Microbiological evaluation and molecular characterization of bifidobacteria strains in commercial fermented milks

The aim of this study was to evaluate the presence of Bifidobacterium animalis subsp. lactis in commercial dairy products using different molecular techniques. We analyzed the microbiological composition of 13 commercial fermented milks available in the Spanish market. Thirteen strains of genus Bifidobacterium were isolated from these products and were identified by genus-specific PCR, by fluorescence in situ hybridization (FISH), by multiplex PCR and amplified ribosomal DNA restriction analysis (ARDRA). The same sets of strains were typed by randomly amplified polymorphic DNA (RAPD) analysis and by amplified fragment length polymorphism technique (AFLP). All strains were identified as B. animalis subsp. lactis using ARDRA and multiplex PCR techniques. Similarity between strains was evaluated based on RAPD and AFLP profiles. The isolated strains showed similar profiles by using these techniques, revealing the reduced genetic variability existing among commercial strains, and all these profiles were reproducible in repeated analysis. ARDRA and multiplex PCR are techniques that allow differentiation of the bifidobacteria at genus and species level, but do not indicate if they are different strains, for which reason the RAPD technique is very useful. All bifidobacteria isolated from commercial fermented milks in Spain belong to the same species B. animalis subsp. lactis. Our results demonstrate the necessity to control the presence of bifidobacteria in commercial fermented milks, not only at species level but also at strain level. Multiplex PCR and RAPDs are the most suitable, rapid and precise techniques to identify all bifidobacteria contained in fermented milk products at genus-, species-, and strain levels.     

Multilocus sequence typing of oenological Saccharomyces cerevisiae strains

This study describes the application of a multilocus sequence typing (MLST) analysis for molecular discrimination at the strain level of Spanish wine yeast strains. The discrimination power of MLST is compared to mitochondrial RFLP analysis. Fragments of the ADP1, ACC1, RPN2, GLN4, and ALA1 genes were amplified by PCR from chromosomal DNA of 18 wine Saccharomyces cerevisiae strains. Ten polymorphic sites were found in the five loci analyzed showing 13 different genotypes, with 11 of them represented by only one strain. RFLP analysis of the same 18 wine yeast strains showed seventeen different mitochondrial patterns. Phylogenetic relationships among the strains analyzed, inferred by MLST data, showed wine isolates of S. cerevisiae as a rather homogeneous group. The discrimination potential of mitochondrial RFLP analysis was superior to the MLST scheme used in this work. However, MLST analysis allowed an easy construction of reliable phylogenetic trees. MLST analysis offers the possibility of typing wine S. cerevisiae strains simultaneously to the study of the genetic relationship among them.     

Microbiological Analyses of Dry and Slurry Yeasts for Brewing

Classical microbiological methods in association with molecular methods (DNA amplification, Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) were used. These methods, developed to rapidly analyze microbial communities on the basis of sequence‐specific separation of DNA amplicons, allowed the detection of DNA differences in the amplicons tested and the identification of the strains analyzed by the comparison of unknown sequences with sequences of known species. TGGE allowed the comparison of the different Saccharomyces cerevisiae strains used in brewing while DGGE allowed the identification of lactic acid bacteria (LAB) in beer. These methods are a reliable tool for fast comparison of strains of Saccharomyces cerevisiae collected from different craft breweries where they were used as starters to check the presence of possible yeast contaminants in the brewing process and for rapid LAB identification.     

Safety and quality of ready-to-eat dry fermented sausages subjected to E-beam radiation

The inactivation kinetics in the death of Listeria innocua NTC 11288 (more radioresistant than five different strains of Listeria monocytogenes) and Salmonella Enterica serovar Enteritidis and S. enterica serovar Typhimurium by E-beam irradiation has been studied in two types of vacuum-packed RTE dry fermented sausages (“salchichon” and “chorizo”) in order to optimize the sanitation treatment of these products. A treatment of 1.29 kGy was calculated to reach the food safety objective (FSO) according to the “zero tolerance” criterion for the three strains. No irradiation treatment was necessary to meet the 10² c.f.u./g microbiological criterion for L. monocytogenes. Dry fermented sausages treated with 2 kGy had negligible sensory (appearance, odour and taste) modifications. Therefore, this treatment produces safe dry fermented sausages with similar sensory properties to the non-irradiated product.     

Identification of yeast strains using the polymerase chain reaction

Commonly used techniques for the identification of industrial yeast strains are usually time-consuming and cumbersome. Moreover, some of these methods may give ambiguous results. A novel strategy has been developed for identifying yeast strain employing polymerase chain reaction technology. Using customised oligonucleotides, some regions of the yeast genome between δ elements are amplified to give an ‘amplified’ sequence polymorphisml (Skolnick and Wallace 1988) characteristic of the strains. With this technique it is possible to identify individual strains of Saccharomyces cerevisiae.     

Intraspecific Diversity of Yeast Associated to Vitis vinifera Albariño Must from Different Vineyard Ecosystems

The genetic diversity of Saccharomyces cerevisiae associated to the must (Vitis vinifera Albariño) from different geographic areas and spontaneous fermentation was studied by analysis of mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP). Colonies were isolated in musts at different stages of fermentation from three different geographic areas, O Val do Salnés, O Rosal and O Condado do Tea from Galicia (NW Spain). A total of 17 different patterns out of 273 S. cerevisiae colonies were identified using mitochondrial DNA restriction analysis. Pattern I from O Rosal, VI from O Val do Salnés and XII from O Condado do Tea were the predominant ones. Only pattern II was common to all fermentations. This molecular study provides useful information about distribution and genetic relationships among several S. cerevisiae strains associated to Vitis vinifera Albariño from different vineyard ecosystems and their influence on certain oenological properties.     

Studies on acetate ester production by non-Saccharomyces wine yeasts

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H. uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.     

Methods for yeast characterization from industrial products

This work compared the efficiency of four methods for the identification of industrial yeast strains and the establishment of a pattern for yeast characterization to be used during industrial fermentation processes, allowing the detection of yeast contaminants. Five strains of yeast currently used in the Brazilian fuel alcohol industry (about 99% of the yeast used for this purpose), and yeast strains isolated from the five major beer industries that represent 95% of the Brazilian beer market were evaluated for their growth and absorption of dyes on differential culture media, their total protein electrophoretic patterns (SDS–PAGE), CHEF chromosome separation patterns, and RAPD profiles. For the identification of brewing yeast, all tested methods were efficient, allowing the identification of at least two different species, one of which wasSaccharomyces cerevisiae . The strains used for the fuel alcohol industries were best characterized by SDS–PAGE and RAPD analysis. Those strains share high level of genetic similarity and they are all known as S. cerevisiae strains.     

Effects of addition of anka rice on the qualities of low-nitrite Chinese sausages

Anka rice (AR), previously inoculated with Monascus purpureus, was added during manufacturing of low-nitrite Chinese sausages. Chemical compositions and water activities of sausages were not affected. “L”, “a”, and “b” values of sausages with less nitrite (25 ppm) and 0.5% AR added were not significantly different from those with more nitrite (100 ppm) added. Colours of the sausages without AR were light red whereas those with AR added were darker red. Addition of AR did not inhibit lipid oxidation. Higher VBN (volatile basic nitrogen) values of the samples with AR added were observed. With addition of AR, the nitrite degrading rate was retarded. Microbial counts of the sausages with AR added were significantly higher than those of the controls (100 ppm nitrite). The low-nitrite Chinese sausage with addition up to 1.5% AR was acceptable when stored at 4 °C for 28 days.     

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.     

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non‐homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR‐amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour‐intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ‐mediated integrative transformation with PCR‐amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.     

Saccharomyces cerevisiae associated with the spontaneous fermentation of tequila agave juice

Saccharomyces cerevisiae dominates the spontaneous fermentation of blue agave juice. Because of the batch heterogeneity, the aim of this work was to determine the strain diversity of S. cerevisiae among fermentations. During January and February 2015, agave juice was sampled in triplicate from four sampling points at a tequila distillery. The heterogeneity of yeast strains and the production of carbon dioxide were assessed during fermentation, whereas the amount of ethanol produced was measured at the end of the process. The fermentation cycle times varied widely (9 to 25 days), as did fermentation efficiency (2.5–45.5%). Yeast isolates were identified at the species level by ITS‐5.8S rRNA restriction fragment length polymorphism and differentiated at the strain level by random amplified polymorphic DNA. A total of 199 isolates were obtained and identified as S. cerevisiae, showing 69 different random amplified polymorphic DNA profiles. There was no clear dominance of any strain during fermentation. However, two strains (P1 and P2) were detected in all fermentation samples, suggesting their residency in the distillery, despite the deep‐cleaning applied to the tanks after each fermentation batch. According to the RAPD profiles, the number of strains isolated from fermentation samples increased from 17 in January to 25 in February. © 2018 The Institute of Brewing & Distilling     

PCR‐TTGE and RAPD‐PCR Techniques to Analyze Saccharomyces cerevisiae and Saccharomyces carlsbergensis Isolated from Craft Beers

In the Friuli Venezia Giulia region (North East of Italy) the production of craft beers has been increasing constantly. Usually microbreweries use yeasts supplied by Italian or foreign industrial breweries for beer production. Yeast species are often not known, moreover the vitality, the viability, the physiological state and the number of generation are not known. To improve the quality of the final product it is important to evaluate the quality of the yeast strain used and the lactic acid bacteria contamination. Various molecular methods have been developed to compare genetic characteristics of yeast strains used in beer and wine production. The methods proposed in this work, PCR‐TTGE and RAPD‐PCR techniques, allow the comparison of specific DNA sequences to identify and/or characterize yeast strains. The molecular methods are faster than traditional methods and they allowed the identification of the strains analysed as S. cerevisiae and the intraspecies differentiation among yeast strains tested within 8 h after cell growth.     

西藏野生油菜遗传多样性分析

利用10对AFLP引物及13对SSR引物,分析了43份来自西藏地区部分野生类型油菜种质的遗传多样性。10对AFLP引物共得到276条清晰的谱带,其中多态性带214条,多态性位点比率为77．5％,平均每对AFLP引物得到21．4条多态性带。13对SSR引物共扩增出57条带,其中多态性带51条,多态性位点比率为89．5％,说明西藏野生油菜遗传多样性丰富。通过对西藏野生类型油菜资源的遗传距离以及聚类分析,可将西藏野生类油菜分为两大类群,分别为白菜型和芥菜型野生油菜种质资源;两种分子标记适合揭示西藏野生油菜的遗传多样性。     

Evaluation of textural properties of channel catfish (Ictalurus punctatus Rafinesque) fillet with the natural contour method

A value-added channel catfish (Ictalurus punctatus Rafinesque) product was being developed to explore more profit for this industry. Textural properties played an important role in the quality control and acceptability of both raw and processed products. Due to insufficient study on this topic a reliable method was needed for textural properties evaluations of catfish and other small-scale fish. Textural properties of raw and smoked channel catfish (I. punctatus) fillets were measured by the “finger” and “tooth” methods with a Texture Analyzer. A novel sampling technique was used to sample thickness contours on the fillets. Indentation force (g) of the “finger” method and shear force (g) of the “tooth” method were measured at different contour levels of four myomere cone bands on the fillets.Shear force and indentation force of the fresh catfish fillets increased with the increasing thickness as measured by the “tooth” and “finger” methods. Both methods could be used to measure the textural properties of catfish fillet. The “finger” method was recommended because of its non-destructive nature and applicability to both raw and smoked catfish samples. The novel sampling technique used in this study was rapid and applicable to irregular fillet shapes including catfish and other fish species. Smoked catfish had decreased indentation force with increased thickness. The dehydration effects and denaturization of fish muscle during the smoking process were main reasons for that.     

Solid-phase microextraction gas chromatography–mass spectrometry (HS-SPME-GC–MS) determination of volatile compounds in orujo spirits: Multivariate chemometric characterisation

A headspace solid-phase microextraction gas chromatography mass spectrometric procedure (HS-SPME-GC–MS) was developed and applied in order to determine 22 volatile compounds (including alcohols, esters, aldehydes and terpenes) in different orujo spirit samples from the Geographic Denomination “Orujo de Galicia/Augardente de Galicia”. The orujo samples considered in this study were elaborated from Albariño variety grapes grown in the Rías Baixas restricted geographical area, and Albariño variety grapes grown in other geographical areas of Galicia (NW Spain) using two of the traditional distillation techniques: alembic and steam distillation. HS-SPME adequate results were obtained using a 65 μm carbowax-divinylbencene fibre during a headspace extraction at 40 °C with constant magnetic stirring for 15 min, and after a 5 min period of pre-equilibrium time. Desorption was performed directly in the gas chromatograph injector port for 5 min at 250 °C using the splitless mode. The applied method was demonstrated to be sensible, accurate, precise, and linear over more than one order of magnitude. Multivariate chemometric techniques (such as cluster analysis, principal component analysis and linear discriminant analysis) were used to characterise the orujo samples according to the geographical origin of the grapes and the distillation system employed in their elaboration on the basis of the chemical information provided for their volatile composition data.     

Biogenic amine production by Gram-positive bacteria isolated from Spanish dry-cured "chorizo" sausage treated with high pressure and kept in chilled storage

We studied the production of biogenic amines by 200 strains of lactic acid bacteria and staphylococci isolated during chilled storage from samples of Spanish dry-cured “chorizo” sausage treated with high-pressure. The presence of biogenic amines in a decarboxylase synthetic broth was confirmed by ion-exchange chromatography. β-phenylethylamine was the biogenic amine more frequently produced (22.5%), followed by tyramine (7.5%). In tyramine producer-strains the presence of a tyrosine decarboxylase gene was confirmed by PCR. Among lactic acid bacteria, the production of tyramine was mainly related to the species Lactobacillus curvatus. Most of the L. curvatus strains were also β-phenylethylamine-producers. In relation to staphylococci, tyramine-production was mainly associated to Staphylococcus carnosus strains. The S. carnosus strains analysed in this study produced β-phenylethylamine or β-phenylethylamine and tyramine simultaneously. RAPD-PCR results indicated that the biogenic amine-producer S. carnosus population changes along storage independently of the high-pressure treatment.     

Development of defined mixed-culture fungal fermentation starter granulate for controlled production of rice wine

As a first step in the development of defined fungal starter granules for controlled winemaking from purple glutinous rice, the interaction of moulds and yeasts isolated from Vietnamese rice wine starters and the effect of some representative oriental herbs on the growth of moulds and yeasts were examined. Amylomyces rouxii and Saccharomyces cerevisae were shown to be compatible in mixed cultures, and the herbs “Tieu Hoi” (Fennel: Foeniculum vulgare Miller) and “Dinh Huong” (Clove: Syzygium aromaticum L.) which are used as supplementary ingredients by some local starter producers, were observed to stimulate the mould and yeast growth. Based on traditional starter manufacturing methods and modified on the basis of optimization experiments, a laboratory-scale manufacturing process for defined mixed-culture starter granules was established. In accordance with the national standard method, the wine produced with new experimental starter granules was found to have superior flavour and overall acceptability, compared with local commercial rice wines.

Industrial relevance

One of the major problems faced by commercial brewers of rice wine in Vietnam, is the variable quality and performance of the traditional starter tablets that are commonly used. The relevance of the present paper is that a stable, granulated starter has been developed, containing a defined mixture of mould and yeast cultures. This has proven to be shelf stable for more than 3 months, producing a very well accepted quality of wine.     

A study on Saccharomyces cerevisiae and Candida utilis cell wall capacity for binding magnesium

The capacity for binding magnesium by bakery's yeast strain Saccharomyces cerevisiae No. 102 (Pure Culture Collection, Faculty Food Technology, Warsaw) and fodder yeast strain Candida utilis (ATCC 9950) was investigated in media supplemented with that element. The capacities of C. utilis (ATCC 9950) and S. cerevisiae (No. 102) biomass for binding magnesium were not statistically different in the first 24 h. In the next 24 h of cultivation the cells of C. utilis (ATCC 9950) were still able to bind magnesium ions, whereas those of S. cerevisiae (No. 102) released a part of previously bound magnesium to the medium. The major part of magnesium bound by the cells of C. utilis (ATCC 9950) was accumulated in cytosole. It was opposite to the cells of bakery yeast S. cerevisiae (No. 102) that accumulated magnesium mainly in the cell wall. The cells of C. utilis (ATCC 9950) yeast were smaller and their cell walls were thinner as compared to those of S. cerevisiae (No. 102) yeast. The thickness of the external mannoprotein layers was similar in both strains analyzed.