Vacuum-packaged Meat Products Research Articles

Spoilage Pseudomonas survive common thermal processing schedules and grow in emulsified meat during extended vacuum storage

Some Pseudomonas species are common meat spoilage bacteria that are often associated with the spoilage of fresh meat. The recently reported ability of these bacteria to also spoil cooked and vacuum packaged meat products has created the need to investigate all potential routes of spoilage they may be able to utilize. The objective of this experiment was to determine if spoilage Pseudomonas spp. survive thermal processing and grow during refrigerated storage under vacuum. Pseudomonas spp. isolates collected from spoiled turkey products were inoculated into a salted and seasoned meat emulsion that was vacuum sealed and thermally treated to final temperatures of 54.4 and 71.1°C to mimic thermal processes commonly used in the meat industry. Samples were stored for a total of 294 days at 4 and 10°C and plated using Pseudomonas spp. specific agar plates. Pseudomonas spp. concentrations were below the detection limit (0.18 log10 CFU/g) immediately after thermal processing and were first recovered from thermally processed samples after 14 days of storage. The final concentration was greater than 2 log10 CFU/g (p<0.05 compared to post-thermal processing) in thermally processed treatment groups at the end of storage, indicating that these Pseudomonas spp. isolates were able to survive thermal processing and grow during extended vacuum storage. This raises concerns about the ability of spoilage bacteria to survive the thermal processing schedules commonly used in the meat industry and confirms that some Pseudomonas spp. are capable of thriving in products other than aerobically stored fresh meat. Practical Application: Spoilage Pseudomonas spp. can survive traditional thermal processing schedules. Heat resistance should be evaluated for commensal and spoilage bacteria to better understand possible ways spoilage of food products may occur.

Isolation and characterization of the new isolated bacteriophage YZU-L1 against Citrobacter freundii from a package-swelling of meat product

Citrobacter freundii is an important foodborne pathogen that can cause urethritis, bacteremia, necrotizing abscess, and meningitis in infants. In this study, a gas-producing isolate from vacuum-packed meat products was identified as C. freundii by 16S rDNA. In addition, a new virulent phage YZU-L1, which could specifically lyse C. freundii, was isolated from sewage samples in Yangzhou. Transmission electron microscopy showed that phage YZU-L1 had a polyhedral head of 73.51 nm in diameter and a long tail of 161.15 nm in length. According to phylogenetic analysis employing the terminase large subunit, phage YZU-L1 belonged to the Demerecviridae family and the Markadamsvirinae subfamily. The burst size was 96 PFU/cell after 30 min of latent period and 90 min of rising period. Phage YZU-L1 could maintain high activity at pH of 4–13, and resist 50 °C for up to 60 min. The complete genome of YZU-L1 was 115,014 bp double-stranded DNA with 39.94% G + C content, encoding 164 open reading frames (ORFs), without genes encoding for virulence, antibiotic resistance, or lysogenicity. Phage YZU-L1 treatment significantly reduced the viable bacterial count of C. freundii in a sterile fish juice model, which is expected to be a natural agent for the biocontrol of C. freundii in foods.

Diagnostic capabilities of modern methods of determination pathogenic listeria in meat products

Annual reports on the morbidity of the adult population report outbreaks of listeriosis caused by Listeria monocytogenes with clinically pronounced febrile gastroenteritis. The main transmission route to humans is believed to be through consumption of contaminated food, especially ready-to-eat meat products. Rapid and specific detection of L. monocytogenes in food is critical for ensuring the safety of consumers. The aim of this work was to study the occurrence of L. monocytogenes in vacuum-packed meat products using modern diagnostic methods. In total, 60 vacuum-packed meat products were studied using real-time PCR (iQ-Check™ realtime PCR kit), immunoassay (VIDAS LMO2 test) and culturing. Thirty-two of 60 (53%) vacuum-packed meat products were L. monocytogenes positive by real-time PCR. The occurrence was especially high in heat-treated sausages (80%). Only two products (two bacons) were VIDAS and culture positive. Additionally, L. monocytogenes was isolated from one heat-treated sausage, which was VIDAS negative. All VIDAS and culture-positive samples were also PCR positive. In two (12%) out of 17 vacuum-packed ready-to-eat products, the number of L. monocytogenes was over 100 cfu/g demonstrating that the shelf life assigned to some vacuum-packed products is not appropriate. Real-time PCR based on iQ-Check™ realtime PCR kit was shown to be very sensitive to detect L. monocytogenes in foods and thus a useful tool for screening.

Genome collection of Shewanella spp. isolated from spoiled lamb.

The diversity of the genus Shewanella and their roles across a variety of ecological niches is largely unknown highlighting the phylogenetic diversity of these bacteria. From a food safety perspective, Shewanella species have been recognized as causative spoilage agents of vacuum-packed meat products. However, the genetic basis and metabolic pathways for the spoilage mechanism are yet to be explored due to the unavailability of relevant Shewanella strains and genomic resources. In this study, whole-genome sequencing of 32 Shewanella strains isolated from vacuum-packaged refrigerated spoiled lamb was performed to examine their roles in meat spoilage. Phylogenomic reconstruction revealed their genomic diversity with 28 Shewanella spp. strains belonging to the same putative novel species, two Shewanella glacialipiscicola strains (SM77 and SM91), Shewanella xiamenensis NZRM825, and Shewanella putrefaciens DSM 50426 (ATCC 8072) isolated from butter. Genome-wide clustering of orthologous gene families revealed functional groupings within the major Shewanella cluster but also considerable plasticity across the different species. Pan-genome analysis revealed conserved occurrence of spoilage genes associated with sulfur and putrescine metabolism, while the complete set of trimethylamine metabolism genes was observed in only Shewanella sp. SM74, S. glacialipiscicola SM77 and SM91 strains. Through comparative genomics, some variations were also identified pertaining to genes associated with adaptation to environmental cues such as temperature, osmotic, salt, oxidative, antimicrobial peptide, and drug resistance stresses. Here we provide a reference collection of draft Shewanella genomes for subsequent species descriptions and future investigations into the molecular spoilage mechanisms for further applications in the meat industry.

Surface Color Variations of Ground Beef Packaged Using Enhanced, Recycle Ready, or Standard Barrier Vacuum Films.

With current meat industry efforts focused on improving environmental influencers, adopting sustainable packaging materials may be an easier transition to addressing the sustainability demands of the meat consumer. With the growing popularity of vacuum-packaged meat products, the current study evaluated instrumental surface color on fresh ground beef using vacuum packaging films, recycle-ready film (RRF), standard barrier (STB) and enhanced barrier (ENB). Ground beef packaged using ENB barrier film was lighter (L*), redder (a*) and more vivid (chroma) than all other packaging treatments during the simulated display period (p < 0.05). By day 12 of the simulated retail display, the ground beef surface color became lighter (L*), more yellow (b*), less red (a*), less vivid (chroma) and contained greater forms of calculated metmyoglobin, oxymyoglobin (p < 0.05). The current results suggest that barrier properties of vacuum packaging film for ground beef are pivotal for extending the surface color during fresh shelf-life conditions.

Comparative genomics of Clostridium species associated with vacuum-packed meat spoilage

Bacterial species belonging to the genus Clostridium have been recognized as causative agents of blown pack spoilage (BPS) in vacuum packed meat products. Whole-genome sequencing of six New Zealand psychrotolerant clostridia isolates derived from three meat production animal types and their environments was performed to examine their roles in BPS. Comparative genome analyses have provided insight into the genomic diversity and physiology of these bacteria and divides clostridia into two separate species clusters. BPS-associated clostridia encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) that enable them to utilize the intramuscular carbohydrate stores and facilitate sporulation. In total, 516 glycoside hydrolases (GHs), 93 carbohydrate esterases (CEs), 21 polysaccharide lyases (PLs), 434 glycosyl transferases (GTs) and 211 carbohydrate-binding protein modules (CBM) with predicted activities involved in the breakdown and transport of carbohydrates were identified. Clostridia genomes have different patterns of CAZyme families and vary greatly in the number of genes within each CAZy category, suggesting some level of functional redundancy. These results suggest that BPS-associated clostridia occupy similar environmental niches but apply different carbohydrate metabolism strategies to be able to co-exist and cause meat spoilage.

A mathematical modeling approach to the quantification of lactic acid bacteria in vacuum-packaged samples of cooked meat: Combining the TaqMan-based quantitative PCR method with the plate-count method

The TaqMan-based quantitative Polymerase Chain Reaction (qPCR) method and the Plate Count (PC) method are both used in combination with primary and secondary mathematical modeling, to describe the growth curves of Leuconostoc mesenteroides and Weissella viridescens in vacuum-packaged meat products during storage under different isothermal conditions. Vacuum-Packaged Morcilla (VPM), a typical cooked blood sausage, is used as a representative meat product, with the aim of improving shelf-life prediction methods for those sorts of meat products. The standard curves constructed by qPCR showed good linearity between the cycle threshold (CT) and log10 CFU/g, demonstrating the high precision and the reproducible results of the qPCR method. The curves were used for the quantification of L. mesenteroides and W. viridescens in artificially inoculated VPM samples under isothermal storage (5, 8, 13 and 18 °C). Primally, both the qPCR and the PC methods were compared, and a linear regression analysis demonstrated a statistically significant linear correlation between the methods. Secondly, the Baranyi and Roberts model was fitted to the growth curve data to estimate the kinetic parameters of L. mesenteroides and W. viridescens under isothermal conditions, and secondary models were used to establish the dependence of the maximum specific growth rate on the temperature. The results proved that primary and secondary models were adequate for describing the growth curves of both methods in relation to both bacteria. In conclusion, the results of all the experiments proved that the qPCR method in combination with the PC method can be used to construct microbial growth kinetics and that primary and secondary mathematical modeling can be successfully applied to describe the growth of L. mesenteroides and W. viridescens in vacuum-packaged morcilla and, by extension, other cooked meat products with similar characteristics.

Modelling the growth of lactic acid bacteria at different temperatures

ABSTRACT Mathematical models are widely used to predict the shelf life of foods. Lactic acid bacteria (LAB), particularly Lactobacillus plantarum, Weissella viridescens and Lactobacillus sakei, are the main spoilage bacteria of refrigerated, vacuum-packed meat products, stored in modified atmosphere, and their growth determines the shelf life length of these products. The objective of this study was to model the growth of L. plantarum, W. viridescens and L. sakei under different isothermal cultivation conditions and establish secondary models to describe the effect of temperature on the growth parameters of these bacteria. The LAB growth was evaluated in culture medium at temperatures of 4, 8, 12, 16, 20 and 30 oC. The fit of Baranyi and Roberts (BAR) and Gompertz (GO) primary models to the growth curves of LAB was compared by statistical indices, in which the BAR model showed slightly better fits to the experimental data. The BAR growth parameters were used to establish the secondary models, µmax and Nmax were established for the three LAB. The power model described the influence of temperature on the parameter λ for L. plantarum, and other bacteria showed no lag phase. The growth of LAB was strongly influenced by storage temperature and the obtained models allow predicting the growth of these bacteria within the temperature range from 4 to 30 oC.

Bacterial evaluation of vacuum packaged meat products

Bacterial evaluation of vacuum packaged meat products

Modeling the growth of Lactobacillus viridescens under non-isothermal conditions in vacuum-packed sliced ham

Lactic acid bacteria (LAB) are responsible for spoiling vacuum-packed meat products, such as ham. Since the temperature is the main factor affecting the microbial dynamic, the use of mathematical models describing the microbial behavior into a non-isothermal environment can be very useful for predicting food shelf life. In this study, the growth of Lactobacillus viridescens was measured in vacuum-packed sliced ham under non-isothermal conditions, and the predictive ability of primary (Baranyi and Roberts, 1994) and secondary (Square Root) models were assessed using parameters estimated in MRS culture medium under isothermal conditions (between 4 and 30°C). Fresh ham piece was sterilized, sliced, inoculated, vacuum-packed, and stored in a temperature-controlled incubator at five different non-isothermal conditions (between 4 and 25°C) and one isothermal condition (8°C). The mathematical models obtained in MRS medium were assessed by comparing predicted values with L. viridescens growth data in vacuum-packed ham. Its predictive ability was assessed through statistical indexes, with good results (bias factor between 0.95 and 1.03; accuracy factor between 1.04 and 1.07, and RMSE between 0.76 and 1.33), especially in increasing temperature, which predictions were safe. The model parameters obtained from isothermal growth data in MRS medium enabled to estimate the shelf life of a commercial ham under non-isothermal conditions in the temperature range analyzed.

Predicting Growth of Weissella Viridescens in Culture Medium under Dynamic Temperature Conditions

The lactic acid bacteria (LAB) are among the main spoilage microorganisms of foods, and the Weissella viridescens (formerly Lactobacillus viridescens) is well known to cause deterioration on the meat surface in vacuum packed meat products, even under refrigerated conditions. Therefore, this study evaluated the predictive ability of Baranyi and Roberts dynamic model to describe W. viridescens growth in culture medium (which simulates a food rich in nutrients), subjected to dynamic temperature conditions. Baranyi and Roberts primary model was fitted to the growth curves of W. viridescens in culture medium under six different isothermal temperatures (4, 8, 12, 16, 20 and 30°C) previously obtained in our laboratory. Four secondary models (linear, square root, exponential and Arrhenius type) were assessed to describe the influence of temperature on the growth parameters. The square root was the best model to describe temperature influence on μmax parameter. For Ymax parameter, the secondary model was considered the mean values obtained experimentally in the studied temperature range. Experimental data were used to evaluate the model predictions under dynamic conditions for two different temperature profiles, NIP-1 (12-16-20-25°C) and NIP-2 (16-12-8-4°C). According to the statistical indexes, the model showed better predictive ability for NIP-1, with RMSE of 0.3341, R2 of 0.9939, bias factor of 1.0046 and accuracy factor of 1.0197; the growth of W. viridescens under NIP-2 conditions was underestimated, indicating a fail dangerous prediction. The results showed that the predictive model can be used to predict the shelf life of meat products spoiled by W. viridescens.

Mathematical Modeling of Lactobacillus Viridescens Growth in Vacuum Packed Sliced Ham under non Isothermal Conditions

Lactic acid bacteria (LAB) are responsible for the spoilage of vacuum packed meat products, as ham. Temperature is the main factor affecting the microbial dynamics and its variation during the production, distribution and storage of foods is considerable. Thus, the use of mathematical models to describe the microbial behavior under variable temperatures can be very useful in predicting the food shelf life. This study evaluated the growth of Lactobacillus viridescens in sliced ham under non isothermal conditions, and assessed the predictive ability of the Baranyi and Roberts model using parameters obtained isothermally in culture medium (MRS). To obtain the BAL growth, the fresh ham piece was sterilized, sliced, inoculated with bacteria and stored in a temperature-controlled incubator. For the establishment of the secondary models, the primary model parameters were obtained isothermally in the culture medium at 4, 8, 12, 16, 20 and 30° C, in which there was no lag phase observed; the square root model was selected to describe the dependence of the μmax parameter (maximum specific growth rate) with the temperature, and the ymax parameter (maximum population) was represented by an average because there was no significant influence of the temperature. The mathematical models were validated with L. viridescens growth data in ham under five variable temperature conditions (NI-1 (4-8-12-16°C), NI-2 (12-16-20-25°C), NI-3 (25-20-16-12-8-4°C), NI-4 (16-12-8-4°C) and NI-5 (12-8-4-8-12°C)), and its predictive ability were assessed through statistical indexes (bias factor, accuracy factor and RMSE), with good results (bias factor between 0.9450 and 1.0326; accuracy factor between 1.0382 and 1.0682, and RMSE between 0.7641 and 1.3317), especially in increasing temperature, where the prediction was safe. The validated model can be used to estimate the shelf life of a commercial ham under different temperature conditions.

Detection of C. perfringens toxins in vacuum packaged meat products by using polymerase chain reaction

Detection of C. perfringens toxins in vacuum packaged meat products by using polymerase chain reaction

Anaerobic spore forming bacteria contaminating some vacuum packaged meat products

The present study was designed to determine the anaerobic spore forming bac­ teria contaminating some vacuum packaged meat products. A total of 45samples of Cocktail , Salami and Pepperoni (15 of each) were collected randomly from different su­ permarkets in Alexandria governorate. The obtained results revealed that the mean values oftotal anaerobic and C.peljringens counts were 9.97x1o3 ± 2.58and 5.13x103 ± 1.08 cfu /g) for Cocktail, 4.12x1o3 ± 0.89 and 1.68x1o3 ± 0.41 (cfu /g). for Salami and 1.55x1o3 ± 0.38 and 6.05x102 ± 1.27 (cfu /g)for Pepperoni, respectively . Anaero­ bic spore forming Clostridial spp. which isolated in different percentages from the ex­ amined samples,included C.peifringens, C.absonum, C.bifermentans, C.butyricum, C.histolyticum, C.pasteurinum, C.putrefaciens, C.sordelli, C.sporogenes. Moreover, Le­cithinase activity ofC.perjringens isolatedfrom the examined samples ofvacuum pack­aged meat products indicated that 5 samples out of 9 Cocktail samples contaminated with C.peljringens were lecithinase +ve while the remaining 4 samples were lecithi­nase +ve. while, 4 Salami samples contaminated with C.peljringens were lecithinase +ve and one sample was lecithinase -ve and 2 samples out of 4 Pepperoni samples contaminated with C.peljringens were lecithinase +ve while the remaining 2 samples were lecithinase -ve.Typiri.g of lecithinase +ve of C.peifringens isolated from the exam­ ined samples of vacuum packaged meat products by intradermal inoculation test of Guinea pig was shown type A,C and Din Coclctail,type A,B and C were detected in Sa­ lami and type A and D were detected in Pepperoni with diffpercentages. The public health importance of the isolated organisms were discussed

Screening of Antibacterial Activity of Lactic Acid Bacteria Against Different Pathogens Found in Vacuum-Packaged Meat Products

The objective of this work was to screen the antibacterial activity of lactic acid bacteria (LAB) isolated from different sources against different pathogens found in ready-to-eat vacuum-packaged meat products (RTE-VPMP). LAB were isolated from human, RTE-VPMP, fermented vegetables, and dairy samples. These isolates were assessed for their antibacterial activity against Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus using spot on lawn technique. Six LAB isolates-three from a human source, two from a RTE-VPMP source, and one from a fermented vegetable source-were found to be effective against all pathogenic strains. Antibacterial activities of cell-free neutral supernatant broths of these isolates were assessed against the different pathogenic strains to confirm bacteriocin production. All six isolates were effective against all pathogenic strains. LAB isolates from the human source had the highest antibacterial activity and were significantly more effective than other LAB isolates, with the inhibition zone ranging from 14 to 22 mm. Inhibition zones of RTE-VPMP LAB isolates were lower than those of human origin (inhibition zone range, 11-17 mm). The lowest activities were for the fermented vegetable isolate, for which inhibition zones ranged from 11 to 15 mm. The three isolates of human origin were identified as L. acidophilus, L. casei, and L. reuteri; the two isolates from RTE-VPMP source were both L. sake; and the one isolate of fermented vegetable origin was L. plantarum. Our results showed that nonmeat product-sourced LAB were effective against several foodborne pathogens, which suggests that they could be used as natural biopreservatives in many RTE-VPMP produced in Jordan.

Prevalence and genetic diversity of Listeria monocytogenes in vacuum-packaged ready-to-eat meat products at retail markets in Latvia.

Nine groups of different retail ready-to-eat vacuum-packaged meat products from 10 Baltic meat processing plants were analyzed for presence and numbers of Listeria monocytogenes at the end of shelf life. A total of 38 (18%) of 211 samples tested positive for L. monocytogenes serotype 1/2a (88%) or 1/2c (12%). The prevalence of L. monocytogenes in cold-smoked, sliced, vacuum-packaged beef and pork products (42%) was significantly higher than in cooked, sliced, vacuum-packaged meat products (0.8%) (P < 0.001). Enumeration of L. monocytogenes showed that 84% of the positive samples contained <100 CFU/g upon expiry of product shelf life. The numbers of L. monocytogenes exceeded 100 CFU/g only in cold-smoked, sliced, vacuum-packaged beef products. Identical pulsed-field gel electrophoresis types were recovered from different production lots of cold-smoked vacuum-packaged beef and pork products produced by the same meat processing plant, demonstrating L. monocytogenes contamination as a recurrent problem within one meat processing plant.

Intraspecific Diversity of Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus sakei, and Leuconostoc mesenteroides Associated with Vacuum-Packed Meat Product Spoilage Analyzed by Randomly Amplified Polymorphic DNA PCR

The intraspecific diversity of Leuconostoc mesenteroides, Lactobacillus curvatus, Lactobacillus sakei, and Lactobacillus plantarum was analyzed by randomly amplified polymorphic DNA (RAPD) PCR with universal primers M13 and T3. The study included 100 reference strains and 210 isolates recovered from two vacuum-packed Spanish meat products, fiambre de magro adobado and morcilla, previously identified by rDNA–restriction fragment length polymorphism profiles. The RAPDM13 profiles identified isolates at species level in L. plantarum and L. mesenteroides, while RAPD-T3 provided profiles in L. sakei. The combination of RAPD-M13 and RAPD-T3 fingerprints revealed a total of 17 profiles in L. mesenteroides, 6in L. sakei, 12 in L. plantarum, and6in L. curvatus. Of these, six profiles corresponding to L. mesenteroides and one corresponding to L. sakei were found in both products. The Shannon-Weaver diversity index (H′), calculated according to RAPD-M13 and RAPD-T3 profiles during storage, revealed that most profiles appeared only in single samplings in both products, indicating a high strain substitution rate during chilled storage of vacuum-packed meat products. When bloating appeared, only one profile corresponding to L. mesenteroides subsp. dextranicum was present throughout the storage period.

Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage: population analysis by rDNA-based methods

To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products. A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, 'morcilla' and 'fiambre de magro adobado' obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing. Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum-packed meat products. Prevention of bloating spoilage in vacuum-packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR).

Contamination of some vacuum-packaged meat productswithListeria monocytogenes

In this study, the contamination of vacuum-packaged processed meat products by L. monocytogenes was investigated in the samples collected from retail markets in Van, Turkey. Of the 100 samples studied, 44 (44%) were positive for Listeria spp., while 14 samples (14%) of Listeria spp. isolates were identified to be contaminated by L. monocytogenes. Listeria spp. and L. monocytogenes were detected in 60% and 20% of Turkish fermented sausage samples, 48% and 12% of sausage samples, 40% and 16% of salami samples, and 28% and 8% of pastirma samples, respectively. Because of high level of L. monocytogenes incidence in examined vacuum-packaged meat products, it is essential to make sure that all necessary sanitary requirements be met to avoid any contamination. It is concluded that inadequte cooking practices or raw consumption of these contaminated products may cause potential risks for public health.

Screening of lactic acid bacteria from Brazilian meats for bacteriocin formation

Twenty samples of vacuum-packaged meat products from Brazil were screened for the presence of bacteriocin-producing lactic acid bacteria, using an agar overlay method. Three bacteriocinogenic isolates were obtained and identified as Enterococcus sp. 18 (from bacon), Leuconostoc sp. 20 (from ham) and Lactobacillus sakei 29 (from hot home made “lingüiça”). Leuconostoc sp. 20 and Lactobacillus sakei 29 presented antilisterial activity, inhibiting all the strains of Listeria monocytogenes tested. However, the strains did not inhibit Brochothrix thermosphacta, Enterobacter sp., Salmonella sp. or Escherichia coli.