Incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia

The purpose of this study was to evaluate the incidence and characterisation of aerobic spore‐forming bacteria originating from dairy milk in Tunisia. The distribution of Bacillus species in raw milk, pasteurised milk and UHT milk were 47.5%, 27.5% and 25%, respectively. Seven Bacillus species, including Bacillus pumilus (10%), Bacillus subtilis (12.5%), Brevibacillus brevis (10%), Bacillus cereus (22.5%), Bacillus sphaericus (7.5%), Bacillus licheniformis (12.5%) and Bacillus sporothermodurans (25%) were identified in different milk samples. Bacillus cereus was predominant in raw and pasteurised milk. Although B. sporothermodurans was the predominant sporogenous micro‐organism in UHT milk, B. cereus, B. sphaericus and B. licheniformis were also present. This study showed that there is a high degree of diversity, both phenotypic and genotypic, among Bacillus isolates from Tunisian milk and the persistence of spoilage risk in UHT milk.     

Multiplex PCR for rapid detection of thermophilic Moorella thermoaceticaand Geobacillus stearothermophilus from canned foods and beverages

A multiplex PCR assay was developed to simultaneously detect the thermophilic spore‐forming bacteria Geobacillus stearothermophilus and Moorella thermoacetica, which are major sources of spoilage in the canned food and beverage industries because of their thermophilic persistence. A total of 294 samples were examined, and none of the samples demonstrated signs of spoilage. Culture‐based assays showed that forty‐five M. thermoacetica isolates were obtained, which was confirmed by multiplex PCR, and G. stearothermophilus was not detected in any of the canned food and beverage, but multiplex PCR analysis identified this species in almost all of the samples. Meanwhile, twelve Bacillus subtilis isolates were found under both aerobic and anaerobic conditions. Multiplex PCR may allow selective detection of species that are present at low levels in complex matrices, or those that cannot be detected by culture‐based methods. Screening for spoilage‐causing micro‐organisms in ingredients and samples from processing lines, together with traditional culture methods, will help risk management and improve hygiene control.     

Pellicle formation,microbial succession and lactic acid utilisation during the aerobic deteriorating process of Sichuan pickle

The pellicles formation, microbial changes and lactic acid utilisation during the aerobic deterioration of Sichuan pickle were discussed in this study. By plate counting, chemical detection and HPLC analysis, the aerobic deterioration of Sichuan pickle was featured by microbial growth, pellicle formation, lactic acid decrease and pH elevation. Bacteria dominated in pellicles and the pellicles present morphological change from smooth to wrinkly along with the aerobic deteriorating process. Nine species of bacteria were identified and responsible for the pellicle formation. Combined with Lactobacillus plantarum, eight pellicle‐forming bacteria were the dominant lactic acid consuming organisms in deteriorating Sichuan pickle. Especially under the harsh acid condition of the early deteriorating period, Lb. plantarum, pellicle‐forming bacteria Bacillus amyloliquefaciens, B. subtilis and Citrobacter freundii participated in the pellicles, utilised lactic acid and increased the brine pH effectively. Sequence analysis of 16S rDNA libraries suggested that the resulting pH of 4.8–5.0 initiated the growth of more undesirable organisms, and the pellicle bacterial diversity changed greatly compared with that in the early deteriorating period. The study indicated the important role of pellicles in the initiation of Sichuan pickle's aerobic deterioration and enhanced our ability to understand and potentially control the aerobic deterioration.     

Occurrence and identification of spore-forming bacteria in skim-milk powders

The different customer and regulatory specifications for mesophilic and thermophilic aerobic and anaerobic spore numbers in skim-milk powder, in addition to some specifications on specific spore-forming bacteria, such as Bacillus cereus, can be challenging for the industry to meet. Twenty-two samples of medium-heat skim-milk spray-dried powder from eight sources were analysed in triplicate with 16 bacterial and spore enumeration tests to understand the variety of spore-forming bacteria population. Using 16S rDNA sequencing, the species were identified for 269 isolates that were representative of the various tests. Of the isolates identified, 68% were Bacillus licheniformis, a facultative anaerobe that can survive and grow at mesophilic and thermophilic temperatures, making it difficult to eliminate in manufacturing environments. Using whole genome sequencing, 16 of 23 isolates identified as B. licheniformis by 16S sequencing were confirmed as B. licheniformis, four were identified as Bacillus paralicheniformis and three were identified as Bacillus sp. H15-1.     

Microbiological and mycotoxicological analyses of processed cereal-based complementary foods for infants and young children from the German market

This study investigated several food safety criteria in 38 different commercial products of processed cereal-based foods (PCF) from the German market. Microbiological assessment, followed by 16S RNA gene sequencing of suspect colonies, included aerobic mesophilic bacteria, moulds, Enterobacteriaceae, Cronobacter spp., and presumptive Bacillus cereus. Mycotoxin analyses were performed by enzyme immunoassays for deoxynivalenol (DON), zearalenone (ZEN), T-2/HT-2 toxins (T-2/HT-2; oat containing products only), ergot alkaloids (EA), and alternariol (AOH). No violative result above existing European Union regulations or international guidelines was obtained. Most samples had very low aerobic mesophilic cell counts (<2.0 × 10¹ CFU/g), the maximum was 9.6 × 10² CFU/g. A few samples contained low numbers of opportunistic pathogens, most notably Cronobacter sakazakii, Acinetobacter spp., Pantoea spp., and enterotoxigenic Bacillus wiedmannii. Levels of mycotoxin contamination were very low, well below European Union maximum limits. DON was found in 10 samples, at levels of 9–35 µg/kg. T-2/HT-2 were found in all 15 oat-based products (1–8 µg/kg). All samples were negative for ZEN and EA. A high number (n = 25) of samples yielded weakly positive results for the nonregulated AOH (0.4–2 µg/kg), but just three samples exceeded a level of 1 µg/kg. No relationship between cereal composition and analytical findings for microbiological parameters and mycotoxins could be found. As long as PCF meals are freshly prepared and consumed immediately after preparation, the risk from sporadically occurring opportunistic bacteria appears to be minimal.     

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.     

New and classical spoilage bacteria causing widespread blowing in Argentinean soft and semihard cheeses

A total of 16 soft and semihard Argentinean cheeses, and 95 pasteurized cheese–milk samples, were analysed for microorganisms responsible for blowing. Lactic acid bacteria (starter microflora) resulted in high numbers (> 10⁷ colony‐forming units (cfu)/g). Coliform and yeast counts were lower than 10⁴ cfu/g. Cremoso cheeses were blown by leuconostocs, lactobacilli and Bacillus polymyxa. Mozzarella was spoiled by B. polymyxa and Bacillus macerans. Semihard cheeses were affected by spore‐forming (Clostridium and Bacillus), propionic and lactobacilli strains. Clostridia, Bacillus, leuconostocs and heterofermentative lactobacilli were detected in pasteurized milk. Bacillus strains were not previously associated with blowing in soft and semihard cheeses.     

Effect of high‐pressure treatments on microbial loads and physicochemical characteristics during refrigerated storage of raw milk Serra da Estrela cheese samples

This work studied the effect of high pressure processing (HPP) at 400, 500 and 600 MPa during 10, 5 and 3 min, respectively, on samples ewe cheese manufactured from raw milk, during storage (100 days) at 5 °C. Total aerobic mesophilic and lactic acid bacteria were slightly affected, decreasing by about 1.0 and 0.82 log CFU g^?1, respectively, immediately after HPP treatment at 600 MPa for 3 min, while Enterobacteriaceae, yeasts and moulds, and Listeria innocua were reduced to below the quantification limits. Lactic acid bacteria decreased further during storage, showing increasing inactivation as the pressure level increased. Physicochemical parameters (water activity, moisture content, pH and titratable acidity) were generally not affected by HPP, while lipid oxidation increased throughout storage, with HPP samples showing lower values (50–66%) at 100 days of storage. The results indicated that HPP has potential to improve cheese microbial safety and shelf‐life, with a lower lipid oxidation level than nonpressurised cheese.     

Effect of dietary humate on the pH,TBARS and microbiological properties of vacuum‐ and aerobic‐packed breast and drumstick meats of broilers

The current trial determined the influence of dietary humate, including humic, fulvic and ulmic acids and some microminerals on the pH, TBARS and microbiological properties of vacuum‐packed and aerobic‐packed breast fillets and drumsticks of broilers. A total of 240 male broiler chicks (Ross‐308) were randomly allocated into four dietary treatments (H₀, H₁, H₂ and H₃ groups). A basal diet (H₀), basal diet plus 0.1 (H₁), 0.2 (H₂) and 0.3% (H₃) humate (Farmagulator DRY^?, Humate, Farmavet International Inc, Kocaeli 41 400, Turkey) were offered during the experimental feeding period. At the end of the trial all chicks were slaughtered. After standard dissection of carcasses, the breasts and drumsticks were divided into two groups for vacuum packaging or aerobic packaging. Packed breasts and drumsticks were stored at 3 °C for 12 days. The breasts and drumsticks were analyzed for pH, TBARS, total mesophilic, total psychrotrophic, lactic acid bacteria and Enterobacteriacea counts at 0, 2, 4, 6, 8, 10 and 12 days of storage. The feeding of humate decreased pH values of breast fillets and drumstick muscles (p < 0.01), especially at the 0.1% level. The TBARS value decreased (p < 0.01) when compared with the without‐humate group (H₀). Total aerobic mesophilic (p < 0.01), total aerobic psychrotrophic (p < 0.01) and lactic acid bacteria (p < 0.05) counts were lower in the H₃ group than the other groups. The effect of meat type on pH, TBARS, total aerobic mesophilic (p < 0.01) and lactic acid bacteria (p < 0.05) and Enterobacteriacea counts was significant. While the pH and TBARS values of breast fillets were lower than those of drumstick meats, the total mesophilic, total psychrotrophic, lactic acid bacteria and Enterobacteriacea counts of drumstick meats were higher than those of breast fillets. The effect of packaging type on pH (p < 0.01), TBARS (p < 0.01), total aerobic psychrotrophic bacteria (p < 0.01) and Enterobacteriacea counts (p < 0.01) was found statistically significant and the maximum values were determined in the aerobic‐packed samples. In addition, investigated parameters were also affected by storage period. While the total aerobic mesophilic count decreased, the other parameters increased when compared with zero day of storage. Copyright © 2005 Society of Chemical Industry     

Active packaging solution to prolong the shelf life of rocket salad

The best packaging conditions for rocket salad were assessed by subsequent experimental trials. In the first step, a preliminary screening of different packaging materials was performed and two micro‐perforated oriented polypropylene films with different micro‐hole diameters (90 and 110 μm) were selected as best packaging solutions. In the subsequent experimental step, modified headspace conditions were applied without any improvement on product quality. In the last step, the effects of an ethylene adsorbent were analysed. Rocket salad packaged in both films with the ethylene adsorbent recorded a shelf life of about 16 days, compared to the control samples that remained acceptable for 13 days. During storage, the microbial quality (mesophilic and psychrotrophic bacteria, pseudomonadaceae, lactic acid bacteria, yeasts, total coliforms and enterobacteriacae), the pH, the colour changes and the main sensory parameters were also monitored.     

Bacillus and Paenibacillus species associated with extended shelf life milk during processing and storage

Characterisation of spore formers associated with extended shelf life milk was performed by analysing the bacteriological quality of milk samples collected at various processing stages and during storage. Isolates were identified with MALDI‐TOF‐MS. Milk had spore counts <2 log₁₀ cfu/mL and 4 log₁₀ cfu/mL during processing and storage, respectively. Bacillus pumilus dominated the bacterial population. Bacterial species were inoculated into sterile milk for a shelf life study, and the population change was observed over 42 days at 7 °C. Although the extended shelf life milk process was effective in reducing bacterial counts and species diversity, the presence of Bacillus cereus shows a potential safety problem in extended shelf life milk.     

Psychrotrophs in dairy products: Their effects and their control

Health concerns and technological effects of psychrotrophic bacteria in dairy products are reviewed, as well as methods to control their presence and development. The various Gram‐negative and Gram‐positive psychrotrophic species are listed and, with respect to pathogenic psychrotrophs, emphasis is given on Listeria monocytogenes, Yersinia enterocolytica, and Bacillus cereus. The influence of psychrotrophic bacteria on the quality of raw milk, pasteurized and UHT milks, butter, ice cream, cheese, and powders is examined. Public health considerations of Listeria monocytogenes, Yersinia enterocolytica, and Bacillus cereus of these various dairy products are also presented. Methods that can be used to eliminate or control the development of psychrotropic bacteria include low or high temperatures, chemicals, gases, the lactoperoxidase system, lactic acid bacteria, microfiltration, bactofugation, lactoferrin‐related proteins, sanitation, flavors, and naturally occurring spore germinants.     

Comparative effect of different test methodologies on Bacillus coagulans spores inactivation kinetics in tomato pulp under isothermal conditions

Heat resistant micro‐organisms are an ongoing challenge to the food industry. Various factors may influence the heat resistance of micro‐organisms including type and strain; the environmental influences during cell and spore formations and during heat exposure; and the equipment and test tools used to perform the experimental process. In an attempt to analyse the influence of different test tools used on the heat inactivation processes, this study aimed to define the isothermal inactivation kinetics of Bacillus coagulans spores in tomato pulp at different temperatures and compare the inactivation of this bacterium when thermal death time (TDT) and capillary tube methods were used. Temperature ranges from 95 °C to 120 °C were studied, and inactivation kinetic parameters were estimated through the application of primary models. TDT inactivation curves consisted of shoulder and linear decline, while capillary method inactivation curves consisted of shoulder, linear decline and long tail. A secondary model was used to describe the influence of the temperature on spore inactivation parameters. The results showed test methods are at least as important in determining thermal processes as the micro‐organisms and media used.     

Survival of micro‐organisms and organic acid profile of probiotic Cheddar cheese from buffalo milk during accelerated ripening

The study aimed to assess the impact of ripening at elevated temperatures on the survival of probiotic micro‐organisms and production of organic acids in Cheddar cheese. Cheese was manufactured from buffalo milk using lactococci starters along with different probiotic bacteria (Lactobacillus acidophilus LA‐5, Bifidobacterium bifidum Bb‐11 and Bifidobacterium longum BB536) as adjunct cultures. The cheeses were ripened at 4–6 °C or 12–14 °C for 180 days and examined for composition, organic acids and microbial survival. The production of organic acids was accelerated at 12–14 °C when compared to normal ripening temperatures. The probiotic bacteria increased production of lactic and acetic acids, compared to cheese made with lactococci alone. The survival of the mesophilic starters was significantly (P < 0.05) reduced in all the cheese samples ripened at the higher temperature. However, the probiotic bacteria remained viable (>7.0 log₁₀ cfu/g) throughout the 180 days of ripening, irrespective of temperature. It was concluded that Cheddar containing additional probiotic cultures can effectively be ripened at elevated temperatures without any adverse effects.     

Isolation and genetic identification of spore-forming bacteria associated with concentrated-milk processing in Nebraska

Spore-forming bacteria are heat-resistant microorganisms capable of surviving and germinating in milk after pasteurization. They have been reported to affect the quality of dairy products by the production of enzymes (lipolytic and proteolytic) under low-temperature conditions in fluid milk, and have become a limiting factor for milk powder in reaching some selective markets. The objective of this research was to isolate and identify the population of spore-forming bacteria (psychrotrophic and thermophilic strains) associated with concentrated milk processing in Nebraska. During 2 seasons, in-process milk samples from a commercial plant (raw, pasteurized, and concentrated) were collected and heat-treated (80°C/12 min) to recover only spore-formers. Samples were spread-plated using standard methods agar and incubated at 32°C to enumerate mesophilic spore counts. Heat-treated samples were also stored at 7°C and 55°C to recover spore-formers that had the ability to grow under those temperature conditions. Isolates obtained from incubation or storage conditions were identified using molecular techniques (16S or rpoB sequencing). Based on the identification of the isolates and their relatedness, strains found in raw, pasteurized, and concentrated milk were determined to be similar. Paenibacillus spp. were associated with both raw and concentrated milk. Due to their known ability to cause spoilage under refrigeration, this shows the potential risk associated with the transferring of these problematic organisms into other dairy products. Other Bacillus species found in concentrated milk included Bacillus clausii, Bacillus subtilis, Lysinibacillus sp., Bacillus safensis, Bacillus licheniformis, Bacillus sonorensis, and Brevibacillus sp., with the last 3 organisms being capable of growing at thermophilic temperatures. These strains can also be translocated to other dairy products, such as milk powder, representing a quality problem. The results of this research highlight the importance of understanding spore-formers associated with the processing of condensed milk, which then may allow for specific interventions to be applied to control these microorganisms in this processing chain. To our knowledge, this is the first study evaluating spore-formers associated with concentrated milk in the United States.     

Nitrite production by thermophilic aerobic bacteria isolated during the manufacture of milk powder

Nitrite contamination in milk powder is hypothesised to be caused by thermophilic bacteria that form biofilms in milk powder manufacturing plants. Regulatory limits in some countries have made nitrite contamination in milk powder an issue influencing product sales. In this study, thermophilic bacteria were isolated from milk samples obtained from a milk processing plant to investigate the potential of different isolates to convert nitrate to nitrite. Eight bacteria species were identified through 16s rDNA gene sequencing (Bacillus licheniformis, Bacillus aerius, Bacillus subtilis, Bacillus sonorensis, Bacillus firmus, Enterococcus faecium, Geobacillus stearothermophilus and Macrococcus caseolyticus). Most bacteria isolated were able to convert nitrate to nitrite, and surprisingly in aerobic conditions. The most interesting species was G. stearothermophilus as its nitrate reducing capability was highly variable between different isolates. This could explain why there was no relationship reported between nitrite level in milk powder and high thermophile count.     

Inactivation of Bacillus spores in batch vs continuous heating systems at sterilisation temperatures

This study was performed to assess the heat resistance of spores of Bacillus species in batch and continuous heating systems under commercial sterilisation conditions. Spores of thermophilic Bacillus smithii and mesophilic Bacillus amyloliquefaciens were found to be highly heat resistant in the batch system. They were able to withstand typical sterilisation temperatures. B. amyloliquefaciens showed tailing in the batch system and, before the onset of the tailing, a higher inactivation rate than in the continuous system at low temperatures. The reason for the tailing might be the presence of spore aggregates which are disrupted in the continuous system.     

Development of an improved extraction and HPLC method for the measurement of ascorbic acid in cows' milk from processing plants and retail outlets

An improved extraction (2.5% HPO₃, 5 mm dithiothreitol) and HPLC quantification methodology using a C–18 column at 35 °C and 0.1 m acetic acid (98%) and acetonitrile (2%) mobile phase was developed to quantify total ascorbic acid (AA) in commercial whole/semi‐skim/skim raw/pasteurised/UHT milk packaged in opaque bags, transparent plastic, cardboard and Tetra Brik^?. AA content ranged from 0.21 to 10 and from 3.4 to 16 mg L^?1 in milk from retail outlets and processing plants, respectively, and was higher in organic milk. For same processor/lot samples, pasteurised milk showed higher AA content than UHT milk. This was not true for retail outlets samples. AA content was similar for whole/semi‐skim and semi‐skim/skim milk, but not for whole/skim comparisons. Among UHT samples, the AA content trend was whole<semi‐skim<skim and lower for UHT milk in opaque plastic and Tetra Brik^? container. After 14 days at 4 °C in the dark, AA losses ranged 35–83% depending on milk type and preservation method with a higher AA retention in unopened containers.     

Microbiological and Fermentative Properties of Baker's Yeast Starter Used in Breadmaking

This study assessed the levels of microbial contaminants in liquid, compressed and dry commercial baker's yeasts used as starters in breadmaking. Eumycetes, Enterobacteriaceae, total and fecal coliforms, Bacillus spp., and lactic acid bacteria (LAB), in particular enterococci, were quantified. Results obtained in this study highlighted that baker's yeast could represent a potential vehicle of spoilage and undesirable microorganisms into the baking environment, even if these do not influence the leavening activity in the dough, as ascertained by rheofermentometer analysis. Different microbial groups, such as spore‐forming bacteria and moulds, were found in baker's yeast starters. Moreover, different species of LAB, which are considered the main contaminants in large‐scale yeast fermentations, were isolated and identified by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rDNA sequencing. The most recurrent species were Lactobacillus plantarum, Enterococcus faecalis, and Enterococcus durans, isolated from both compressed and dry starters, whereas strains belonging to Leuconostoc and Pediococcus genera were found only in dry ones. Nested‐Polymerase Chain Reaction (Nested‐PCR) and Randomly Amplified Polymorphic DNA–PCR (RAPD‐PCR) were also used to highlight the biodiversity of the different commercial yeast strains, and to ascertain the culture purity.     

Detection and quantification of Escherichia coli and Pseudomonas aeruginosa in cow milk by near‐infrared spectroscopy

This work investigated the potential of NIR technology to be applied in the dairy industry for the detection of micro‐organisms. To this end, two types of cow milk samples were studied, one in which only bacterial biomass was considered and the other in which bacteria were cultured and grown in milk for 24 h. The study was carried out using two micro‐organisms Escherichia coli and Pseudomonas aeruginosa. Both types of samples with different counts of both micro‐organisms were analysed by a NIR analyser in the range 10 000–4000 cm^?1 based on transmittance measurements. Multivariate models indicated that a better discrimination between micro‐organisms was attained in those milk samples in which micro‐organisms have been grown.