M.J. Robert Nout

Biogenic amine formation and degradation by potential fish silage starter microorganisms

Abstract Fish waste can be advantageously upgraded into animal feed by fermentation with lactic acid bacteria (LAB). This procedure is safe, economically advantageous and environment friendly. The pH value of the fish pastes decreases to below 4.5 during ensilage. This pH decrease is partly responsible for preservation. Decreased pH values and relatively low oxygen concentrations within the silage facilitate decarboxylase activity. Biogenic amines may constitute a potential risk in this kind of product since their precursor amino acids are present in fish silage. It is of great importance to ensure that the LAB strains chosen for starters do not produce biogenic amines. Some bacteria, among which some LAB species, are able to degrade these metabolites by means of amino oxidases. This could be of interest for fish silage production, to control biogenic amine build-up in this product. Seventy-seven LAB cultures isolated from fish pastes submitted to natural fermentation at two temperatures (15 and 22°C) and selected combinations of these isolates were examined for histamine, tyramine, cadaverine and putrescine production. Of the isolates tested, 17% were found to produce one or more of these biogenic amines. The behaviour of diamine oxidase was tested under the conditions present in fish silage. Addition of 12% sucrose or 2% NaCl did not affect histamine degradation. Addition of 0.05% cysteine decreased histamine degradation. Degradation occurred at all temperatures tested (15, 22 and 30°C), but not at pH 4.5. Forty-eight potential fish silage starters were tested for histamine degradation in MRS broth containing 0.005 g l−1 histamine and incubated at 30°C. Indications were found that five of these isolates could degrade as much as 20–56% of the histamine added to the medium within 30 h, when used as pure cultures. No histamine degradation was observed with combinations of cultures. Histamine degradation (50–54%) by two of these isolates was also observed in ensiled fish slurry.

Complex microbiota of a Chinese “Fen” liquor fermentation starter (Fen-Daqu), revealed by culture-dependent and culture-independent methods

Daqu is a traditional fermentation starter that is used for Chinese liquor production. Although partly mechanized, its manufacturing process has remained traditional. We investigated the microbial diversity of Fen-Daqu, a starter for light-flavour liquor, using combined culture-dependent and culture-independent approaches (PCR-DGGE). A total of 190 microbial strains, comprising 109 bacteria and 81 yeasts and moulds, were isolated and identified on the basis of the sequences of their 16S rDNA (bacteria) and 26S rDNA and ITS regions (fungi). DGGE of DNA extracted from Daqu was used to complement the culture-dependent method in order to include non-culturable microbes. Both approaches revealed that Bacillus licheniformis was an abundant bacterial species, and Saccharomycopsis fibuligera, Wickerhamomyces anomalus, and Pichia kudriavzevii were the most common yeasts encountered in Fen-Daqu. Six genera of moulds (Absidia, Aspergillus, Mucor, Rhizopus, Rhizomucor and Penicillium) were found. The potential function of these microorganisms in starters for alcoholic fermentation is discussed. In general the culture-based findings overlapped with those obtained by DGGE by a large extent. However, Weissella cibaria, Weissella confusa, Staphylococcus saprophyticus, Enterobacter aerogenes, Lactobacillus sanfranciscensis, Lactobacillus lactis, and Bacillus megaterium were only revealed by DGGE.

Microbiological safety and quality of commercial sufu – a Chinese fermented soybean food

Abstract In this study, the microbiological safety and quality of commercial sufu were investigated. Twenty-three samples of three different types of sufu were obtained, mainly in China and some in The Netherlands. Chemical parameters analysed included moisture, pH, free amino N, NaCl, ethanol, sucrose, glucose, and fructose. Concentrations of NaCl, ethanol, glucose and fructose varied from 6.2%, 0.5%, 0% and 0% to 14.8%, 6.3%, 6.2% and 4.8%, respectively. Microbiological analyses were done for total count of mesophilic aerobic bacteria (TMAB), bacterial endospores, total count of halotolerant bacteria at 10% (THB10) and at 17.5% NaCl (THB17.5), lactic acid bacteria (LAB), fungi, Enterobacteriaceae, and the following pathogens: Bacillus cereus , Clostridium perfringens , Staphylococcus aureus and Listeria monocytogenes . High levels (>10 5 CFU/g) of TMAB and bacterial endospores were found in most samples, and 85% of TMAB was identified as Gram-positive. Considerable levels (10 5 and 10 7 CFU/g) of LAB were detected in two samples of white sufu, and isolates of LAB were identified as most probably Lb. casei . One-third of the samples contained less then 10 3 CFU/g B. cereus , but three samples had over 10 5 CFU/g indicating potential hazard to consumers. All samples had less than 10 3 CFU/g C. perfringens , except sample R11 (∼10 5 CFU/g). S. aureus could not be detected in any of the samples tested since the competitive microflora (usually bacilli) disturbed typical features on the selective medium used; however S. aureus enterotoxin A was detected in some of the white and grey sufu samples. Fungi, Enterobacteriaceae, and L. monocytogenes were not detected in any of the samples. Based on these results, a microbiological guideline for safe commercial sufu is proposed.

Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus on the metabolite profile of set-yoghurt

Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L. delbrueckii subsp. bulgaricus was investigated in terms of microbial growth, acidification and changes in the biochemical composition of milk during set-yoghurt fermentation. A complementary metabolomics approach was applied for global characterization of volatile and non-volatile polar metabolite profiles of yoghurt associated with proteolytic activity of the individual strains in the starter cultures. The results demonstrated that only non-proteolytic S. thermophilus (Prt-) strain performed proto-cooperation with L. delbrueckii subsp. bulgaricus. The proto-cooperation resulted in significant higher populations of the two species, faster milk acidification, significant abundance of aroma volatiles and non-volatile metabolites desirable for a good organoleptic quality of yoghurt. Headspace SPME-GC/MS and (1)H NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Furthermore, multivariate statistical analysis allows discriminating set-yoghurts fermented by different types of starter cultures according to their metabolite profiles. Our finding underlines that selection of suitable strain combinations in yoghurt starters is important for achieving the best technological performance regarding the quality of product.

Effects of temperature and relative humidity on growth and enzyme production by Actinomucor elegans and Rhizopus oligosporus during sufu pehtze preparation

Sufu is a Chinese soybean cheese obtained after maturation of solid-state mould-fermented tofu. Ambient temperatures of 30-35degreesC during the summer season prohibit the use of the usual starter Actinomucor elegans. We compared the properties of the latter with a potential alternative starter Rhizopus oligosporus that could be used at higher temperatures. The effects of temperature and relative humidity on growth rate of Actinomucor elegans and Rhizopus oligosporus were optimum at 25degreesC at RH 95-97%, and 35degreesC at RH 95-97%, respectively. Yields of protease (108 U/g pehtze), lipase (172 U/g) and glutaminase (176 U/g) by A. elegans were maximum after 48 h at 25degreesC and RH 95-97%, and for alpha-amylase (279 U/g pehtze) and alpha-galactosidase (227 U/g) at 30degreesC and RH 95-97% after 48 and 60 h of incubation. Highest protease (104 U/g pehtze), and lipase (187 U/g) activities of R. oligosporus were observed after 48 h at 35degreesC and RH 95-97%, while maximum alpha-amylase (288 U/g pehtze) and glutaminase (187 U/g) activities were obtained after 36 h at 35degreesC and RH 95-97%. Maximum alpha-galactosidase activity (226 U/g) by R. oligosporus was found after 36 h at 30degreesC and RH 95-97%. It is concluded that R. oligosporus is a potential alternative to A. elegans as sufu pehtze starter during hot seasons

Microbial changes during the production of Sufu - a Chinese fermented soybean food

Abstract Sufu is a Chinese soybean cheese obtained by solid-state fungal fermentation of tofu followed by ripening in dressing mixture. The aim of this study was to quantify microflora changes during the sufu process, which is carried out under non-sterile conditions. From tofu to pehtze (tofu overgrown with fungus) stages, total counts of mesophilic aerobic bacteria (TMAB), bacterial endospores (spores), Bacillus cereus, lactic acid bacteria (LAB), Enterobacteriaceae and fungi increased. All of them decreased after the salting of pehtze. TMAB and spores in sufu with 8% and 11% salt content decreased to around 106 cfu/g during the ripening. B. cereus remained at around 103 cfu/g. LAB in sufu with 8% and 11% salt content decreased gradually to

Microbiota dynamics related to environmental conditions during the fermentative production of Fen-Daqu, a Chinese industrial fermentation starter

Chinese Daqu is used as a starter for liquor and vinegar fermentations. It is produced by solid state fermentation of cereal-pulse mixtures. A succession of fungi, lactic acid bacteria and Bacillus spp. was observed during the production of Daqu. Mesophilic bacteria followed by fungi, dominated the first phase of fermentation. Next, lactic acid bacteria increased in relative abundance, resulting in an increase of the acidity of Daqu. At the final stages of fermentation, Bacillus spp. and thermophilic fungi became the dominant groups, possibly due to their tolerance to low water activity and high temperature. Both culture-dependent and culture-independent analyses confirmed that Bacillus spp. were ubiquitous throughout the process. Yeast species such as Wickerhamomyces anomalus, Saccharomycopsis fibuligera and Pichia kudriavzevii were present throughout almost the entire fermentation process, but the zygomycetous fungus Lichtheimia corymbifera proliferated only during the final stages of fermentation. Canonical correspondence analysis (CCA) revealed the significance of acidity, moisture content and temperature in correlation with the composition of the microbial communities at different stages.

Effect of sublethal preculturing on the survival of probiotics and metabolite formation in set-yoghurt

The objective of this study was to investigate the effect of preculturing of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB12 under sublethal stress conditions on their survival and metabolite formation in set-yoghurt. Prior to co-cultivation with yoghurt starters in milk, the two probiotic strains were precultured under sublethal stress conditions (combinations of elevated NaCl and low pH) in a batch fermentor. The activity of sublethally precultured probiotics was evaluated during fermentation and refrigerated storage by monitoring bacterial population dynamics, milk acidification and changes in volatile and non-volatile metabolite profiles of set-yoghurt. The results demonstrated adaptive stress responses of the two probiotic strains resulting in their viability improvement without adverse influence on milk acidification. A complementary metabolomic approach using SPME-GC/MS and (1)H-NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Principal component analysis revealed substantial impact of the activity of sublethally precultured probiotics on metabolite formation demonstrated by distinctive volatile and non-volatile metabolite profiles of set-yoghurt. Changes in relative abundance of various aroma compounds suggest that incorporation of stress-adapted probiotics considerably influences the organoleptic quality of product. This study provides new information on the application of stress-adapted probiotics in an actual food-carrier environment.

Characterization of the microbial community in different types of Daqu samples as revealed by 16S rRNA and 26S rRNA gene clone libraries

Daqu is a fermentative saccharification agent that is used to initiate fermentation in the production of Chinese liquor and vinegar. Different types of Daqu can be distinguished based on the maximum fermentation temperature, location of production, and raw materials used. We aimed to characterize and distinguish the different types of Daqu using a culture-independent cloning method. The lowest microbial diversity was found in Daqu produced at high-temperature. Principal component analysis (PCA) was used to compare the bacterial composition of Daqu from different regions (i.e., northern Daqu and southern Daqu). Staphylococcus gallinarum and Staphylococcus saprophyticus were found in southern Daqu, and were absent in northern Daqu. The fungi Saccharomycopsis fibuligera and Lichtheimia ramosa dominated in low/medium-temperature Daqu, whereas Thermomyces lanuginosus occurred in high-temperature Daqu. Our study identified potential biomarkers for the different types of Daqu, which can be useful for quality control and technology development of liquor or vinegar production.

Mucoraceous moulds involved in the commercial fermentation of Sufu Pehtze

Sufu is a fermented cheese-like soybean product in China and Vietnam, obtained by fungal solid-state fermentation of soybean curd (tofu), which results in moulded tofu or ‘pehtze’. The final product sufu is obtained by maturing pehtze in a brine containing alcohol and salt during a period of several months. The present report deals with the identity and phylogenetic relationships of mould starter cultures used for the preparation ofpehtze. Starter cultures used in commercial pehtze fermentation were obtained from factories located in several provinces of China and Vietnam, isolated from their pehtze and some were obtained from culture collections. They were identified as Actinomucor repens, Actinomucor taiwanensis, Mucor circinelloides, Mocur hiemalis, Mocur racemosus, and Rhizopus microsporus var. microsporus. Phylogenetic relations based on sequencing of genomic DNA of these starters and of relevant control strains from collections indicate that the genera Mucor, Actinomucor and Rhizopus form distinct and homogenous clusters, with Mucor and Actinomucor showing a slightly closer relationship with each other than with Rhizopus.