A simplified procedure for fractionating plant materials.

Sequential extraction of plant materials with methanol–chloroform–water and phenol–acetic acid–water mixtures gave good yields of water-soluble low-molecular-mass substances, lipids, proteins and polysaccharides in separate fractions unlaboriously and with little chemical damage. Results with potato tubers (Solanum tuberosum L.), leafy shoots of lucerne (Medicago sativa L.) and an oilseed (Pentaclethra macrophylla Benth.) are presented.     

Exploiting heterozygosity in industrial yeasts to create new and improved baker's yeasts

The main aim of the work was to utilize heterozygosity of industrial yeast strains to construct new baker's yeast strains. Commercial baker's yeast strain ALKO 743, its more ethanol tolerant descendant ALKO 554 selected initially for growth over 300 generations in increasing ethanol concentrations in a glucose medium, and ALKO 3460 from an old domestic sour dough starter were used as starting strains. Isolated meiotic segregants of the strains were characterized genetically for sporulation ability and mating type, and the ploidy was determined physically. Heterozygosity of the segregant strains was estimated by a variety of molecular characterizations and fermentation and growth assays. The results showed wide heterozygosity and that the segregants were clustered into subgroups. This clustering was used for choosing distantly or closely related partners for strain construction crosses. Intrastrain hybrids made with segregants of ALKO 743 showed 16–24% hybrid vigour or heterosis. Interstrain hybrids with segregants of ALKO 743 and ALKO 3460 showed a wide variety of characteristics but also clear heterosis of 27–31% effects as assayed by lean and sugar dough raising. Distiller's yeast ALKO 554 turned out to be a diploid genetic segregant and not just a more ethanol tolerant mutant of the tetraploid parent strain ALKO 743.     

Technical note: A simplified procedure for myofibril hydrophobicity determination

A simple and reliable method for the determination of surface hydrophobicity of nonsolubilized myofibrils (from pig M. longissimus dorsi) was developed and validated. This method is based on the interaction of the hydrophobic chromophore bromophenol blue (BPB) with myofibrillar proteins and the separation of free and bound BPB by centrifugation. The titration of bound BPB is performed by absorption spectroscopy, and the amount of bound BPB is considered as an index of protein hydrophobicity. Heating, which is known to increase protein hydrophobicity, was performed in order to validate this method. Fixation of BPB to myofibrils increased with heating time and temperature, strongly suggesting that it may be closely related to protein hydrophobicity.     

几种酒类酿造酵母产酒精以外的糖消耗的研究

以葡萄酒1#酵母、葡萄酒6#生产酵母和啤酒酵母为研究对象,采用3,5-二硝基水杨酸光度法和比重瓶法分别测还原糖和酒精,对酒精以外的糖消耗进行研究.结果表明,酒类生产酵母在酒精发酵进入减速阶段初时达到峰值,对于控制压榨酒的风味具有指导意义.     

Technical note: A simplified procedure for myofibril hydrophobicity determination

A simple and reliable method for the determination of surface hydrophobicity of nonsolubilized myofibrils (from pig M. longissimus dorsi) was developed and validated. This method is based on the interaction of the hydrophobic chromophore bromophenol blue (BPB) with myofibrillar proteins and the separation of free and bound BPB by centrifugation. The titration of bound BPB is performed by absorption spectroscopy, and the amount of bound BPB is considered as an index of protein hydrophobicity. Heating, which is known to increase protein hydrophobicity, was performed in order to validate this method. Fixation of BPB to myofibrils increased with heating time and temperature, strongly suggesting that it may be closely related to protein hydrophobicity.     

Orthogonal-field-alternation gel electrophoresis banding patterns of DNA from yeasts

Chromosomal DNAs from various yeast species were separated by orthogonal-field-alternation gel electrophoresis (OFAGE). To this end we developed a spheroplasting and lysis method to obtain intact DNA from both ascomycetous and basidiomycetous yeasts. The OFAGE banding patterns of 22 ascomycetous and four basidiomycetous yeast strains were compared. The strains represented species from the genera: Brettanomyces, Candida, Cryptococcus, Filobasidiella, Geotrichum, Hansenula, Kluyveromyces, Pachysolen, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes, Saccharomycopsis, Schizosaccharomyces and Zygosaccharomyces. Variations occurred in the number of bands and their positions in the gel, not only among strains of different genera but also among species from the same genus and even between varieties of the same species. The ascomycetous yeasts, with the exception of Saccharomyces cerevisiae, only showed one to five bands of DNA larger than 1000 kilobase pairs (kb) in general none smaller. The patterns of the four basidiomycetous yeasts revealed also a few large DNA bands but in addition one to six bands ranging in size from 500 to 1000 kb, with the exception of a single smaller chromosome in Rhodotorula mucilaginosa. From the OFAGE banding patterns of strains studied here it appears that in Sacch. cerevisiae the partitioning of DNA over chromosomes is unique. But rather than the large number of chromosomes, the presence of four chromosomes with less than 500 kb of DNA is characteristic for Sacch. cerevisiae.     

夏黑葡萄附生酵母菌分离及多相分类鉴定

葡萄野生酵母是酿造优质葡萄酒的重要菌种来源,为发现有价值的野生葡萄酵母,本研究以夏黑葡萄为研究对象,采用平板划线分离的方法得到3株葡萄野生酵母YYMPT-1、YYMPT-2和YYMPT-3,利用扫描电子显微镜（SEM）进行形态观察,PCR扩增26S核糖体DNA的D1/D2区（26S r DNA D1/D2）、核糖体内转录间隔区（ITS1-5.8SITS2）和肌动蛋白基因（Actin gene）区域,并构建系统发育树,将3株酵母菌分别鉴定为有孢汉逊酵母（Hanseniaspora uvarum）、近玫色锁掷酵母（Sporidiobolus pararoseus）和假丝酵母（Candida zemplinina）。本研究结果为优质葡萄酒酿造和改善葡萄酒风味提供了3株具有潜在工业化应用价值的资源菌。      

Linear DNA plasmid pPK2 of Pichia kluyveri: distinction between cytoplasmic and mitochondrial linear plasmids in yeasts

The linear plasmids frequently found in plants and filamentous fungi are associated with mitochondria or chloroplasts. In contrast, all the linear plasmids known in yeasts are cytoplasmic elements. From a strain of the yeast Pichia kluyveri, we have isolated a new linear plasmid, pPK2, which was found to be associated with mitochondria. This 7·1 kilobase pairs‐long DNA contained only two genes, which code for DNA and RNA polymerases, as judged from their nucleotide sequences translated by a mitochondrial genetic code. When we examined several recently isolated yeast plasmids for their subcellular localization, we found that two linear plasmids, pPH1 from Pichia heedii, as well as pPK1 from another strain of P. kluyveri, were also localized in mitochondria. These plasmids are the first examples of mitochondria‐associated linear plasmids in yeast. All other linear plasmids we examined were of cytoplasmic origin. Whilst the cytoplasmic type linear plasmids were efficiently eliminated by ultraviolet irradiation of host cells, the mitochondria‐associated plasmids were highly resistant. The mitochondrial pPK2 plasmid was rapidly lost by treatment of the host cells with ethidum bromide. Copyright © 1999 John Wiley & Sons, Ltd.     

Efficiency of mitochondrial DNA restriction analysis and RAPD-PCR to characterize yeasts growing on dry-cured Iberian ham at the different geographic areas of ripening

The efficiency of mitochondrial DNA (mtDNA) restriction analysis and random amplification of polymorphic DNA (RAPD)-PCR to characterize yeasts growing on dry-cured Iberian ham was evaluated. Besides, the distribution of the main species and biotypes of yeasts in the different ripening areas of this product was investigated. MtDNA restriction analysis allowed yeast characterization at species and strain level. RAPD-PCR with the primers (GACA)₄ and (GAC)₅ was inappropriate for characterization at species level. Most of the mtDNA restriction patterns detected in dry-cured Iberian ham were consistent with Debaryomyces hansenii. Several yeasts biotypes were associated to specific geographic areas of dry-cured Iberian ham ripening.     

DNA typing methods for differentiation of yeasts related to dry-cured meat products

RFLP analysis of the ITS and 18S rDNA, RAPD-PCR using mini- and microsatellite primers and RFLP analysis of mitochondrial DNA were examined to discriminate yeasts related to dry-cured meat products at species and strain level. Seven species and 35 strains of yeasts usually found in dry-cured meat products were tested. RFLP analysis of the ITS1-5.8S rDNA-ITS2 and 18S rDNA did not allow the separation at species level of all of the species tested. RAPD with a M13 primer was found to be useful for differentiation of Rhodotorula mucilaginosa, Candida zeylanoides, Yarrowia lipolytica, Debaryomyces hansenii and Saccharomyces cerevisiae. However, no differences were observed between Debaryomyces polymorphus and Pichia carsonii. RAPD analysis with microsatellite primers (GACA)(4), (GTG)(5) and (GAC)(5) enabled discrimination at species and strain level. However, the degree of discrimination by means of RAPD-PCR depends highly on the primers used. Thus, the PCR fingerprinting with primer (GACA)(4) enabled a higher level of discrimination than primers (GAC)(5) and (GTG)(5). The RFLP analysis of mtDNA allowed the discrimination at the species and strain level except for R. mucilaginosa, where no polymorphisms were observed in the strains tested. RAPD analysis with primer (GACA)(4) and the restriction analysis of mtDNA used in the present work are useful for the differentiation at species and strain level of yeasts related to dry-cured meat products.     

Differentiation of yeasts growing on dry-cured Iberian ham by mitochondrial DNA restriction analysis, RAPD-PCR and their volatile compounds production

The efficiency of mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR and volatile compounds analysis to differentiate yeast biotypes involved in flavour development of dry-cured Iberian ham throughout the ripening process is evaluated. For this purpose, 86 yeasts isolated from Iberian hams in the main ripening stages at different industries of the four Protected Designations of Origin of this product, were used. The combination of mtDNA restriction analysis and RAPD-PCR using the primer (GACA)₄ showed a higher variability in the yeast species detected than obtained using only mtDNA restriction analysis. Only two species, Debaryomyces hansenii and Candida zeylanoides, were identified throughout the whole ripening process and a wide diversity of biotypes was found in these two species, with those of D. hansenii predominating. Clear differences between biotypes were detected in the generation of volatile compounds, with the biotype C2-2 of D. hansenii showing the highest concentrations of volatiles. The combined use of mtDNA restriction analysis and RAPD-PCR distinguishes yeast biotypes with different production of volatile compounds. In addition, analysis of the production profile of volatile compounds is needed to differentiate yeast strains of the same biotype recovered at different stages of ripening. Thus, the combination of these three methods could be very useful to select or monitor yeasts as starter cultures in dry-cured meat products.     

A simplified X-ray fluorescence (XRF) procedure for the determination of sulphur in graminaceous materials

An automated, rapid and much simplified XRF procedure for assessing total sulphur in graminaceous plant materials is described. Sample preparation has been refined and microprocessor control incorporated to provide considerable savings in analytical time. The procedure has been tested using samples from two grass-silage cuts, taken from an ADAS (Agricultural Development and Advisory Service) trial studying responses, under intensive grassland management, to sulphur and nitrogen applications. Good correlation between XRF and wet chemical analysis was achieved, and sample turnround was markedly increased. The method produced accurate results for both cuts with high precision and reproducibility where careful attention was paid to sample homogeneity and disc preparation. The potential for wider application of XRF techniques to elemental analysis of agricultural materials is discussed.     

Comparison of DNA sequencing analysis with phenotyping test for identification of yeasts isolated from foods

The identification of 20 strains of yeasts isolated from foods by means of DNA sequence analysis with two kinds of universal primers for the rDNA region was examined, and the results were compared with those of the conventional phenotyping test using API 20C AUX. In the analysis of the 26S region, all 20 yeast strains tested were identified at the species level. In the ITS1 region, 16 strains were also classified at the species level. In addition, all results of DNA sequence analysis were consistent with those of the phenotyping test at the genus level. Furthermore, DNA sequence analysis was able to identify causative yeasts observed in two suspect foods, though phenotyping tests alone failed to identify them.     

Contribution of different yeasts isolated from musts of monastrell grapes to the aroma of wine.

Volatile substances of wines obtained by fermentation of musts from 'Monastrell' grapes (Alicante, Spain) was studied for yeast isolated from such musts. The results of the statistical treatment performed show the importance of yeasts of low fermentative power, particularly Kloeckera apiculata, in the production of volatile substances. Saccharomyces cerevisiae var. chevalieri was found to be the most important yeast of high fermentative power.     

A simplified procedure for quantification of potato glycoalkaloids in tuber extracts by H.p.l.c.; comparison with ELISA and a colorimetric method

An improved high-performance liquid chromatography (h.p.l.c.) procedure for determination of glycoalkaloid levels in potato tubers has been developed in which Sep-Pak cartridges replace the commonly used alkaline precipitation for clean-up of tuber extracts. Glycoalkaloids are extracted from fresh tuber tissue into an aqueous medium, the extract is submitted to clean-up and α-solanine and α-chaconine are quantitatively separated on a reversed phase column with ethanolamine modifier added to the mobile phase. Potato tubers were comparatively analysed for their glycoalkaloid content by this and two other methods: a recently developed immunoassay (ELISA) and a colorimetric procedure representing the traditional chemical approach. Agreement between the h.p.l.c. and the other methods was good.     

Detection of cows' milk in goats' cheeses inferred from mitochondrial DNA polymorphism

A new polymerase chain reaction (PCR)-based method was developed to detect cows milk in goat cheese. This method is based on mitochondrial DNA (mtDNA) control region sequence variations. DNA extractions from 150 mg of cheese were carried out using a spin column-based method. Subsequent PCR amplifications of DNA were performed with cow specific primers, demonstrating the ability to detect cows' milk in a variety of cheeses. This simple approach provides high quality DNA, and is shown to be very sensitive, with a detection limit of less than 0.1% of cows' milk. Analysis of an agarose gel digital image allows a rough estimation of the percentage of cows' milk used in adulteration.     

Inhibition of yeasts isolated from traditional Turkish cheeses by Lactobacillus spp.

Antifungal activity of some Lactobacillus strains was determined. Yeasts were isolated and identified as Saccharomyces cerevisiae (10 of 17) and one each of Candida pseudotropicalis, C. krusei, C. lipolytica, C. lusitaniae, C. ciferrii, Torulopsis glabrata and Rhodotorula rubra . Lactobacillus strains were found to have fairly strong antifungal activity against S. cerevisiae . Of the 19 bacteria tested, L. plantarum Lp 21 seemed to have the most inhibitory effect against all of the S. cerevisiae strains except for S. cerevisiae 13. We suggest that L. plantarum Lp 21 can be used with starter cultures for cheese production.     

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.