Biomedical subjects

W H Holzapfel

Publications and source records attributed to W H Holzapfel.

At least 19 recordsLinked to original sources

PubMed2006

Characterisation and biochemical properties of predominant lactic acid bacteria from fermenting cassava for selection as starter cultures.

A total of 375 lactic acid bacteria were isolated from fermenting cassava in South Africa, Benin, Kenya and Germany, and were characterised by phenotypic and genotypic tests. These could be divided into five main groups comprising strains of facultatively heterofermentative rods, obligately heterofermentative rods, heterofermentative cocci, homofermentative cocci and obligately homofermentative rods, in decreasing order of predominance. Most of the facultatively heterofermentative rods were identified by phenotypic tests as presumptive Lactobacillus plantarum-group strains, which also comprised the most predominant bacteria (54.4% of strains) isolated in the study. The next predominant group of lactic acid bacteria (14.1% of total isolates) consisted of obligately heterofermentative rods belonging either to the genus Lactobacillus or Weissella, followed by the heterofermentative cocci (13.9% of isolates) belonging to the genera Weissella or Leuconostoc. Homofermentative cocci were also isolated (13.3% of isolates). Biochemical properties such as production of alpha-amylase, beta-glucosidase, tannase, antimicrobials (presumptive bacteriocin and H(2)O(2)-production), acidification and fermentation of the indigestible sugars raffinose and stachyose, were evaluated in vitro for selection of potential starter strains. A total of 32 strains with one or more desirable biochemical properties were pre-selected and identified using rep-PCR fingerprinting in combination with 16S rRNA sequencing of representative rep-PCR cluster isolates. Of these strains, 18 were identified as L. plantarum, four as Lactobacillus pentosus, two each as Leuconostoc fallax, Weissella paramesenteroides and Lactobacillus fermentum, one each as Leuconostoc mesenteroides subsp. mesenteroides and Weissella cibaria, while two remained unidentified but could be assigned to the L. plantarum-group. These strains were further investigated for clonal relationships, using RAPD-PCR with three primers, and of the 32 a total of 16 strains were finally selected for the development as starter cultures for Gari production.

DNA, Ribosomal↗

PubMed2006

The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods.

Export of cocoa beans is of great economic importance in Ghana and several other tropical countries. Raw cocoa has an astringent unpleasant taste and a spontaneous fermentation is the first step in a process leading to cocoa beans with the characteristic cocoa flavour and taste. The microbiology of Ghanaian cocoa fermentations was investigated using culture-dependent and culture-independent methods. Samples were collected at 12 hour intervals during 96-144 hour tray and traditional heap fermentations. Yeast, Lactic Acid Bacteria (LAB), Acetic Acid Bacteria (AAB) and Bacillus spp. were enumerated on suitable substrates and identified using phenotypic and molecular methods. The yeast and bacterial micro-populations involved in the cocoa fermentation were further investigated using the culture-independent method Denaturing Gradient Gel Electrophopresis (DGGE). A microbiological succession was observed during the fermentations. At the onset of fermentation yeasts were the dominating microorganisms. Lactic Acid Bacteria became dominant after 12-24 h of fermentation and remained predominant throughout the fermentations with AAB reaching high counts in the mid phase of fermentation. Bacillus spp. were only detected during heap fermentations where they reached high numbers during the later stages of fermentation. Hanseniaspora guilliermondii was the predominant yeast during the initial phase and Pichia membranifaciens during the later phases of fermentation. A number of other yeast species including three putatively undescribed species were isolated during the fermentations. Lactobacillus fermentum was the dominant LAB in most samples. Several other LAB including Lactobacillus plantarum, Leuconostoc pseudomesenteroides, Leuconostoc pseudoficulneum, Pediocococcus acidilactici and a putatively undescribed LAB species were detected during the fermentations. Acetobacter syzygii, Acetobacter pasteurianus and Acetobacter tropicalis were the predominant AAB in all investigated fermentations. During the later stages of heap fermentation Bacillus licheniformis and occasionally other Bacillus spp. were detected in high numbers. In general the culture-based findings were confirmed using DGGE. However, DGGE indicated that Lc. pseudoficulneum plays a more important role during the fermentation of cocoa than expected from the culture-based findings as it yielded a strong band in most DGGE fingerprints. Cluster analysis of the DGGE fingerprints revealed that the DGGE fingerprints clustered according to fermentation site. Within each fermentation site the profiles clustered according to fermentation time. The DGGE method seems to offer a relatively fast and reliable tool for studying yeast and bacterial dynamics during cocoa fermentations.

Bacteria↗

PubMed2006

Biological degradation of aflatoxin B1 by Rhodococcus erythropolis cultures.

Aflatoxin contamination of food and grain poses a serious economic and health problem worldwide, but particularly in Africa. Aflatoxin B(1) (AFB(1)) is extremely mutagenic, toxic and a potent carcinogen to both humans and livestock and chronic exposure to low levels of AFB(1) is a concern. In this study, the biodegradation of aflatoxin B(1) (AFB(1)) by Rhodococcus erythropolis was examined in liquid cultures using thin layer chromatography (TLC), high performance liquid chromatography (HPLC), electro spray mass spectrometry (ESMS) and liquid chromatography mass spectrometry (LCMS). AFB(1) was effectively degraded by extracellular extracts from R. erythropolis liquid cultures. Results indicated that the degradation is enzymatic and that the enzymes responsible for the degradation of AFB(1) are extracellular and constitutively produced. Furthermore, the biodegradation of AFB(1) when treated with R. erythropolis extracellular fraction coincided with a loss of mutagenicity, as evaluated by the Ames test for mutagenicity.

Aflatoxin B1↗

PubMed2005

Degradation of aflatoxin B(1) by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. nov. DSM44556(T).

Biological degradation of aflatoxin B(1) (AFB(1)) by Rhodococcus erythropolis was examined in liquid cultures and in cell-free extracts. Dramatic reduction of AFB(1) was observed during incubation in the presence of R. erythropolis cells (17% residual AFB(1) after 48 h and only 3-6% residual AFB(1) after 72 h). Cell-free extracts of four bacterial strains, R. erythropolis DSM 14,303, Nocardia corynebacterioides DSM 12,676, N. corynebacterioides DSM 20,151, and Mycobacterium fluoranthenivorans sp. nov. DSM 44,556(T) were produced by disrupting cells in a French pressure cell. The ability of crude cell-free extracts to degrade AFB(1) was studied under different incubation conditions. Aflatoxin B(1) was effectively degraded by cell free extracts of all four bacterial strains. N. corynebacterioides DSM 12,676 (formerly erroneously classified as Flavobacterium aurantiacum) showed the lowest degradation ability (60%) after 24 h, while >90% degradation was observed with N. corynebacterioides DSM 20,151 over the same time. R. erythropolis and M. fluoranthenivorans sp. nov. DSM 44,556(T) have shown more than 90% degradation of AFB(1) within 4 h at 30 degrees C, whilst after 8 h AFB(1) was practicably not detectable. The high degradation rate and wide temperature range for degradation by R. erythropolis DSM 14,303 and M. fluoranthenivorans sp. nov. DSM 44,556(T) indicate potential for application in food and feed processing.

Aflatoxin B1↗

PubMed2005

Molecular characterization, technological properties and safety aspects of enterococci from 'Hussuwa', an African fermented sorghum product.

AIMS: To identify enterococci from Hussuwa, a Sudanese fermented sorghum product, and determine their technological properties and safety for possible inclusion in a starter culture preparation. METHODS AND RESULTS: Twenty-two Enterococcus isolates from Hussuwa were identified as Enterococcus faecium by using phenotypic and genotypic tests such as 16S rDNA gene sequencing, RAPD-PCR and restriction fragment length polymorphism of the 16S/23S intergenic spacer region fingerprinting. Genotyping revealed that strains were not clonally related and exhibited a considerable degree of genomic diversity. Some strains possessed useful technological properties such as production of bacteriocins and H2O2 or utilization of raffinose and stachyose. None produced alpha-amylase or tannase. A safety investigation revealed that all strains were susceptible to the antibiotics ampicillin, gentamicin, chloramphenicol, tetracycline and streptomycin, but some were resistant to ciprofloxacin, erythromycin, penicillin and vancomycin. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some strains. CONCLUSIONS: Enterococcus faecium strains are associated with fermentation of Sudanese Hussuwa. Some strains exhibited useful technological properties such as production of antimicrobial agents and fermentation of indigestible sugars, which may aid in stabilizing and improving the digestibility of the product respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococci were shown to play a role in the fermentation of African foods. While beneficial properties of these bacteria are indicated, their presence in this food may also imply a hygienic risk as a result of antimicrobial resistances or presence of virulence determinants.

Biogenic Amines↗

PubMed2004

Mycobacterium fluoranthenivorans sp. nov., a fluoranthene and aflatoxin B1 degrading bacterium from contaminated soil of a former coal gas plant.

Mycobacterium strain FA4T was isolated with fluoranthene as the single carbon source from soil of a former coal gas plant, polluted with polycyclic aromatic hydrocarbons. The physiological properties, fatty acid pattern, and the 16S ribosomal RNA gene sequence indicated membership to the genus Mycobacterium, but were different from all type strains of Mycobacterium species. Based on comparative 16S rRNA gene sequence analyses strain FA4T could be assigned to the Mycobacterium neoaurum taxon showing 98% sequence similarity to M. diernhoferi as its closest neighbour. The occurrence of epoxymycolate in the cell wall differentiates FA4 from all members of this taxon which synthesize wax-ester mycolates in addition to alpha-mycolates. Strain FA4T is able to degrade aflatoxin B1. This biological attribute might be useful in biological detoxification processes of foods and feeds. From the investigated characteristics it is concluded that strain FA4T represents a new species, for which we propose the name Mycobacterium fluoranthenivorans sp. nov. The type strain of Mycobacterium fluoranthenivorans is FA4T (DSM 44556T = CIP 108203T).

Aflatoxin B1↗

PubMed2003

Effect of potentially probiotic lactobacilli on faecal enzyme activity in minipigs on a high-fat, high-cholesterol diet-a preliminary in vivo trial.

Minipigs were fed a "Western-style", high-cholesterol diet for a baseline period, followed by the diet containing a mixture of three Lactobacillus strains with potential probiotic features, after which a normal pig diet was followed. The faecal enzyme activity for beta-glucuronidase and azoreductase, which are commonly considered as markers for procarcinogenic activity, was significantly reduced during the 5 weeks of "probiotic" supplementation. During the period of Lactobacillus administration, the cell counts for total anaerobes increased, whereas the total number of aerobes showed no change.

Animals↗

PubMed2003

Identification of heterofermentative lactobacilli isolated from pig faeces by numerical analysis of total soluble cell protein patterns and RAPD-PCR.

AIMS: The study deals with a number of heterofermentative Lactobacillus strains isolated from pig faeces and their identification. METHODS AND RESULTS: SDS-PAGE of total soluble cell proteins and RAPD-PCR profiles were used to identify the strains isolated from pig faeces. Protein profiles obtained with SDS-PAGE revealed that 15 strains clustered at r >or= 0.78 with Lactobacillus buchneri and nine strains at r >or= 0.77 with two reference strains of Lactobacillus reuteri. The identity of the strains was confirmed with RAPD-PCR. CONCLUSIONS: Numerical analysis of protein profiles and RAPD-PCR proved valuable in the differentiation of Lactobacillus spp. isolated from pig faeces. SIGNIFICANCE: This is the first report on the association of Lact. buchneri with pig faeces.

Animals↗

PubMed2002

Appropriate starter culture technologies for small-scale fermentation in developing countries.

Modern food biotechnology has moved a long way since ancient times of empirical food fermentations. Preservation and safeguarding of food are, however, still major objectives of fermentation. In addition, other aspects, such as wholesomeness, acceptability and overall quality, have become increasingly important and valued features to consumers even in developing countries where old traditions and cultural particularities in food fermentations are generally well maintained. Due to limitations in infrastructure and existing low technologies, rural areas in most developing countries have not been able to keep abreast of global developments toward industrialisation. At the same time, fermented foods play a major role in the diet of numerous regions in Africa and Asia. In many traditional approaches, the advantages of some form of inoculation of a new batch, e.g. by back-slopping or the repeated use of the same container (e.g. a calabash) is appreciated and generally practised. Still, the benefits of small-scale starter culture application as a means of improved hygiene, safety and quality control, in support of HACCP approaches, are not yet realised in small-scale fermentation operations. Approaches and considerations for the selection of pure cultures for small-scale, low-tech applications may differ in some respects from the large-scale industrial approaches practised since 100 years. Selection criteria should take account of the substrate, technical properties of the strain, food safety requirements and quality expectations. Lack of experience in the application of starter cultures in small-scale operations and under rural conditions presents a major obstacle but also an exciting challenge to food microbiologist and technologist. Culture preservation, maintenance and distribution demand special logistic and economic considerations. Quality, safety and acceptability of traditional fermented foods may be significantly improved through the use of starter cultures selected on the basis of multifunctional considerations, also taking into account the probiotic concept and possibilities offered for improved health benefits.

Bacteria↗

PubMed2001

Efficacy of nisin in combination with protective cultures against Listeria monocytogenes Scott A in tofu.

Nisin can be used as a biopreservative to control growth of Listeria monocytogenes in various minimally processed foods. Tofu is an example of a non-fermented soybean product, which may allow growth of Listeria at refrigeration temperatures and in which nisin may be applied to prevent multiplication of Listeria. The efficacy of nisin against Listeria may be compromised by the emergence of spontaneous nisin-resistant mutants. Exposure of L. monocytogenes Scott A to nisin in a culture medium or in a food product results in an initial reduction of Listeria population which is followed by regrowth of survivors to nisin during further incubation. In vitro studies using Standard I Nutrient broth showed that Enterococcus faecium BFE 900-6a and Lactobacillus sakei Lb 706-1a used as protective cultures in combination with nisin were able to suppress proliferation of Listeria cells not killed by nisin at 10 degrees C. Growth and bacteriocin production of these two strains and a third protective culture, Lactococcus lactis BFE 902 was also observed in soymilk and tofu at 10 degrees C. Inoculation studies with tofu prepared with nisin and protective cultures showed that lower amounts of nisin are required for an effective inhibition of L. monocytogenes Scott A when either E. faecium BFE 900-6a or Lc. lactis BFE 902 are used in addition. The combination of nisin with these bacteriocinogenic lactic acid bacteria (LAB) resulted in a complete suppression of listerial growth in homemade tofu stored at 10 degrees C for 1 week. Lb. sakei Lb 706-1a was less effective and did not prevent a slight increase of L. monocytogenes Scott A numbers during storage.

Anti-Bacterial Agents↗

PubMed2001

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↗

PubMed2001

Characterization of Penicillium roqueforti strains used as cheese starter cultures by RAPD typing.

Seventy-six strains of Penicillium roqueforti used as starter cultures for mould ripened blue cheeses have been analysed for their RAPD genotype by using three different primers. A comparison of the RAPD patterns within each primer group revealed that the genetic constitution of the strains was similar, as most of the strains showed very similar overall patterns. Despite these similarities with each primer, distinct RAPD genotype groups could be identified. With one of the primers, it was possible to detect two heteropolymorphic DNA regions resulting in 13 different groups. With the other two primers, three or four groups could be identified. Between the groups of the different primers marked correspondence with respect to strain distribution could be observed, indicating that the polymorphisms detected by the primers were not independent. The RAPD patterns were compared to the production of secondary metabolites. A correlation was observed between the RAPD patterns of all primers and the production of mycophenolic acid. In addition, one of the primer (ari1) was able to distinguish between P. roqueforti strains producing larger or smaller numbers of metabolites.

Cheese↗

PubMed2001

Taxonomy of obligately homofermentative and facultatively heterofermentative lactobacilli in pig faeces.

AIMS: Identification and characterization of obligately homofermentative and facultatively heterofermentative strains of Lactobacillus spp. isolated from the faeces of pigs that had been raised under different conditions. METHODS AND RESULTS: The phenotypic relatedness of the isolated strains and reference strains were determined by numerical analysis of total soluble cell protein patterns and simple physiological and biochemical tests. Of the 23 strains isolated from faeces, nine were obligately homofermentative and 14 facultatively heterofermentative. The strains clustered at r > or = 0.61 with Lactobacillus amylovorus (seven strains), Lactobacillus crispatus (one strain), Lactobacillus plantarum (14 strains) and Lactobacillus intestinalis (one strain). CONCLUSIONS: Results obtained from the physiological and biochemical tests confirmed the identity of the isolates as determined by numerical analysis of total soluble cell protein profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the association of Lact. crispatus and Lact. intestinalis with the gastro-intestinal tract of pigs.

Animals↗

PubMed2001

Taxonomy and important features of probiotic microorganisms in food and nutrition.

Lactic acid bacteria are among the most important probiotic microorganisms typically associated with the human gastrointestinal tract. Traditionally, lactic acid bacteria have been classified on the basis of phenotypic properties, eg, morphology, mode of glucose fermentation, growth at different temperatures, lactic acid configuration, and fermentation of various carbohydrates. Studies based on comparative 16S ribosomal RNA sequencing analysis, however, showed that some taxa generated on the basis of phenotypic features do not correspond with the suggested phylogenetic relations. Thus, some species are not readily distinguishable by phenotypic characteristics. This is especially true for the so-called Lactobacillus acidophilus group, the Lactobacillus casei and Lactobacillus paracasei group, and some bifidobacteria, strains of which have been introduced in many probiotic foods, eg, the novel yogurt-like commodities. Consequently, modern molecular techniques, including polymerase chain reaction-based and other genotyping methods, have become increasingly important for species identification or for the differentiation of probiotic strains. Probiotic strains are selected for potential application on the basis of particular physiologic and functional properties, some of which may be determined in vitro. The classification and identification of a probiotic strain may give a strong indication of its typical habitat and origin. The species, or even genus name, may also indicate the strain's safety and technical applicability for use in probiotic products. Molecular typing methods such as pulsed-field gel electrophoresis, repetitive polymerase chain reaction, and restriction fragment length polymorphism are extremely valuable for specific characterization and detection of such strains selected for application as probiotics.

Bifidobacterium↗

PubMed2001

Incidence of virulence factors and antibiotic resistance among Enterococci isolated from food.

The incidence of virulence factors among 48 Enterococcus faecium and 47 Enterococcus faecalis strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of E. faecium strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of E. faecalis strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.

Anti-Bacterial Agents↗

PubMed2001

Bile salt hydrolase activity of Enterococci isolated from food: screening and quantitative determination.

One hundred seventeen enterococcal strains isolated from food (47 Enterococcus faecium, 48 Enterococcus faecalis, 16 Enterococcus durans, 2 Enterococcus gallinarum, 3 Enterococcus casseliflavus, and 1 Enterococcus malodoratus) were screened for bile salt hydrolase (BSH) activity on de Man, Rogosa, and Sharpe agar medium containing taurocholic acid and calcium chloride. The highest incidence of BSH-active strains was observed for E. faecalis (81%) followed by E. faecium (50%) and E. durans (44%). Isolates were grouped into four putative activity groups (no, low, medium, and high activity) based on the size of precipitation zones observed in the screening experiment. Our results showed that assumptions on BSH activity based on the size of bile precipitation zones in screening experiments did not correlate with actual activity as quantified by high-pressure liquid chromatography, but the screening assay is useful for assessing the presence or absence of BSH activity.

Amidohydrolases↗

PubMed2000

The use of a starter culture in the fermentation of cassava for the production of "kivunde", a traditional Tanzanian food product.

Three cassava fermentation methods (spontaneous fermentation, back-slopping and the use of starter culture) for the production of kivunde, executed in three trials at 30 degrees C, were compared in terms of cyanide level reduction, microbiology and product quality improvement. Among the isolates from spontaneously fermented cassava batches, four strains were selected on the basis of their enzymatic activities and acid production. All were identified as Lactobacillus plantarum and were used as starters in this study. Lowest residual cyanide levels were detected after 120 h fermentation time in samples fermented with the starter culture and were below the maximum value of 10 mg/kg recommended by the Codex/FAO for cassava flour. This finding seems to be related to the alpha-glucosidase activity of the inoculated strains of which API-zyme (Bio-Merieux) tests showed activities of between 20 and > or = 40 nmol/4 h. The total residual cyanide levels of the spontaneous and back-slopping fermentations at 96 h were respectively 43.5 and 47.7 mg/kg dry weight of cassava. Extension of the fermentation period to 5 days, lead to further substantial reduction in the residual cyanide level in both these processes, but not below the recommended maximum value as in the case of starter culture fermented products. The spontaneous and back-slopping fermented cassava showed signs of deterioration after 3 days of fermentation. There was a sharp drop of pH and an increase of titratable acidity for all three batches during the first 48 h followed by a slow rise of pH and drop in titratable acidity towards the end of fermentation. The samples fermented with the starter culture had a smooth texture and pleasant fruity aroma, as opposed to the course and dull appearance and more complex flavour of the samples of spontaneous and back-slopping batches. During fermentation with starter culture, Enterobacteriaceae and yeasts and moulds could not be isolated throughout the period of fermentation (detection limit: 10 colony forming units/g). The present findings indicate the suitability of these Lb. plantarum strains as starter cultures for cassava fermentation in the kivunde process. The paper highlights the potential for the improvement of a traditional African fermented food (kivunde) through the use of a starter culture.

Bacteriological Techniques↗

PubMed2000

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces.

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by alpha-chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 3.4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.

Animals↗