Spoilage Bacteria In Milk Research Articles

The Essential Oil from the Spices and Herbs Have Antimicrobial Activity Against Milk Spoilage Bacteria

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.

A rapid and efficient DNA isolation method for qPCR-based detection of pathogenic and spoilage bacteria in milk

The objective of this study was to find an efficient, rapid, simple, and cost-effective method of pretreating raw milk samples to produce PCR-ready DNA for subsequent microbial detection using the strains of eight bacterial species. A total of 17 in-house protocols and three commercial kits were evaluated in three steps from scientific, practical, and economic perspectives. The results showed that an in-house procedure involving Triton X-100-based pretreatment and an inhibitor removal resin was superior to all other methods tested in terms of DNA yield, sensitivity, ease of sample handling, time efficiency, and cost per sample. Overall, this simplified preanalytical protocol was shown to have a great potential for use in rapid detection of dairy-related bacterial species, thereby enabling early intervention in the food chain and thus reducing the risk of negative economic and health outcomes. • Methods of pretreating raw milk samples to yield PCR-ready bacterial DNA were tested. • Of 20 procedures, a Triton X-100-based in-house protocol showed the best results. • This approach has a great potential for use in detection of dairy-related bacteria. • With further optimization, the protocol may be included in milk quality guidelines.

Photodynamic inactivation of Pseudomonas fluorescens in Minas Frescal cheese using curcumin as a photosensitizer

The dairy industries demand innovative technologies for food preservation and elimination of psychrotrophic bacteria like the Photodynamic Inactivation of Microorganisms (PDIM) modalit, which is based on the generation of reactive oxygen species using photosensitizers. The objective was to determine in vitro the phototoxic potential of Curcumin (CUR) solubilized in different media against Pseudomonas fluorescens. In Minas Frescal cheese inoculated with Pseudomonas fluorescens, CUR acted as an antimicrobial agent and photosensitizers when incorporated in an edible and biodegradable coating. The coatings were characterized in terms of mechanical properties and permeability. The cheeses were evaluated on days 0, 3, 6, 9 and 12 by microbiological, physical-chemical and mechanical analysis. The efficiency of PDIM was influenced by solubilizers and concentrations of CUR, observing the synergistic effect of CUR with irradiation. Dimethyl sulfoxide and ethanol were the solubilizers that allowed greater CUR phototoxic activity and reduced 7 log CFU/mL of Pseudomonas fluorescens, the main spoilage bacteria in milk and dairy products. In cheeses, the photoinhibition of microorganisms was hampered by the complexity of the surface. The samples treated with CUR showed lower bacterial count and proteolysis, and consequently, greater hardness. Therefore, PDIM can be an alternative technique for use in dairy products.

Ultrasound on Milk Decontamination: Potential and Limitations Against Foodborne Pathogens and Spoilage Bacteria

ABSTRACT Ultrasound is an emerging non-thermal and environment-friendly technology with high potential applications for milk and dairy products. However, several bacterial factors still limited industrial adoption. Bacterial morphological aspects in ultrasound efficacy and mechanisms promote the lack of this technology and restrict the improvement of bacterial inactivation. Hence, this review focused on the capacity of ultrasound in the inactivation of pathogenic and spoilage bacteria in milk. In addition, this paper presents the key scientific limitations on the use of ultrasound on an industrial scale. The knowledge of the limitations allows (i) the study of optimal conditions for the application of ultrasound in milk in different bacterial genera and (ii) the eligibility of other inactivation methods aiming at the combined use to increase the efficiency according to the hurdle theory. Thus, reaching higher levels of microbiological safety with environmental sustainability may be in the near future.

Antimicrobial effects of a bioactive glycolipid on spore-forming spoilage bacteria in milk

The growth of psychrotolerant aerobic spore-forming bacteria during refrigerated storage often results in the spoilage of fluid milk, leading to off-flavors and curdling. Because of their low toxicity, biodegradability, selectivity, and antimicrobial activity over a range of conditions, glycolipids are a novel and promising intervention to control undesirable microbes. The objective of this study was to determine the efficacy of a commercial glycolipid product to inhibit spore germination, spore outgrowth, and the growth of vegetative cells of Paenibacillus odorifer, Bacillus weihenstephanensis, and Viridibacillus arenosi, which are the predominant spore-forming spoilage bacteria in milk. For spore germination and outgrowth assays, varying concentrations (25-400 mg/L) of the glycolipid product were added to commercial UHT whole and skim milk inoculated with ∼4 log10 spores/mL of each bacteria and incubated at 30°C for 5 d. Inhibition of spore germination in inoculated UHT whole milk was only observed for V. arenosi, and only when glycolipid was added at 400 mg/L. However, concentrations of 400 and 200 mg/L markedly inhibited the outgrowth of vegetative cells from spores of P. odorifer and B. weihenstephanensis, respectively. No inhibition of spore germination or outgrowth was observed in inoculated UHT skim milk for any strain at the concentrations tested (25 and 50 mg/L). The effect of glycolipid addition on vegetative cell growth in UHT whole and skim milk when inoculated with ∼4 log10 cfu/mL of each bacteria was also determined over 21 d of storage at 7°C. Glycolipid addition at 50 mg/L was bactericidal against P. odorifer and B. weihenstephanensis in inoculated UHT skim milk through 21 d of storage, whereas 100 mg/L was needed for similar control of V. arenosi. Concentrations of 100 and 200 mg/L inhibited the growth of vegetative cells of B. weihenstephanensis and P. odorifer, respectively, in inoculated UHT whole milk, whereas 200 mg/L was also bactericidal to B. weihenstephanensis. Additional studies are necessary to identify effective concentrations for the inhibition of Viridibacillus spp. growth in whole milk beyond 7 d. Findings from this study demonstrate that natural glycolipids have the potential to inhibit the growth of dairy-spoilage bacteria and extend the shelf life of milk.

Modelling Growth and Decline in a Two-Species Model System: Pathogenic Escherichia coli O157:H7 and Psychrotrophic Spoilage Bacteria in Milk.

Shiga toxin-producing Escherichia coli O157:H7 is a food-borne pathogen and the major cause of hemorrhagic colitis. Pseudomonas is the genus most frequent psychrotrophic spoilage microorganisms present in milk. Two-species bacterial systems with E. coli O157:H7, non-pathogenic E. coli, and P. fluorescens in skimmed milk at 7, 13, 19, or 25 °C were studied. Bacterial interactions were modelled after applying a Bayesian approach. No direct correlation between P. fluorescens’s growth rate and its effect on the maximum population densities of E. coli species was found. The results show the complexity of the interactions between two species in a food model. The use of natural microbiota members to control foodborne pathogens could be useful to improve food safety during the processing and storage of refrigerated foods.

Species-Level Discrimination of Psychrotrophic Pathogenic and Spoilage Gram-Negative Raw Milk Isolates Using a Combined MALDI-TOF MS Proteomics-Bioinformatics-based Approach.

Identification of psychrotrophic pathogenic and spoilage Gram-negative bacteria using rapid and reliable techniques is important in commercial milk processing, as these bacteria can produce heat-resistant proteases and act as postprocessing contaminants in pasteurized milk. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a proven technology for identification of bacteria in food, however, may require optimization for identification of pathogenic and spoilage bacteria in milk and dairy products. The current study evaluated the effects of various culture conditions and sample preparation methods on assigning of raw milk isolates to the species level by MALDI-TOF MS. The results indicated that culture media, incubation conditions (temperature and time), and sample preparation significantly affected the identification rates of bacteria to the species level. Nevertheless, the development of spectral libraries of isolates grown on different media using a web tool for hierarchical clustering of peptide mass spectra (SPECLUST) followed by a ribosomal protein based bioinformatics approach significantly enhanced the assigning of bacteria, with at least one unique candidate biomarker peak identified for each species. Phyloproteomic relationships based on spectral profiles were compared to phylogenetic analysis using 16S rRNA gene sequences and demonstrated similar clustering patterns with significant discriminatory power. Thus, with appropriate optimization, MALDI-TOF MS is a valuable tool for species-level discrimination of pathogenic and milk spoilage bacteria.

Inhibition of bacterial growth in sweet cheese whey by carbon dioxide as determined by culture-independent community profiling

Whey is a valuable co-product from cheese making that serves as a raw material for a wide range of products. Its rich nutritional content lends itself to rapid spoilage, thus it typically needs to be pasteurised and refrigerated promptly. Despite the extensive literature on milk spoilage bacteria, little is known about the spoilage bacteria of whey. The utility of carbon dioxide (CO2) to extend the shelf-life of raw milk and cottage cheese has been well established, but its application in whey preservation has not yet been explored. This study aims to characterise the microbial populations of fresh and spoiled sweet whey by culture-independent community profiling using 454 pyrosequencing of 16S rRNA gene amplicons and to determine whether carbonation is effective in inhibiting bacterial growth in sweet whey. The microbiota of raw Cheddar and Mozzarella whey was dominated by cheese starter bacteria. After pasteurisation, two out of the three samples studied became dominated by diverse environmental bacteria from various phyla, with Proteobacteria being the most dominant. Diverse microbial profiles were maintained until spoilage occurred, when the entire population was dominated by just one or two genera. Whey spoilage bacteria were found to be similar to those of milk. Pasteurised Cheddar and Mozzarella whey was spoiled by Bacillus sp. or Pseudomonas sp., and raw Mozzarella whey was spoiled by Pseudomonas sp., Serratia sp., and other members of the Enterobacteriaceae family. CO2 was effective in inhibiting bacterial growth of pasteurised Cheddar and Mozzarella whey stored at 15°C and raw Mozzarella whey stored at 4°C. The spoilage bacteria of the carbonated samples were similar to those of the non-carbonated controls.

Adhesive properties of predominant bacteria in raw cow’s milk to bovine mammary gland epithelial cells

Various bacteria have been found in raw cow's milk, and identifying milk microflora and its functions is critical for maintaining cow health and farm hygiene. Although studies on pathogens and spoilage bacteria in milk have been widely reported, the relationship between milk bacteria, including nonpathogenic bacteria, and the bovine udder is poorly understood. We investigated milk microflora over 1year using a culture-dependent method and culture-independent analysis by denaturing gradient gel electrophoresis. Among 240 isolates, Lactococcus lactis (81/240) was predominant. The predominant genera were Lactococcus, Stenotrophomonas, Microbacterium, Chryseobacterium, Serratia and Pseudomonas. Among seven strains belonging to these predominant genera, two strains of L. lactis (ssp. lactis and ssp. cremoris) exhibited the highest adherence to bovine mammary gland epithelial cells (BMECs) derived from the bovine udder; 3.4% of the inoculated bacteria adhered to BMECs. This was followed by Serratia sp. (1.6%), Microbacterium sp. (0.8%), Stenotrophomonas maltophilia (0.5%), Pseudomonas sp. (0.3%) and Chryseobacterium sp. (0.1%). The two L. lactis isolates exhibited higher adherence to BMECs than type strains and isolates of various origins.

Molecular analysis of bacterial population structure and dynamics during cold storage of untreated and treated milk

Spoilage bacteria in milk are controlled by treatments such as thermization, microfiltration and addition of carbon dioxide. However, little information is known about the changes in microbial communities during subsequent cold storage of treated milk. Culture-dependent methods and a direct molecular approach combining 16S rRNA gene clone libraries and quantitative PCR (Q-PCR) were applied to obtain a better overview of the structure and the dynamics of milk microbiota. Raw milk samples were treated by the addition of carbon dioxide (CO 2), thermization (TH) or microfiltration (MF) and stored at 4 °C or 8 °C up to 7 d. Untreated milk (UT) was used as a control. Psychrotrophic and staphylococci bacteria were enumerated in the milk samples by culture methods. For the molecular approach, DNA was extracted from milk samples and 16S rRNA gene was amplified by PCR with universal primers prior to cloning. The Q-PCR method was used to evaluate the dynamics of dominant bacterial species revealed by clone library analysis of 16S rRNA gene. Comparison of the 16S rRNA gene sequence indicated that the two most abundant operational taxonomic units (OTU), determined at 97% identity, belonged to the class Gammaproteobacteria (40.3% of the 1415 sequences) and Bacilli (40%). Dominant bacterial species in UT, CO 2 and TH milk samples at day 3 were affiliated with Staphylococcus, Streptococcus, Clostridia, Aerococcus, Facklamia, Corynebacterium, Acinetobacter and Trichococcus. Dominant bacterial species detected in MF milk were Stenotrophomonas, Pseudomonas and Delftia, while Pseudomonas species dominated the bacterial population of UT, CO 2 and MF milk samples at day 7. Staphylococcus and Delftia were the dominant bacterial species in thermized milk. Q-PCR results showed that populations of S. aureus, A. viridans, A. calcoaceticus, C. variabile and S. uberis were stable during 7 d of storage at 4 °C. Populations of P. fluorescens, S. uberis and total bacteria increased in UT and CO 2 milk samples during 7 d of storage at 8 °C and were noticeable from day 3. This study shows new microbial species which can develop during cold storage after milk treatment and contributes to identifying causes of reduced shelf life and deterioration of technological properties of milk during storage.

Rapid On-chip flow Cytometric Detection of Listeria monocytogenes in Milk

Listeria monocytogenes (L. monocytogenes) is a Gram-positive, intracellular bacterium that can cause severe infections in humans. Contamination by L. monocytogenes in foods represents a potential public health problem. Here we describe a rapid method for the detection of L. monocytogenes in milk by flow cytometry based on microfluidics (on-chip flow cytometry). This rapid identification of L. monocytogenes is based on fluorescence in situ hybridization (FISH) with a Cy5-labeled rRNA-targeting oligonucleotide probe. FISH experiments were successfully analyzed using a commercially available on-chip flow cytometry. FISH results with bacterial cultures indicated that the L. monocytogenes probe RL-2 was hybridized with 4 major strains of L. monocytogenes (serotype 1/2a, 1/2b, 1/2c and 4b) but not with other Listeria strains or milk spoilage bacteria. Specific FISH detection of L. monocytogenes was accomplished using the probe RL-2 when the milk contained a mixture of other bacterial species. The positive identification of L. monocytogenes in milk was completed within 5 hr (milk clearing: 40 min, hybridization: 3.5 hr, on-chip analysis: 30 min). The method presented in this study allows the specific and rapid identification of L. monocytogenes in milk.

Tracking Heat-Resistant, Cold-Thriving Fluid Milk Spoilage Bacteria from Farm to Packaged Product

Control of psychrotolerant endospore-forming spoilage bacteria, particularly Bacillus and Paenibacillus spp., is economically important to the dairy industry. These microbes form endospores that can survive high-temperature, short-time pasteurization; hence, their presence in raw milk represents a major potential cause of milk spoilage. A previously developed culture-dependent selection strategy and an rpoB sequence-based subtyping method were applied to bacterial isolates obtained from environmental samples collected on a New York State dairy farm. A total of 54 different rpoB allelic types putatively identified as Bacillus (75% of isolates), Paenibacillus (24%), and Sporosarcina spp. (1%) were identified among 93 isolates. Assembly of a broader data set, including 93 dairy farm isolates, 57 raw milk tank truck isolates, 138 dairy plant storage silo isolates, and 336 pasteurized milk isolates, identified a total of 154 rpoB allelic types, representing an extensive diversity of Bacillus and Paenibacillus spp. Our molecular subtype data clearly showed that certain endospore-forming bacterial subtypes are present in the dairy farm environment as well as in the processing plant. The potential for entry of these ubiquitous heat-resistant spoilage organisms into milk production and processing systems, from the dairy farm to the processing plant, represents a considerable challenge that will require a comprehensive farm-to-table approach to fluid milk quality.

Application of gas-sensor array technology for detection and monitoring of growth of spoilage bacteria in milk: A model study

The aim of this study was to develop a novel, rapid system for detection and monitoring of growth of undesirable bacteria in food using gas-sensor array technology. Three spoilage bacteria isolated from a cheese-processing hall were identified as Serratia marcescens, Serratia proteamacufans and Pseudomonas putida. The growth of these bacteria in milk was investigated using a commercial solid state based gas-sensor array system. On the basis of the temporal sensor readings of the pure cultures, bacterial growth could be monitored and the individual strains identified and followed throughout the complete growth cycle in both single and mixed culture. The gas-sensor signals could be used as early indicators of the onset of bacterial growth. Start detection of volatile bacterial metabolites coincided with the start of the exponential growth phase taking place around 7 h after inoculation and corresponding to bacterial numbers of 10 4 (cfu/ml). The results were confirmed by comparing the gas profiles with the cell counts and by headspace gas chromatography mass spectrometry (GC/MS) of volatile microbial metabolites. High correlation ( r > 0.90, p < 0.001) was found between the gas-sensor readings and major secondary volatile metabolites. Using the sensor readings, cell numbers of single strain cultures could be predicted with an error of less than 5%. The results show that it is possible to monitor growth of individual strains of spoilage bacteria in a mixed culture in milk on the basis of the type and amount of volatile compounds which they produce, using a gas-sensor array system. The system thus affords possibilities for further development for quick, more accurate and full scale determinations of shelf life, the design of spoilage indicators, rapid identification of undesired microorganisms and rapid measurements of spoilage.

A Comparison of Ozonation and Chlorination for the Disinfection of Stainless Steel Surfaces

Ozonated water and chlorinated sanitizer were compared for effectiveness against biofilms of milk spoilage bacteria. Stainless steel plates were incubated in UHT-pasteurized milk inoculated with pure cultures of either Pseudomnas fluorescens (ATCC 949) or Alcaligenes faecalis (ATCC 337). After incubation, the plates were removed and rinsed in sterile PBS. A control rinsed stainless steel plate was swabbed and plated on standard plate count agar. A second rinsed stainless steel plate was covered and treated for 2min with a commercial chlorinated sanitizer (dichloro-s-triazinetrione), prepared according to the manufacturer's recommendations; after treatment, the plate was rinsed twice in sterile PBS, swabbed, and plated on standard plate count agar. A third rinsed stainless steel plate from the culture was placed in ozonated deionized H20 (.5ppm of ozone) for 10min, rinsed twice as described, swabbed, and plated. Both ozonation and chlorination reduced bacteria populations by >99% at initial cell densities in the range of approximately 1.24 × 105 to 8.56 × l05 cfu/cm2 for P. flourescens and 1.53 × 104 to 8.56 × 105 cfu/cm2 for A. faecalis in milk films on stainless steel surfaces.

Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay.

Pseudomonas fluorescens P1 is a psychrotrophic bacterium isolated from milk. Proteinase P1, the main extracellular heat-stable proteinase fraction of P. fluorescens P1, has been purified to homogeneity. A procedure with a sandwich enzyme-linked immunosorbent assay, using microplates and alkaline phosphatase conjugate was shown to detect 0.25 ng of proteinase P1 in 1 ml of reconstituted skim milk or defatted cream. The method offers the combination of sensitivity and specificity for the detection of these enzymes in milk and dairy products. In reconstituted skim milk cultures proteinase P1 was detectable when the CFU approached 10(7)/ml. Cultures in milk diluted 1:10, 1:30, or 1:100 with water showed detectable proteinase at population densities close to 10(6) CFU/ml. Aeration stimulated proteinase production; thus, a skim milk culture with shaking at 5 degrees C had a proteinase level of 36,000 ng/ml after 7 days as compared to 80 ng/ml in a stationary culture. The rate of inactivation of proteinase P1 at 150 and 55 degrees C as expressed by residual antigenic activity determined by the enzyme-linked immunosorbent assay was somewhat different from the rate determined on the basis of residual proteolytic activity. The specificity of the enzyme-linked immunosorbent assay with proteinase P1 antibodies was identical for proteinase P1 and for enzymes from six other strains of P. fluorescens, one Chromobacterium strain, and one Flavobacterium strain. Some psychrotrophic strains produced immunologically unrelated proteinase(s). These preliminary observations indicate that proteinase P1-related enzymes are common among psychrotrophs appearing as spoilage bacteria in milk.