Application of Emerging Non-Thermal Processing Technologies: Impact on Characteristics, Efficacy, and Safety of Foods.

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 [...].

Drastic Microbial Count Reduction in Soy Milk Using Continuous Short-Wave Ultraviolet Treatments in a Tubular Annular Thin Film UV-C Reactor.

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.

Ultraviolet-C inactivation and hydrophobicity of Bacillus subtilis and Bacillus velezensis spores isolated from extended shelf-life milk.

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 sub-populations 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.

Evaluation of Continuous UVC Treatments and its Combination with UHPH on Spores of Bacillus subtilis in Whole and Skim 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.

Screening Method to Evaluate Amino Acid-Decarboxylase Activity of Bacteria Present in Spanish Artisanal Ripened Cheeses.

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.

High Hydrostatic Pressure as a Tool to Reduce Formation of Biogenic Amines in Artisanal Spanish Cheeses.

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.

Influence of ultra-high pressure homogenisation on antioxidant capacity, polyphenol and vitamin content of clear apple juice.

Ultra-high pressure homogenisation (UHPH) is a recently developed technology and is still under study to evaluate its effect on different aspects of its application to food products. The aim of this research work was to evaluate the effect of UHPH treatments on quality characteristics of apple juice such as antioxidant capacity, polyphenol composition, vitamin C and provitamin A contents, in comparison with raw (R) and pasteurised (PA) apple juice. Several UHPH treatments that include combinations of pressure (100, 200 and 300MPa) and inlet temperatures (4 and 20°C) were assayed. Apple juice was pasteurised at 90°C for 4min. Antioxidant capacity was analysed using the oxygen radical antioxidant capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP) assay while total phenolic content was determined by the Folin-Ciocalteau assay. According to the FRAP and DPPH assays, UHPH processing did not change apple juice antioxidant capacity. However, significant differences were detected between samples analysed by TEAC and ORAC assays. In spite of these differences, high correlation values were found between the four antioxidant capacity assays, and also with total polyphenol content. The analysis and quantification of individual phenols by HPLC/DAD analytical technique reflects that UHPH-treatment prevented degradation of these compounds. Vitamin C concentrations did not change in UHPH treated samples, retaining the same value as in raw juice. However, significant losses were observed for provitamin A content, but lower than in PA samples. UHPH-treatments at 300MPa can be an alternative to thermal treatment in order to preserve apple juice quality.

Fate of Escherichia coli strains inoculated in model cheese elaborated with or without starter and treated by high hydrostatic pressure.

The aim of this research was to study high hydrostatic pressure inactivation of two strains of Escherichia coli (E. coli O59:H21 [CECT 405] and E. coli O157:H7 [CECT 5947]) inoculated in washed-curd model cheese elaborated with and without starter and the ability of these strains for survival, recovery, and growth. Samples were treated at 300, 400, and 500 MPa for 10 min at 20 degrees C and analyzed after the treatment and after 1, 7, and 15 days of storage at 8 degrees C to study the behavior of Escherichia populations. Cheeses elaborated with starter showed the maximum lethality at 400 and 500 MPa, and no significant differences in the baroresistant behavior of either strains were detected, except for E. coli O157:H7 at 400 MPa in cell counts obtained with thin agar layer method medium, where the decrease value was significantly lower. In cheese elaborated without starter, the highest decrease value was observed at 500 MPa, except for E. coli O59:H21 in cell counts obtained with selective culture medium, where the highest decrease value was also found at 400 MPa. The ability to repair and grow was not observed in model cheese elaborated with starter, as cell counts of treated samples decreased after 15 days of storage at 8 degrees C. By contrast, in cheese elaborated without starter, all pressurized samples showed the trend to repair and grow during the storage period in both strains. These results suggest that the presence of starter and low pH values are the main factors that control the ability of Escherichia strains inoculated in this type of cheese and treated by high hydrostatic pressure to recover and grow.

Inactivation of Mycobacterium avium subsp. paratuberculosis in cow's milk by means of high hydrostatic pressure at mild temperatures.

Two strains of Mycobacterium avium subsp. paratuberculosis (3644/02 and ATCC 19698) were inoculated (approximately 6 log CFU/ml) into sterilized milk to evaluate inactivation by high hydrostatic pressure. Reductions of M. avium subsp. paratuberculosis increased with pressure level. Significant differences were also found between M. avium subsp. paratuberculosis strains and between the media used. Average reductions of 4 log CFU/ml after treatment with 500 MPa are comparable to those caused by thermal treatments.

Inactivation by ultrahigh-pressure homogenization of Escherichia coli strains inoculated into orange juice.

The aim of this work was to evaluate the efficacy of ultrahigh-pressure homogenization (UHPH) for inactivation and/or sublethal injury of two strains of Escherichia coli (O58:H21 ATCC 10536 and O157:H7 CCUG 44857) inoculated into orange juice (pH 3.6). The effects of orange juice inlet temperature (6 and 20 degrees C) on the lethality values and the capacity of these strains for survival, repair, and growth during refrigerated storage after UHPH treatment also was evaluated. Samples of orange juice that had been treated with ultrahigh temperatures were inoculated with E. coli in the stationary phase of growth until a final concentration of approximately 7.0 log CFU/ml was reached. These samples were then treated for one cycle with a double-valve UHPH machine, with 300 MPa at the primary homogenizing valve and 30 MPa at the secondary valve. Counts of viable and injured bacterial cells were obtained for samples taken 2 h after UHPH treatment and after 3, 6, 9, 12, 15, 18, 21, 27, and 33 days of storage at 4 degrees C. The inlet temperature and the strain type both influenced significantly (P < 0.05) the lethality effect on E. coli, which was higher when the inlet temperature was 20 degrees C. No sublethal injuries were detected after any treatment. The changes in viable counts over time for both strains in pressurized and control samples were similar. The viable counts remained high from day 0 to day 18 and then tended to decrease. After 27 days of storage at 4 degrees C, E. coli O157: H7 was more resistant in orange juice samples pressurized at inlet temperatures of 6 and 20 degrees C, with viable counts of 3.41 and 3.20 log CFU/ml, respectively.

Inactivation of Listeria innocua in milk and orange juice by ultrahigh-pressure homogenization.

The aim of this work was to evaluate the bactericidal efficacy of ultrahigh-pressure homogenization (UHPH) against Listeria innocua ATCC 33090 inoculated into milk and orange juice. We also intended to study the effect of inlet temperature on the lethality and production of sublethal injuries in this microorganism and its ability to survive, repair, and grow in refrigerated storage after UHPH treatment. Samples of ultrahigh-temperature whole milk and ultrahigh-temperature orange juice inoculated at a concentration of approximately 7.0 log (CFU per milliliter) were immediately pressurized at 300 MPa on the primary homogenizing valve and at 30 MPa on the secondary valve, with inlet temperatures of 6.0 +/- 1.0 degrees C and 20 +/- 1.0 degrees C. L. innocua viable counts and injured cells were measured 2 h after UHPH treatment and after 3, 6, and 9 days of storage at 4 degrees C for milk and after 3, 6, 9, 12, 15, 18, and 21 days of storage at 4 degrees C for orange juice. Both the inlet temperature and the food matrix influenced significantly (P < 0.05) the inactivation of L. innocua, which was higher in whole milk at the 20 degrees C inlet temperature. The UHPH treatment caused few or no sublethal injuries in L. innocua. During storage at 4 degrees C after treatments, counts increased by approximately 2 logarithmic units from day 0 to 9 in whole milk, whereas in orange juice counts diminished by approximately 2.5 logarithmic units from day 0 to 18.

Survival and growth of Yersinia enterocolitica strains inoculated in skimmed milk treated with high hydrostatic pressure.

Four human pathogenic strains of Yersinia enterocolitica (serotypes O:1, O:3, O:8, and O:9) were inoculated (7-8 log CFU/ml) in UHT skimmed milk and treated at 300, 400, and 500 MPa for 10 min at 20 degrees C, and then kept at 8 degrees C to assess their evolution for 15 days. Treatments at 400 and 500 MPa caused the highest lethality, generally reaching counts below detection level (1 CFU/ml) in the culture media. At 300 MPa, the most baroresistant serotypes were O:3 and O:8. After 15 days of storage at 8 degrees C, Y. enterocolitica showed growth over 8 log (CFU/ml) in all treatments. Kinetic study of microbial inactivation in skimmed milk was performed with serotype O:8 at 300 MPa, showing a tailing after 35 min of pressure treatment.

Behavior of Yersinia enterocolitica strains inoculated in model cheese treated with high hydrostatic pressure.

The effects of high hydrostatic pressure treatment and the ability for survival, repair, and growth of three human pathogenic serotypes (O:1, O:3, O:8) of Yersinia enterocolitica were investigated in washed-curd model cheese made with pasteurized bovine milk. Samples were treated at 300, 400, and 500 MPa for 10 min at 20 degrees C and analyzed at 0, 1, 7, and 15 days to assess the viability of the Yersinia population. A long-term study (up to 60 days of ripening after high hydrostatic pressure treatment) was also undertaken. Treatments at 400 and 500 MPa caused maximum lethality, and only the treatment at 300 MPa showed significant differences (P < 0.05) between serotypes; the most baroresistant was O:3. Ability to repair and grow was not observed after 15 days of storage at 8 degrees C. Yersinia counts in untreated cheese samples also decreased below the detection limit at day 45 in the long-term study. These results suggest that the cheese environment did not allow recovery of injured cells or growth. A primary contributing factor to this effect seemed to be the low pH resulting from the production of lactic acid during cheese ripening.

Evaluation of biogenic amines and microbial counts throughout the ripening of goat cheeses from pasteurized and raw milk.

The effect of the hygienic quality of milk on changes in microbial counts and biogenic amine content was evaluated during ripening of goat cheeses manufactured from pasteurized and raw milks at 1, 14, 30, 60 and 90 d. The original milk, rennet, curd and whey were also included in the study. The pH, salt content and extent of proteolysis in the cheese were also evaluated. Spermidine and spermine were the main amines in raw milk, while they were minor amines in cheeses. Other amines increased markedly during ripening, tyramine being the main amine in cheese made from raw milk and cadaverine and putrescine in those produced from pasteurized milk. Enterobacteriaceae counts decreased during ripening whereas those of lactic acid bacteria increased, especially lactobacilli and enterococci. Cheese made from raw milk showed higher microbial counts during ripening than those made from pasteurized milk, especially for Enterobacteriaceae and enterococci, counts being 2 or 3 log units higher. Raw milk cheese showed remarkably higher biogenic amines compared with pasteurized milk cheeses. Therefore, pasteurization of milk causes a decrease in final biogenic amine content of cheese as a result of the reduction of its microbial counts.

Comparison of biogenic amine profile in cheeses manufactured from fresh and stored (4 degrees C, 48 hours) raw goat's milk.

In this study, the evolution of microbial counts, biogenic amine contents, and related parameters (pH, moisture, and proteolysis) in goat cheese made from fresh raw milk or raw milk stored for 48 h at 4 degrees C was examined. In both cases the milk was nonpasteurized. This study was designed to evaluate the effect of milk quality on the profile of biogenic amines in relation to the evolution of the microbial population during cheese making. Cheese made from raw milk stored for 48 h at 4 degrees C showed the highest microbial counts and biogenic amine levels. The storage of milk under refrigeration caused significant increases in the levels of some microbial and biogenic amines during ripening, but not initially. Tyramine was the main biogenic amine in the two cheeses tested, followed by cadaverine. However, the main differences in amine contents between batches were found for putrescine, histamine, and beta-phenylethylamine, whose levels were more than twofold higher in samples from raw milk refrigerated for 48 h than in samples from fresh milk.

Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme.

The objective of this work was to study high hydrostatic pressure (HHP) inactivation of spores of Bacillus cereus ATCC 9139 inoculated in model cheeses made of raw milk, together with the effects of the addition of nisin or lysozyme. The concentration of spores in model cheeses was approximately 6-log10 cfu/g of cheese. Cheeses were vacuum packed and stored at 8 degrees C. All samples except controls were submitted to a germination cycle of 60 MPa at 30 degrees C for 210 min, to a vegetative cells destruction cycle of 300 or 400 MPa at 30 degrees C for 15 min, or to both treatments. Bacillus cereus counts were measured 24 h and 15 d after HHP treatment. The combination of both cycles improved the efficiency of the whole treatment. When the second pressure-cycle was of 400 MPa, the highest inactivation (2.4 +/- 0.1 log10 cfu/g) was obtained with the presence of nisin (1.56 mg/L of milk), whereas lysozyme (22.4 mg/L of milk) did not increase sensitivity of the spores to HHP. For nisin (0.05 and 1.56 mg/L of milk), no significant differences were found between counts at 24 h and 15 d after treatment. Considering that mesophilic spore counts usually range from 2.6 to 3.0 log10 cfu/ml in raw milk, HHP at mild temperatures with the addition of nisin may be useful for improving safety and preservation of soft curd cheeses made from raw milk.

Histidine Decarboxylase Activity of Enterobacter cloacae S15/19 during the Production of Ripened Sausages and Its Influence on the Formation of Cadaverine.

The histidine decarboxylase activity of Enterobacter cloacae S15/19 was studied during the production process of salchichón , a Spanish ripened sausage. Counts of fecal coliform and histidine decarboxylase bacteria decreased during the production process, showing a good correlation in both inoculated and control samples. In the samples inoculated with Enterobacter cloacae S15/19, fecal coliforms were undetectable the last day of the survey, while the population of histidine decarboxylase bacteria was over 2 log MPN/g. Despite the fact that inoculation with Enterobacter cloacae S15/19 increased histidine decarboxylase bacteria counts, no differences were observed in the histamine concentration reached, which was undetectable in most of the control and inoculated samples. In contrast, cadaverine concentration increased significantly (P < 0.01) in the inoculated samples, suggesting that cadaverine could be used as a hygienic-quality indicator of the raw materials employed in sausage processing.

Sensory Quality and Histamine Formation during Controlled Decomposition of Tuna ( Thunnus thynnus ).

Histamine production was studied during controlled tunafish decomposition at 0, 8, and 20°C. The influence of the location of the anatomic section on the amount of histamine formed and the incidence of histidine decarboxylating bacteria were also considered. By the time of sensory rejection, histamine levels in tunafish sections stored at 0 and 20°C were still below the hazard levels and the allowable levels established by both the U.S. Food and Drug Administration (FDA) and the European Union. Toxic amounts were only formed after the tunafish was considered organoleptically unsuitable for human consumption. However, at 8°C, levels of histamine between 100 and 200 mg/l00 g of fish were found before tuna reached the rejection point. Hence, physical appearance was not a good criterion for estimating the shelf life and especially the histamine-related health hazard when tuna was stored at 8°C, a common temperature in many home refrigerators.

Histidine Decarboxylase Activity of Bacteria Isolated from Raw and Ripened Salchichón , a Spanish Cured Sausage.

Twenty samples of raw sausages (before the ripening process) and 10 of ripened sausages of two different sizes of salchichón , one of the most-consumed ripened meat products in Spain, were microbiologically analyzed. Histidine decarboxylase activity of different isolates obtained from different culture media was evaluated by a decarboxylase agar medium and confirmed afterwards by an enzymatic method. Of the isolates obtained from raw sausages, 15.8% showed histamine-forming activity, most of them (78.1%) belonging to the Enterobacteriaceae family. Klebsiella oxytoca , Enterobacter aerogenes (one isolate), and Enterobacter cloacae (six isolates) showed a powerful histamine-forming activity, producing more than 3,000 µg/ml. In spite of histamine-forming activity being detected in eight of ten analyzed samples of ripened sausages, only one histamine-forming bacterium, identified as Lactobacillus curvatus , was isolated; it showed an ability to form 1,994 µg/ml of histamine in experimental conditions.

Bacteriological Quality of Tuna Fish ( Thunnus thynnus ) Destined for Canning: Effect of Tuna Handling on Presence of Histidine Decarboxylase Bacteria and Histamine Level.

The present paper analyzes the bacteriological quality and histamine content of tuna fish samples destined for canning. Raw material used in the canning process was of good quality. Histamine-producing bacteria counts were only found in three samples from the last step of the canning process before sterilization. Most of the bacteria identified as histamine formers were gram negative, and nearly all of which belonged to the Enterobacteriaceae family. Morganella morganii was the most frequent and active histamine former in tuna fish destined for canning. Other powerful histamine-producing bacteria isolated during the canning operation were Klebsiella oxytoca , Klebsiella pneumoniae and some strains of Enterobacter cloacae and Enterobacter aerogenes . They all were able to produce more than 500 ppm of histamine in experimental conditions. Most of these species might be expected to be found as a result of contamination of fish during capture and subsequent unhygienic handling in the canning plant. An increase in histamine content in tuna meat was not expected through the canning process. Histamine content always was acceptable in accordance with the maximum allowable levels of histamine fixed by both the European Economic Community and Food and Drug Administration.