Bacterial spore inactivation at 45-65 °C using high pressure processing: Study of Alicyclobacillus acidoterrestris in orange juice

High pressure processing (HPP) is a new non-thermal technology commercially used to pasteurize fruit juices and extend shelf-life, while preserving delicate aromas/flavours and bioactive constituents. Given the spoilage incidents and economic losses due to Alicyclobacillus acidoterrestris in the fruit juice industry, the use of high pressure (200 MPa – 600 MPa) in combination with mild temperature (45 °C–65 °C) for 1–15 min, to inactivate these spores in orange juice were investigated. As expected, the higher the temperature, pressure and time, the larger was the A. acidoterrestris inactivation. The survival curves were described by the first order Bigelow model. For 200 MPa, D_45 °C = 43.9 min, D_55 °C = 28.8 min, D_65 °C = 5.0 min and z-value = 21.3 °C. At 600 MPa, D_45 °C = 12.9 min, D_55 °C = 7.0 min, D_65 °C = 3.4 min and z-value = 34.4 °C. Spores were inactivated at 45 °C and 600 MPa, and at 65 °C only 200 MPa was needed to achieve reduction in spore numbers.     

Molecular identification and pectate lyase production by Bacillus strains involved in cocoa fermentation

We have previously reported the implication of Bacillus in the production of pectinolytic enzymes during cocoa fermentation. The objective of this work was to identify the Bacillus strains isolated from cocoa fermentation and study their ability to produce pectate lyase (PL) in various growth conditions. Ninety-eight strains were analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Four different banding patterns were obtained leading to the clustering of the bacterial isolates into 4 distinct ARDRA groups. A subset of representative isolates for each group was identified by 16S rRNA gene partial sequencing. Six species were identified: Bacillus subtilis, Bacillus pumilus, Bacillus sphaericus, Bacillus cereus, Bacillus thuringiensis, together with Bacillus fusiformis which was isolated for the first time from cocoa fermentation. The best PL producers, yielding at least 9 U/mg of bacterial dry weight, belonged to B. fusiformis, B. subtilis, and B. pumilus species while those belonging to B. sphaericus, B. cereus and B. thuringiensis generally showed a low level of activity. Two kinds of PL were produced, as revealed by isoelectrofocusing: one with a pI of 9.8 produced by B. subtilis and B. fusiformis, the other with a pI of 10.5 was produced by B. pumilus. Strains yielded about 2 fold more PL in a pectic compound medium than in glucose medium and maximum enzyme production occurred in the late stationary bacterial growth phase. Together all these results indicate that PL production in the bacilli studied is modulated by the growth phase and by the carbon source present in the medium.     

Detecting single Bacillus spores by surface enhanced Raman spectroscopy

Detection of Bacillus spores is of considerable importance to the food industry since they can survive standard processing procedures and sanitation treatments. Surface enhanced Raman spectroscopy (SERS) coupled with gold SERS-active substrates was used to detect and discriminate among five Bacillus spores (B. cereus ATCC 13061, B. cereus ATCC 10876, B. cereus sp., B. subtilis sp., and B. stearothermophilus sp.). Tremendously enhanced Raman signals of Bacillus spores deposited onto the gold substrates were detected, allowing the limit of detection (LOD) of SERS to reach the level of a single spore. Distinct spectral differences were observed between different Bacillus spores. Hierarchical cluster analysis (HCA) and principle component analysis (PCA) show clear data segregations at the species level between five Bacillus spores. Principle component (PC) values indicate that the Raman shift range between 900 and 1200 cm⁻¹ contributed significantly to the total data variance in the PCA results. In particular, a prominent band of dipicolinic acid (DPA) was observed at 998 cm⁻¹ and served as a biomarker for bacterial spores. This study demonstrates that SERS coupled with gold nanosubstrates is able to detect and discriminate single Bacillus spores non-destructively and with minimum sample preparation. SERS method is a promising tool for rapid, ultra-sensitive, and selective detection of bacterial spores, potentially in foods and other complex biological matrices.     

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R² values of 0.9969 and 0.9958 respectively. Linear correlations between the log₁₀ input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10¹ CFU/mL to 1.65 × 10⁶ CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.     

Inhibition of Bacillus cereus spore outgrowth and multiplication by chitosan

Bacillus cereus is an endospore-forming bacterium able to cause food-associated illness. Different treatment processes are used in the food industry to reduce the number of spores and thereby the potential of foodborne disease. Chitosan is a polysaccharide with well-documented antibacterial activity towards vegetative cells. The activity against bacterial spores, spore germination and subsequent outgrowth and growth (the latter two events hereafter denoted (out)growth), however, is poorly documented. By using six different chitosans with defined macromolecular properties, we evaluated the effect of chitosan on Bacillus cereus spore germination and (out)growth using optical density assays and a dipicolinic acid release assay. (Out)growth was inhibited by chitosan, but germination was not. The action of chitosan was found to be concentration-dependent and also closely related to weight average molecular weight (M_w) and fraction of acetylation (F_A) of the biopolymer. Chitosans of low acetylation (F_A = 0.01 or 0.16) inhibited (out)growth more effectively than higher acetylated chitosans (F_A = 0.48). For the F_A = 0.16 chitosans with medium (56.8 kDa) and higher M_w (98.3 kDa), a better (out)growth inhibition was observed compared to low M_w (10.6 kDa) chitosan. The same trend was not evident with chitosans of 0.48 acetylation, where the difference in activity between the low (19.6 kDa) and high M_w (163.0 kDa) chitosans was only minor. In a spore test concentration corresponding to 10²-10³ CFU/ml (spore numbers relevant to food), less chitosan was needed to suppress (out)growth compared to higher spore numbers (equivalent to 10⁸ CFU/ml), as expected. No major differences in chitosan susceptibility between three different strains of B. cereus were detected. Our results contribute to a better understanding of chitosan activity towards bacterial spore germination and (out)growth.     

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.     

Tracking spore-forming bacterial contaminants in fluid milk-processing systems

The presence of psychrotolerant Bacillus species and related spore formers (e.g., Paenibacillus spp.) in milk has emerged as a key biological obstacle in extending the shelf life of high-temperature, short-time pasteurized fluid milk beyond 14 d. A recently developed rpoB DNA sequence-based subtyping method was applied to characterize spoilage bacteria present in raw milk supplies for 2 processing plants, and to assess transmission of these organisms into pasteurized products. Thirty-nine raw milk samples and 11 pasteurized product samples were collected to represent the processing continuum from incoming truck loads of raw milk to packaged products. Milk samples were held at 6°C for up to 16 d and plated for bacterial enumeration at various times throughout storage. Among the 88 bacterial isolates characterized, a total of 31 rpoB allelic types representing Bacillus and Paenibacillus spp. were identified, including 5 allelic types found in both raw milk and finished product samples. The presence of the same bacterial subtypes in raw and commercially pasteurized milk samples suggests that the raw milk supply represents an important source of these spoilage bacteria. Extension of the shelf life of high-temperature, short-time pasteurized fluid milk products will require elimination of these organisms from milk-processing systems.     

Characterisation and profiling of Bacillus subtilis,Bacillus cereus and Bacillus licheniformis by MALDI-TOF mass fingerprinting

The Bacillus genus includes species such as Bacillus cereus, Bacillus licheniformis and Bacillus subtilis, some of which may be pathogenic or causative agents in the spoilage of food products. The main goal of this work was to apply matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass fingerprinting to the classification of these Bacillus species. Genetic analyses were also compared to phyloproteomic analyses. A collection of 57 Bacillus strains isolated from fresh and processed food and from culture collections were studied and their mass spectra compiled. The resulting mass fingerprints were compared and characteristic peaks at the strain and species levels were assigned. The results showed that MALDI-TOF was a good complementary approach to 16S rRNA sequencing and even a more powerful tool in the accurate classification of Bacillus species, especially for differentiating B. subtilis and B. cereus from Bacillus amyloliquefaciens and Bacillus thuringiensis, respectively. MALDI-TOF was also found to provide valuable information at both intra- and interspecies levels in the Bacillus species studied.     

Clostridial spore germination versus bacilli: genome mining and current insights

Bacilli and clostridia share the characteristic of forming metabolically inactive endospores. Spores are highly resistant to adverse environmental conditions including heat, and their ubiquitous presence in nature makes them inevitable contaminants of foods and food ingredients. Spores can germinate under favourable conditions, and the following outgrowth can lead to food spoilage and foodborne illness. Germination of spores has been best studied in Bacillus species, but the process of spore germination is less well understood in anaerobic clostridia. This paper describes a genome mining approach focusing on the genes related to spore germination of clostridia. To this end, 12 representative sequenced Bacillus genomes and 24 Clostridium genomes were analyzed for the distribution of known and putative germination-related genes and their homologues. Overall, the number of ger operons encoding germinant receptors is lower in clostridia than in bacilli, and some Clostridium species are predicted to produce cortex-lytic enzymes that are different from the ones encountered in bacilli. The in silico germination model constructed for clostridia was linked to recently obtained experimental data for selected germination determinants, mainly in Clostridium perfringens. Similarities and differences between germination mechanisms of bacilli and clostridia will be discussed.     

Short communication: Bacterial ecology of high-temperature, short-time pasteurized milk processed in the United States

To determine the microbial ecology of pasteurized milk within the United States, 2% fat pasteurized fluid milk samples were obtained from 18 dairy plants from 5 geographical areas representing the Northeast, Southeast, South, Midwest, and West. Of the 589 bacterial isolates identified using DNA sequence-based subtyping methods, 346 belonged to genera characterized as gram-positive endospore-forming bacteria (i.e., Bacillus and Paenibacillus). Of the 346 gram-positive endospore-forming bacteria isolated in the present study, 240 were classified into 45 allelic types identical to those previously identified from samples obtained in New York State, indicating the widespread presence of these microbes in fluid milk production and processing systems in the United States. More than 84% of the gram-positive spore-forming isolates characterized at d 1, 7, and 10 were of the genus Bacillus, whereas more than 92% of isolates characterized at d 17 of shelf life were of the genus Paenibacillus, indicating that the predominant gram-positive spoilage genera shifts from Bacillus spp. to Paenibacillus spp. during refrigerated storage.     

Use of Fourier transform infrared spectroscopy for rapid comparative analysis of Bacillus and Micrococcus isolates

The identification of foodborne microorganisms and their endospores in food products are important for food safety. The present work compares Bacillus (Bacillus licheniformis, Bacillus circulans and Bacillus subtilis) and Micrococcus (Micrococcus luteus) species with Fourier transform infrared (FTIR) spectroscopy. Our results show that there are several characteristic peaks belonging to both the Micrococcus and Bacillus species which can be used for the identification of these foodborne bacteria and their endospores. For Micrococcus species, a new band was observed at 1338 cm⁻¹ which may be due to acetate oxidation via the carboxylic acid cycle. The bands at 1313 cm⁻¹ and 1256 cm⁻¹ can be explained by an exopolymer formation and the other bands at 1074 cm⁻¹ and 550 cm⁻¹, may be due to the glycogen-like storage material in Micrococcus spp. There are also characteristic peaks at 993 cm⁻¹ and 801 cm⁻¹ for these bacterial species. Different Bacillus species also showed characteristic peaks at 1000–500 cm⁻¹ region. Dipicolinic acid (DPA) bands at ∼728 cm⁻¹ and ∼703 cm⁻¹ seen only in B. circulans were the marker of an endospore formation.     

Identification of surfactin producing strains in Soumbala and Bikalga fermented condiments using Polymerase Chain Reaction and Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry methods

In this study, 67 strains were isolated from two fermented condiments from Burkina Faso: Soumbala and Bikalga. Phenotypical methods, biochemical tests and molecular approaches were used to determinate their genus or species. Twenty-two of them belong to the Bacillus genus. Six strains were selected for their antibacterial or antifungal properties. Their ability to produce lipopeptides synthesized by Non Ribosomal Peptide Synthetases was investigated using two different approaches: PCR with specific degenerated primers and Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-ToF MS) performed on whole cells cultivated on a solid medium. PCR revealed that the six strains contain genes involved in the biosynthesis of surfactins whereas surfactins C₁₄ and C₁₅ were only detected by MALDI-ToF MS in two of the six strains. For the first time, the presence of surfactins C₁₄ and C₁₅ was also identified by MALDI-ToF MS analyses directly performed on Soumbala methanolic crude extracts. The structure of these compounds was confirmed by + MS2 and + MS3 of sample and reference surfactins.     

Origin of bacterial spores contaminating foods

Bacterial spores (= endospores) are common contaminants in foods. Sources of contamination in the food chain may include soil, faeces, animal feeds and food ingredients and processing chain themselves. Sporulation may occur in very diverse environments. The environment of sporulation has a strong influence on spore properties relevant for food quality and safety.     

High temperature, short time pasteurization temperatures inversely affect bacterial numbers during refrigerated storage of pasteurized fluid milk

The grade A Pasteurized Milk Ordinance specifies minimum processing conditions of 72°C for at least 15 s for high temperature, short time (HTST) pasteurized milk products. Currently, many US milk-processing plants exceed these minimum requirements for fluid milk products. To test the effect of pasteurization temperatures on bacterial numbers in HTST pasteurized milk, 2% fat raw milk was heated to 60°C, homogenized, and treated for 25 s at 1 of 4 different temperatures (72.9, 77.2, 79.9, or 85.2°C) and then held at 6°C for 21 d. Aerobic plate counts were monitored in pasteurized milk samples at d 1, 7, 14, and 21 postprocessing. Bacterial numbers in milk processed at 72.9°C were lower than in milk processed at 85.2°C on each sampling day, indicating that HTST fluid milk-processing temperatures significantly affected bacterial numbers in fluid milk. To assess the microbial ecology of the different milk samples during refrigerated storage, a total of 490 psychrotolerant endospore-forming bacteria were identified using DNA sequence-based subtyping methods. Regardless of processing temperature, >85% of the isolates characterized at d 0, 1, and 7 postprocessing were of the genus Bacillus, whereas more than 92% of isolates characterized at d 14 and 21 postprocessing were of the genus Paenibacillus, indicating that the predominant genera present in HTST-processed milk shifted from Bacillus spp. to Paenibacillus spp. during refrigerated storage. In summary, 1) HTST processing temperatures affected bacterial numbers in refrigerated milk, with higher bacterial numbers in milk processed at higher temperatures; 2) no significant association was observed between genus isolated and pasteurization temperature, suggesting that the genera were not differentially affected by the different processing temperatures; and 3) although typically present at low numbers in raw milk, Paenibacillus spp. are capable of growing to numbers that can exceed Pasteurized Milk Ordinance limits in pasteurized, refrigerated milk.     

Use of endospore-forming bacteria as an active oxygen scavenger in plastic packaging materials

The incorporation of active oxygen scavengers in polymer packaging materials is essential to allow packaging of oxidation sensitive products. Opposed to the currently available chemical oxygen scavengers, systems based upon natural and biological components could have advantages towards consumer perception and sustainability. A modelsystem for a new oxygen scavenging poly(ethylene terephthalate) (PET) bottle is proposed using an endospore-forming bacteria genus Bacillus amyloliquefaciens as the active ingredient. Spores were incorporated in poly(ethylene terephthalate, 1,4-cyclohexane dimethanol) (PETG), an amorphous PET copolymer having a considerable lower processing temperature and higher moisture absorption compared to PET. To asses spore viability after incorporation, a method was optimized to extract spores from PETG using a chloroform/water mixture. Samples were also analyzed using a Live/Dead BacLight Bacterial Viability kit. It was shown that endospores were able to survive incorporation in PETG at 210 °C. Incorporated spores could actively consume oxygen for minimum 15 days, after an activation period of 1–2 days at 30 °C under high humidity conditions.

Industrial relevance

The study describes a modelsystem for the use of incorporated spores genus Bacillus amyloliquefaciens as an active oxygen scavenger in PET multilayer bottles using PETG as the middle layer material. Industrially, oxygen scavengers using incorporated viable spores as the active compound could have advantages towards consumer perception, recyclability, safety, material compatibility, production costs, … compared to currently available chemical oxygen scavengers.     

Toxin profile, antibiotic resistance, and phenotypic and molecular characterization of Bacillus cereus in Sunsik

Sunsik, a ready-to-eat food in Korea, is comprised of various agricultural and marine products, and has been an important concern in Bacillus cereus food poisoning. The aim of this study was to investigate the toxin profiles, genotypic and phenotypic patterns as well as antibiotic resistance of B. cereus strains isolated from Sunsik. A subtyping method known as automated repetitive sequence-based PCR system (DiversiLab™) was used to assess the intraspecific biodiversity of these isolates. Thirty-five B. cereus strains were isolated from 100 commercial Sunsik samples, all of which harbored at least 1 enterotoxin gene. The detection rates of nheABC, hblCDA, cytK, and entFM enterotoxin gene among all isolates were 97%, 86%, 77%, and 100%, respectively. Most strains also produced corresponding enterotoxins such as HBL (83%) and NHE (94%). One strain (2.9%) carried the emetic toxin genes, including ces and EM1, and was positive for the HEp-2 cell emetic toxin assay. Most strains were positive for various biochemical tests such as salicin hydrolysis (86%), starch fermentation (89%), hemolysis (89%), motility test (100%) and lecithinase hydrolysis (89%). All isolates were susceptible to most antibiotics although they were highly resistant to β-lactam antibiotics. By using the automated rep-PCR system, all isolates were successfully differentiated, indicating the diversity of B. cereus strains present in Sunsik.     

Technological and probiotic role of adjunct cultures of non-starter lactobacilli in soft cheeses

The influence of two cheese-isolated Lactobacillus strains on cheese composition, acceptability and probiotic capacity was assessed. Soft cheeses with and without the addition of Lactobacillus plantarum I91 or Lactobacillus paracasei I90 were prepared. Gross composition was assessed and secondary proteolysis was described by soluble fractions and free amino acids profiles. Acceptability was determined by a panel of 98 non-trained consumers. Cheeses harboring added Lactobacillus strains were also studied in vivo to evaluate their probiotic capacity. Gross composition of the cheeses was similar for control and treated (Lactobacillus-added) cheeses. Peptidolysis increased in cheeses with added lactobacilli, which was evidenced by a higher free amino acid content. Overall, the acceptability of the cheeses was good: 65%–80% of the consumers said that they “liked very much” or “liked” the cheeses. Cheeses with L. plantarum I91 showed the highest changes in composition and proteolysis and were the most accepted ones. On the contrary, composition of cheeses with L. paracasei I90 was similar to that of the controls, but these samples were less accepted than cheeses without lactobacilli. The oral administration of cheese containing L. plantarum I91 or L. paracasei I90 proved to be safe and able to enhance the number of IgA + cells in the small intestine lamina propria of mice. The use of selected strains of NSLAB exerted a technological and probiotic role: it contributed to the standardization of cheese quality and induced benefic health effects at the gut mucosa in vivo.     

Effect of microencapsulation on survival of Lactobacillus plantarum in simulated gastrointestinal conditions, refrigeration, and yogurt

In the present research the survival of free and microencapsulated cells of a new strain of Lactobacillus plantarum BL011 under stress conditions was tested in sodium alginate or pectin, coated with sodium alginate or chitosan. Results for the simulated gastrointestinal medium (SGT) showed no change in viability of cells in relation to the control. However, the simulated gastric medium (GM) drastically reduced the viability under the tested conditions, with no significant differences between free and immobilized cells. Under refrigerated storage viability of immobilized cells were greatly enhanced compared to the free microorganisms, and the treatments showing the lowest loss of viability were those of 4% (w/v) pectin, 3% (w/v) sodium alginate coated with chitosan and a mixture of 2% (w/v) sodium alginate and 2% (w/v) pectin, respectively. Loss of viability of immobilized L. plantarum in 3% alginate coated with chitosan in yogurt was of 0.55 log cycles during 38 days of storage. The results of this study suggest the efficiency of immobilization techniques to increase the survival of lactobacilli in yogurt under refrigerated storage.