Artemisia afra essential oils inclusion in diets induces desirable effects on meat quality and fatty acid profiles of broilers chickens.

The effects of Artemisia afra (African wormwood) essential oil incorporation in diets on meat quality and fatty acid profilers of broilers was assessed. Four hundred Cobb500 day-old chicks were obtained and placed randomly into the following 4 diets replicated 10 times: NC-Negative control (broiler diet without antibiotics), PC-positive control (broiler diet with zinc bacitracin), AA1-broiler diet + 0.1 % A. afra essential oil and AA2 - broiler diet + 0.3% A. afra essential oil. A pen having 10 chicks was regarded as the experimental unit. From the results, all carcass traits were influenced (P < 0.01) by diet apart from the dressing out percentage. The highest slaughter weights (1846g) and hot carcass weight (1427.36g) were obtained in birds fed the AA1 diet. Highest ultimate pH value (6.24) was obtained from the birds fed the AA1 while the highest values for meat lightness (L*) were obtained in birds offered the AA1 and NC diets (55.73 and 55.82). Cooking loss was highest in birds fed the NC (31.01%) diet. Birds fed the AA2 diet (23.7mg/g)) had the highest intramuscular fat followed by the AA1 fed birds. Inclusion of A. afra essential oils in diets also increased the proportion of PUFAs, n-6 and n-3 fatty acids and the PUFA/SFA ratios. In conclusion, the incorporation of A. afra essential oil in diets improved meat quality and fatty acid composition of broiler meat for the benefit of consumers.

Novel Cosmetic Ingredient CS-AA Polyion Complex and Skin Moisturizing Effect.

PURPOSE: The study explored the enhanced skin moisturizing capabilities and moisture retention effects achieved by forming a polyion complex using sulfated glycosaminoglycan (GAG), specifically chondroitin sulfate (CS), and amino acids (AA) such as glutamine (Q) and arginine (R). The overall hydration effect of this CS-AA complex was examined. METHODS: After analyzing the CS-AA polyion complex structure using spectroscopic methods, the ex vivo moisture retention ability was assessed under dry conditions using porcine skin samples. Additionally, the efficacy of the CS-AA polyion complex in reducing transepidermal water loss (TEWL) and improving skin hydration was evaluated on human subjects using a digital evaporimeter and a corneometer, respectively. RESULTS: Validating a systematic reduction in particle size, the following order was observed: CS > CS/AA simple mixture > CS-AA complex based on dynamic light scattering (DLS) and transmission electron microscopy (TEM) analysis. Furthermore, observations revealed that the CS-AA complex exhibits negligible surface charge. Additionally, Fourier-transform infrared spectroscopy (FT-IR) analysis demonstrated a distinct peak shift in the complex, confirming the successful formation of the CS-AA complex. Subsequently, the water-holding effect through porcine skin was assessed, revealing a notable improvement in moisture retention (weight loss) for the CS-Q complex: 40.6% (1 h), 20.5% (2 h), and 18.7% (4 h) compared to glycerin. Similarly, the CS-R complex demonstrated enhancements of 50.2% (1 h), 37.5% (2 h), and 33% (4 h) compared to glycerin. Furthermore, TEWL improvement efficacy on human skin demonstrated approximately 25% improvement for both the CS-Q complex and CS-R complex, surpassing the modest 12.5% and 18% improvements witnessed with water and glycerin applications, respectively. Finally, employing a corneometer, hydration changes in the skin were monitored over 4 weeks. Although CS alone exhibited nominal alterations, the CS-Q complex and CS-R complex showed a significant increase in moisture levels after 4 weeks of application. CONCLUSION: In this study, polyion complexes were successfully formed between CS, a sulfated GAG, and AA. Comparisons with glycerin, a well-known moisturizing agent, confirmed that the CS-AA complex exhibits superior moisturizing effects in various aspects. These findings suggest that the CS-AA complex is a more effective ingredient than CS or AA alone in terms of efficacy.

Efficient Inhibition of Ice Recrystallization During Frozen Storage: Based on the Diffusional Suppression Effect of Silk Fibroin.

Effectively controlling ice recrystallization (IR) during the frozen storage of food remains highly challenging. Inspired by the structural characteristics of antifreeze proteins in nature, silk fibroin (SF) derived from silk fibers has been developed. Through dual validation using the "splat" assay and "sucrose sandwich" assay, the IR inhibition activity of SF at various concentrations was confirmed, revealing that its regular alternating hydrophilic/hydrophobic domains endow SF with the potential to inhibit the axial growth of single ice crystal and significantly reduce the average maximum crystal size by approximately 67%. Additionally, the quality stability of frozen muscle foods treated with SF was comprehensively evaluated. In stark contrast to traditional commercial antifreeze agents (4% sucrose and 4% sorbitol), prepared steaks with the addition of 2% SF maintained rich juiciness and excellent color acceptability over a three-month frozen storage period. Thus, SF holds promise as a potential protective agent for frozen muscle foods, enhancing their quality during storage.

Production, Purification, and Characterization of a Novel Exopolysaccharide from Probiotic Lactobacillus amylovorus: MTCC 8129.

Extracellular polysaccharides (EPS) produced by Lactic Acid Bacteria have an individual effect on the flavour and consistency of novel food materials, as well as potential therapeutic applications. The purpose of this study was to create, improve, and characterise EPS from Lactobacillus amylovorus MTCC 8129. FTIR examination showed the compound's composition (acetyl group, hydroxy group, ring structure) as well as the numerous interlinks between sugar residues, which were then validated by Nuclear Magnetic Resonance Spectroscopy. Thermogravimetric examination showed that the EPS exhibited resistance to heat at a temperature of 640 °C, with antioxidant levels ranging from 70 to 85% and emulsification activity above 50%. Furthermore, it has 180% water holding capacity and 140% oil holding capacity. Based on these findings, it seems that the EPS that was reviewed might potentially be an advantageous addition to the food processing industry.

Relationship between tree species diversity and water holding capacity of litter layer in subtropical region.

Litter layer, serving as the "skin" of forest soil, plays a crucial role in conserving water resources and maintaining soil and water conservation. We analyzed the relationship of tree species richness, community weighted mean traits, and functional diversity with the standing mass, maximum water holding rate, and effective water sto-rage capacity of litters from various tree species including Liquidambar formosana, Mytilaria laosensis, Castanopsis sclerophylla, Castanopsis hystrix, Cunninghamia lanceolata, Pinus massoniana, Fokienia hodginsii, Taxus wallichiana and their combinations of mixed forests in subtropical region. The results showed that across various tree species combinations, the ranges of maximum water holding rate, standing litter mass and effective water storage capacity of undecomposed layer were 0-419%, 0-0.58 t·hm-2, and 0-1.66 t·hm-2, respectively. For the semi-decomposition layer, these values spanned in 0-375%, 0-6.14 t·hm-2, and 0-16.03 t·hm-2, respectively. Tree species richness and community weighted mean specific leaf area had significantly positive effects on standing mass of litter and effective water storage capacity, while community weighted mean leaf N content had significantly negative effect on standing mass of litter. The maximum water holding rate increased with the increases of functional diversity of specific leaf area and community weighted mean specific leaf area, decreased with the increase of community weighted mean leaf thickness. Results of structural equation model showed that tree species richness increased litter water holding capacity by increasing functional diversity of specific leaf area. The community weighted mean specific leaf area increased the water holding capacity of litter layer by increasing standing mass of litter and the maximum water holding rate. It is necessary to consider planting mixed forest with higher community weighted mean specific leaf area in the management of subtropical artificial forest, so as to improve the water holding capacity of litter layer.

Microplastics originated from agricultural mulching films affect enchytraeid multigeneration reproduction and soil properties.

Microplastics (MPs) are increasingly entering agricultural soils, often from the breakdown of agricultural plastics (e.g., mulching films). This study investigates the effects of realistic MPs from different mulching films: two conventional polyethylene (PE-1 and PE-2) and two biodegradable (starch-blended polybutylene adipate co-terephthalate; PBAT-BD-1 and PBAT-BD-2). MPs were mixed into Lufa 2.2 soil at a concentration range from 0.005 % to 5 % (w/w dry soil), wide enough to reflect both realistic environmental levels and "worst-case scenarios". Effects on Enchytraeus crypticus reproduction over two generations and six important soil properties were studied. PBAT MPs notably reduced enchytraeid reproduction in the F0 generation, with a maximum decrease of 35.5 ± 9.6 % at 0.5 % concentration. F1 generation was unaffected by PBAT contamination. PE MPs had a more substantial reproductive impact, with up to a 55.3 ± 9.7 % decrease at 5 % PE-1 concentration compared to the control, showing a dose-related effect except for 1 %. Both MP types also significantly affected soil water holding capacity, pH, and total carbon. Other soil properties remained unaffected. Our results highlight the potential negative impacts of MPs originating from real agricultural plastics on soil health and raise concerns about the role of agricultural plastics in sustainable agriculture and food safety.

Find alternative for bovine and porcine gelatin: Study on physicochemical, rheological properties and water-holding capacity of chicken lungs gelatin by ultrasound treatment.

In this study, the study on physicochemical, rheological properties and water-holding capacity of gelatin of chicken lungs was investigated to replace bovine and porcine gelatin. The extraction rates of chicken, bovine and porcine lung gelatin by ultrasound assisted alkaline protease were 52.12 %, 69.06 % and 70 %, respectively. Three lung gelatins had similar molecular weight distribution in SDS-PAGE with low content of high molecular weight subunits. The amino acid content of bovine lung gelatin (18.03 %) was higher than in chicken (16.62 %) and porcine lung (15.30 %). The highest intensity of 2θ = 7.5° diffraction peak in bovine lung gelatin was observed, which indicated that the triple helix content of bovine lung gelatin was higher than that of chicken and porcine lung gelatin. The lowest apparent viscosity of chicken lung gelatin was 0.253 mPa·s, but the highest water holding capacity of chicken lung gelatin was 331.72 %. Therefore, chicken lung gelatin can be used as a substitute for bovine and porcine gelatin in some functional properties.

A green extraction method for agar with improved thermal stability and water holding capacity.

The conventional agar extraction method has drawbacks such as high energy consumption, low yield, poor quality, and possible residual harmful factors, which greatly limit its application in high-end fields such as biomedicine and high-end materials. This work explored a new freezing-thawing-high-temperature coupling technique for agar extraction. It increased the yield and the strength of agar by 10.6 % and 13.7 %, respectively, as compared to direct high-temperature extraction of agar (HA). The greater molecular weight and lower sulfate content of agar obtained from freeze-thaw cycles combined with high temperature extraction (FA) may be attributed to the desulfurization effect caused by freeze-thaw cycles and the preservation of the molecular chain structure. The reduction in sulfate content decreases the steric hindrance resistance of the polysaccharide chains, enhances their interactions, and promotes the regularity and density of the agar structure, while also improving its water retention and thermal stability. In conclusion, this research can offer a theoretical basis and guidance for the eco-friendly extraction of agar with improved agar characteristics and expended its applications.

Enhancing Solanum lycopersicum Resilience: Bacterial Cellulose Alleviates Low Irrigation Stress and Boosts Nutrient Uptake.

The use of natural-origin biomaterials in bioengineering has led to innovative approaches in agroforestry. Bacterial cellulose (BC), sharing the same chemical formula as plant-origin cellulose (PC), exhibits significantly different biochemical properties, including a high degree of crystallinity and superior water retention capacity. Previous research showed that natural-origin glucose-based chitin enhanced plant growth in both herbaceous and non-herbaceous plants. In this study, we produced BC in the laboratory and investigated its effects on the substrate and on Solanum lycopersicum seedlings. Soil amended with BC increased root growth compared with untreated seedlings. Additionally, under limited irrigation conditions, BC increased global developmental parameters including fresh and dry weight, as well as total carbon and nitrogen content. Under non-irrigation conditions, BC contributed substantially to plant survival. RNA sequencing (Illumina®) on BC-treated seedlings revealed that BC, despite its bacterial origin, did not stress the plants, confirming its innocuous nature, and it lightly induced genes related to root development and cell division as well as inhibition of stress responses and defense. The presence of BC in the organic substrate increased soil availability of phosphorus (P), iron (Fe), and potassium (K), correlating with enhanced nutrient uptake in plants. Our results demonstrate the potential of BC for improving soil nutrient availability and plant tolerance to low irrigation, making it valuable for agricultural and forestry purposes in the context of global warming.

Evaluation of low-temperature ultrasonic marination of pork meat at various frequencies on physicochemical properties, myoglobin levels, and volatile compounds.

This study aims to evaluate the pork meat quality after ultrasonic brining at different frequencies, thereby providing a more comprehensive understanding of the effects of ultrasound marination on meat. The texture profile analysis showed that ultrasonic curing at various frequencies significantly improved the textural properties of samples, especially at 26.8 kHz, resulting in a reduction of tenderness, hardness, and chewiness values by 44%, 43%, and 44%, respectively. The cooking loss of samples marinated by ultrasound decreased from 27% without ultrasonic treatment to 22%, indicating a significant improvement in water-holding capacity, while the changes in pH had only a subtle impact on pork quality. Meanwhile, the color of pork became more rosy hue due to decreased L⁎ values and increased a⁎ values, which was mainly attributed to an elevated proportion of oxymyoglobin and reduced metmyoglobin content. Additionally, ultrasonic marination did not exert a negative impact on the oxidation of pork protein and lipids. After roasting, samples marinated by ultrasound exhibited a significantly higher abundance of volatile flavor compounds compared to static marinated meat (with an increase of 16 flavor substances) and fresh pork (with an increase of 24 flavor substances), demonstrating the efficacy of ultrasonic marination in enhancing the overall flavor and taste profile of pork. Consequently, the application of ultrasonic technology holds great potential for the "home kitchen type" rapid marination.

Electrical stimulation and wet ageing effects on the carcass and meat quality of Australian rangeland goats.

Medium voltage electrical stimulation and wet ageing periods effects on the carcasses and meat quality of Australian rangeland goats were investigated. 64 rangeland goats were slaughtered as 2 equal consignments, wherein half were exposed to post-dressing electrical stimulation (300 V, 500 ms pulse width, 45 s) and half were not (control). Carcass and pH decline parameters and glycogen concentration were recorded. At 24 h post-mortem, fresh colour was measured and longissimus lumborum muscles (LL) removed. Within carcass, paired LL were halved and allocated to each of 4 ageing periods (1, 2, 4, or 21 d). There were no significant effects from electric stimulation on LL pH at 18 °C, LL temperature at pH 6, LL pH at 24 h post-mortem, semitendinosus pH at 24 h post-mortem, and LL glycogen concentration. There were no significant interactions between stimulation and ageing period on cooking loss, particle size, purge loss, sarcomere length, shear force, and ultimate pH. Independent to stimulation, shear force, particle size, and cooking values declined as ageing period increased. Purge loss was highest after 21 d of ageing. Colour stability was unaffected by ageing, although all CIE colour coordinates varied across the 74 h total retail display period. There were negative linear relationships found between LL glycogen concentrations and LL temperature at pH 6, ultimate pH, and sarcomere length. These findings suggest there to be little benefit to post-dressing electrical stimulation of rangeland goat carcasses, when applied independent to or in combination with ageing.

Effect of bittern immersion on textural properties and water-holding capacity in beef.

Hard meat has low market value; hence, we used bittern as a novel meat tenderizer for bovine M. semitendinosus, one of a hard muscle. We investigated the effects of beef immersion in bittern, a basic solution primarily comprising MgCl2, on textural properties and water-holding capacity. Muscle samples from M. semitendinosus of Holstein steers were immersed in seven different solutions (RO, NaCl, MgCl2, red wine, pH 3, bittern, and pH 8) and heated at 80°C for 5min. The pH of the beef and immersion solutions, water-holding capacity, and maximum load of the meat were measured. Although beef immersed in red wine (pH 3) had a lower pH and water-holding capacity, that immersed in bittern (pH 8.4) had a higher pH and higher water holding capacity. These results indicate that immersion in acidic red wine may harden beef and that immersion in basic bittern may be more effective in maintaining water-holding capacity and softening beef.

Impact of frozen storage on physicochemical parameters and quality changes in cooked crayfish.

Customers are increasingly opting for ready-to-eat and easy-to-prepare food products, such as cooked crayfish. It is highly valued for its unique taste, tender meat, and nutritional properties. Therefore, we conducted an investigation into its quality parameters over an 8-week period at -20 °C. Parameters such as water distribution, oxidation reactions, color, microstructure, texture properties, and physicochemical parameters were examined. The physicochemical results indicated that as the storage time increased, the levels of pH and TVB-N (total volatile basic nitrogen) showed a significant increase, while the water holding capacity decreased significantly (P < 0.05). After two months of frozen storage, the carbonyl content and TBARS (thiobarbituric acid reactive substances) increased to 4.15 ± 0.16 nmol/mg protein and 1.6 ± 0.00 mg/kg, respectively. Additionally, the total sulfhydryl content decreased to 4.91 ± 0.10 mol/105 g protein, which had an impact on the quality of the crayfish. Electron microscopy revealed that with increasing storage time, the fiber structure gradually deteriorated due to water crystallization, leading to severe damage and breakage of muscle fibers. Interestingly, these changes related to storage affected color and texture parameters, thereby influencing the overall quality of the crayfish.

Use of sustainable packaging materials for fresh beef vacuum packaging application and product assessment using physicochemical means.

Packaging material should guarantee the longest possible shelf life of food and help to maintain its quality. The aim of the study was to assess the physicochemical changes taking place during 28-day ageing of beef steaks packed in two types of multilayer films containing biodegradable polymers - polylactic acid (NAT/PLA) and Mater-Bi® (NAT/MBI). The control group consisted of steaks packed in synthetic polyamide/polyethylene (PA/PE) film. The samples stored in NAT/PLA had significantly lower purge loss than the control samples and the lowest expressible water amount after 14 and 21 days. Following blooming, the most favourable colour was shown in steaks stored in NAT/MBI, with the highest values for the L*, a* and C* parameters and the R630/580 ratio, a high proportion of oxymyoglobin, and the lowest share of metmyoglobin. All steaks, regardless of the type of packaging material, had acceptable tenderness and were stable in terms of lipid oxidation.

The carcass characteristics and quality of meat from lambs grazing perennial wheat with different companion legumes (clover, serradella, lucerne) or a mineral supplement.

This study compared carcasses as well as the quality and mineral concentration of meat from lambs extensively grazing perennial wheat with clover (PW + C), serradella (PW + S), lucerne (PW + L), or a mineral salt supplement (PW + Min). A split-plot design was used, wherein 3 crossbred ewe lambs (n = 72 in total) (sub-plots) grazed each of 4 forage types (plots), that were replicated across 6 locations (blocks). The feeding study concluded after 96 d, when all the lambs were slaughtered. The left longissimus lumborum muscles (LL) were collected and wet aged for either 5 or 56 d post-mortem. Lambs grazing PW + Min were found to produce carcasses with lower dressing percentage values to those grazing the other forage types (P = 0.037). The LL of lambs grazing PW + L had the lowest crude protein values (P = 0.015). Forage type by ageing period interactions did not affect meat quality. The 56 d ageing period resulted in higher purge loss (P < 0.001) and TVB-N values (P < 0.001) and a decline in shear force (P < 0.001) compared to the 5 d ageing period. The other carcass and meat quality parameters were not affected by forage type; including hot carcass weight, pH decline parameters, eye muscle area, cooking loss, intramuscular fat, sarcomere length, colour stability, and concentrations of calcium, iron, magnesium, sodium, and zinc in the LL. These findings confirm that perennial cereal production systems, that include legume forages with contrasting protein, energy, and micronutrient profiles, can deliver comparable lamb carcasses and meat quality.

Combined Relaxation Spectra for the Prediction of Meat Quality: A Case Study on Broiler Breast Fillets with the Wooden Breast Condition.

This study evaluated the potential of using combined relaxation (CRelax) spectra within time-domain nuclear magnetic resonance (TD-NMR) measurements to predict meat quality. Broiler fillets affected by different severities of the wooden breast (WB) conditions were used as case-study samples because of the broader ranges of meat-quality variations. Partial least squares regression (PLSR) models were established to predict water-holding capacity (WHC) and meat texture, demonstrating superior CRelax capabilities for predicting meat quality. Additionally, a partial least squares discriminant analysis (PLS-DA) model was developed to predict WB severity based on CRelax spectra. The models exhibited high accuracy in distinguishing normal fillets from those affected by the WB condition and demonstrated competitive performance in classifying WB severity. This research contributes innovative insights into advanced spectroscopic techniques for comprehensive meat-quality evaluation, with implications for enhancing precision in meat applications.

Natural aromatic extract of black tea improved the water retention of pork meat batter.

The effect of varying proportions (w/w) of natural aromatic extract of black tea (NAEBT) with pre-emulsification on the water-holding capacity (WHC) of pork meat batter was investigated. The addition of NAEBT significantly reduced the cooking loss (CL) of pork meat batter from 23.95 % to 18.30 % (P < 0.05). Furthermore, NAEBT with pre-emulsification significantly improved the color stability and increased the springiness (P < 0.05). The results of TBARS and carbonyls indicated that NAEBT with pre-emulsification significantly alleviated oxidative damage to proteins (P < 0.05), resulting in an increased level of ß-sheet (P < 0.05), as confirmed by FT-IR analysis. As a result, the water mobility of pork meat batter was restricted (P < 0.05), resulting in an increase in the energy storage modulus (P < 0.05) and a decrease in the pore size. In summary, the WHC of pork meat batter was improved by the antioxidant effect of the NAEBT.

Alterations in the fillet quality of myostatin-knockout red sea bream Pagrus major: Preliminary insights into nutritional, compositional, and textural properties.

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and a popular target for enhancing the productivity of farmed fish. We previously developed an mstn-knockout breed of the aquaculture fish red sea bream (Pagrus major) using genome editing technology. However, little is known about the effects of mstn disruption on the fillet quality of red sea bream and other fish species. In this study, we used fillets of mstn-deficient red sea bream to evaluate their compositional and textural changes during refrigeration. Compared to the wild type, the mutant fillets exhibited an increase in moisture content and a decrease in drippings, indicating an enhanced water-holding capacity. Furthermore, the mutant fillets showed increased water retention and marginally lower collagen content, resulting in lower breaking force, an index of texture. In conclusion, we demonstrated that mstn disruption alters the compositional and textural properties of red sea bream fillets.

Carcass Characteristics and Meat Quality of Wild-Living Mallard (Anas platyrhynchos L.) Originating from Croatia.

The aim of this study was to determine the effects of sex and hunting location on carcass characteristics and meat quality of wild-living mallard (Anas platyrhynchos) from Croatia. Twenty-eight mallards (14 ♂; 14 ♀) were hunted at two hunting locations (HL I = 8 ♂, 8 ♀; HL II = 6 ♂, 6 ♀) in the Croatian lowlands. The carcasses were eviscerated, dressed, and dissected, and the individual internal organs and carcass parts were weighed. The breast muscle (m. pectoralis major) was sampled and used to determine color, pH, drip loss, cooking loss, shear force, and proximate chemical and fatty acid composition. Sex and HL had a significant effect on the majority of carcass characteristics analyzed, but they had no effect on the physical meat parameters. The protein and ash content of mallard meat was significantly higher in HL II (23.16% vs. 22.67%; 1.45% vs. 1.36%, respectively) and the moisture content in females (72.40% vs. 71.59%). HL had a significant effect on SFA (II 33.96% vs. I 29.91%), PUFA n-3 (II 3.55% vs. I 2.69%), PUFA/SFA and n-6/n-3 ratios, and all lipid indices. Females had a significantly higher C22:6n-3 content, a higher PI index and a lower n-6/n-3 ratio. The data presented in this study contribute to a better understanding of game-bird meat quality originating from different regions.

Correlation analysis of phosphorylation of myofibrillar protein and muscle quality of tilapia during storage in ice.

In this study, the correlation between protein phosphorylation and deterioration in the quality of tilapia during storage in ice was examined by assessing changes in texture, water-holding capacity (WHC), and biochemical characteristics of myofibrillar protein throughout 7 days of storage. The hardness significantly decreased from 471.50 to 252.17 g, whereas cooking and drip losses significantly increased from 26.5% to 32.6% and 2.9% to 9.1%, respectively (P < 0.05). Myofibril fragmentation increased, while myofibrillar protein sulfhydryl content and Ca2+-ATPase activity decreased from 119.33 to 89.29 µmol/g prot and 0.85 to 0.46 µmolPi/mg prot/h, respectively (P < 0.05). Correlation analysis revealed that the myofibrillar protein phosphorylation level was positively correlated with hardness and Ca2+-ATPase activity but negatively correlated with WHC. Myofibrillar protein phosphorylation affects muscle contraction by influencing the dissociation of actomyosin, thereby regulating hardness and WHC. This study provides novel insights for the establishment of quality control strategies for tilapia storage based on protein phosphorylation.