Identification of the contaminating psychrotrophic bacteria in refrigerated bulked raw milk and the assessment of their deteriorating potential

This work aimed to characterize, identify, and determine the deteriorating potential of the contaminating psychrotrophic bacteria in refrigerated raw milk. Samples were submitted to serial dilutions and plated in specific culture media to form a bacterial culture collection. The isolates were characterized for their morphology and biochemical characteristics. The deteriorating potential of the isolates was determined according to the proteolytic, lipolytic and lecithinase activities at 4.0 ºC, 6.5 ºC, and 25.0 ºC. The results obtained for deterioration potential were assessed by the multivariate statistical method and by the principal components analysis (PCA). A total of 159 isolates were characterized, and of these, 46 strongly proteolytic Gram-negative isolates were selected for identification using the API 20 NE kit. The predominant bacteria were Gram-negative and oxidase and catalase positive, with a predominance of bacteria of the genus Pseudomonas. Using PCA, it was shown that the bacteria with the greatest deterioration potential were lecithinase producers, and that, in the autumn, proteolytic bacteria predominated at 4.0 ºC. Of the 46 isolates identified, more than 80% belonged to the species Pseudomonas fluorescens. Thus, attention should be given to the importance of implementing microbial contamination prevention measures in the bulking process, since, even under refrigeration, psychrotrophic bacteria multiply and produce enzymes that deteriorate lipids and proteins, with consequent quality losses of the milk and its derivatives, yield losses in the production of dairy products, and economic losses.

Probiotic Potential of a Folate-Producing Strain Latilactobacillus sakei LZ217 and Its Modulation Effects on Human Gut Microbiota

Folate is a B-vitamin required for DNA synthesis, methylation, and cellular division, whose deficiencies are associated with various disorders and diseases. Currently, most folic acid used for fortification is synthesized chemically, causing undesirable side effects. However, using folate-producing probiotics is a viable option, which fortify folate in situ and regulate intestinal microbiota. In this study, the folate production potential of newly isolated strains from raw milk was analyzed by microbiological assay. Latilactobacillus sakei LZ217 showed the highest folate production in Folic Acid Assay Broth, 239.70 ± 0.03 ng/μL. The folate produced by LZ217 was identified as 5-methyltetrahydrofolate. LZ217 was tolerant to environmental stresses (temperature, pH, NaCl, and ethanol), and was resistant to gastrointestinal juices. Additionally, the in vitro effects of LZ217 on human gut microbiota were investigated by fecal slurry cultures. 16S rDNA gene sequencing indicated that fermented samples containing LZ217 significantly increased the abundance of phylum Firmicutes and genus Lactobacillus, Faecalibacterium, Ruminococcus 2, Butyricicoccus compared to not containing. Short-chain fatty acids (SCFAs) analysis revealed that LZ217 also increased the production of butyric acid by fermentation. Together, L. sakei LZ217 could be considered as a probiotic candidate to fortify folate and regulate intestinal microecology.

The Quality of Five Natural, Historical Italian Cheeses Produced in Different Months: Gross Composition, Fat-Soluble Vitamins, Fatty Acids, Total Phenols, Antioxidant Capacity, and Health Index

Five natural historic cheeses of Southern Italy were investigated—Caciocavallo Palermitano (CP), Casizolu del Montiferru (CdM), Vastedda della Valle del Belìce (VVB), Pecorino Siciliano (PS), and Caprino Nicastrese (CN)—which are produced with raw milk and with traditional techniques and tools, from autochthonous breeds reared under an extensive system. The effects of the month of production on gross composition, MUFA, PUFA, PUFA-ω6, PUFA-ω3, α-tocopherol, retinol, cholesterol, TPC, TEAC, and GHIC were evaluated. In CP, CLA, TPC, and GHIC were higher in April than in February. CdM showed higher values in terms of fat, saturated fatty acids, PUFA-ω3, α-tocopherol, TEAC, and GHIC in May than in February and September, while low values in terms of protein, moisture, and CLA were found. In VVB, MUFA, PUFA-ω6, and α-tocopherol increased in June compared with April; conversely, protein, FRAP, and TEAC were higher in April. In PS, protein, CLA, PUFA, PUFA-ω3, α-tocopherol, and GHIC increased in May compared with January; on the contrary, moisture, NaCl, and TEAC showed high values in January. CN showed higher values in terms of PUFA, PUFA-ω6, PUFA-ω3, TPC, TEAC, and GHIC in April and June compared with January. It is shown that each cheese is unique and closely linked to the production area. Cheeses produced in the spring months showed a high nutritional quality due to the greatest presence of healthy compounds originating from an extensive feeding system.

Identification of Bovine miRNAs with the Potential to Affect Human Gene Expression

Milk and other products from large mammals have emerged during human evolution as an important source of nutrition. Recently, it has been recognized that exogenous miRNAs (mRNA inhibited RNA) contained in milk and other tissues of the mammalian body can enter the human body, which in turn have the ability to potentially regulate human metabolism by affecting gene expression. We studied for exogenous miRNAs from Bos taurus that are potentially contain miRNAs from milk and that could act postprandially as regulators of human gene expression. The interaction of 17,508 human genes with 1025 bta-miRNAs, including 245 raw milk miRNAs was studied. The milk bta-miR-151-5p, bta-miR-151-3p, bta-miRNA-320 each have 11 BSs (binding sites), and bta-miRNA-345-5p, bta-miRNA-614, bta-miRNA-1296b and bta-miRNA-149 has 12, 14, 15 and 26 BSs, respectively. The bta-miR-574-5p from cow’s milk had 209 human genes in mRNAs from one to 25 repeating BSs. We found 15 bta-miRNAs that have 100% complementarity to the mRNA of 13 human target genes. Another 12 miRNAs have BSs in the mRNA of 19 human genes with 98% complementarity. The bta-miR-11975, bta-miR-11976, and bta-miR-2885 BSs are located with the overlap of nucleotide sequences in the mRNA of human genes. Nucleotide sequences of BSs of these miRNAs in 5′UTR mRNA of human genes consisted of GCC repeats with a total length of 18 nucleotides (nt) in 18 genes, 21 nt in 11 genes, 24 nt in 14 genes, and 27–48 nt in nine genes. Nucleotide sequences of BSs of bta-miR-11975, bta-miR-11976, and bta-miR-2885 in CDS mRNA of human genes consisted of GCC repeats with a total length of 18 nt in 33 genes, 21 nt in 13 genes, 24 nt in nine genes, and 27–36 nt in 11 genes. These BSs encoded polyA or polyP peptides. In only one case, the polyR (SLC24A3 gene) was encoded. The possibility of regulating the expression of human genes by exogenous bovine miRNAs is discussed.

Detection and quantification of Bacillus cereus and its spores in raw milk by qPCR, and distinguish Bacillus cereus from other bacteria of the genus Bacillus

Introduction The raw milk is the basic raw material of dairy products, Bacillus cereus is a typical conditional pathogenic bacteria and cold-phagocytic spoilage bacteria in raw milk. This study established a qPCR method for detecting B. cereus in raw milk Materials and Methods In this study, a qPCR method for detecting B. cereus in raw milk was established. The specificity of the method was verified by using other Bacillus bacteria and pathogenic bacteria, the sensitivity of the method was evaluated by preparing recombinant plasmids and simulated contaminated samples, and the applicability of the method was verified by using pure spore DNA. The actual sample detection was completed by using the established qPCR method Results The qPCR established in this study can specifically detect B. cereus in raw milk. The LOD of the method was as low as 200 CFU/mL, and the LOQ ranged from 2 × 10 2 to 2 × 10 8 CFU/ml, the amplification efficiency of qPCR was 96.6% Conclusins The method established in this study can distinguish B. cereus from other Bacillus bacteria, and spore DNA can be used as the detection object. This method has the advantages of strong specificity, high sensitivity, wide application range and short detection time, which is expected to be applied in the dairy industry.

Complete Genome Sequencing and Comparative Genomics of Three Potential Probiotic Strains, Lacticaseibacillus casei FBL6, Lacticaseibacillus chiayiensis FBL7, and Lacticaseibacillus zeae FBL8

Lacticaseibacillus casei, Lacticaseibacillus chiayiensis, and Lacticaseibacillus zeae are very closely related Lacticaseibacillus species. L. casei has long been proposed as a probiotic, whereas studies on functional characterization for L. chiayiensis and L. zeae are some compared to L. casei. In this study, L. casei FBL6, L. chiayiensis FBL7, and L. zeae FBL8 were isolated from raw milk, and their probiotic properties were investigated. Genomic analysis demonstrated the role of L. chiayiensis and L. zeae as probiotic candidates. The three strains were tolerant to acid and bile salt, with inhibitory action against pathogenic bacterial strains and capacity of antioxidants. Complete genome sequences of the three strains were analyzed to highlight the probiotic properties at the genetic level, which results in the discovery of genes corresponding to phenotypic characterization. Moreover, genes known to confer probiotic characteristics were identified, including genes related to biosynthesis, defense machinery, adhesion, and stress adaptation. The comparative genomic analysis with other available genomes revealed 256, 214, and 32 unique genes for FBL6, FBL7, and FBL8, respectively. These genomes contained individual genes encoding proteins that are putatively involved in carbohydrate transport and metabolism, prokaryotic immune system for antiviral defense, and physiological control processes. In particular, L. casei FBL6 had a bacteriocin gene cluster that was not present in other genomes of L. casei, resulting in this strain may exhibit a wide range of antimicrobial activity compared to other L. casei strains. Our data can help us understand the probiotic functionalities of the three strains and suggest that L. chiayiensis and L. zeae species, which are closely related to L. casei, can also be considered as novel potential probiotic candidate strains.

Inactivation Kinetics of Coxiella burnetii During High-Temperature Short-Time Pasteurization of Milk

The Gram-negative, obligate intracellular bacterium Coxiella burnetii is the causative organism of the zoonosis Q fever and is known for its resistance toward various intra- and extracellular stressors. Infected ruminants such as cattle, sheep, and goats can shed the pathogen in their milk. Pasteurization of raw milk was introduced for the inactivation of C. burnetii and other milk-borne pathogens. Legal regulations for the pasteurization of milk are mostly based on recommendations of the Codex Alimentarius. As described there, C. burnetii is considered as the most heat-resistant non-spore-forming bacterial pathogen in milk and has to be reduced by at least 5 log10-steps during the pasteurization process. However, the corresponding inactivation data for C. burnetii originate from experiments performed more than 60 years ago. Recent scientific findings and the technological progress of modern pasteurization equipment indicate that C. burnetii is potentially more effectively inactivated during pasteurization than demanded in the Codex Alimentarius. In the present study, ultra-high heat-treated milk was inoculated with different C. burnetii field isolates and subsequently heat-treated in a pilot-plant pasteurizer. Kinetic inactivation data in terms of D- and z-values were determined and used for the calculation of heat-dependent log reduction. With regard to the mandatory 5 log10-step reduction of the pathogen, the efficacy of the established heat treatment regime was confirmed, and, in addition, a reduction of the pasteurization temperature seems feasible.

Establishment and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pseudomonas fluorescens in Raw Milk

Raw milk is susceptible to microbial contamination during transportation and storage. Pseudomonas fluorescens producing heat-resistant enzymes have become the most common and harmful psychrophilic microorganisms in the cold chain logistics of raw milk. To rapidly detect P. fluorescens in raw milk, the protease gene aprX was selected as a detection target to construct a set of primers with strong specificity, and a loop-mediated isothermal amplification (LAMP) assay was established. The detection thresholds of the LAMP assay for pure cultured P. fluorescens and pasteurized milk were 2.57 × 102 and 3 × 102 CFU/mL, respectively. It had the advantages over conventional method of low detection threshold, strong specificity, rapid detection, and simple operation. This LAMP assay can be used for online monitoring and on-site detection of P. fluorescens in raw milk to guarantee the quality and safety of dairy products.

Fethiye Bölgesinde Açıkta Satılan Tüketime Hazır Bazı Gıdalarda Koliform Bakteri Sayısının Belirlenmesi

Since coliform bacteria are common both in the intestine and in nature (soil, plant, etc.), they are considered as a sanitation indicator in the food industry. It is known that the majority of bacteria defined as fecal coliform in the coliform group are Escherichia coli. The presence of E. coli or fecal coliform bacteria in any sample is an indication that the necessary hygienic measures are not taken during production, storage, and sale. That means the sample is directly or indirectly contaminated with faeces, and/or other intestinal pathogens may also exist. In this study, raw milk, freshly squeezed fruit juices, unpackaged ice cream, shaved ice, and ice-cold samples were purchased from the famous touristic destination Fethiye and analyzed for coliform bacteria. For this purpose, the samples were purchased from local marketplaces, buffets, cafes, patisseries, restaurants, and roadsides at Fethiye. In total 60 samples were analyzed using Violet Red Bile (VRB) Agar. The results of coliform bacteria ranged

High Hydrostatic Pressure Processing of Human Milk Increases Apelin and GLP-1 Contents to Modulate Gut Contraction and Glucose Metabolism in Mice Compared to Holder Pasteurization

Background: High hydrostatic pressure (HHP) processing is a non-thermal method proposed as an alternative to Holder pasteurization (HoP) for the sterilization of human breast milk (BM). HHP preserves numerous milk bioactive factors that are degraded by HoP, but no data are available for milk apelin and glucagon-like peptide 1 (GLP-1), two hormones implicated in the control of glucose metabolism directly and via the gut–brain axis. This study aims to determine the effects of HoP and HHP processing on apelin and GLP-1 concentrations in BM and to test the effect of oral treatments with HoP- and HHP-BM on intestinal contractions and glucose metabolism in adult mice. Methods: Mice were treated by daily oral gavages with HoP- or HHP-BM during one week before intestinal contractions, and glucose tolerance was assessed. mRNA expression of enteric neuronal enzymes known to control intestinal contraction was measured. Results: HoP-BM displayed a reduced concentration of apelin and GLP-1, whereas HHP processing preserved these hormones close to their initial levels in raw milk. Chronic HHP-BM administration to mice increased ileal mRNA nNos expression level leading to a decrease in gut contraction associated with improved glucose tolerance. Conclusion: In comparison to HoP, HPP processing of BM preserves both apelin and GLP-1 and improves glucose tolerance by acting on gut contractions. This study reinforces previous findings demonstrating that HHP processing provides BM with a higher biological value than BM treated by HoP.