DNA typing methods for differentiation of Debaryomyces hansenii strains and other yeasts related to surface ripened cheeses

The discriminative power of ITS-PCR, ITS-PCR RFLP and mitochondrial (mt)-DNA RFLP were evaluated for differentiation of yeasts of importance for surface ripened cheeses. In total 60 isolates were included. Of these, 40 strains of the following species, Debaryomyces hansenii var. hansenii, D. hansenii var. fabryi, Saccharomyces cerevisiae, Candida zeylanoides, Kluyveromyces lactis and Yarrowia lipolytica, were obtained from culture collections and 20 isolates of D. hansenii representing six different phenotypes were collected from seven Danish producers of surface ripened cheeses. ITS-PCR was evaluated for differentiation at species level on the 40 strains obtained from culture collections. Ten strains of each variety of D. hansenii and five strains of each of the above mentioned species were analysed. For each of the investigated species, a specific ITS1-5.8S rDNA-ITS2 region size was observed. Accordingly ITS-PCR was found valuable for differentiation at species level of yeasts of importance for surface ripened cheeses. ITS-PCR RFLP was investigated for the purpose of strain typing of D. hansenii. Ten CBS strains of each variety of D. hansenii were analysed. Only one enzyme (TaqI) out of several investigated (BamHI, DpnI, Fnu4HI, HaeIII, HindIII, HpaII, NlaII, Sau3AI, TaqI) demonstrated genetic diversity within the strains. This enzyme divided the 20 strains in three groups. Sequence analysis of the ITS1-5.8S rDNA-ITS2 region for the type strains of each variety of D. hansenii showed an identity of 99.84%, corresponding to a difference in one basepair. Based on these results, ITS-PCR RFLP was found ineffective for strain typing of D. hansenii. MtDNA RFLP using HaeIII and HpaII was evaluated for strain typing of D. hansenii on the 20 CBS strains of D. hansenii. The CBS strains were divided into 16 groups according to their restriction profiles, which proved the method useful for typing of D. hansenii at subspecies level. The 20 dairy isolates showed a lower genetic variability than the CBS strains as they were divided into eight groups. Cluster analysis of the 20 CBS strains and the 20 dairy isolates based on their mtDNA restriction profiles showed (max. similarity level = 52%) that the dairy isolates only clustered with the CBS strains of D. hansenii var. hansenii. For some of the dairies more than one strain of D. hansenii were found to be involved in the ripening process, indicating that the method could be useful for subspecies typing and investigation of the microbial succession between strains of D. hansenii during the ripening process of surface ripened cheeses.     

Technologies to shorten the drying period of dry-cured meat products

Dry-cured meat products are well-known for their unique sensory characteristics. However, the traditional process is very time consuming. The process can be shortened especially by accelerating the drying period, which is the most time consuming. This paper deals with some technological, safety and sensorial aspects for producing fermented sausages and dry-cured hams when the process time is shortened. Different techniques, such as temperature increase and thickness reduction, and the effects of some ingredients and additives are discussed. A Quick-Dry-Slice process based on a continuous system that combines both convective and vacuum drying could accelerate the drying of slices after the desired pH is reached in fermented sausages. There are safety concerns when processes are shortened, but possible additional hurdles, such as the introduction of bacteriocin-producing starter cultures and high-pressure treatments at the end of the process, could reduce them. Methods to speed up the development of typical colour, texture and flavour and their limitations are also discussed.     

Species differentiation of heated meat products by DNA hybridization

A method for quantitation of pork in heat-treated meat products is described. The procedure involves isolation of DNA from meat samples followed by a determination of the average size of DNA fragments by agarose gel electrophoresis. The DNA is then immobilized on nylon membranes and hybridized with a (32)P-labelled probe made from genomic porcine DNA. The signal intensity from filter-bound DNA probe is determined by laser densitometry of the autoradiographs. Functional relationships between the signal intensity and the fragment size of the DNA as well as signal intensity and the amount of pork DNA have been established. Pork DNA can therefore be quantified using DNA standards with the same fragment size as the actual sample DNA. Samples of known composition and heat treatment have been investigated. The detection limit has been determined to approximately 0·1% pork in beef whereas for heat-treated samples the detection limit has been determined to approximately 0·5% pork in beef. Examples are given in which the present method has been applied on commercial canned and cold-stored foods. The results of the investigation indicate that the DNA-hybridization technique applying (32)P-labelled probes can be used for quantitative determinations in quality control of heat-treated meat products.     

温和条件下的美拉德反应对干腌肉品风味的贡献

为了研究干腌肉制品风味形成机理,建立了核糖与氨基酸(Leu,Ile,Val,Met)在温和(微酸性pH及非加热)条件下的美拉德反应模型.经过SPME-GC/MS检测,表明产生的主要挥发性产物分别是3-甲基丁醛、2-甲基丁醛、2-丁酮、2-甲基-2-丁烯醛、2-甲基丙醛、二甲基二硫化物、3-甲基硫代丙醛,以上挥发性化合物已在一些不同的干腌火腿中检测到.     

Selection of antifungal protein-producing molds from dry-cured meat products

To control unwanted molds in dry-cured meats it is necessary to allow the fungal development essential for the desired characteristics of the final product. Molds producing antifungal proteins could be useful to prevent hazards due to the growth of mycotoxigenic molds. The objective has been to select Penicillium spp. that produce antifungal proteins against toxigenic molds. To obtain strains adapted to these products, molds were isolated from dry-cured ham. A first screening with 281 isolates by the radial inhibition assay revealed that 166 were active against some of the toxigenic P. echinulatum, P. commune, and Aspergillus niger used as reference molds. The activity of different extracts from cultured medium was evaluated by a microspectroscopic assay. Molds producing active chloroform extracts were eliminated from further consideration. A total of 16 Penicillium isolates were screened for antifungal activity from both cell-free media and the aqueous residues obtained after chloroform extraction. The cell-free media of 10 isolates that produced a strong inhibition of the three reference molds were fractionated by FPLC on a cationic column. For protein purification, the fractions of the three molds that showed high inhibitory activity were further chromatographed on a gel filtration column, and the subfractions containing the highest absorbance peaks were assayed against the most sensitive reference molds. One subfraction each from strains AS51D and RP42C from Penicillium chrysogenum confirmed the inhibitory activity against the reference molds. SDS-PAGE revealed a single band from each subfraction, with estimated molecular masses of 37 kDa for AS51D and 9 kDa for RP42C. Although further characterisation is required, both these proteins and the producing strains can be of interest to control unwanted molds on foods.     

Advances in the ultrasound characterization of dry-cured meat products

In this work, the feasibility of using non-contact ultrasonic techniques (air-coupled and scanning acoustic microscopy, SAM) for characterizing different dry-cured meat products was assessed. Air-coupled ultrasonic measurements were performed on vacuum packaged sliced dry-cured ham, and compared with contact measurements. The average ultrasonic velocity in dry-cured ham was 1846 ± 49 m/s and 1842 ± 42 m/s for air-coupled and contact measurements, respectively. The deviation (1% relative error) between both techniques was related to the influence of the heterogeneous structure and composition of dry-cured ham and the transducer focusing. The SAM was used to characterize dry-cured ham and chorizo samples. B-scan images for dry-cured ham and chorizo showed two dominant reflections from the sample, linked to reflections in the lean and fatty tissues. The results indicate that contact ultrasonic measurements could be replaced by the air-coupled technique, reducing the measuring time and the material handling. On the other hand, SAM technique allows the microscopic characterization of dry-cured meat products.     

The mycobiota of three dry-cured meat products from Slovenia

The surface mycobiota of three types of Slovenian dry-cured meat products were isolated from a total of 75 items of product that were sampled periodically during the drying/ripening stage of processing. The predominant filamentous fungal genus isolated was Penicillium. Eurotium spp., Aspergillus versicolor and Cladosporium spp. were isolated from only two of the products. Eight Penicillium species were identified. Penicillium nordicum was recovered frequently. Penicillium nalgiovense was recovered less frequently, from one product only (a salami), while a yet-to-be described species Penicillium “milanense” was isolated from 21 items. The other penicillia were rarely isolated. Of the isolated and identified species, those that can produce mycotoxins are: A. versicolor, Penicillium brevicompactum, Penicillium chrysogenum, P. nordicum, and Penicillium polonicum. Their growth on dry-cured meat products is undesirable.     

Reduction of ochratoxin A in dry-cured meat products using gamma-irradiation

This study investigated the efficiency of gamma (γ)-irradiation in the reduction of ochratoxin A (OTA) present in dry-cured meat products prepared from intentionally contaminated raw materials from OTA-treated pigs. OTA concentrations determined in the samples (n = 24) ranged from 25.8 μg kg^–1 in bacon to 17.8 μg kg^–1 in smoked ham. After γ-irradiation at doses of 3, 7 and 10 kGy (i.e. the doses used in the food industry), a dose-depended OTA reduction was observed; however, it was not statistically significant. The mean OTA reduction achieved with 3-, 7- and 10-kGy γ-doses was approximated to 8.5%, 13.9% and 22.5%, respectively. The storage of irradiated samples (1 month, 4°C) did not significantly affect OTA levels. Based on the correlation between the OTA reduction level and basic chemical composition of dry-cured meat samples, OTA reduction may be linked to the samples’ fat content. The results indicate that γ-irradiation can reduce OTA levels in dry-cured meat products, but only to a limited extent due to the complexity of the matrix.     

核糖作为干腌肉制品中具有反应活性的风味前体物的几点证据

肉中ATP代谢形成的产物中,只有核糖与4种氨基酸(Leu、Ile、Val、Met)在温和条件下反应才检测到挥发性风味化合物生成,说明核糖是肉中具有高反应活性的风味前体物质.通过对鲜肉、干腌肉块和干腌火腿核糖含量的测定,表明在干腌肉品生产过程中核糖含量呈下降的趋势,提示核糖含量变化的发生可能与干腌肉品中风味物质的产生存在一定的相关性.     

Ochratoxin A in traditional dry-cured meat products produced from sub-chronic-exposed pigs

The presence of ochratoxin A (OTA) was determined in traditional dry-cured meat products made from sub-chronically OTA-exposed pigs. The experimental group of pigs (n = 5) was treated with 300 µg OTA kg^–1 of feed during 30 days, whereas the control group (n = 5) remained untreated. After the household production of six types of dry-cured meat products based on traditional recipes, OTA residues were determined in final products produced from each treated and untreated animal using an immunoenzymatic technique (ELISA) and HPLC with fluorescence detection (HPLC-FD). The analytical methods showed acceptable analytical performance results and high correlation coefficients. Mean OTA concentrations ranged from 4.51 ± 0.11 µg kg^–1 in smoked ham to 6.87 ± 2.01 µg kg^–1 in home-made Slavonian sausage. The study demonstrated that pig exposure to OTA leads to the accumulation of OTA residues in muscle and adipose tissue used for the production, and consequently results in contamination of the final meat products.     

肉品冷冻工艺及冻结方法

畜禽肉的冷冻在现代肉及肉制品加工工业中起着很重要的作用,常见的肉品冷冻的工艺有两阶段冻结工艺和直接冻结工艺法两种,其又有各自的优缺点。肉品的冻结方法有很多,其中包括许多常用冻结方法和新兴冻结方法。本文对肉品冷冻的工艺及冻结方法及其各自特点进行了总结。     

The role of muscle enzymes in dry-cured meat products with different drying conditions

Several muscle proteases (cathepsins, calpains, peptidases and aminopeptidases) and lipases (lysosomal acid lipase, acid phospholipase and adipose tissue lipase) are involved in important biochemical mechanisms taking place during the processing of dry-cured meat products which are directly related to the final quality. These enzymes are affected by the conditions typically found in the processing of dry-cured meat products, being dehydration one of the most important factors. This work is presenting the effect of different drying conditions, typical in the processing of dry-cured meat products, on the activity of muscle proteases and lipases as well as its relevance for the final product quality.     

Comparison of different methods for total lipid quantification in meat and meat products

This study was aimed to evaluate the efficiency of six extraction methods for the quantification of total lipid content in meat and meat products: standard Soxhlet method (with and without previous acid hydrolysis), continuous Soxhlet method (with and without previous acid hydrolysis), and those methods based in the use of a mixture of chloroform and methanol, and described by Folch, Less, and Sloane (1957) and Bligh and Dyer (1959). Lipid content was determined in nine different meat products with different fat contents and physico-chemical features: cooked turkey breast, fresh pork loin, cooked ham, dry-cured ham, mortadella, beef burger, fresh sausage, dry-cured sausage and salami. The most effective methods for determining fat content in the studied meat products were the method described by Folch et al. (1957) and the Soxhlet with previous acid hydrolysis method. The Soxhlet method without previous acid hydrolysis adequately extracted lipids only in those meat products with very high fat content. The use of the method described by Bligh and Dyer (1959) gave rise to the lowest lipid contents in all the studied meat products.     

Occurrence and growth of yeasts in processed meat products - Implications for potential spoilage

Spoilage of meat products is in general attributed to bacteria but new processing and storage techniques inhibiting growth of bacteria may provide opportunities for yeasts to dominate the microflora and cause spoilage of the product. With the aim of obtaining a deeper understanding of the potential role of yeast in spoilage of five different processed meat products (bacon, ham, salami and two different liver patés), yeasts were isolated, enumerated and identified during processing, in the final product and in the final product at the end of shelf life. Yeasts were isolated along the bacon production line in numbers up to 4.2 log (CFU/g). Smoking of the bacon reduced the yeast counts to lower than 1.0 log (CFU/g) or non-detectable levels. In general, yeasts were only isolated in low numbers during the production of salami, cooked ham and liver paté. In the final products yeasts were detected in low numbers in a few samples (3 out of 30) samples, 1.0-1.3 log (CFU/g). By the end of storage, yeasts were only detected in 1 out of 25 investigated samples 1.8 log (CFU/g). A combination of phenotypic and genotypic methods was used to identify the yeast microflora present during production of the processed meat products. The yeast microflora was complex with 4-12 different species isolated from the different production sites. In general, Candida zeylanoides, Debaryomyces hansenii and the newly described Candida alimentaria were found to be the dominant yeast species. In addition, three putatively previously undescribed yeast species were isolated. Fourteen isolates, representing seven different species isolated during the production of the processed meat products and one species isolated from spoiled, modified atmosphere packed, sliced ham, were screened for their ability to grow in a meat model substrate under a low oxygen/high carbon-dioxide atmosphere (0.5% O(2), 20% CO(2), 79.5% N(2)) at two different temperatures (5 and 8°C). Eleven out of the tested 14 strains were able to grow in the meat model substrate with C. zeylanoides, D. hansenii, Pichia guilliermondii and Candida sake reaching levels of 10(5)-5×10(6) log (CFU/g), where sensoryical changes appear.     

DNA条形码技术在肉及其制品中的应用

DNA条形码技术作为一种新的分子生物学检测技术在鉴定和区分各物种和物种间亲缘关系方面得到了广泛应用,该技术能实现对肉的快速、准确检测。DNA条形码已成为生物学领域发展最迅速的一种技术,该技术基于广泛的物种基因数据库信息,在肉品研究中具有良好的应用前景。本文简述DNA条形码技术的基本原理,并基于DNA水平上与其他相关技术进行比较,综述其在物种鉴定、肉品质量安全、商业欺诈等方面的应用,并对该技术在今后肉品科学研究中的应用进行展望。      

生产乳化型肉制品应该注意的问题

主要从原材料的选择、预处理、配方设计、乳化过程、熟制等方面,分析生产乳化型肉制品应该注意的问题。     

Characterization of molds from dry-cured meat products and their metabolites by micellar electrokinetic capillary electrophoresis and random amplified polymorphic DNA PCR

Molds are common contaminants of dry-cured meat products in which mycotoxins could be synthesized if stored under favorable conditions. Thus, efficient and accurate characterization of the toxigenic molds from dry-cured meat products is necessary. A micellar electrokinetic capillary chromatography (MECC) method was tested to analyze secondary metabolites produced by 20 mold strains commonly found in dry-cured meat products. In addition, their random amplified polymorphic DNA (RAPD) genotypes were determined by using a PCR method. Although peak profiles of the secondary metabolites differed among mold strains of different species, they were similar in the same species. MECC analysis showed that 10 of the 20 molds tested produced mycotoxins, including patulin, penicillic acid, cyclopiazonic acid, mycophenolic acid, aflatoxin B1, sterigmatocystin, and griseofulvin. The RAPD analysis yielded a different pattern for each of the mold species tested. However, strains of the same species showed similar RAPD profiles. A high correlation between RAPD analysis and MECC was observed, since strains of the same species that showed similar RAPD patterns had similar profiles of secondary metabolites. RAPD patterns with primer GO2 and MECC profiles, either singly or combined, could be of great interest to distinguish toxigenic from nontoxigenic molds in dry-cured meat products.     

Investigation of methods to detect mechanically recovered meat in meat products - IV: Immunology

The possibility of using immunological techniques as a method for the detection of mechanically recovered chicken meat in meat products has been investigated in this preliminary study. Antibodies were raised against a low molecular weight fraction (≤ 30 kDa) of chicken bone marrow proteins and an enzyme-linked immunosorbent assay (ELISA) developed. The system was used to test for the presence of mechanically recovered meat (MRM) in a range of product types, from raw chicken meat through to heat processed samples. The results show that it is possible to raise antibodies to chicken bone marrow proteins which show a strong reactivity with chicken and turkey MRM but show little reaction with extracts of MRM and hand deboned meat of other common meat species. However, blood, skin and soya all affected the accuracy of the ELISA.

This study has demonstrated the potential for the use of an immunological procedure as a rapid test for MRM. The selectivity of the antiserum would, however, have to be increased before this procedure could be considered as a suitable technique for the detection of MRM in meat products.     

Investigation of methods to detect mechanically recovered meat in meat products - III: Microscopy

Carcasses from 36 Large White gilts, 70–80 kg live weight, were randomly allocated to three experimental groups. Pigs in the first group were electrically stimulated with low voltage during bleeding (85v, 14Hz for 64 s) and split before cooling. The left sides were rapidly chilled in air at -15°C for 75 min and then at 1°C until 24 h post-slaughter; right sides were chilled conventionally in air at 1°C for 24 h. In the second group, two different treatments were used 20 min post-slaughter: left sides were stimulated with low voltage, and right sides with high voltage (700 v, 12·5 Hz for 90 s). Both sets of sides were chilled rapidly. Carcasses from the third group were not stimulated, and sides chilled either rapidly or conventionally. Drip loss, colour and texture were measured in M. longissimus thoracis et lumborum at 3 and 10 days post-slaughter.

At 3 days post-slaughter the high voltage, treatment gave meat which was the most tender, was not pale and lost no more drip than unstimulated controls. Low voltage stimulation during bleeding gave meat which was 18% more tender than the unstimulated controls, but the improvement in tenderness was not as great as the 28% achieved with high voltage. Unexpectedly, low voltage stimulation applied 20 min after slaughter, was almost as effective in improving tenderness (by 17%) as low voltage applied during bleeding.

Tenderness improved from 3 days to 10 days in all stimulated samples, but not in unstimulated controls. The results suggest a degree of coldtoughening in the latter, even with conventional chilling, and a positive effect of electrical stimulation on tenderness, independent of its protective action against cold-shortening.