scholarly journals Quantification of psychrotrophic bacteria and molecular identification of Pseudomonas fluorescens in refrigerated raw milk

2019 ◽  
Vol 86 ◽  
Author(s):  
Camila Lampugnani ◽  
Mykaella Zanatta Was ◽  
Maike Taís Maziero Montanhini ◽  
Luis Augusto Nero ◽  
Luciano dos Santos Bersot
Keyword(s):  
Pseudomonas Fluorescens ◽  
Proteolytic Activity ◽  
Molecular Identification ◽  
Raw Milk ◽  
Chain Reaction ◽  
Psychrotrophic Bacteria ◽  
Milk Samples ◽  
16S Gene ◽  
Polymerase Chain ◽  
Proteolytic Capacity

ABSTRACT In this study, we investigated the contamination of refrigerated raw milk produced in the western region of Paraná, southern Brazil, with psychrotrophic microorganisms, aiming to assay the proteolytic activity of the isolates and to identify Pseudomonas fluorescens, the main proteolytic species associated with the spoilage of milk products. Raw milk samples from 50 dairy farms were submitted to the counting of psychrotrophic microorganisms, being the microbiota characterized by its mesophilic behavior and proteolytic capacity, besides molecular identification of P. fluorescens. Of the samples evaluated, 94% had psychrotrophic counts ranging from 3 to 7.1 log CFU mL-1, and 48.5% of these showed mesophilic behavior. Of the isolates, 48.0% had proteolytic activity in at least one evaluated temperature (21 and 30°C), and 39.3% had proteolytic activity in both temperatures. Among the 61 isolates submitted to molecular identification by polymerase chain reaction (PCR), 86.8% contained the expression of the 16S gene characteristic for P. fluorescens. In this study, we demonstrated that P. fluorescens is the most prevalent psychrotrophic bacteria species in raw refrigerated milk and their proteolytic ability poses high risks to the dairy industry.

LISTERIA MONOCYTOGENES IN COW’S MILK PRODUCED BY FAMILY-OWNED DAIRY FARMS

2020 ◽  
Vol 27 ◽  
pp. 1-10
Author(s):  
Benedito Menozzi
Keyword(s):  
Listeria Monocytogenes ◽  
Animal Health ◽  
Dairy Farms ◽  
Microbiological Analysis ◽  
Chain Reaction ◽  
Psychrotrophic Bacteria ◽  
Milk Samples ◽  
Negative Results ◽  
Preservation Method ◽  
Polymerase Chain

Refrigeration is an important milk preservation method. However, milk quality may deteriorate if the product is refrigerated for long periods, mainly due to the growth of psychrotrophic bacteria. This group of microorganisms includes pathogenic genera, most notably Listeria monocytogenes. The detection of this bacterium in food is important, given its pathogenic effects on human and animal health and also its economic relevance. This study focused on detecting the presence of L. monocytogenes in milk samples collected at small family-owned dairy farms. Samples were cultivated on PALCAM and ALOA agars for microbiological analysis and a molecular analysis by polymerase chain reaction was performed for the detection of L. monocytogenes. Despite the negative results obtained in both these analyses, further studies are recommended to confirm or refute the negligible effect of L. monocytogenes on small dairy farms.

scholarly journals Microbial analysis and virulence genes detection of milk preserved using heat-assisted pulsed electric field

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Suci Yuliangsih ◽  
Diana Elizabeth Waturangi ◽  
Yogiara
Keyword(s):  
Electric Field ◽  
Virulence Genes ◽  
Pulsed Electric Field ◽  
Raw Milk ◽  
Chain Reaction ◽  
Log Reduction ◽  
Milk Samples ◽  
Microbial Analysis ◽  
Polymerase Chain ◽  
Thermal Pasteurization

Abstract Objective Microbial analysis in milk preserved using heat-assisted Pulsed Electric Field (PEF) need to be assessed. In this study we analyze the microbial quality and virulence-associated genes in milk samples preserved using heat-assisted PEF from several producers in Indonesia. Results Milk samples were collected consisting of raw milk, milks taken after the heating, PEF, mixing, cooling, and packaging. Microbiological and Polymerase Chain Reaction (PCR) detection for virulence genes were performed. Heat-assisted PEF treatment gave 2.7–7.47 log reduction for TPC; 1.6–2.56 log reduction for MPN number; 3.13–6.48 log reduction for S. aureus; and for B. cereus there was an increase of 0.76 log and a reduction of 0.46 log. While milk samples from thermal pasteurization gave log reduction numbers of TPC, MPN, and S. aureus respectively 5.28; 2.56; and 4.73, for B. cereus was increasing 2.4 log. Producer C performed the best results with significant reduction compared with others (p < 0.005). There were no colonies of L. monocytogenes found in all of the samples. PCR results showed that milk samples possessed virulence genes 17.5% (10/57) of invA genes, 54.4% (31/57) of nheA genes, 68.4% (39/57) of cytK genes, 38.6% (22/57) of nuc genes, 63.2% (36/57) of ileS genes, while hly and actA genes were not detected.

scholarly journals Characterization of Campylobacter jejuni An Important Zoonotic Food Borne Pathogen from Mastitis Milk and Raw Milk Samples

2021 ◽  
Author(s):  
Sumedha Bobade ◽  
K. Vijayarani ◽  
K.G. Tirumurugaan ◽  
A. Thangavelu ◽  
S. Vairamuthu
Keyword(s):  
Polymerase Chain Reaction ◽  
Campylobacter Jejuni ◽  
Morphological Characteristics ◽  
Raw Milk ◽  
Phenotypic Characterization ◽  
Chain Reaction ◽  
Milk Samples ◽  
Food Borne ◽  
Polymerase Chain ◽  
Pcr Targeting

Background: Campylobacter has emerged as an important zoonotic food borne pathogen of human and animals worldwide. Campylobacter is one of the most common bacterial enteropathogens of food borne origin in industrialized countries with C. jejuni being the most common species followed by C. coli. There are very few cases reported from mastitis therefore this study was aimed to determine the incidence of Campylobacter jejuni from from mastitis milk and raw milk samples. Methods: Total of 72 milk samples comprising mastitis milk (20) and raw milk (52) were collected. The samples were subjected to cultural examination, biochemical as well as molecular identification. The isolates were further subjected to phenotypic characterization by biochemical test and genotypic characterization by Polymerase Chain Reaction. The isolates were subjected to PCR targeting hip O and MAP A genes. Result: The 52 samples showed growth on modified Blood Free Charcoal Cefoperazone Deoxycholate agar media and 18 (34.61%) samples showed typical morphological characteristics. The result revealed that 10 (19.23%) isolates were positive by phenotypic characteristic and 7(70%) by Polymerase chain reaction for C. jejuni. The outcome result showed that importance of Campylobacter jejuni in cattle, especially raw milk and milk from mastitis cows, as a potential source for transmission of Campylobacteriosis in human and dairy farm environment. This can cause acute bacterial gastroenteritis in humans and associated with foodborn infection, food safety and a serious public health threat.

scholarly journals Detection of coagulase gene in Staphylococcus aureus from several dairy farms in East Java, Indonesia, by polymerase chain reaction

2019 ◽  
Vol 12 (1) ◽  
pp. 68-71 ◽  
Author(s):  
Mustofa Helmi Effendi ◽  
Mirza Atikah Madarina Hisyam ◽  
Poedji Hastutiek ◽  
Wiwiek Tyasningsih
Keyword(s):  
Staphylococcus Aureus ◽  
Polymerase Chain Reaction ◽  
Dairy Farms ◽  
Raw Milk ◽  
Chain Reaction ◽  
Milk Samples ◽  
Epidemiological Tool ◽  
Gene Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Coa Gene

Aim: This study was conducted to study the coagulase (coa) gene-based genetic diversity of Staphylococcus aureus, isolated from different samples of cattle from three different regions in East Java Province, Indonesia. Materials and Methods: A total of 160 raw milk samples collected in East Java Province, Indonesia, were screened for the presence of S. aureus. The presumptive isolates were confirmed by coa test. The confirmed S. aureus isolates were subjected to coa gene polymerase chain reaction. Results: Of 160 different samples, 20 (12.5%) isolates of S. aureus were confirmed by positive coa test. Of 20 S. aureus isolates, 19 (95%) isolates carried coa gene. Six different genotypes of coa gene, i.e., 440 bp, 510 bp, 547 bp, 680 bp, 740 bp, and 820 bp were obtained. One coa genotypes, 510 bp (10 isolates) were observed in polymorphism to be more prevalent than the others, and the genotype was present in at least one isolates from every region. Conclusion: It can be concluded that coa gene is easily epidemiological tool for detection of variation strain from S. aureus.

Incidence and Polymerase Chain Reaction Assay of Listeria monocytogenes from Raw Milk in Gyeongnam Province of Korea

2002 ◽  
Vol 65 (1) ◽  
pp. 111-115 ◽  
Author(s):  
KWANG-SOO HA ◽  
SEON-JA PARK ◽  
SOOK-JAE SEO ◽  
JUNG-HYUN PARK ◽  
DUCK-HWA CHUNG
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Raw Milk ◽  
Food Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
Milk Samples ◽  
Polymerase Chain ◽  
Pcr Assays

A total of 50 raw milk samples from Gyeongnam Province of Korea were examined for the incidence of Listeria monocytogenes between July 1998 and August 1998. L. monocytogenes isolated by biochemical test was confirmed by polymerase chain reaction (PCR) with two sets of primers designed from the invasion-associated protein (iap) gene. After standard PCR with external primers, the amplified DNA was confirmed by a second round of PCR with internal primers (nested PCR). Both the external and internal primers generated 468-bp and 287-bp products, respectively. Only one (G9 strain) of the three suspect samples that tested positive in biochemical tests for L. monocytogenes from 50 raw milk samples was also PCR positive. Following this procedure, PCR-positive G9 strain was confirmed by Southern blot using the 287-bp internal iap probe again. The detection limit of G9 strain by standard PCR assay was as few as 102 cells, equivalent to approximately 1 pg of L. monocytogenes DNA. These PCR assays may be useful for novel detection as well as rapid confirmation for L. monocytogenes from food samples and the field.

Use of Novel Polymerase Chain Reaction Primers for the Specific Detection of Heat-Labile Toxin I, Heat-Stable Toxin I and II Enterotoxigenic Escherichia coli in Milk

1996 ◽  
Vol 59 (8) ◽  
pp. 795-802 ◽  
Author(s):  
HAU-YANG TSEN ◽  
WAN-RONG CHI ◽  
CHIEN-KU LIN
Keyword(s):  
Raw Milk ◽  
Pcr Primers ◽  
Enterotoxigenic Escherichia Coli ◽  
Target Cells ◽  
Specific Detection ◽  
Chain Reaction ◽  
E Coli ◽  
Milk Samples ◽  
Heat Stable ◽  
Polymerase Chain

Enterotoxigenic Escherichia coli (ETEC) strains which produce heat-labile and/or heat-stable toxins (LT and ST) have been some of the most important microorganisms causing food- and waterborne diseases. Rapid and sensitive methods for the specific detection of enterotoxigenic E. coli are thus important. Although quite a few polymerase chain reaction (PCR) primers have been developed for the specific detection of ETEC genes coding for LT I, ST Ia, and ST Ib, only a few primers have been designed for the detection of ST II and ST Ia, together with ST Ib ETEC. By gene-sequence comparison and serial PCR assay studies, we were able to develop novel PCR primers specific for the detection of LT I and ST Ia as well as ST Ib and ST II enterotoxin-coding genes of E. coli cells. The DNA sequences of these PCR primers are different from those reported by other laboratories. Studies on the detection sensitivities of these PCR primers showed that when cell lysate rather than the total DNA obtained by the phenol-chloroform-isoamyl alcohol extraction method was used for PCR, a lower detection limit, i.e., 101 or 102 CFU target cells per assay could be obtained. When such a cell-lysis method was used for the PCR detection of ETEC cells in a variety of milk samples, such as whole, &lt;2% fat, skim, and raw milk samples, it was found that if target cells in these milk samples were precultured in MacConkey broth for 8 h prior to cell lysis, as few as 100 cells per g of whole, &lt;2% fat, or skim milk samples, and 102 cells per g of raw milk sample could be detected.

Detection of proteolysis in raw milk stored at low temperature by an inhibition ELISA

1994 ◽  
Vol 61 (3) ◽  
pp. 395-404 ◽  
Author(s):  
Catherine Picard ◽  
Isabelle Plard ◽  
Dominique Rongdaux-Gaida ◽  
Jean-Claude Collin
Keyword(s):  
Proteolytic Activity ◽  
Limit Of Detection ◽  
Bacterial Count ◽  
Raw Milk ◽  
Enzyme Linked Immunosorbent Assay ◽  
Psychrotrophic Bacteria ◽  
Milk Samples ◽  
Inhibition Elisa ◽  
Different Strains

SummaryAn inhibition ELISA (enzyme-linked immunosorbent assay) was developed for the determination of caseinomacropeptide (CMP) in order to estimate the proteolysis of κ-casein due to the enzymes of psychrotrophic bacteria in bulk raw milk. The CMP present in milk was quantified specifically by an antibody. The limit of detection was ∽ 0·1 μg/ml and the CV was < 10%. This method was used to study the proteolytic activity of three strains of psychrotrophic Pseudomonas fluorescens in raw milk and to analyse different raw milk samples supplied by four dairy plants. The proteolytic activity for different strains of psychrotrophs and for different milk samples varied considerably, but no correlation was established between the level of microbial flora and κ-casein proteolysis. It is thus not possible to determine the extent of proteolysis from the bacterial count alone. However, by CMP determination in bulk raw milk samples after 6 d storage at 4°C, the mean κ-casein proteolysis was ∽ 4%. Among the milk samples analysed that contained < 107 cfu psychrotrophs/ml, 30% exhibited a proteolysis of κ-casein < 0·5%, i.e. < 5μg CMP/ml.

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