Effect of curing agents and water activity on pork muscle and adipose subcutaneous tissue lipolytic activity

The effect of chemical agents (salt, nitrate, ascorbic acid and glucose) and water activity (a_w) on pork muscle and adipose subcutaneous tissue upases and esterases has been studied. In muscle, salt above 20 g/L strongly inhibited neutral lipase and acid esterase but activated acid lipase. In adipose subcutaneous tissue, 7.5 g/L salt inhibited basic lipase and strongly inhibited acid esterase (around 40% recovery). Nitrate, ascorbic acid and glucose had a negligible or slightly inhibitory effect. A decrease in muscle a_w affected both neutral and basic lipase down to negligible activities at a_w=0.80. A decrease in adipose tissue a_w (down to 0.66) affected both acid and neutral esterase (around 40 and 65% of recovered enzyme activity, respectively). An in-vitro study representing three stages of the dry-curing process revealed that muscle acid lipase and acid esterase might play an important role throughout the process, whereas the rest of the studied enzymes in muscle would be less important. Neutral lipase might be the most important enzyme in the subcutaneous adipose tissue lipolysis of dry-cured ham.`

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Der Einfluß von Pökelhilfsstoffen und Wasseraktivität auf die Lipase-Aktivität in Muskeln und Fettgewebe von Schweinefleisch

Zusammenfassung Die Wirkung von Pökelhilfsstoffen (Salz, Nitrat, Ascorbinsäure und Glucose) und der Wasseraktivität auf die Lipase- und Esterase-Aktivität im Fettgewebe von Schweinefleisch wurde untersucht. Salz (20 g/L Lösung) hemmt die neutrale Lipase und saure Esterase sehr stark, während sie die saure Lipase fördert. In Fettgewebe hemmt Salz (7,5 g/L) die basische Lipase und die neutrale Esterase sehr stark (ca. 40%). Die Enzym-Aktivität wird von Nitrat, Ascorbinsäure und Glucose nicht beeinflußt. Bei Abnahme der Wasseraktivität (a_w=0,99–0,80) im Fleisch wird die Aktivität der neutralen und basischen Lipase sehr stark gehemmt. Bei Abnahme der Wasseraktivität (a_w=0,66) im Fettgewebe werden die Aktivitäten der sauren und der neutralen Esterase um ca. 40 und 65% verringert. Eine Untersuchung in vitro mit den drei Phasen des Reifens zeigt, daß die saure Lipase und die saure Esterase insgesamt in Fleisch eine wichtige Rolle spielen. Im Fettgewebe war die neutrale Lipase-Aktivität ca. 100%.

     

Assay of lipase and esterase activities in fresh pork meat and dry-cured ham

Summary Lipase and esterase activities in post-mortem pork muscle and adipose tissue were assayed. Acid lipases showed optimal activity in the presence of 0.8 mg bovine serum albumin (BSA)/ml and 0.05% (by vol.) Triton X-100, while neutral/basic lipases required 5 mg BSA/ml and no addition of Triton X-100. All lipases had an optimal temperature of 37° C except neutral muscle lipase, which was optimally active at 45° C. A wider range of optimal temperatures of 30–45 and 15–45° C was found for muscle acid esterase activity and neutral esterase activity, respectively. In adipose tissue, higher temperatures of 60° C and 45–75° C were found for maximal acid esterase and neutral esterase activities, respectively. Lipolytic and esterolytic activity assays in muscle and adipose tissue were conducted at four different stages in the processing of Spanish Serrano dry-cured ham. Recovered activities of muscle enzymes were more than 40% of the original activity, even at the end of the drycuring process. In adipose tissue, recovered esterase activity was also around 50% of the original activity at the end of the process, while lipolytic activity was significant only during the post-salting stage.

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Untersuchung der Lipase- und Esteraseaktivität in frischem Schweinefleisch und trockengepökeltem Schinken

Zusammenfassung Die Lipase- und Esteraseaktivität wurde im Muskel und Fettgewebe eines Schweines getestet. Die Lipaseaktivität war optimal bei einem Gehalt von 0,8 mg BSA/ml Rinderalbumin und 0,05% (in Volumen) X-100 Triton, wogegen die neutral-basischen Lipasen 5 mg BSA/ml und keinen Zusatz von X-100 Triton benötigten. Alle Lipasen hatten eine optimale Wirkungstemperatur von 37 °C, mit Ausnahme der neutralen Muskellipase, deren optimale Wirkung bei 45 °C lag. Im Fettgewebe lag die optimale Temperatur für saure und neutrale Esterase höher (60 °C bzw. 45 bis 75 °C). Es wurden die Lipase- und Esteraseaktivitäten im Muskel und Fettgewebe in vier Stufen der Trocknung des Schinkens getestet. Im Muskel wurde mehr als 40% der Anfangsaktivität am Ende des Trocknungsverfahrens wiedergewonnen. Im Fettgewebe betrug die zurückgewonnene Esteraseaktivität um die 50% am Ende des Prozesses, wogegen die Lipaseaktivität erst nach der Salzung signifikant war.

     

EFFECT OF MYOGLOBIN ON THE MUSCLE LIPASE SYSTEM

The effect of different concentrations of myoglobin on the enzyme systems involved in lipid degradation were determined. Lipases, including both acid and neutral lipases and acid phospholipase, were inhibited by myoglobin. The neutral lipase was more susceptible to the inhibitory effect of myoglobin (35% of its initial activity at 1 mg/mL of myoglobin), followed closely by acid phospholipase; acid lipase activity was affected to a lesser extent (less than 20% inhibition). This inhibition was also observed on the esterase activities, being greater on the neutral esterase activity (38% of its initial activity at 1 mg/mL of myoglobin) than on the acid one. In view of these results, myoglobin could be considered as an endogenous inhibitor which would determine the lipolytic activity in different type of muscles and therefore, the rate of generation of the free fatty acids.     

Comparison of muscle proteolytic and lipolytic enzyme levels in raw hams from Iberian and White pigs

Muscle enzyme activities in Biceps femoris from Iberian and White crossbreeds were quantified in vitro to determine possible differences due to crossbreed and the different slaughtering age (12-month-old Iberian pigs and 6-month-old White pigs). Large differences were found between proteolytic and lipolytic enzyme levels from the skeletal muscle of Iberian pig breed and White pig crossbreed. In raw hams from Iberian pig, calpain and cathepsin (B, L and H) were significantly lower, while cathepsin D was significantly higher than in White pigs. Significantly lower dipeptidyl peptidase (II and IV), aminopeptidase B, leucyl and pyroglutamyl aminopeptidase activities were found in Iberian pig breed compared with White pig crossbreed. Significantly higher levels of dipeptidyl peptidase III and alanyl aminopeptidase and lower activities of acid lipase and neutral esterase were found in Iberian breed. It was concluded that lower muscle enzyme activities and in consequence slower proteolysis and lipolysis should be desirable to obtain higher quality in dry-cured products. © 1998 SCI     

Activity of Aminopeptidase and Lipolytic Enzymes in Five Skeletal Muscles with Various Oxidative Patterns

The activity of muscle aminopeptidase B, alanyl aminopeptidase, acid lipase and acid esterase were assayed in pork muscles of different metabolic type. The muscles were Masseter , Trapezius , Semimembranosus , Biceps Femoris and Longissimus dorsi and some significant differences (P>0·05) were found in enzyme activities. Alanyl aminopeptidase showed lower activity in Masseter and Biceps femoris while aminopeptidase B had lower activity in Biceps femoris . Acid lipase and acid esterase showed lower activity in Trapezius and higher in Masseter . However, no clear relationship could be established between the assayed enzyme activities and the metabolism of the muscles.     

SUBCUTANEOUS ADIPOSE TISSUE LIPOLYSIS IN THE PROCESSING OF DRY-CURED HAM

ABSTRACT The activities of subcutaneous adipose tissue lipases and esterases were assayed at different stages (0 to 15 months) in the processing of dry-cured ham. The formation of free fatty acids during the process was also determined. Maximal generation of free fatty acids occurred during the first 10 months. Simultaneously, the triglyceride content decreased while the diglycerides increased during the aging period. Neutral and basic lipases showed maximal activity at the beginning of the process but only neutral lipase remained as the main enzyme responsible for the reported lipolysis during the drying ripening stages. Adipose tissue esterases showed excellent stability but the generation of volatile free fatty acids was negligible, suggesting a minor role of these enzymes.     

Effects of the terminal sire type and sex on pork muscle cathepsins (B, B+L and H), cysteine proteinase inhibitors and lipolytic enzyme activities

Pork muscle cathepsins (B, B+L, and H), cysteine proteinase inhibitors and lipolytic enzyme activities were measured in the offspring of five different genetic sire types: Danish Duroc (DU), Dutch Large White (LW(D)), English Large White (LW(E)), Belgian Landrace × Landrace (BL×LR) and Belgian Landrace (BL). Cathepsin B and B+L activities were higher for LW(E) and LW(D) sires than for BL×LR and BL. Cathepsin H activity showed an opposite evolution, being higher for BL and BL×LR sires than for DU, LW(D) and LW(E). Cysteine proteinase inhibitor activity was higher for LW(E) sires than for DU and BL. In lipolytic enzymes, BL sires had a lower acid lipase activity than DU and LW(E) sires and also a lower neutral esterase activity than LW(E) and LW(D) sires. Significant differences between sexes were found for cathepsin H activity only, being higher for females.     

Cyanidin-3-O-β-glucoside improves obesity and triglyceride metabolism in KK-Ay mice by regulating lipoprotein lipase activity

BACKGROUND: Cyanidin‐3‐O‐β‐glucoside (Cy‐3‐g)‐rich foods have been reported to inhibit the onset of obesity, but whether the pure anthocyanin supplementation affects obesity remains uncertain. RESULTS: Cy‐3‐g supplementation significantly reduced obesity, accumulation of fat in visceral adipose and liver tissues, and plasma triglyceride levels. Furthermore, adenosine monophosphate (AMP)‐activated protein kinase phosphorylation (pAMPK) in the skeletal muscle and visceral adipose were significantly increased by Cy‐3‐g consumption. This was followed by the activation of lipoprotein lipase (LPL) in plasma and skeletal muscle but the suppression of this enzyme in visceral adipose. LPL activation in skeletal muscle cells and its suppression in adipocytes by Cy‐3‐g were blocked by inhibition of pAMPK. CONCLUSION: Our present data thus demonstrate that Cy‐3‐g improves obesity and triglyceride metabolism in KK‐Ay mice. The underlying mechanism is found to be partly related to the activation of LPL in plasma and skeletal muscle, and inhibition of LPL in adipose tissue following the activation of pAMPK. Copyright © 2011 Society of Chemical Industry     

Effect of Antioxidants on Malonaldehyde Production and Fatty Acid Composition in Pieces of Bovine Muscle and Adipose Tissue Stored Fresh and Frozen

TBA-reactive material was produced in pieces of bovine semitendinosus muscle and adipose tissue during storage at 2° 2°C and ?10° 2°C. The process was faster in muscle than in adipose tissue and the total content, higher at 2°C than at ?10°C. The effect of sprying butylated hydroxytoluene and a citric acid-EDTA-ascorbic acid mixture on the production of malonaldehyde was studied. Declines in both saturated and unsaturated fatty acid proportions of the polar lipids without increases in the content of free fatty acids suggests that enzymes involved in lipid catabolism remain active at low temperatures. Whereas lipid breakdown was unaffected, malonaldehyde production was inhibited by sprying antioxidants in early stages of the slaughtering process.     

Lipolysis in dry-cured ham maturation

Thirty light Parma hams were tested for muscle lipolytic activity (acid and neutral lipase activity) and free fatty acid (FFA) amounts in M. semimembranosus and biceps femoris, during progressive phases (0, 3, 6, 10 months) of dry-cured ham manufacturing. No correlation was found between the activities of acid and neutral lipases in fresh M. semimembranosus, while during processing the activities were positively related (p<0.1), probably due to effects of muscle composition changes on lipolytic activities. In each processing step tested, acid lipase activities were higher in the M. semimembranosus than in the M. biceps femoris, and FFA amounts varied accordingly, the only exception being for the very dehydrated 10-month old M. semimembranosus, which yielded lower FFA than in the corresponding M. biceps femoris. FFAs in the end product correlated positively with acid and neutral lipase activities of green ham, suggesting that FFA production could be influenced by both raw meat properties and muscle composition during processing.     

Efficient enzymatic synthesis of amide with (aminomethyl)trimethylsilane

Hydrolase-catalyzed amide synthesis using a silicon-containing amine, (aminomethyl)trimethylsilane, as the substrate was studied. Six hydrolases (lipase OF 360, lipase Novo, lipase KLIP-001, lipoprotein lipase Type A, cholesterol esterase Type A, and cholesterol esterase III) were capable of forming the amide of (aminomethyl)trimethylsilane with octanoic acid in 2,2,4-trimethylpentane. Lipoprotein lipase Type A and cholesterol esterase Type A showed particularly the high levels of activity. From a comparative study of (aminomethyl)trimethylsilane and its carbon analog, 2,2-dimethylpropylamine, the former was found to be a better substrate for the hydrolases than the carbon analog. This difference is considered to arise from the specific properties of the silicon atom. (Aminomethyl)trimethylsilane exhibited a homotropic effect at concentration under 100 mM in amide synthesis by lipoprotein lipase Type A, while the carbon analog showed no such effect.     

The role of muscle enzymes in dry-cured meat products with different drying conditions

Several muscle proteases (cathepsins, calpains, peptidases and aminopeptidases) and lipases (lysosomal acid lipase, acid phospholipase and adipose tissue lipase) are involved in important biochemical mechanisms taking place during the processing of dry-cured meat products which are directly related to the final quality. These enzymes are affected by the conditions typically found in the processing of dry-cured meat products, being dehydration one of the most important factors. This work is presenting the effect of different drying conditions, typical in the processing of dry-cured meat products, on the activity of muscle proteases and lipases as well as its relevance for the final product quality.     

Calpain and cathepsin activities in post mortem fish and meat muscles

Post mortem tenderization is one of the most unfavourable quality changes in fish muscle and this contrasts with muscle of mammalian meats. The tenderization can be partly attributed to the acid lysosomal cathepsins and cytosolic neutral calcium-activated calpains. In this study, these proteases from fish and bovine muscles were quantified and compared. The cathepsin B and L activities were in more important amounts in sea bass white muscle than in bovine muscle. On the other hand, cathepsin D activity was 1.4 times higher in meat that in fish muscle, while cathepsin H was negligible in both muscles. Calpain activities were similar in both types of muscle. Moreover, calpastatin (calpain endogenous inhibitor) level is 3.9 times higher in sea bass white muscle. These differential activities are considered in relation to their probable involvement in post mortem degradation of muscle.     

Utilization of extruded linseed to modify fatty composition of intensively-reared lamb meat: Effect of associated cereals (wheat vs. corn) and linoleic acid content of the diet

Sixty male lambs were used in two trials to study the efficiency of transfer and elongation of linolenic acid (ALA) in muscle and caudal adipose tissue and to assess factors affecting this process and related changes in fatty acid (FA) profile. In experiment 1, lambs were fed a control diet or extruded linseed (L) diet either with wheat (W, rapid starch) or corn (C, slow starch). In experiment 2, lambs were fed L with “normal” rapeseed, or high-oleic rapeseed, or soybean. In experiment 1, L increased ALA proportion and total n-3 PUFA in muscle and adipose tissue. In adipose tissue but not in muscle, LC lambs had higher proportion of ALA than LW lambs. In experiment 2, increasing linoleic acid (LA) intake increased LA proportion in muscle and adipose tissue but did not modify ALA proportion. Moreover, in muscle, it did not change the desaturation and elongation processes of ALA to long-chain n-3 PUFA.     

糖添加量对广式腊肠脂质氧化稳定性及感官品质的影响研究

研究了不同糖添加量(3%、6%、9%、12%,m/m)对广式腊肠脂质水解酶活、脂质氧化稳定性及产品感官品质的影响规律。结果表明中性脂肪酶、酸性脂肪酶和磷脂酶活力在加工过程中呈下降趋势(p0.05)。中性脂肪酶活力高于酸性脂肪酶和磷脂酶活力。糖添加量显著影响三种脂肪酶活力在加工过程中的变化,高糖添加组的三种脂肪酶活力略高于低糖添加组;糖添加量为3%时,烘烤结束后中性脂肪酶、酸性脂肪酶和磷脂酶活力分别下降77%、81%和96%,而添加量为12%时,其分别下降67%、65%和78%。烘烤结束后低糖添加组的过氧化值、羰基值和己醛含量较高,表明糖添加量的降低导致脂质氧化的加剧。感官分析表明糖添加量的降低致使咸味更加突出且出现一定的脂质哈败味,降低了广式腊肠产品的接受度。     

Antioxidant, lipolytic and proteolytic enzyme activities in pork meat from different genotypes

Oxidative processes in meat lead to meat quality deterioration. Meat has endogenous antioxidants and prooxidants, but information on factors influencing the activity of antioxidant enzymes in meat is limited. Lipolytic and proteolytic enzymes are involved in important aspects of meat quality. Our objective was to find differences between five different genotypes on the activity of antioxidant, lipolytic and proteolytic enzymes in meat. Forty Psoas major muscles of females of five different pig genotypes were used, Pietrain, Landrace, Large-White, lberian, and lberian×Duroc. Pre slaughter conditions were similar for all the genotypes. After slaughter, muscles were vacuum packed and frozen at -20?°C until required. Differences between genotypes were found for the activity of catalase and SOD, while GSH-Px showed no differences. The highest differences between breeds were found for the lberian breed where catalase had the highest activity. Catalase activity also showed differences between the white pigs, with large values for LR and lower activities in P. There were no differences in neutral lipase activities between the different genotypes while acid lipase and phospholipase showed significant differences. The activities of cathepsin B and H were significantly lower for Iberian pigs compared with other breeds except LR, while the ratio of cathepsin B+L/cathepsin B was higher in Iberian. The differences between genotypes found in enzyme activities suggest some genetic effects on the antioxidant, lipolytic and proteolitytic activity of pork meat.     

Esterolytic and lipolytic activities of lactic acid bacteria isolated from ewe's milk and cheese

In the present work, we report on the esterase and lipase activities of lactic acid bacteria representing the genera Lactococcus, Leuconostoc, Lactobacillus, and Enterococcus isolated from ewe's milk and cheeses. Esterase activity was studied using alpha- and beta-naphthyl derivatives of 2 to 12 carbon atoms and postelectrophoretic detection. The lactic acid bacteria evaluated had intracellular esterase activities, which preferentially degraded the alpha- and beta-naphthyl derivatives of 2 to 6 carbon atoms. By studying postelectrophoretic patterns, it was found that some strains presented more than one esterase. Lactobacillus plantarum O236 showed four enzymes that hydrolyze carboxyl ester linkages with different specificity. Lipase activity was studied in intracellular and extracellular fractions using tributyrin, tricaprylin, triolein, and milk fat as substrates. The intracellular and extracellular fractions of Leuconostoc mesenteroides O257, Lactobacillus plantarum O236, and Lactobacillus acidophilus O177 were able to hydrolyze tributyrin. L. plantarum O186, L. acidophilus O252, Enterococcus faecium O174 and O426, and Enterococcus faecalis Ov409 showed lipase activity associated with the intracellular fraction on tributyrin. Lactococcus lactis O233, L. plantarum O155, and Lactobacillus casei O190 did not hydrolyze triglycerides. Not all strains that showed esterase activity exhibited high activity on triglycerides. Esterase and lipase activities were species- and strain-specific. Wide variations in activity between strains highlight the need for selecting appropriate starters to produce enzyme-modified cheese as well as accelerated ripened cheese.     

Muscle and Adipose Tissue Aminopeptidase Activities in Raw and Dry-Cured Ham.

Free amino acids have been analyzed in biceps femoris muscle and adipose tissue from raw and dry-cured ham. A high increase was observed for all amino acids except glutamine and taurine. Major increases were in glutamic acid, arginine, alanine, valine, leucine, and lysine. A survey of five aminopeptidase activities of muscle and adipose tissue from raw and dry-cured ham was performed by using 7-amino-4-methylcoumarin derivatives of five amino acids (Leu, Arg, Ala, Tyr and pGlu) as substrates. Optimum activity was found at neutral pH and around 37°C, except the leucyl hydrolyzing activity which was 45°C. High recoveries of activity (25–75%) were obtained in the dry-cured ham. These enzymes might be responsible for free amino acids increasing during dry-curing.     

The experimental obesity by high-fat feeding — A model for evaluation of the biological value of fats

The experimental obesity by high-fat feeding of rats, introduced after weaning, was found to be a suitable model for evaluation of the biological value of nutritional fats with different fatty acid composition: butter (group B), lard (L), partially hydrogenated oil (H), lard + sunflower oil 2:1 (LS). The differences in the fatty acid composition of these regimens affect: the efficiency in creating the model of obesity; the hormonal pattern of blood plasma; some metabolic pathways in liver and adipose tissue (especially in group H); the fatty acid composition of some structural, reserve and transport lipids; some biological tests indicative of membrane's phospholipid and fatty acid composition, i.e. the rate of platelets aggregation. A special attention should be paid to the striking differences in the cellularity and morphogenesis of adipose tissue in group B (hyperplastic obesity) in comparison with all other high-fat groups (hypertrophic obesity), irrespective of the identical energy and protein content of the diets. Thus, the early administration of a diet with butterfat (50% of energy) promoted a model of hyperplastic obesity, while the isocaloric diet with lard + sunflower oil caused a hypertrophic type of obesity. The authors have proved the regenerating capacity of the periepididymal fat pad in adult rats after partial lipectomy. The relative contribution of endogenic and exogenic (nutritional) factors in this process is discussed and the modifications in the cellularity of adipose tissue on these conditions are evaluated.     

Fat deposition, fatty acid composition and meat quality: A review

This paper reviews the factors affecting the fatty acid composition of adipose tissue and muscle in pigs, sheep and cattle and shows that a major factor is the total amount of fat. The effects of fatty acid composition on meat quality are also reviewed. Pigs have high levels of polyunsaturated fatty acids (PUFA), including the long chain (C20-22) PUFA in adipose tissue and muscle. The full range of PUFA are also found in sheep adipose tissue and muscle whereas cattle ‘conserve’ long chain PUFA in muscle phospholipid. Linoleic acid (18:2n − 6) is a major ingredient of feeds for all species. Its incorporation into adipose tissue and muscle in relation to the amount in the diet is greater than for other fatty acids. It is deposited in muscle phospholipid at a high level where it and its long chain products eg aracidonic acid (20:4n − 6) compete well for insertion into phospholipid molecules. Its proportion in pig adipose tissue declines as fat deposition proceeds and is an index of fatness. The same inverse relationships are not seen in ruminant adipose tissue but in all species the proportion of 18:2n − 6 declines in muscle as fat deposition increases. The main reason is that phospholipid, where 18:2n − 6 is located, declines as a proportion of muscle lipid and the proportion of neutral lipid, with its higher content of saturated and monounsaturated fatty acids, increases. Oleic acid (18:1cis − 9), formed from stearic acid (18:0) by the enzyme stearoyl Co-A desaturase, is a major component of neutral lipid and in ruminants the same enzyme forms conjugated linoleic acid (CLA), an important nutrient in human nutrition. Like 18:2n − 6, -linolenic acid (18:3n − 3) is an essential fatty acid and is important to ruminants since it is the major fatty acid in grass. However it does not compete well for insertion into phospholipid compared with 18:2n − 6 and its incorporation into adipose tissue and muscle is less efficient. Greater biohydrogenation of 18:3n − 3 and a long rumen transit time for forage diets also limits the amount available for tissue uptake compared with 18:2n − 6 from concentrate diets. A positive feature of grass feeding is that levels of the nutritionally important long chain n − 3 PUFA are increased ie EPA (20:5n − 3) and DHA (22:6n − 3). Future research should focus on increasing n − 3 PUFA proportions in lean carcasses and the use of biodiverse pastures and conservation processes which retain the benefits of fresh leafy grass offer opportunities to achieve this. The varying fatty acid compositions of adipose tissue and muscle have profound effects on meat quality. Fatty acid composition determines the firmness/oiliness of adipose tissue and the oxidative stability of muscle, which in turn affects flavour and muscle colour. Vitamin E is an essential nutrient, which stabilises PUFA and has a central role in meat quality, particularly in ruminants.