Biocatalysis in Modification of Lipids for Food Industry

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1 Biocatalysis in Modification of Lipids for Food Industry Casimir C. Akoh Department of Food Science and Technology The University of Georgia Athens, Georgia USA Latin American Congress & Exhibition on Fats and Oils (AOCS), Chile August 22, 2013

2 Biotechnology and Biocatalysis Biotechnology refers to the integrated application of the disciplines of biochemistry, microbiology, genetics, and process engineering, particularly recombinant DNA technology and genetic engineering, to biologically derived molecules such as cells, organisms, or enzymes to produce useful products Biocatalysis or enzyme technology is a major part of the technological areas of biotechnology. Involves use of enzymes to catalyze reactions that yield useful products

3 Biocatalytic Modifications Terpenes Carbohydrates Fats and Oils Phytosterols

4 Enzymes are Work Horses We employ enzymes to do a job Conditions for employment must be right Provide tools to do the job Right conditions of: temp, cations, water, ph, enough time enzyme will accept the job and is done Wrong conditions yo-yo job, underemployment, or no product

5 Enzymes Cyclases Monoterpenes (C10) - GPP (menthol, pinene) flavor compounds Sesquiterpenes (C15) - FPP (patchoulol, caryophyllene) fragrance compounds Proteases chymotrypsin, subtilisin (serine hydrolase) Proteins but can synthesize lipids Lipases (esterases, phospholipases) - lipids, many substrates Carbohydrases (e.g., amylase family) carbohydrate hydrolysis and synthesis

6 acetyl-coa HMG-CoA mevalonic acid IPP DMAPP GPP menthol -PP = Pyroor diphosphate FPP patchoulol Mevalonic acid pathway to different terpenes Source: McCaskill and Croteau (1997) Adv Biochem Eng Biotechnol

7 GPP acyclic Fenchol cyclic Cyclization requires dication Mg++ or Mn++ Cyclization of GPP to monoterpenes by cyclases Source: McCaskill and Croteau (1997) Adv Biochem Eng Biotechnol

8 patchoulol t-germacradienyl cation caryophyllene t,t-fpp Cyclization of FPP to sesquiterpenes by cyclases Source: McCaskill and Croteau (1997) Adv Biochem Eng Biotechnol

9 Patchoulol Biosynthesis and Mechanism t,t-fpp Hydride shift mechanism, no germacrene or bulnesene intermediates (from 14C and 3H labeled FPP) Patchoulol cyclase patchoulol

10 Lipase-Catalyzed Flavor Synthesis Time course of enzymatic synthesis of geranyl and citronellyl acetates in n-hexane catalyzed by Candida antarctica lipase, SP435 Source: Claon and Akoh, 1994

11 Terpene Butyrates Using Various Substrates Catalyzed by Lipases Source: Claon and Akoh, 1994

12 Our Lipid Research Interests Structured lipids (SL) Production of healthier lipids (lower cholesterol, improve immune system, reduce weight and obesity) Lipids for better functionality in foods (salad dressing, margarine, shortenings, spreads, cocoa butter alternatives, cooking and frying oils) Trans-free fats Incorporation of omega-3 fatty acids from fish oil, algae, SDA soybean (infant formula, better n-3/n-6 ratios) GLA-containing modified lipids CLA-containing modified lipids Infant formula (human milk fat substitutes) Oxidation of SL and SL emulsions Value addition to palm oil and other lipids Phytosteryl esters Terpene esters as flavor materials General fats & oils chemistry and biochemistry

13 SL Production Direct esterification: Glycerol + FA TAG + Water Acidolysis: TAG 1 + FA 1 TAG 2 + FA 2 Alcoholysis: TAG 1 + Alcohol 1 TAG 2 + Alcohol 2 Interesterification: TAG 1 + TAG 2 TAG 3 + TAG 4 Chemical Enzymatic Non-specific Poor control over the final product Harsh reaction conditions Both specific and non-specific Good control Milder reaction conditions

14 Lipase Reactions in Organic Solvent (Synthesis) Source: Weete, Lai and Akoh (2008) In Food Lipids Book

15 Reactions in Organic Solvent (Interesterification and Hydrolysis) Source: Weete, Lai and Akoh (2008) In Food Lipids Book

16 Cocoa Butter Substitutes SOS and POS Source: Weber & Mukherjee (2008) In Food Lipids Book

17 Moisture Absorption by Lipid Coated Crackers Source: Sellappan and Akoh (2000), JAOCS 77:

18 Human Milk fat Analogs (HMF) Human milk fat TAG Vegetable oil TAG sn-1 UFA MCFA / LCFA / PUFA SFA sn-2 SFA (Palmitic acid >60 %) UFA sn-3 UFA MCFA / LCFA / PUFA SFA

Ca P U sn-3 Triacylglycerol (TAG) sn-2 monopalmitin U P TAG P Ca P P

19 Background and significance Loss of nutrients U P Human milk fat sn-1 Pancreatic lipase sn-2 P U Vegetable oil P U Ca 2+ Pancreatic lipase P U P Ca P U sn-3 Triacylglycerol (TAG) sn-2 monopalmitin U P TAG P Ca P P Calcium dipalmitate P= palmitic acid U= unsaturated fatty acids Nagachinta and Akoh, 2013

20 Human Milk Fat Analogs OPO DOD or GOG Source: Weber & Mukherjee (2008) In Food Lipids Book

21 Modified Lipids for Infant Formula SL resembling human milk fat (HMF) containing GLA SL resembling HMF containing EPA and DHA Lipozyme RM IM or lipozyme TL IM as biocatalysts Substrates: Tripalmitin + Hazelnut oil FAs + GLA OR Tripalmitin + Fish oil fatty acids Sahin et al. (2005) J Agric Food Chem

22 Optimal Condition and Results Temp = 55 ºC, Time = 24 h, substrate molar ratio = 14 Results: HMF enriched with EPA and DHA (45.5% palmitic, 37.5% oleic, 4.4% linoleic, 6.2% EPA and DHA) HMF enriched with GLA (10% GLA, 45% oleic) Sn-2 position mostly occupied by palmitic acid (>60%) Sahin et al. (2005) J Agric Food Chem

23 Phytosteryl Esters Plant sterols Lower plasma total cholesterol and LDL-chol by (10-20%) in humans Used in margarine, spread, milk, salad dressing Solubility problem with free sterol Phytosteryl esters preferred in food formulations (lower m.p., increased solubility) Chemical or enzymatic synthesis In organic solvent or oil/water phase Kim and Akoh (2007) Food Chem

24 O O Esterification with Free Fatty Acids phytosterol - crystalline powder - restricted fat-solubility - melting range C HO Sitosterol Lipase, CRL Oleic acid + H 2 O phytosteryl ester - similar fat properties as edible fats - melting range C Sitosteryl oleate Kim and Akoh (2007) Food Chem

25 Candida rugosa Lipase-Catalyzed Synthesis Phytosterol + oleic acid in hexane (monophasic media) No water removal RSM Factors considered Temp = 35-55ºC; Time = 4-24 h; substrate molar ratio (oleic acid:phytosterol = 1-3; lipase = 2-10 weight% Measure degree of esterification (mol%) Optimal Condition: Temp = 51.3ºC Time = 17 h Substrate molar ratio = 2.1 Lipase = 7.2% Degree of esterification = 97 mol% Kim and Akoh (2007) Food Chem

26 Enzymes Can be Trained to Perform Better But first Normal Reactions Substrate specific Regiospecific Optimal reaction conditions very good job Genetic engineering Okay, will take another substrate Will tolerate higher temperature Will handle unusual ph Cut and paste amino acid regions chimeric or recombinant enzymes Protein engineering Fusion enzymes more than one enzyme (e.g., linear and debranching amylases)

27 Lipases Esterify carbohydrates sugar fatty acid esters as emulsifiers, antimicrobials, crystal inhibitors Esterify terpene alcohols Modify TAGs, DAG, MAG, PLs Esterify primary and secondary alcohols Peptide bond formation?

28 Mono- and Diacylglycerols partial hydrolysis esterification Source: Weber & Mukherjee (2008) In Food Lipids Book

29 Sugar and Alkyl Glycosides Esters sugar ester alkyl glycoside ester Source: Weber & Mukherjee (2008) In Food Lipids Book

30 Phospholipid Synthesis Source: Weber & Mukherjee (2008) In Food Lipids Book

31 Enzyme Capabilities are Limitless! Not Really Esterification of 3º (tertiary) alcohols not easy If you motivate me, I will do more of the same job (enzyme reuse, wash and regenerate) Immobilize me I will not go away, will stay and work for you for a long time ph memory remembers the last aqueous solution exposed to for immobilized enzymes

32 Applied Biocatalysis Trans-free fats for margarine, shortening, and spreads Infant formula or milk fat substitutes or analogs Phytosteryl esters Sugar esters Pro-drugs Chiral intermediates Resolve racemic mixtures, e.g., (±)-menthol Flavor esters terpene esters and other short chain esters Cocoa butter equivalents, coating lipids Healthful lipids omega-3, CLA-, GLA-containing Esterify branched alcohols Biodiesel

33 You can stop enzyme Economic or Quality Reasons? Filter to remove immobilized enzyme Pour cold water on enzyme - you get a rain check Throw enzyme out of office terminate reaction Make the conditions for catalysis a living hell Confuse enzyme wrong substrate or conditions

34 Optimization and Scale-up One factor at a time time consuming Response Surface Methodology, RSM better, less # of experiments Bioreactor and types: Stirred tank (liquid or semisolid substrates) large scale Packed bed (pump liquid substrates) large scale Test tube/flask small scale

substrate Jacketed bed with temperature maintained by a circulating water bath

35 Packed Bed Bioreactor Source: Willis and Marangoni (2008) In Food Lipids Book Stainless steel column bed 47 mm (internal diameter) x 50 cm long Pump to feed the substrate Jacketed bed with temperature maintained by a circulating water bath Temperature and pressure probes at inlet and outlet, connected to a computer for monitoring Dr Akoh s Lab

36 Source: Willis and Marangoni (2008) In Food Lipids Book Akoh s Lab

37 Recovery/Downstream Processing Very important will determine purity, yield, application, cost Filter enzyme out Acidification TLC and column chromatography small scale Short-path or molecular still distillation large scale Deodorization large scale, necessary for food application

38 Short-Path Distillation UIC KDL-4 (UIC Inc., Joliet, IL) First stage - removal of caprylic acid (heating oil temp, 75ºC; cooling water 15ºC; mix holding temp 40ºC) Second stage - removal of long-chain FA (heating oil temp 185ºC; cooling water 20ºC; mix holding 40ºC) Pressure in both cases - below 0.02 Torr Dr Akoh s Lab

39 Post Recovery/Processing Enzymatic Products - to Applications Physical, chemical, stability, and sensory properties Food and product application Animal study Human feeding trial/clinical for some Goal! Product in the market

40 trans-free Margarines Prepared with Enzymatically Synthesized Structured Lipids

41 Traditional margarine fats Margarine fats are traditionally produced by partial hydrogenation process Contain trans fatty acids (TFAs) (10-36% of total FA) TFAs are a risk factor for coronary heart disease (CHD)

42 Structured lipids (SLs) are a good alternative to partially hydrogenated fats The use of structured lipids (SLs) is one of the most successful alternatives to the partial hydrogenation process to reduce or eliminate TFAs in margarines SLs are restructured fats/oils where the composition and positional distribution of FAs are modified from the native state by chemical or enzymatic methods

43 High diversity in FA profile contributes to the formation of β crystal polymorphic form High diversity in FA composition contributes to the predominant presence of β crystal polymorphic form over other forms (α and β) β form is the most desirable in margarines because it gives smooth texture to the products β form is undesirable in margarines because it gives grainy texture to the products

44 Commercial margarine fats separated from CTMF: commercial trans margarine fat CTFMF: commercial trans-free margarine fat

45 FA profiles of lipid substrates (w/w%) canola oil palm stearin palm kernel oil fatty acid total sn-2 sn-1,3 total sn-2 sn-1,3 total sn-2 sn-1,3 C6:0 62% C8:0 trace C10:0 trace C12: C14:0 97% 97% C16: C16:1n trace 0.2 C18: C18:1t trace 0.1 C18:1n C18:1n C18:2t,t C18:2n C18:3n C20: C20: trace SFA USFA TFA trace 0.1

46 SLs synthesis SLs were produced by Lipozyme TL IMcatalyzed interesterification of the blends of: Canola oil (CO) - provide oleic acid (cholesterol-lowering effects) Palm stearin (PS) - contribute to rigidity Palm kernel oil (PKO) - contribute to high diversity in FA and TAG profiles Blending ratio (CO:PS:PKO, w/w/w) 40:60:0 SL460 40:50:10 SL451 40:40:20 SL442 40:30:30 SL433 50:30:20 SL532 60:25:15 SL621

47 Margarine formulation Ingredient w/w % Lipid phase 80.5 Commercial margarine fats or SLs 80.0 Soy lecithin fluid 0.5 TBHQ 0.01 Aqueous phase 19.5 Distilled water 17.7 Table salt 1.8

48 Total FA profile (w/w%) commercial margarine fat structured lipids fatty acid CTMF CTFMF SL460 SL451 SL442 SL433 SL532 SL621 C6:0 trace trace trace C8:0 trace C10:0 trace C12: C14: C16: C16:1n-7 trace C18: C18:1t 14.9 C18:1n C18:1n C18:2t,t 0.1 C18:2n C18:3n C20: C20: SFA USFA TFA 15.0

49 Melting completion temperature (DSC) commercial margarine fat CTMF CTFMF structured lipids SL460 SL451 SL442 SL433 SL532 SL621 melting completion temperature ( C) 40.1 c 32.0 f 43.5 a 40.6 b 37.0 d 33.5 e 33.9 e 31.7 f

50 Solid fat content (pulsed NMR)

51 Polymorphic form of fat crystal at 4 C (X-ray diffractometer) polymorphic form level of β and β forms commercial margarine fat CTMF α + β + β β >> β CTFMF α + β + β β = β structured lipids SL460 α + β + β β = β SL451 α + β + β β > β SL442 α + β + β β > β SL433 α + β + β β > β SL532 α + β + β β > β SL621 α + β + β β > β

52 SL532 and SL621 were most suitable for formulating trans-free margarines with desirable nutritional and textural properties Trans-free Lower and similar atherogenicity compared to CTMF and CTFMF, respectively Good cold-spreadability Smooth texture Similar textural properties to CTMF and CTFMF margarines, respectively

53 Food and Functional Applications of SL Beverages, infant formula, milk drinks Margarine, butter, spreads, shortening, dips all trans free Salad dressing, sauces Frying oil, snack foods Confectionaries, coating lipid, soft candies Cocoa butter substitutes, alternatives, equivalents, chocolates Dairy products, ice cream Improve melting and crystallization properties of fats Baking chips, baked goods Capsules, e.g., DHA, EPA, CLA-containing SL as nutritional supplements or delivery mechanism

54 Potential Health Benefits of SL Enhanced absorption of sn-2 fatty acid Prevention of thrombosis Improved immune function Calorie reduction Improved absorption of other fats Reduction in serum TAG, LDL cholesterol, and total cholesterol Lipid emulsion for enteral and parenteral feeding Incorporation into mainstream diets Healthful and digestible fats

55 Thank You!! Thank you!

Overview of the food science behind fatty acid technology

Overview of the food science behind fatty acid technology Pamela J. White, Ph.D. Food Chemist/Scientist Food Science and Human Nutrition Dept. & Center for Crops Utilization Research Iowa State University