Frequency of DLK1 c.639C>T polymorphism and the analysis of MEG3/DLK1/PEG11 cluster expression in muscle of swine raised in Poland

DLK1 — (Drosophila like element 1) is a paternally expressed gene, associated with the callipyge phenotype in sheep. In a present study we designed a new real-time PCR alleleic discrimination assay for genotyping of a silent C/T mutation (c.639C>T) in DLK1 gene in swine. The DLK1 c.639C>T mutation was highly polymorphic in all breeds analyzed and C allele was predominant in Landrace and Duroc while T allele was more frequent in Pietrain and Pu?awska breed. Moreover, we analyzed mRNA expression of DLK1 and adjacent genes — MEG3 and PEG11 in muscles of swines of different breeds raised in Poland. We did not observe significantly different expression of DLK1, MEG3 or PEG11 mRNA in any of analyzed breeds. We also attempted to assess the effect of DLK1 (c.639C>T) on the expression of genes in callipyge locus but did not find significant differences between animals with alternate genotypes (C/C and T/T homozygotes).     

Comparison of the most odour-active volatiles in different hop varieties by application of a comparative aroma extract dilution analysis

Application of a comparative aroma extract dilution analysis on the volatiles isolated from five different hops (Hallertau Perle, Hallertau Hersbrucker Spät, Slowenian Golding, Hallertau Smaragd, US Cascade) revealed linalool and myrcene with the highest Flavour Dilution (FD)-factors in all varieties, followed by 2-isopropyl-3-methoxypyrazine, 3-methylbutanoic acid and geraniol. Some odourants, however, showed high FD-factors only in certain varieties, for example, (5Z)-octa-1,5-dien-3-one and germacrene B in Hersbrucker Spät, (3E,5Z)-undeca-1,3,5-triene in Hersbrucker Spät and Cascade and nonanal in Cascade. The overall odour profile of the Cascade sample clearly differed from the other varietes, and was dominated by a black currant like odour note. The identification experiments revealed 4-methyl-4-sulfanylpentan-2-one, so far unknown as hop constituent, as key contributor to this odour. In addition, an odour-active undecatetraene was present, in particular, in Perle and Cascade. Synthesis and structural assignment of the four stereoisomers of (3E)-undeca-1,3,5,9-tetraene allowed the identification of the fresh, pineapple-like smelling compound as (3E,5Z,9E)-undeca-1,3,5,9-tetraene. Among the four isomers synthesised, this compound showed by far the lowest odour threshold of 0.01 ng/L in air.     

Scanner-based color measurement in L * a * b * format with artificial neural networks (ANN)

A computerized inspection system (CIS) that uses a flat-bed scanner, a computer, and an algorithm and graphical user interface coded and designed in Matlab^® 7.0 was developed to determine food color based on CIE L ^* a ^* b ^*, a color format. The USA Federal Color Standard printouts (SP) comprised of 456 different colors were used to train and test the artificial neural network (ANN) integrated CIS. Strong correlations were found between the results estimated from ANN-integrated CIS and those obtained from spectrophotometer (R ², 0.991, 0.989, and 0.995 for L ^*, a ^*, and b ^*, respectively) for test images data set. Various food samples were also evaluated to test the performance of the CIS. A good agreement, R ², 0.958, 0.938, and 0.962 for L ^*, a ^*, and b ^*, respectively, was found between color measurement with CIS and a spectrophotometer. CIS with a mean error of 0.60% and 2.34% for test and various food samples, respectively, has an ability to imitate the results obtained from a spectrophotometer. CIS allows users to store the captured picture for further use and estimate the overall color or the color of selected region of the samples either heterogeneous in color or amorphous in shape.     

Genetic typification by pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) of wild <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> wine strains

Genetic typification of 120 bacterial isolates of Lactobacillus plantarum and Oenococcus oeni from different Rioja musts and wines was performed by numerical analysis of pulsed-field gel electrophoresis (PFGE) patterns with endonuclease SfiI, and 46 of them were also studied by randomly amplified polymorphic DNA (RAPD)-PCR. A comparative study of both typification methods applied to L. plantarum and O. oeni oenological strains was performed. Bacterial species was determined both by biochemical identification methods and by specific PCR analysis. A wide variety of restriction digest patterns were detected by PFGE among L. plantarum strains (36 unrelated patterns and one closely related pattern, out of 48 isolates), as well as among O. oeni strains (18 unrelated patterns out of 72 isolates). PFGE was shown to be a suitable method for strain differentiation and to determine which strains are present in wine fermentations, with a discriminatory power to type L. plantarum and O. oeni strains higher than that of RAPD-PCR.     

Optimization of ultrasound-assisted extraction of melanin from Auricularia auricula fruit bodies

In this paper, ultrasound-assisted extraction (UAE) conditions of natural melanin from dried fruit bodies of Auricularia auricula (A. auricula) were investigated. By single-factor experiments, ultrasound power was determined at 250 W and the other factors (temperature, liquid–solid ratio and duration) were chosen to further optimize extraction conditions using response surface methodology (RSM). The Box–Behnken experimental results showed the optimum extraction conditions as follows: a temperature of 63 °C, a liquid–solid ratio of 43 mL/g and a duration of 36 min. Under these conditions, two extractions sufficiently reached the maximal melanin yield (120.05 mg/100 g). Light absorbance, solubility and redox properties of the melanin extracted from A. auricula were similar to those of the typical melanin.Industrial relevanceAuricularia auricula (A. auricula) has been used as food and drug in China for a long time. The annual production of A. auricula in China was 1.44 million tons in 2007 (http://www.mushroommarket.net/news/show.asp?id=19769). However, most of this precious macro-fungus product was only used as cuisine materials, and many of its functional components, such as melanin, a healthful natural food colorant, were not fully developed and employed. In this paper, ultrasound-assisted extraction (UAE) was applied to melanin extraction from A. auricula fruit bodies to improve the extraction efficiency. This research helps to develop a new and economical method of extracting natural melanin and to fully use this edible mushroom.     

Quantitative determination of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. in milk using a series piezoelectric quartz crystal sensor

A series piezoelectric quartz crystal (SPQC) sensor was developed for quantitative determination of Lactobacillus spp. populations in milk. When the electrodes were immersed in a reaction cell with bacterial inoculum, the change of frequency was caused by the impedance change of the microbial metabolism. A significant frequency decrease was found due to the coagulation of milk when the Lactobacillus spp. was cultivated in the media. The SPQC sensor system established in this study demonstrated the specificity and selectivity for detection of Lactobacillus spp. in milk sample. The calibration curve of detection time against density of Lactobacillus spp. shows a linear correlation coefficient (R ² = 0.8453) over the range of 10²–2.4 × 10⁵ CFU ml⁻¹. The detection time was influenced by the addition of peptone and glucose. The sensor exhibited rapid (within 4 h) and enabled real time monitoring of Lactobacillus spp. growth. This system is potentially applicable to detect Lactobacillus spp. concentration at local farm when a suitable temperature control device is adapted.     

Production of biogenic amines by<Emphasis Type="Italic"> Morganella morganii</Emphasis>,<Emphasis Type="Italic"> Klebsiella pneumoniae</Emphasis> and<Emphasis Type="Italic"> Hafnia alvei</Emphasis> using a rapid HPLC method

The histidine decarboxylating activity and production of biogenic amines by Morganella morganii (NCIMB, 10466), Klebsiella pneumoniae (NCIMB, 673) and Hafnia alvei (NCIMB, 11999) were investigated using a rapid HPLC method. Derivatisation of the bacterial samples was carried out using benzoyl chloride. A gradient elution system was used for analysis with a mixture of acetonitrile and HPLC grade water. Bacterial strains not only produce histamine in histidine-enriched broth but also the other biogenic amines. The chromatographic results show that bacterial strains are also capable of producing spermine and spermidine in histidine-enriched broth. Bacterial ammonia production by all three strains was clearly detected since ammonia is generated during the degradation of histidine. The study demonstrates that the highest histamine production was obtained by Morganella morganii, followed by Klebsiella pneumoniae, and the lowest with the Hafnia alvei. Therefore, Morganella morganii and Klebsiella pneumoniae have strong histidine decarboxylase activity since they are prolific histamine-forming bacteria     

Synergistic effects of insect-resistant maize and Teretrius nigrescens on the reduction of grain losses caused by Prostephanus truncatus (Horn.)

Prostephanus truncatus is a pest that causes serious losses in stored maize (Zea mays L.) especially in developing countries. This research was conducted to investigate the use of post-harvest insect resistance maize in combination with biological control of P. truncatus by the predator Teretrius nigrescens to reduce maize storage losses. We studied the population dynamics of P. truncatus with and without a predator in combination with susceptible maize and resistant maize to insects under laboratory conditions. This study confirms that P84c3 is a resistant variety against P. truncatus. Maize resistant kernels had a reduction of 30% losses in comparison with susceptible kernels. Significant and favorable interactions were observed between P84c3 maize and presence of T. nigrescens. A dramatic reduction of 80% in progeny number, 81% grain weight loss, and 75% frass production caused by P. truncatus was observed when the predator was used in combination with P84c3. Resistant maize reduced the prey development time and consequently the insect density allowing the predator to control more effectively the population. Prey/predator proportion on resistant maize was significantly higher in comparison with susceptible kernels; thus, giving a more effective pest population control by the predator. These results demonstrated that the combination of post-harvest insect resistance maize with the predator T. nigrescens reduces grain maize losses by P. truncatus.     

Association studies on the porcine RETN, UCP1, UCP3 and ADRB3 genes polymorphism with fatness traits

Searching for effects of candidate gene polymorphisms on fatness traits is an important goal for pig industry. In this study we evaluated polymorphism of four porcine genes involved in energy metabolism (RETN, UCP1, UCP3 and ADRB3). Moreover, their association with fat deposition traits was analyzed in two breeds (Polish Landrace, Polish Large White) and a Polish synthetic line (L990). Altogether, five SNPs were identified, including two novel ones in the 5′-flanking region of the RETN gene and a novel missense substitution in the UCP3. Distribution of these polymorphisms in the studied five breeds and the synthetic line was not uniform. Two of the analyzed SNPs: g.−178G > A in the RETN and g.946C > T in the UCP3 gene revealed a significant association with abdominal fat weight or backfat thickness. Such associations were not observed for the UCP1 or ADRB3 gene polymorphisms. Our study showed that polymorphisms of the UCP3 and RETN genes are potentially associated with porcine fatness traits.     

Genes involved in novel adaptive aluminum resistance in Rhodotorula glutinis

Rhodotorula glutinis IFO1125 acquired increased aluminum (Al) resistance from 50 μM to more than 5 mM by repetitive culturing with stepwise increases in Al concentration at pH 4.0. In our previous study we performed differential display to find that three genes (RgFET3, RgGET3, and RgCMK) encoding proteins homologous to Saccharomyces cerevisiae FET3p, GET3p, and CMK1p and CMK2p, respectively, were up-regulated in the Al-resistant cells. In this study we cloned these genes and found they were indeed up-regulated in Al-resistant strains. The cloned genes were introduced into S. cerevisiae and corresponding mutants to test their relevance to Al resistance. The introduction of RgFET3 and RgGET3 conferred Al resistance to the host, but that of RgCMK did not. Green fluorescent protein (GFP)-tagged RgFet3p was localized at the cell periphery in the host. GFP-tagged RgGet3p formed more punctate bodies in the host under Al stress than in the absence of Al. Different growth responses to Fe (III), Cu (II), Ca ions, and cyclosporin A in the wild type and resistant cells of R. glutinis suggested the involvement and possible links of the three genes in Al resistance.     

Xylanase from Marine Filamentous Fungus Pestalotiopsis sp. AN-7 Was Activated with Diluted Salt Solution Like Brackish Water

The genus Pestalotiopsis are endophytic fungi that have recently been identified as cellulolytic system producers. We herein cloned a gene coding for a xylanase belonging to glycoside hydrolase (GH) family 10 (PesXyn10A) from Pestalotiopsis sp. AN-7, which was isolated from the soil of a mangrove forest. This protein was heterologously expressed by Pichia pastoris as a host, and its enzymatic properties were characterized. PesXyn10A was produced as a glycosylated protein and coincident to theoretical molecular weight (35.3 kDa) after deglycosylation by peptide-NfF-glycosidase F. Purified recombinant PesXyn10A exhibited maximal activity at pH 6.0 and 50 °C, and activity was maintained at 90 % at pH 5.0 and temperatures lower than 30 °C for 24 h. The substrate specificity of PesXyn10A was limited and it hydrolyzed glucuronoxylan and arabinoxylan, but not β-glucan. The final hydrolysis products from birchwood xylan were xylose, xylobiose, and 1,2³-α-D-(4-O-methyl-glucuronyl)-1,4-β-D-xylotriose. The addition of metallic salts (NaCl, KCl, MgCl₂, and CaCl₂) activated PesXyn10A for xylan degradation, and maximal activation by these divalent cations was approximately 160 % at a concentration of 5 mM. The thermostability of PesXyn10A significantly increased in the presence of 50 mM NaCl or 5 mM MgCl₂. The present results suggest that the presence of metallic salts at a low concentration, similar to brackish water, exerts positive effects on the enzyme activity and thermal stability of PesXyn10A.     

Extracts of plant cell cultures of <Emphasis Type="Italic">Lavandula vera</Emphasis> and <Emphasis Type="Italic">Rosa damascena</Emphasis> as sources of phenolic antioxidants for use in foods

Ethanolic extracts of plant cell cultures of lavender (Lavandula vera) and rose (Rosa damascena) have been examined as potential food antioxidants. The L. vera cell extract quenched the radicals Fremy’s salt, DPPH (2,2-diphenyl-1-picrylhydrazyl radical), and ABTS^·+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic) radical) more efficiently than the R. damascena extract. Also the L. vera extract inhibited lipid oxidation in a methyl linoleate emulsion more efficiently than the R. damascena extract. However, the L. vera extract had a prooxidative effect on the iron-based Fenton reaction in an aqueous model system. A similar effect was observed for pure rosmarinic acid, but not for the R. damascena extract. The addition of L. vera extract to minced chicken meat reduced lipid oxidation (measured as thiobarbituric acid reactive species) and the loss of α-tocopherol during cold storage after the meat was cooked. This suggests the antioxidative properties of L. vera extracts dominate in a real food system.     

Novel anticoagulant compound from fermented red alga <Emphasis Type="Italic">Pachymeniopsis elliptica</Emphasis>

Natural fermentation was tested as a method of releasing active compounds during screening for potential anticoagulant activity in three types of algae (Pachymeniopsis elliptica, Sargassum horneri, and Ulva pertusa). Freeze dried algae samples (2.5 g) were fermented by adding 75 g of sugar and 500 mL of water and thereafter kept at room temperature (25 °C) for 3 months. Activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) were measured every 2 weeks for 3 months to determine the optimum time for the highest activity. Fermented P. elliptica, (which had the highest activity) was subjected to anion exchange chromatography (DEAE-cellulose) and sepharose 4B gel permeation chromatography. The purified sample was analyzed by agarose-gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) to confirm the purification and to determine the molecular mass, respectively. The 360 μg/mL of purified compound (Mwt > 500,000 Da) had both APTT and PT activities (>1,000 s). However, at the concentrations of 180 μg/mL, purified compound and heparin showed 540 and >1,000 s APTT activity, respectively. Though, the purified compound of P. elliptica considered as a weaker anticoagulant than heparin, this purified anticoagulant polysaccharide could be considered as a good alternative source as an anticoagulant. Moreover, the technique of fermentation is an inexpensive and feasible, this purified anticoagulant polysaccharide compound could be used in pharmaceutical and biomedical industry. Further investigations need to be performed to determine the mechanism of this novel anticoagulant compound. The authors Prashani Mudika Ekanayake and Chamilani Nikapitiya contributed equally to this work.     

Influence of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> on<Emphasis Type="Italic"> Staphylococcus aureus</Emphasis> growth in a fresh vegetable model system

The influence of a Lactobacillus plantarum (B₄) on the growth of Staphylococcus aureus (Sa₄) was verified by impedometric methods in a suitable model reproducing the characteristics of fresh vegetables. The inoculum size of the single strains and their growth temperature were varied according to a Central Composite Design. The results obtained via statistical analysis showed that the temperature affected the growth of both S. aureus and L. plantarum strains. The pathogenic strain, independently of its inoculum size, was inhibited by L. plantarum at all the tested temperatures. A proper combination of specific lactic acid bacteria and storage temperature should improve the safety of the vegetable products.     

Barrier property and penetration traces in packaging films against Plodia interpunctella (Hübner) larvae and Tribolium castaneum (Herbst) adults

The barrier property of different types of plastic film (with or without pinholes) against two insects, Plodia interpunctella (Hübner) larvae and Tribolium castaneum (Herbst) adults, and the morphology of damage produced in these insects were investigated. Using a penetration apparatus, four types of plastic films varying in thickness were used for insect-penetration tests: casted polypropylene, 20 μm and 25 μm (CPP20 and CPP25); oriented polypropylene, 20 μm and 30 μm (OPP20 and OPP30); linear low-density polyethylene, 40 μm and 50 μm (LLDPE40 and LLDPE50); and polyethylene terephthalate 12 μm and 16 μm (PET12 and PET16). After being fixed and tested in the penetration apparatus, each film was cut into a disc shape and 10 holes (200 μm diameter) were made by a pin. The shape of film damage and the mouthparts of insects were observed using scanning electronic microscopy. Plodia interpunctella larvae could penetrate all films with pinholes, while T. castaneum adults were unable to penetrate any of the films tested, even those with pinholes. The penetration-percentages by P. interpunctella larvae were 38% (LLDPE40), 3% (LLDPE), 53% (CPP20), 37% (CPP25), 63% (OPP20), 43% (OPP30), 83% (PET12) and 63% (PET16). The elongation value, tensile strength and thickness of film were important factors in the penetration test. LLDPE, which has the highest elongation value and the lowest tensile strength value, was the film that best protected against insect penetration. In CPP and LLDPE films, there were many scratches and tears around the holes. In comparison, much less damage was observed around the holes in OPP and PET films. By observing the mouthparts of insects, it was determined that P. interpunctella larvae had sharper mandibles than those of T. castaneum adults.     

Production of -aminobutyric acid (GABA) by Lactobacillus paracasei isolated from traditional fermented foods

Lactobacillus strains that accumulated γ-aminobutyric acid (GABA) in culture medium were screened to determine strains with high GABA-producing ability. One strain, NFRI 7415, which was isolated from a Japanese traditional fermented fish (funa-sushi), showed the highest GABA-producing ability among the screened strains. Identification tests (i.e., 16S rDNA sequencing and sugar assimilation ability) indicated that NFRI 7415 belongs to Lb. paracasei. The GABA production was further improved by the addition of pyridoxal phosphate to the culture medium and pH regulation of culture medium at pH 5.0. Under optimal cultivation conditions, strain NFRI 7415 produced GABA at a concentration of 302 mm when the glutamate concentration in the culture medium was 500 mm.     

Large-scale production and application of leucine aminopeptidase produced by <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> LL1 for hydrolysis of chicken breast meat

Leucine aminopeptidase (LAP) production by Aspergillus oryzae LL1 (LAP LL1) was scaled up by varying fermenting conditions. The maximal LAP activity for 5, 20, 250, and 2,000 L fermenters were 0.22, 0.16, 0.19, and 0.12 U mL⁻¹, respectively. LAP LL1 production coincides with increasing pH for all scale cases. A pilot plant scale hydrolysis experiment using LAP LL1 was prepared by removing the cells from 1,000 L of A. oryzae LL1 culture and concentrating the supernatant to ∼60 L, with a LAP LL1 activity yield of ∼20% and a specific LAP activity of 0.054 U mg⁻¹, comparable to Flavourzyme. Using a casein substrate, LAP LL1 specific protease activity (50.9 U mg⁻¹) is 43% higher than Flavourzyme (35.6 U mg⁻¹). We believe LAP LL1 has potential use as an industrial enzyme for food processing. Comparing the hydrolysis effects on chopped chicken breast meat (100 g), the degree of hydrolysis (D_H) of LAP LL1 (50.6%) was about twice as effective as Flavourzyme (25.6%). On a pilot-plant scale hydrolysis experiment of chopped chicken breast meat (54 kg), the D_H of LAP LL1 was 45.6%. Adding 30 μmol L⁻¹ zinc increased D_H by 33.3% for LAPs LL1 but zinc did not enhance Flavourzyme hydrolysis. Together, this study provides possibility that LAP LL1 has significant potential for application in the food industry. Shie-Jea Lin and Yi-Hong Chen have contributed equally to this work.     

Potential aromatic compounds as markers to differentiate between Tuber melanosporum and Tuber indicum truffles

The Tuber indicum (Chinese truffle) and Tuber melanosporum (Black truffle) species are morphologically very similar but their aromas are very different. The black truffle aroma is much more intense and complex, and it is consequently appreciated more gastronomically. This work tries to determine whether the differences between the aromatic compounds of both species are sufficiently significant so as to apply them to fraud detection. An olfactometric evaluation (GC–O) of T. indicum was carried out for the first time. Eight important odorants were identified. In order of aromatic significance, these were: 1-octen-3-one and 1-octen-3-ol, followed by two ethyl esters (ethyl isobutyrate and ethyl 2-methylbutyrate), 3-methyl-1-butanol, isopropyl acetate, and finally the two sulfides dimethyldisulfide (DMDS) and dimethylsulfide (DMS). A comparison of this aromatic profile with that of T. melanosporum revealed the following differences: T. indicum stood out for the significant aromatic contribution of 1-octen-3-one and 1-octen-3-ol (with modified frequencies (MF%) of 82% and 69%, respectively), while in the case of T. melanosporum both had modified frequencies of less than 30%. Ethyl isobutyrate, ethyl 2-methylbutyrate and isopropyl acetate were also significantly higher, while DMS and DMDS had low MF (30–40%) compared to T. melanosporum (>70%). The volatile profiles of both species were also studied by means of headspace solid-phase microextraction (HS-SPME-GC–MS). This showed that the family of C8 compounds (3-octanone, octanal, 1-octen-3-one, 3-octanol and 1-octen-3-ol) is present in T. indicum at much higher levels. The presence of 1-octen-3-ol was higher by a factor of about 100, while 1-octen-3-one was detected in T. indicum only (there was no chromatographic signal in T. melanosporum). As well as showing the greatest chromatographic differences, these two compounds were also the most powerful from the aromatic viewpoint in the T. indicum olfactometry. Therefore, either of the two chromatographic methods (GC–O or HS-SPME-GC–MS), together or separately, could be used as a screening technique to distinguish between T. indicum and T. melanosporum and thus avoid possible fraud.     

Engineering of protease-resistant phytase from Penicillium sp.: High thermal stability, low optimal temperature and pH

A new and simple method based on the mechanism of detoxification of metallothionein was developed by using a water-soluble porphyrin and Zn(II)-bound metallothionein for evaluating heavy metal toxicity. Labile Zn(II) ions were released when toxic metal ions such as Cu(II), Pb(II), Bi(III), Cd(II), Hg(II), Co(II), Ag(I), and Ni(II) bound to Zn(II)-bound metallothionein. The water-soluble porphyrin, 5,10,15,20-tetraphenyl-21H,23H-porphinetetrasulfonic acid, a chromogenic reagent that is highly sensitive to Zn(II), formed a complex with the labile Zn(II) ions. The absorption change at 423 nm resulting from the formation of the Zn(II)–porphyrin complex was used to evaluate the toxicity of sample solutions containing different metal ions. The absorption change was well correlated with the toxicity, which was evaluated by a bioluminescence inhibition assay using the bioluminescent bacteria Vibrio fischeri. This observation indicated that the absorption change determined by our method was a good indicator of heavy metal toxicity. The proposed method was more sensitive than conventional bioassays and could be used to detect metal toxicity at submicromolar concentrations of toxic metal ions.