and the acceleration of ripening would result in a reduction of the storage time and would increase the profit margin and the competitiveness of the end product. The different strategies that have been assayed with this purpose include ripening at elevated temperature, use of genetically modified starter bacteria, addition of enzymes and addition of slurry systems. Over the last decade, numerous studies have been carried out, especially on the addition of enzymes. The aim of this paper is to review and update the knowledge on this topic. The more recent approaches in this field, such as the use of microbial extracts, are also presented.     

Influence of starter cultures on the generation of antioxidant nitrogen compounds in Iberian dry‐fermented sausages

The purpose of this work was to investigate the influence of a starter culture on the generation of nitrogen compounds with antioxidant activity during the ripening of Iberian dry‐fermented sausages. Starters P200S34 (P. acidilactici M200 and S. vitulus RS34) and P198S34 (P. acidilactici MS198 and S. vitulus RS34) were used to make the Iberian dry‐fermented sausages ‘salchichón’ and chorizo; then, the physicochemical and microbial properties were determined during the ripening process. The oxygen radical absorbance capacity (ORAC) assay was employed to evaluate the antioxidant potential of the nitrogen extracts obtained during ripening of the sausages. This activity was correlated with the most relevant compounds detected by HPLC‐ESI‐MS in the final ripened extracts. Although a relevant part of the antioxidant activity was attributed to the predominant natural nitrogen fraction, the microbial population found in fermented sausages and the fermentation conditions significantly influenced the low molecular weight nitrogen profile and antioxidant activities. Inoculation with the starter culture P200S34 increased free amino acids and amines, such as methionine and tyramine, but other nitrogen compounds also increased the antioxidant activity of the low molecular weight nitrogen extracts. Thus, these starter cultures in Iberian sausages can contribute to delaying oxidative changes during storage.     

Effects of Lactic and Acetic Acid on Survival of Salmonella enteritidis During Refrigerated and Frozen Storage of Chicken Meats

Chicken leg and breast meat samples inoculated with Salmonella enteritidis [4–5 log most probable number (MPN)/cm²] were dipped into lactic acid (LA; 1% and 3%) and acetic acid (AA; 1% and 2%) solutions for 10 min. After packaging, samples were stored at 4 °C (10 days) or −18 °C (6 months). Immediately after dipping into 1% LA, 3% LA, 1% AA, and 2% AA solutions, S. enteritidis counts on leg meat samples were reduced by 0.75, 1.21, 0.85, and 0.95 log MPN/cm², while the reductions were 0.97, 1.72, 0.92, and 1.58 log MPN/cm² on breast meat samples, respectively. The differences between the water-washed control and the acid-treated groups for Salmonella counts were statistically significant (P < 0.05). Salmonella counts on leg meat samples treated with 1% LA, 1% AA, and 2% AA were reduced by 0.54–1.52 log MPN/cm² (P < 0.05) during storage at 4 °C. However, for the breast meat samples, only Salmonella counts of water-washed controls were significantly reduced during refrigerated storage (P < 0.05). S. enteritidis counts on organic acid-treated samples were reduced by 0.13–0.55 log MPN/cm² during storage at −18 °C for 6 months, while the reduction on the water-washed controls was 0.64 log MPN/cm². It can be concluded that lactic or acetic acid treatment could be useful especially for reducing the initial Salmonella contamination. On the other hand, this pathogen could survive on poultry meats during refrigerated and frozen storage even following lactic or acetic acid decontamination.     

A Capillary Electrophoresis Method for the Determination of Hydroxyproline as a Collagen Content Index in Meat Products

The addition of collagen or its hydrolysates as a protein source or water binding agent is a common practice in the production of meat products. However, its level of addition is restricted by international regulations. Glycine, proline, and hydroxyproline are the most abundant amino acids in collagen, which is the main constituent of connective tissue. Thus, the objective of this study was to develop a method for the determination of hydroxyproline content in meat products by capillary electrophoresis (CE) as a collagen content index. Acid hydrolysis of samples and amino acid derivatization with fluorescamine prior to CE analysis were conducted. Separations were carried out using an uncoated capillary at 33 °C, using tetraborate buffer 0.05 M, pH 9.3 with 100 mM SDS. Hydroxyproline was detected at 214 nm. The assay was reproducible (RSD < 3% for normalized peak areas and migration times) and had good correlation (r = 0.975, p < 0.001) with the AOAC official method. From the samples tested (36), commercial ham showed the lowest average collagen content (<7.16 g per 100 g protein) by the CE method. On the other hand, frankfurter and Mexican “chorizo” sausages showed average collagen contents of 12.88 and 11.17 g per 100 g protein, respectively. The CE method developed could be used by regulatory agencies to ensure compliance with maximum levels of collagen addition in processed meats.     

Detection of DNA in soybean oil

 The presence of DNA in foodstuffs which are, or contain, genetically modified organisms (GMO) is the basic requirement for labelling of GMO food in Switzerland and is also being discussed as a requirement for labelling in the European Union. The present work presents data indicating that no genetic material can be recovered after the first processing steps of soybean oil, i.e. when crude soybean oil is simply centrifuged. This fact is of some relevance because centrifugation is one of the first steps in industrial oil processing. A nested PCR system utilising a single copy gene (lectin 1) showed that centrifugation purified the oil from the genetic material by at least a factor of 10 000. The same results were observed when industrially processed oil fractions were analysed. Thus, with respect to the presence of DNA, soybean oil from GMO soybeans is identical to traditional oil and does not need to be labelled as a GMO product in Switzerland. Received: 21 January 1998 / Revised version: 30 March 1998     

Detection of DNA in soybean oil

 The presence of DNA in foodstuffs which are, or contain, genetically modified organisms (GMO) is the basic requirement for labelling of GMO food in Switzerland and is also being discussed as a requirement for labelling in the European Union. The present work presents data indicating that no genetic material can be recovered after the first processing steps of soybean oil, i.e. when crude soybean oil is simply centrifuged. This fact is of some relevance because centrifugation is one of the first steps in industrial oil processing. A nested PCR system utilising a single copy gene (lectin 1) showed that centrifugation purified the oil from the genetic material by at least a factor of 10 000. The same results were observed when industrially processed oil fractions were analysed. Thus, with respect to the presence of DNA, soybean oil from GMO soybeans is identical to traditional oil and does not need to be labelled as a GMO product in Switzerland. Received: 21 January 1998 / Revised version: 30 March 1998