Effect of heat treatment on the antimicrobial properties of tef dough, injera, kocho and aradisame and the fate of selected pathogens

A known population from each of a 24h culture of Bacillus cereus, Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Klebsiella spp. and Staphylococcus aureus was inoculated into tef flour–water/kocho–water mixtures in screw-capped flasks and allowed to ferment for 30h at room temperature (18–21°C). The flasks were then heat-treated. Cultures of the test bacteria were inoculated into tubes containing graded volumes of 30-h-fermented tef dough/kocho extracts which had been heat-treated at 45, 61 and 80°C in assay broth containing aqueous extracts from injera and aradisame. They were incubated for 24h at 32°C and optical densities determined. Populations of the major indigenous bacteria, yeasts and moulds in fermented tef dough (30h), kocho samples, injera and aradisame were determined from other control portions of the same samples. Higher temperature (80°C) heat-treatment promoted the inhibitory potential of extracts from doughs of both foods as compared with lower temperature heat-treatments (45 and 61°C). Asporogenous test bacteria were affected more than the spore-formers. Better efficacy of extracts from injera and aradisame suggested improved antimicrobial properties of the baked products than in doughs. Heat of baking inactivated all vegetative cells although spores of B.cereus, the yeasts and moulds survived the heat (100°C) applied for 5min. The c.f.u./g of food for B. cereus was below the disease-causing level (0.5×101 and 1.5×103, in injera and aradisame, respectively). Actual baking temperatures in homes are higher than the ones used here; if post-baking contamination is minimized or prevented, the products would be microbiologically safe with respect to the asporogenous pathogens when served fresh. Further studies on aflatoxins and improved storage conditions for kocho are recommended.     

Experiences with the use of a starter culture in the fermentation of maize for ‘kenkey’ production in Ghana

Controlled fermentation of maize was carried out using six strains of Lactobacillus fermentum and one strain of yeast, Saccharomyces cerevisiae, isolated from traditionally fermented maize dough as starter cultures for inoculum enrichement. The fermentations were monitored by pH, acidity, microbiological analysis and taste panel evaluation of two products, kenkey and koko, prepared from the fermented doughs. The strains of L. fermentum used as starter culture dominated the microflora during fermentation and in most inoculated doughs the required pH was attained by 24 h instead of 48 h of dough fermentation. Higher contents of lactic acid bacteria and yeasts were observed in inoculated doughs at the initial stages of fermentation but the spontaneously fermented doughs attained similar lactic acid bacteria and yeasts counts by 24 h of dough fermentation. The organoleptic quality of kenkey and koko prepared from doughs fermented with starter culture for 48 h was not significantly different from the traditional products. Kenkey prepared from doughs fermented for 24 h with starter culture were found to be unacceptable by the taste panel although similarly produced koko was acceptable.The authors are with the Food Research Institute, Council for Scientific and Industrial Research, P.O Box M 20. Accra, Ghana.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27·5°C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10⁴ spores/g whereas with 800 g loaves survival occurred with doughs containing 5·0 times 10³ spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0·2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

The effect of fermentation on the growth and survival ofSalmonella typhimurium,Staphylococcus aureus,Bacillus cereus andPseudomonas aeruginosa in fermenting tef (Eragrostis tef)

Summary Growth ofSalmonella typhimurium, Staphylococcus aureus, Bacillus cereus andPseudomonas aeruginosa was inhibited when the pH of fermenting tef approached 5.0, 5.0, 5.5 and 5.0, respectively. However the test organisms grew in far more acidic conditions in broth than in fermenting tef and this is due to antimicrobial substance(s) being produced by some of the lactic acid bacteria. Except forBacillus cereus spores, all the test organisms were heat-inactivated during the baking process of the final tef injera.

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Effet de la fermentation sur la croissance et la survie de Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus et Pseudomonas aeruginosa dans le tef (Eragrostis tef) en fermentation

Résumé La croissance deSalmonella typhimurium, deStaphylococcus aureus, deBacillus cereus et dePseudomonas aeruginosa est inhibée lorsque les pH du tef en fermentation approchent respectivement 5:0, 5.0, 5.5 et 5.0. Toutefois, les organismes tests croissent dans des conditiones bien plus acides que dans le tef en fermentation. Ceci est dû à des substances antimicrobiennes produites par certaines bactéries lactiques. A l'exception des spores deBacillus cereus, tous les organismes tests sont inactivés par la chaleur durant le processus de cuisson.

     

A polyphasic study on the taxonomic position of industrial sour dough yeasts

The sour dough bread making process is extensively used to produce wholesome palatable rye bread. The process is traditionally done using a back-slopping procedure. Traditional sour doughs in Finland comprise of lactic acid bacteria and yeasts. The yeasts present in these doughs have been enriched in the doughs due to their metabolic activities, e.g. acid tolerance. We characterized the yeasts in five major sour bread bakeries in Finland. We found that most of the commercial sour doughs contained yeasts which were similar to Candida milleri on the basis of 18S rDNA and EF-3 PCR-RFLP patterns and metabolic activities. Some of the bakery yeasts exhibited extensive karyotype polymorphism. The minimum growth temperature was 8 degrees C for C. milleri and also for most of sour dough yeasts.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27.5 degrees C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10(4) spores/g whereas with 800 g loaves survival occurred with doughs containing 5.0 X 10(3) spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0.2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

The contribution of moulds and yeasts to the fermentation of 'agbelima' cassava dough

Agbelima, a fermented cassava meal widely consumed in Ghana, Togo and Benin, is produced by fermenting grated cassava with one of several types of traditional cassava dough inoculum. During fermentation a smooth textured sour dough is produced, the toxicity of cassava is reduced and there is a build up of volatile aroma compounds. Four types of inocula were included in the present investigation. In one type moulds were found to form a dominant part of the microbiota, the species present being Penicillium sclerotiorum, P. citrinum, P. nodulum, Geotrichum candidum and a basidiomycete. All these moulds were found to possess cellulase activity which was responsible for the hydrolysis of cassava tuber cellulose during fermentation leading to a breakdown of the coarse texture of cassava dough. The yeasts Candida krusei, C. tropicalis and Zygosaccharomyces spp. were present in high numbers in the four types of inocula including themouldy inoculum. The yeasts C. tropicalis and some strains of Zygosaccharomyces, all ofwhich possessed cellulase activity, were also found to contribute to the modification of cassavatexture during fermentation. All yeasts and moulds exhibited linamarase activity and were therefore capable of breaking down the cyanogenic glucosides present in cassava.     

A note on the leavening activity of yeasts isolated from Nigerian palm wine

The role of the yeast flora of Nigerian palm wine in the leavening activity of the beverage was investigated by subjecting organisms from the wine to dough-raising tests. Those with appreciable leavening activity were identified as Saccharomyces cerevisiae and Candida spp. They produced maximum dough volumes in 3–4 h at 37°C. The study has provided experimental evidence that yeasts contribute to the leavening activity of palm wine and has identified strains which have potential utility in commercial bread baking.     

Randomly amplified polymorphic DNA (RAPD) for discrimination of Pediococcus pentosaceus and Ped. acidilactici and rapid grouping of Pediococcus isolates

In a taxonomic study of the known Pediococcus species, together with 116 isolates from fermenting tef dough and fermented kocho using a PCR-based RAPD procedure, all the different species developed well differentiated agarose gel electrophoresis profiles. Analyses of the images with the Pearson product moment correlation coefficient (r) and upgma clustering algorithm in the GelCompar version 4·0 software gave a distinct identification scheme within and between type strains and food isolates of Pediococcus species. The procedure is simple, rapid for grouping of isolates, applicable to all species of pediococci and particularly useful for differentiating between strains of Ped. pentosaceus and Ped. acidilactici .     

Amylase production by a Gram-positive bacterium isolated from fermenting tef (Eragrostis tef)

Bacillus sp. A-001, which produced large amounts of amylase, was isolated from fermenting tef ( Eragrostis tef ) on tryptone soya agar supplemented with 1% starch. The organism grew between pH 4.5 and 10.5 with an optimum at 7–7.5. Growth occurred between 20 and 55°C but the optimum was about 35–40°C. At optimum medium pH (7.5) and a temperature of 35°C the organism entered the stationary phase after about 72 h and amylase production was at its highest (9.6 units ml^-1) at this time. Enzyme activity was optimal at pH 5.5 and 40°C and showed good thermal stability; it required 110 min to lose 50% of its activity at 70°C. The enzyme hydrolysed native starch (flour from tef, corn and kocho) to various oligosaccharides, including maltotriose, maltose and glucose.     

Multi Fermentation Screening System (MFSS): Computerized simultaneous evaluation of carbon dioxide production in twenty-four yeasted broths or doughs

Multi Fermentation Screening System (MFSS) is a 24-channel computerized instrument for the study of yeast anaerobic fermentation in broths or bread doughs. The evolved carbon dioxide is registered as the increasing pressure at a constant volume. MFSS works with either 1.52g doughs or 1.2–2.4 ml broths, which are kept in a commercially available multidish during the measurement. By the use of one stainless steel ball-bearing in each chamber, and by attaching the multidish to a rotary shaker the doughs are kneaded, or the broths are kept homogenous. MFSS was performed in order to determine the reproducibility between the channels, the repeatability of dough fermentation in triplicate, the possibility to work under sterile conditions, the ability to work at measurement temperatures ranging from 5 to 45°C, and the suitable volumes of yeasted broths. The reproducibility is illulstrated by a SD of less than ±1.90%, and the repeatability by a SD of less than ±3.40% in dough measurements at 30°C, while the repeatability was even better for the 2.4 ml broths, as illustrated by a SD of less than ±1.70%.     

Sources of microorganisms in pozol,a traditional Mexican fermented maize dough

Freshly prepared pozol, a traditional Mexican fermented maize dough, contained (c.f.u./g wet wt): lactic acid bacteria, 10⁴ to 10⁶; aerobic mesophiles, 10⁴ to 10⁵; Enterobacteriaceae, 10² to 10³; yeasts, 10² to 10⁴; and mould propagules, <10³. After 30 h at 28°C the numbers were, respectively: 10⁹, 7×10⁶, 5×10⁵, 10⁶ and 10⁴. Soaking alkali-treated grains overnight allowed lactic acid bacteria, aerobic mesophiles and Enterobacteriaceae to grow and these then constituted the primary microbial flora of the pozol dough. Grinding in a commercial mill inoculated the dough with lactic acid bacteria, aerobic mesophiles, Enterobacteriaceae and yeasts. Other processing stages, including the nature of the surface upon which the balls were made, handling of the dough, and air, contributed only minor numbers of microbes compared with the two major sources, soaking and grinding. The pH of pozol fell from an initial value of 7.3 to 4.6 after 30 h incubation at 28°C. The numbers of Enterobacteriaceae and other aerobic mesophilic bacteria remained constant between 11 and 30 h incubation and there was no evidence of the acidic conditions having any lethal effects on these organisms.     

Leavening ability and freeze tolerance of yeasts isolated from traditional corn and rye bread doughs.

Strains of Saccharomyces cerevisiae and Torulaspora delbrueckii isolated from traditional bread doughs displayed dough-raising capacities similar to the ones found in baker's yeasts. During storage of frozen doughs, strains of T. delbrueckii (IGC 5321, IGC 5323, and IGC 4478) presented approximately the same leavening ability for 30 days. Cell viability was not significantly affected by freezing, but when the dough was submitted to a bulk fermentation before being stored at -20 degrees C, there was a decrease in the survival ratio which depended on the yeast strain. Furthermore, the leavening ability after 4 days of storage decreased as the prefermentation period of the dough before freezing increased, except for strains IGC 5321 and IGC 5323. These two strains retained their fermentative activity after 15 days of storage and 2.5 h of prefermentation, despite showing a reduction of viable cells under the same conditions. The intracellular trehalose content was higher than 20% (wt/wt) in four of the yeasts tested: the two commercial strains of baker's yeast (S. cerevisiae IGC 5325 and IGC 5326) and the two mentioned strains of T. delbrueckii (IGC 5321 and IGC 5323). However, the strains of S. cerevisiae were clearly more susceptible to freezing damages, indicating that other factors may contribute to the freeze tolerance of these yeasts.     

Application of Cell-Surface Engineering for Visualization of Yeast in Bread Dough: Development of a Fluorescent Bio-Imaging Technique in the Mixing Process of Dough

Cell-surface engineering (Ueda et al., 2000) has been applied to develop a novel technique to visualize yeast in bread dough. Enhanced green fluorescent protein (EGFP) was bonded to the surface of yeast cells, and 0.5% EGFP yeasts were mixed into the dough samples at four different mixing stages. The samples were placed on a cryostat at ?30 °C and sliced at 10 μm. The sliced samples were observed at an excitation wavelength of 480 nm and a fluorescent wavelength of 520 nm. The results indicated that the combination of the EGFP-displayed yeasts, rapid freezing, and cryo-sectioning made it possible to visualize 2-D distribution of yeast in bread dough to the extent that the EGFP yeasts could be clearly distinguished from the auto-fluorescent background of bread dough.     

Significance of Molecular Identification and Antifungal Susceptibility of Clinically Significant Yeasts and Moulds in a Global Antifungal Surveillance Programme

The increasing diversity of opportunistic fungi causing serious invasive fungal infections (IFI) has been documented. Accurate identification (ID) is important in guiding therapy, determining prognosis for IFIs and in epidemiological surveys. We assessed the utility of PCR-based methods for the ID of yeasts and moulds that either were uncommon, failed conventional ID, or represented unusual biochemical or phenotypic profiles of common species. Among 1,790 viable fungal clinical isolates received during the SENTRY Program in 2010, 322 strains from 40 study sites had ID confirmed by molecular methods. Isolates were previously identified in participant institutions. Yeasts that were not confirmed by morphology on CHROMagar, growth at 45?°C (Candida albicans/dubliniensis), or assimilation of trehalose (C. glabrata) as well as non-Candida yeasts and all moulds were amplified and sequenced using primers amplifying one or more of the following genes: ITS, 28S, β-tubulin (Aspergillus spp.), TEF (Fusarium spp.), IGS (Trichosporon spp.). The isolates selected for molecular ID included 149 isolates of Candida species, 77 of Aspergillus species, 73 non-Candida yeasts, and 23 other moulds (a total of 41 different species). Overall, the ID determined by the submitting site was confirmed for 189 isolates (58.7?%): Aspergillus spp. (64.1?% correct); Candida spp. (60.1?% correct); non-Candida yeasts (58.9?% correct); non-Aspergillus moulds (30.4?% correct). Species with high levels of concordance between conventional and molecular ID included A. fumigatus (95.0 %), C. lusitaniae (100?%), C. dubliniensis (92.3?%), C. kefyr (100?%), and C. neoformans (90.2?%). Only 50.0?% of isolates of C. albicans and 59.1?% of C. glabrata selected due to unusual phenotypic or biochemical features were found to be correctly identified by the submitting site. Molecular methods for the identification of fungal pathogens are an important adjunct to the conventional identification of many less common clinically relevant yeasts and moulds including species of Candida with unusual or erroneous phenotypic or biochemical profiles. Molecular confirmation of fungal identification is essential in epidemiological surveys such as SENTRY.     

The antimicrobial activity of extracts of the lichen Cladonia foliacea and its (-)-usnic acid, atranorin, and fumarprotocetraric acid constituents

The antimicrobial activity of the chloroform, diethyl ether, acetone, petroleum ether, and ethanol extracts of the lichen Cladonia foliacea and its (-)-usnic acid, atranorin, and fumarprotocetraric acid constituents against 9 bacteria and fungi has been investigated. The extracts and pure compounds alone were found active against the same bacteria and the same yeasts. Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, Proteus vulgaris, Listeria monocytogenes, Aeromonas hydrophila, Candida albicans, and Candida glabrata growth were inhibited. In addition, the MICs of the extracts, (-)-usnic acid, atranorin and fumarprotocetraric acid were determined.     

Effect of Microalgae (Arthrospira platensis and Chlorella vulgaris) Addition on 3D Printed Cookies

Rheological and textural characteristics of cookie doughs were measured to characterise the effect of two microalgae biomasses additions (Arthrospira platensis and Chlorella vulgaris) in 3D printed cookies. The rheological characteristics determined the addition of microalgae lead to a greater mechanical resistance and a predominance of the elastic component ahead of the viscous component, this behaviour was confirmed by the textural analysis that showed a greater force for the extrusion of microalgae-enriched doughs. Besides, the influence of processing parameters, including filament diameter (26 and 27 mm) and layer height (1.3 and 1.5 mm), on the geometric accuracy of a 3D printed food structure made of cookie dough before and after baking process, was evaluated. The addition of microalgae biomass in dough, for 3D printed cookies, improves the printability in terms of dimensional properties, achieving 3D structures more stable and resistant to baking.

     

The microbiological quality of hot water-washed broccoli florets and cut green beans

AIMS: To determine the effect of hot water washing on the microbiological quality of cut broccoli florets and trimmed green beans. METHODS AND RESULTS: Broccoli florets and trimmed beans were washed for 90 s in tap water at either 20 degrees C or 52 degrees C and stored at 7 and 10 degrees C. The numbers of naturally occurring aerobic mesophilic organisms, Pseudomonas spp., Enterobacteriaceae, yeast and moulds and lactobacilli or lactic acid bacteria were enumerated at intervals for up to 2 weeks. The ability of Listeria monocytogenes, Bacillus cereus and Escherichia coli O157:H7 inoculated onto the tissue post heat treatment to survive or grow was also measured to mimic the effect of postprocess contamination. Using a hot wash treatment improved the initial appearance of the vegetables and resulted in a small, but significant, reduction in populations of all groups of endogenous flora measured. The number of yeast and moulds on the vegetables washed at 52 degrees C remained below the levels observed on the 20 degrees C washed vegetables throughout the observation period, but Pseudomonas spp., lactobacilli and Enterobacteriaceae were better able to grow on the hot-washed vegetables such that the counts at the end of storage were greater on hot-washed than ambient-washed vegetables. All three of the pathogens tested were better able to grow on hot-washed broccoli and beans than on equivalent product washed at 20 degrees C. CONCLUSIONS: Hot water washing can be used to control enzymic browning or yeast and moulds growth but it can also allow more rapid and extensive growth by pathogens and spoilage organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Reduced protection against growth by pathogens means that the hot wash treatment of vegetables should be used with caution and requires careful assessment of risk.     

Rheological and breadmaking properties of wheat samples infected with<Emphasis Type="Italic">Fusarium spp.</Emphasis>

Rheological and breadmaking properties of untreated and suboptimally stored wheat samples (grain moisture: 20%, temperature: 20°C) and also of wheat which was inoculated withFusarium spp. were investigated. The deoxynivalenol (DON) content of the stored and inoculated wheat samples ranged between 820–12,000 μg/kg. Gluten proteins were isolated with different extraction solutions and the fractions obtained were analysed by means of RP-HPLC. Microextension tests and micro-baking tests were used for the determination of dough properties (maximum resistance (MR) and extensibility (EX)) and bread volume, respectively. In spite of the extremely high DON concentrations of some wheat samples contaminated withFusarium spp. they showed only a slight decrease of the amount of gluten proteins. Extension tests of dough led to a slight decrease of MR, bread volumes stayed almost the same compared with the non-contaminated grain. The contamination of wheat withAspergillus andPenicillium led to a high decrease of gluten proteins, which resulted in an extremely decreased MR of the dough and a very low bread volume.     

Microbiology of pozol,a Mexican fermented maize dough

Mexican fermented maize dough, pozol, including traditional banana leaf-wrapped samples and material in plastic bags, was purchased. All samples were pH 4.7 to 5.7 approx. 12 h after preparation, pH declining to 3.6 to 3.9 after 6 to 9 days storage at ambient temperature. These latter samples had dry matter contents of 31% to 48% (w/w), 0.35% to 0.75% titratable acidity as lactic acid and lactic acid bacteria as predominant microbial flora at about 10⁸ c.f.u./ml. The lactic acid bacteria included strains of Leuconostoc mesenteroides, Lactobacillus plantarum, Lactobacillus confusus, Lactococcus lactis and Lactococcus raffinolactis. Fungi were not found in the samples stored in plastic bags. The samples wrapped in banana leaf, however, developed a large surface mycoflora within 2 days. This included Geotrichum candidum, yeasts and moulds. The majority of the lactic acid bacteria and approx. 50% of yeasts hydrolysed starch to some extent. No Geotrichum isolate hydrolysed starch. Lactate was assimilated by all the Geotrichum isolates and by 17 of 39 yeast strains.