A method for assessing the bacterial retention ability of hydrophobic membrane filters

This paper is the 19th in a series of articles on the hygienic design of food processing equipment. It is based on a report prepared by the Test Methods Subgroup of the European Hygienic Equipment Design Group (EHEDG). A method for validating the bacterial retention ability of sterilizing grade hydrophobic membrane filters is described. The procedure was developed at The TNO Nutrition and Food Research Institute, Zeist, The Netherlands. The bacterial aerosol challenge test was found to be sufficiently sensitive to determine filter efficiency up to 99.9995% for the types of micro-organisms used.¹     

The influence of hydrophobic substances on water vapor permeability of fish gelatin films modified with transglutaminase or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)

The effect of different hydrophobic substances on water vapor permeability (WVP) of unmodified fish gelatin films and cross-linked with transglutaminase (TGase) or with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was determined. Both unmodified and cross-linked films were characterized by very poor water barrier properties. Amaranth oil, rapeseed oil, lanolin, beeswax and ozococerite at concentration of 10% decreased WVP of unmodified gelatin films by 42, 15, 37, 53 and 36%, respectively. Increasing concentration of these substances up to 60% caused further improving of the water barrier properties. Addition of lecithin into film-forming emulsions prevented separation of lipid layer on the film surface. Among unmodified films with lecithin and 60% of lipids the highest decrease of WVP was found in case of amaranth oil and beeswax, by 73 and 87%, respectively, in comparison to only-gelatin films. WVP of chemically modified films in the presence of 60% of beeswax with addition of lecithin was decreased by about 65%. Enzymatically modified films with beeswax were very brittle and broke during analyzes, similarly as those with amaranth oil and lecithin. WVP of enzymatically modified films with lecithin and 60% of rapeseed oil and lanolin was respectively, about 60 and 47% lower than that of films without hydrophobic substances.     

A multiplex-PCR assay for the authentication of mackerels of the genus Scomber in processed fish products

In this study, we have developed a novel multiplex-PCR assay for the authentication of mackerels of the genus Scomber in processed food. The method consists of two novel Scomber japonicus- (104 bp) and Scomber australasicus-specific (143 bp) amplicons, respectively, corresponding to the mitochondrial control region. It also includes the previously described Scomber colias-specific product (159 bp) corresponding to the 5S ribosomal DNA, the Scomber scombrus-specific fragment (123 bp) from the mitochondrial NADH dehydrogenase subunit 5, and finally a positive amplification control corresponding to the small 12S rRNA subunit (188 bp). The system was assayed in fresh samples as well as in a total of 40 commercial samples including 28 different canned products and 12 unprocessed fresh fillets. A positive identification was observed in all cases according to their commercial labelling. Overall, this methodology reveals as a potential molecular tool for direct application in the authentication of Scomber mackerels in the seafood industry.     

Dynamic passive dosing for studying the biotransformation of hydrophobic organic chemicals: microbial degradation as an example

Biotransformation plays a key role in hydrophobic organic compound (HOC) fate, and understanding kinetics as a function of (bio)availability is critical for elucidating persistence, accumulation, and toxicity. Biotransformation mainly occurs in an aqueous environment, posing technical challenges for producing kinetic data because of low HOC solubilities and sorptive losses. To overcome these, a new experimental approach based on passive dosing is presented. This avoids using cosolvent for introducing the HOC substrate, buffers substrate depletion so biotransformation is measured within a narrow and defined dissolved concentration range, and enables high compound turnover even at low concentrations to simplify end point measurement. As a case study, the biodegradation kinetics of two model HOCs by the bacterium Sphingomonas paucimobilis EPA505 were measured at defined dissolved concentrations ranging over 4 orders of magnitude, from 0.017 to 658 μg L(-1) for phenanthrene and from 0.006 to 90.0 μg L(-1) for fluoranthene. Both compounds had similar mineralization fluxes, and these increased by 2 orders of magnitude with increasing dissolved concentrations. First-order mineralization rate constants were also similar for both PAHs, but decreased by around 2 orders of magnitude with increasing dissolved concentrations. Dynamic passive dosing is a useful tool for measuring biotransformation kinetics at realistically low and defined dissolved HOC concentrations.     

Protein denaturation in frozen fish. 13. A modified cell fragility method insensitive to the pH of the fish

A modified cell fragility method is described for estimating the deterioration which occurs during the storage of frozen fish. A sample of only 100 mg is needed and this is homogenised in a medium consisting of 2% trichloroacetic acid in 1.2% formaldehyde. The results obtained by the new modification are almost unaffected by the pH of the fish, so can give a more truthful estimate of the time-temperature history than those of the original method. Experimental points on optical density/time curves obtained by the new modification show less scatter than those by the original method. Values decrease during storage rather more rapidly than by the original method, to an asymptote which is lower. Modified cell fragility values do not correlate with the length of the fish or its water content. It appears that the escape of myofilaments from the fibrils of high-pH cod accounted for the drop in optical density (scattering) seen in the original method, while in the modified method such proteinaceous material is precipitated and contributes to the optical density of the homogenate. Good curves of optical density/storage time were obtained with other non-fatty fish species, but fatty fish were not satisfactory. Advanced bacterial spoilage of the fish before freezing reduces the optical density in both methods.     

A highly sensitive method to determine the washing resistance of artificial hair colours

Citation: IFSCC Magazine, 11 (2008) (2) 139–142 Abstract: Due to the improved performance of modern hair colorants, the high natural variability in hair qualities and the individual influences of manual product applications, colour care effects of cosmetic treatments are very difficult to detect. A new, highly sensitive test method to study the colour fading behaviour of human hair was established for a large variety of cosmetic treatments. This method is based on an automatic multistage application of standard wool tissues combined with automatic colour evaluation by means of CIE L × a × b × measurements (DIN 5033). The delta E values are the main interest because they include all information regarding the L (black vs. white), a (red‐green) and b (blue‐yellow) axes defined in the Hunter Lab colour space. For validation of this new method, different permanent and non‐permanent hair colour shades were applied to undamaged light brown Caucasian hair strands. The hair strands were washed manually stepwise 30 times and the colour loss compared with the results obtained on wool tissues using an automatic application system. For most of the investigated hair colours, a coefficient of determination of r2 > 0.99 was achieved. Modern permanent hair colours show a high resistance to cosmetic treatments. Over 90% of the initial colour result was retained after 30 product applications. Shampoo and conditioner formulations induce different degrees of colour loss in hair. In basic formulas significant influences of single surfactants could be detected. The new method using wool tissues correlates very well with that using manually washed Caucasian hair strands. This automatic method is very time‐effective and offers an excellent reproducibility with a high sensitivity for assessing product influences on artificial hair colours. Keywords: Color fading, color protection, color retention, hair color resistance, hair wash simulation Paper presented at the IFSCC Conference 2007, Amsterdam, The Netherlands     

Direct evidence of sequestration in sediments affecting the bioavailability of hydrophobic organic chemicals to benthic deposit-feeders

In contrast to equilibrium partitioning model (EqP) calculations, biota to sediment accumulation factors (BSAF) of hydrophobic organic compounds for deposit-feeders are highly variable. Recent literature suggests that this variability can be attributed to differences in sequestration or the presence of slowly desorbing fractions in the sediment. In the present study, we investigated whether the observed relationship between bioavailability and sequestration is causal. We determined BSAF values and sequestration status, measured as the distribution over rapidly and slowly desorbing fractions, of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in a manipulated sediment as well as in the original, unmanipulated sediment The manipulation, 48 h suspending with Tenax, resulted in reduction of the rapidly desorbing fraction, while other factors such as contact time and sediment properties remained constant. Contrary to expectations based on EqP, BSAF values did not remain constant but were reduced by a factor of 2-27, proportional to the reduction in rapidly desorbing fractions. The results provide direct evidence of a causal relationship between sequestration and bioavailability to deposit-feeders. Furthermore, the present study demonstrates the need to modify traditional use of the equilibrium partitioning model to account for variation in the sequestration status of HOC in sediments.     

Use of B-mode, linear array ultrasonography for evaluating the technique of bovine artificial insemination

B-Mode, linear array ultrasonography was used to evaluate the site of semen deposition in live cows. The insemination sheath was modified to hold a brass bead that was deposited at the simulated site of semen deposition. The position of the bead was identified by ultrasonography and the bead was recovered by pulling a nylon line that was attached to the bead. An ultrasound technician identified placement of the bead in the site designated for semen deposition by a skilled inseminator in 24 of 25 cases. This procedure improves methods for inseminator training by providing a method of evaluating insemination technique using live animals rather than excised reproductive tracts. The ultrasound equipment used for inseminator evaluation is affordable, portable, and does not pose a human health risk.     

Development of an ultrasensitive PCR assay for polycyclic musk determination in fish

Polycyclic musks (PCMs) in the aquatic environment and organisms have become an emerging environmental issue because of their potential risk. The most used method for polycyclic musk determination is gas chromatography-mass spectrometry (GC-MS) with different sample extractions, which are somewhat expensive to operate, complex and laborious. In this study, a novel and ultrasensitive real-time polymerase chain reaction (PCR) assay with multiple signal amplification of carboxylic-DNA by gold nanoparticle-polyamidoamine conjugation (Au-PAMAM) was developed for determining polycyclic musks in fish. Hapten and immunogen were specially prepared. Polyclonal antibodies were produced based on the optimal immunisation, and the antibodies were characterised. Due to PAMAM’s unique nanostructure of numerous functional amino groups, polyclonal antibody and carboxylic-DNA were immobilised by Au-PAMAM conjugation to develop the antibody-Au-PAMAM-DNA probes, which were used as a signal DNA amplifier in the PCR system. Compared with real-time immuno-PCR, this biological probe-amplified immuno-PCR (BPAI-PCR) assay had higher sensitivity due to the probes’ higher ratio of signal DNA. Finally, the BPAI-PCR assay was applied to analyse AHTN (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene,Tonalide) concentrations in fish samples in the range from 1 pg/L to 10 ng/L, giving an of LOD 0.61 pg/L. In general, due to the specificity of the antibody and novel nanoprobe design, this BPAI-PCR assay provided a potential way for trace analysis of AHTN in the aquatic organisms. The high concentrations of AHTN found in cultivated fish should encourage further toxicological studies.     

A detection method of CryIAc protein for identifying genetically modified rice using the lateral flow strip assay

We examined the lateral flow strip assay for identifying unauthorized genetically modified (GM) rice. The GM rice expresses the Bacillus thuringiensis (Bt) toxin, CryIAc protein, which confers tolerance to insects. The recombinant CryIAc protein was prepared from the inclusion bodies of an E. coli. strain into which the CryIAc gene had been inserted, using gel filtration chromatography. The lateral flow strip assay for the identification of GM cotton which also expresses CryIAc protein, was applied to unpolished rice and polished rice spiked with recombinant CryIAc protein. The spiked recombinant CryIAc protein was clearly detected at the level of 0.012 microg/g in both the unpolished and polished rice. After loading of the extract on the strip, a 60 -minute stand time is necessary to clearly detect CryIAc protein. The detection limit was approximately 12 ng CryIAc protein per gram of rice. These results suggest that the lateral flow strip assay for GM cotton can be used to detect CryIAc protein expressed in GM rice.     

Absence of Trichinella infection in adult pigs slaughtered in Palmas, State of Parana (Brazil), detected by modified artificial digestion assay

Samples of 2,490 adult pigs, slaughtered under federal inspection between May 2004 and February 2005 in the county of Palmas, State of Paraná, Brazil, were examined by pooled sample artificial digestion with magnetic stirrer assay for a survey of Trichinella spp. larvae. Animals originated from 53 counties in three states of southern Brazil. Test sensitivity was increased with modifications of the European standard for artificial digestion. In this survey, a 5-g sample of tongue and 5-g sample of diaphragm pillar were collected from each pig into a pool of 100 g (up to 10 animals for each assay). A 355-microm mesh sieve was used, but no larvae were detected in the pigs, indicating that trichinellosis does not occur in the examined stocks.     

A rapid and improved method for the detection of Vibrio parahaemolyticus and Vibrio vulnificus strains grown on hydrophobic grid membrane filters

DNA probe-based detection methods were developed and characterized as an alternative to time-consuming and less specific conventional protocols. Digoxigenin-labeled probes were prepared by polymerase chain reaction amplification of the targeted sequences in the specific amplicons generated from genomic DNA. Specific probes with high yields were generated for the detection of the tlh gene of Vibrio parahaemolyticus and the cth gene of V. vulnificus. Colony (Southern) hybridization analyses were carried out using hydrophobic grid membrane filters (HGMFs) to allow biotype-specific differentiation of the two species. Eight strains of V. vulnificus and five strains of V. parahaemolyticus, including one standard (ATCC) strain of each biotype, were examined. Colony lysis, hybridization, and nonradioactive detection parameters were optimized for identification of the target biotypes arranged on the same HGMF and also on a conventional nylon membrane, thereby confirming the specificity of the probes and the comparative usefulness of the HGMFs. The experimental procedure presented here can be completed in 1 day. The protocol was designed specifically to identify the target Vibrio spp. and could potentially be used for the enumeration and differentiation of V. parahaemolyticus and V. vulnificus in foods.     

Comparison of a modified digestion assay with trichinoscopy for the detection of Trichinella larvae in pork

A pepsin-HCl digestion assay and two compressorium techniques (trichinoscopy) for the identification of swine muscle tissue containing low levels of Trichinella larvae were compared as part of the test validation process for quality assurance purposes. Compressoria read with a stereomicroscope detected more larvae (P < 0.0001, n = 57) and more tissues (P = 0.0047, n = 57) than did compressoria read with a projection microscope (trichinoscope). The digestion assay evaluated was 3.2 times as likely as the best compressorium technique to identify a positive tissue when these procedures were used to test 1 g of infected muscle (P < 0.001; 95% confidence interval for the odds ratio, 2.0 to 5.4; n = 161 and n = 189, respectively). Detection by trichinoscopy improved as the number of larvae in tissues increased to > 2 larvae per g, but trichinoscopy was less sensitive than the digestion assay regardless of the tissue larval load. These data indicate that the quality controlled digestion assay used in this study is more sensitive than trichinoscopic techniques in the detection of tissues containing low levels of Trichinella larvae.     

高效改性AKD乳液在瓦楞原纸生产中的应用

介绍了高效改性AKD乳液用于抄遣高强瓦楞原纸的生产实践，并对其结果进行了讨论。结果表明：采用高效改性AKD乳液进行的中性施胶，施胶成本低于普通AKD乳液，成纸的各项指标均能达到A级瓦楞原纸的标准，而熟化时间能明显缩短。