“Framboisé” spoilage in French ciders: Zymomonas mobilis implication and characterization

Biodiversity of 17 Zymomonas mobilis strains isolated from French “framboisé” ciders from 7 different geographical areas of France was analysed using random amplified polymorphic DNA (RAPD) and 6 different strain fingerprints were observed. One of the fingerprints was identical for 10 of the 17 cases. No correlation between geographical origin and strain fingerprint was observed. In parallel, the cider-making process was followed from the orchard to the final product for Z. mobilis presence. Detection of the bacterium only occurred during the fermentation process. The French isolate, strain AN0101, recently proposed as a novel subspecies (Z. mobilis subsp. francensis) was characterized in a synthetic medium using various growth conditions that can be encountered during cider-making (T (°C), pH, ethanol, SO₂, polyphenols) to determine growth limits. Results clearly showed that cider is in a risk zone for Z. mobilis contamination as this bacterium was able to grow at temperatures as low as 4 °C, in pH range from 3.5 to 6.0, in 0-8 ml/100 ml ethanol, at the legal limit of 200 mg/l SO₂ and in the presence of a cider marc polyphenol extract up to 3 g/l. These results should allow for a better understanding of Z. mobilis conditions of contamination in cider and lead to possible preventive measures in the future.     

DISCOVERY OF THE “CIDER SICKNESS” BACTERIUM ZYMOMONAS ANAEROBIA IN APPLE PULP

After a lapse of nearly 20 years the “cider sickness” organism has been rediscovered. On this occasion it was found in apple pulp, this being the first time it has been isolated from a source other than “sick cider”. In spite of its presence in the pulp, no outbreak of the disorder occurred in the factory from which it was isolated, thus reaffirming the soundness of the cider-making methods specifically designed to prevent the growth of this bacterium. The characteristics of this isolate are compared with the two known species, namely, Zymomonas anaerobla and Z. mobilis and it is concluded that the new isolate is a strain of the former.     

Alcohol Production from Raisin Extracts: Volatile By products

The formation of methanol, acetaldehyde, 2-methyl-butanol-I and 3-methyl-butanol-1 in Zytnomonas mobilis alcoholic fermentation of raisin extracts, in comparison with Sacharomyces cerevisiae, was studied. Z. mobilis fermentations gave lower acidities and about 30% less of the volatile compounds than S. cerevisiae. Thus, acidity and volatiles should not be a problem in industrial fermentations with Z. mobilis. The effect of pH, initial cell concentration (ICC) and initial sugar content (ISC) were also investigated. Z. mobilis produced the lowest concentration of volatiles at DH 3.5, ISC 150 d/L and ICC 40 g/L. An increase of ISC leads to a reduction of volatiles per g EtOH. Bacteria form 2-methyl-bulanol-1 and 3-methyl-butanol-1 in the raisin extract fermentation medium, as do S. cerevisiae.     

Comparative Performance of Zymomonas mobilis and Saccharomyces cerevisiae in Alcoholic Fermentation of Saccharified Wheat Starch B in a Continuous Dynamic Immobilized Biocatalyst Bioreactor

Glucose derived from an industrial, enzymatically saccharified wheat starch fraction (B starch) was investigated for ethanol production using free and immobilized cells of Saccharomyces cerevisiae and Zymomonas mobilis. Batch cultures of these organisms could successfully ferment this substrate within 24 h giving 7% ethanol by volume and over 96% sugar conversion. Fermentation with S cerevisiae and Z. mobilis immobilized on rotating fiber discs in a continuous dynamic biocatalyst bioreactor (CDIBB) resulted in very rapid and efficient fermentation and concurrent high ethanol productivity. Z. mobilis loaded at 30g/I cell density (based on bioreactor volume, 31) fermented saccharified wheat starch B slurry at 15% solids and having 128g/I glucose, in 0.9 h with 92% sugar utilization; and volumetric productivity based on liquid hold up was 65.5 g/I-h, being threefold higher than that obtained with immobilized yeast cells. Z. mobilis appears to be the preferred organism for the rapid fermentation of glucose rich solutions in the CDIBB. Advantages and the possible application of this bioreactor system to alcoholic fermentation of industrial derived substrates associated with high suspended solids are discussed. Die aus einer industriellen, enzymatisch verzuckerten Weizen-B-Stärke gewonnene Glucose wurde unter Verwendung von freien und immobilisierten Zellen von Saccharomyces cerevisiae und Zymomonas mobilis hinsichtlich der Ethanolgewinnung untersucht. Chargenweise Kulturen dieser Organismen vergoren dieses Substrat erfolgreich 24h; sie ergaben 7 Vol.% Ethanol bei 96% iger Zuckerumwandlung. Die Vergärung mit auf rotierenden Faserplatten immobilisierten S. cerevisiae und Z. mobilis in einem kontinuierlichen, dynamischen Biokatalysator-Bioreaktor (CDIBB) führte zu sehr schneller und wirksamer Gärung und vergleichbar hoher Ethanolproduktivität. Mit einer Zelldichte von 30g/1 (bez. auf das Bioreaktorvolumen, 31) gepackte Z. mobilis vergor eine Weizen-B-Stärkesuspension mit 15% Feststoff und 128g Glucose/1 in 0,9h bei 92% Zuckerverwertung. Die Volumenproduktivität, bezogen auf die Flüssigkeitsrückhaltung, betrug 65,5 g/1-h und war damit dreimal so hoch wie bei immobilisierten Hefezellen. Z. mobilis dürfte der bevorzugte Organismus für die rasche Vergärung von glucosereichen Lösungen im CDIBB sein. Die Vorteile und die mögliche Anwendung dieses Bioreaktorsystems für die alkoholische Vergärung von industriellen Substraten in Verbindung mit hoch suspendierten Feststoffen werden diskutiert.     

Quantitative detection of lactic acid bacteria in dried sourdoughs using real-time PCR

A real-time PCR system with 16S rRNA gene-targeted group-specific primers was developed to quantitatively detect lactic acid bacteria (LAB) of the genera Lactobacillus, Leuconostoc, Pediococcus, and Weissella in different types of commercially available dried sourdoughs. Despite a high degree of degradation in the DNA isolated from the doughs, the 341-bp 16S rRNA gene fragment of the sourdough LAB biota could specifically be amplified. For dried sourdoughs, the resulting calculated LAB cell counts were determined to be up to 3.7 × 10⁷ cells/g fresh dough, whereas in non-fermented dough acidifiers, used as a control, the calculated LAB cell counts did not exceed 3.6 × 10⁴ cells/g fresh dough. Moreover, the effect of low pH and/or high lactic acid concentrations prevailing in the doughs on the detectability of LAB cells in spray- and roller-dried sourdoughs by PCR was investigated. Drying of non-acidified sourdoughs still permitted to detect the LAB cells by PCR, whereas drying of acidified doughs reduced the detectable cell counts by approximately 5 (spray dried) and 2 (roller dried) orders of magnitudes. Incubation of acidified doughs for 24 h did not affect the detectability of LAB cells in spray-dried doughs but further reduced the detectable cell counts in roller-dried doughs by additional 2 orders of magnitude.     

Isolation and Identification of Lactic Acid Bacteria from Fermented Red Dragon Fruit Juices

Abstract: Red dragon fruit or red pitaya is rich in potassium, fiber, and antioxidants. Its nutritional properties and unique flesh color have made it an attractive raw material of various types of food products and beverages including fermented beverages or enzyme drinks. In this study, phenotypic and genotypic methods were used to confirm the identity of lactic acid bacteria (LAB) appeared in fermented red dragon fruit (Hylocereus polyrhizus) beverages. A total of 21 isolates of LAB were isolated and characterized. They belonged to the genus of Enterococcus based on their biochemical characteristics. The isolates can be clustered into two groups by using the randomly amplified polymorphic DNA method. Nucleotide sequencing and restriction fragment length polymorphism of the 16S rRNA region suggested that they were either Enterococcus faecalis or Enterococcus durans. Practical Application: Current research revealed the use of biochemical analyses and molecular approaches to identify the microbial population particularly lactic acid bacteria from fermented red dragon fruit juices.     

PCR-based methodology for the authentication of the Atlantic mackerel Scomber scombrus in commercial canned products

A multiplex-PCR assay for the authentication of the Atlantic mackerel Scomber scombrus in commercial canned products has been developed. This novel method consists of a S. scombrus-specific fragment (123 bp) corresponding to the mitochondrial NADH dehydrogenase subunit 5, and a positive amplification control corresponding to the small rRNA 12S subunit (188 bp). The system was assayed using six different canned products labeled as S. scombrus. We observed a positive identification in all samples, revealing this methodology as a potential molecular tool for direct application in the authentication of S. scombrus canned products.     

ISOLATION OF ZYMOMONAS MOBILIS FROM PRIMED BEER*

Isolation from beer of a sucrose-fermenting species of Zymomonas confirmed as Z. mobilis, is recorded. Motility and H₂S production were variable and sensitive to precise environmental conditions. The beer-spoilage potential of this species was established as limited. A strain of Z. anaerobia was isolated which acquired sucrose-fermenting ability after growth in fructose. It is doubtful, therefore, whether the creation of two distinct species within this genus is valid.     

Genetic interrelationship among species of the genus Zygosaccharomyces as revealed by small-subunit rRNA gene sequences

The phylogenetic interrelationship of species of the genus Zygosaccharomyces was examined by 18S rRNA gene sequencing. Comparative analysis of the sequence data revealed the genus to consist of a number of distinct subdivisions. The most prevalent species associated with food spoilage, Z. bailii, Z. bisporus and Z. rouxii, along with Z. mellis were found to form one subdivision. Zygosaccharomyces cidri and Z. fermentati formed a distinct species pair, as did Z. microellipsoides and Z. mrakii. Zygosaccharomyces florentinus formed a separate line displaying no specific relationship with any of the other Zygosaccharomyces species examined. Comparison with nine published ascosporogenous yeast 18S rRNA gene sequences showed that Z. microellipsoides and Z. mrakii were genealogically very close to Torulaspora delbrueckii (both displaying 99·8% 18S rRNA sequence similarity), raising the possibility that these two Zygosaccharomyces species should be moved to the genus Torulaspora. The topologies of trees derived from complete 18S rRNA gene sequences and from individual domains within the gene were compared and the implications of using partial sequence data for inferring phylogenetic relationships discussed.     

Salmon and Trout Analysis by PCR-RFLP for Identity Authentication

Raw and smoked Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) were differentiated by PCR amplification of a 950 bp conserved fragment of the mitochondrial 16S rRNA gene followed by restriction site analysis with the endonucleases Hpa I and Bst Ell. Salmon PCR products digested with Hpa I yielded two fragments of 804 bp and 146 bp, while trout PCR products were not cleaved by this enzyme. However, Bst Ell did not cleave salmon PCR products while two bands of 513 bp and 437 bp were produced when trout samples were cleaved with this enzyme. This genetic marker could be very useful for detecting fraudulent substitution of lower valued smoked trout.     

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.     

Production of ethanol from liquefied cassava starch using co-immobilized cells of Zymomonas mobilis and Saccharomyces diastaticus

Co-immobilized cells of Saccharomyces diastaticus and Zymomonas mobilis produced a high ethanol concentration compared to immobilized cells of S. diastaticus during batch fermentation of liquefied cassava starch. The co-immobilized cells produced 46.7 g/l ethanol from 150 g/l liquefied cassava starch, while immobilized cells of yeast S. diastaticus produced 37.5 g/l ethanol. The concentration of ethanol produced by immobilized cells was higher than that by free cells of S. diastaticus and Z. mobilis in mixed-culture fermentation. In repeated-batch fermentation using co-immobilized cells, the ethanol concentration increased to 53.5 g/l. The co-immobilized gel beads were stable up to seven successive batches. Continuous fermentation using co-immobilized cells in a packed bed column reactor operated at a flow rate of 15 ml/h (residence time, 4 h) exhibited a maximum ethanol productivity of 8.9 g/l/h.     

INCIDENCE OF INDIGENOUS MICROBIAL FLORA FROM UTENSILS AND SURROUNDING AIR IN TRADITIONAL FRENCH CIDER MAKING

Lactic acid bacteria and yeasts were numerated, isolated and identified from two ciders obtained from similar raw materials used under different conditions. One cider was produced in a cellar of a traditional producer while the second one was produced under laboratory conditions. In both ciders alcoholic fermentation was achieved mainly by Saccharomyces cerevisiae. In the traditional cider, among the lactic acid bacteria (LAB) the species Leuconostoc oenos was predominant and malolactic conversion was observed. By contrast no development of LAB occurred in the second cider and therefore malolactic conversion was not achieved .     

Effects of Inhibitory Environmental Factors on Growth of Oenococcus oeni CCSYU2068 for Malolactic Fermentation of Cider Production

The effects of several inhibitory factors (sulfur dioxide, pH and ethanol) on the growth of lactic acid bacteria and the subsequent malolactic fermentation (MLF) were studied by inoculation of different culture strains of Oenococcus oeni, the major lactic acid bacteria (LAB) in cider production. After comparing their organoleptic properties, three strains of Oenococcus oeni were selected from indigenous and commercial sources and their inhibitory effects on cell growth and MLF examined. The malolactic bacteria expressed variations in tolerance to the environmental conditions of pH, sulfur and ethanol concentration. Isolated from an indigenous cider production facility, O. oeni L4 had a better capacity with constant growth even when the concentration of SO₂ was 50 ppm, ethanol 10% (v/v) and pH 3.0. O. oeni L4 showed better properties for metabolizing the major acids: malic, lactic and acetic acid. The decomposition mean rate of malic acid was as high as 228.52 mg/L per day with a low acetic acid concentration of 101.78 mg/L under the stress conditions of cider production.     

Microbial Populations and Malolactic Fermentation of Apple Cider using Traditional and Modified Methods

The evolution of yeasts and lactic acid bacteria was examined during two methods of cider making: the traditional employed unwashed apples of different varieties and the modified used a sole washed acidic apple variety with temperature control during the cider making process. The alcoholic fermentation was carried out mainly by Kloeckera apiculata and Sacharomyces cerevisiae species, and their distributions were similar in the two methods. In the traditional, the malolactic fermentation proceeded at the same time as alcoholic fermentation and the “piqure lactique” occurred. In the modified method, no malolactic fermentation took place. In all musts, a fundamentally heterofermentative lactic flora was found, with Lactobacillus species most abundant in the traditional process.     

Quantitative duplex real-time polymerase chain reaction assay with TaqMan probe detects and quantifies Crocodylus porosus in food chain and traditional medicines

Consumption and exploitation of crocodiles have been rampant for their exotic, nutritive and medicinal attributes. These depredations are alarming and although they have continued to be monitored by wildlife and conservation agencies, unlawful trading of crocodiles shows an increasing trend worldwide. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays for crocodile have been documented but they are only suitable for identification and cannot quantify adulterations. We described here a quantitative duplex real-time PCR assay with probes to quantify contributions from Crocodylus porosus materials simultaneously. A very short amplicon size of 127bp was used because longer targets could have been broken down in samples, bringing considerable uncertainty in molecular analysis. We have validated a TaqMan probe-based duplex real-time PCR (qPCR) assay for the detection of 0.004 ng DNA in pure state and 0.1% target meat in model chicken meatball. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 12 model chicken meatballs adulterated with C. porosus reflected 96.3?120.2% target recovery at 0.1?10% adulterations. A validation test of 21 commercial food and traditional medicine (TM) crocodile-based products showed 100% effectiveness. Short amplicon sizes, alternative complementary target, exceptional stability and superior sensitivity suggested the assay could be used for the identification and quantitative determination of C. porosus in any food or TM samples even under degraded conditions.     

Multi-Loci Gene Sequencing and Identification of Bifidobacteria Strains Isolated from Dairy and Pharmaceutical Sources in South Africa

The 16S rRNA gene sequence analysis of Bifidobacterium species reveals high interspecies sequence similarity in the range of 87.7–99.5%. This study illustrated the extent of superiority of a multigenic approach, involving protein-coding genes, in comparison to the 16S rRNA gene, to precisely delineate presumptive Bifidobacterium isolates obtained from probiotic milk beverages, culture collections and pharmaceutical probiotic preparations. Oligonucleotide pairs PurF-rev/PurF-uni; RpoC-uni/RpoC-rev; DnaB-uni/DnaB-rev; DnaG-uni/DnaG-rev; and ClpC-uni/ClpC-rev amplified housekeeping genes while 27F/1492R amplified the 16S rRNA gene of the presumptive bifidobacteria in a polymerase chain reaction. Sequences of 16S rRNA gene and some protein-coding genes effectively identified the isolates. Phylogenetic analysis together with concatenation showed that clpC, purF and dnaG genes had over 8-fold better discriminatory power than the 16S rRNA gene in discriminating between Bifidobacterium isolates. However, phylogenetic analysis involving dnaB and rpoC gene sequences or their concatenated trees showed discrepancies in clustering isolates with designated type strains.     

Genetic differentiation between sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) by PCR–RFLP analysis of a 12S rRNA gene fragment

PCR–RFLP analysis was applied to the identification of two closely related flatfish species: sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides). Amplification of DNA isolated from fish muscle samples was carried out using a set of primers flanking a region of 321 base pairs (bp) from the mitochondrial 12S rRNA gene. Restriction endonuclease analysis based on sequence data of this DNA fragment revealed the presence of polymorphic sites for AciI and MwoI endonucleases. The restriction profiles obtained by agarose gel electrophoresis when amplicons were cut with AciI and MwoI enzymes allowed the unequivocal identification of sole and Greenland halibut species. © 2000 Society of Chemical Industry