Probiogenomic In-Silico Analysis and Safety Assessment of Lactiplantibacillus plantarum DJF10 Strain Isolated from Korean Raw Milk.

The whole genome sequence of Lactiplantibacillus plantarum DJF10, isolated from Korean raw milk, is reported, along with its genomic analysis of probiotics and safety features. The genome consists of 29 contigs with a total length of 3,385,113 bp and a GC content of 44.3%. The average nucleotide identity and whole genome phylogenetic analysis showed the strain belongs to Lactiplantibacillus plantarum with 99% identity. Genome annotation using Prokka predicted a total of 3235 genes, including 3168 protein-coding sequences (CDS), 59 tRNAs, 7 rRNAs and 1 tmRNA. The functional annotation results by EggNOG and KEGG showed a high number of genes associated with genetic information and processing, transport and metabolism, suggesting the strain's ability to adapt to several environments. Various genes conferring probiotic characteristics, including genes related to stress adaptation to the gastrointestinal tract, biosynthesis of vitamins, cell adhesion and production of bacteriocins, were identified. The CAZyme analysis detected 98 genes distributed under five CAZymes classes. In addition, several genes encoding carbohydrate transport and metabolism were identified. The genome also revealed the presence of insertion sequences, genomic islands, phage regions, CRISPR-cas regions, and the absence of virulence and toxin genes. However, the presence of hemolysin and antibiotic-resistance-related genes detected in the KEGG search needs further experimental validation to confirm the safety of the strain. The presence of two bacteriocin clusters, sactipeptide and plantaricin J, as detected by the BAGEL 4 webserver, confer the higher antimicrobial potential of DJF10. Altogether, the analyses in this study performed highlight this strain's functional characteristics. However, further in vitro and in vivo studies are required on the safety assurance and potential application of L. plantarum DJF10 as a probiotic agent.

Broad-spectrum antimicrobial activity of cinnamoyl esterase-producing Lactobacilli and their application in fermented rice bran.

BACKGROUND: Cinnamoyl esterase (CE) can release antioxidant phenolic acids from its non-digestible ester-linked form. Fermentation using CE-producing lactic acid bacteria (LAB) can be useful in the food industry because of its ability to produce bioactive compounds and antibacterial metabolites. The purpose of this study was to confirm the food applicability of LAB with CE-producing ability and broad-spectrum antibacterial activity. RESULTS: Among the 219 bacterial strains identified in infant feces, five Lactobacillus gasseri and six Limosilactobacillus fermentum with a high CE activity were isolated. The survival rate of all selected LABs was > 95% at pH 2.5 for 3 h and > 70% when treated with 0.3% bile salt for 4 h. Moreover, cell-free supernatants of all strains strongly inhibited five food-borne bacterial pathogens (Listeria monocytogenes, Salmonella enterica, Escherichia coli O157:H7, Bacillus cereus, and Staphylococcus aureus) and three toxin-producing fungal pathogens (Aspergillus niger, Penicillium sp., and Fusarium oxysporum). To improve phenolic acid content and rice bran preservation, Limosilactobacillus fermentum J2 with the strongest CE activity and Lactobacillus gasseri N2 with the strongest antibacterial activity were used in rice bran fermentation, respectively. FRB-J2 (fermented rice bran with Limosilactobacillus fermentum J2) and FRB-N2 (fermented rice bran with Lactobacillus gasseri N2) significantly increased caffeic acid and ferulic acid (P < 0.01). FRB-J2 and FRB-N2 artificially inoculated with F. oxysporum showed no visible fungal growth during the test period (21 days). CONCLUSION: Fermentation by Limosilactobacillus fermentum J2 and Lactobacillus gasseri N2 can help extend the shelf life of rice bran-based products and produce bioactive compounds. © 2021 Society of Chemical Industry.

Effects of Queso Blanco cheese containing Bifidobacterium longum KACC 91563 on fecal microbiota, metabolite and serum cytokine in healthy beagle dogs.

The study describes the effects of administration of Queso Blanco cheese containing Bifidobacterium longum on the fecal microbiota, metabolite and serum cytokine in healthy beagle dogs. Twelve healthy beagle dogs were randomly divided in three groups of four dogs each:a control group, not fed with any cheese, and groups fed with Queso Blanco cheese with B. longum KACC 91563 (QCB) or without B. longum (QC) for 8 weeks. Fecal microbiota was analyzed using a culture-based method and 16s rRNA gene sequencing. Serum cytokine levels, activation of natural killer cells, and proliferation of peripheral blood mononuclear cells were determined. SPME-GC-MS method was used to determine the concentrations of short chain fatty acids and indole in dog feces. Administration of QCB for 4 weeks significantly increased the Bifidobacterium. QCB supplementation for 8 weeks reduceds Enterobacteriaceae and Clostridium perfringens (p < 0.05). The abundance of Fusobacterium, Blautia and Collinesella in QCB group were reduced as compared with the control group. Serum TNF-α and IL-6 levels at 8 weeks significantly increased in QCB group as compared with QC group. There was no change in the concentrations of total short chain fatty acids by B. longum at 0 and 4 weeks. At week 8, the acetic acid, propionic acid and butyric acid of the QCB and QC groups were significantly decreased compared to the control group. In conclusion, our results demonstrate that administration of QCB had positive effects on fecal microbiota and immune response in beagle dogs. We suggest that Queso Blanco cheese containing B. longum KACC 91563 could be used as a functional food for companion animals.

Characterisation of fungal contamination sources for use in quality management of cheese production farms in Korea.

OBJECTIVE: This study was conducted to determine the composition and diversity of the fungal flora at various control points in cheese ripening rooms of 10 dairy farms from six different provinces in the Republic of Korea. METHODS: Floor, wall, cheese board, room air, cheese rind and core were sampled from cheese ripening rooms of ten different dairy farms. The molds were enumerated using YM petrifilm, while isolation was done on yeast extract glucose chloramphenicol agar plates. Morphologically distinct isolates were identified using sequencing of internal transcribed spacer region. RESULTS: The fungal counts in 8 out of 10 dairy farms were out of acceptable range, as per hazard analysis critical control point regulation. A total of 986 fungal isolates identified and assigned to the phyla Ascomycota (14 genera) and Basidiomycota (3 genera). Of these Penicillium, Aspergillus, and Cladosporium were the most diverse and predominant. The cheese ripening rooms was overrepresented in 9 farms by Penicillium (76%), while Aspergillusin a single farm. Among 39 species, the prominent members were Penicillium commune, P. oxalicum, P. echinulatum, and Aspergillus versicolor. Most of the mold species detected on surfaces were the same found in the indoor air of cheese ripening rooms. CONCLUSION: The environment of cheese ripening rooms persuades a favourable niche for mold growth. The fungal diversity in the dairy farms were greatly influenced by several factors (exterior atmosphere, working personnel etc.,) and their proportion varied from one to another. Proper management of hygienic and production practices and air filtration system would be effective to eradicate contamination in cheese processing industries.

Extracellular vesicle-derived protein from Bifidobacterium longum alleviates food allergy through mast cell suppression.

BACKGROUND: The incidence of food allergies has increased dramatically during the last decade. Recently, probiotics have been studied for the prevention and treatment of allergic disease. OBJECTIVE: We examined whether Bifidobacterium longum KACC 91563 and Enterococcus faecalis KACC 91532 have the capacity to suppress food allergies. METHODS: B longum KACC 91563 and E faecalis KACC 91532 were administered to BALB/c wild-type mice, in which food allergy was induced by using ovalbumin and alum. Food allergy symptoms and various immune responses were assessed. RESULTS: B longum KACC 91563, but not E faecalis KACC 91532, alleviated food allergy symptoms. Extracellular vesicles of B longum KACC 91563 bound specifically to mast cells and induced apoptosis without affecting T-cell immune responses. Furthermore, injection of family 5 extracellular solute-binding protein, a main component of extracellular vesicles, into mice markedly reduced the occurrence of diarrhea in a mouse food allergy model. CONCLUSION: B longum KACC 91563 induces apoptosis of mast cells specifically and alleviates food allergy symptoms. Accordingly, B longum KACC 91563 and family 5 extracellular solute-binding protein exhibit potential as therapeutic approaches for food allergies.

Development of a new lactic acid bacterial inoculant for fresh rice straw silage.

OBJECTIVE: Effects of newly isolated Lactobacillus plantarum on the fermentation and chemical composition of fresh rice straw silage was evaluated in this study. METHODS: Lactic acid bacteria (LAB) from good crop silage were screened by growing them in MRS broth and a minimal medium with low carbohydrate content. Selected LAB (LAB 1821) were Gram-positive, rods, catalase negative, and were identified to be Lactobacillus plantarum based on their biochemical characteristics and a 16S rRNA analysis. Fresh rice straw was ensiled with two isolated LAB (1821 and 1841), two commercial inoculants (HM/F and P1132) and no additive as a control. RESULTS: After 2 months of storage at ambient temperature, rice straw silages treated with additives were well-preserved, the pH values and butyric and acetic acid contents were lower, and the lactic acid content and lactic/acetic acid ratio were higher than those in the control (p<0.05). Acidity (pH) was lowest, and lactic acid highest, in 1821-treated silage (p<0.05). The NH3-N content decreased significantly in inoculant-treated silage (p<0.05) and the NH3-N content in 1821-treated silage was lowest among the treatments. The dry matter (DM) content of the control silage was lower than that of fresh rice straw (p<0.05), while that of the 1841- and p1174-inoculant-treated silages was significantly higher than that of HM/F-treated silage. Microbial additives did not have any significant (p>0.05) effect on acid detergent fiber or neutral detergent fiber contents. Crude protein (CP) content and in vitro DM digestibility (IVDMD) increased after inoculation of LAB 1821 (p<0.05). CONCLUSION: LAB 1821 increased the CP, IVDMD, lactic acid content and ratio of lactic acid to acetic acid in rice straw silage and decreased the pH, acetic acid, NH3-N, and butyric acid contents. Therefore, adding LAB 1821 improved the fermentation quality and feed value of rice straw silage.

The Immune-Stimulating and Anti-Diabetic Effects of Allium hookeri Leaves Grown in a Plant Factory with Artificial Lights in Immunosuppressed Obese C57BL/6 Mice.

We investigated the immune-stimulating and anti-diabetic effects of Allium hookeri leaves grown in a plant factory with artificial lights. The immunomodulatory effects of A. hookeri leaves' ethanol extracts were evaluated with immune-related hematological factors in blood, the proliferation of splenocytes, NK cell activity, IgG and cytokine levels, and their mechanisms in immunosuppressed obese mice. Anti-diabetic effects were determined by the inhibitory activity against α-amylase and α-glucosidase in vitro and fasting blood glucose levels and biochemical factors in the serum of immunosuppressed obese mice. A. hookeri leaf extracts increased WBC and LYM counts, the proliferation of splenocytes, and serum IgG and IL-1ß concentrations compared to those of the NC group, which was used as a negative control. A. hookeri leaf extracts also improved serum HDL levels while they decreased the activities of digestive enzymes, fasting blood glucose, and biochemical factors (ALT, AST, T-Chol, TG, LDL, and GLU). The expressions of IL-1ß, JNK, c-Jun, p65, and iNOS in the thymus of immunosuppressed mice were activated by the treatment of A. hookeri leaf extracts. The results suggest that A. hookeri leaves grown in a plant factory with artificial lights also have immune-stimulatory and anti-diabetic effects and can be used as novel functional supplements to control related diseases and to improve public health.

Simple Purification and Antimicrobial Properties of Bacteriocin-like Inhibitory Substance from Bacillus Species for the Biopreservation of Cheese.

Bacteriocins may be used as natural preservatives and antibiotic substitutes in various foods. However, the multistep purification process of bacteriocins results in high production costs, which is an obstacle to their commercial use and consumer accessibility. In this study, a bacteriocin-like inhibitory substance (BLIS) from Bacillus spp. isolated from Korean fermented foods was partially purified using the aqueous two-phase system (ATPS). The maximum activity of the BLIS was achieved for ATPS composed of PEG 1000 (15% [w/w])/ammonium sulfate (20% [w/w])/sodium chloride (2% [w/w]), which caused BLIS activity to increase by 3 times with a 99% recovery rate. In particular, B. amyloliquefaciens Y138-6 BLIS exhibited broad antibacterial activity, high resistance to acid-base stress, and excellent thermal stability. This antibacterial substance inhibited the growth of aerobic bacteria and fungi on the walls of cheese and ripening rooms. These antibacterial properties have been shown to increase food safety and have the potential for use as biopreservatives. Moreover, considering that the execution of the ATPS requires only salts and PEG, it is a simple, environmentally friendly, and cost-effective process and may have industrial applications in the recovery of BLIS from fermentation broth.

Genome sequence of Lactobacillus mucosae LM1, isolated from piglet feces.

Lactobacillus mucosae LM1, isolated from stool samples of a healthy piglet, displays good in vitro mucin adhesion and antimicrobial activity against pathogenic bacteria. To elucidate its antimicrobial effects and to find its epithelial cell and mucin adhesion genes, the genomic sequence of L. mucosae LM1 was investigated.

Development of Desiccation-Tolerant Probiotic Biofilms Inhibitory for Growth of Foodborne Pathogens on Stainless Steel Surfaces.

Lactic acid bacteria biofilms can be used to reduce foodborne pathogen contamination in the food industry. However, studies on growth inhibition of foodborne pathogens by inducing biofilm formation of antagonistic microorganisms on abiotic surfaces are rare. We developed a desiccation-tolerant antimicrobial probiotic biofilm. Lactobacillus sakei M129-1 and Pediococcus pentosaceus M132-2 isolated from fermented Korean foods were found to exhibit broad-spectrum antibacterial activity against Bacillus cereus, Escherichia coli O157:H7, Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica. Their biofilm levels were significantly (p < 0.05) higher on stainless steel than on polyethylene or ceramic. Biofilms of both isolates showed significantly (p < 0.05) enhanced resistance against desiccation (exposure to 43% atmospheric relative humidity) as compared with the isolates not in the biofilm form. The antimicrobial activity of the isolates was sustained in dried biofilms on stainless steel surface; the initial number of foodborne pathogens (average 7.0 log CFU/mL), inoculated on stainless steel chips containing L. sakei M129-1 or P. pentosaceus M132-2 biofilm decreased to less than 1.0 log CFU within 48 h. The lactic acid bacteria antibacterial biofilms developed in this study may be applied to desiccated environmental surfaces in food-related environments to improve microbiological food safety.

Identification and Characterization of a Bacteriocin from the Newly Isolated Bacillus subtilis HD15 with Inhibitory Effects against Bacillus cereus.

Natural antimicrobial substances are needed as alternatives to synthetic antimicrobials to protect against foodborne pathogens. In this study, a bacteriocin-producing bacterium, Bacillus subtilis HD15, was isolated from doenjang, a traditional Korean fermented soybean paste. We sequenced the complete genome of B. subtilis HD15. This genome size was 4,173,431 bp with a G + C content of of 43.58%, 4,305 genes, and 4,222 protein-coding genes with predicted functions, including a subtilosin A gene cluster. The bacteriocin was purified by ammonium sulfate precipitation, Diethylaminoethanol-Sepharose chromatography, and Sephacryl gel filtration, with 12.4-fold purification and 26.2% yield, respectively. The purified protein had a molecular weight of 3.6 kDa. The N-terminal amino acid sequence showed the highest similarity to Bacillus subtilis 168 subtilosin A (78%) but only 68% similarity to B. tequilensis subtilosin proteins, indicating that the antimicrobial substance isolated from B. subtilis HD15 is a novel bacteriocin related to subtilosin A. The purified protein from B. subtilis HD15 exhibited high antimicrobial activity against Listeria monocytogenes and Bacillus cereus. It showed stable activity in the range 0-70°C and pH 2-10 and was completely inhibited by protease, proteinase K, and pronase E treatment, suggesting that it is a proteinaceous substance. These findings support the potential industrial applications of the novel bacteriocin purified from B. subtilis HD15.

Genome sequence of Lactobacillus johnsonii PF01, isolated from piglet feces.

Lactobacillus johnsonii PF01, an autochthonous bacterium of the gastrointestinal tract, was isolated from a fecal sample from a piglet. The strain adhered specifically to the duodenal and jejunal epithelial cells of the piglet and had high bile resistance activity. Here we report the genomic sequence of L. johnsonii PF01.

Complete genome sequence of Bifidobacterium longum subsp. longum KACC 91563.

Bifidobacterium longum strains predominate in the colonic microbiota of breast-fed infants. Here we report the complete genome sequence of B. longum subsp. longum KACC 91563, isolated from feces of neonates. A single circular chromosome of 2,385,301 bp contains 1,980 protein-coding genes, 56 tRNA genes, and 3 rRNA operons.

Genome sequence of Lactobacillus salivarius GJ-24, a probiotic strain isolated from healthy adult intestine.

The draft genome sequence of Lactobacillus salivarius GJ-24 isolated from the feces of healthy adults was determined. Its properties, including milk fermentation activity and bacteriocin production, suggest its potential uses as a probiotic lactic acid bacterium and start culture for dairy products.

Genome sequence of Lactobacillus salivarius NIAS840, isolated from chicken intestine.

Lactobacillus salivarius is a well-known lactic acid bacterium to which increasing attention has been paid recently for use as probiotics for humans and animals. L. salivarius NIAS840 was first isolated from broiler chicken feces, displaying antimicrobial activities against multidrug-resistant Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Here, we report the genome sequence of L. salivarius NIAS840 (2,046,557 bp) including a small plasmid and two megaplasmids.

Inactivation of Listeria monocytogenes on agar and processed meat surfaces by atmospheric pressure plasma jets.

An apparatus for generating atmospheric pressure plasma (APP) jet was used to investigate the inactivation of Listeria monocytogenes on the surface of agar plates and slices of cooked chicken breast and ham. He, N2 (both 7 L/min), and mixtures of each with O2 (0.07 L/min) were used to produce the plasma jets. After treatment for 2 min with APP jets of He, He + O2, N2, or N2 + O2, the numbers of L. monocytogenes on agar plates were reduced by 0.87, 4.19, 4.26, and 7.59 log units, respectively. Similar treatments reduced the L. monocytogenes inoculated onto sliced chicken breast and ham by 1.37 to 4.73 and 1.94 to 6.52 log units, respectively, according to the input gas used with the N2 + O2 mixture being the most effective. Most APP jets reduced the numbers of aerobic bacteria on the meat surfaces to <10² CFU/g, and the numbers remained below that level of detection after storage at 10 °C for 7 days. The results indicate that APP jets are effective for the inactivation of L. monocytogenes on sliced meats and for prolonging the shelf-life of such foods.

Quantitative Analysis of Biogenic Amines in Different Cheese Varieties Obtained from the Korean Domestic and Retail Markets.

To evaluate the safety and risk assessment of cheese consumption in the Republic of Korea, sixty cheese samples purchased from the farmstead and retails markets (imported) were analyzed for their biogenic amine (BA) contents. The BA profiles and quantities of eight amines (tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) were determined using high-performance liquid chromatography (HPLC). Spermine was the only amine detectable in all the samples. The BAs of fresh cheeses from both farmstead and retail markets were mostly undetectable, and comparatively at lower levels (<125 mg/kg) than ripened samples. Putrescine was undetectable in all the domestic ripened cheeses. The sum of BA levels in the imported ripened cheeses of Pecorino Romano (1889.75 mg/kg) and Grana Padano (1237.80 mg/kg) exceeds >1000 mg/kg, of which histamine accounts nearly 86 and 77% of the total levels, respectively. The tolerable limits of the potential toxic amines, histamine and tyramine surpassed in four and three imported ripened samples, respectively. Furthermore, the presence of potentiators (putrescine and cadaverine) together in samples even with a lower level of toxic amines alarms the risk in consumption. Therefore, adoption of strict hygienic practices during the entire chain of cheese production, along with obligatory monitoring and regulation of BA in cheeses seems to be mandatory to ensure the safety of the consumers.

Rapid Discrimination and Authentication of Korean Farmstead Mozzarella Cheese through MALDI-TOF and Multivariate Statistical Analysis.

Geographical origin and authenticity are the two crucial factors that propel overall cheese perception in terms of quality and price; therefore, they are of great importance to consumers and commercial cheese producers. Herein, we demonstrate a rapid, accurate method for discrimination of domestic and import mozzarella cheeses in the Republic of Korea by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The protein profiles' data aided by multivariate statistical analysis successfully differentiated farmstead and import mozzarella cheeses according to their geographical location of origin. A similar investigation within domestic samples (farmsteads/companies) also showed clear discrimination regarding the producer. Using the biomarker discovery tool, we identified seven distinct proteins, of which two (m/z 7407.8 and 11,416.6) were specific in farmstead cheeses, acting as potential markers to ensure authentication and traceability. The outcome of this study can be a good resource in building a database for Korean domestic cheeses. This study also emphasizes the combined utility of MALDI-TOF MS and multivariate analysis in preventing fraudulent practices, thereby ensuring market protection for Korean farmstead cheeses.

Pig Skin Gelatin Hydrolysates Attenuate Acetylcholine Esterase Activity and Scopolamine-induced Impairment of Memory and Learning Ability of Mice.

The protective effect of pig skin gelatin water extracts (PSW) and the low molecular weight hydrolysates of PSW generated via enzymatic hydrolysis with Flavourzyme® 1000L (LPSW) against scopolamine-induced impairment of cognitive function in mice was determined. Seventy male ICR mice weighing 20-25 g were randomly assigned to seven groups: Control (CON); scopolamine (SCO, 1 mg/kg B.W., intraperitoneally (i.p.); tetrahydroaminoacridine 10 [THA 10, tacrine; 10 mg/kg B.W. per oral (p.o.) with SCO (i.p.)]; PSW 10 (10 mg/kg B.W. (p.o.) with SCO (i.p.); PSW 40 (40 mg/kg B.W. (p.o.) with SCO (i.p.); LPSW 100 (100 mg/kg B.W. (p.o.) with SCO (i.p.); LPSW 400 (400 mg/kg B.W. (p.o.) with SCO (i.p.). All treatment groups, except CON, received scopolamine on the day of the experiment. The oxygen radical absorbance capacity of LPSW 400 at 1 mg/mL was 154.14 µM Trolox equivalent. Administration of PSW and LPSW for 15 weeks did not significantly affect on physical performance of mice. LPSW 400 significantly increased spontaneous alternation, reaching the level observed for THA and CON. The latency time of animals receiving LPSW 400 was higher than that of mice treated with SCO alone in the passive avoidance test, whereas it was shorter in the water maze test. LPSW 400 increased acetylcholine (ACh) content and decreased ACh esterase activity (p<0.05). LPSW 100 and LPSW 400 reduced monoamine oxidase-B activity. These results indicated that LPSW at 400 mg/kg B.W. is a potentially strong antioxidant and contains novel components for the functional food industry.

1H HRMAS-NMR based metabolic fingerprints for discrimination of cheeses based on sensory qualities.

In this study, the 1H HRMAS-NMR (High-resolution Magic Angle Spinning-Nuclear Magnetic Resonance) spectra of 52 cheese samples obtained from the South Korean dairy farms were evaluated for their metabolic profiling and intensities associating with the sensory qualities. The NMR profiles displayed a broad range of compounds comprising amino acids, carbohydrates, organic acids, and phospholipids. Afterwards, the cheese samples were categorized into three groups (more likeness - G1, moderate likeness - G2, less likeness - G3), in relating to their sensory scores. The NMR data of the samples were later investigated through multivariate statistical tools to define the variations in metabolic fingerprints of every cheese sample and their intensities hailing in individual sensory groups. The unsupervised PCA employing all cheese samples unveiled the uniqueness in metabolite profiles of the brown and cheddar type cheeses (outliers). Moreover, Gouda and other types of cheeses displayed samples positioning in respective of their metabolite profiles. The pairwise comparison of sensory groups in the supervised models perceived better separation in OPLS-DA than PLS-DA. The corresponding VIP (PLS-DA) and loading (OPLS-DA) plots revealed amino acids and organic acids (lactate, citrate) as significant variables. The discrimination of G 1 Gouda type of cheeses against G 2 and G 3 was highly associated with their citrate levels. Further investigation using heatmaps displayed clear differentiation between each sensory group in terms of the levels of amino acids, lactate, citrate, phospholipids, and glycerol, conveying these variations are likely due to proteolytic and metabolic processes in cheese ripening. This study concluded that 1H HRMAS-NMR metabolite profile of the Korean cheeses is consistence with their sensory qualities. Further, the candidate metabolites identified in this study confers their potential application as biomarkers in cheese industries for faster and effective validation of sensory characteristics.