Sources of propionic acid bacteria contamination in the milking parlor environment on Alpine Dairy Farms.

High quality raw milk is an important prerequisite for the production of long ripened raw milk cheeses. This implies not only the absence of pathogenic microorganisms in raw milk, but also low levels of spoilage bacteria, including dairy propionic acid bacteria (dPAB), that can cause blowing and sensory defects in cheese, resulting in severe economic losses for producers. Raw milk contamination with dPAB has been primarily associated with improperly cleaned milking systems, but they have been detected in feed, soil, feces and on the teat skin. The objective of this study was to identify potential sources of raw milk contamination with dPAB in the barn and milking parlor environments. We also wanted to know more about the prevalence of the dPAB species in these environments and the levels of contamination. For this purpose, 16 small scale Alpine dairy farms were visited in August 2022: samples were taken from the barn environment (e.g., swab samples, air, feed, bedding), the milking system (swab samples, residual cleaning water, cleaning sponges, milk filters) and milk samples were collected at various sampling points along the milking system. Samples were analyzed for dPAB contamination, and results showed contamination at multiple sampling locations. We observed potential adverse effects of improperly set cleaning parameters of the milking system, as well as of farm specific practices. In addition, we identified cleaning water residues as an important source of contamination. Based on these findings, we propose potential mitigation strategies to reduce the risk of raw milk contamination with cheese spoilage bacteria, thereby contributing to a more sustainable food production.

The gut bacterial microbiome of Nile tilapia (Oreochromis niloticus) from lakes across an altitudinal gradient.

BACKGROUND: Microorganisms inhabiting the gut play a significant role in supporting fundamental physiological processes of the host, which contributes to their survival in varied environments. Several studies have shown that altitude affects the composition and diversity of intestinal microbial communities in terrestrial animals. However, little is known about the impact of altitude on the gut microbiota of aquatic animals. The current study examined the variations in the gut microbiota of Nile tilapia (Oreochromis niloticus) from four lakes along an altitudinal gradient in Ethiopia by using 16S rDNA Illumina MiSeq high-throughput sequencing. RESULTS: The results indicated that low-altitude samples typically displayed greater alpha diversity. The results of principal coordinate analysis (PCoA) showed significant differences across samples from different lakes. Firmicutes was the most abundant phylum in the Lake Awassa and Lake Chamo samples whereas Fusobacteriota was the dominant phylum in samples from Lake Hashengie and Lake Tana. The ratio of Firmicutes to Bacteroidota in the high-altitude sample (Lake Hashengie, altitude 2440 m) was much higher than the ratio of Firmicutes to Bacteroidota in the low altitude population (Lake Chamo, altitude 1235 m). We found that the relative abundances of Actinobacteriota, Chloroflexi, Cyanobacteria, and Firmicutes were negatively correlated with altitude, while Fusobacteriota showed a positive association with altitude. Despite variability in the abundance of the gut microbiota across the lakes, some shared bacterial communities were detected. CONCLUSIONS: In summary, this study showed the indirect influence of altitude on gut microbiota. Altitude has the potential to modulate the gut microbiota composition and diversity of Nile tilapia. Future work will be needed to elucidate the functional significance of gut microbiota variations based on the geographical environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study determined the composition and diversity of the gut microbiota in Nile tilapia collected from lakes across an altitude gradient. Our findings greatly extend the baseline knowledge of fish gut microbiota in Ethiopian lakes that plays an important role in this species sustainable aquaculture activities and conservation.

Propionic acid bacteria in the food industry: An update on essential traits and detection methods.

Propionic acid bacteria (PAB) is an umbrella term for a group of bacteria with the ability to produce propionic acid. In the past, due to this common feature and other phenotypic similarities, genetically heterogeneous bacteria were considered as a single genus, Propionibacterium. Members of this genus ranged from "dairy propionibacteria," which are widely known for their role in eye and flavor formation in cheese production, to "cutaneous propionibacteria," which are primarily associated with human skin. In 2016, the introduction of two new genera based on genotypic data facilitated a clear separation of cutaneous (Cutibacterium spp.) from dairy PAB (Propionibacterium spp., Acidipropionibacterium spp.). In light of these taxonomic changes, but with particular emphasis on dairy PAB, this review describes the current state of knowledge about metabolic pathways and other characteristics such as antibiotic resistance and virulence factors. In addition, the relevance of dairy PAB for the food industry and cheese production in particular is highlighted. Furthermore, methods for cultivation, detection, and enumeration are reviewed, incorporating the current taxonomy as well as the potential for routine applications.

Bacterial growth dynamics and corresponding metabolite levels in the extraction area of an Austrian sugar beet factory using antimicrobial treatment.

BACKGROUND: During the manufacture of sucrose from sugar beet, different microorganisms originating from the plant material as well as from the soil enter the process. Due to the formation of polysaccharide-based slimes, these contaminants may induce several adverse effects such as filtration problems during juice purification. Certain microorganisms also metabolize sucrose, leading to product losses with financial consequences. To better understand and to prevent these negative effects, the aim of the study was to investigate the evolution of relevant bacterial groups, including their metabolites appearing during the extraction process. For this purpose, one production cycle was monitored to identify the major contamination steps and to clarify how they relate to the processing conditions. Traditionally, different antimicrobial agents such as formaldehyde, sulfur dioxide, hypochlorous acid, sodium hypochlorite, and chlorine dioxide have been added to inhibit microbial growth. In the present study, a rosin-based product derived from pine trees was applied as an alternative to those substances. RESULTS: Press water, raw juice, and mid-tower juice were identified as being highly contaminated with bacteria, and processing conditions such as time, temperature and pH level significantly influenced bacterial levels and the corresponding metabolites. Among the contaminants identified, lactic acid bacteria, and mesophilic and thermophilic aerobic bacteria played a dominant role, whereas lactic acid, acetic acid, butyric acid, and ethanol were identified as typical metabolites. CONCLUSION: Bacterial growth during production could be reduced by shock dosing of the rosin-based material in the extraction area. © 2020 Society of Chemical Industry.

The Influence of Meat Batter Composition and SausageDiameter on Microbiota and Sensory Traits of ArtisanalWild Boar Meat Sausages.

In this study, the influence of meat batter composition and sausage diameter on the development of microbiota and sensory traits of traditional, spontaneously fermented wild boar meat sausages are evaluated. This research also demonstrates how principal component analysis (PCA) can be used to relate product sensory properties to particular microbial genotype and to select potential starter or adjunct culture. Generally, similar microbiological results were obtained in all types of products. The undesirable microbiota was either not detected at any sausage production stage or its number decreased below the detection limit in ripened sausages. The low growth rate of lactic acid bacteria (LAB) was consistent with the obtained pH and slow acidification rate. Although no differences in the composition of LAB species were noticed between sausage types (50S=50% wild boar meat in small casing, 50L=50% wild boar meat in large casing, 100S=100% wild boar meat in small casing), a clear separation based on LAB genotypes could be observed. Upon quantitative descriptive analysis, significant differences in sensory attributes between sausage types were established. According to the PCA, the overall acceptability traits of sausages are closely linked to one Leuconostoc mesenteroides genotype (LM_4). Of all tested technological properties, LM_4 strains showed remarkable acidification ability, lowering the pH from pH=5.41 to 3.74, and pronounced proteolytic activity on skimmed milk as well as antagonistic activity against Staphylococcus aureus (DSM 20231) and Brochothrix thermosphacta (LMG 17208). Lipolytic and haemolytic activities were not detected, and all analyzed strains were susceptible to tested antibiotics and possessed no biogenic amine genes.

Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii.

Cronobacter sakazakii is a foodborne pathogen that can cause a rare, septicemia, life-threatening meningitis, and necrotizing enterocolitis in infants. In general, standard methods for pathogen detection rely on culture, plating, colony counting and polymerase chain reaction DNA-sequencing for identification, which are time, equipment and skill demanding. Recently, nanoparticle- and surface-based immunoassays have increasingly been explored for pathogen detection. We investigate the functionalization of gold nanoparticles optimized for irreversible and specific binding to C. sakazakii and their use for spectroscopic detection of the pathogen. We demonstrate how 40-nm gold nanoparticles grafted with a poly(ethylene glycol) brush and functionalized with polyclonal antibodies raised against C. sakazakii can be used to specifically target C. sakazakii. The strong extinction peak of the Au nanoparticle plasmon polariton resonance in the optical range is used as a label for detection of the pathogens. Individual binding of the nanoparticles to the C. sakazakii surface is also verified by transmission electron microscopy. We show that a high degree of surface functionalization with anti-C. sakazakii optimizes the detection and leads to a detection limit as low as 10 CFU/mL within 2 h using a simple cuvette-based UV-Vis spectrometric readout that has great potential for further optimization.

Fermentability of a Novel Galacto-Oligosaccharide Mixture by Lactobacillus spp. and Bifidobacterium spp.

This study aimed to investigate the specific growth stimulation of certain desired intestinal bacteria by a novel galacto-oligosaccharide mixture, which was produced with a ß-galactosidase from a potential probiotic Lactobacillus isolate that contained mainly oligosaccharides of ß-1,3 and ß-1,6 glycosidic linkages (termed Lb-GOS) using single-strain fermentations. The composition of this Lb-GOS mixture was 33.5% disaccharides, 60.5% trisaccharides, 4.8% tetrasaccharides, and 1.0% pentasaccharides with a negligible amount of monosaccharides, lactose, and lactobionic acid (0.3%). Eight Lactobacillus spp. strains and three Bifidobacterium spp. strains were used in single-strain fermentations to determine the fermentation activity scores of this Lb-GOS preparation compared to two commercially available prebiotic mixtures, 4'GOS-P and Vivinal GOS (V-GOS). The highest scores were obtained when L. reuteri Lb46 and the two Bifidobacterium strains, B. animalis subsp. lactis Bif1 and Bif3, were grown on these galacto-oligosaccharide mixtures. In addition, the Lb-GOS mixture was found to have higher fermentation activity scores; hence, it stimulated the growth of these probiotic strains more than 4'GOS-P and V-GOS, which may be attributed to the different glycosidic linkage types that are found in the Lb-GOS mixture compared to the other two commercial preparations. These findings suggested that the Lb-GOS mixture that is described in this work should be of interest for the formulations of new carbohydrate-based functional food ingredients.

Lactococci of Local Origin as Potential Starter Culturesfor Traditional Montenegrin Cheese Production.

The aim of this study is to characterise and examine the biochemical properties of 40 Lactococcus lactis strains isolated from indigenous Montenegrin dairy products in order to explore their potential to be used as starter cultures for producing typical Montenegrin cheese, such as 'bijeli sir', 'masni sir' and 'njeguski sir'. Their safety regarding the production of biogenic amines, the presence of antimicrobial resistance and the antibacterial activity against relevant pathogens and spoilage microorganisms has also been tested. Based on the characterisation, all strains belong to L. lactis ssp. lactis. Out of these 40 strains, 23 displayed rapid acidification ability and proteolysis. However, none of the strains exhibited the ability of lipid degradation. Most of the strains were not associated with any health risk investigated. Summing up, a large percentage (27.5%) of the tested strains showed good properties. These strains should be further examined for their possible application as specific starter cultures in the production of indigenous cheese in Montenegro.

Celeribacter persicus sp. nov., a polycyclic-aromatic-hydrocarbon-degrading bacterium isolated from mangrove soil.

A Gram-stain-negative, mesophilic bacterial strain, designated SBU1T, which degrades polycyclic aromatic hydrocarbons was isolated from the sediments of the mangrove forests of Nayband Bay in the Iranian Persian Gulf during a bioremediation experiment. The 16S rRNA gene sequence of strain SBU1T exhibited highest similarities with Celeribacter indicus P73T (98.52%) and Celeribacter neptunius H 14T (97.05%). Phylogenetic analysis, based on 16S rRNA gene sequences, demonstrated that strain SBU1T fell within a cluster consisting of the type strains of species of the genus Celeribacter and formed a stable clade with C. indicus P73T in trees generated with three algorithms. The fatty acid profile of strain SBU1T consisted of the major fatty acids C18:1ω7c/ω6c and C18:1ω7c 11-methyl. The major compounds in the polar lipid profile were one phosphatidylglycerol and four unidentified phospholipids. The quinone system exclusively comprised ubiquinone (Q-10). The DNA G+C content was 60.4 mol%. A combination of phylogenetic analysis, DNA-DNA hybridization estimation, average nucleotide identity results and differential phenotypic and chemotaxonomic characteristics demonstrated that strain SBU1T could be distinguished from its close relatives. Therefore, strain SBU1T is considered to represent a novel species of the genus Celeribacter for which the name Celeribacter persicus sp. nov. is proposed. The type strain is SBU1T (=MCCC 1A00672T=DSM 100434T).

The intestinal microbiota of piglets fed with wheat bran variants as characterised by 16S rRNA next-generation amplicon sequencing.

The intestinal microbiota of piglets fed with a Control diet low in dietary fibre and modified wheat bran variants as an additional source of insoluble dietary fibre was characterised. In this context, variances in the microbiota of three different gut segments were assessed. Wheat bran was either included in its native form or modified by fermentation and extrusion before added at 150 g/kg to a basal diet for 48 piglets (12 animals per treatment). Total DNA was extracted from digesta samples from the jejunum, the end of the ileum and the colon ascendens. Samples were prepared accordingly for subsequent sequencing with the Illumina MiSeq. The obtained results revealed distinct location-specific differences in microbial composition. While Firmicutes were most predominant in all three gut segments, Bacteroidetes were additionally found in the colon at high abundance. The parameters of alpha and beta diversity analysis showed significant differences (p < 0.01) between the colon and the other two gut segments. Specialised bacterial groups like Prevotella and Ruminococcaceae were among the most predominant ones found in the colon, as they possess cellulolytic properties to degrade (at least partially) non-starch polysaccharides, while their abundance was negligible in the jejunum and the ileum. Conversely, the genera Lactobacillus, Bifidobacterium and Veillonella, for example, were among the most predominant groups in the jejunum and ileum, while in the colon they were hardly found. Although statistical taxonomical evaluation, following p-value correction, did not reveal pronounced differences in abundance related to bran modification, alpha and beta diversity analysis showed an influence regarding the various feeding strategies applied. Based on these findings, a more in-depth view on intestinal microbial composition within the gastrointestinal tract of young pigs fed with low- and high-fibre diets was generated.

Fermented and extruded wheat bran in piglet diets: impact on performance, intestinal morphology, microbial metabolites in chyme and blood lipid radicals.

The aim of the present study was to evaluate the influence of native, fermented and extruded wheat bran on the performance and intestinal morphology of piglets. Additionally, short-chain fatty acids (SCFA), biogenic amines, ammonia, lactic acid, pH as well as E. coli and lactic acid bacterial counts were analysed in digesta samples from three gut sections. Furthermore, the antioxidant potential in blood samples was evaluated based on the lipid radicals formed. For this purpose, 48 newly weaned piglets (28 d old) were allocated to one of the four different dietary treatment groups: no wheat bran (Control), native wheat bran, fermented wheat bran as well as extruded wheat bran. Wheat bran variants were included at 150 g/kg into the diets. All diets were mixed to reach the calculated isonitrogenic nutrient contents. Gut tissue and digesta samples were collected from the proximal jejunum, the terminal ileum and the colon ascendens, blood samples directly at slaughter. Although none of the dietary interventions had an impact on performance parameters, the amount of goblet cells in the ileum was increased upon feeding native and extruded wheat bran, compared to fermented bran (p < 0.05). The E. coli counts in colonic chyme were significantly lower (p < 0.05) in the Control group compared to the groups fed with wheat bran. The concentration of SCFA showed differences for minor compounds (p < 0.05), while linear contrast analyses revealed a reduced concentration of total SCFA in the colon following the feeding of modified wheat bran compared to native wheat bran. This may suggest that several compounds are more easily digested already in the ileum, resulting in a reduced nutrient flow into the large intestine and therefore less unexploited digesta is available as substrate for the microorganisms there. Fermentation also resulted in a significant decrease of methylamine in the colon (p < 0.05), while other biogenic amines in the ileum and colon showed no statistically significant differences. The formation of lipid radicals was decreased (p < 0.05) after feeding native wheat bran compared to the Control group. These results suggest that fermentation and extrusion of wheat bran exert some different impact regarding their physiological mode of action.

Rectal Lactobacillus species and their influence on the vaginal microflora: a model of male-to-female transsexual women.

INTRODUCTION: Based on Lactobacillus species co-colonizing the vagina and rectum, it has been hypothesized that the rectum may be an important reservoir for vaginal colonization by lactobacilli. There are no data on this issue in male-to-female transsexual women. AIM: We undertook this observational study to characterize the Lactobacillus species present in the neovagina and rectum of male-to-female transsexual women and to determine the degree of neovaginal-rectal co-colonization in order to gain a better understanding of the potential role of the gut as a reservoir for genital lactobacilli. METHODS: Sixty-one male-to-female transsexual women with penile skin lined neovagina without clinical signs of infection were recruited on an ongoing basis from among male-to-female transsexual outpatients. Neovaginal and rectal smears were taken for molecular Lactobacillus species profiling by denaturing gradient gel electrophoresis (PCR-DGGE). MAIN OUTCOME MEASURES: Matching Lactobacillus species between neovagina and rectum. RESULTS: Forty-three of the 61 male-to-female transsexual women (70.5%) simultaneously harbored the same lactobacilli in both the neovagina and rectum. We found 276 neovaginal and 258 rectal DGGE bands representing 11 Lactobacillus species, with 201 matches of the same Lactobacillus species in neovagina and rectum. 37 of the 61 women (61%) had two or more matching Lactobacillus species. CONCLUSION: These data support the hypothesis that the rectum may play an important role as source of Lactobacillus species that colonies neovagina of male-to-female transsexual women. In view of the specific anatomical circumstances of the study population, these findings may be extended to the general population of women.

One-Year Monitoring of Prevalence and Diversity of Dairy Propionic Acid Bacteria in Raw Milk by Means of Culture-Dependent and Culture-Independent Methods.

Even low levels of dairy propionic acid bacteria (dPAB) can cause cheese defects, resulting in severe economic losses for the producers of selected raw milk cheeses. Therefore, routine quality control of raw cheese milk for dPAB contamination is essential if propionic acid fermentation is undesired. Although knowledge of dPAB contamination of raw milk is important to understand cheese spoilage, long-term dPAB screening data are outdated, and studies taking into account different farm management parameters and their potential influence on dPAB levels are scarce. This study aims to provide insight into the dPAB levels of raw milk over time, to identify farm management factors that potentially influence dPAB levels, and to compare a cultural yeast extract lactate agar (YELA) and lithium glycerol agar (LGA) and a culture-independent method (qPCR) for dPAB quantification with respect to their applicability in routine quality control for the dairy industry. For this purpose, bulk tank milk from 25 dairy farms was screened for dPAB contamination over a one-year period. We were able to identify significant differences in the dPAB contamination levels in raw milk depending on selected farm-specific factors and observed relationships between the different types of milking systems and dPAB contamination levels in raw milk. When dPAB were quantified by cultivation on YELA, strong overgrowth of commensal microbiota impeded counting. Therefore, we conclude that quantification on LGA or by qPCR is preferable. Both methods, colony counting on LGA as well as quantification of dPAB using qPCR, have advantages for the application in (routine) quality control of raw milk, one being low-tech and inexpensive, the other being fast and highly specific, but the detection of (low level) dPAB contamination in raw milk remains a challenge.

Specificity of the AMP-6000 Method for Enumerating Clostridium Endospores in Milk.

Enumeration of endospores of butyric acid-forming clostridia in cheese milk is an essential part of milk quality monitoring for cheese producers to avoid late blowing, severe spoilage caused by clostridia during ripening. However, due to the lack of an internationally standardized method, different methods are used and it is important to consider how the choice of method affects the results. This is particularly relevant when clostridial spore counts in milk are considered for quality payments. The aim of this study was to evaluate the specificity of the AMP-6000 method for the enumeration of endospores of cheese spoiling clostridia in milk. First, to assess the prevalence of Clostridium diversity and to determine potential non-target species, we identified isolates from positive reactions of the AMP-6000 method used to quantify clostridial endospores in raw milk and teat skin samples by MALDI-TOF MS. Based on these results, a strain library was designed to evaluate method inclusivity and exclusivity using pure cultures of target and non-target strains according to ISO 16140-2:2016. Most target Clostridium tyrobutyricum strains, as well as all tested C. butyricum and C. sporogenes strains were inclusive. However, C. beijerinckii may be underestimated as only some strains gave positive results. All non-target strains of bacilli and lysinibacilli, but not all paenibacilli, were confirmed to be exclusive. This study provides performance data to better understand the results of microbiological enumeration of butyric acid-forming clostridia in milk and serves as a basis for future methodological considerations and improvements.

Aerobic spore-forming bacteria associated with ropy bread: Identification, characterization and spoilage potential assessment.

Aerobic spore-forming (ASF) bacteria have been reported to cause ropiness in bread. Sticky and stringy degradation, discoloration, and an odor reminiscent of rotting fruit are typical characteristics of ropy bread spoilage. In addition to economic losses, ropy bread spoilage may lead to health risks, as virulent strains of ASF bacteria are not uncommon. However, the lack of systematic approaches to quantify physicochemical spoilage characteristics makes it extremely difficult to assess rope formation in bread. To address this problem, the aim of this study was to identify, characterize and objectively assess the spoilage potential of ASF bacteria associated with ropy bread. Hence, a set of 82 ASF bacteria, including isolates from raw materials and bakery environments as well as strains from international culture collections, were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and their species identity confirmed by 16S rRNA and gyrA or panC gene sequencing. A standardized approach supported by objective colorimetric measurements was developed to assess the rope-inducing potential (RIP) of a strain by inoculating autoclaved bread slices with bacterial spores. In addition, the presence of potential virulence factors such as swarming motility or hemolysis was investigated. This study adds B. velezensis, B. inaquosorum and B. spizizenii to the species potentially implicated of causing ropy bread spoilage. Most importantly, this study introduces a standardized classification protocol for assessing the RIP of a bacterial strain. Colorimetric measurements are used to objectively quantify the degree of breadcrumb discoloration. Furthermore, our results indicate that strains capable of inducing rope spoilage in bread often exhibit swarming motility and virulence factors such as hemolysis, raising important food quality considerations.

Clostridium strain FAM25158, a unique endospore-forming bacterium related to Clostridium tyrobutyricum and isolated from Emmental cheese shows low tolerance to salt.

The genus Clostridium is a large and diverse group of species that can cause food spoilage, including late blowing defect (LBD) in cheese. In this study, we investigated the taxonomic status of strain FAM25158 isolated from Emmental cheese with LBD using a polyphasic taxonomic and comparative genomic approach. A 16S rRNA gene sequence phylogeny suggested affiliation to the Clostridium sensu stricto cluster, with Clostridium tyrobutyricum DSM 2637T being the closest related type strain (99.16% sequence similarity). Average Nucleotide Identity (ANI) analysis revealed that strain FAM25158 is at the species threshold with C. tyrobutyricum, with ANI values ranging from 94.70 to 95.26%, while the digital DNA-DNA hybridization values were below the recommended threshold, suggesting that FAM25158 is significantly different from C. tyrobutyricum at the genomic level. Moreover, comparative genomic analysis between FAM25158 and its four closest C. tyrobutyricum relatives revealed a diversity of metabolic pathways, with FAM25158 differing from other C. tyrobutyricum strains by the presence of genes such as scrA, srcB, and scrK, responsible for sucrose utilization, and the absence of many important functional genes associated with cold and osmolality adaptation, which was further supported by phenotypic analyses. Surprisingly, strain FAM25158 exhibited unique physiologic traits, such as an optimal growth temperature of 30°C, in contrast to its closest relatives, C. tyrobutyricum species with an optimal growth temperature of 37°C. Additionally, the growth of FAM25158 was inhibited at NaCl concentrations higher than 0.5%, a remarkable observation considering its origin from cheese. While the results of this study provide novel information on the genetic content of strain FAM25158, the relationship between its genetic content and the observed phenotype remains a topic requiring further investigation.

Importance of Pre-Milking Udder Hygiene to Reduce Transfer of Clostridial Spores from Teat Skin to Raw Milk.

Butyric acid producing clostridia (BAPC) cause the so-called late-blowing defect, a serious quality problem in semi-hard and hard cheeses. Late-blown cheeses are characterized by undesired slits and cracks, irregular eyes, and off-flavors due to excessive amounts of gas and organic acids produced by clostridia. Clostridial transfer to raw milk can occur during milking through dirty teats. Therefore, teat cleaning before milking is a key factor in preventing clostridial contamination of the milk. However, different cleaning methods are used, and little information is available on the efficacy of routine teat cleaning in reducing clostridial endospores. The main objectives of this study were to assess the extent of udder contamination with BAPC spores and to investigate the efficacy of routine teat cleaning on BAPC spore counts in milk. In a longitudinal study, eight dairy farms were visited during five sampling events. Clostridial spore counts were quantified from teat skin before and after routine teat cleaning, in pooled quarter milk samples from individual cows, and in bulk tank milk samples using a most probable number method. In addition, farm management data were collected periodically through a survey, and average cow cleanliness was assessed by a veterinarian. On average, teat cleaning resulted in a 0.6 log unit reduction in BAPC spores on teat skin, and a strong positive correlation was found between BAPC spore concentrations on teat skin after cleaning and in pooled quarter milk samples. Seasonal variations and the potential influence of differences in farm management were also noted. Interestingly, average cow cleanliness correlated strongly with BAPC spore levels in milk, suggesting the potential for a quick and rough estimation method of clostridial contamination that could be implemented by farmers.

Development of a DNA Metabarcoding Method for the Identification of Insects in Food.

Insects have the potential to become an efficient and reliable food source for humans in the future and could contribute to solving problems with the current food chain. Analytical methods to verify the authenticity of foods are essential for consumer acceptance. We present a DNA metabarcoding method that enables the identification and differentiation of insects in food. The method, developed on Illumina platforms, is targeting a 200 bp mitochondrial 16S rDNA fragment, which we found to be suitable for distinguishing more than 1000 insect species. We designed a novel universal primer pair for a singleplex PCR assay. Individual DNA extracts from reference samples, DNA extracts from model foods and food products commercially available were investigated. In all of the samples investigated, the insect species were correctly identified. The developed DNA metabarcoding method has a high potential to identify and differentiate insect DNA in the context of food authentication in routine analysis.

Comparing the Efficacy of MALDI-TOF MS and Sequencing-Based Identification Techniques (Sanger and NGS) to Monitor the Microbial Community of Irrigation Water.

In order to intensify and guarantee the agricultural productivity and thereby to be able to feed the world's rapidly growing population, irrigation has become very important. In parallel, the limited water resources lead to an increase in usage of poorly characterized sources of water, which is directly linked to a higher prevalence of foodborne diseases. Therefore, analyzing the microorganisms or even the complete microbiome of irrigation water used for food production can prevent the growing numbers of such cases. In this study, we compared the efficacy of MALDI-TOF Mass spectrometry (MALDI TOF MS) identification to 16S rRNA gene Sanger sequencing of waterborne microorganisms. Furthermore, we analyzed the whole microbial community of irrigation water using high-throughput 16S rRNA gene amplicon sequencing. The identification results of MALDI-TOF MS and 16S rRNA gene Sanger sequencing were almost identical at species level (66.7%; 64.3%). Based on the applied cultivation techniques, Acinetobacter spp., Enterobacter spp., Pseudomonas spp., and Brevundimonas spp. were the most abundant cultivable genera. In addition, the uncultivable part of the microbiome was dominated by Proteobacteria followed by Actinobacteria, Bacteroidota, Patescibacteria, and Verrucomicrobiota. Our findings indicate that MALDI-TOF MS offers a fast, reliable identification method and can act as an alternative to 16S rRNA gene Sanger sequencing of isolates. Moreover, the results suggest that MALDI-TOF MS paired with 16S rRNA gene amplicon sequencing have the potential to support the routine monitoring of the microbiological quality of irrigation water.

Characterization of bifidobacteria suitable for probiotic use in calves.

In our previous experiment, the ten calves originated bifidobacterial strains were administered to calves and re-isolated. Fingerprinting techniques used in this study enabled us to distinguish the surviving and non-surviving strains. Only the species Bifidobacterium animalis ssp. animalis and Bifidobacterium longum ssp. suis were found to survive in the intestine.