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New developments in the study of the microbiota of naturally fermented sausages as determined by molecular methods: a review.

The microflora of different types of fermented sausages has been defined by isolation and biochemical identification of the microorganisms commonly found in these products. It is generally agreed that the main microbial groups involved in such products are lactic acid bacteria and coagulase-negative cocci. In addition, and depending on the product, other groups may play a role, such as yeasts and enterococci. Since it appears that the types of microbial groups, or even the specific strains of a given microbial group, that dominate the fermentation, significantly affect the organoleptic profile of the final product, there is an increasing interest in the description of the microbiota that are found in different fermented sausages. More recently, new tools, based on molecular methods, allowing fast and unequivocal identification of strains, isolated from fermented sausages, became available. These methods have been successfully applied and, in general, biochemical and molecular identification compared well. However, new information comes to light when molecular methods are applied to DNA and/or RNA extracted directly from sausages. This approach eliminates problems related to traditional isolation. This review deals with the recent findings and results of the application of molecular methods, in a culture-dependent and culture-independent manner, on the study of the microflora of fermented sausages.

Animals↗

Application of fluorescence in situ hybridisation (FISH) to the analysis of yeast population dynamics in winery and laboratory grape must fermentations.

To analyse the yeast population diversity during wine fermentations, specific fluorescein-labelled oligonucleotide probes targeted to the D1/D2 region of the 26S rRNA of different yeast species known to occur frequently in this environment were designed and tested with reference strains. The probes were then used to identify wine must isolates and to follow, in combination with plate counts, the evolution of yeast populations in two winery fermentations of white and red grape musts. In both cases, a high diversity of non-Saccharomyces yeast species was detected, including Candida stellata, Hanseniaspora uvarum, H. guilliermondii, Kluyveromyces marxianus, K. thermotolerans and Torulaspora delbrueckii. Some of these species (e.g., K. marxianus, K. thermotolerans and T. delbrueckii) were present in significant amounts during the tumultuous fermentation stage, despite the predominance of Saccharomyces cerevisiae cells following the inoculation of the wine musts with a starter strain. To further clarify the yeast population dynamics at the late phase of the fermentations, and because winery conditions do not allow a reliable control of experimental variables, strains isolated from the industrial musts were used to conduct two laboratory microvinifications in synthetic grape juice, using different ratios of S. cerevisiae/non-Saccharomyces in the inocula. Under these conditions, the results were similar to those obtained in the winery, showing a yeast profile with mixed species throughout the first fermentation stage, i.e. until about 40-50% of the total sugar was consumed. Non-Saccharomyces yeasts were outgrown by S. cerevisiae only after ethanol reached concentrations around 4-5% (v/v), which argues in favour of a potential important role of non-Saccharomyces in the final organoleptic characteristics of the wine.

Coculture Techniques↗

Microbiological and sensory quality of dry fermented sausages containing alginate-microencapsulated Lactobacillus reuteri.

The probiotic bacterium Lactobacillus reuteri was added to dry sausage batter, without or after being microencapsulated in alginate using either extrusion or emulsion technology. Pediococcus pentosaceus and Staphylococcus carnosus were added at 7 log cfu/g as starter cultures for fermentation. The sausage batter was stuffed in 55 mm fibrous casings and fermented, with smoking, at <or=26 degrees C and 88% relative humidity (RH) for 72 h. During fermentation as well as drying at 75% RH and 13 degrees C for 25 days, the pH and water activity (a(w)) of the sausages were measured. The numbers of the three organisms in sausages during processing were determined. A consumer taste panel study was conducted to determine if addition of L. reuteri as unencapsulated cells or in microcapsules added at 1% (w/w) affected the sensory quality of the dry fermented sausages. Changes in sausage pH from 5.8 to 4.8, and a(w) from 0.97 to 0.89, were the same for both control and microcapsule-containing sausages. The numbers of unencapsulated L. reuteri cells dropped by 2.6 log units, whereas microencapsulated L. reuteri were reduced by only <or=0.5 log unit after drying. No significant difference in sensory quality was found between control and sausages containing either unencapsulated or microencapsulated L. reuteri. Thus, microencapsulation of probiotics like L. reuteri may be an option for formulation of fermented meat products with viable heath-promoting bacteria.

Animals↗

Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum.

The effect of pH (between 5.0 and 6.3) on butyric acid fermentation of xylose by Clostridium tyrobutyricum was studied. At pH 6.3, the fermentation gave a high butyrate production of 57.9 g l(-1) with a yield of 0.38-0.59 g g(-1) xylose and a reactor productivity up to 3.19 g l(-1)h(-1). However, at low pHs (<5.7), the fermentation produced more acetate and lactate as the main products, with only a small amount of butyric acid. The metabolic shift from butyrate formation to lactate and acetate formation in the fermentation was found to be associated with changes in the activities of several key enzymes. The activities of phosphotransbutyrylase (PTB), which is the key enzyme controlling butyrate formation, and NAD-independent lactate dehydrogenase (iLDH), which catalyzes the conversion of lactate to pyruvate, were higher in cells producing mainly butyrate at pH 6.3. In contrast, cells at pH 5.0 had higher activities of phosphotransacetylase (PTA), which is the key enzyme controlling acetate formation, and lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate. Also, PTA was very sensitive to the inhibition by butyric acid. Difference in the specific metabolic rate of xylose at different pHs suggests that the balance in NADH is a key in controlling the metabolic pathway used by the cells in the fermentation.

Acetate Kinase↗

A fermentation assay to evaluate the effectiveness of antimicrobial agents on gut microflora.

The measurement of gas produced as a fermentation end product in vitro was correlated with absorbance as a measure of bacterial growth and was used as a rapid screening procedure to test the antimicrobial activity of certain essential oil and tannin secondary plant metabolites on gastrointestinal microorganisms from chickens. The assay was optimised using Clostridium perfringens and Lactobacillus fermentum, and tested in antimicrobial assays against C. perfringens; the minimum inhibitory concentration for each essential oil and condensed tannin was determined. The effect of penicillin-G on C. perfringens, in both growth and fermentation assays, was similar, and for all secondary metabolites tested, concentrations that inhibited fermentation were also bacteriocidal. The assay was also used to demonstrate the effect of dietary composition and enzyme supplementation on fermentation of mixed gut microflora in vitro; results are compared with in vivo results for the same dietary treatments. The data demonstrate that the effects of bioactive secondary plant products and feed composition on individual organisms or mixed gut microflora can be tested by analysis of fermentative activity in vitro, and that this provides a rapid assay for testing potential poultry feed additives before in vivo trials.

Animals↗

Ecology of lactic acid bacteria in Italian fermented sausages: isolation, identification and molecular characterization.

In this study, the ecology of the lactic acid bacteria (LAB) of three naturally fermented sausages produced in the Friuli-Venezia-Giulia region, in the North East of Italy, was investigated. A total of 465 strains isolated from three fermentations were identified by molecular methods and 12 different species of LAB were detected. Lactobacillus curvatus and Lactobacillus sakei were the most numerous (67 and 353 strains isolated, respectively) and they were subjected to RAPD-PCR. Clusters containing strains isolated from different plants were observed, underlining a coherent population distribution in three different fermentations. However, we also observed clusters formed by strains isolated from a specific fermentation, only. This could be explained considering the different technologies and recipes used for the production in three plants. Ingredient composition, fermentation and maturation parameters could play an important role in the selection of specific populations adapted in a specific environment.

Animals↗

Acoustical experiment of yogurt fermentation process.

One of the important factors through food manufacturing is hygienic management. Thus, food manufactures prove their hygienic activities by taking certifications like a Hazard Analysis and Critical Control Point (HACCP). This concept also applies to food monitoring. Acoustical measurements have advantage for other measurement in food monitoring because they make it possible to measure with noncontact and nondestructive. We tried to monitor lactic fermentation of yogurt by a probing sensor using a pair of acoustic transducers. Temperature of the solution changes by the reaction heat of fermentation. Consequently the sound velocity propagated through the solution also changes depending on the temperature. At the same time, the solution change its phase from liquid to gel. The transducers usage in the solution indicates the change of the temperature as the change of the phase difference between two transducers. The acoustic method has advantages of nondestructive measurement that reduces contamination of food product by measuring instrument. The sensor was inserted into milk with lactic acid bacterial stain of 19 degrees C and monitored phase retardation of propagated acoustic wave and its temperature with thermocouples in the mild. The monitoring result of fermentation from milk to Caspian Sea yogurt by the acoustic transducers with the frequency of 3.7 MHz started to show gradient change in temperature caused by reaction heat of fermentation but stop the gradient change at the end although the temperature still change. The gradient change stopped its change because of phase change from liquid to gel. The present method will be able to measure indirectly by setting transducers outside of the measuring object. This noncontact sensing method will have great advantage of reduces risk of food contamination from measuring instrument because the measurement probes are set out of fermentation reactor or food containers. Our proposed method will contribute to the hygienization for the food manufacture industry.

Acoustics↗

Direct capture of product from fermentation broth using a cell-repelling ion exchanger.

A new technique for treating anion exchangers has been proposed allowing direct capture of the fermentation product, shikimic acid directly from the cell-containing fermentation broth. A layer of hydrophilic polymer, poly(acrylic acid) (PAA) has been physically adsorbed on the anion exchanger followed by a covalent cross-linking of PAA. The PAA layer is penetrable for small molecules despite being negatively charged as PAA is, but the polymer layer repels large negatively charged structures like cell debris and cells preventing them from adsorption to the chromatographic matrix. The binding capacity for pure shikimic was about 81 mg/ml adsorbent for both cross-linked PAA-Amberlite and native Amberlite in the fluidized mode of column operation. Binding capacity dropped to 17 and 15 mg per ml adsorbent, respectively, when using filtrated fermentation broth and to about 10 mg/ml adsorbent for cross-linked PAA-Amberlite when using directly the fermentation broth containing cells. Native Amberlite cannot be used for the direct capture of shikimic acid due to the immediate clogging of the column and the collapse of the expanded bed. The cross-linked PAA-Amberlite was used repeatedly for the direct adsorption of shikimic acid from the industrial fermentation broth.

Acrylic Resins↗

Comparison of isotopic fractionation in lactic acid and ethanol fermentations.

Pure D(-) and L(+) enantiomers of lactic acid were prepared by fermentation reactions with specific bacteria. In addition, naturally deuterated ethanol was prepared and converted into diastereoisomers using mandelic acid. Various sugars and nutrients were fermented into lactic acid in water having different deuterium contents and ethanol samples were obtained from yeast fermentation of sugars from different botanical origins. The methine and methylene groups in lactic acid and ethanol respectively show similar deuterium contents which are related to that found in the fermentation water. However, the methyl groups of both molecules are significantly different whatever the botanical origin of the carbon source in the fermentation medium.

Biotransformation↗

A new selective medium for the isolation of glucose non-fermenting bifidobacteria from hen caeca.

Avian caeca contain a large and diverse population of bacteria. Certain genera, including Bifidobacterium, are thought to exert health-promoting effects. Two media were evaluated to determine their sensitivity and selectivity for bifidobacteria in the hen caecal samples: modified Wilkins-Chalgren agar (MW; Oxoid) with the addition of glacial acetic acid (1 ml/l) and mupirocin (100 mg/l) and modified TPY agar (MTPY; ADSA, Spain) with glacial acetic acid (1 ml/l) and mupirocin (100 mg/l). The colonies arising on the plates inoculated with caecal samples were Gram stained, screened for the presence of fructose-6-phosphate phosphoketolase activity, and tested for fermentation patterns using ANAEROtest (Lachema, Czech Republic) and API 50 CHL (BioMérieux, France) kits. Both agars were selective for bifidobacteria, however, MTPY agar showed higher cfu/g than MW agar. Bifidobacterial counts were higher than 10(10) cfu/g of caecal contents using MTPY agar. Most of strains isolated from this medium fermented melibiose, sucrose, and raffinose, but not glucose. Soya peptone (5 g/l; Oxoid) stimulated the growth of glucose non-fermenting strains in complex liquid media. The results suggest that the media for selective enumeration and isolation of bifidobacteria in poultry caecal samples should not contain glucose as the sole carbon source. It can be concluded that MTPY medium is highly selective and permits the growth of both glucose fermenting and glucose non-fermenting bifidobacteria.

Animals↗

Biochemical characterization of lactic acid bacteria isolated from spontaneous fermentation of 'Almagro' eggplants.

A total of 149 strains of lactic acid bacteria isolated from the spontaneous fermentation of 'Almagro' eggplants were characterized and identified. Of the isolates, 148 were determined as belonging to the genus Lactobacillus. A coccoid, gram-positive database-negative isolate was obtained in the early stages of fermentation. The Lactobacillus strains were divided into six groups based on sugar fermentation patterns and other physiological and morphological characteristics, and were identified as Lactobacillus plantarum biotype 1 (54.4%), Lactobacillus brevis biotype 2 (19.5%), Lactobacillus fermentum (9.4%), Lactobacillus brevis biotype 3 (5.4%), Lactobacillus pentosus (4.7%) and nine strains, which were not included in the previous species, were grouped as Lactobacillus spp. (6.0%). Fermentation was initiated by Lactobacillus brevis biotype 2 and Lactobacillus fermentum. During the fermentation Lactobacillus plantarum became the predominant species.

Cluster Analysis↗

A Chinese fermented soybean food.

Sufu or furu is a fermented soybean product originating in China. It is a cheese-like product with a spreadable creamy consistency and a pronounced flavour. Sufu is a popular side dish consumed mainly with breakfast rice or steamed bread. It has a long history and written records date back to the Wei Dynasty (220-265 AD). Sufu is made by fungal solid state fermentation of tofu (soybean curd) followed by aging in brine containing salt and alcohol. The present review is based on scientific data published in Chinese and international sources. Several types of sufu can be distinguished, according to processing method or according to colour and flavour. Choice of processing can result in mould fermented sufu, naturally fermented sufu, bacterial fermented sufu, or enzymatically ripened sufu. Depending on the choice of dressing mixture, red, white or grey sufu may be obtained. The stages of the process are discussed and include the preparation of tofu, the preparation of pehtze, salting and ripening. Fungal starters include Actinomucor spp., Mucor spp. and Rhizopus spp. The chemical composition is discussed with particular reference to the proximate composition, the amino acid content and profile, as well as the volatile flavour components of various types of sufu.

Biotechnology↗

Field survey and literature review on traditional fermented milk products of Ethiopia.

The wide variety and the socio-economic and dietary importance of traditional fermented milk products of Ethiopia are discussed in this paper. Information on the microbiology of these products is sparse and has relevance to those organisms associated with spoilage and to those considered desirable for fermentation. There is a clear need to improve the production of African foods and beverages [Int. J. Food Microbiol. 18 (1993) 85]. The objective of this review was to document traditional technology used and information on the microbiology of the products, and to identify various constraints to the development and commercialisation of fermented milk products. Thereby the major problems and potential areas for improvement are pointed out. Ergo, the most important traditional product resembles yoghurt and, as the other traditional products, is prepared by "spontaneous" fermentation, commonly initiated by either "back slopping" or by repeated use of the same utensil. Other products include traditional fermented curd or ititu, traditional butter or kibe, neter kibe or traditional ghee, ayib resembling cottage cheese, arrera or defatted buttermilk and augat or traditional whey.

Animals↗

New efficient amylase-producing strains of Lactobacillus plantarum and L. fermentum isolated from different Nigerian traditional fermented foods.

Amylolytic lactic acid bacteria (ALAB) were isolated from Nigerian traditional fermented foods (fufu, burukutu, ogi-baba and kunu-zakki) with the aim of selecting efficient amylase-producing strains. Nine isolates were characterized on the basis of their phenotypic and taxo-molecular characteristics. Three groups could be distinguished by their fermentation profiles and this was confirmed by DNA restriction analysis. Though fermentation profiles gave good identification of strain K9 (unique representative of group III) as Lactobacillus fermentum, they could not be used to ascertain the taxonomic position of strains of groups I and II. Analysis of partial 16S rRNA sequences led to the identification of these groups as L. plantarum strains and confirmed the species of strain K9 as L. fermentum. The two distinct phenotypic groups of L. plantarum differed in their use of D-xylose, L-arabinose, melibiose and were different from the previously described amylolytic L. plantarum A6 isolated from retted cassava in Congo. L. fermentum K9 was different from L. fermentum OgiE1 and Mw2 isolated from Benin maize sourdough and it is the first amylolytic L. fermentum described from Nigerian fermented products. Enzymatic profiles showed some differences between the strains of a similar fermentation group. One of the most relevant characteristics of the isolates was a higher yield of amylase production than those reported for previously described ALAB grown under the same conditions. Furthermore, all isolates were tolerant to an exposure at pH 2 and to bile salts.

Amylases↗

Effect of freezing on the spontaneous fermentation and sensory attributes of Almagro eggplants.

A study of the physico-chemical and microbiological processes taking place during the spontaneous fermentation of eggplant fruits that had been frozen after blanching revealed a delay in the onset of the active fermentation stage, which has been ascribed to the adverse effect of the freezing temperature employed on the lactic acid bacteria involved in fermentation. The species most affected by freezing were L. fermentum and L. brevis biotype 2. Despite this finding, the pattern of fermentation was similar to the fermentation process in unfrozen eggplants. Freezing of the fruits did not significantly affect the sensory acceptability of the product and, in fact, in some cases, Almagro eggplants made from frozen fruits were preferred to commercially manufactured products.

Fermentation↗

Fermented soymilk with a monoculture of Lactococcus lactis.

Lactococcus lactis strain (LL3) isolated from mothers' milk was used to produce fermented soymilk. The strain survived at levels of over 7 log cfu/ml for 3 weeks in the fermented soymilk. A consumer survey was carried out to compare the acceptability of the fermented product with a similar product made with L. lactis ATCC11545 originally isolated from cow's milk. Blind samples produced by fermentation with the two strains were rated equally attractive, whereas information on the origin of the strains significantly enhanced the pleasantness of the fermented soymilk.

Animals↗

Biogenic amines in dry fermented sausages: a review.

Biogenic amines are compounds commonly present in living organisms in which they are responsible for many essential functions. They can be naturally present in many foods such as fruits and vegetables, meat, fish, chocolate and milk, but they can also be produced in high amounts by microorganisms through the activity of amino acid decarboxylases. Excessive consumption of these amines can be of health concern because their not equilibrate assumption in human organism can generate different degrees of diseases determined by their action on nervous, gastric and intestinal systems and blood pressure. High microbial counts, which characterise fermented foods, often unavoidably lead to considerable accumulation of biogenic amines, especially tyramine, 2-phenylethylamine, tryptamine, cadaverine, putrescine and histamine. However, great fluctuations of amine content are reported in the same type of product. These differences depend on many variables: the quali-quantitative composition of microbial microflora, the chemico-physical variables, the hygienic procedure adopted during production, and the availability of precursors. Dry fermented sausages are worldwide diffused fermented meat products that can be a source of biogenic amines. Even in the absence of specific rules and regulations regarding the presence of these compounds in sausages and other fermented products, an increasing attention is given to biogenic amines, especially in relation to the higher number of consumers with enhanced sensitivity to biogenic amines determined by the inhibition of the action of amino oxidases, the enzymes involved in the detoxification of these substances. The aim of this paper is to give an overview on the presence of these compounds in dry fermented sausages and to discuss the most important factors influencing their accumulation. These include process and implicit factors as well as the role of starter and nonstarter microflora growing in the different steps of sausage production. Moreover, the role of microorganisms with amino oxidase activity as starter cultures to control or reduce the accumulation of biogenic amines during ripening and storage of sausages is discussed.

Amine Oxidase (Copper-Containing)↗

The microbial ecology of cocoa bean fermentations in Indonesia.

Cocoa beans are the principal raw material of chocolate manufacture. The beans are subject to a microbial fermentation as the first stage in chocolate production. The microbial ecology of bean fermentation (Forastero and Trinitario cultivars) was investigated at three commercial fermentaries in East Java, Indonesia by determining the populations of individual species at 12-h intervals throughout the process. The first 2-3 days of fermentation were characterised by the successional growth of various species of filamentous fungi, yeasts, lactic acid bacteria and acetic acid bacteria. The principal species found were Penicillium citrinum, an unidentified basidiomycete, Kloeckera apis, Saccharomyces cerevisiae, Candida tropicalis, Lactobacillus cellobiosus, Lactobacillus plantarum and Acetobacter pasteurianus. The later stages of fermentation were dominated by the presence of Bacillus species, mostly, Bacillus pumilus and Bacillus licheniformis. Glucose, fructose, sucrose and citric acid of the bean pulp were utilised during fermentation, with the production of ethanol, acetic acid and lactic acid that diffused into the beans. The filamentous fungi were notable for their production of polygalacturonase activity and probably contributed to the degradation of bean pulp.

Bacillus↗