Whey-cheese production using freeze-dried kefir culture as a starter

AIMS: The aim of the present study was to evaluate the use of a freeze-dried kefir culture in the production of a novel type of whey-cheese similar to traditional Greek Myzithra-cheese, to achieve improvement of the quality characteristics of the final product and the extension of shelf-life. METHODS AND RESULTS: The use of kefir culture as a starter led to increased lactic acid concentrations and decreased pH values in the final product compared with whey-cheese without starter culture. The effect of the starter culture on production of aroma-related compounds responsible for cheese flavour was also studied using the solid phase microextraction gas chromatography/mass spectrometry technique. Spoilage in unsalted kefir-whey-cheese was observed on the thirteenth and the twentieth day of preservation at 10 and 5 degrees C, respectively, while the corresponding times for unsalted whey-cheese preservation were 11 and 14 days. CONCLUSIONS: The cheeses produced were characterized as high-quality products during the preliminary sensory evaluation. An indication of increased preservation time was attributed to the freeze-dried kefir culture, which also seemed to suppress growth of pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggested the use of kefir culture as a means to extend the shelf-life of dairy products with reduced or no salt content.     

Direct detection of lactococcal bacteriophages in cheese whey using DNA probes

Abstract We have designed a new method for the rapid detection of lactococcal phage directly in milk samples. The method was based on a dot blot analysis and did not require a biological assay with sensitive indicator strains. Culture media or whey permeate samples containing phage were spotted directly onto a nylon membrane and the phage were lysed in situ prior to hybridization. For skim milk, whole milk and whey samples, the samples were first treated with 50 mM EDTA, 20% Triton X-100 and heated at 60°C for 5 min, prior to spotting on the membrane. The detection limit was approximately between 10⁵ and 10⁷ pfu/spot. A large number of samples could be processed simultaneously and the results were obtained within 24 h.     

Online optical detection of food contaminants in microdroplets

To accommodate the considerable increase of disease based on microbial food contaminants in the last decade, a modulated, fast optical fluorescence detection combined with microdevices is created. This method, which consists of five different steps, first selects contaminants, mainly bacteria, in the food matrix. This process is based on a biomagnetic separation technique developed by our collaborators at the Technical University of Dresden. By the steps of binding antibody functionalized magnetic beads and fluorescent capsules on the target cell, a magnetic bead‐target cell‐microcapsule complex (MTM) is generated. The well‐established pipe‐based bioreactors (pbb) platform enables the generation of droplets with a volume between 60 and 160 nL and the detection of the target cell with an integrated microscopic and spectroscopic detection system. The module used for generating droplets is based on the segmented flow principle and is chip‐ or probe‐based. In this context, the successful use of polydimethylsiloxane (PDMS) as a cost‐effective alternative to the well‐established glass‐chips is introduced. To quantify the detection based on a yes‐ or no‐decision, the most important step is to separate one MTM‐complex per droplet. This equalized the quantity of the fluorescent signals with the quantity of the contaminants in the cell sample. The feasibility of microscopic and spectroscopic detection with only one fluorescent capsule per droplet is shown. Also the first results of a special prototyping optical detection set‐up that is already in an advanced stage of development, will be presented. This easy‐to‐use device implemented a software‐controlled, automatic documentation for every fluorescent signal of a droplet to guarantee the quality control. Here are the advantages of an integration of microdevices in a rapid detection of food pathogens presented. Obviously, the modular set‐up of this detection platform enables a wide range of high‐throughput applications.     

Single-molecule DNA sequencing of widely varying GC-content using nucleotide release,capture and detection in microdroplets

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.     

Rapid detection of low‐level HeLa cell contamination in cell culture using nested PCR

HeLa cells are a commonly used cell line in many biological research areas. They are not picky for culture medium and proliferate rapidly. HeLa cells are a notorious source of cell cross‐contamination and have been found to be able to contaminate a wide range of cell lines in cell culture. In this study, we reported a simple and efficient method for detecting the presence of HeLa cell contamination in cell culture. HPV‐18 was used as a biomarker. The cell culture supernatant was used directly as the template for nested PCR without extracting nucleic acid. By PCR amplification of the cell culture supernatant with the designed primers, we were able to detect the presence of HeLa cells in the culture. The sensitivity of this method can reach 1%, which is 10‐fold higher than Short tandem repeat sequence (STR) profiling. This simple, rapid, and “noninvasive” quality checking method should find applications in routine cell culture practice.     

Rapid tissue culture method for detection of Mycoplasma hyorhinis

An easy, rapid tissue culture method for detection of Mycoplasma hyorhinis is described. The organism induces morphological changes in mink S + L - cells. This effect was not observed in eight other animal cell lines infected by M. hyorhinis and it did not occur in the mink cells infected with six other strains of mycoplasma. This cell system should be useful in research laboratories which do not have other standard techniques available for monitoring the presence of M. hyorhinis.     

Rapid detection of Bacillus stearothermophilus using impedance-splitting

An impedance splitting method was used to detect Bacillus stearothermophilus in suspension and attached to stainless steel surfaces. The effects of bacterial metabolism on the impedance of the culture medium and the ionic layers of the measuring electrodes were recorded using the BacTrac 4000 microorganism growth analyser. Impedance changes were measured at 55 degrees C. Seven of the eight media produced changes in the electrode impedance (E-value) and all media produced negligible changes in the impedance of the culture medium (M-value). Good correlations were obtained between cell numbers and the E-value measured over 18 h (r > 0.9) for the two strains of B. stearothermophilus tested in trypticase soy broth. The E-value correlations were used to estimate the numbers of both vegetative and spore forms of B. stearothermophilus as either planktonic or adhered cells. For the detection of B. stearothermophilus using impedance, only methods where the E-value impedance is recorded, can be used.     

Strain-specific differentiation of lactococci in mixed starter culture populations using randomly amplified polymorphic DNA-derived probes.

A hydrophobic grid membrane filtration (HGMF) colony hybridization assay was developed that allows strain-specific differentiation of defined bacterial populations. The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was used to identify potential signature nucleic acid sequences unique to each member of a commercial cheese starter culture blend. The blend consisted of two closely related Lactococcus lactis subsp. cremoris strains, 160 and 331, and one L. lactis subsp. lactis strain, 210. Three RAPD primers (OPX 1, OPX 12, and OPX 15) generated a total of 32 products from these isolates, 20 of which were potential strain-specific markers. Southern hybridization analyses revealed, that the RAPD-generated signature sequences OPX15-0.95 and a 0.36-kb HaeIII fragment of OPX1-1.0b were specific for strains 331 and 210, respectively, within the context of the test starter culture blend. These strain-specific probes were used in a HGMF colony hybridization assay. Colony lysis, hybridization, and nonradioactive detection parameters were optimized to allow specific differentiation and quantitation of the target strains in the mixed starter culture population. When the 210 and 331 probes were tested at their optimal hybridization temperatures against single cultures, they detected 100% of the target strain CFUs, without cross-reactivity to the other strains. The probes for strains 210 and 331 also successfully detected their targets in blended cultures even with a high background of the other two strains.     

On-chip impedimetric detection of bacteriophages in dairy samples

Bacteriophage infection of starter cultures constitutes a major problem in the dairy fermentation industry, which may bring about important economic losses. In this study, a rapid detection method of bacteriophages was developed based on analysis of impedance changes occurred upon infection of a host-biofilm established onto metal microelectrodes. Bacteriophage PhiX174 and Escherichia coli WG5 were chosen as models for bacteriophage and host strain, respectively, because of their easiness of manipulation. Impedimetric changes occurring at the microelectrode surface, caused by bacteriophage infection and subsequent lysis of the host strain, were monitored over a 6-h period after the initial inoculation of phages by non-faradic impedance spectroscopy (IS) in PBS and milk samples. Analysis of data was performed by two different approaches: (1) the equivalent circuit modelling theory, where a decrease in the magnitude of both the double layer and the biofilm capacitances due to the bacteriophage infection process was recorded, and (2) analysis of the impedance value, specially the impedance imaginary component (Z(i)) at selected frequencies. Z(i) is related to the capacitance of the circuit and also showed a decrease with respect to the control sample (without bacteriophages). The simplicity of the assay and the possibility of miniaturization of the system as well as its wide application, being able of detecting any bacteriophage as long as a suitable bacterial host is available, increase the number of applications to which this system could be used for.     

Bacteriocin-producing Lactobacillus sake as starter culture in dry sausages

One hundred and fifty-two strains of Lactobacillus spp and Micrococcus spp, isolated from dry sausages, were screened for inhibitory activity. Two of the strains assayed of the genus Lactobacillus showed bactericidal activity. They were able to inhibit Listeria monocytogenes, Listeria seeligeri, Listeria innocua, Lactobacillus alimentarius and Lactobacillus bavaricus. The strains of Escherichia coli, Salmonella bradford and Salmonella newlands, Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Serratia marcescens were resistant. Their antimicrobial activity was due to peptides detectable in the culture broths and inactivated by treatment with proteolytic enzymes. Using bacteriocin-producing Lactobacillus sake as starter cultures in dry sausages could be promising in the food industry.     

Preparation of fungal starter culture in liquid fluidized bed reactor

Summary Pregerminated Trichoderma reesei (Rut C-30) spores were grown on corncob particles in a liquid fluidized bed reactor (LFBR). Hyphal mass covered particles were recovered from the top of the reactor in 24 h and used as starters for solid substrate fermentation. The starter from LFBR gave better biomass production than spore or mycelial inoculum.     

Rapid detection of conserved regions in protein sequences using wavelets

We present an algorithm to detect protein sub-structural motifs from primary sequence. The input to the algorithm is a set of aligned multiple protein sequences. It uses wavelet transforms to decompose protein sequences represented numerically by different indices (such as polarity, accessible surface area or electron-ion integration potentials of the amino acids). The numerical representation of a protein sequence has significant correlation with its biological activity, thus common motifs are expected to be observable from the wavelet spectrum. The decomposed signals are then up-sampled and similarity search techniques are used to identify similar regions across all the proteins at multiple scales. Results indicate that wavelet transform techniques are a promising approach for rapid motif detection.     

Evolutionary engineering to enhance starter culture performance in food fermentations

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Rapid detection of phylloplane bacterium Enterobacter cloacae based on chitinase gene transformation and lytic infection by specific bacteriophages

AIMS: To establish a rapid and efficient method for detecting Enterobacter cloacae based on chitinase gene transformation and lytic infection by virulent bacteriophages. METHODS AND RESULTS: A phylloplane strain of E. cloacae was isolated from tomato leaves and transformed with a chitinase gene. Transformed bacteria were collected from single colonies and infected with newly isolated, virulent bacteriophages in the presence of the chitinase substrate 4-methylumbelliferon (4MU)-(GlcNac)3. To assay chitinase activity in the lysates, the product 4MU was measured spectrofluorophotometrically or visibly detected under u.v. irradiation. Chitinase gene-transformed bacteria obtained from single colonies could be specifically identified in 30 min by the emission of 4MU fluorescence following lysis caused by phage infection. CONCLUSIONS: The chitinase gene was used as a reporter gene to construct a new system for easy and rapid monitoring of transgenic strains of E. cloacae released in the environment, in combination with specific recognition by virulent bacteriophages. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is simple, rapid, inexpensive, easy to perform and applicable to other strains. The system can be used for the routine monitoring of bacteria, which is important because of the increased use of transgenic strains of E. cloacae as an antagonistic biological control agent for plant diseases.     

Microorganisms in Daqu: a starter culture of Chinese Maotai-flavor liquor

Maotai-flavor liquor, a famous traditional Chinese drink, is distilled from fermented sorghum in the southern province of China. Moreover, it is of interest as one of the few examples of liquor distilled from the product of a fermentation using a wild microflora starter. Daqu is the starter of this fermentation process. Daqu is a mixture of two components: milled wheat and a complex microbial community. The composition and the effects of this microbial group are largely unknown. In this study, we have analysed the constituents of the microbial community and the development of microorganisms in the industrial Daqu preparation and ripening process. More than 200 colonies were isolated and characterized. The isolates were discriminated by phenotypic and conventional biochemical taxonomic methods. The results revealed the presence of bacteria, moulds and yeasts. Bacteria consist of Bacillus, Acetobacter, Lactobacillus, and Clostridium, among which Bacillus strains were found to be predominant. Moulds consisted of Aspergillus, Mucor, Rhizopus, Monascus and Trichoderma, and Aspergillus strains were found to be predominant in the six different biotypes. Yeasts comprised Saccharomyces, Hansenula, Candida, Pichia, and Torulaspora. The most frequently isolated yeasts belonged to the genus Saccharomyces. The microbial diversity shift showed that the microbial genera changed with increasing ripening time. Knowledge of the microbial diversity in Daqu provides a basis for microflora management and understanding of the role of microbes in the Daqu production process, and the contribution of Daqu performed as a starter culture to Maotai-flavor liquor.     

多功能悬浮点阵技术快速检测肠道致病菌的初步应用研究

目的建立以多功能悬浮点阵技术为基础的临床常见肠道致病菌的快速检测方法。方法以细菌16S rDNA基因保守区序列设计1对通用引物,采用不对称PCR扩增7种临床常见肠道致病菌标准菌株,多功能悬浮点阵技术对不同菌株的PCR产物进行检测以验证相应菌种探针的特异性,最后对48份粪便标本进行肠道致病菌高通量快速检测。结果 7种临床常见肠道致病标准菌株的不对称PCR得到了大量单链产物,其产物用多功能悬浮点阵技术的检测特异性为100%,48份粪便标本不对称PCR产物可与相应探针发生特异性结合,且在多功能悬浮点阵技术的相应检测信号大于阴性对照3倍以上,5种细菌的多功能悬浮点阵技术检测结果与培养鉴定结果符合率100%,48份标本PCR产物均与志贺菌属探针发生杂交反应(阳性率100%)。结论 16S rDNA可以作为细菌快速鉴定的靶序列,不对称PCR产物可以显著提高与悬浮芯片杂交检测的灵敏度,多功能悬浮点阵技术在鉴定细菌方面具有简单快速、高通量、高检出率等特点,可以作为细菌快速鉴定的一种新方法,但无法鉴别志贺菌属和大肠埃希菌属。     

Rapid detection of Staphylococcus aureus using bioluminescent enzyme immunoassay

A bioluminescent enzyme immunoassay (BLEIA) method for detecting protein A-bearing Staphylococcus aureus was developed using biotinylated firefly luciferase. The BLEIA was able to detect protein A at one pg ml-1 and 103 cfu ml-1 level of Staph. aureus. The BLEIA showed significant signals with overnight cultures of all 24 Staph. aureus strains, and the BLEIA did not show any significant signals with overnight cultures of all 44 strains of coagulase-negative staphylococci and the other genus bacteria. After 5 h cultivation beginning at approximately 50 cfu ml-1, the BLEIA was able to detect all 35 Staph. aureus strains isolated from healthy humans.     

Rapid detection of DNA-damaging agents using repair-deficient CHO cells

A screening method is introduced to detect and claissify DNA-damaging agents using DNA repair-deficient strains of Chinese hamster ovary cells. Differential cytotoxicity (relative growth) of the mutant cells compared to the wild-type cells was interpreted as a measure of lethal, potentially repairable damage to DNA. The assay consists of exposing the wild-type cells and three mutant strains to the test compound in a 24-well tray and using staining intensity to estimate growth after 72 h. The battery of mutants consists of two UV-sensitive strains (UV4 and UV5) that are deficient in different aspects of nucleotide excision repair, and strain EM9, which is defective in DNA-strand-break rejoining. The assay was highly reproducible, and the magnitude of the differential cytotoxicity response compared favorably with the amount of differential killing measured by colony-formation survival curves for several chemicals. 15 direct-acting and 7 metabolism-dependent agents that were expected to produce bulky, covalent DNA adducts were tested in the assay, and all produced a differential cytotoxicity response in at leastwo of the test UV5 showed a response to all of the test compounds whereas EM9 showed a response to 7 of the test compounds. Thus, the pattern of mutant responses presumably reflects the types of DNA damage produced by a compound. Although this aspect is still under development, these results indicate the potential of a larger battery of mutants to classify a wide spectrum of chemicals according to the lesions they produce. 13 non-DNA damaging agents were also tested and none produced a differential cytotoxicity response, suggesting that this endpoint is specific for DNA damage. We conclude that this assay may be a cost-effective alternative or adjunct to the existing short-term tests.     

Surface plasmon resonance detection of E. coli and methicillin-resistant S. aureus using bacteriophages

Early diagnosis and appropriate treatment of Escherichia coli (E. coli) O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) are key elements in preventing resultant life-threatening illnesses, such as hemorrhagic colitis, hemolytic uremic syndrome, and septicemia. In this report, we describe the use of surface plasmon resonance (SPR) for the biodetection of pathogenic bacteria, using bacteriophages as the recognition elements. T4 bacteriophages were used to detect E. coli, while a novel, highly specific phage was used to detect MRSA. We found that the system permits label-free, real-time, specific, rapid and cost-effective detection of pathogens, for concentrations of 10(3) colony forming units/milliliter, in less than 20 min. This system promises to become a diagnostic tool for bacteria that cause major public concern for food safety, bioterrorism, and nosocomial infections.