Evaluation of a novel antimicrobial (lauric arginate ester) substance against biofilm of Escherichia coli O157:H7, Listeria monocytogenes,and Salmonella spp.

The purpose of this study was to evaluate the activity of a novel antimicrobial substance lauric arginate ester (LAE) against selected foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp.) in biofilm. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined and showed that LAE exhibits a strong antimicrobial activity. Biofilms were grown on abiotic stainless steel, rubber, MBEC biofilm device) and biotic (lettuce) surfaces. The efficacy of LAE (50, 100 and 200 ppm) at reducing the biofilm cells on these surfaces was examined by applying LAE for 2 h. Results revealed that LAE exhibited the reduction in biofilm bacteria up to 7 log CFU cm^?2, 3.5 log CFU cm^?2, 4.0 log CFU peg^?1 and 1.5 log CFU cm^?2 on stainless steel, rubber, MBEC and lettuce surfaces, respectively. Overall, these results suggest that LAE has been shown to be a potential alternative to control bacteria in biofilm mode in food industry.     

Enhanced antimicrobial activity of nisin in combination with allyl isothiocyanate against Listeria monocytogenes,Staphylococcus aureus,Salmonella Typhimurium and Shigella boydii

This study was designed to evaluate the synergistic antimicrobial effect of nisin and allyl isothiocyanate (AITC) against Listeria monocytogenes, Staphylococcus aureus, Salmonella Typhimurium and Shigella boydii. The synergistic interactions between nisin and AITC were observed against all foodborne pathogens, showing the fractional inhibitory concentrations <1. The populations of L. monocytogenes and S. aureus at the combined treatment of nisin and AITC were decreased to below 1 log CFU mL^?1 after 10‐h incubation at 37 °C. The changes in fatty acid profiles of all strains were substantially influenced by nisin alone and the combined treatment of nisin and AITC. A good agreement was observed among cell viability, membrane permeability and depolarisation activity in response to nisin and AITC. The results suggest that nisin and AITC as synergistic inhibitors could be an effective approach to achieve satisfactory antimicrobial activity against a wide range of foodborne pathogens.     

Antilisterial effect of two bioprotective cultures in a model system of Iberian chorizo fermentation

This study reports the different effects of two bioprotective cultures on the growth of different Listeria monocytogenes strains by a rapid assay simulating the first stage of Iberian chorizo fermentation. Ground pork with or without protective cultures was inoculated with L. monocytogenes and incubated under two different conditions simulating the traditional, slow fermentation temperature (7 °C, 1 day) and a high, fast fermentation temperature (20 °C, 1 day), followed in both cases by storage at 7 °C for 13 days. Both bioprotective cultures reduced the growth of L. monocytogenes by at least 2 log CFU g^?1 at the end of both incubation periods compared with a noninoculated culture control lot. The best results were obtained with the strain Lactobacillus sakei CTC494, which exerted a bactericidal effect on L. monocytogenes under both conditions assayed, achieving a 5.4‐log reduction after 14 days compared with the control when the initial temperature was 20 °C.     

Efficacy of bovicin HC5 and nisin combination against Listeria monocytogenes and Staphylococcus aureus in fresh cheese

Fresh and soft cheeses provide a suitable medium for the growth of many microorganisms and have been frequently associated with foodborne diseases. This study aimed to evaluate the effects of the bacteriocins bovicin HC5 and nisin on Listeria monocytogenes and Staphylococcus aureus in Minas Frescal cheese, a traditional Brazilian fresh product. The cheese was inoculated with 10⁴ CFU g^?1 of both pathogens, and the bacteriocins were added at 600 AU g^?1 of each one. After 9 days of storage at 4 °C, L. monocytogenes Scott A were not detected in 25 g samples. Although the bacteriocins reduced the population of S. aureus ATCC 6538, increasing numbers were detected after 15 days of storage at 4 °C. Staphylococcal enterotoxin C was detected in cheeses added of bacteriocins and stored under abusive temperature of 15 °C. The results demonstrate the potential application of combination of bovicin HC5 and nisin in controlling the pathogens growth assessed in Minas Frescal cheese.     

Effect of partial replacement of potassium lactate and sodium diacetate by natural green tea and grape seed extracts and postpackaging thermal treatment on the growth of Listeria monocytogenes in hotdog model system

Low‐ (5%) and high‐fat (20%) chicken and turkey hotdogs were formulated in three groups: no antimicrobials (control), chemical preservatives (potassium lactate and sodium diacetate) alone and partial replacement of chemical preservatives by green tea (GTE) and grape seed extracts (GSE), surface inoculated (c. 10³ CFU g^?1) with Listeria monocytogenes, treated with or without heat treatment (65 °C for 104 s) to determine the growth of L. monocytogenes until spoilage (28 days). Maximum growth inhibitions (c. 2.0 CFU g^?1) were observed in the treatments having chemical preservatives and plant extracts regardless of the meat and fat type. Furthermore, plant extracts along with chemical preservatives demonstrated additional inhibitory effect on the growth of L. monocytogenes survivors in chicken hotdog samples followed by postpackaging heat treatment. Results demonstrated that natural GTE and GSE can partially replace the chemical preservatives and further enhance the antilisterial activities when combined with heat treatment.     

Effect of Mentha spicata L. and Mentha aquatica L. essential oils on the microbiological properties of fermented dairy product,kashk

The antibacterial activity of Mentha spicata and Mentha aquatica essential oils (EO) was tested against Staphylococcus aureus, Lactobacillus reuteri, Bifidobacterium animalis and Clostridium perfringens using agar well and disc diffusion techniques. Results showed that M. spicata EO had the highest inhibition activity against the studied microorganisms. Then, the antibacterial activity of both EO at 1500 and 2500 ppm was examined in industrial liquid kashk during the storage at 4 °C for 20 days. Both EO reduced the S. aureus viable count below 5 log CFU g^?1 after 4 days; however, the population of C. perfringens, L. reuteri and B. animalis decreased <1 log CFU g^?1 during the storage time. The least deteriorative effect on the lactic acid bacteria was related to M. aquatica. As revealed by organoleptic studies, kashk samples containing M. aquatica EO at 1500 and 2500 ppm were the most preferred samples.     

Application of enterocins A and B, sakacin K and nisin to extend the safe shelf-life of pressurized ready-to-eat meat products

The efficiency of food preservation systems is determined by the technologies that are combined, the intrinsic properties of the food products and the target microorganisms. In the present study, the bacteriocins nisin, enterocins A and B and sakacin K were applied to cooked and dry cured ham spiked with Listeria monocytogenes, Salmonella enterica and Staphylococcus aureus and submitted to a high pressure treatment of 600 MPa. Before pressurization nisin produced significant reductions to the counts of L. monocytogenes and S. aureus, especially in dry cured ham. After the pressurization, Salmonella and L. monocytogenes were not detected in 25 g of both cooked and dry cured ham and remained at this level during the entire storage (57 days at 4 °C + 63 days at 15 °C). S. aureus levels, in contrast, only decreased below the detection limit (1 log CFU/g) in the nisin batches. Afterward, when storage was performed at an abusive temperature, the ability of S. aureus to grow was dependant on the bacteriocin applied and the kind of meat product. Thus, at the end of storage, while S. aureus counts were <1 log CFU/g in all dry cured ham batches, only nisin could inhibit its growth in cooked ham.     

Reduction of Listeria monocytogenes in queso fresco cheese by a combination of listericidal and listeriostatic GRAS antimicrobials

Single and combined effects of three GRAS (generally recognized as safe) antimicrobials including, bacteriophage P100 (phage P100), lauric arginate (LAE), and potassium lactate-sodium diacetate mixture (PL-SD) were evaluated against Listeria monocytogenes cold growth in queso fresco cheese (QFC). The fate of phage P100 when exposed to LAE (200 ppm) or PL-SD (2.8% PL and 0.2% SD) was determined at 4°C and 30°C in a broth model. Phage P100 was found to be stable in the presence of these antimicrobial agents as plaque forming units (PFU) did not vary between control, LAE or PL-SD treatments. When 9 log CFU/ml of stationary phase cells of L. monocytogenes was exposed to these antimicrobials in tryptic soy broth, there was a 3 to 5 log CFU/ml reduction with phage P100 and a complete 9 log CFU/ml reduction with LAE but no measurable reduction with PL-SD after 24h at 4°C or 30°C. In QFC, the L. monocytogenes populations increased from the initial 3.5 log CFU/cm(2) to 7.7 log CFU/cm(2) in 28 days at 4°C. Treatment with 7.8 log PFU/cm(2) of phage P100 or 200 ppm of LAE showed strong listericidal effect initially by reducing L. monocytogenes counts by 2 to 3.5-4 log CFU/cm(2) while there was a subsequent regrowth of L. monocytogenes at 4°C. Treatment with PL-SD showed strong listeriostatic effect without decreasing L. monocytogenes counts but growth was prevented for 28 days at 4°C. Only the combined treatment of listericidal phage P100 or LAE with listeriostatic PL-SD reduced the initial L. monocytogenes counts by 2-4 log CFU/cm(2) and also kept the L. monocytogenes counts at that reduced level in QFC for 28 days at 4°C.     

Bioactive action of β‐glucosidase enzyme of Bifidobacterium longum upon isoflavone glucosides present in soymilk

Bioconversion of isoflavone glucosides and antioxidant activity by probiotic strain (Bifidobacterium longum) during soymilk fermentation was investigated, as well as partial characterisation of the produced enzyme β‐glucosidase. The enzyme has higher affinity for genistin than for other substrates assayed. Maximum activity occurred at 42 °C and at pH 6.0; keeping 70–80% of activity for 60 days stored at low temperatures. Bifidobacterium longum grew well in soymilk (8.26 log CFU mL^?1 and pH of 3.9 at 24 h) and were produced in good quantities of organic acids. High hydrolysis degree of isoflavone glucosides (81.2%) was observed at 24 h. Enhancements in bioactivity were assessed in fermented soymilk by monitoring the radical‐scavenging activity, antioxidant activity and DNA protective action. The use of probiotic Bifidobacterium strain as β‐glucosidase producer increased bioactive isoflavone content and demonstrated that this enzyme plays a key role in the bioavailability of soymilk isoflavones, reducing the bioconversion time compared to other studies.     

Effect of gums on viability and β‐galactosidase activity of Lactobacillus spp. in milk drink during refrigerated storage

The viability and β‐galactosidase activity of four Lactobacillus strains in milk drink containing gums during 28 days of refrigerated storage at 4 °C were assessed. The population of Lactobacillus rhamnosus GGB101 and Lactobacillus rhamnosus GGB103 were maintained, whereas the population of Lactobacillus reuteri DSM20016 and Lactobacillus reuteri SD2112 significantly decreased. The recommended level of 6 log CFU g^?1 was exceeded for all tested trains throughout storage. The highest viable number of Lactobacillus rhamnosus GGB103 (8.76 ± 0.03 log CFU mL^?1) was obtained in the product containing carrageenan–maltodextrin. The addition of guar–locust bean–carrageenan led to 20‐fold increase in the level of β‐galactosidase activity for L. rhamnosus GGB101 (1208 ± 2.12 Miller units mL^?1) compared to the control (61 ± 2.83 Miller units mL^?1). Our results suggested that gums could be added to milk to improve viability and enhance β‐galactosidase activity of Lactobacillus.     

Polyphenolic and phytochemical content of Cucumis sativus seeds and study on mechanism of preservation of nutritional and quality outcomes in enriched mayonnaise

Oxidative rancidity in food emulsions leads to a reduction in shelf life. Synthetic antioxidants are widely used in food industry to prevent the development of rancidity. The present study was focussed on investigating the antioxidant potential of Cucumis sativus seeds (CSS) and correlates these findings with mayonnaise enrichment and extends its shelf life. CSS exhibited the highest abundance in phenolic compounds (93.5 ± 0.1 mgGAE g^?1), flavonoids (57.4 ± 0.1 mgQE g^?1), β‐carotene (19.46 ± 0.4 mg carotenoids per 100 g) and high free radical scavenging activity. CSS (200 ppm) and butylated hydroxyanisole (200 ppm) were incorporated in mayonnaise and the oxidative stability was evaluated by peroxide, p‐anisidine and TBARS values during storage at different temperatures. Organoleptic evaluations indicated that CSS enriched sample was recorded the highest overall acceptability. The results from our study will provide scientific basis for CSS as natural preservative against lipid oxidation or food enrichment while developing functional foods.     

Antimicrobial Activity of Vanillin and Mixtures with Cinnamon and Clove Essential Oils in Controlling <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 in Milk

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of vanillin against Listeria monocytogenes Scott A and Escherichia coli O157:H7 was determined in tripticase soy broth (TSB), pH 7 and 6, incubated at 35 °C/24 h and in semi-skim milk incubated at 35 °C/24 h and 7 °C/14 days. The influence of the fat content of milk on the antimicrobial activity of vanillin was tested in whole and skim milk incubated at 7 °C/14 days. Mixtures of clove and cinnamon with vanillin were also evaluated in semi skim milk incubated at 7 °C. The MICs for L. monocytogenes were 3,000 ppm in TSB (pH 7) and 2,800 ppm in TSB (pH 6). The MICs for E. coli O157:H7 were 2,800 ppm in TSB (pH 7) and 2,400 ppm in TSB (pH 6). The MBCs in TSB were 8,000 ppm for L. monocytogenes and 6,000 ppm for E. coli O157:H7. The pH values assayed did not influence significantly the MIC or MBC in TSB. The MICs in semi-skim milk for L. monocytogenes and E. coli O157:H7 were 4,000 and 3,000 ppm at 35 °C/24 h, and 2,500 and 1,000 ppm at 7 °C/7 days, respectively. The MBCs were 20,000 ppm for L. monocytogenes and 11,000 ppm for E. coli O157:H7. High incubation temperatures did not affect the MBC but increased the MIC of the vanillin in milk. This effect could be attributed to the increased membrane fluidity and to the membrane perturbing activity of vanillin at low temperatures. The fat in milk reduced significantly the antimicrobial activity of vanillin, probably due to effect protective of the fat molecules. Mixtures of clove and cinnamon leaves inhibited the growth of L. monocytogenes in a similar way that vanillin alone but had a synergistic effect on the E. coli O157:H7. Mixtures of cinnamon bark and vanillin had always a synergistic effect and some of the combination assayed showed bactericidal activity on the population of L. monocytogenes and E. coli O 157:H7.     

Effects of Pseudomonas chlororaphis and gaseous chlorine dioxide on the survival of Salmonella enterica on tomatoes

Control of Salmonella enterica on tomatoes is important for food safety. The aim of this research was to evaluate the survival of Salmonella enterica serovars Montevideo (SM) and Typhimurium (ST) on tomatoes exposed to gaseous chlorine dioxide and Pseudomonas chlororaphis (Pc). Pc was applied to stem scars of tomatoes prior to inoculations with SM and ST. Tomatoes were treated with gaseous ClO₂ at 0.4 mg L^?1 for 2 and 4 h (90% R.H. 13 °C), respectively. At 4 h of ClO₂ treatment, SM and ST populations were reduced to 0.82 and <0.30 log CFU g^?1, respectively. Tomatoes treated with SM and ST had 5.42 and 5.37 log CFU g^?1 of Salmonella. Tomatoes treated with Pc + Salmonella count was 2.59 (treated) and 5.83 log CFU g^?1 (control). Salmonella survival was similar at 2 and 4 h of ClO₂ treatment. Application of ClO₂ and Pc may reduce contamination of tomatoes by Salmonella serovars.     

Characterisation of malolactic conversion by Oenococcus oeni to reduce the acidity of apple juice

The high concentration of malic acid is responsible for the acidity and sourness in apple juice. Bio‐conversion of malic acid to lactic acid through malolactic conversion (MC) in apple juice using Oenococcus oeni was investigated. When apple juice was inoculated with O. oeni (1 × 10⁶ CFU mL^?1), over 90% of malic acid was converted into lactic acid within 96 h at room temperature. When pH of apple juice was adjusted to 4.1 prior to inoculation, MC was completed within 60 h. MC was enhanced at a higher temperature (30°C) when compared with room temperature. The rate of MC was directly proportional to the number of bacteria added and MC was completed within 24 h at 1 × 10⁹ CFU mL^?1 initial cell density. MC occurred equally under aerobic and anaerobic conditions. The sensory analysis of partial MC‐applied juice when compared against control revealed potential for use of MC for manufacture of low‐acid apple juice.     

Bactericidal activity of lauric arginate in milk and Queso Fresco cheese against Listeria monocytogenes cold growth

Lauric arginate (LAE) at concentrations of 200 ppm and 800 ppm was evaluated for its effectiveness in reducing cold growth of Listeria monocytogenes in whole milk, skim milk, and Queso Fresco cheese (QFC) at 4°C for 15 to 28 d. Use of 200 ppm of LAE reduced 4 log cfu/mL of L. monocytogenes to a nondetectable level within 30 min at 4°C in tryptic soy broth. In contrast, when 4 log cfu/mL of L. monocytogenes was inoculated in whole milk or skim milk, the reduction of L. monocytogenes was approximately 1 log cfu/mL after 24 h with 200 ppm of LAE. When 800 ppm of LAE was added to whole or skim milk, the initial 4 log cfu/mL of L. monocytogenes was nondetectable following 24 h, and no growth of L. monocytogenes was observed for 15 d at 4°C. With surface treatment of 200 or 800 ppm of LAE on vacuum-packaged QFC, the reductions of L. monocytogenes within 24 h at 4°C were 1.2 and 3.0 log cfu/g, respectively. In addition, the overall growth of L. monocytogenes in QFC was decreased by 0.3 to 2.6 and by 2.3 to 5.0 log cfu/g with 200 and 800 ppm of LAE, respectively, compared with untreated controls over 28 d at 4°C. Sensory tests revealed that consumers could not determine a difference between QFC samples that were treated with 0 and 200 ppm of LAE, the FDA-approved level of LAE use in foods. In addition, no differences existed between treatments with respect to flavor, texture, and overall acceptability of the QFC. Lauric arginate shows promise for potential use in QFC because it exerts initial bactericidal activity against L. monocytogenes at 4°C without affecting sensory quality.     

Encapsulation of probiotic Lactobacillus acidophilus by ionic gelation with electrostatic extrusion for enhancement of survival under simulated gastric conditions and during refrigerated storage

The probiotic Lactobacillus acidophilus was encapsulated in biodegradable and biocompatible capsules prepared by ionic gelation between phytic acid (PA) and chitosan (CS) with an electrostatic extrusion method. Calcium carbonate (CaCO₃) and starch were used as co‐encapsulants for improvement of capsule stability. Capsules were characterised and evaluated for survival of encapsulated L. acidophilus cells in simulated gastric fluid (SGF) and during refrigerated storage. Loading capacity values of PA‐CS capsules, PA‐CS‐starch capsules and PA‐CS‐CaCO₃ capsules were 8.20, 8.12 and 7.81 log CFU g^?1 of wet capsule, respectively. Capsules showed particle sizes of 1.3–1.5 mm and a uniform spherical shape. PA‐CS‐CaCO₃ capsules were the most stable vehicles for the protection of probiotic cells against acidic damage, particularly at pH 1.5 and pH 2. L. acidophilus cells from PA‐CS‐CaCO₃ capsules showed only a 0.64 log CFU reduction in numbers after 2 h in pH 1.5 SGF conditions. The numbers of L. acidophilus encapsulated in PA‐CS‐CaCO₃ capsules were decreased by only 0.69 log CFU g^?1, while PA‐CS capsules and PA‐CS‐starch capsule numbers were reduced by more than 1.45 log CFU g^?1 after 4 weeks at 4 °C. Addition of calcium carbonate to PA‐CS capsules provided protection against acid injury via antacid and buffering effects for encapsulation of L. acidophilus.     

Synergistic antimicrobial effect of lactocin AL705 and nisin combined with organic acid salts against Listeria innocua 7 in broth and a hard cheese

The effectiveness of antimicrobial mixtures against Listeria innocua 7, used as a L. monocytogenes surrogate, was investigated in broth and a food system. Synergistic effects were found for nisin (Nis), potassium sorbate (PS), calcium propionate (CP) and sodium lactate (SL), Nis + PS being the most effective binary mixture that exhibited listericidal activity in broth. To assess the effect of adding lactocin AL705 (AL705) to Nis + organic acid salt combinations, tridimensional isobolograms were generated. Sub-MIC combinations of the antimicrobials exerted bactericidal activity against L. innocua 7 after AL705 addition to the binary mixtures. However, when applied on Sardo cheese contaminated with L. innocua 7 (initial inoculum 4.45 ± 0.06 CFU g⁻¹), only Nis + PS + AL705 produced count reductions respect to the control, reaching 3.04 ± 0.35 CFU g⁻¹ counts after 15 days at 15 °C. Ternary combinations containing AL705 showed potential to reduce antimicrobial usages for L. innocua 7 inhibition.     

Control of Listeria monocytogenes in whole milk using antimicrobials applied individually and in combination

Dairy product recalls and dairy-related illnesses are often the result of contamination with Listeria monocytogenes, which can occur throughout the dairy production and supply chains. The use of antimicrobial compounds is one practical approach for controlling pathogen survival and growth in foods. The goal of this study was to use fluid milk as a model system to identify listeristatic or listericidal treatments that show promise for application in fluid milk and for further evaluation in other dairy products (e.g., cheese). Caprylic acid (CA), ε-polylysine (EPL), hydrogen peroxide, lauric arginate (LAE), and sodium caprylate (SC) were added individually or in combination to whole milk inoculated with L. monocytogenes at ?4 log₁₀ cfu/mL. Samples were stored at 7°C for 21 d, and L. monocytogenes counts were determined weekly. Inhibitory concentrations of LAE (800 mg/L) and EPL (100–400 mg/L), as well as SC and CA (3,200 mg/L each), were identified. The addition of EPL at 800 mg/L reduced L. monocytogenes counts by >3 log₁₀ cfu/mL from initial inoculation levels after 21 d. Addition of hydrogen peroxide to milk reduced counts by >3 log₁₀ cfu/mL from initial inoculation within 24 h (400 and 800 mg/L) or by d 7 (200 mg/L). Although the combinatory treatments of EPL + CA, EPL + LAE, and LAE + SC were characterized as indifferent, EPL + SC worked synergistically to reduce L. monocytogenes populations in milk over 21 d. Overall, these data identify potential antimicrobial treatments to control L. monocytogenes in milk and serve as a foundation for the continued development of antimicrobial controls for L. monocytogenes in dairy products.     

Evaluation of parameters affecting modified atmosphere packaging engineering design for pomegranate arils

This study evaluated the effects of passive modified atmosphere packaging design parameters as a function of the amount of product (g), temperature (°C) and time (days) on two pomegranate cultivars. Arils (75, 100 and 125 g) were packed in trays, heat sealed with polylid film and stored at 5, 10 and 15 °C for 14 days, and analysed for physicochemical parameters viz headspace gas composition, weight loss, total soluble solids, titratable acidity, pH, anthocyanin, aerobic‐mesophilic bacterial and fungal load (log CFU g^?1). At the highest temperature and weight, O₂ concentration continuously decreased below the critical limit (2%) after 4 days, while at 5 °C, this lower limit was not reached. Shelf life of arils was limited to 10, 7 and 3 days by fungal growth ≥2 log CFU g^?1 at 5, 10 and 15 °C, respectively. Using unsteady‐state equation, a good agreement was found between simulated and experimental gas composition data.     

Antimicrobial activities of high molecular weight water‐soluble chitosans against selected gram‐negative and gram‐positive foodborne pathogens

Antimicrobial activities of high molecular weight water‐soluble chitosans (HMWWS) against selected Gram‐negative and Gram‐positive foodborne pathogens (initial inoculation of ca. 6.5 Log CFU mL^?1) were evaluated. Chitosans with 789 kDa and/or 1017 kDa were dissolved in aspartic acid (AS) to obtain 1–4% w/v solutions. Among HMWWS, only 4% 789 kDa AS chitosan reduced E. coli counts by 2 Log CFU mL^?1 from 7.33 at 0 h to 5.16 Log CFU mL^?1 at 96 h, and they were not effective against S. Typhimurium. Depending on the concentrations, HMWWS completely inhibited V. cholerae, V. vulnificus and V. parahaemolyticus as well as B. cereus and L. monocytogenes after 48 h or 96 h of incubation. Compared with the control (no HMWWS), 2% or 3% 1017 kDa AS chitosans showed about 3 Log CFU mL^?1 lower (4.72–4.86 vs. 7.71) for S. aureus at 96 h of incubation.