Thermoduric psychrotrophic proteolytic microbiota from refrigerated raw milk

Thermoduric microorganisms may withstand high temperatures during the pasteurization of milk. Therefore, the microbiota in pasteurized milk consist of thermoduric microbes and directly influence the shelf-life of the milk. The aim of this study was to identify thermoduric psychrotrophic proteolytic microbiota in refrigerated raw milk. Twenty samples, previously heat-treated, were streaked and incubated at 7°C for 10 days. The strains isolated were streaked on milk agar to assess proteolytic activity and were initially analyzed morphologically by light microscopy and then by molecular techniques to identify the species. In 40% of the samples analyzed was observed only one bacterial growth and others 10 thermoduric psychrotrophic fungi. All isolates were proteolytic. The sequencing of 16S rRNA gene identified the bacterial strain as Bacillus pumilus and analysis of the ITS1-5.8S-ITS2 region of fungal isolates revealed the Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), and the 3 remaining strains were identified as Geotrichum candidum (30%). This is a fist description of B. pumilus in Brazilian raw milk. Considering the spoilage potential of all isolates and of the fungi present in raw milk and their survival in pasteurized milk, it is extremely important to carry out further studies to your resistant to heat, their impact on the shelf-life of pasteurized milk, ultra-high temperature (UHT) milk and dairy products.

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Spoilage potential of spore-forming bacteria from refrigerated raw milk

Semina Ciências Agrárias ◽

10.5433/1679-0359.2018v39n5p2049 ◽

2018 ◽

Vol 39 (5) ◽

pp. 2049 ◽

Cited By ~ 2

Author(s):

José Carlos Ribeiro Júnior ◽

Ronaldo Tamanini ◽

André Luís Martinez de Oliveira ◽

Juliane Ribeiro ◽

Vanerli Beloti

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Shelf Life ◽

Lipolytic Activity ◽

Raw Milk ◽

Rrna Gene ◽

16S Rrna Gene Analysis ◽

Pasteurized Milk ◽

Bacillus Spp ◽

Aerobic Spores

Aerobic bacterial spores are an important group of microorganisms in raw milk. These microbes are thermoduric, whereas the vegetative forms are thermophilic, thermoduric and psychrotrophic and reduce the shelf life of pasteurized milk. In Brazil, there are a lack of studies on the load of aerobic spores in raw milk; thus, little is known about the spoilage activity of these organisms. The aim the present study was to quantify the aerobic spores in Brazilian refrigerated raw milk of dairy region of Castro, Paraná state, assess the potential proteolytic and/or lipolytic isolates and identify the microorganisms derived from the germination. Twenty milk samples were evaluated, and the aerobic spore count was performed after plating the samples following heat treatment at 80°C for 12 min. The activity proteolytic and lipolytic isolates were evaluated through subculture on milk agar and tributyrin agar, respectively, and these microorganisms were identified using partial 16S rRNA gene sequences that were compared through GenBank. The aerobic spore counts ranged from 1 to 3.7 log CFU.mL-1, with a mean of 1.75 (± 0.59) log CFU.mL-1. After spore germination, 137 aerobic bacterial isolates were obtained, 40 of which (29.2%) showed milk spoilage activity. Among these, 31 isolates (77.5%) were proteolytic and lipolytic, seven isolates (17.5%) were exclusively lipolytic and two isolates (5%) were only proteolytic. Based on the 16S rRNA gene analysis, Bacillus licheniformis (55%), Bacillus spp. (27.5%), Paenibacillus spp. (7.5%), Bacillus pumilus (5%), Bacillus circulans (2.5%) and Brevibacillus spp. (2.5%) were identified. Studies of Brazilian raw milk microbiota have not yet described B. circulans which are frequently detected in milk from other countries. Among the 22 B. licheniformis isolates, 21 microbes (95.5%) showed proteolytic and lipolytic activity, and one isolate (4.5%) exhibited only proteolytic activity. The two B. pumilus isolates were proteolytic and lipolytic, whereas the B. circulans isolate was only lipolytic. Among the 11 Bacillus spp. isolates, eight isolates (72.7%) were proteolytic and lipolytic, one isolate (9.1%) was proteolytic and the other two isolates (18.2%) were lipolytic. The three Paenibacillus spp. and Brevibacillus spp. isolates were primarily lipolytic. Therefore, to extend the shelf life of pasteurized milk, preventive measures must be adopted to reduce contamination with spores because one-third of these microorganisms exhibited proteolytic and/or lipolytic activity.

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Spoilage potential of Paenibacillussp. in Brazilian raw milk

Ciência Rural ◽

10.1590/0103-8478cr20150810 ◽

2016 ◽

Vol 46 (4) ◽

pp. 637-640 ◽

Cited By ~ 1

Author(s):

José Carlos Ribeiro Júnior ◽

Brígida Kussumoto de Alcântara ◽

Vanerli Beloti

Keyword(s):

Shelf Life ◽

Dna Sequences ◽

Thermal Conditions ◽

Raw Milk ◽

Bacterial Spores ◽

Rrna Gene ◽

Pasteurized Milk ◽

Paenibacillus Sp ◽

Spore Forming Bacteria ◽

The 16S Rrna Gene

ABSTRACT: Bacterial spores are widespread in the environment and can contaminate milk. Spores are resistant to thermal conditions and your germination reduces milk shelf-life because the aerobic bacteria that are sporulated produce proteases and lipases. The aim of this study was identify Paenibacillus sp., the spoilage microbiota, arising from the germination of spores in raw milk and your spoilage potential. Twenty different milk samples were treated at 80°C/12min and plated to isolate spore-forming bacteria. These strains were picked in milk agar and tributyrin agar for verification of their potential proteolytic and lipolytic activities, respectively. Amplification and sequencing of the 16S rRNA gene of the strains for identification by similarity to the DNA sequences deposited in GenBank was performed. One hundred and thirty-seven isolates were obtained, of which 40 (29.2%) showed spoilage activity for milk. Of these, three (7.5%) were identified as strains of Paenibacillus sp., and all were lipolytic. Paenibacillus sp. have been identified as primarily responsible for the spoilage of pasteurized milk with a long shelf-life in other countries. To increase the shelf-life of Brazilian pasteurized milk, it is important to identify the sporulated microbes to determine their origin and to control the contamination of milk by vegetative forms such as spores.

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Occurrence of Listeria monocytogenes in Milk and Dairy Products Produced or Imported Into Morocco

Journal of Food Protection ◽

10.4315/0362-028x-56.3.256 ◽

1993 ◽

Vol 56 (3) ◽

pp. 256-259 ◽

Cited By ~ 10

Author(s):

A. EL MARRAKCHI ◽

A. HAMAMA ◽

F. EL OTHMANI

Keyword(s):

Listeria Monocytogenes ◽

Dairy Products ◽

Raw Milk ◽

Listeria Innocua ◽

Pasteurized Milk ◽

Listeria Species ◽

Fresh Cheese ◽

Milk And Dairy Products

Examination of 227 samples of milk and dairy products for Listeria monocytogenes showed that raw milk and some Moroccan traditionally made dairy products such as Iben and raib (fermented milks) and jben (fresh cheese) were contaminated with this pathogen. L. monocytogenes was the only Listeria species isolated except in one case in which it was associated with Listeria innocua. Pasteurized milk, fresh cream, and fresh and ripened cheeses (industrially made) were free from L. monocytogenes.

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Influence of the microbiological quality of raw milk on the shelf life of pasteurized milk

Semina Ciências Agrárias ◽

10.5433/1679-0359.2019v40n4p1469 ◽

2019 ◽

Vol 40 (4) ◽

pp. 1469

Author(s):

José Carlos Ribeiro Júnior ◽

Aline Marangon de Oliveira ◽

Fernando Godoi Silva ◽

Lorena Natalino Haber Garcia ◽

Cátia Maria de Oliveira Lobo ◽

...

Keyword(s):

Shelf Life ◽

Microbiological Quality ◽

Raw Milk ◽

Poor Quality ◽

Pasteurized Milk ◽

Microbial Counts ◽

Legal Standards ◽

Milk Samples ◽

Brazilian Standard

The dairy industry strives to produce high quality products with high nutritional value as well as to meet the legal standards for longer shelf life. However, these goals are made unfeasible by the poor quality of raw milk produced in some regions of Brazil. Others Brazilian dairy regions, however, already succeed in producing milk with low microbial counts, such as the municipality of Castro, Paraná state, designated as the ‘Brazilian dairy capital’. In order to evaluate the effect of raw milk quality on microbial counts during the shelf life of pasteurized milk, samples were collected from two dairy regions of Paraná: the northern and Castro region, characterized by milk production with high and low microbiological counts, respectively. Samples were experimentally pasteurized and the total microorganism counts were analyzed for 18 days at 7°C, using the Brazilian standard microbiological count limit for pasteurized milk (8 x 104 CFU/mL) as the end of the shelf life. Low microbiological counts in raw milk (Castro) resulted in significantly lower counts shortly after pasteurization and over the entire shelf life, meeting the pasteurized milk standard for 18 days. The temporal evolution in the counts over 18 days for the milks of high and low microbiological count was similar; however, the disparity between the absolute counts between the regions was significant (p < 0.05). Of the milk samples from northern Paraná, four (44.4%) already had counts higher than that of the legislative limit for pasteurized milk immediately after pasteurization. The others (five) reached the maximum microbiological count limit for pasteurized milk on the 6th day after pasteurization. In contrast, the milk from the Castro region remained below the limit throughout the analysis period. Thus, it can be stated that the microbiological quality of raw milk is directly related to the initial count of microorganisms after pasteurization, and that pasteurized milk produced from raw milk with low microbiological counts complies with the Brazilian legislation for 18 days following thermal processing.

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Paenibacillus lactis sp. nov., isolated from raw and heat-treated milk

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.02822-0 ◽

2004 ◽

Vol 54 (3) ◽

pp. 885-891 ◽

Cited By ~ 80

Author(s):

Patsy Scheldeman ◽

Karen Goossens ◽

Marina Rodriguez-Diaz ◽

Annelies Pil ◽

Johan Goris ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dairy Farm ◽

16S Rrna Gene Sequencing ◽

Raw Milk ◽

Rrna Gene ◽

Heat Treated ◽

Dairy Plant ◽

Rrna Gene Sequencing ◽

High Degree

Endospore-forming bacteria were recovered from individual packages from different processing lines in a dairy plant during a tenacious periodical contamination of their UHT-milk production. Two colony types were seen, one of which was identified as Bacillus sporothermodurans. Analysis of the 16S rRNA gene of the second colony type placed these isolates within the genus Paenibacillus, with Paenibacillus lautus as the closest known relative. Moreover, over 99 % similarity was observed to the 16S rDNA sequence of MB 2035, a strain isolated previously from raw milk during a survey at dairy farms for very heat-resistant spore-forming bacteria. Nine other potentially closely related strains among the dairy farm isolates were found using rep-PCR typing. The taxonomic positions of these 19 isolates were further investigated using 16S rRNA gene sequencing and DNA–DNA hybridizations of representative strains. All 19 isolates shared a high degree of phenotypic similarity and were easily distinguished from closely related members of the genus. Anteiso-C15 : 0, C16 : 0 and iso-C15 : 0 were among the major fatty acids and the genomic DNA G+C content was 51·6–51·7 mol%. Therefore, based on their phenotypic, phylogenetic and genomic distinctiveness, these 19 strains, isolated from both raw and heat-treated milk, are placed in the genus Paenibacillus as Paenibacillus lactis sp. nov. The type strain is MB 1871T (=LMG 21940T=DSM 15596T).

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Proteolytic and lipolytic microbiota of refrigerated raw milk from northeast and southern regions of Brazil

Semina Ciências Agrárias ◽

10.5433/1679-0359.2015v36n6supl2p4289 ◽

2015 ◽

Vol 36 (6Supl2) ◽

pp. 4289 ◽

Cited By ~ 1

Author(s):

Jose Carlos Ribeiro Junior ◽

Joyce Bitencourt Atayde Lima ◽

Kleydejany Lima de Lemos ◽

Livia Cavaletti Corrêa da Silva ◽

Ronaldo Tamanini ◽

...

Keyword(s):

Shelf Life ◽

Microbiological Quality ◽

Raw Milk ◽

The State ◽

Advanced Technology ◽

Daily Production ◽

Pasteurized Milk ◽

Hygiene Practices ◽

Spoilage Microorganisms

<p>The shelf life of milk and milk derivatives is directly related to the microbiological quality of refrigerated raw milk. Spoilage microorganisms with proteolytic and/or lipolytic properties are primarily responsible for the decrease in the quality of milk, which is reflected in the shelf life of pasteurized milk and all derivatives. The aim of this study was to determine the spoilage microbial load of refrigerated raw milk from the northeast and southern regions of Brazil, which have different climatic and technological conditions of production. We evaluated 46 samples of milk from the state of Paraná in the southern region, and 10 samples of milk from the state of Maranhão in the northeast region, totaling 56 samples collected from November 2013 to November 2014. The producers of Paraná were divided into large (20) or small (26) according to the average daily production. All producers of Maranhão were considered small (<500L/day). The proteolytic and lipolytic microorganism counts were conducted in milk agar and tributyrin agar, respectively. Milk from the large producers of Paraná had average counts of 1.4 × 104 CFU/mL for proteolytic microorganisms and 1.2 × 103 CFU/mL for lipolytics microorganisms, significantly (p <0.05) lower than the small producers in the same state, and the producers of Maranhão. Producers of Maranhao had counts of 1.1 × 105 CFU/mL for proteolytic microorganisms and 2 × 105 CFU/mL for lipolytic microorganisms, with the proteolytic count significantly lower than that of small Paraná producers. The amount of proteolytic and lipolytic spoilage microorganisms in milk is influenced by the adaptation of the microorganisms to cold, promoted by the cooling of milk, which is practiced less frequently in the country’s northeastern region. The amount of spoilage microorganisms is also affected by the implementation of milking hygiene practices, which reduce contamination. Such practices are more frequently and efficiently implemented among large producers with more advanced technology, which can improve the microbiological quality of raw milk, thus increasing the shelf life of pasteurized milk, and reducing problems in UHT milk as well as milk derivatives. </p>

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Proteolytic and lipolytic microbiota of refrigerated raw milk from northeast and southern regions of Brazil

Semina Ciências Agrárias ◽

10.5433/1679-0359.2015v36n6sup2p4289 ◽

2015 ◽

Vol 36 (6Supl2) ◽

pp. 4289

Author(s):

Jose Carlos Ribeiro Junior ◽

Joyce Bitencourt Atayde Lima ◽

Kleydejany Lima de Lemos ◽

Livia Cavaletti Corrêa da Silva ◽

Ronaldo Tamanini ◽

...

Keyword(s):

Shelf Life ◽

Microbiological Quality ◽

Raw Milk ◽

The State ◽

Advanced Technology ◽

Daily Production ◽

Pasteurized Milk ◽

Hygiene Practices ◽

Spoilage Microorganisms

The shelf life of milk and milk derivatives is directly related to the microbiological quality of refrigerated raw milk. Spoilage microorganisms with proteolytic and/or lipolytic properties are primarily responsible for the decrease in the quality of milk, which is reflected in the shelf life of pasteurized milk and all derivatives. The aim of this study was to determine the spoilage microbial load of refrigerated raw milk from the northeast and southern regions of Brazil, which have different climatic and technological conditions of production. We evaluated 46 samples of milk from the state of Paraná in the southern region, and 10 samples of milk from the state of Maranhão in the northeast region, totaling 56 samples collected from November 2013 to November 2014. The producers of Paraná were divided into large (20) or small (26) according to the average daily production. All producers of Maranhão were considered small (<500L/day). The proteolytic and lipolytic microorganism counts were conducted in milk agar and tributyrin agar, respectively. Milk from the large producers of Paraná had average counts of 1.4 × 104 CFU/mL for proteolytic microorganisms and 1.2 × 103 CFU/mL for lipolytics microorganisms, significantly (p <0.05) lower than the small producers in the same state, and the producers of Maranhão. Producers of Maranhao had counts of 1.1 × 105 CFU/mL for proteolytic microorganisms and 2 × 105 CFU/mL for lipolytic microorganisms, with the proteolytic count significantly lower than that of small Paraná producers. The amount of proteolytic and lipolytic spoilage microorganisms in milk is influenced by the adaptation of the microorganisms to cold, promoted by the cooling of milk, which is practiced less frequently in the country’s northeastern region. The amount of spoilage microorganisms is also affected by the implementation of milking hygiene practices, which reduce contamination. Such practices are more frequently and efficiently implemented among large producers with more advanced technology, which can improve the microbiological quality of raw milk, thus increasing the shelf life of pasteurized milk, and reducing problems in UHT milk as well as milk derivatives.

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The freezing point of raw and heat treated sheep milk and its variation during lactation

Acta Veterinaria Brno ◽

10.2754/avb201382020187 ◽

2013 ◽

Vol 82 (2) ◽

pp. 187-190

Author(s):

Bohumíra Janštová ◽

Pavlína Navrátilová ◽

Michaela Králová ◽

Lenka Vorlová

Keyword(s):

Freezing Point ◽

Chemical Properties ◽

Raw Milk ◽

Important Indicator ◽

Pasteurized Milk ◽

Milk Samples ◽

Sheep Milk ◽

Physico Chemical ◽

Heat Treated ◽

Time Specific

The freezing point of milk is an important indicator of the adulteration of the milk with water, but heat treatment may also affect its value. The aim of this study was determine freezing point of raw and heat treated sheep milk and its variation during lactation. The freezing point was determined in 42 bulk tank raw sheep milk samples and 42 pasteurized milk samples collected during lactation of sheep at one ecofarm in Moravian Walachia (Valašsko in the Czech Republic). The freezing point was determined in accordance with the standard ČSN 57 0538 using a thermistor cryoscope. The average freezing point of raw milk was -0.617 ± 0.052 °C, with a range from -0.560 to -0.875 °C. The freezing point was lower in the first months of lactation and increased at the end of lactation. The freezing point correlated (r = 0.8967) with the content of total non-fat solids. The average freezing point of sheep milk pasteurized at 65 °C for 30 min was -0.614 ± 0.053 °C, with a range from -0.564 to -0.702 °C. The median of freezing point differences between raw and pasteurized milk was 0.004 °C. Our study extends data about physico-chemical properties of sheep milk and registers for the first time specific changes in the freezing point value of sheep milk by heating.

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Microbiological Safety of Cheese Made from Heat-Treated Milk, Part III. Technology, Discussion, Recommendations, Bibliography

Journal of Food Protection ◽

10.4315/0362-028x-53.7.610 ◽

1990 ◽

Vol 53 (7) ◽

pp. 610-623 ◽

Cited By ~ 35

Author(s):

ERIC A JOHNSON ◽

JOHN H. NELSON ◽

MARK JOHNSON

Keyword(s):

Heat Treatment ◽

Whey Protein ◽

Raw Milk ◽

Toxin Production ◽

Ph Control ◽

Heat Treatments ◽

Low Risk ◽

Pasteurized Milk ◽

Psychrotrophic Bacteria ◽

Heat Treated

Heat treatment or pasteurization does not adversely affect the cheesemaking process or the resulting physical properties of the cheese. Both types of heat-treatments can correct chemical changes that occur in cold stored raw milk. Thermization on the farm may help control psychrotrophic bacteria in cold stored milk. Some denaturation of whey protein does occur during pasteurization. Heat treatments slightly above current minimum pasteurization requirements can cause body/texture and moisture control problems in cheese. Loss of functionality can adversely affect the marketing of whey protein products. Cheeses made from pasteurized milk ripen more slowly and usually do not exhibit the flavor intensity of cheeses made from raw or heat-treated milk. Swiss and hard Italian type cheese, whose traditional flavor results in part from native milk enzymes and microflora, would also be adversely affected if milk pasteurization for cheesemaking were mandatory. The quality of cheese made from pasteurized milk is consistently better than cheese made from raw milk as evidenced by fewer body and flavor defects consequent to the growth of undesirable bacteria. Either pasteurization or heat-treatment enables improved uniform process control and quality during cheesemaking. Pathogens were prioritized as high, medium, or low risk in cheese. Three organisms, Salmonella, Listeria monocytogenes and enteropathogenic Escherichia coli were judged to be high risk threats to the cheese industry. Staphylococcus aureus was listed as low risk because growth and toxin production is readily suppressed by lactic cultures and acidity (pH) control in cheese. Three actions are recommended:Establish a guideline for minimum heat-treatment of milk for cheesemaking: 64.4°C (148°F) for 16 s or equivalent with adequate process control.Evaluate current safety technology and practices used for cheese manufacture. Support research with primary emphasis on the combined effect of heat-treatment and other current cheese technologies.Evaluate technologies not currently utilized in cheese manufacture for safety potential.

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Reactivation of the Lactoperoxidase System during Raw Milk Storage and its Effect on the Characteristics of Pasteurized Milk

Journal of Food Protection ◽

10.4315/0362-028x-51.7.558 ◽

1988 ◽

Vol 51 (7) ◽

pp. 558-561 ◽

Cited By ~ 12

Author(s):

CECILIA E. MARTINEZ ◽

PATRICIA G. MENDOZA ◽

FRANCISCO J. ALACRON ◽

HUGO S. GARCIA

Keyword(s):

Shelf Life ◽

Raw Milk ◽

Taste Panel ◽

Pasteurized Milk ◽

Lactoperoxidase System ◽

Off Flavors ◽

Control Milk ◽

Milk Storage

The Lactoperoxidase (LP) system was activated periodically during raw milk storage, and after pasteurization. Raw milk was stored at 4°C for 4, 6 or 8 d, and after pasteurization at 8 or 16°C, until a laboratory-trained taste panel reported the presence of off-flavors. When treated milk was stored raw for 4 d, then pasteurized it maintained its quality for 12 d at 8°C; pasteurized control milk had a shelf-life of 9 d only. Shelf-life of pasteurized milk stored at 16°C after pasteurization was 6 d for LP-treated and 5 d for control milk. LP-treated milk that was stored raw for 6 d retained its quality after pasteurization for 11 d at 8°C and 5 d at 16°C, while shelf-life for control milk of this experiment was 5 d at 8°C and 1 d at 16°C. When storage of raw milk was extended to 8 d, control milk showed off-flavors right after pasteurization, whereas LP-treated milk developed a “stored” or “unclean” taste after 1 d of pasteurized storage. Reactivation of the LP system is proposed to extend the shelf-life of pasteurized milk, when raw milk storage for over 2 d is necessary.

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