Academic literature on the topic 'Milk spoilage'

The reason for the reported difference in spoilage behaviour of skim and whole pasteurised milks was investigated. The rates of growth of psychrotrophic bacteria were not significantly different in the two milks and the bacterial types, all pseudomonads, present at spoilage were also similar. However, when representative spoilage organisms were cultured into freshly pasteurised skim and whole milks, skim milks exhibited predominantly bitter flavours while whole milk showed mostly sour flavours. The different spoilage behaviours can be largely explained by greater proteolysis in skim milk than in whole milk, caused by higher production of protease and greater susceptibility of the protein to protease attack. In addition, lipolysis in whole milk, caused by the substantial quantities of lipase produced by spoilage pseudomonads in this milk, also contributes to the different flavours produced during cold storage of these milk types.

Standard plate counts (SPC) and psychrotrophic plate counts (PPC) from chocolate milk samples were compared with those of unflavored milk samples plated within 24 h of processing and at 7, 10, and 14 days of storage at 6°C using matched samples collected over four time periods from four milk-processing plants. Bacterial numbers within 24 h of processing were not significantly different in unflavored and in chocolate milk samples (P > 0.001), with SPC less than 1,000 CFU/ml and PPC below 10 CFU/ml for both types of products. SPC and PPC were higher in chocolate milk samples than in unflavored milk samples collected from all four plants after 14 days of storage (P < 0.001). To examine the effects of chocolate milk components on bacterial numbers, SPC for days 0, 7, 14, and 21 were monitored in samples of experimentally prepared unflavored milk, milk with chocolate powder and sucrose (chocolate milk), milk with sucrose only, and milk containing chocolate powder only. At days 14 and 21, SPC were higher in both chocolate milk and in milk with chocolate powder only, than in either the unflavored milk or milk with sucrose only (P < 0.001). These findings suggest that the addition of chocolate powder to milk can contribute to a greater relative increase in bacterial numbers in pasteurized chocolate milk than in identically processed unflavored milk at 14 days postprocessing.

Milk spoilage is an inevitable occurrence, which generates waste and can result in food poisoning. When milk spoils, the off-flavor and curdling are due to excessive proliferation of various bacteria which causes pH changes. Time, temperature, environment, and previous handling practice all affect the spoilage rate. There is a need for a fast reliable and accurate method that can identify in situ early spoilage of milk. Here we show the ability of attenuated total reflection Fourier transformed infrared spectroscopy (ATR FT-IR) in conjunction with multivariate data analysis to predict the age of milk. We found that dried films vastly increased the absorbance of important biomolecules within milk such as lipids, proteins, and sugars, compared to an unchanged milk sample. This allowed us to note the minor discrepancies that happened in spoilage. Spoilt milk was characterized by bands associated with increased lipids, proteins, and lactic acid and a decrease in carbohydrates. A semi-quantitative prediction model for milk spoilage at room temperature demonstrated ATR FT-IR spectroscopy can predict milk age with a root mean square error of prediction of approximately 14 h. The model showed poor performance in the first 40 h but the predictions improved significantly after this time. The experimental procedure proposed for detecting biomolecules within milk has the potential to improve common practice. Furthermore, the model would be a starting point for newer and improved methods to predict the spoilage date of milk, with potential commercial uses to reduce food waste and costs to the milk industry.

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.

ABSTRACT Milk spoilage caused by psychrotrophic bacteria and their heat-stable enzymes is a serious challenge for the dairy industry. In many studies, spoilage has been explored based on the simplistic view of undesirable enzymes produced by planktonic cells. Recently, biofilms and quorum sensing (QS) have been suggested as important factors in the deterioration of milk, which opens new avenues for investigation of the processes and challenges. Production and heat stability of enzymes are enhanced in biofilms, mainly because of inherent differences in physiological states and protective shielding by extracellular polymeric substances. QS plays a key role in modulating expression of hydrolytic enzymes and biofilm formation. To date, few studies have been conducted to investigate the complex interplays of enzyme production, biofilm formation, and QS. This review provides novel insights into milk spoilage with particular emphasis on the roles of biofilms and QS and summarizes potential effective strategies for controlling the spoilage of milk.

Spices and herbs are usually added to milk to impart a particular flavour and medicinal purposes. Moreover, it is reported that additional spices and herbs extend the shelf life of milk. Contemporary use of essential oils from spices and herbs show promising results against various food spoilage microorganisms. Therefore, the essential oils from the spices and herbs from Zanzibar were used to assess antimicrobial activity against milk spoilage bacteria. The essential oils were extracted using steam distillation by a Clevenger apparatus. Minimum inhibitory concentrations (MICs) were determined by the microdilution method. All the extracts from cardamom, cinnamon, ginger, and lemongrass showed antimicrobial activity against Streptococcus thermophillus, Lactobacillus plantarum, and Escherichia coli. The essential oils exhibited higher antimicrobial activity than gentamycin. The MICs ranged between 0.004 and 0.125 µg/µl. Therefore, spices and herbs added to milk have the potential to inhibit the growth of milk spoilage bacteria.

M. Tech. (Biotechnology, Department of Biosciences, Faculty of Applied and Computer Sciences), Vaal University of Technology.
The Organization for Economic Co-operation and Development (OECD) and the Food and Agriculture Organization (FAO) of the United Nations predict that milk production and the dairy sector will remain one of the fastest-growing agricultural subsectors over the coming decade. The global milk production is projected to expand over the 2011-2020 period at an annual rate of 2%. In South Africa alone, approximately 14 – 15 million litres of milk are wasted annually due to microbial spoilage. Therefore, the identification of the spoilage microorganisms in the milk products is necessary. This will contribute towards the design of appropriate measures to prevent wastage due to spoilage and in turn contribute towards sustainability of the sector. Accordingly, one hundred samples of spoiled full cream UHT milk were collected from two plants of each of the two largest milk processors. These samples were examined visually, and the pH was measured. A presumptive identification up to genus level was conducted by examining morphological features and conducting Gram-stain, catalase and oxidase tests. Species-specific identification was done by using the Analytical Profile Index and Biolog system. Molecular profiling was done by sequencing the rDNA genes. The main spoilage organisms identified in the samples were Pseudomonas, Micrococcus, Bacillus, Enterococcus and Lactobacillus. All organisms belonging to the five genera were psychrotrophs, which are commonly found in biofilms in UHT milk processing equipment. Therefore, according to the study, the spoilage bacteria apparently entered into the milk due to inadequate cleaning-in-place (CIP) processes. More importantly, further studies should be conducted in order to identify the spoilage microbes and how CIP processes can be improved.

Milk is an important nutritious component of our diet consumed by most humans on a daily basis. Microbiological spoilage affects its safe use and consumption, its organoleptic properties and is a major part of its quality control process. European Union legislation and the Hazard Analysis and the Critical Control Point (HACCP) system in the dairy industry are therefore in place to maintain both the safety and the quality of milk production in the dairy industry. A main limitation of currently used methods of milk spoilage detection in the dairy industry is the time-consuming and sometimes laborious turnover of results. Attenuated total reflectance (ATR) and high throughput (HT) Fourier transform infrared (FTIR) spectroscopy metabolic fingerprinting techniques were investigated for their speed and accuracy in the enumeration of viable bacteria in fresh pasteurized cows' milk. Data analysis was performed using principal component-discriminant function analysis (PC-DFA) and partial least squares (PLS) multivariate statistical techniques. Accurate viable microbial loads were rapidly obtained after minimal sample preparation, especially when FTIR was combined with PLS, making it a promising technique for routine use by the dairy industry. FTIR and Raman spectroscopies in combination with multivariate techniques were also explored as rapid detection and enumeration techniques of S. aureus, a common milk pathogen, and Lactococcus lactis subsp cremoris, a common lactic acid bacterium (LAB) and potential antagonist of S. aureus, in ultra-heat treatment milk. In addition, the potential growth interaction between the two organisms was investigated. FTIR spectroscopy in combination with PLS and kernel PLS (KPLS) appeared to have the greatest potential with good discrimination and enumeration attributes for the two bacterial species even when in co-culture without previous separation. Furthermore, it was shown that the metabolic effect of L. cremoris predominates when in co-culture with S. aureus in milk but with minimal converse growth interaction between the two microorganisms and therefore potential implications in the manufacture of dairy products using LAB. The widespread and high consumption of milk make it a target for potential financial gain through adulteration with cheaper products reducing quality, breaking labeling and patent laws and potentially leading to dire health consequences. The time consuming and laborious nature of currently used analytical techniques in milk authentication enabled the study of FTIR spectroscopy and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) as rapid analytical techniques in quantification of milk adulteration, using binary and tertiary fresh whole cows', goats' and sheep's milk mixture samples. Chemometric data analysis was performed using PLS and KPLS multivariate analyses. Overall, results indicated that both techniques have excellent enumeration and detection attributes for use in milk adulteration with good prospects for potential use in the dairy industry.

The effects of natamycin, oxygen scavengers and a 25% CO₂:75% N₂ modified atmosphere on the growth of Penicillium roqueforti in shredded cheddar and mozzarella cheese stored at 10°C for 0, 60, 120, or 180 day was studied. Microbiological and sensory testing was assessed on 0, 7, 14 and 21 days after opening. Carbon dioxide decreased (P<0.05) as O₂ (P>0.05) and N₂ (P>0.05) increased throughout storage. Cheddar and mozzarella cheeses were stored for 180 and 60 days, respectively without significant (P> 0.05) increases in yeast and mold populations. Fungal populations increased significantly (P< 0.05) after packages were opened. Differences in yeast and mold (YM) counts during storage and once the packages were opened were independent of natamycin application and presence, O₂ scavengers and inoculated Penicillium roqueforti for both types of cheeses. Growth of Penicillium roqueforti, Aspergillus niger, Geotrichum candidum and Neosartorya fischeri were evaluated in atmospheres of 0:30:70, 0.5:29.5:70, 1:29:70, 2:28:70, and 5:25:70, O₂:CO₂:N₂ over a 5-day period. Spores were cultured on antibiotic-supplemented potato dextrose agar (pH 5.6, a_w 0.95) and incubated at 25°C. All four molds germinated and grew at 0.5:29.5:70. Extent of mycelia growth diameter (mm) increased significantly (P<0.05) as oxygen concentration increased from 0.5% to 5%. All growth was inhibited at 0:30:70, but germination and growth occurred once cultures were exposed to 20.9% atmospheric O₂, indicating that a modified atmosphere containing no residual O₂ is fungistatic. Yeast and mold growth was seen in ultra-pasteurized (UP) extended shelf-life fluid milk stored at (7.2°C). Ten half-pint, pint, quart and half gallon filled cartons were randomly selected from all UP products available. Samples, pulled at random on day 0, 15, 30, 45, and 60, were plated on Yeast and Mold Petrifilm™. Forty-seven percent of the UP products stored for 45 days tested positive for mold. Fungal growth was apparent down the side and along the bottom of the 5th panel. Contamination was traced to the presence of yeast and mold spores in paperboard cartons. Pinholes were present in the polyethylene coating and wicking occurred at the unskived 5th panel. Fungi of similar origin and fatty acid profile were isolated from UP milk products and the paperboard cartons.
Ph. D.

Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d₂, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.

Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d2, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
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RIASSUNTO I batteri psicrotrofi sono i principali responsabili del deterioramento del latte crudo o termotrattato poiché sono in grado di sintetizzare proteasi e lipasi extracellulari termostabili, causa di formazione di odori e sapori sgradevoli, fenomeni di gelificazione, riduzione delle proprietà schiumogene del latte, perdita di qualità sensoriale e riduzione della shelf-life. Ad oggi, non esiste ancora una sufficiente conoscenza degli specifici pathways proteolitici e lipolitici di questi enzimi termostabili. Inizialmente questo lavoro ha riguardato la caratterizzazione dell’attività enzimatica di 80 ceppi di psicrotrofi isolati da latte crudo. I batteri appartenenti al genere Pseudomonas sono stati i più isolati (78.75%) e Pseudomonas fluorescens è risultata la specie predominante (30.16%); tra le Enterobacteriaceae (21.25%), Serratia marcescens è stata la specie più frequentemente isolata (52.94%). Quarantuno ceppi di psicrotrofi mostravano tutte le attività enzimatiche. Il più alto numero di ceppi positivi a tutte le temperature di incubazione è stato osservato per l’attività lipolitica (59) e, a seguire, proteolitica (31) e lecitinasica; i tratti enzimatici variavano tra i ceppi di Pseudomonas e Enterobacteriaceae ed erano marcatamente influenzati dalla temperature di incubazione, essendo 30 °C quella ottimale. Il gene aprX è stato ritrovato in 19 ceppi di Pseudomonas ed è risultato esser diffuso tra i ceppi di P. fluorescens (15 su18). La seconda parte della ricerca è stata focalizzata sulla valutazione della produzione di composti organici volatili (VOCs) e del rilascio di acidi grassi liberi (FFAs) da ceppi di batteri psicrotrofi. Diverse specie del genere Pseudomonas e il ceppo Serratia marcescens hanno mostrato profili di VOCs complessi e dipendenti dal ceppo batterico inoculato in campioni di latte UHT, durante le differenti condizioni di incubazione. In particolare, sono stati identificati 56 VOCs appartenenti a aldeidi, chetoni, acidi grassi, alcoli, composti solforati e idrocarburi. Generalmente, il numero di VOCs tendeva ad incrementare con il progredire del tempo di incubazione, sia nel latte controllo non inoculato sia nei campioni di latte contaminati. Tra i VOCs rilevati, alcune molecole sono state individuate solo quando il latte era contaminato da uno specifico ceppo microbico. Nel dettaglio, 3-metilbutan-2-olo, 3-metilesan-2-olo, pentan-1-olo e 3,3-dimetilesano sono stati rilevati solo a seguito dello sviluppo di P. fragi. P. rhodesiae è stata l’unica specie a produrre pentano-2,3-dione, eptano e 3-metilesano, mentre l’esano è stato rilasciato solo in campioni di latte contaminati con P. fluorescens. La maggior produzione di composti solforati e alcoli è stata individuata nello spazio di testa del latte contaminato con P. mosselii e P. fragi, rispettivamente. Lo sviluppo e l’attività di P. rhodesiae e S. marcescens sono risultati associati ad un più alto numero di acidi grassi e chetoni mentre P. fluorescens ha mostrato la più scarsa produzione di composti volatili. Alcuni VOCs come 3-metilbutan-1-olo, 2-metilpropan-1-olo, 3-idrossibutan-2-one, butano-2,3-dione, acido butanoico ed acido esanoico potrebbero perciò rappresentare potenziali marker per il riconoscimento dell’attività enzimatica di batteri psicrotrofi e per la precoce individuazione del deterioramento del prodotto. Per quanto riguarda il rilascio di FFAs, diverse quantità di questi composti sono state rilasciate dai batteri psicrotrofi appartenenti a specie diverse e alla stessa specie conseguentemente alla diversa capacità di produrre lipasi. Gli acidi palmitico (16:0), oleico (18:1) e linoleico (18:2) sono risultati i più presenti tra gli acidi grassi saturi, monoinsaturi e polinsaturi. P. fluorescens PS73 e P. fluorescens PS81 sono state le specie che hanno prodotto la maggior quantità di FFAs, a 24 h e 4 giorni di incubazione, rispettivamente. Al contrario, H. alvei PS57 e P. fragi PS55 hanno rilasciato la minor quantità di FFAs ad entrambi i tempi di incubazione. Le lipasi dei ceppi di psicrotrofi hanno mostrato una specificità variabile nei riguardi degli esteri degli acidi grassi con diversa lunghezza della catena carboniosa. P. fragi PS55, P. putida PS17, P. fluorescens PS14 and P. fulva PS10 sono risultate le specie più attive nell’idrolisi dei trigliceridi. La lipasi del ceppo di P. rhodesiae PS62 ha mostrato la più scarsa capacità idrolitica verso tutti i trigliceridi testati. Infine, è stata effettuata la caratterizzazione proteomica di proteasi extracellulari di ceppi di P. fluorescens. Una proteasi termostabile di circa 45 kDa è stata individuata su casein zymogram gel in ciascun surnatante dei ceppi batterici selezionati. L’estratto enzimatico del ceppo P. fluorescens PS19, concentrato per ultrafiltrazione (10 kDa), ha mostrato un’elevata attività proteolitica e due ulteriori bande proteolitiche di circa 15 e 25 kDa. I risultati delle analisi nLC/MS/MS dopo digestione sia in gel che in solution hanno evidenziato che la proteasi di 45 kDa corrisponde ad una AprX metalloproteasi di P. fluorescens (acc. no. C9WKP6, UniProt). La proteasi di 15 kDa è stata riconosciuta come un frammento della stessa AprX metalloproteasi, mentre la proteasi di 25 kDa non ha mostrato nessuna omologia con alcuna delle proteine note di Pseudomonas. La caratterizzazione tramite LC/MS del profilo peptidico generato dall’azione delle proteasi termostabili dello stesso ceppo sulle frazioni caseiniche del latte è in corso di studio. In generale, questo studio fornisce ulteriori conoscenze per la lo studio delle attività enzimatiche di batteri psicrotrofi nel latte.
ABSTRACT Psychrotrophic bacteria are responsible for the highest spoilage of unprocessed or heated milk during storage because of their capacity to synthesize thermostable extracellular proteases and lipases. The activities of these enzymes lead to formation of off-odours/flavours, gelation of milk, lowering of milk foaming properties, loss of sensory quality and shortening of the shelf life. To date, still little is known about the specific proteolytic and lipolytic pathways of these thermostable enzymes. Initially we evaluated the enzymatic traits of 80 raw milk-associated psychrotrophic strains. Among psychrotrophic isolates, Pseudomonas were the most commonly occurring contaminants (78.75%) being Pseudomonas fluorescens the predominant isolated species (30.16 %), along with Enterobacteriaceae (21.25%), primarily Serratia marcescens (52.94 %). Forty-one of the psychrotrophic strains were positive for all the enzymatic activities. The highest number of positive strains for all incubation temperatures was found for the lipolytic activity (59), followed by proteolytic (31) and lecithinase (28) activities. The enzymatic traits varied among the Pseudomonas and Enterobacteriaceae strains and were markedly influenced by incubation temperature being 30 °C the optimal one. The aprX gene was detected in 19 out of 80 psychrotrohic strains and it resulted widespread among P. fluorescens strains (15 out of 18). The second part of the research was focused on the evaluation of spoilage potential of psychrotrophic strains by analyzing the production of volatile organic compounds (VOCs) and the release of free fatty acids (FFAs). From results of SPME-GC/MS analysis, different species of the genus Pseudomonas and Serratia marcescens produced a complex and strain-dependent VOCs profiles in UHT milk samples at different storage and time conditions. Fifty-six VOCs belonging to 7 chemical groups (aldehydes, ketones, fatty acids, esters, alcohols, sulphur compounds and hydrocarbons) were identified. Generally, the VOCs went to increase during the storage time both in the control and contaminated milk samples, some compounds being detected only in the latter samples. Compounds such as 3-methylbutan-2-ol, 3-methylhexan-2-ol, pentan-1-ol and 3,3-dimethylhexane were detectable only for P. fragi. P. rhodesiae was the only species producing pentane-2,3-dione, heptane and 3-methylhexane while hexane was released only by P. fluorescens. P. mosselii and P. fragi produced the highest number of sulphur compounds and alcohols, respectively. The highest number of FFAs and ketons was detected in the headspace of milk samples contaminated by P. rhodesiae and S. marcescens. P. fluorescens provided the lowest development of VOCs. 3-methylbutan-1-ol, 2 methylpropan-1-ol, 3-hydroxybutan-2-one, butane-2,3-dione and butanoic and hexanoic acids could be regarded as potential markers of psycrotrophic contamination useful for the early detection of milk bacterial spoilage. Regarding the release of FFAs, different quantities of these compounds have been released from milk fat by tested bacteria, between and within species, in relation to diverse capacity for production of lipolytic enzymes. Palmitic (16:0), oleic (18:1) and linoleic (18:2) acids levels were found to be the highest among the SFAs, MUFAs and PUFAs, respectively. P. fluorescens PS73 and P. fluorescens PS81 were the major FFAs producers, at 24 h and 4 days of incubation, respectively while H. alvei PS57 and P. fragi PS55 were the less active in lipid breakdown at both the incubation conditions. Lipases from psychrotrophic strains showed a variable range of specificity toward fatty acid esters with different fatty acid chain lengths, being P. fragi PS55, P. putida PS17, P. fluorescens PS14 and P. fulva PS10 the more active to hydrolyse triglycerides. Lipase from P. rhodesiae PS62 showed the highest hydrolytic resistance toward all tested fatty acid triglycerides. Finally, proteomic characterization of extracellular proteases of P. fluorescens strains has been performed. One thermostable protease of approximately 45 kDa was detected in each of the cell-free supernatant of the selected strains on a casein zymogram gel. After concentration by ultrafiltration (10 kDa), the protease extract of P. fluorescens PS19 showed a high proteolytic activity and two additional proteolytic bands with molecular masses of approximately 15 and 25 kDa on casein zymography. This extract was subjected to proteomic characterization by nLC/MS/MS analysis of both in gel and in solution digestion. Results showed the protease of 45 kDa to correspond to P. fluorescens AprX metalloprotease (acc. no. C9WKP6, UniProt). In addition, the same results leaded to recognize the 15 kDa protease as a fragment of this AprX metalloprotease. On the contrary, the 25 kDa protease showed no homology to any known protein of Pseudomonas spp. The characterization by LC/MS of the peptides profile generated by the action of thermostable proteases of the same strain on milk caseins is still under investigation. Overall, this study provides a better understanding of the enzymatic activities of psychrotrophic bacteria in milk.

Thesis (M.S.)--University of Wisconsin--Madison, 2004.
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Several lactic acid bacteria (LAB) of the Lactococcus, Lactobacillus, Leuconostoc and Pediococcus genera were screened for inhibition of food-borne pathogens and spoilage microorganisms in raw milk and dairy products. Listeria monocytogenes was killed by Lactococcus lactis subsp. lactis and Pediococcus pentosaceus due to their production of bacteriocin-type inhibitors. Staphylococcus aureus was not able to grow in raw milk at temperatures below 5°C even without LAB being present. Gram negative Salmonella enteritidis. Salmonella typhimurium and Escherichia coli, along with spoilage bacteria of the genus Pseudomonas were dramatically inhibited by a Lactobacillus species, designated AS-1, in raw and pasteurized milk as well as in cottage cheese. However, other LAB were not able to inhibit these organisms. Lactobacillus AS-1, did not produce hydrogen peroxide but carbon dioxide was produced. The AS-1 strain was a gram positive coccobacillus, catalase and oxidase negative and produced DL-lactic acid. It deaminated arginine and grew over a temperature range of 5°C to 45°C. It was also able to ferment glucose, galactose, fructose and lactose in addition to 17 other carbohydrates. High numbers (107 CFU/ml) of AS-1 were required to obtain complete inhibition of gram negative bacteria. A selective medium (ASLM) for Listeria monocytogenes was developed to follow the fate of this particular pathogen in association with LAB in raw milk; other selective media were not able to inhibit the growth of background flora of raw milk. ASLM was superior to four other media in allowing only the growth of the target pathogen. For the Lactococcus genus, a selective and differential agar medium (Alsan) was formulated to selectively allow growth of Lactococcus spp. and to differentiate between Lactococcus lactis subsp. lactis and the biovariety diacetylactis, based on citrate utilization.
Graduation date: 1993

Milk, which is a basic component of the human diet since millennia worldwide, is rich in all kind of nutrients, thus comprises an excellent growth substrate for both beneficial and harmful microorganisms. As a result, milk contains numerous microorganisms deriving either from the animal itself or from the environment during the collection and storage of milk. These microbes can play a number of roles, such as facilitating dairy fermentations, promoting health, causing spoilage or even disease. For decades, the microbial ecology of dairy products has been based on the study of microbial isolates through conventional culture-dependent microbiological analysis. However, culture-independent approaches, and, most importantly, high throughput sequencing coupled with the omics-based techniques enable a thorough taxonomic fingerprinting of the dairy microbiota. This chapter provides a broad overview on the microbiota of raw cow's milk, as this has been described so far by both culture-dependent and -independent approaches.

Examines aspects of the microbiology of the major food commodity groups, e.g. milk and meat and their products, describing the microflora with particular emphasis on spoilage. How this might be changed by common preservation/processing techniques is also discussed.

Milk spoilage is a common issue in remote dairy farms due to the unavailability of power grid. Many farmers rely on diesel engines to power their milk chillers. A sustainable approach is to replace the environmentally harmful diesel generators with solar powered chillers. Solar energy is attractive in such application because the peak cooling demand occurs at the peak solar irradiance. Solar energy can be converted to cooling through PV powered chiller or through thermally driven chillers. This concept was applied to a Modular Solar Bulk Milk Chiller (MilkPod™) which uses the solar energy to generate electricity in 6 kWp PV panels. The system cools 600 liters of milk per day as well as produces hot water for cleaning the milk tank. A detailed model was used in the design process and the system equipment were selected such that the system uses about 98% of its energy from the sun throughout the year. The effect of the solar energy utilization ratio, equipment supplied current as well as milk loading were investigated on the system performance.

In the preserved grain, urea, when subjected to hydrolysis, secretes am-MIAC, protecting it from self-heating and spoilage, and the other part of it goes to ammonium compounds, thereby increasing the protein nutrition of the grain. Processing of grain with urea contributed to a decrease in the concentration of protein, both soluble — by 3.24%, and cleavable-by 4.01%, compared with the control. The cost of processing 1 ton of grain with urea is 1.72 times cheaper than conventional drying. The profitability of milk production increased by 26.62%.

A qualidade do leite cru está associada às boas práticas de ordenha proveniente de rebanhos sadios e com refrigeração obrigatória do leite cru na granja leiteira entre 4 e 7° C, podendo chegar até, no máximo, 9° C durante a coleta final (BRASIL 2018). Embora a refrigeração seja eficaz para aumentar a vida útil do leite cru, a manutenção das baixas temperaturas favorece a multiplicação de bactérias psicrotróficas, que produzem proteases e lipases termorresistentes, que permanecendo ativas mesmo após a pasteurização, podem afetar negativamente, tanto o próprio leite, quanto seus derivados (RIBEIRO et al., 2018). Visando reduzir a contagem de microrganismos psicrotróficos no leite cru, o presente trabalho avaliou a eficácia da aplicação de ozônio, considerado um bactericida verde por não ser poluente e não deixar resíduos no alimento, diretamente no tanque de resfriamento. As amostras de leite cru foram obtidas de um rebanho saudável e recolhidas diretamente do tanque de resfriamento da propriedade e armazenadas em frascos estéreis com volume de 6 litros sob refrigeração (4 e 9° C). A aplicação do ozônio seguiu delineamento fatorial com variáveis independentes sendo as temperaturas do leite (4 e 9° C) e os tempos de exposição ao ozônio (5 e 15 minutos) utilizando Gerador de Ozônio N202F Bivolt 500mg/h O³ Disinfector - Diluka Power. Foram retiradas amostras antes e após a ozonização. As amostras foram então armazenadas por 48 horas sob refrigeração (4° C) para permitir a proliferação da flora psicrotrófica. O teor de psicrotróficos e a contagem bacteriana total das amostras e do grupo controle foram avaliados em meio ágar bacteriológico PCA por 10 dias a 7° C e a 36° C por 24 horas, respectivamente. A aplicação de ozônio foi eficaz em reduzir a contagem de microrganismos psicrotróficos no tempo de 5 minutos a 4° C, esse resultado é extremamente satisfatório, visto que, a temperatura de aplicação é a mesma aplicada aos tanques de refrigeração nas propriedades, o que permitiria, facilmente, a aplicação nas próprias fazendas. Agradecimentos Agradecemos à equipe do Serviço de Orientação à Alimentação Pública da FMVZ-Unesp e ao proprietário da fazenda por disponilibizar o material utilizado na pesquisa. Referências BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Instrução normativa nº 76, de 26 de novembro de 2018. Regulamentos Técnicos que fixam a identidade e as características de qualidade que devem apresentar o leite cru refrigerado, o leite pasteurizado e o leite pasteurizado tipo A. Diário Oficial da União, Brasília, 2018. RIBEIRO JÚNIOR, J. C.et al. The main spoilage1 related psychrotrophic bacteria in refrigerated raw milk. Journal of dairy science, v. 101, n. 1, p. 75-83, 2018. PALAVRAS-CHAVE: Microbiologia dos alimentos, Qualidade do leite, Tecnologia verde