The inactivation of Listeria monocytogenes and Escherichia coli on stainless steel surfaces was evaluated using a combination of ultraviolet light-emitting diodes (UVC-LED) and ultrasonic aerosolization with generally recognized as safe (GRAS) agents: sodium hypochlorite (NaOCl), acetic acid (AA), and hydrogen peroxide (H2O2). Stainless steel coupons (2 cm in diameter) inoculated with 6-7 log CFU/coupons of both pathogens were used as bioindicators. Coupons were treated with: UVC-LED (275 nm) at 2 and 4 cm distance for 5, 10, and 15 min; ultrasonic aerosolization with NaOCl, AA, or H2O2 for 5, 10, and 15 min or with combinations of both technologies. Lethality, sublethal damage, and cell leakage were evaluated. Results showed that UVC-LED treatments were more effective at doses of 30.42 mJ/cm2 (shorter distance at 2 cm and longer exposure time 15 min), achieving significant reductions in L. monocytogenes (2.27 log CFU/coupon). The aerosolized GRAS agents were also effective, especially at the highest concentrations and longer exposure times, achieving complete inactivation of both bacteria. Combining UVC-LED (5, 10, 15 min) with 0.3% H2O2 aerosolization resulted in a more effective inactivation at lower doses, highlighting the potential of these combined technologies to improve food safety on food contact surfaces in the food industry.

The development of thermal treatments based on the precepts proposed by Nicolas Appert at the beginning of the 19th century is one of the main milestones achieved to prolong the conservation of food and guarantee its supply to the population even if they are at long distances from production sites [...]

Vegetative cells of Listeria monocytogenes and Escherichia coli and spores of Bacillus subtilis and Aspergillus niger were inoculated in soy milk at an initial concentration of ≈5 log CFU/mL. Inoculated and control (non-inoculated) soy milk samples were submitted to three types of treatments using a tubular annular thin film short-wave ultraviolet (UV-C) reactor with 1 mm of layer thickness. Treatments applied depended on the flow rate and the number of entries to the reactor, with UV-C doses ranging from 20 to 160 J/mL. The number of entries into the reactor tube (NET) was established as the most determining parameter for the efficiency of the UV-C treatments. Conidiospores of A. niger were reported as the most resistant, followed by B. subtilis spores, while vegetative cells were the most sensible to UV-C, with Listeria monocytogenes being more sensible than Escherichia coli. Treatments of just 80 J/mL were needed to achieve a 5 log CFU/mL reduction of L. monocytogenes while 160 J/mL was necessary to achieve a similar reduction for A. niger spores.

High-pressure processing (HPP) is a non-thermal treatment in which, for microbial inactivation, foods are subjected to isostatic pressures (P) of 400-600 MPa with common holding times (t) from 1.5 to 6 min. The main factors that influence the efficacy (log(10) reduction of vegetative microorganisms) of HPP when applied to foodstuffs are intrinsic (e.g. water activity and pH), extrinsic (P and t) and microorganism-related (type, taxonomic unit, strain and physiological state). It was concluded that HPP of food will not present any additional microbial or chemical food safety concerns when compared to other routinely applied treatments (e.g. pasteurisation). Pathogen reductions in milk/colostrum caused by the current HPP conditions applied by the industry are lower than those achieved by the legal requirements for thermal pasteurisation. However, HPP minimum requirements (P/t combinations) could be identified to achieve specific log(10) reductions of relevant hazards based on performance criteria (PC) proposed by international standard agencies (5-8 log(10) reductions). The most stringent HPP conditions used industrially (600 MPa, 6 min) would achieve the above-mentioned PC, except for Staphylococcus aureus. Alkaline phosphatase (ALP), the endogenous milk enzyme that is widely used to verify adequate thermal pasteurisation of cows' milk, is relatively pressure resistant and its use would be limited to that of an overprocessing indicator. Current data are not robust enough to support the proposal of an appropriate indicator to verify the efficacy of HPP under the current HPP conditions applied by the industry. Minimum HPP requirements to reduce Listeria monocytogenes levels by specific log(10) reductions could be identified when HPP is applied to ready-to-eat (RTE) cooked meat products, but not for other types of RTE foods. These identified minimum requirements would result in the inactivation of other relevant pathogens (Salmonella and Escherichia coli) in these RTE foods to a similar or higher extent.

The effect of ultra-high-pressure homogenization (UHPH) treatments at 300 MPa at inlet temperatures (Ti) between 45 and 75 °C on the microbiological, physical, and sensorial characteristics of fish broth was evaluated. Before the application of UHPH treatments, different fish broth formulations were tested, selecting the formula with the best organoleptic and nutritional characteristics and the lowest cost, containing 45% monkfish heads and rock fish in the same proportion. The microbiological shelf-life of fish broth during cold storage at 4 and 8 °C was extended by a minimum of 20 days by applying UHPH treatments at inlet temperatures (Ti) between 45 and 65 °C. Fish broth UHPH-treated at Ti = 75 °C was microbiologically sterile during storage at 4 °C, 8 °C, and room temperature. Fish broth UHPH-treated was physically stable, significantly reducing the particle size. Color showed higher luminosity and lower yellowness as the inlet temperature increased. In fish broth UHPH-treated at Ti = 75 °C, selected for its microbiological stability, no differences were observed in the nutritional composition, antioxidant activity, and sensorial perception compared to untreated fish broth. Hence, UHPH treatments showed to be an alternative to preserving fish broth with an improved microbiological shelf-life and good sensorial characteristics.

Ultrahigh-pressure homogenization (UHPH) is a promising technology that is gaining importance over time as a potential technology to replace or complement the traditional heat processing of liquid foods. UHPH consists of forcing a pressurized fluid to flow through a small gap, offering the possibility of combining homogenization and preservation into a single-unit operation. This technology has been tested on different liquid food products of vegetal and animal origins enabling pasteurized or sterilized products with good sensorial, nutritional, functional, and technological properties. The aims of this chapter are to present the potential application of UHPH with respect to conventional homogenization and heat treatments applied to milk and dairy products, and to discuss their effects on the microbiology, main structures, and components of milk, and on their techno-functional properties of dairy products.

This work addresses the physicochemical, enzymatic and sensory changes in cloudy apple juice treated by ultrahigh pressure homogenization (UHPH) and short-wave ultraviolet radiation (UV-C) applied at 20 degrees C. Those technologies were applied in single and combined treatments at different UHPH pressures (200-300 MPa) and UV-C doses (14.3-27.8 J/mL). UV-C treatments could not effectively inactivate enzymes, but treatments at 300MPa UHPH reduced pectin methylesterase activity to a 24.6%, and polyphenol oxidase activity was not detected. Those samples presented a higher antioxidant capacity (283% measured by FRAP, and 286.4% by DPPH) than in non-treated juice, and after a combination with 28.7 J/mL of UV-C the polyphenols content augmented to 277.6%. Sensory evaluation revealed that UHPH at 300 MPa and UV-C at 21.5 J/mL significantly changed perceptible odour and overall flavour of cloudy apple juice, while treatments at 200 MPa didn't produce any significant changes in the different parameters. The results obtained in this study give a promising perspective of what a combination of both technologies can bring about in terms of obtaining stabilized fruit juices with improved antioxidant activity and polyphenol availability.

Liquid foods might present interferences in their optical properties that can reduce the effectiveness of short-wave ultraviolet radiation (UV-C) treatments used for sterilization purposes. The effect of turbidity as UV-C interference factor against the inactivation of bacterial spores was analysed by using phosphate-buffered saline solutions (PBS) of different turbidity values (2000, 2500, and 3000 NTU) which were adjusted with the addition of apple fibre. These suspensions were inoculated with spores of Bacillus subtilis and Alicyclobacillus acidoterrestris. While higher UV-C doses increased the inactivation rates of spores, these were reduced when turbidity values increased; a dose of 28.7 J/mL allowed inactivation rates of B. subtilis spores of 3.96 Log in a 2000-NTU suspension compared with 2.81 Log achieved in the 3000-NTU one. Spores of B. subtilis were more UV-C-resistant than A. acidoterrestris. Cloudy apple juice inoculated with A. acidoterrestris spores was processed by UV-C at different doses in a single pass and with recirculation of the matrix through the reactor. Inactivation increased significantly with recirculation, surpassing 5 Log after 125 J/mL compared with 0.13 Log inactivation after a single-pass treatment at the same UV-C dose. UV-C treatments with recirculation affected the optical properties (absorption coefficient at 254 nm and turbidity) of juice and increased browning as UV-C doses became higher.

Bacterial spores are important in food processing due to their ubiquity, resistance to high temperature and chemical inactivation. This work aims to study the effect of ultraviolet C (UVC) on the spores of Bacillus subtilis and Bacillus velezensis at a molecular and individual level to guide in deciding on the right parameters that must be applied during the processing of liquid foods. The spores were treated with UVC using phosphate buffer saline (PBS) as a suspension medium and their lethality rate was determined for each sample. Purified spore samples of B. velezensis and B. subtilis were treated under one pass in a UVC reactor to inactivate the spores. The resistance pattern of the spores to UVC treatment was determined using dipicolinic acid (Ca-DPA) band of spectral analysis obtained from Raman spectroscopy. Flow cytometry analysis was also done to determine the effect of the UVC treatment on the spore samples at the molecular level. Samples were processed for SEM and the percentage spore surface hydrophobicity was also determined using the Microbial Adhesion to Hydrocarbon (MATH) assay to predict the adhesion strength to a stainless-steel surface. The result shows the maximum lethality rate to be 6.5 for B. subtilis strain SRCM103689 (B47) and highest percentage hydrophobicity was 54.9% from the sample B. velezensis strain LPL-K103 (B44). The difference in surface hydrophobicity for all isolates was statistically significant (P < 0.05). Flow cytometry analysis of UVC treated spore suspensions clarifies them further into subpopulations unaccounted for by plate counting on growth media. The Raman spectroscopy identified B4002 as the isolate possessing the highest concentration of Ca-DPA. The study justifies the critical role of Ca-DPA in spore resistance and the possible sub-populations after UVC treatment that may affect product shelf-life and safety. UVC shows a promising application in the inactivation of resistant spores though there is a need to understand the effects at the molecular level to design the best parameters during processing.

Alicyclobacillus acidoterrestris is a spore-forming bacterium that can survive thermal pasteurization and acidic conditions. It produces changes in the odour and flavour of fruit juices leading to economical loses. A. acidoterrestris CECT 7094 spores were inoculated in clarified and cloudy apple juices (Golden delicious var.) in the range of 5-6 log(10) spores/mL and submitted to different short-wave ultraviolet light (UV-C) doses (7.2-28.7 J/mL) and ultra-high pressure homogenisation (UHPH) treatments (100-300 MPa), including their combination. A. acidoterrestris could be inactivated in clarified apple juice at a level of 4.8 log(10) CFU/mL by a 300 MPa-UHPH treatment when the inlet temperature was 80 degrees C. UV-C treatments showed to be more efficient achieving a lethality of 5.5 log(10) CFU/mL with a dose of 21.5 J/mL at 20 degrees C. In cloudy apple juice (2357 NTU) UV-C treatments were less efficient with a maximum lethality of 4.07 CFU/mL after a dose of 28.7 J/mL. A previous application of UHPH contributed with UV-C to obtain higher reductions of A. acidoterrestris spores at the doses of 14.3 and 21.5 J/mL compared with UV-C single treatments. On the other hand, this previous treatment also changed the properties of particles in the matrix which apparently reduced the effectiveness of UV-C at 28.7 J/mL.

Bacterial spores are of concern in food processing due to their ubiquity and resistance. This study seeks to determine the effect of ultraviolet C (UV-C) in the inactivation of spores of Bacillus subtilis and Bacillus velezensis that can result in enzymatic spoilage in foods using PBS as the suspension medium. Purified spore samples were treated under 1 pass in a UV-C reactor using 10 mL of spore inoculum with one dose of the radiation (410 mJ/cm2) for 10secs at room temperature. Aliquots of the treated samples were plated on tryptone soy agar supplemented with 0.6% glucose and the colonies counted. Flow cytometry analysis was done using 500 μL of both treated and control samples with a cell concentration of a ≥106 CFU/ml with propidium iodide (15 μM) and SYTO 9 (500 nM) used as live/dead stains. Samples were processed for microscopy (SEM and Raman-AFM Imaging). The maximum lethality is 2.5 for B. velezensis and the minimum is 0.1 for B. subtilis. Microscopic imaging of treated spores shows significant morphological disruption of the spore structure. The Raman spectroscopy analysis reveals the B. subtilis isolates to have the highest concentrations of dipicolnic acid (Ca+2DPA) as well as other compounds belonging to other functional groups. Flow cytometric analysis of treated spores reveals sub-populations unaccounted for by plate count. UV-C shows a promising application in the inactivation of resistant spores during processing of liquid foods such as milk.

The aim of this study was to evaluate the effectiveness of different UVC treatments, alone or in combination with ultra-high pressure homogenization (UHPH) on Bacillus subtilis spores in milk. Spores of B. subtilis (CECT4002) were inoculated in whole and skim milk to an initial concentration about 6 log CFU/mL. Milk was subjected to different ultraviolet radiation treatments at 254 nm (UVC) using a concentric tubular reactor in a dose ranging from 10 to 160 J/mL. Different number of passes were used to adjust the final dose received by the matrix. In general, increasing the number of passes (defined as number of entries to the tunnel-NET) increased the inactivation of spores of B. subtilis. The best lethality results (above 4 Log CFU/mL) were obtained by applying doses from 100 J/mL with several NET. When the same doses were achieved with a single pass lethality in most cases did not exceed 1 log CFU/mL. Increasing the NET also increased the likelihood for the spores to remain longer in the effective distance from the UVC source, estimated as 0.02 mm for whole milk and 0.06 mm for skim milk. Combination of UHPH and UVC did not clearly increase the efficiency of a single UVC treatment, and a lower lethality was even observed in some cases. UHPH treatments increased the turbidity and absorption coefficient (254 nm) of both whole and skim milk.

This work addresses the use of a thin-film short wave ultraviolet radiation (UVC) reactor and its combination with ultra-high pressure homogenization (UHPH) for the inactivation of Talaromyces macrosporus (CBS 130.89) and Neosartorya spinosa (CBS 586.90), fungal species that can cause food spoilage in products made of fruits. Ascospores of these microorganisms were inoculated in phosphate buffer solution (PBS) and clarified apple juice at a concentration between 5 and 6 Log10 spores/mL before treatments. Ascospores of both microorganisms were not detected in PBS after UV-C treatments even at the lowest assayed dose (1.8 J/mL), but in apple juice T. macrosporus showed to be significantly more UV-C resistant than N. spinosa. A UV-C dose of 21.5 J/mL in a single pass achieved decimal reductions of 2.15 Log10 and 5.4 Log10 respectively. Decimal reductions of both strains significantly increased in many cases when the same UV-C dose was applied in 2 or 3 passes throughout the UV-C reactor achieving T. macrosporus a maximum reduction of 3.88 Log10 after a UV-C dose of 21.5 J/mL applied in 3 passes. UHPH treatments at 100 and 200 MPa were ineffective in inactivating ascospores of both microorganisms, However, UHPH left ascospores more vulnerable to UV-C resulting in a synergistic effect achieving a maximum reduction of 3.6 Log10 on T. macrosporus after a combined treatment of UHPH at 200 MPa and a single pass of UV-C at 21.5 J/mL. The use of scanning electron microscopy revealed that only UHPH caused small changes in the cell wall structure of ascospores, but that might have left cells better exposed to UV-C.

Short wave ultraviolet light (UV-C) was studied in honey to inactivate vegetative cells of Escherichia coil (CECT 405) and spores of Bacillus subtilis (CECT 12) and Clostridium sporogenes (CECT 553) inoculated at a level of 10(4)-10(5) CFU/g. UV-C doses ranging from 1.5 to 21.6 J/mL were used passing inoculated honey samples through an UV-C reactor up to 4 times. Lethal effect increased with both the final dose applied and number of passes through the LTV-C reactor. E. coli was the most sensitive obtaining maximum reductions above 5 Log(10) CFU/g at 14.4 J/mL in treatments with 2 passes while for B. subtilis spore reductions of just 2.7 Log(10) CFU/g were obtained after the same treatment. For spores of CL sporogenes maximum reduction of 2.5 Log(10) CFU/g was observed after an 18 J/mL treatment. No significant differences (P > 0.05) were observed when treatments were applied with three passes or less, but after 4 passes, spore reduction above 3.5 Log(10) CFU/g was achieved. Effect of UV-C on some quality parameters of honey, such as hydroxymethylfurfural, pH and color, was also assessed. UV-C light made changes in most of these parameters although this not necessarily implied a reduction in the quality of honey.

A qualitative microplate screening method, using both low nitrogen (LND) and low glucose (LGD) decarboxylase broths, was used to evaluate the biogenic amine (BA) forming capacity of bacteria present in two types of Spanish ripened cheeses, some of them treated by high hydrostatic pressure. BA formation in decarboxylase broths was later confirmed by High Performance Liquid Chromatography (HPLC). An optimal cut off between 10⁻25 mg/L with a sensitivity of 84% and a specificity of 92% was obtained when detecting putrescine (PU), tyramine (TY) and cadaverine (CA) formation capability, although these broths showed less capacity detecting histamine forming bacteria. TY forming bacteria were the most frequent among the isolated BA forming strains showing a strong production capability (exceeding 100 mg/L), followed by CA and PU formers. Lactococcus, Lactobacillus, Enterococcus and Leuconostoc groups were found as the main TY producers, and some strains were also able to produce diamines at a level above 100 mg/L, and probably ruled the BA formation during ripening. Enterobacteriaceae and Staphylococcus spp., as well as some Bacillus spp. were also identified among the BA forming bacteria isolated.

Lethality rates of Bacillus subtilis, Geobacillus stearothertnophilus, Alicyclobacillus acidoterrestris, and Aspergillus niger spores were studied into phosphate-buffer saline (PBS) and PBS supplemented with caramel (PBSC) after Ultra-High Pressure Homogenization (UHPH) and UV-C treatments applied separately and combined. UHPH procedures were carried out at different pressures (100, 200 and 300 MPa) and inlet temperatures (20, 50 and 70 degrees C), while UV-C treatments consisted in a combination of doses (from 0.9 to 21.5 J/mL) and eventually temperatures (20 and 50 degrees C) or cycles. UHPH treatments were efficient at inactivating A. niger conidiospores in PBS but much less efficient in the inactivation of bacterial spores. Efficacy of UHPH against bacterial spores was increased pre-heating the samples at 70 degrees C. UV-C was more efficient against bacterial spores in PBS, but much less in PBSC. When UV-C treatments were applied through several cycles their efficacy against B. subtilis spores increased. Combination of UHPH at 200 MPa and UV-C moderately increased the lethal effect on B. subtilis spores than when these treatments were applied separately. Nevertheless, the combination of both technologies has a complementary effect allowing acting on a wide range of microorganisms, increasing the possibilities to obtain a safer and durable product.

Two artisanal varieties of cheese made in Spain, one made of ewes' raw milk and the other of goats' raw milk were selected to evaluate the effect of a high hydrostatic pressure (HHP) treatment at 400 MPa during 10 min at 2 °C on the formation of biogenic amines (BA). These conditions were applied at the beginning of the ripening (before the 5th day; HHP1) and in the case of ewes' milk cheeses also after 15th days (HHP15). BA formation was greatly influenced by HHP treatments in both types of cheese. HHP1 treatments significantly reduced the amounts of BA after ripening, being tyramine and putrescine the most affected BA in goats' milk cheeses and tyramine and cadaverine in ewes' milk cheeses. The BA reduction in the HHP1 samples could be explained by the significant decrease in microbiological counts, especially in the LAB, enteroccocci and enterobacteria groups at the beginning of ripening. The proteolysis in these samples was also affected reducing the amount of free amino acids. Although proteolysis in ewes' milk cheeses HHP15 was similar than in control samples a reduction of BA was observed probably because the decrease caused on microbial counts.

The need for enhancing microbial food safety, shelf-life, and quality, without negatively altering the sensory, functional, technological, and nutritional characteristics of foods, has led to increased interest in innovative low-temperature technologies for food preservation. Among these technologies, high-pressure homogenization has been identified as a particularly promising technology for processing liquid foods. The aim of this chapter is to present the potential application of ultrahigh-pressure homogenization with respect to classical homogenization and heat treatments applied to animal and vegetable milks, dairy products (cheese and yogurt), and fruit juices, and to discuss their effects on the main structures and components of these products and on their technological properties.

Current knowledge of the main changes induced in milk (including goat, ewe, and buffalo milks) and milk products when treated by high hydrostatic pressure (HHP) is presented. The effects of HHP on casein micelles, whey proteins, lipids, indigenous enzymes, mineral equilibrium, and microorganisms are described. The significance of these effects on the technological properties of milk, particularly in cheese-and yogurt-making applications, and functional properties is also discussed.

Microbial and volatile amines changes during processing steps and storage in optimal and abuse temperatures of semipreserved cuttlefish and their efficiency for quality was investigated. After osmotic treatment the initial microbial contamination and the total volatile basic nitrogen (TVBN) decreased sharply. The microbiota changed to Gram positive bacteria in which species of Bacillus and Staphylococcus were dominant. Mesophilic and psychrotrophic microorganisms increased markedly especially in abuse storage temperature and consequently chemical substances derived from its activity as total volatile basic nitrogen and ammonia. Among these parameters, mesophilic and psycrotrophic counts higher than 104 and 105 cfu/g, respectively, and TVBN contents higher than 100 mg per kilogram (mantle) or per liter (brine) would indicate a rupture of cold chain. Spanish legal microbial limit established is not appropriate for quality evaluation of semi preserved cuttlefish. A value of ammonia in muscle cuttlefish higher than 130 mg/kg would be useful spoilage indicator. Key words: Cuttlefish, semi preserved microorganisms, total volatile basic nitrogen (TVBN), ammonia, Trimethylamine (TMA), Sepia.