Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

Lipoprotein lipase and lipolysis in milk

Bovine milk contains a lipoprotein lipase that accounts for most, if not all, of its lipolytic activity. The total lipase activity in raw milk is sufficient to cause rapid hydrolysis of a large proportion of the fat. However, in reality this does not happen, because the lipase is prevented from accessing the fat by the milkfat globule membrane. Physical damage to this membrane in raw milk initiates lipolysis. Furthermore, simply cooling certain individual milks soon after secretion can initiate the so-called spontaneous lipolysis. The biochemical basis of spontaneous lipolysis is still poorly understood, but it appears to be related to a balance between activating and inhibiting factors in the milk. Lipolysis in milk and milk products causes rancid off-flavours and other problems, and is a constant concern in the dairy industry. A thorough understanding of the mechanism of lipolysis and constant vigilance by operatives is required to minimize lipase-related problems.     

Synergistic effects of various lipases and phospholipase C on milk fat

The enzyme, phospholipase C, from a number of psychrotrophs isolated from milk was shown to survive HTST pasteurization and UHT sterilization processes. The phospholipase C of Bacillus cereus was able to increase lipolytic activity of a commercial lipase preparation and native milk lipoprotein lipase in raw milk by making the substrate more susceptible to hydrolysis: phospholipase C had no effect on the lipase activity of a fluorescent Pseudornonas spp. The enhancement of lipolysis by phospholipase C was affected by pronase and papain, but not by trypsin. Homogenization of the raw milk had the same effect on lipolysis by lipoprotein lipase as addition of phospholipase C.     

Lipolytic Enzyme Activity of Macrophages in Bovine Mammary Gland Secretions

The ability of macrophages isolated from the involuted bovine mammary gland and pooled raw milk to secrete lipolytic enzymes was investigated. Macrophages obtained from the involuted gland and maintained in cell culture secreted lipolytic enzymes into culture medium for up to 120 h. Leukocytes in pooled raw milk were separated using Ficoll discontinuous density gradients. Macro-phages secreted lipolytic enzymes into the gradient while fractions containing polymorphonuclear leukocytes and lymphocytes did not possess lipolytic activity. Enzyme activity of macrophages from pooled raw milk averaged .1% of the total milk lipoprotein lipase activity present in the original milk samples. Fresh raw milk with a macrophage concentration increased to 2.5 × 10⁶ cells/ml contained 11.6% higher milk lipoprotein lipase activity after storage for 48 h at 4°C. These results indicate that macrophages isolated from bovine mammary secretions produce lipolytic enzymes that could influence milk lipoprotein lipase activity in raw milk over storage.     

Lipoprotein lipase of goat mammary tissue: comparison of extraction methods

Mammary lipoprotein lipase of lactating goats was extracted by 3 methods: homogenization of tissue acetone-ether powders; aqueous homogenization of crude tissue using an Ultra-Turrax apparatus; aqueous homogenization of crude tissue using a Sorvall Omni-mixer microhomogenizer. Although there were differences between absolute values obtained by the 3 methods, each type of homogenate had a lipolytic activity with lipoprotein lipase characteristics (i.e. more than 90% inhibition by serum omission or NaCl addition). Furthermore, the 3 methods were highly correlated and presented similar variations with the stage of lactation, in parallel with long-chain fatty acid secretion into milk. Repeatability of the measure of homogenate lipolytic activities was about 8%, whereas day-to-day repeatability of enzyme extraction and assay was about 20% for each method.     

Characterization of lipase of Pseudomonas fluorescens 27 based on fatty acid profiles

The objectives of this research were to isolate lipase from Pseudomonas fluorescens 27, to compare the purity of the partially purified lipase preparation with crude extract, and to determine if bands of lipase activity revealed by disc gel electrophoresis liberated different free fatty acids from milk fat. Lipases were isolated from a shaken skim milk culture of P. fluorescens 27 by using ion-exchange chromatography on DEAE cellulose (Whatman DE 32) and gel filtration on Sephadex G-150. The principal lipase-rich fractions from gel filtration represented 6.2% of total lipolytic activity. Disc gel electrophoresis of partially purified enzyme revealed two protein bands. These protein bands were cut from disc electrophoresis gels and used as an enzyme source for reaction with butter oil. Free fatty acids were isolated from the assay mixture, separated, and quantified by gas chromatography. Data from gas chromatographic analysis indicated that P. fluorescens 27 produces at least two different lipases.     

Isolation and identification of lipolytic, psychrotrophic, spore forming bacteria from raw milk

Out of 100 samples of raw milk, 48% were found to contain lipolytic, psychrotrophic, spore forming bacilli. The lipase activity of 59 selected isolates ranged from 8.5 to 42 units/ml. Forty one per cent of the total isolates exhibited enzyme activity in the range of 21–25 units/ml. On the basis of morphological and biochemical characteristics, the 59 lipolytic isolates were identified as Bacillus cereus (19), Bpolymyxa (12), B licheniformis (10), B circulans (7), B subtilis (5), B laterosporus (4) and B coagulans (2). B cereus (32.2%) was found to be the predominant organism.     

Determination of the extracellular and cell-associated hydrolase profiles of Pseudomonas fluorescens sp. using the Analytab API ZYM system

The extracellular and cell-associated hydrolase profiles of a number of Pseudomonas fluorescens strains were examined with the Analytab API ZYM system. Esterase/lipase was the only strong extracellular enzyme activity detected (mean 3.33): weak esterase, lipase, and leucine aminopeptidase activities were found with some strains (mean activities of 1.08, 1.53, and 1.40, respectively). Very strong leucine aminopeptidase activity (4.5) was associated with the cells. Cell-associated trypsin, esterase/lipase, acid phosphatase, and phosphoamidase were also found. Neither extracellular nor cell-associated hydrolase profiles changed significantly when cells were grown in skim milk or mineral salts medium at either 5 or 20 degrees C. Similarly, added calcium did not seem required for synthesis of any of the enzymes. The extracellular enzyme profiles differed considerably from those of the cell-associated enzymes for all strains tested. An extracellular proteinase-deficient mutant of strain 32A (RM14) failed to produce significant quantities of extracellular esterase/lipase activity. Production of cell-associated enzymes was unaffected by the mutation. These results suggest that the Analytab API ZYM system may be useful in identifying psychrotrophs isolated from milk.     

Isolation and some properties of extracellular heat-stable lipases from Pseudomonas fluorescens strain AFT 36

Aeration increased the growth and lipase production in milk by Pseudomonas fluorescens strain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented approximately 71% of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8.0 and 35 degrees C; it had a Km on tributyrin of 3.65 mM and was inhibited by concentrations of substrate greater than approximately 17 mM. The enzyme was very stable over the pH range 6-9; it was relatively heat-labile in phosphate buffer in the temperature range 60-80 degrees C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100-150 degrees C: the D values at 150 degrees C were approximately 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the corresponding Z values in the temperature range 100-150 degrees C were approximately 40 and approximately 42 degrees C and the Ea for inactivation were 7.65 X 10(4) J mol-1 and 6.97 X 10(4) J mol-1 respectively.     

Inhibition of Lipolytic Activity in Milk by Polysaccharides,

The effect of gums on the activity of milk lipase and a Pseudomonas lipase in milk was investigated. Gums were hydrated in water and mixed with whole milk. Lipase was added to the gum-milk mixture and hydrolysis was determined after 48 h at 4°C by the acid degree value method. Of the gums tested, the anionically charged λ-, ι- and κ-carrageenan, furcellaran, and sodium alginate significantly inhibited milk lipase activity by 93.7, 81.2, 46.8, 50.6, and 62.1%, respectively. Furthermore, λ-carrageenan was 87.6% effective in inhibiting lipolysis by a purified Pseudomonas fluorescens MC50 lipase in milk. The other gums tested, tragacanth, carboxymethyl cellulose, locust bean, propylene glycol alginate, xanthan, microcrystalline cellulose, guar, and arabic did not significantly inhibit milk lipase. Commonly used stabilizers can inhibit lipolytic activity in milk.     

产低温脂肪酶菌株的筛选、纯化及其部分酶学性质研究

从大黑山(大连)和海水中筛出59株低温细菌,从中筛选出10株产低温脂肪酶的菌株,分别对其进行紫外诱变,筛出一株高酶活的低温脂肪酶菌株。通过形态学、生理生化和分子生物学鉴定,菌株CZWOO1属于芽孢杆菌属(BacillusCohn),与Bacillusthuringien-si(sNR 043403.1)进化关系近,且同源性达到100%,故将其命名为Bacillus thuringiensis CZWOO1。苏云金芽孢杆菌CZW001脂肪酶发酵上清液经硫酸铵沉淀、透析、超滤离心、DEAE-纤维素-52离子交换层析和Sephadex G-100凝胶过滤层析获得到电泳纯的脂肪酶,纯化倍数为75.5倍,活性回收率为37.6%,12%SDS-PAGE电泳估计其分子量约40ku。对纯化后的低温脂肪酶进行二级结构预测,结果表明其二级结构成分:α-螺旋10.6%,β-折叠40.1%,β-转角18.8%,无规则卷曲30.5%。对该菌脂肪酶的酶学性质初步研究表明,酶的最适作用温度为25℃,对热敏感,60℃处理30min仅残留30%酶活性,酶的适宜作用pH范围在7~9,最适pH值为8。     

Purification and Partial Characterization of Caprine Milk Lipoprotein Lipase

Lipoprotein lipases of caprine and bovine milks were isolated, purified, and partially characterized from raw whole milk. Fractions with enzyme activity were two for caprine and one for bovine lipase. The milk lipoprotein lipase enzyme fractions were purified further by hydroxylapatite and intervent dilution chromatography. Caprine enzyme fractions were purified 142,000 and 87,000-fold from the isolates compared to the bovine enzyme, which was purified 5500-fold. For the two caprine enzyme fractions were three distinct electrophoretic bands of molecular weights 66,100; 58,900; and 55,000. Electrophoretic patterns of the two fractions were similar. Isoelectric focusing of the highly purified caprine fractions also revealed heterogeneity with minor differences in isoelectric points, pH 5.02 and 5.14. Bovine enzyme had two distinct electrophoretic bands with molecular weights of 74,100 and 66,100, but poly aery lamide gel electrophoresis in the absence of sodium dodecylsulfate or β-mercaptoethanol revealed only one major band indicating a quaternary structure. The pH optimum of the caprine milk lipoprotein lipase was 8.7 compared to bovine enzyme of 8.5. Increase of yield of enzyme from both species during purification was attributed to several factors, the most important being the removal of an inhibitor. If in milk, this inhibitor may play a role in susceptibility of milk to hydrolytic rancidity.     

Milk lipoprotein lipases: a review.

Lipoprotein lipase activity has been found in the milks from severals species where it is assumed to result from leakage from the mammary gland into milk. The function of the enzyme in the gland is apparently to assist in the transfer of blood lipoprotein triacylglycerol fatty acids into milk triacylglycerols. Bovine skim milk is one of the richest sources of lipoprotein lipase and this enzyme has been purified extensively (7000 fold) by affinity chromatography. The lipase has a molecular weight of about 62000, is inhibited by protamine sulfate, 1.0 M sodium chloride, apolipoprotein C-I (apolipoprotein-serine), and apolipoprotein C-III (apolipoprotein-alanine). The enzyme is activated by apolipoprotein C-II (apolipoprotein-glutamic acid), serum, and by heparin to which it also binds. The lipase is highly specific for the primary esters of acylglycerols and exhibits a slight stereospecificity for the sn-1 ester in preference to the sn-3-ester. Bovine milk also has separate activity toward 1-monoacylglycerols. Human milk contains a serum stimulated lipoprotein lipase with many of the characteristics of the enzyme in bovine milk, as well as an enzyme stimulated by bile salts which resembles the sterol ester hydrolase of rat pancreatic juice. The assay, function, purification, characteristics, and substrate specificities of these enzyme are discussed.     

A review of nutritional and physiological factors affecting goat milk lipid synthesis and lipolysis

Although the effect of lactation stage is similar, the responses of milk yield and composition (fat and protein contents) to different types of lipid supplements differ greatly between goats and cows. Milk fat content increases with almost all studied fat supplements in goats but not in cows. However, the response of milk fatty acid (FA) composition is similar, at least for major FA, including conjugated linoleic acid (CLA) in goats and cows supplemented with either protected or unprotected lipid supplements. Goat milk CLA content increases sharply after either vegetable oil supplementation or fresh grass feeding, but does not change markedly when goats receive whole untreated oilseeds. Important interactions are observed between the nature of forages and of oil supplements on trans-10 and trans-11 C18:1 and CLA. Peculiarities of goat milk FA composition and lipolytic system play an important role in the development of either goat flavor (release of branched, medium-chain FA) or rancidity (excessive release of butyric acid). The lipoprotein lipase (LPL) activity, although lower in goat than in cow milk, is more bound to the fat globules and better correlated to spontaneous lipolysis in goat milk. The regulation of spontaneous lipolysis differs widely between goats and cows. Goat milk lipolysis and LPL activity vary considerably and in parallel across goat breeds or genotypes, and are low during early and late lactation, as well as when animals are underfed or receive a diet supplemented with protected or unprotected vegetable oils. This could contribute to decreases in the specific flavor of goat dairy products with diets rich in fat.     

Survival of Lipase during Manufacture of Nonfat Dry Milk

Extracellular lipases from several strains of Pseudomonas fluorescens were active against milk fat. The activity of P. fluorescens strain B52 lipase in reconstituted NDM using β-naphthyl caprylate as substrate was 70% of that found with skim milk; however, hydration of the NDM at 4°C for 12 h resulted in 85% recovery of activity. As little as .0025% (vol/vol) of P. fluorescens spent media was detected in reconstituted skim milk powder using β-naphthyl caprylate. The process of freeze-drying or spray-drying without heating had little effect on either native or P. fluorescens B52 lipase. The bacterial lipase activity was reduced by 12 to 21% and 55 to 59% in NDM treated with low heat (72°C/16 s) and high heat (110°C/2 min), respectively, while the native lipases were completely inhibited by these treatments. Both native and bacterial enzymes were stable when stored in NDM for extended periods at 20°C. In a limited study of commercial NDM samples, microbial lipase was not detected, suggesting that the quality of the raw milk was sufficiently high to restrict the presence of heat-stable lipolytic enzymes.     

Chemiluminescent determination of xanthine oxidase activity in milk.

A chemiluminescent method for determining xanthine oxidase (XOD) activity was developed and applied to the assay of milk enzyme activity using a photomultiplier luminometer. Various kinds of milk and cream samples were analysed for XOD content. In pasteurized milk, XOD activity depended on the fat content and in UHT milk it disappeared owing to the heat treatment. Milk sample preparation was very simple, requiring only homogenization at 40 degrees C followed by a 1:10 dilution with UHT ('XOD-free') milk. The assay was carried out at 25 degrees C. The response obtained from XOD standard solutions in milk was linear from 0.1 to 500 enzyme units (U) l-1, but for the actual milk samples values ranged only from 1 to 135 U l-1. The detection limit at 2 SD was 0.1 U l-1 in milk, while in buffer it was 100 times lower. The intra-assay and interassay CV for XOD activity in milk were 6-12%.     

The influence of the enzymatic composition of lamb rennet paste on some properties of experimentally produced PDO Fiore Sardo cheese

An experiment on PDO (Protected Designation of Origin) Fiore Sardo cheese making was carried out to assess the influence of rennet paste on cheese quality. Four different preparations of lamb rennet paste (A, B, C and D) were used. Rennet A came exclusively from suckling lambs slaughtered immediately after suckling, rennet B from suckling lambs not fed for 12 h, and rennets C and D from suckling lambs allowed to graze and slaughtered in the same way as A and B, respectively. In vitro lipolysis and multiple cheese-making trials were carried out for each rennet. The enzymatic composition of the prepared rennets was different. Lipolytic activity was assessed in vitro by measuring the quantity of free fatty acids released by hydrolysis of a sheep milk cream substrate. Rennet A had the greatest lipolytic activity, which had a strong preference for esters of short-chain fatty acids. The cheeses made using the different rennets did not differ significantly in their moisture, protein and fat content or in the nitrogen fractions which resulted from proteolysis. The cheese produced with rennet A had the highest lipolysis. Short-chain fatty acids, in particular butyric acid, were prevalent, as they were in the in vitro trials. The diet and the presence of milk in the abomasa was responsible for the enzymatic peculiarities of rennet A. Suckling stimulated the secretion of pre-gastric lipase from the pharingeal tissue. Thus, if one wishes to produce a rennet with the particular enzymatic composition necessary for a particular cheese, the diet and the slaughtering conditions of the lambs should be controlled.     

Some aspects of the periurban dairy system in Cameroon

A survey to establish baseline data for future intervention in milk and dairy production in the peri-urban areas of Garoua, Maroua and Bamenda was carried out. Dairy products are still produced in a traditional way in areas around Maroua and Garoua with milk from native zebu cattle (Red and White Fulani breeds). The important dairy products are pendidam and kindirmu, both of which are fermented milks. Naturally sour milk was found occasionally. Around Bamenda, milk is produced by herds comprising pure Holsteins, Jerseys and their crosses with local zebu cows; cross-breeding is being intensified to increase rapidly the size of the dairy herds. Appreciable volumes of milk are produced daily, so allowing the industrial processing of fresh milk into pasteurized milk, yogurt and cheese. The potential for expansion of the dairy industry, and the possible constraints, were identified and solutions proposed.     

Effect of the size and interface composition of milk fat globules on their in vitro digestion by the human pancreatic lipase: Native versus homogenized milk fat globules

Although the bioavailability of dietary lipids is of primary importance in human nutrition and health, the mechanisms involved in lipid digestion are not fully understood and are of growing interest. The objective of this study was to determine the effect of the size of milk fat globules and of the composition of their interface on the activity of the human pancreatic lipase (PL). Native milk fat globules of various sizes covered by their biological membrane (MFGM) and homogenized fat globules of various sizes covered by milk proteins were prepared from whole milk and underwent lipolysis by the human PL with colipase and bile salts. A lag phase preceding the hydrolysis of milk TAG occurred with all native milk fat globules samples but not with homogenized milk samples. The kinetic parameters of human PL were determined by measuring the enzyme activity either after the lag phase for native milk fat globules samples or immediately after the addition of the enzyme for homogenized milk samples. The catalytic efficiency of human PL is 4.6-fold higher on small (1.8 μm) than large (6.7 μm) native milk fat globules, related to a 3.6-fold larger available surface. Despite the 25-fold larger available surface, milk TAG from homogenized milk are only 2-fold better hydrolyzed compared to native milk fat globules, as a possible result of a less favourable interface covered by milk proteins. The potential mechanisms involved in native vs. homogenized milk fat globules digestion by the human PL are discussed. Our study highlights the crucial role of the MFGM in the efficient digestion of milk fat globules and brings new insight for the design of dairy products and infant formulas.     

On-line determination of the total lipolytic activity in a four-phase system using a lipase adsorption law

Lipases have a very well known affinity for organic-aqueous interfaces. A previously developed on-line turbidimetric analyser can only analyse the lipase activity present in the aqueous phase of a culture broth. An adsorption law of Langmuir type has been derived to determine the lipolytic activity remaining in the organic-aqueous interface formed between oleic acid and culture broth in Candida rugosa lipase production fermentation. In the concentration range of oleic acid (0-8 g.l(-1)) and lipolytic activity (0-35 U.ml(-1)) tested, lipases were not adsorbed in a multilayer form and the Brunauer-Emmet-Teller (B.E.T.) law was not applicable. The Langmuir adsorption law has been shown to be the most suitable to describe the adsorption process involved. The methodology employed enables on-line determination of the total lipolytic activity produced by the microorganism, using the adsorption law determined. This finding, in combination with the on-line measurement of variables such as biomass, aqueous lipolytic activity, oleic acid concentration and specific growth rate, permits the development of control systems for ensuring improved throughput, quality and repeatability of the process.