Fungal metabolism of butter and shea oils by Penicillium roquefortii in solid state cultures

Monoacylglycerols (monoglycerides, MAGs) were produced from butter oil and shea stearin fraction (shea oil) by two strains of Penicillium roquefortii, FRR 2456 (isolated from a spoilt melon) and Wisbey PJ (a commercial dairy strain), at pH 7.0 at 10 and 25 °C. The system was designed as a model of cheese using modified Czapek medium in solid state cultures. Shea oil with its unique fatty acid profile (stearic, oleic and palmitic acids) was used for comparison with butter oil. Yields of MAGs, which ranged from 3 to 14 g kg^?1 oil, were higher with butter than with shea oil and higher when the spoilage strain FRR 2456 was used. Monoacylglycerols produced by mycelium‐bound lipases from both fungal strains were mainly sn‐1(3) or α isomers (60–70 mol%). Monopalmitin was the major MAG produced from both butter and shea oils. The production and use of MAGs alone or in combination with free fatty acids (FFAs) as food preservatives are discussed. It is implied that sn‐1(3) MAGs together with free fatty acids may be part of a natural antimicrobial system in relatively high‐pH foods such as blue mould‐ripened cheese where growth of foodborne pathogens such as Listeria monocytogenes can be a problem. Copyright © 2003 Society of Chemical Industry     

Screening of microbes for lipases specific for saturated medium and long-chain fatty acids of milk fat

Over 250 microbial strains were screened for lipase activity specific towards saturated medium and long-chain fatty acids. Strains showing trimyristin (C14:0) hydrolysis on agar medium were further streaked on tricaprylin (C8:0) and on olive oil (mostly C18:1) agar to identify strains hydrolysing C12–C15 fatty acids with modest or no activity towards short-chain and unsaturated fatty acids. The twenty strains showing the desired specificity were grown as liquid cultures and analysed for p-nitrophenyl (p-NP) myristate hydrolysis in the presence of Triton X-100. Cells from the cultures showing the highest activity towards p-NP myristate were analysed for specificity towards p-NP esters of different fatty acid chain lengths. On the basis of the p-NP ester profiling, four lipase samples were selected for hydrolysis experiments on butter oil. Of these, the sample obtained from the cultivation of Didpodascus capitatus met the screening criteria: C12:0–C16:0 specificity and no preference towards C18:0 and C18:1 fatty acids.     

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.     

Characterization of anti-listerial lactic acid bacteria isolated from Thai fermented fish products

Thai fermented fish products were screened for lactic acid bacteria capable of inhibitingListeriasp. (Listeria innocua). Of 4150 assumed lactic acid bacteria colonies from MRS agar plates that were screened by an agar-overlay method 58 (1.4%) were positive. Forty four of these strains were further characterized and 43 strains were inhibitory againstListeria monocytogenes. The strains were inhibitory to other Gram-positive (lactic acid) bacteria probably because of production of bacteriocins. All 44 strains inhibited bothVibrio choleraeandVibrio parahaemolyticusand 37 were inhibitory to a mesophilic fish spoilage bacterium (anAeromonassp.). Inhibition of Gram-negative bacteria was attributed to production of lactic acid. Most strains were identified asLactobacillusspp., and all grew well at ambient temperatures (25–37°C) and tolerated up to 6.5% NaCl. Glucose was fermented rapidly in laboratory media whereas pH decreased only very slowly in fish juice supplemented with 4% glucose and 3.5% NaCl or in a rice–fish mixture. Only four of 44 isolates could degrade and ferment complex carbohydrates such as rice, potatoes and maize starch. This indicates that other types of bacteria may be responsible for the rapid spontaneous fermentation of the products or that other yet-unknown factors ensure rapid fermentation. Overall anti-listerial lactic acid bacteria do occur in fermented fish products and the antibacterial activity against pathogenic bacteria indicates that they may be important in product safety.     

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.     

Esterification of polyunsaturated fatty acids with lipases from different sources

Enzymatically catalyzed esterification between glycerol and polyunsaturated fatty acids were studied for six lipases of different biological origin. Most efficient was lipase from Mucor miehei (yeast) and Chromobacterium viscosum (bacterium) which incorporated free fatty acids in the glycerol to 75% and 80%, respectively. Both lipases showed a slight preference for oleic acid. M. miehei lipase incorporated eicosapentaenoic acid at the same level as the acid occurred in the free fatty acid fraction while C. viscosum lipase incorporated the acid at a lesser level. Both lipases esterified less docosahexaenoic acid.     

Proteinase and Peptidase Activities of Cell-Free Extracts from Mutant Strains of Lactic Streptococci

Cell-free extracts prepared from two lactose-negative, proteinase-negative mutant strains of lactic streptococci, Streptococcus lactis (25Sp), and S. cremoris (KHA2), and the parent strains, S. lactis C2 and S. cremoris (KH), were evaluated for proteinase and peptidase activities. Of the mutant cultures tested, the extract from S. lactis (25Sp) had the highest aminopeptidase activity as determined with the substrates L-lysyl-para-nitroanilide, L-leucyl-para-nitroanilide, and L-prolyl-para-nitroanilide. Extract from the same strain also had the highest dipeptidase (DL-alanylglycine and DL-leucylglycine served as substrates) activity when cells were initially grown in a suitable medium with pH maintained at 6 or 6.5 by addition of NH₄OH. The cell-free extracts from S. lactis (25Sp) and S. cremoris (KHA2) had higher enzyme activities (aminopeptidase, and dipeptidase) and had lower intracellular proteinase activity (N,N-dimethyl casein served as substrate) than did extracts of the parent strains when they were grown at pH 6.5. Electrophoretic patterns of cell-free extracts indicated different bands with various enzyme specifities. Both mutant strains had similar specificity for different aminoacyl-β-naphthylamides. Minor differences were noted for dipeptidase activities.     

Effect of yeast and bacterial adjuncts on the CLA content and flavour of a washed-curd,dry-salted cheese

Three strains of Propionibacterium freudenreichii ssp. shermanii that converted free linoleic acid to conjugated linoleic acid (CLA) in laboratory media were used as adjunct strains, together with strains of Geotrichum candidum and Yarrowia lipolytica, to make a dry-salted, washed-curd cheese. Lactobacillus fermentum was included to produce ethanol (from lactose), a potential substrate for ethyl ester synthesis, while Lactobacillus rhamnosus was used to control the adventitious non-starter lactic acid bacteria population. The total (esterified plus free) level of CLA was similar in the control and experimental cheeses and remained unchanged over 4 months of ripening. Addition of linoleic acid-rich safflower oil to the cheese curd increased the concentration of free linoleic acid generated in the cheese but the CLA content did not change. Free linoleic acid was released by the yeast lipase(s) but there was no conversion to CLA. High concentrations of ethyl esters were produced in the cheeses made with added yeast, giving a fruity flavour.     

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.     

Studies on rumen metabolism. VI. In vitro hydrolysis of triglyceride and isolation of a lipolytic fraction

Hydrolysis of groundnut oil and synthetic glyceryl tri(oleate-1-¹⁴C) by bovine rumen contents was demonstrated under in vitro conditions. Fractionation by differential centrifugation of total rumen contents obtained from a cow grazing on fresh pasture showed that the organisms responsible for hydrolysis of triglycerides were closely associated with the particulate material in the rumen and homogenisation released much of the lipolytic activity. Cell-free extracts of mixed rumen bacteria were prepared by subjecting mixed rumen bacteria to high frequency sonication or osmotic shock followed by isolation of the released lipolytic enzymes by high-speed centrifugation. A method for assaying lipase activity is described which uses glyceryl tri(oleate-1-¹⁴C) as substrate, and thin-layer chromatography to separate the free fatty acids, mono-, di- and tri-glycerides prior to radio-chemical determination. The method was developed using pancreatic lipase and was applied to the isolation of lipases from rumen micro-organisms.     

Microbiological and biochemical changes during ripening of salchichón,a Spanish dry cured sausage

Development of the microflora and changes in the lipid and protein fractions during the ripening of salchichón, a kind of Spanish dry cured sausage, were studied. A commercial mixture of Pediococcus pentosaceus andMicrococcus varians was used as starter culture. Total aerobic bacteria, lactic acid bacteria, Micrococcaeae, Staphylococcus aureus, Entero-bacteriaceae , sulphite reducer Clostridium , yeasts and moulds were determined. The sarcoplasmic and myofibrillar solubility, denaturation and proteolysis index, parameters related to fat stability and the free fatty acid composition were analysed throughout the curing process: after mincing, after the fermentation stage, after the 2nd week of drying and in the final product (4th week of drying). The major changes in the characteristics of salchichón took place during the fermentation stage. This was due to proteolysis and lipolysis phenomena derived from high counts of lactic acid bacteria and Micrococcaeae in this stage. These changes were shown in the increase of the total free amino acid content, the peroxide index and the carbonyl compound content, and in the decrease of the protein solubility and the percentage of polyunsaturated free fatty acids, thus achieving the organoleptic characteristics of the final product.     

The absence of endogenous lipase from oil palm mesocarp

Oil palm (Elaeis guineensis Jacq.) fruit mesocarp had higher levels of free fatty acids at the ends of the fruits than in the middle, and microscopy showed that yeasts penetrate the mesocarp via the scars at each end. Very low levels of esterase activity, probably not due to a lipase, were detected in mesocarp extracts by using 4-methyl umbelliferone esters as substrates, but no activity against triacylglycerols could be found. In bruising and storage trials, the only variable that correlated (P<0.01) with free fatty acid level was yeast and mould count. It is concluded that yeasts and moulds are the source of lipolytic activity in oil palm mesocarp, and there is no evidence for an endogenous lipase.     

Discrimination against diacylglycerol ethers in lipase-catalysed ethanolysis of shark liver oil

Lipase-catalysed ethanolysis of squalene-free shark liver oil was investigated. The mentioned shark liver oil was comprised mainly of diacylglycerol ether and triacylglycerols. In order to test discrimination against diacylglycerol ether, up to 10 different lipases were compared. The ratio of oil to ethanol and lipase stability were also evaluated. Surprisingly, lipase from Pseudomonas stutzeri was the fastest biocatalyst among all assayed, although poor discrimination against diacylglycerol ether was observed. The best results in terms of selectivity and stability were obtained with immobilised lipase from Candida antarctica (Novozym 435). Ethanolysis reaction after 24 h in the presence of Novozym 435 produced total disappearance of triacylglycerol and a final reaction mixture comprised mainly of diacylglycerol ethers (10.6%), monoacylglycerol ethers (32.9%) and fatty acid ethyl esters (46.0%). In addition, when an excess of ethanol was used, diacylglycerol ethers completely disappeared after 15 h, giving a final product mainly composed of monoacylglycerol ethers (36.6%) and fatty acid ethyl esters (46.4%).     

Concentration of n‐3 polyunsaturated fatty acids in cholesterol‐reduced cod‐liver oil by lipases

This study was conducted to elucidate the effects of different lipases originated from Candida rugosa (CR), porcine pancreas (PP) and Aspergillus niger (AN) on the degree of hydrolysis (DH) in cholesterol‐reduced cod‐liver oil (87.5%) and evaluate the changes in n‐3 polyunsaturated fatty acid concentrations in the oil hydrolysed by the lipases. The lipase‐catalysed hydrolysis of cholesterol‐reduced cod‐liver oil was performed at 37 °C for 8 h. Among all the lipase samples studied, DH in the oil after lipase‐catalysed hydrolysis followed the decreasing order: CR lipase (70.01%) > PP lipase (26.18%) > AN lipase (18.57%). Triacylglycerol levels in the oil hydrolysed by all the lipases studied decreased, while mono‐ and diacylglycerol levels increased during lipase‐catalysed hydrolysis. The eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations in cholesterol‐reduced cod‐liver oil hydrolysed by the CR lipase were remarkably higher than those by the PP or AN lipase. Thus, it is suggested in this study that the CR lipase appears to be most suitable for producing and n‐3 polyunsaturated fatty acids including EPA and DHA concentrates from cholesterol‐reduced cod‐liver oil.     

Isolation and Screening of Lipase-Producing Fungi with Hydrolytic Activity

Lipases are enzymes that can be secreted by several microorganisms, making interesting the biodiversity exploration for searching new microorganisms able to produce these enzymes. Many agro-industrial residues can be used as potential substrates for production of enzymes. The main objective of this work was the isolation and screening of microorganisms with potential to produce lipases. Among 24 fungi, five were selected as good lipase producers using tributyrin on agar plates and solid state fermentation of soybean bran. Two of them were isolated from soil samples, another two from soybean bran, and one from dairy products. These fungi were identified by microcultivation technique as from Penicillium and Aspergillus genera. Through random amplified polymorphic DNA technique, the most promising strains could be genetically discriminated, selecting two fungi as good lipase producers but genetically different. One isolated from soybean bran could hydrolyze efficiently triglycerides with fatty acids with different chain length. Another isolated from dairy products was only effective to hydrolyze triglycerides with long-chain fatty acids. Two distinct groups could be verified by means of this technique, comprising the most productive strains and the lowest or nonproductive ones in terms of hydrolytic activity.     

Ester synthesis by lactic acid bacteria isolated from goat’s and ewe’s milk and cheeses

The present work evaluates the ability of lactic acid bacteria isolated from goat’s and ewe’s milk and cheeses to synthesise short-chain fatty acid esters. In order to elucidate the mechanisms of ester synthesis involved, cell-free extracts were incubated in sodium phosphate buffer containing triglyceride plus ethanol (alcoholysis) and free fatty acid plus ethanol (esterification). After 24 h incubation at 37 °C esters were extracted and determined by gas chromatography. Strains evaluated were able to synthesise ethyl esters from 2 to 10 carbon atoms, mainly ethyl butanoate and ethyl hexanoate. A great variability amongst strains was observed. In general, higher ester-forming activities by esterification were detected. In enterococci strains the alcoholysis mechanism was also involved.     

Lipase-catalysed production of triacylglycerols enriched in pinolenic acid at the sn-2 position from pine nut oil

BACKGROUND: The purpose of this study was to produce triacylglycerols (TAGs) enriched in pinolenic acid (PLA) at the sn‐2 position using the principle of acyl migration, from the pine nut oil containing PLA esterified exclusively at the sn‐3 position. RESULTS: Two types of lipase‐catalysed reactions, i.e. redistribution and reesterification of fatty acids, were successively performed using seven commercially available lipases as biocatalysts. Of the lipases tested, Novozym 435 and Lipozyme TL IM were effective biocatalysts for positioning PLA at the sn‐2 location. These biocatalysts were selected for further evaluation of the effects of reaction parameters, such as temperature and water content on the migration of PLA residues to the sn‐2 position and TAG content. For both lipases, a significant decrease in TAG content was observed after the lipase‐catalysed redistribution of fatty acids for both lipases. The reduced TAG content could be enhanced up to approx. 92%, through lipase‐catalysed re‐esterification of the hydrolysed fatty acids under vacuum. CONCLUSION: TAG enriched in PLA at the sn‐2 position was synthesised from pine nut oil via lipase‐catalysed redistribution and re‐esterification of fatty acid residues using Lipozyme TL IM and Novozym 435 as biocatalysts. Copyright © 2011 Society of Chemical Industry     

Evaluation of microbial dynamics during the ripening of a traditional Taiwanese naturally fermented ham

Isolation and identification of the autochthonous starter from a naturally fermented meat allows control of the fermentation process and promises microbiological safety for this specialty. Thus the purpose of this study was to identify the lactic acid bacteria and coagulase-negative cocci present in a traditional Taiwanese naturally fermented ham (TNFH) and to study the microbial dynamics at different ripening stages; the approach was a combination of conventional microbiological cultivation, polymerase chain reaction-denaturing gradient gel electrophoresis and DNA sequencing. In total, twelve different strains of lactic acid bacteria and three Staphylococcus strains were identified in the TNFH samples, whereas only 5 dominant strains were observed in the TNFH samples when the DGGE as a culture-independent method was applied. The bacterial ecology on the surface of the samples was mainly characterized by the stable presence of Lactobacillus sakei and Staphylococcus saprophyticus; nonetheless Leuconostoc mesenteroides and Carnobacterium divergens were the most abundant bacteria found in the final product. These results are also agreed with the findings of the culture-independent method. In addition, Microbacterium spp., Carnobacterium spp., Enterobacter spp., Brochothrix spp., Enterococcus spp., and Bacillus spp. were also present at the beginning of the ripening, but few bacteria were found at the center of the TNFH samples during the early ripening stages. However, after 30 days of ripening, the microbial ecology at the center of the TNFH samples paralleled that of the surface. Finally, as far as we have been able to determine, our report is the first to investigate the microbiological dynamics in fermented meat products using combination of cultivation, the Harrison disc method, DGGE and DNA sequencing as the culture-dependent method. Our report is also the first to show the presence of Staphylococcus arlettae in a fermented sausage and ham product.     

Gel Matrix Influence on Hydrolysis of Triglycerides by Immobilized Lipases

The hydrolytic activities and specificities of gel-entrapped C. cylindracae lipase (CCL) and R. arrhizus lipase (RAL) toward olive oil and tributyrin were investigated. Lipases in hydrophobic gels with the longest chain lengths generally displayed highest activity. The optimal temperature was 30–35° for free and 37–40° for gel-entrapped lipases. The ratio of the activity on tributyrin to that of olive oil (expressed as T/O ratio), an indicator of substrate specificity, increased from 0.3 for free lipases to 12.3 ± 2.3 for CCL lipase in ENTP-2000-formed gel and 16.2 ± 0.3 for RAL lipase in ENTP-4000-formed gel.     

无溶剂系统脂肪酶催化POMF转酯生产类可可脂的研究

研究了在无溶剂系统中，利用1，3位置特异性脂肪酶催化POMF与硬脂酸甲酯进行酯交换反应制取类可可脂。探讨了反应温度、振荡速度、底物配比、酶浓度、加水量等因素对酶促酯交换反应的质量产率和特异性酯交换速率的影响，并对经减压蒸馏得到的产品进行了有关性能鉴定。