Flowering phenophases influence the antibacterial and anti-biofilm effects of Thymus vulgaris L. essential oil.

BACKGROUND: Essential oils are becoming increasingly popular in medicinal applications because of their antimicrobial effect. Thymus vulgaris L. (Lamiaceae) is a well-known and widely cultivated medicinal plant, which is used as a remedy for cold, cough and gastrointestinal symptoms. Essential oil content of thyme is responsible for its antimicrobial activity, however, it has been reported that the chemical composition of essential oils influences its biological activity. In order to explore flowering phenophases influence on the chemical composition of thyme essential oil and its antibacterial and anti-biofilm activity, plant materials were collected at the beginning of flowering, in full bloom and at the end of flowering periods in 2019. METHODS: Essential oils from fresh and dried plant materials were distilled and analyzed with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID). The antibacterial activity was performed by broth microdilution and thin layer chromatography-direct bioautography (TLC-DB) assays and the anti-biofilm effect by crystal violet assay, respectively. Scanning electron microscopy was applied to illustrate the cellular changes of bacterial cells after essential oil treatment. RESULTS: Thymol (52.33-62.46%) was the main component in the thyme essential oils. Thyme oil distilled from fresh plant material and collected at the beginning of flowering period exerted the highest antibacterial and anti-biofilm activity against Haemophilus influenzae, H. parainfluenzae and Pseudomonas aeruginosa. CONCLUSION: The different flowering periods of Thymus vulgaris influence the antibacterial and anti-biofilm activity of its essential oils, therefore, the collection time has to be taken into consideration and not only the full bloom, but the beginning of flowering period may provide biological active thyme essential oil.

Activity of Binary Combinations of Natural Phenolics and Synthetic Food Preservatives against Food Spoilage Yeasts.

Natural compounds are a suitable alternative to synthetic food preservatives due to their natural origin and health-promoting properties. In the current study, phenolic-phenolic and phenolic-synthetic combinations were tested for their antibiofilm formation, anti-planktonic growth, and anti-adhesion properties against Debaryomyces hansenii, Wickerhamomyces anomalus (formerly Pichia anomala), Schizosaccharomyces pombe, and Saccharomyces cerevisiae. The phenolics were vanillin and cinnamic acid, while the synthetic preservatives were sodium benzoate, potassium sorbate, and sodium diacetate. The vanillin-cinnamic acid combination had synergistic effect in all the tested yeasts for the biofilm inhibition with a fractional inhibitory concentration index (FICI) of ≤0.19 for W. anomalus, 0.25 for S. pombe, 0.31 for S. cerevisiae, and 0.5 for D. hansenii. Most of the phenolic-synthetic combinations had indifferent interaction regarding biofilm formation. The vanillin-cinnamic acid combination also had higher activity against spoilage yeasts adhesion on the abiotic surface and planktonic growth compared to the phenolic-synthetic combinations. For the phenolic-synthetic anti-planktonic activity, synergistic interaction was present in all the vanillin-synthetic combinations in S. pombe, vanillin-sodium benzoate and vanillin-potassium sorbate in S. cerevisiae, vanillin-sodium benzoate in W. anomalus, and cinnamic acid-sodium diacetate in S. pombe. These results suggest a novel antimicrobial strategy that may broaden the antimicrobial spectrum and reduce compound toxicity against food spoilage yeasts.

Eradication of multiple-species biofilms from food industrial and domestic surfaces using essential oils.

Microbial biofilm formation represents a serious problem for both food industry and households. Natural biofilms are formed mostly by multiple species, and show resistance against most of the usual sanitizers. In this study, the effects of cinnamon (Cinnamomum zeylanicum), marjoram (Origanum majorana) and thyme (Thymus vulgaris) essential oils (EOs) and their main components (cinnamaldehyde, terpinene-4-ol, and thymol) were investigated on four-species biofilms of Escherichia coli, Listeria monocytogenes, Pseudomonas putida and Staphylococcus aureus. Minimum bactericide concentration (MBC) and killing time were determined by means of the microdilution method. MBC of the investigated EOs and components was between 0.5 mg/mL (cinnamaldehyde) to 25 mg/mL (terpinene-4-ol). Killing times for the four-species suspension were 5 or 10 min, time spans usable in the food industry. For eradication of the mixed-population biofilm from stainless steel (SS), polypropylene (PP), tile and wood surfaces, EO- or EO component-based disinfectant solutions were developed, and their effects were compared to a peracetic acid-based industrial sanitizer (HC-DPE). Total eradication of biofilms (99.9%) was achieved, with solutions containing cinnamon and thyme EO and EO components, from SS and PP, but not from tile or wood surfaces. Apparently, cinnamon EO, terpinene-4-ol and thymol have better disinfectant activity than HC-DPE.

Immortelle (Helichrysum italicum (Roth) G. Don) Essential Oil Showed Antibacterial and Biofilm Inhibitory Activity against Respiratory Tract Pathogens.

The biofilm formation of bacteria in different parts of the human body can influence the success of antibiotic therapy. Essential oils (EOs) and their components are becoming increasingly popular in point of view of medicinal applications, because of their antibacterial efficacy. The immortelle EO has been used traditionally as an expectorant; however, there are no studies summarizing its antibacterial effect against respiratory tract bacteria. Our aim was to investigate the antibacterial and biofilm inhibitory activity of immortelle (Helichrysum italicum) EO against respiratory tract pathogens such as Haemophilus influenzae, H. parainfluenzae, Pseudomonas aeruginosa and Streptococcus pneumoniae. In order to prove the antibacterial effect of the immortelle EO, broth microdilution and biofilm inhibition tests, and membrane damage assay were investigated. Scanning electron microscopy was used to identify the structural modifications in bacterial cells. Our results showed that immortelle EO has antibacterial and anti-biofilm effects against respiratory tract bacteria used in this study. H. parainfluenzae was the most sensitive to each treatment, however, P. aeruginosa was the most resistant bacteria. In conclusion, the studied EO may have a role in the treatment of respiratory tract infections due to their antibacterial and anti-biofilm activity.

Biodegradation of two persistent aromatic compounds by using oil shale.

Low-cost oil shale was investigated as a biodegradation promoter material, in order to exploit its potential for more widespread and efficient usage in the elimination of pollution. Degradation of two model pollutants, 4-nitrophenol and phenol, was examined in the presence of oil shale in a batch system. In order to investigate the role of the natural microflora of the oil shale in degradation, sodium azide was added to inhibit microbial growth. The effect of metal ions was also investigated. In the sodium azide-free solutions the model pollutants were completely degraded up to 2000 µmol/L concentration in a dose-dependent way, while the addition of sodium azide delayed greatly but did not stop the degradation. Manganese(II) ions increased the rate of the degradation of 4-nitrophenol, and given quantities of iron(II), manganese(II) or zinc(II) ions were also effective in degradation of phenol. Our results suggest that oil shale is not only an adsorbent but has an active role in the degradation of pollutants by its natural microflora. Utilizing these features of oil shale, it is a suitable candidate as an ameliorating agent, which can also be used in industrial size.

In Vitro Activity of Selected Phenolic Compounds against Planktonic and Biofilm Cells of Food-Contaminating Yeasts.

Phenolic compounds are natural substances that can be obtained from plants. Many of them are potent growth inhibitors of foodborne pathogenic microorganisms, however, phenolic activities against spoilage yeasts are rarely studied. In this study, planktonic and biofilm growth, and the adhesion capacity of Pichia anomala, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Debaryomyces hansenii spoilage yeasts were investigated in the presence of hydroxybenzoic acid, hydroxycinnamic acid, stilbene, flavonoid and phenolic aldehyde compounds. The results showed significant anti-yeast properties for many phenolics. Among the tested molecules, cinnamic acid and vanillin exhibited the highest antimicrobial activity with minimum inhibitory concentration (MIC) values from 500 µg/mL to 2 mg/mL. Quercetin, (-)-epicatechin, resveratrol, 4-hydroxybenzaldehyde, p-coumaric acid and ferulic acid were also efficient growth inhibitors for certain yeasts with a MIC of 2 mg/mL. The D. hansenii, P. anomala and S. pombe biofilms were the most sensitive to the phenolics, while the S. cerevisiae biofilm was quite resistant against the activity of the compounds. Fluorescence microscopy revealed disrupted biofilm matrix on glass surfaces in the presence of certain phenolics. Highest antiadhesion activity was registered for cinnamic acid with inhibition effects between 48% and 91%. The active phenolics can be natural interventions against food-contaminating yeasts in future preservative developments.

Plant Phenolics and Phenolic-Enriched Extracts as Antimicrobial Agents against Food-Contaminating Microorganisms.

Phenolic compounds and extracts with bioactive properties can be obtained from many kinds of plant materials. These natural substances have gained attention in the food research as possible growth inhibitors of foodborne pathogenic and spoilage bacteria. Many phenolic-enriched plant extracts and individual phenolics have promising anti-quorum sensing potential as well and can suppress the biofilm formation and toxin production of food-related pathogens. Various studies have shown that plant phenolics can substitute or support the activity of synthetic food preservatives and disinfectants, which, by the way, can provoke serious concerns in consumers. In this review, we will provide a brief insight into the bioactive properties, i.e., the antimicrobial, anti-quorum sensing, anti-biofilm and anti-enterotoxin activities, of plant phenolic extracts and compounds, with special attention to pathogen microorganisms that have food relation. Carbohydrase aided applications to improve the antimicrobial properties of phenolic extracts are also discussed.

Anti-Biofilm Effect of Selected Essential Oils and Main Components on Mono- and Polymicrobic Bacterial Cultures.

Biofilms are surface-associated microbial communities resistant to sanitizers and antimicrobials. Various interactions that can contribute to increased resistance occur between the populations in biofilms. These relationships are the focus of a range of studies dealing with biofilm-associated infections and food spoilage. The present study investigated the effects of cinnamon (Cinnamomum zeylanicum), marjoram (Origanum majorana), and thyme (Thymus vulgaris) essential oils (EOs) and their main components, i.e., trans-cinnamaldehyde, terpinen-4-ol, and thymol, respectively, on single- and dual-species biofilms of Escherichia coli, Listeria monocytogenes, Pseudomonas putida, and Staphylococcus aureus. In dual-species biofilms, L. monocytogenes was paired with each of the other three bacteria. Minimum inhibitory concentration (MIC) values for the individual bacteria ranged between 0.25 and 20 mg/mL, and trans-cinnamaldehyde and cinnamon showed the highest growth inhibitory effect. Single-species biofilms of L. monocytogenes, P. putida, and S. aureus were inhibited by the tested EOs and their components at sub-lethal concentrations. Scanning electron microscopy images showed that the three-dimensional structure of mature biofilms embedded in the exopolysaccharide matrix disappeared or was limited to micro-colonies with a simplified structure. In most dual-species biofilms, to eliminate living cells from the matrix, concentrations exceeding the MIC determined for individual bacteria were required.

Anti-Haemophilus Activity of Selected Essential Oils Detected by TLC-Direct Bioautography and Biofilm Inhibition.

Essential oils (EOs) are becoming increasingly popular in medical applications because of their antimicrobial effect. Direct bioautography (DB) combined with thin layer chromatography (TLC) is a screening method for the detection of antimicrobial compounds in plant extracts, for example, in EOs. Due to their lipophilic character, the common microbiological assays (etc. disk diffusion) could not provide reliable results. The aim of this study was the evaluation of antibacterial and anti-biofilm properties of the EO of cinnamon bark, clove, peppermint, thyme, and their main components against Haemophilus influenzae and H. parainfluenzae. Oil in water (O/W) type Pickering nano-emulsions stabilized with silica nanoparticles from each oil were prepared to increase their water-solubility. Samples with Tween80 surfactant and absolute ethanol were also used. Results showed that H. influenzae was more sensitive to the EOs than H. parainfluenzae (except for cinnamon bark oil). In thin layer chromatography-direct bioautography (TLC-DB) the ethanolic solutions of thyme oil presented the best activity against H. influenzae, while cinnamon oil was the most active against H. parainfluenzae. Pickering nano-emulsion of cinnamon oil inhibited the biofilm formation of H. parainfluenzae (76.35%) more efficiently than samples with Tween80 surfactant or absolute ethanol. In conclusion, Pickering nano-emulsion of EOs could inhibit the biofilm production effectively.

Effects of clary sage oil and its main components, linalool and linalyl acetate, on the plasma membrane of Candida albicans: an in vivo EPR study.

The effects of clary sage (Salvia sclarea L.) oil (CS-oil), and its two main components, linalool (Lol) and linalyl acetate (LA), on cells of the eukaryotic human pathogen yeast Candida albicans were studied. Dynamic and thermodynamic properties of the plasma membrane were investigated by electron paramagnetic resonance (EPR) spectroscopy, with 5-doxylstearic acid (5-SASL) and 16-SASL as spin labels. The monitoring of the head group regions with 5-SASL revealed break-point frequency decrease in a temperature dependent manner of the plasma membrane between 9.55 and 13.15 °C in untreated, in CS-oil-, Lol- and LA-treated membranes. The results suggest a significant increase in fluidity of the treated plasma membranes close to the head groups. Comparison of the results observed with the two spin labels demonstrated that CS-oil and LA induced an increased level of fluidization at both depths of the plasma membrane. Whereas Lol treatment induced a less (1 %) ordered bilayer organization in the superficial regions and an increased (10 %) order of the membrane leaflet in deeper layers. Acute toxicity tests and EPR results indicated that both the apoptotic and the effects exerted on the plasma membrane fluidity depended on the composition and chemical structure of the examined materials. In comparison with the control, treatment with CS-oil, Lol or LA induced 13.0, 12.3 and 26.4 % loss respectively, of the metabolites absorbing at 260 nm, as a biological consequence of the plasma membrane fluidizing effects. Our results confirmed that clary sage oil causes plasma membrane perturbations which leads to cell apoptosis process.

Anti-listerial effect of selected essential oils and thymol.

The anti-listerial effect of marjoram, thyme essential oils (EOs) and thymol on Listeria monocytogenes inoculated chicken breast fillets was investigated. Before inoculation the fillets were pretreated by washing or not under running tap water. Inoculated samples were kept at 6 °C for 24 h to allow the growth of L. monocytogenes. After this, the fillets were put in marinating solutions containing salt (5%) and EOs or thymol in MIC/2 concentration established in vitro. Total germ count (TGC) and L. monocytogenes count was monitored on the meat surface and in the marinating solutions following 24 and 48 h storage at 6 °C. Thyme and thymol reduced significantly Listeria cell count (1-3 log CFU) in both samples. They also gave good flavour to the fried meat. The doses of EOs used were optimal for antimicrobial efficiency and had a pleasant flavour effect. Washing was not efficient in reducing total germ count.

Latest about Spoilage by Yeasts: Focus on the Deterioration of Beverages and Other Plant-Derived Products.

Food and beverage deterioration by spoilage yeasts is a serious problem that causes substantial financial losses each year. Yeasts are able to grow under harsh environmental conditions in foods with low pH, low water activity, and high sugar and/or salt content. Some of them are extremely resistant to the traditional preservatives used in the food industry. The search for new methods and agents for prevention of spoilage by yeasts is ongoing, but most of these are still at laboratory scale. This minireview gives an overview of the latest research issues relating to spoilage by yeasts, with a focus on wine and other beverages, following the interest of the research groups. It seems that a better understanding of the mechanisms to combat food-related stresses, the characteristics leading to resistance, and rapid identification of strains of yeasts in foods are the tools that can help control spoilage yeasts.

Effects of essential oil combinations on pathogenic yeasts and moulds.

Essential oils (EOs) can be used as alternative or complementary antifungal agents against human pathogenic moulds and yeasts. To reduce the effective dose of antimicrobial agents, EOs are combined which can lead to synergistic or additive effect. In this study the anti-yeast and anti-mould activities of selected EOs were investigated, alone and in combinations, against clinical isolates of Candida albicans, C. parapsilosis, Aspergillus fumigatus, A. terreus, Rhizopus microsporus, Fusarium solani and Lichtheimia corymbifera. Minimum inhibitory concentrations (MICs) were determined for the EOs of cinnamon, citronella, clove, spearmint and thyme. To investigate the combination effect of the EOs, fractional inhibitory concentrations (FICs) were defined by the checkerboard method and the type of interaction was determined by the FIC index (FICI). FIC index below 0.5 was considered as synergism and between 0.5 and 1 as additive effect. Strongest antifungal activity was showed by thyme EO with MIC values below 1.0 mg/ml. Combination of EOs resulted in additive or indifferent effect, with occasional "borderline synergism". The best combination was cinnamon with clove leading to additive effect in all cases.

Green tea and vitamin C ameliorate some neuro-functional and biochemical signs of arsenic toxicity in rats.

BACKGROUND/OBJECTIVES: Nervous system damage is one of the consequences of oral exposure to waterborne inorganic arsenic. In this work, the role of oxidative status in the neurotoxicity of arsenic and the possible role of two foodborne antioxidants in ameliorating arsenic-related oxidative stress were investigated. METHODS: Male Wistar rats were given 10 mg/kg b.w. of trivalent inorganic arsenic (in the form of NaAsO2), 5 day/week for 6 weeks by gavage, combined with vitamin C solution (1 g/l) or green tea infusion (2.5 g in 500 ml boiled water) as antioxidants given in the drinking fluid. RESULTS: Body weight gain was reduced by arsenic from the second week and the antioxidants had no effect on that. Cortical evoked potentials had increased latency, tail nerve conduction velocity was reduced, and this latter effect was counteracted by the antioxidants. The effect of green tea was stronger than that of vitamin C, and green tea also diminished lipid peroxidation induced by As. There was fair correlation between brain As levels, electrophysiological changes, and lipid peroxidation, suggesting a causal relationship. DISCUSSION: Natural antioxidants might be useful in the protection of the central nervous system against the toxicity of oral As.

Enhanced production of industrial enzymes in Mucoromycotina fungi during solid-state fermentation of agricultural wastes/by-products.

Cellulolytic, lipolytic and proteolytic enzyme production of zygomycetes Mucor corticolus, Rhizomucor miehei, Gilbertella persicaria and Rhizopus niveus were investigated using agro-industrial wastes as substrates. Solid-state cultures were carried out on untreated corn residues (stalk and leaf) as single substrate (SSF1) or corn residues and wheat bran in mixed fermentation (SSF2). Rapid production of endoglucanase (CMCase) was observed with maximal activity reaching after about 48-h fermentation, while cellobiohydrolase (CBH) and ß-glucosidase enzymes generally had their peak after 72-h incubation. Highest filter paper degrading (FPase), CMCase, CBH and ß-glucosidase activities obtained were (U g⁻¹ dss) 17.3, 74.1, 12.2 and 158.3, for R. miehei, G. persicaria, M. corticolus and Rh. niveus, respectively. M. corticolus proved to be the best lipolytic enzyme producer in SSF1 presenting 447.6 U g⁻¹ dss yield, while R. miehei showed 517.7 U g⁻¹ dss activity in SSF2. Rh. niveus exhibited significantly greater protease production than the other strains. Suc-AAPF-pNA hydrolyzing activities of this strain were 1.1 and 1.96 U g⁻¹ dss in SSF1 and SSF2, respectively. We conclude that the used corn stalk and leaf residues could potentially be applicable as strong inducers for cellulase and lipase production by Mucoromycotina fungi.

Characterization of an extracellular laccase of Leptosphaerulina chartarum.

Laccase-producing fungi were isolated from air, using selective media with a chromogenic substrate to indicate enzyme activity. The best laccase producer strain proved to be a Leptosphaerulina chartarum isolate. Laccase production was investigated in the presence of various inducers in different cultivation conditions. The extracellular laccase was purified for further investigations. SDS-PAGE showed that this laccase is a monomeric protein of 38 kDa molecular weight. The enzyme is active in the pH-range of 3.5-6, with an optimum at pH 3.8. It is active in the 10-60 °C temperature range, with an optimum at 40 °C. After 20 min incubation at temperatures above 70 °C the enzyme lost its activity. Degradation of seven aniline and phenol compounds (2,4-dichlorophenol; 2-methyl-4-chlorophenol; 3-chloroaniline; 4-chloroaniline; 2,6-dimethylaniline; 3,4-dichloroaniline and 3-chloro-4-methylaniline) was investigated, with or without guaiacol (2-methoxyphenol) as mediator molecule. Addition of a mediator to the system significantly increased the degradation levels. These results confirmed that the isolated laccase is able to convert these harmful xenobiotics at in vitro conditions.

Isolation and characterization of antagonistic Bacillus strains capable to degrade ethylenethiourea.

In this study, more than 150 bacteria showing antagonistic properties against bacterial and fungal pathogens of the tomato plant were isolated and characterized. The most efficient agents against these phytopathogenic microorganisms belong to the genus Bacillus: the best biocontrol isolates were representatives of Bacillus subtilis, B. mojavensis and B. amyloliquefaciens species. They intensively produced fengycin or/and surfactin depsipeptide antibiotics and also proved to be excellent protease secretors. It was proved, that the selected strains were able to use ethylenethiourea (ETU) as sole nitrogen source. These antagonistic and ETU-degrading Bacillus strains can be applied as biocontrol and also as bioremediation agents.

Characterization of a ß-glucosidase with transgalactosylation capacity from the zygomycete Rhizomucor miehei.

An extracellular ß-glucosidase from the zygomycete Rhizomucor miehei NRRL 5282 cultivated in a wheat bran-based solid state fermentation system was characterized. The purified enzyme exhibited an optimum temperature of 68-70 °C and pH of 5.0. It efficiently hydrolyzed oligosaccharides having ß-(1→4) glycosidic linkages and exhibited some ß- and α-galactosidase activity. The V(max) for p-nitrophenyl-ß-d-glucopyranoside and cellobiose was 468.2 and 115.5 U/mg, respectively, while the K(m) was 0.12 mM for both substrates. The enzyme had transglucosylation and transgalactosylation activities resulting in the formation of glycosides from cellobiose, lactose and ethanol. The enzyme increased the amounts of free phenolic antioxidants in sour cherry pomace indicating that its hydrolyzing activity could potentially be applicable to improve the bioavailability of these compounds.

Anti yeast activities of some essential oils in growth medium, fruit juices and milk.

The anti-yeast activities of four essential oils (EOs) from clary sage, juniper, lemon and marjoram against wild-type isolates of the food-related yeasts Geotrichum candidum, Pichia anomala, Saccharomyces cerevisiae and Schizosaccharomyces pombe in malt extract (ME) medium, apple juice and milk were investigated. Minimal inhibitory concentrations (MICs) for the EOs and their main components were determined and the checkerboard method was used to calculate fractional inhibitory concentration (FIC) indices for the combinations of EOs or components. The most sensitive yeast was S. pombe (MICs of 0.0625-0.125 µl/ml) while G. candidum proved to be the most insensitive (MICs of 0.5-2 µl/ml). In general, the lag phases were lengthened by increasing EO concentrations, while significant reduction of growth rates was obtained only at the highest EO concentrations. The anti-yeast effects of the EOs were good in the acidic pH range optimal for yeasts growth. Combinations of juniper and clary sage EOs resulted in additive effects in the case of S. cerevisiae and G. candidum, but all other combinations showed no interaction. The combination of α-pinene and limonene led to synergism, while the combination of α-pinene with linalool resulted in an additive effect. Cloudy apple juice protected the yeasts against the effect of lemon EO: the lag phases were shorter and the growth rates higher than in clear apple juice. Lemon EO decreased the growth rate of G. candidum in skimmed milk in a dose-independent manner. Our results show that by adding lemon EO to clear apple juice a new, harmonic taste can be achieved and open storage time could be prolonged.

Identification of acid- and thermotolerant extracellular beta-glucosidase activities in Zygomycetes fungi.

Extracellular beta-glucosidase activity of 94 strains, representing 24 species of the genera Gilbertella, Mucor, Rhizomucor , and Rhizopus was evaluated in submerged culture and under solid state fermentation on wheat bran. Gilbertella persicaria G1 isolate showed the highest activity (70.9 U ml -1 ) followed by other Gilbertella (58.6-59.0 U ml -1 ) and Rhizomucor miehei isolates (29.2-42.0 U ml -1 ). Optimum temperature for enzyme production was 25 degrees C for Gilbertella and Mucor , and 30 degrees C for Rhizomucor and Rhizopus strains. Enzymes of R. miehei strains proved to be thermotolerant preserving up to 92.8% residual activity after heating to 75 degrees C in the presence of cellobiose substrate. Enzymes of Mucor racemosus f. chibinensis, R. miehei and Rhizopus microsporus var. oligosporus strains were activated at acidic condition (pH 4). Glucose was a strong inhibitor for each fungal beta-glucosidase tested but some of them showed ethanol tolerance up to 20% (v/v). Ethanol also activated the enzyme in these strains suggesting glycosyl transferase activity.