Effect of the addition of pancreatic lipase on the ripening of dry-fermented sausages - Part 1. Microbial, physico-chemical and lipolytic changes

The effect of the addition of nine different amounts (3-500 units) of pancreatic lipase on the microbial and physico-chemical parameters and lipid fractions during the ripening of dry fermented sausages has been studied. No differences between conventional and lipase-added sausages were found for pH, dry matter and water activity. The addition of lipase caused a greater accumulation of products resulting from the triglyceride breakdown, mainly diglycerides and free fatty acids (FFA). The maximum rate of lipolysis was observed during the first week of the ripening process, specially in the fermentation phase. The greater the pancreatic lipase added, the higher lipolysis observed. At the end of the ripening, the levels of total FFA were clearly higher (1·5 to 5-fold) in all lipase-added batches than in the controls. This fact gave rise to the accumulation of a great amount of FFA, which can contribute either by themselves to the flavour of the sausage or can be available as substrates for further transformations which may generate other flavour compounds.     

Effect of the addition of pancreatic lipase on the ripening of dry-fermented sausages - Part 2. Free fatty acids, short-chain fatty acids, carbonyls and sensory quality

The effect of the addition of nine different amounts (3–500 units) of pancreatic lipase on the composition of free fatty acids (FFA), short-chain fatty acids and carbonyls, as well as the sensory quality, has been studied on dry fermented sausages. The lipase produced a greater release of all fatty acids in relation to the control sausages. The greater the pancreatic lipase content, the higher the release of these fatty acids, the most important ones being myristic, palmitoleic and oleic acids. A lower release of linoleic acid was observed, probably because of its oxidative degradation. At the end of the ripening in all lipase-added batches, a clear increase of the carbonyl content was noticed in relation to conventional sausages. No consistent changes were observed in the short-chain fatty acid fraction. In the sensory evaluation, the highest significant differences (P < 0·01) between control sausages and lipase-added batches were observed when 60 and 90 lipase units were used. According to the chemical and sensory analyses, it can be concluded that the addition of 60 and 90 units to sausages seems to be useful to enhanced the flavour of these products.     

Transport of organic acids through a supported liquid membrane driven by lipase-catalyzed reactions

We have developed a lipase-facilitated supported liquid membrane. Lipase-catalyzed reactions were coupled with a supported liquid membrane (SLM) to transport organic acids through the SLM. We succeeded in the rational transport of organic acids through the SLM using lipase-catalyzed reactions and observed that there were differences in the transport behavior of various organic acids due to the substrate specificity of lipase. Subsequently, various parameters, such as the alcohol concentration in the feed phase, the pH in each aqueous phase, an organic solvent in the SLM, and the kind of lipase, were investigated. We found that the optimum conditions were 65 vol% alcohol concentration, pH 6.3 in each aqueous phase, isooctane as the liquid membrane phase and Candida rugosa lipase as the esterification biocatalyst.     

Use of lipase from Rhizomucor miehei in dry fermented sausages elaboration: Microbial, chemical and sensory analysis

Three different amounts of lipase (0.075, 0.100 and 0.150 LU/g) from Rhizomucor miehei (Palatase M 200L Novo Nordisk) were used to determine the correct amount to use in dry fermented sausages. Determination of acidity values through fourteen days of ripening showed that 0.100 LU/g was the most appropriate. Two types of fermented sausages were manufactured, addition of the enzyme being the only difference between them. Addition of Palatase did not affect product stability (pH and A(w)), and the growth of micro-organisms. In spite of the increase in acidity value, no rancidity developed as determined by both chemical and sensory analysis. Increases in the liberation of palmitic, palmitoleic, stearic, oleic and linoleic acids were found when lipase was used. Juiciness and taste were slightly better in the sausages with Palatase than in those without, but these differences were not reflected in the overall acceptability.     

Dry fermented sausages elaborated with lipase from Candida cylindracea. Comparison with traditional formulations

The addition of microbial lipase to fermented sausages was studied. A sausage with lipase from Candida cylindracea and a control sausage with starter (Lactobacillus plantarum and Staphylococcus carnosus) were produced in a pilot plant. The acidity value and the amounts of the different free fatty acids (FFA) showed a higher intensity of lipolytic activity in sausages with lipase than in sausages with starter. In sausages with lipase, the percentage of saturated FFA was greater and that of polyunsaturated FFA was lower than in sausage with starter. Mono-unsaturated FFA percentage was similar in both sausages. TBA and peroxide values indicated that the increase of FFA produced by lipase action did not increase the rancidity. A slight increase in acetic, propionic and butyric acids was observed in sausage with lipase but this was not sufficient to develop excessive acidity in the product.     

Enzymatic synthesis of structured lipids using a novel cold-active lipase from Pichia lynferdii NRRL Y-7723

Structured lipids (SL) were synthesized by the acidolysis of borage oil with caprylic acid using lipases. Six commercial lipases from different sources and a novel lipase from Pichia lynferdii NRRL Y-7723 were screened for their acidolysis activities and Lipozyme RM IM and NRRL Y-7723 lipase were selected to synthesize symmetrical SL since recently NRRL Y-7723 lipase was identified as a novel cold-active lipase. Both lipases showed 1,3-regiospecifity toward the glycerol backbone of borage oil. The effects of enzyme loading and temperature on caprylic acid incorporation into the borage oil were investigated. For Lipozyme RM IM and NRRL Y-7723 lipase, the incorporation of caprylic acid increased as enzyme loading increased up to 4% of total weight of the substrate, but significant increases were not observed when enzyme loading was further increased. The activity of NRRL Y-7723 lipase was higher than that of Lipozyme RM IM in the temperature range between 10 and 20 °C.     

Modulator-mediated synthesis of active lipase of Pseudomonas sp. 109 by Escherichia coli cell-free coupled transcription/translation system

Catalytically active lipase was synthesized using Escherichia coli S30 extract from the signal-deleted lipL gene (lipL) in the presence of its N-terminal hydrophobic fragment-truncated modulator (rLimL) that was purified from the overexpressing E. coli cells. The specific activity of the lipase thus synthesized was 125 times higher than that of the purified one from Pseudomonas sp. 109. No lipase activity was detected in the absence of rLimL, even though the lipase protein itself was synthesized. Active lipase was also produced in vitro by coexpression of rlipL and the modulator gene (rlimL), although a much smaller amount of the lipase was formed. In the absence of rLimL, aggregates of the lipase were formed during its folding process. The addition of rLimL proportionally raised both lipase solubility and enzyme activity. An unstable but high activity peak of the lipase was found during its folding process.     

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。     

Changes in lipid fractions and sensory properties of Idiazabal cheese induced by lipase addition

This work studied the addition of an adequate lipase to enhance lipolysis reactions and the development of piquant flavour and sharp odour in Idiazabal cheese, as an alternative to the use of lamb rennet paste. Cheeses were manufactured from bulk raw ewes' milk in 50 l vats with commercial bovine rennet and 80 lipase units of pregastric or 180 lipase units of fungal lipase and ripened for 180 days. A higher lipolytic activity was induced by lipase addition promoting strong changes in odour and flavour attributes. Both fungal and pregastric lipases increased the content of total free fatty acids (FFA), but the fungal lipase released mainly medium- and long-chain FFA. In contrast, the pregastric lipase preferably released short-chain FFA. Diglyceride (DG) content was considerably higher in cheeses made with added pregastric lipase compared with those made with fungal lipase or with no lipase. Monoglycerides (MG) were detected only in cheeses made with either lipase added, reaching comparable concentrations after ripening for 180 days. The cheeses made with pregastric lipase had the highest scores for odour and flavour intensity, and sharp and rennet odours, desirable attributes for the Idiazabal cheese made with lamb rennet paste. None of the texture attributes were significantly influenced by the concentrations of MG and DG in the cheeses made with either lipase. Thus, the pregastric lipase was more appropriate than the fungal lipase to develop a more traditionally-flavoured Idiazabal cheese.     

Effect of Rice Hemicelluloses on Pancreatic Lipase Activity In Vitro

The influence of rice bran water-soluble hemicellulose on hydrolysis of fat by pancreatic lipase with and without bile salts was examined in vitro. Bile salts are required for emulsification of the oil. Three of the major salts found in human bile (sodium cholate, glycocholate, and taurocholate) were tested for emulsifying properties with and without rice hemicellulose. The lipase activity decreased with increased addition of hemicellulose to the bile salt-enzyme reaction mixture. The results suggest that rice hemicellulose affects lipase activity indirectly by rendering the bile salts less available for emulsification of the oil and subsequent hydrolysis by the lipase.     

Effect of hydrocarbon-water interfaces on synthetic and hydrolytic activities of lipases

We have developed a new method of lipase activation for interesterification, in which lipase, in contact with a hydrocarbon-water interface, has been found to have high interesterification activity in an anhydrous solvent. We have applied this activation method to various lipase and obtained high synthetic activity in n-hexane, and have investigated the effect of various hydrocarbon-water interfaces on the synthetic and hydrolytic activities of lipases. The esterification and/or interesterification activity of lipases tested was improved by this activation method, using an n-tetradecane-water interface. From the initial group of lipases, three representative lipases (from Rhizopus japonicus, Chromobacterium viscosum and porcine pancreas) were selected for further study. The effect of various hydrocarbon-water interfaces on synthetic (interesterification or esterification) activity was studied. We demonstrated that the resulting synthetic activity was affected by the choice of hydrocarbon-water interface and that there were differences in the effects of interfaces on the synthetic activity of these lipases.     

Enhancement of Blue cheese flavour using sodium dodecylsulphate and lipase

Accelerated Blue cheese ripening as affected by addition of sodium dodecylsulphate (SDS) in levels of 0.05 or 0.075% alone or SDS (0.05 or 0.075%) + lipase was studied. There were no effects on the cheese chemical composition by addition to the cheese curd of SDS or SDS/lipase combination, except for an increase in titratable acidity of cheese. Proteolysis, lipolysis and carbonyl formation were enhanced in Blue cheese treated with SDS alone. Addition of lipase with SDS greatly increased lipolysis and carbonyl formation but it did not increase proteolysis as SDS alone. Organoleptic evaluation indicated that addition of SDS alone or in combination with lipase enhanced the development of texture and flavour of cheese and improved Penicillium roqueforti distribution throughout the cheese. Cheese treated with SDS (0.075%) and lipase showed higher quality than the other cheeses.     

脂肪酶催化大豆卵磷脂和亚油酸酯交换反应的研究

采用Lipozyme TL IM酶催化大豆卵磷脂(90%)和亚油酸(95%)进行酯交换反应,以制备富含亚油酸的卵磷脂.探讨了大豆卵磷脂质量浓度(甲苯为溶剂)、底物摩尔比(n(亚油酸):n(大豆卵磷脂))、酶添加量(以底物总质量为基准)、水分添加量(以酶质量为基准)、反应时间、反应温度对酯交换反应的影响.优化后的反应条件为:大豆卵磷脂质量浓度0.25g/ml,底物摩尔比6:1,酶添加量20%,水分添加量3%,反应时间60h,反应温度65℃.在最优反应条件下最终获得82.85%亚油酸含量的卵磷脂产品.     

外源酶缩短羊肉发酵香肠成熟期的效果研究

对脂酶、酸性蛋白酶和风味蛋白酶缩短羊肉发酵香肠成熟期的效果进行研究。通过三因子二次通用旋转设计,确定3 种酶的最优组合,并测定添加外源酶后发酵羊肉香肠成熟过程中理化指标和感官指标的变化情况。结果表明：3 种酶的最优添加质量分数为脂酶0.0022%、酸性蛋白酶0.0011% 和风味蛋白酶0.0027%;加酶组成熟到第14 天时游离脂肪酸(FFA)的含量、非蛋白氮(NPN)的含量分别达到1.437%、0.647%,与对照组第35 天的FFA 含量1.382%、NPN 含量0.634% 无显著差异(P ＞ 0.05);而在整个发酵成熟过程中,加酶组和对照组的挥发性盐基氮(TVB-N)值和硫代巴比妥酸(TBA)值没有显著差异(P＞ 0.05);加酶组成熟第21 天时已与对照组成熟第35 天的感官质量相似。说明外源酶的添加可以使羊肉发酵香肠的成熟期缩短40%。     

Isolation and analysis of lipase-overproducing mutants of Serratia marcescens

We have isolated a lipase-overproducing mutant, GE14, from Serratia marcescens 8000 after three rounds of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutant GE14 produced 95 kU/ml of extracellular lipase in the lipase medium, which was about threefold higher than that of produced by the original strain 8000. Enzymatic characteristics including specific activity of purified lipases from culture supernatants of GE14 and 8000 were almost same. The lipase gene (lipA) of GE14 contained two base substitutions; one in the promoter region and another in the N-terminal region of the lipA gene without an amino acid substitution. Promoter analysis using lipA-lacZ fusion plasmids revealed that these substitutions were responsible for the increase in the lipA expression level, independently. In contrast, no base substitution was found in the genes encoding the lipase secretion device, the Lip system. In addition, the genes coding for metalloprotease and the cell surface layer protein which are both secreted through the Lip system and associated with extracellular lipase production, also contained no base substitution. The strain GE14 carrying a high-copy-number lipA plasmid produced a larger amount of the extracellular lipase than the recombinant strains of 8000 and other mutants also did, indicating that GE14 was not only a lipase-overproducing strain, but also an advantageous host strain for overproducing the lipase by a recombinant DNA technique. These results suggest that the lipase-overproducing mutant GE14 and its recombinant strains are promising candidates for the industrial production of the S. marcescens lipase.     

Effect of brine thawing/salting on endogenous enzyme activity and sensory quality of Iberian dry-cured ham

Simultaneous brine thawing/salting process was applied as an alternative to traditional pile salting process using 51 frozen Iberian hams. The effect of this type of salting process on endogenous enzyme activity and sensory quality of Iberian dry-cured hams was analysed. The frozen hams were simultaneously thawed and salted with saturated brine, with and without vacuum pulses, and were compared to hams thawed under refrigeration and traditionally salted. The peptidase and lipase activities were measured at the end of salting and post-salting stages. The activity of cathepsin B+L was reduced in the two brine salted batches while few differences among batches were observed for the other peptidases. Several lipase activities were significantly reduced in the two brine salted batches. The brine thawed processing affected the free fatty acid content at the different stages although the differences were more appreciated at the beginning of the process and no differences were observed at the end. The long ripening time makes these differences negligible and the consumer did not appreciate any differences between the sensory quality of Iberian brine/thawed hams and traditional Iberian thawed pile salted hams.     

脂肪酶预处理及鸡油添加对蚕蛹呈味基料风味的影响

分别以桑蚕蛹和柞蚕蛹为原料,采用脂肪酶预处理、添加鸡油两种方式对原料进行处理,研究不同处理对蚕蛹酶解产物的水解度、氨基酸、感官风味等指标的影响。结果表明:桑蚕蛹与柞蚕蛹的脂肪酶预处理和鸡油的添加均能显著提高呈味肽的风味(p<0.05),脂肪酶处理组与对照组相比,在组织形态和滋味方面存在显著性优势(p<0.05),鸡油添加组与对照组相比,在滋味方面存在显著性优势(p<0.05)。其中柞蚕蛹加脂肪酶酶解,再加鸡油进行美拉德反应后的产物风味最好,评分为81.8,酶解液水解度及鲜甜味氨基酸占总氨基酸的比例均为最高,分别为26.21%和45.58%。结果表明,脂肪酶预处理和鸡油的添加两者共同作用,能显著提高蚕蛹呈味基料的风味(p<0.05)。     

Lipolytic activity of Trichothecium roseum on hazelnut

Trichothecium roseum is a mould which grows on hazelnut after harvesting. It produces lipase enzyme which hydrolyses fat to produce free fatty acids (FFA) and partial glycerides. Free fatty acid content (which causes bitterness) is an important criterion, determining the quality and taste of hazelnut. The lipase production characteristics ofT. roseum have been investigated on agar and hazelnut in this study. Trichothecium roseum -inoculated hazelnuts have been stored at 25°C at 90%, 85% and 80% relative humidity. Free fatty acids as a percentage of the total fat content were found to be 1·6%, 1·4% and 1·4% respectively. Free fatty acids forT. roseum -inoculated hazelnut whose water content had been increased by water addition, was found to be 2% after 4 days, and 25% after 28 days storage. This is due to rapid hydrolysis under this condition. Partial glycerides were also observed after 4 days of storage.     

Modulator-mediated synthesis of active lipase of Pseudomonas sp. 109 by Escherichia coli cell-free coupled transcription/translation system

Catalytically active lipase was synthesized using Escherichia coli S30 extract from the signal-deleted lipL gene (lipL) in the presence of its N-terminal hydrophobic fragment-truncated modulator (rLimL) that was purified from the overexpressing E. coli cells. The specific activity of the lipase thus synthesized was 125 times higher than that of the purified one from Pseudomonas sp. 109. No lipase activity was detected in the absence of rLimL, even though the lipase protein itself was synthesized. Active lipase was also produced in vitro by coexpression of rlipL and the modulator gene (rlimL), although a much smaller amount of the lipase was formed. In the absence of rLimL, aggregates of the lipase were formed during its folding process. The addition of rLimL proportionally raised both lipase solubility and enzyme activity. An unstable but high activity peak of the lipase was found during its folding process.     

Differences in hydrolytic abilities of two crude lipases from Geotrichum candidum 4013

The fungus Geotrichum candidum 4013 produces two types of lipases (extracellular and cell‐bound). Both enzymes were tested for their hydrolytic ability to p‐nitrophenyl esters and compounds having a structure similar to the original substrate (triacylglycerols). Higher lipolytic activity of extracellular lipase was observed when triacylglycerols of medium‐ (C12) and long‐ (C18) chain fatty acids were used as substrates. Cell‐bound lipase preferentially hydrolysed trimyristate (C14). The differences in the abilities of these two enzymes to hydrolyse p‐nitrophenyl esters were observed as well. The order of extracellular lipase hydrolysis relation velocity was as follows: p‐nitrophenyl decanoate > p‐nitrophenyl caprylate > p‐nitrophenyl laurate > p‐nitrophenyl palmitate > p‐nitrophenyl stearate. The cell‐bound lipase indicates preference for p‐nitrophenyl palmitate. The most striking differences in the ratios between the activity of both lipases (extracellular : cell‐bound) towards different fatty acid methyl esters were 2.2 towards methyl hexanoate and 0.46 towards methyl stearate (C18). The Michaelis constant (K_m) and maximum reaction rate (V_max) for p‐nitrophenyl palmitate hydrolysis of cell‐bound lipase were significantly higher (K_m 2.462 mM and V_max 0.210 U/g/min) than those of extracellular lipase (K_m 0.406 mM and V_max 0.006 U/g/min). Copyright © 2010 John Wiley & Sons, Ltd.