Starter Population Research Articles

Dynamics of Starter and Non-Starter Lactic Acid Bacteria Populations in Long-Ripened Cheddar Cheese Using Propidium Monoazide (PMA) Treatment.

The microbial community of industrially produced Canadian Cheddar cheese was examined from curd to ripened cheese at 30–32 months using a combination of viable plate counts of SLAB (GM17) and NSLAB (MRSv), qPCR and 16S rRNA gene amplicon sequencing. Cell treatment with propidium monoazide excluded DNA of permeable cells from amplification. The proportion of permeable cells of both Lactococcus spp. and Lacticaseibacillus spp. was highest at 3–6 months. While most remaining Lacticaseibacillus spp. cells were intact during later ripening stages, a consistent population of permeable Lactococcus spp. cells was maintained over the 32-month period. While Lactococcus sequence variants were significant biomarkers for viable cheese curd communities at 0–1 m, Lacticaseibacillus was identified as a distinctive biomarker for cheeses from 7 to 20 months. From 24 to 32 months, Lacticaseibacillus was replaced in significance by four genera (Pediococcus and Latilactobacillus at 24 m and at 30–32 m, Secundilactobacillus and Paucilactobacillus). These results underscore the importance of monitoring potential defects in cheeses aged over 24 months, which could be diagnosed early through microbial DNA profiling to minimize potential waste of product. Future perspectives include correlating volatile flavor compounds with microbial community composition as well as the investigation of intra-species diversity.

Improving SCOBY starter using co-culture of tapai and bakery yeast

Abstract. Urbahillah A, Jayus J, Nurhayati N. 2021. Improving SCOBY starter using co-culture of tapai and bakery yeast. Biodiversitas 22: 4617-4624. Kombucha is a beverage fermented by a symbiotic bacteria and yeast known as SCOBY (Symbiotic Culture of Bacteria and Yeast). Bacteria and yeast contribute to the formation of organic acids, aroma, taste, and flavor of kombucha. The commercial yeasts used in Indonesian are baker’s yeast and tapai yeast. This study was conducted to develop SCOBYco-culture with tapai yeast and baker’s yeast and evaluate its activity. The ingredients for the kombucha were cascara, water, and sugar, which were fermented with three formula starter, i.e. original SCOBY 10% w/v (SN), co-culture SCOBY 10% w/v with 0.1% w/v of baker’s yeast (SNR), and co-culture SCOBY 10% w/v with tapai yeast 0.1% w/v (SNT). The starter activity were determined based on the OD (Optical Density) value. Yeast screening was carried out on the dominant starter population. Furthermore, morphologically yeast was identified based on colony type, color, and shape of cell. Then yeast was identified by their fermentation profile using API 20C Aux Kit. Isolate A showed white colony with convex elevation and the cell was round-shaped. Colony of isolate B and isolate C were creamy in color and oval cell shaped. The API results revealed that the first isolate was identified as Candida famata, second isolate was as Candida krusei, and the third isolate was as Candida magnoliae. Three types of fungi were found from SCOBY, namely Mucor sp., Trichoderma sp., and Fusarium sp. Mucor sp. has non-septate hyphae, and round black spores. Trichoderma sp. has septate hyphae, greenish-white spores, and the conidia have the shape of globose to ellipsoidal Fusarium sp. has a mold with septate hyphae, yellowish-white colonies, and the conidia have the shape of obovoid. Bacteria, yeast, and mold present in the medium form a powerful symbiosis for produce metabolite.

Effectiveness of edible coating based on chitosan and Natamycin on biological, physico-chemical and organoleptic attributes of Iranian ultra-filtrated cheese

Edible coatings can be applied on the surface of fruits and food pieces to improve their nutritional and sensory qualities. Present study focuses on the effects of applied edible coating formulation containing 1.6% W/V chitosan and 18.5 ppm Natamycin, on the surface of Iranian Ultra-filtrated (UF) cheese and evaluates biological, physico-chemical and organoleptic attributes of the coated cheeses as compared to those of the control samples, during 6 weeks of storage at temperature of 4 ± 2 °C. Coated and control samples were analyzed for bacterial total count, molds and yeasts population, coliform count and starter populations, as biological characteristics. pH, acidity, firmness, and salt, moisture, dry mater, fat and fat in dry mater contents were evaluated for both samples, as their physico-chemical properties. Appearance, odor and flavor, taste, texture and overall acceptability were evaluated for both coated and control cheese samples at weeks 3 and 6 of storage, as their sensorial attributes. Sensorial analysis indicated that, however there were insignificant differences between the values of sensory parameters for both samples at week 6, but, provided coating could significantly maintained sensory attributes of the coated cheese as compared to those of the control samples, at week 6 of storage.

Mapping Brain-Wide Afferent Inputs of Parvalbumin-Expressing GABAergic Neurons in Barrel Cortex Reveals Local and Long-Range Circuit Motifs

Parvalbumin (PV)-expressing GABAergic neurons are the largest class of inhibitory neocortical cells. We visualize brain-wide, monosynaptic inputs to PV neurons in mouse barrel cortex. We develop intersectional rabies virus tracing to specifically target GABAergic PV cells and exclude a small fraction of excitatory PV cells from our starter population. Local inputs are mainly from layer (L) IV and excitatory cells. A small number of inhibitory inputs originate from LI neurons, which connect to LII/III PV neurons. Long-range inputs originate mainly from other sensory cortices and the thalamus. In visual cortex, most transsynaptically labeled neurons are located in LIV, which contains a molecularly mixed population of projection neurons with putative functional similarity to LIII neurons. This study expands our knowledge of the brain-wide circuits in which PV neurons are embedded and introduces intersectional rabies virus tracing as an applicable tool to dissect the circuitry of more clearly defined cell types.

Effect of Greek-style yoghurt manufacturing processes on starter and probiotic bacteria populations during storage

This study compared viable counts of Streptococcus thermophilus (starter) and Lactobacillus helveticus R0052 (probiotic), as a function of two manufacturing processes of Greek-style (GS) yoghurt: centrifugation of a curd (GS-CF process) or ultrafiltration of milk prior to fermentation (GS-UF process). Fresh GS-UF and GS-CF yoghurts had between 3 and 7 times higher counts of Lb. helveticus R0052 and S. thermophilus than the regular stirred yoghurt (Control). Strain R0052 was three times more stable in the GS-CF yoghurt than in the Control over the 44 d storage period. The highest lactose content (44 g L−1) was obtained in the Control, while 60% less was obtained in the GS-UF yoghurt; the opposite trend was observed for lactic acid and galactose. Free amino acid levels were 33% higher in the GS products. GS yoghurt can be 10 times better than traditional yoghurt to deliver probiotic bacteria, as a function of the process used.

Induced Pluripotent Stem Cell Therapies for Cervical Spinal Cord Injury.

Cervical-level injuries account for the majority of presented spinal cord injuries (SCIs) to date. Despite the increase in survival rates due to emergency medicine improvements, overall quality of life remains poor, with patients facing variable deficits in respiratory and motor function. Therapies aiming to ameliorate symptoms and restore function, even partially, are urgently needed. Current therapeutic avenues in SCI seek to increase regenerative capacities through trophic and immunomodulatory factors, provide scaffolding to bridge the lesion site and promote regeneration of native axons, and to replace SCI-lost neurons and glia via intraspinal transplantation. Induced pluripotent stem cells (iPSCs) are a clinically viable means to accomplish this; they have no major ethical barriers, sources can be patient-matched and collected using non-invasive methods. In addition, the patient’s own cells can be used to establish a starter population capable of producing multiple cell types. To date, there is only a limited pool of research examining iPSC-derived transplants in SCI—even less research that is specific to cervical injury. The purpose of the review herein is to explore both preclinical and clinical recent advances in iPSC therapies with a detailed focus on cervical spinal cord injury.

Compromised Lactobacillus helveticus starter activity in the presence of facultative heterofermentative Lactobacillus casei DPC6987 results in atypical eye formation in Swiss-type cheese

Nonstarter lactic acid bacteria are commonly implicated in undesirable gas formation in several varieties, including Cheddar, Dutch-, and Swiss-type cheeses, primarily due to their ability to ferment a wide variety of substrates. This effect can be magnified due to factors that detrimentally affect the composition or activity of starter bacteria, resulting in the presence of greater than normal amounts of fermentable carbohydrates and citrate. The objective of this study was to determine the potential for a facultatively heterofermentative Lactobacillus (Lactobacillus casei DPC6987) isolated from a cheese plant environment to promote gas defects in the event of compromised starter activity. A Swiss-type cheese was manufactured, at pilot scale and in triplicate, containing a typical starter culture (Streptococcus thermophilus and Lactobacillus helveticus) together with propionic acid bacteria. Lactobacillus helveticus populations were omitted in certain vats to mimic starter failure. Lactobacillus casei DPC6987 was added to each experimental vat at 4 log cfu/g. Cheese compositional analysis and X-ray computed tomography revealed that the failure of starter bacteria, in this case L. helveticus, coupled with the presence of a faculatively heterofermentative Lactobacillus (L. casei) led to excessive eye formation during ripening. The availability of excess amounts of lactose, galactose, and citrate during the initial ripening stages likely provided the heterofermentative L. casei with sufficient substrates for gas formation. The accrual of these fermentable substrates was notable in cheeses lacking the L. helveticus starter population. The results of this study are commercially relevant, as they demonstrate the importance of viability of starter populations and the control of specific nonstarter lactic acid bacteria to ensure appropriate eye formation in Swiss-type cheese.

In vitro passage selects for Chlamydia muridarum with enhanced infectivity in cultured cells but attenuated pathogenicity in mouse upper genital tract.

Although modern Chlamydia muridarum has been passaged for decades, there are no reports on the consequences of serial passage with strong selection pressure on its fitness. In order to explore the potential for Pasteurian selection to induce genomic and phenotypic perturbations to C. muridarum, a starter population was passaged in cultured cells for 28 generations without standard infection assistance. The resultant population, designated CMG28, displays markedly reduced in vitro dependence on centrifugation for infection and low incidence and severity of upper genital tract pathology following intravaginal inoculation into mice compared to the parental C. muridarum population, CMG0. Deep sequencing of CMG0 and CMG28 revealed novel protein variants in the hypothetical genes TC0237 (Q117E) and TC0668 (G322R). In vitro attachment assays of isogenic plaque clone pairs with mutations in either TC0237 and TC0668 or only TC0237 reveal that TC0237(Q117E) is solely responsible for enhanced adherence to host cells. Paradoxically, double mutants, but not TC0237(Q117E) single mutants, display severely attenuated in vivo pathogenicity. These findings implicate TC0237 and TC0668 as novel genetic factors involved in chlamydial attachment and pathogenicity, respectively, and show that serial passage under selection pressure remains an effective tool for studying Chlamydia pathogenicity.

Detection and viability of Lactococcus lactis throughout cheese ripening.

Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese.

Organic acids profiles in lactose-hydrolyzed yogurt with different matrix composition

The composition of the dairy matrix used in yogurt manufacture could influence the metabolic activity of yogurt starter and the compounds produced during fermentation. In this way, the lactose hydrolysis with a β-galactosidase enzyme and the supplementation with dairy powders to obtain different types of delactosed yogurts for lactose-intolerant people modifies the milk base composition. In this work, we studied the influence of the addition of different doses of β-galactosidase and levels of dairy powders on organic acids profile during the manufacture and storage of two varieties of yogurt (natural and sweetened). Lactose, glucose, galactose, titratable acidity, fermentation time, and microbiological counts were also evaluated. The mean proportions of lactose/glucose/galactose in relation to the total sugars were 18:36:46 in hydrolyzed yogurts, while they were 81:4:15 in unhydrolyzed ones. In supplemented yogurts, the content of lactose, citric, orotic, and hippuric acids were significantly increased. The starter population was similar in all yogurts, but some changes in their activity were evidenced due to both factors studied. The fermentation time was slightly increased, and the lactic acid content and the titratable acidity were significantly increased due to fortification while they were decreased by enzyme addition. These results are probably due to the increase in the buffer properties of the milk base by the dairy powder addition and due to an inhibition of the starter activity caused by changes in the carbohydrate profile. The results demonstrated that the changes in the matrix composition of the yogurt affected the organic acids profile, above all the lactic acid content.

Effect of grape indigenous Saccharomyces cerevisiae strains on Montepulciano d'Abruzzo red wine quality

The growth of selected, indigenous Saccharomyces cerevisiae added as starters (SRS1, MS72 and RT73) was monitored during Montepulciano d'Abruzzo wine production. In all the fermentations the addition of the starter, caused a decrease of the non-Saccharomyces yeasts. When strains MS72 and RT73 were used as starters they were detected in the first phases of fermentations, while strain SRS1 competed successfully with native yeasts during all the process. Wines obtained by fermentation with the indigenous starters showed some different characteristics, according to the chemical and sensory analyses. This study highlighted that among selected starters with high fermentative capacity, some are able to dominate better than other natural wine yeast biota, whereas some strains can interact and survive besides native yeast populations during the fermentation. As a consequence, the dominance character can have a positive or negative effect on wine quality and has to be considered in the frame of yeast selection in order to improve or characterize traditional wines. Winemakers could choose among different degrees of yeast dominance to modulate the interaction among starter and native wine yeast population.

Identification of the limitations on recombinant gene expression in CHO cell lines with varying luciferase production rates

Mammalian cell lines are currently employed as one of the main cellular factories for the expression of recombinant protein-based drugs. The establishment of high-producing cell lines typically begins with a heterogeneous starter population of cells, from which the highest producing cells are selected via empirical approaches. This approach is time consuming, and is likely to encounter natural upper limits imposed by the inherent biology of the cell lines in question. In an attempt to understand both the nature of the variability in populations of cells transfected with recombinant protein encoding DNA and the natural mechanisms of productivity limitation, we developed protocols for the detailed investigation of gene expression pathways in such cell lines. This novel approach was then applied to a set of clonal CHOK1 cell lines producing recombinant luciferase with varying productivities. Our results show that the initial limitation in these cell lines is at the transcriptional level, however in the highest producing cell line post-translational mechanisms affecting both protein turnover and protein folding become severely limiting. The implications for the development of strategies to engineer cells for enhanced recombinant protein production levels are discussed.

Monitoring of Leuconostoc mesenteroides DRC starter in fermented vegetable by random integration of chloramphenicol acetyltransferase gene

In 2004, Leuconostoc mesenteroides DRC was first used as a starter culture for achieving higher organoleptic effects in Korean kimchi manufacture. For a better understanding of starter growth in a mixed culture system, and for predicting starter predominance in kimchi, a monitoring system for the starter was established. The chloramphenicol resistance marker gene (cat) was randomly integrated into chromosomal DNA of L. mesenteroides DRC using a viral transposon and transposase. The DRC mutant, tDRC2, had a similar growth pattern to the host strain, with no major alteration in phenotypic characteristics. The mutant strain was inoculated into real kimchi, and monitoring of the starter population was successfully achieved. The overall predominance of Leuconostoc in kimchi inoculated with DRC followed the general growth pattern of this genus during kimchi fermentation. Our results also demonstrate the competitive ability of the DRC starter against Leuconostoc from natural flora, maintaining its predominance above 88% during the whole fermentation period. Based on this experiment, the random gene integration method using a transposon was shown to be of utility in transferring any commercial starter into a selectable and monitorable strain for simulation purposes.

Mini soft cheese as a simple model for biochemical studies on cheese-making and ripening

A new miniature cheese model obtained under controlled microbiological conditions was proposed, characterized and tested for reproducibility. Optimal heat treatment of cheesemilk was defined, as well as maximal ripening time. Miniature cheeses were obtained with batch pasteurized milk (65 °C, 30 min) and ripened at 5 °C. Lactic and nonlactic microbial populations were monitored by plate counts. Proteolysis was assessed by nitrogen fractions, electrophoresis and liquid chromatography, and a sniffing test was applied to evaluate aroma. Coliform bacteria decreased during ripening but moulds and yeasts increased up to 10 4 cfu/g after 60 d, which defined the end of ripening period. Starter population remained constant during all ripening (10 9 cfu/g), while nonstarter lactic acid bacteria increased from ∼10 2 to 10 4 cfu/g. Soluble nitrogen levels at pH 4.6, in trichloracetic acid (0.73 mol/l) and in phosphotungtic acid (0.009 mol/l) were 151, 67, and 10 g/1000 g of the total nitrogen, respectively, after 60 d of ripening, which are usual values for soft cheeses. Proteolytic patterns as measured by electrophoresis were also similar to those of standard cheeses, as well as the aroma of the products. Peptide profiles revealed that the areas of most peaks increased with ripening time. The proposed model showed to be suitable for the production of mini cheese specimens for laboratory testing of cultures and enzymes in similar conditions to their real environment in the food matrix.

Loci affecting flowering time in oat under short-day conditions

Flowering time (or days to heading) is an important characteristic in crop plants that affects adaptation to cropping cycles and growing seasons. The objectives of this study were to identify molecular markers associated with flowering time in 3 oat populations developed from Brazilian oat varieties, and to compare their map locations with those of other loci that might influence flowering time. Flowering time was studied in recombinant inbred lines from 3 hexaploid oat populations: UFRGS 8 x Pc68/5*Starter; UFRGS 881971 x Pc68/5*Starter; and UFRGS 8 x UFRGS 930605. Bulked segregant analysis, using amplified fragment length polymorphism, was followed by selective mapping in each population and in a reference population, 'Kanota' x 'Ogle' (KxO). One quantitative trait locus (QTL) with major effects on flowering time was identified in each cross. Comparative mapping showed that a major QTL, with earliness alleles originating from UFRGS 8 and UFRGS 881971, is in a region with close homology to KxO linkage group 17 and to a locus that reportedly confers day-length insensitivity in oat (Di1). This is the first report to identify the map location of the Di1 locus, and putatively confirm the presence of Di1 alleles in new germplasm. Further comparative mapping and the alignment of mapped oat markers with the sequenced rice genome suggest that this QTL and (or) Di1 is orthologous to the Hd1 locus in rice and the CONSTANS gene in Arabidopsis and other species. A different QTL with major effects segregated in the UFRGS 8 x UFRGS 930605 cross, where the early-flowering allele for Di1 was probably fixed. Two additional QTLs with smaller effects were identified in the UFRGS 8 x Pc68/5*Starter population. These results suggest that the Brazilian oat line UFRGS 8 contains an optimal set of alleles conditioning earliness under the short-day conditions of the Brazilian winter growing season, and that molecular selection could be used to introgress these alleles into other breeding material.

Isolation of neuronal progenitor cells from the adult human neocortex

The reliability of harvesting neuronal progenitor cells (NPCs) from the adult human neocortex has not been established, with respect to preparing autologous cell cultures for transplantation in stroke and traumatic brain injured patients. Enriched NPC cultures have been generated from nonneurogenic regions of the adult rodent brain by buoyancy-dependent fractionation, but the feasibility of using such a method to isolate NPCs from the adult human cortex has not been reported previously. To determine if a starter population of human adult cortical NPCs could be isolated for in vitro expansion using this method, tissue samples from five patients undergoing cortical resection for either epilepsy or trauma were assayed. Cultured cells generated from all patients predominately expressed both the NPC marker nestin and neuron-specific beta-tubulin III. The presence of NPCs was verified by in vitro BrdU/beta-tubulin III co-labeling and increasing beta-tubulin expression in differentiating conditions. Despite the formation of aggregates in monolayer culture, cell proliferation as measured by BrdU incorporation was not as prevalent as that reported from rodent cultures generated by this protocol. NPCs isolated from the adult human neocortex using this method expressed beta-tubulin III in larger percentages than has been previously reported for NPCs isolated using other methods. As such, these data suggest the possibility of culturing dividing neuroblasts from the adult neocortex for further manipulation as transplantable cells.

Follicular dendritic-like cells derived from human monocytes

BackgroundFollicular dendritic cells (FDCs) play a central role in controlling B-cell response maturation, isotype switching and the maintenance of B-cell memory. These functions are based on prolonged preservation of antigen and its presentation in its native form by FDCs. However, when entrapping entire pathogens, FDCs can turn into dangerous long-term reservoirs that may preserve viruses or prions in highly infectious form.Despite various efforts, the ontogeny of FDCs has remained elusive. They have been proposed to derive either from bone marrow stromal cells, myeloid cells or local mesenchymal precursors. Still, differentiating FDCs from their precursors in vitro may allow addressing many unsolved issues associated with the (patho-) biology of these important antigen-presenting cells. The aim of our study was to demonstrate that FDC-like cells can be deduced from monocytes, and to develop a protocol in order to quantitatively generate them in vitro.ResultsEmploying highly purified human monocytes as a starter population, low concentrations of Il-4 (25 U/ml) and GM-CSF (3 U/ml) in combination with Dexamethasone (Dex) (0.5 μM) in serum-free medium trigger the differentiation into FDC-like cells. After transient de-novo membrane expression of alkaline phosphatase (AP), such cells highly up-regulate surface expression of complement receptor I (CD35). Co-expression of CD68 confirms the monocytic origin of both, APpos and CD35pos cells. The common leukocyte antigen CD45 is strongly down-regulated. Successive stimulation with TNF-α up-regulates adhesion molecules ICAM-1 (CD54) and VCAM (CD106). Importantly, both, APpos as well as APneg FDC-like cells, heterotypically cluster with and emperipolese B cells and exhibit the FDC characteristic ability to entrap functionally preserved antigen for prolonged times. Identical characteristics are found in monocytes which were highly expanded in vitro by higher doses of GM-CSF (25 U/ml) in the absence of Dex and Il-4 before employing the above differentiation cocktail.ConclusionIn this work we provide evidence that FDC-like cells can be derived from monocytes in vitro. Monocyte-derived FDC-like cells quantitatively produced offer a broad utility covering basic research as well as clinical application.

Development of genetic differentiation and postzygotic isolation in experimental metapopulations of spider mites.

We studied the development of genetic differentiation and postzygotic isolation in experimental metapopulations of the two-spotted spider mite, Tetranychus urticae Koch. A genetically diverse starter population was made by allowing six inbred sublines to interbreed. Then three migration patterns were tested: no migration, or one or three immigrants per subpopulation per generation. Variations in four traits were investigated: allozymes, acaricide resistance, diapause, and hatchability. In the allozymes, acaricide resistance, and diapause, genetic variation among subpopulations became high in metapopulations with no migration, but not in the others, which showed that one immigrant is enough to prevent genetic differentiation. Hatchability, which was decreased by interbreeding among the six sublines, gradually recovered in succeeding generations. In metapopulations with no migration, hatchability was reduced again after in-migration at the 15th generation. Different karyotypes or coadapted gene complexes can survive in different subpopulations by genetic drift, and both Wolbachia-infected and -noninfected subpopulations may be selected, which would lead to postzygotic isolation between isolated subpopulations. Our results indicate that sampling effects such as genetic drift or stochastic loss of Wolbachia produce postzygotic isolation in laboratory populations of spider mite.

Female Fertility and Single Nucleotide Polymorphism Comparisons in Cylindrocladium pauciramosum.

Cylindrocladium pauciramosum is well established in South America, and has recently been collected from nurseries in South Africa, Italy, and the United States. Isolates were compared with respect to the percentages of hermaphrodites and the respective mating types in the different samples. Based on these data, the effective population size could be determined for the different areas studied. All nurseries had mating type ratios significantly different from an idealized 1:1 ratio. In the South African nursery, the MAT-1 mating type was dominant, while the MAT-2 mating type dominated in other samplings. This is consistent with an introduction of a small starter population. High percentages of hermaphrodites also agreed with recent introductions into nurseries in Italy and the United States. Variability of DNA sequences of the 5' end of the β-tubulin gene from a set of C. pauciramosum isolates from different geographic regions was low to high. Isolates from South Africa, the United States, and Australia had identical β-tubulin DNA sequences; this sequence was also found in the Italian sample, along with another unique group. Finally, a group of isolates obtained from South and Central America had the highest variation of all isolates investigated, and also included isolates that shared single nucleotide variations with another species, C. candelabrum. These findings suggest that C. pauciramosum most likely has a Central or South American center of origin.

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.