Production and nutritional aspects of human milk fat substitutes

Human milk fat substitute (HMFS), one of the vital ingredients of infant formula, constitutes about 50% of energy intake for many infants. The composition of HMFS depends on the raw material and the production technology. Both human and animal studies have shown that absorption of fats and minerals are affected by triacylglycerol structures and fatty acid compositions of HMFS. Additionally, the level of other micronutrients, such as fat‐soluble vitamins, can also be affected by production processes. The ultimate goal in the design of infant formula is to mimic human milk, regarding both lipids and other micronutrients. This paper covers human milk fat and selected HMFS as well as their impact on absorption of fat and calcium and illustrates recent progresses and findings.     

Preparation and Characterization of Human Milk Fat Substitutes Based on Triacylglycerol Profiles

Human milk fat substitutes (HMFS) having similarity in (TAG) composition to human milk fat (HMF) were prepared by Lipozyme RM IM‐catalyzed interesterification of lard blending with selected oils in a packed bed reactor. Four oil blends with high similarity in fatty acid profiles to HMF were first obtained based on the blending model and then the blending ratios were screened based on TAG composition similarity by enzymatic interesterification in a batch reactor. The optimal ratio was determined as lard:sunflower oil:canola oil:palm kernel oil:palm oil:algal oil:microbial oil = 1.00:0.10:0.50:0.13:0.12:0.02:0.02. This blending ratio was used for a packed bed reactor and the conditions were then optimized as residence time, 1.5 h; reaction temperature, 50 °C. Under these conditions, the obtained product showed high degrees of similarity in fatty acid profile with 39.2 % palmitic acid at the sn‐2 position, 0.5 % arachidonic acid (n‐6) and 0.3 % docosahexaenoic acid (n‐3) and the scores for the degree of similarity in TAG composition was increased from 58.4 (the oil blend) to 72.3 (the final product). The packed bed reactor could be operated for 7 days without significant decrease in activity. The final product presented similar melting and crystallization profiles to those of HMF. However, due to the loss of tocopherols during deacidification process, the oxidative stability was lower than that of the oil blend. This process for the preparation of HMFS from lard with high similarity in TAG composition by physical blending and enzymatic interesterification, as optimized by mathematical models in a packed bed reactor, has a great potential for industrialization.     

Biotechnological and Novel Approaches for Designing Structured Lipids Intended for Infant Nutrition

Human milk fat (HMF) is a perfect nutritional source that includes all the required ingredients which are necessary for the growth of infants up to 6 months. Although its composition may differ among mothers or during lactation stage, its unique triacylglycerol (TAG) structure remains constant which is characterized by the presence of palmitic acid (PA) at the sn‐2 position. Previous reports provided convincing information of higher PA and calcium absorption and efficient use of dietary energy when at this specific position in the TAG moiety than when PA is at the sn‐1,3 positions. During the design of structured lipids (SLs) intended for infant nutrition, this unique property is taken into consideration. Human milk fat substitutes (HMFS) enriched with important fatty acids such as omega‐3 and omega‐6 fatty acids are intended to better mimic the functions of HMF as well as provide associated health benefits. The use of microencapsulation technology and novel technologies such as ultrasound technology in conjunction with SL production and enzyme‐catalyzed reactions are evolving and ongoing issues in infant formula production. Therefore, further studies should be directed towards new process improvements in order to increase the functional properties and oxidative stabilities of HMFS. Novel technologies in lipid biotechnology related to HMFS preparation should also be explored.     

Characterization and Oxidative Stability of Structured Lipids: Infant Milk Fat Analog

Structured lipids (SLs) for infant milk formulation were produced by enzymatic interesterification of tripalmitin with vegetable oil blends and fish oil. The SLs were characterized by fatty acid content and structure, melting profiles, oxidative stability index, free fatty acid (FFA) concentration, and tocopherol content. Oxidative stability was studied using accelerated methods by quantifying FFA, peroxides (peroxide value) and aldehydes (p-anisidine value) production. Total oxidation (TOTOX value) was calculated as 2 × (peroxide value) + (p-anisidine value). The structured lipids after purification by distillation and addition of antioxidants had melting profiles, oxidative stability index, and initial FFA concentration that were similar to that of the starting oil blends, while the fatty acid composition and structure of the SLs were similar to that of human milk fat. Oxidative stability of the SLs was improved with tocopherol addition as antioxidants and was comparable to that of the vegetable oils and oil blends.     

Lipase‐catalyzed interesterification reactions for human milk fat substitutes production: A review

The synthesis of human milk fat substitutes (HMFS) which show a high degree of similarity to human milk fat (HFM) is of great interest to ensure a supply in infant nutrition with a triacylglyceride composition as close as possible to mothers milk. Biocatalyzed modifications of natural oils using microbial or plant‐derived enzymes enable the production of high‐value HMFS. Due to the mild reaction conditions and the exceptionally high sn‐1,3‐regioselectivity of these enzymes, they are preferred over chemical catalysis in the development of these lipids with desired nutritional and functional properties. In this article, research spanning over 20 years of lipase‐catalysis for the production HMFS is reviewed. Specific attention is paid to the evaluation of the regiospecificity of the biocatalysts, choice of natural oils and acyl donors, production processes, purification of the HMFS, and analytical procedures for their characterization.     

Formulation and Characterization of Human Milk Fat Substitutes Made from Blends of Refined Palm Olein,and Soybean,Olive, Fish,and Virgin Coconut Oils

The simplest and the most cost-effective way of human milk fat substitute (HMFS) production is formulating of suitable vegetable oils at proper ratios. To do this, the D-optimal mixture design was used to optimize the HMFS formulation. The design included 25 formulations made from refined palm olein (35–55%), soybean oil (5–25%), olive oil (5–20%), virgin coconut oil (5–15%), and fish oil (0–10%). Samples were produced in laboratory and characterized in terms of fatty acid and triacylglycerol (TAG) compositions, free fatty acid content, peroxide value, iodine value, and oxidative stability index (OSI). HMFS samples were also compared with Codex Alimentarius (CA) and Iran National Standards Organization (INSO) standards. Each characteristic of HMFS samples was then expressed as a function of ingredient ratio using regression models. Finally, using numerical optimization, four optimized blends (PB₁-PB₄) were selected, made in the laboratory (HMFS₁-HMFS₄), characterized, and compared with CA and INSO standards. The properties of all the optimized blends (except the palmitic acid content of HMFS₂ and the monounsaturated fatty acid [MUFA] content of HMFS₃) met the standards. HMFS₄ showed the highest OSI in Rancimat and the lowest oxidation rate in Schaal oven tests. POL (19.53–21.73%), PPO (20.77–21.73%), OOO (9.11–11.16%), and OPO (8.84–9.46%) were the main (totally about 60%) TAG species found in HMFS samples. In conclusion, the HMFS₄ formula (55% palm olein, 13.5% soybean oil, 16% refined olive oil, 15% virgin coconut oil, and 0.5% fish oil) was suggested as the best formula for HMFS production.     

Characterization and Oxidative Stability of Human Milk Fat Substitutes Enzymatically Produced from Palm Stearin

Production of human milk fat substitutes (HMFSs) from three types of palm stearin with palmitic acid (PA) of 91.3, 70.3 and 62.6 %, respectively, was scaled up to a kilogram scale. The physiochemical properties of these products including fatty acid profiles, triacylglycerol compositions, tocopherol contents, oxidative stability and melting and crystallization profiles were compared with those of HMFSs from lard, butterfat and tripalmitin and fats from infant formulas. Based on their chemical compositions, HMFSs from palm stearin with PA contents of 70.3 and 62.6 % produced by enzymatic acidolysis were found to have the highest degree of similarity to human milk fat, which indicated that these HMFSs were the most suitable for use in infant formulas. However, HMFSs from palm stearin with PA content of 91.3 % had the highest tocopherol contents. By investigation of the primary and secondary oxidation products during accelerated oxidation, the oxidative stability of HMFSs was found to be positively correlated to the contents of tocopherols, and the volatile oxidation compounds with the highest relative contents in HMFSs were aldehydes analyzed by solid-phase microextraction-GC–MS. All HMFSs had final melting points lower than body temperature.     

Production of Human Milk Fat Substitutes by Interesterification of Tripalmitin with Ethyl Oleate Catalyzed by Candida parapsilosis Lipase/Acyltransferase

In human milk fat, the saturated fatty acids, namely palmitic acid, are located at the sn-2 position of triacylglycerols (TAG) while unsaturated fatty acids (e.g. oleic acid) are esterified at position sn-1,3. Thus, sn-1,3-dioleoyl-2-palmitoylglycerol (OPO) is the target TAG to be used as human milk fat substitutes (HMFS) in infant formulas. In this study, the noncommercial recombinant lipase/acyltransferase from Candida parapsilosis (CpLIP2) was immobilized in Accurel MP1000, and used as a biocatalyst for the interesterification of tripalmitin with ethyl oleate in a solvent-free medium, to obtain structured lipids used as HMFS. Different molar ratios (MR) of ethyl oleate to tripalmitin (2:1–8:1) were used. After 4 h reaction at 60°C, about 30 mol% of oleic acid incorporation was already observed for all tested MR. An apparent equilibrium was reached after 8–24 h, with 32–51 mol% final incorporation, increasing with the MR. The incorporation of oleic acid into TAG was compared with the maximum predicted values when a random or a sn-1,3-regioselective biocatalyst was used. The obtained values are consistent with the maximum incorporation expected for a sn-1,3-regioselective enzyme. In fact, the amount of oleic acid at position sn-2 was approximately 15% for all the MR tested, which is explained by the acyl migration phenomenon. CpLIP2 exhibited higher activity than most commercial immobilized lipases (e.g. faster reaction in solvent-free media, low enzyme load, and low MR needed), and showed a recognized sn-1,3 regioselective behavior.     

Preparation of Human Milk Fat Substitutes from Lard by Lipase‐Catalyzed Interesterification Based on Triacylglycerol profiles

Human milk fat substitutes (HMFSs) with triacylglycerol profiles highly similar to those of human milk fat (HMF) were prepared from lard by physical blending followed by enzymatic interesterification. Based on the fatty acid profiles of HMF, different vegetable and single‐cell oils were selected and added to the lard. Blend ratios were calculated based on established physical blending models. The blended oils were then enzymatically interesterified using a 1,3‐regiospecific lipase, Lipozyme RM IM (RML from Rhizomucor miehei immobilized on Duolite ES562; Novozymes A/S, Bagsværd, Denmark), to approximate HMF triacylglycerol (TAG) profiles, particularly with respect to the distribution of palmitic acid in the sn?2 position. The optimized blending ratios were determined to be: lard:sunflower oil:canola oil:palm kernel oil:palm oil:algal oil:microbial oil = 1.00:0.10:0.50:0.13:0.12:0.02:0.02. The optimized reaction conditions were determined to be: enzyme load of 11 wt%, temperature of 60 °C, water content of 3.5 wt%, and reaction time of 3 hours. The resulting product was evaluated for total and sn?2 fatty acids, polyunsaturated fatty acids, and TAG composition. A high degree of similarity was obtained, indicating the great potential of the product as a fat alternative for use in infant formulas.     

The modification and analysis of vegetable oil for cheese making

Objectives of this study were (i) to incorporate short-chain fatty acids (SCFA) in vegetable oils to obtain a bland product that could be used as a milk fat substitute in cheese making and (ii) to improve the methods for fatty acid analysis of vegetable oils modified with SCFA. Short-chain triglycerides (SCTG) were synthesized by esterifying SCFA with glycerol, and using a toluene azeotrope to remove the water of esterification. SCFA from two sources were used: (i) commercial acids and (ii) acids isolated by double distillation of milk fat methyl esters. The SCTG had a bitter, unacceptable flavor, but after interesterification with high-oleic sunflower oil (HOSO) and deodorization, the flavor was quite acceptable. SCTG were incorporated into HOSO at 100 and 120% of the levels found in the milk fat, by sodium methoxide-catalyzed interesterification. For fatty acid analysis, milk fat and simulated milk fat were converted to their decyl ester derivatives and analyzed by gas chromatography without further purification. The method was accurate and rapid for fatty acid analysis of fats containing a wide range of fatty acid chain lengths. All modified HOSO gave bland and acceptable flavors and had a SCFA composition close to that of milk fat. Results from using the modified HOSO in cheese making are reported in a later paper.     

Lipids and human milk

To support the growth and development of the breast‐fed infant, human milk provides the dietary essential fatty acids, linoleic acid (LA; 18:2n‐6), α‐linolenic acid (ALA, 18:3n‐3), as well as longer‐chain polyunsaturated fatty acids including arachidonic acid (20:4n‐6) and docosahexanoic (DHA 22:6n‐3). The linoleic acid, alpha‐linolenic acid, DHA and arachidonic acid concentration of pasteurized and unpasteurized human milk remains stable during the first month of storage at –20°C and –80°C. However after the first month, a slow decrease in concentration progresses until the end of 6 months of storage at both temperatures. The levels of n‐6 and n‐3 fatty acids, particularly linoleic acid, alpha‐linolenic acid and DHA, in human milk vary widely within and among different populations, and are readily changed by maternal dietary intake of the respective fatty acid. The present paper reviews recent understanding from key researchers of maternal diet and human milk fat composition and form our work the effect of milk fat composition on storage conditions. It is important to understand that maternal diet can affect human milk fat composition and subsequently infant development and growth.     

The influence of dietary lipids on the composition and membrane fluidity of rat hepatocyte plasma membrane

Weanling male Wistar rats were fed for five weeks on standard rat chow (23 g fat/kg diet) or one of four synthetic diets with butterfat, coconut oil, corn oil, or fish oil as the main lipid source (100 g fat/kg diet). In all diets, 10% of the fat was provided as corn oil to prevent essential fatty acid deficiency. Significant differences were observed in the saturated, monounsaturated, and polyunsaturated fatty acid composition, and in the ratio of cholesterol to phospholipid, in the hepatocyte membranes. The fluidity of hepatocyte plasma membranes was assessed using the fluorescence recovery after photobleaching technique and steady-state fluorescence anisotropy of diphenylhexatriene. No significant differences were found in the fluidity of plasma membranes between animals on the different fat diets, despite diet-induced changes in their fatty acid composition. However, the proportion of lipid free to diffuse in the plasma membrane varied with diet, being significantly greater (P<0.05) in animals fed chow (63.7%), coconut oil (61.5%), and butterfat (57.6%) diets than in those fed the corn oil (47.3%) diet. Animals fed fish oil showed an intermediate (50.0%) proportion of lipid free to diffuse. The data support the hypothesis that dietary lipids can change both the chemical composition and lateral organization (lipid domain structure) of rat hepatocyte plasma membranes.     

Dietary fat and fatty acids modulate cholesterol cholelithiasis in the hamster

We tested two hypotheses, i) whether the type and the amount of fat in the diet will affect the formation of cholesterol gallstones in the hamsters, and ii) whether palmitic acid, a major fatty acid component of butterfat, can act as a potentiator of cholesterol cholelithiasis in the hamster. Young, male golden Syrian hamsters (Sasco) were fed a semipurified diet containing casein, corn starch, cellulose and cholesterol (0.3%) to which various types and amounts of fat (butterfat, olive oil, menhaden oil, corn oil) were added. All diets contained 2% corn oil to supply essential fatty acids to the growing hamsters. No deaths or illness occurred during the experiment. Animals fed the semipurified diet plus 4% butterfat (group 1) had a gallstone incidence of 63%. Replacement of butterfat with either olive oil, corn oil or menhaden oil prevented the formation of cholesterol gallstones entirely (groups 2–4). When total butterfat was increased from 4% to 8% (group 8), the incidence of cholesterol gallstones increased to 80%. Substitution of 4% olive oil (group 5), corn oil (group 6), or menhaden oil (group 7) for the additional 4% butterfat significantly reduced gallstones to 35%, 45% and 30%, respectively. The replacement of 4% butterfat with 1.2% palmitic acid gave the highest incidence of cholesterol gallstones (95%). These results suggest that butterfat (and one of its components, palmitic acid) intensifies gallstone formation in this model whereas mono- and polyunsaturated fats act as inhibitors of cholesterol cholelithiasis. A fatty acid, possibly palmitic acid, appears to act as lithogen in our model.     

Adulterated butterfat: Fatty acid composition of triglycerides and 2-monoglycerides

Beef tallow and cottonseed oil were mixed with a pure butterfat in the ratios of 2%, 4% and 6% to obtain admixtures of beef tallow with butterfat and cottonseed oil with butterfat. The hydrolysis of individual triglycerides was carried out using the lipase to obtain 2-monoglycerides. The results indicated that butterfat had a higher percentage of C14:0 and C16:0 acids than found in the triglycerides and 2-monoglycerides of beef tallow and cottonseed oil. Beef tallow contained a higher proportion of C18:0 and C18:1 acids than butterfat and cottonseed oil triglycerides or 2-monoglycerides. Cottonseed oil had a higher percentage of C18:2 acid located in triglyceride or 2-monoglyceride than found in butterfat or beef tallow triglycerides and 2-monoglycerides. The analysis of the samples of butterfat containing 2%, 4% and 6% beef tallow revealed that the addition of beef tallow to butterfat affected the fatty acid composition of butterfat triglycerides and 2-monoglycerides with C18:0 and C18:1 acids; the effect was increased with increasing percentages of beef tallow. The addition of cottonseed oil to butterfat in the ratios of 2%, 4% and 6% affected the fatty acid composition of butterfat triglycerides and 2-monoglycerides. It was found that both C18:1 and C18:2 increased as the added cottonseed oil percentages increased.     

Triglyceride structure of milk fats

The enantiomeric nature of the triglycerides of bovine milk fat was reinvestigated by determining the stereospecific distribution of fatty acids in rearranged butterfat, following partial hydrolysis with pancreatic lipase, and in certain molecular distillates of native butterfat, following Grignard degradation. The results with rearranged butterfat confirmed the validity of pancreatic lipase hydrolysis as a means of generating representative diglycerides from milk fat triglycerides. The Grignard degradation and lipolysis gave identical distributions for fatty acids when included as part of the assay system in the stereospecific analysis. Characteristically, butyric acid and the other short chain acids occupied the 3 position in the native butterfat, while in the rearranged oil they were distributed more or less randomly. Gas chromatographic analysis of the short chain glycerides on polyester columns allowed an effective resolution of butyryl, caproyl and caprylyl glycerides of identical numbers of total acyl carbons and double bonds. The method was especially well suited for resolution of the 2,3-diglycerides, which were recovered either as the more polar fraction from thin layer chromatography of the X-1,2-diacylglycerols, or by acetolysis of the residual phenolphosphatides resulting from phospholipase A digestion. It was shown that butyric acid in the 3 position was preferentially paired with myristic, palmitic and oleic acid in the 2 position, and palmitic and oleic acid in the 1 position, which was also characteristic of the other short chain acids. One of eight papers presented in the symposium “Milk Lipids,” AOCS Meeting, Ottawa, September 1972.     

Einsatz von Soja- und Sonnenblumen-Extraktionsschrot und Sonnenblumensamen als Eiweißfuttermittel fü Milchkühe

Use of soybean and sunflower meal and sunflower seed as a protein source for milking cows. In a feeding trial with 37 cows of Brown Swiss the impact of 10% soybean meal (Soja), 16% sunflower meal (Soblex) and 30% sunflower seed (Soblsa) in the feeding stuff on dry matter intake, milk yield, milk ingredients and also on the conditions of milk fat was investigated. Soybean meal and sunflower meal are similar protein sources for cows. Sunflower seeds caused a lower dry matter intake, milk yield and content of milk protein, but showed positive effects on the consistency of butterfat. Butterfat of the sunflower seed group had a higher iodine value (p<0.01) and a higher content of trans fatty acids (p<0.01) than the Soja- and Soblex-groups and resembled the composition of butterfat in the grazing periode.     

Zusammensetzung und Aufbau der Triglyceride von Muttermilch und einiger Rohstoffe für Säuglingsnahrung

Composition and Structures of Triglycerides of Human Milk and Some Base Components for Infant Milk Formulas Following HPLC separation of human milk triglycerides with a silver-loaded ion exchange and an RP-18-phase column, the 50 substantial fractions obtained were characterized by gas chromatography of triglycerides and fatty acid methyl esters as well as by the analysis of intramolecular distribution on a reduced scale using ethyl magnesium bromide. Exploitation of all data available resulted in the detailed structures of 106 triglycerides, representing 81 g/100 g human milk fat. A substitute for producing infant milk formulas should be adapted to the composition and fatty acid distribution of those 14 substantial triglycerides which are present in human milk fat with more than 1 g/100 g, amounting to a total of 42 g/100 g. Among several fats and oils analyzed, only lard and a recent product were found to contain palmitic acid also predominantly in the 2-position. Therefore, other base components would require a directed fractionation and/or interesterification to be best adapted to human milk fat.     

Solvent‐free preparation of phytosteryl esters with fatty acids from butterfat in equimolecular conditions in the presence of a lipase from Candida rugosa

BACKGROUND: Enzymatic esterification of phytosterols with fatty acids from butterfat in equimolecular conditions to produce phytosteryl esters was performed in solvent‐free medium. Commercial and immobilized Candida rugosa lipases were used as biocatalysts for the reaction. RESULTS: By this methodology, under simple and mild reaction conditions (without solvents, 50 °C and short reaction times), 94% and 99% (w/w) of phystosteroyl esters were obtained in 48 h and 9 h with the commercial and the immobilized lipase, respectively. The effects of temperature, fatty acid specificity, enzyme amount and residual activity of each lipase were also evaluated. CONCLUSIONS: The phytosteryl esters from butterfat produced in this study are expected to have lower melting point, improved oil and fat solubility and bioavailability compared to that of their corresponding free phytosterols. Copyright © 2008 Society of Chemical Industry     

Human milk in disease: Lipid Composition

Differences in the lipid composition of human milk have been described in maternal diseases known to affect fat metabolism. Diseases such as diabetes, cystic fibrosis, hypobetalipoproteinemia and Type I hyperlipoproteinemia affect the quantity and quality of human milk fat. Increased fatty acid chain elongation and changes in desaturation (especially Δ6 desaturase), as well as changes in lipid class composition, have been shown in diabetes and cystic fibrosis, whereas compensatory increases in medium-chain fatty acids have been described in hypobetalipoproteinemia and Type I hyperlipoproteinemia. It is important to realize that these observations were made either on single women or on very small groups of women. In infant diseases, such as breast milk jaundice and ectopic eczema, changes in polyunsaturated fatty acids in maternal milk have been described. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Process optimization of simultaneous enzymatic production of 1,3-dioleoyl-2-palmitoylglycerol and 1-oleoyl-2-palmitoyl-3-linoleoylglycerol

1,3-dioleoyl-2-palmitoylglycerol (OPO), a structured triacylglycerols (TAG) containing palmitic acid at sn-2 position, is widely used as breast milk fat substitutes in infant formula. Interestingly, the main component of breast milk fat in Western countries is OPO while Chinese breast milk contains both 1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) and OPO. Thus, OPL is additionally essential for Chinese infants. In the present study, we developed a process for simultaneous production of OPL and OPO. According to the fatty acid (FA) composition of Chinese breast milk, soy oil, palm kernel stearin, and palm stearin were selected as the base oils for random interesterification, and the appropriate ratio was determined as 2:3:15. Then interesterification at sn-1,3 position was conducted, four reaction parameters were examined, where 1.6 molar ratio of oleic acid to linoleic acid (OLA/LNA), 10% enzyme quantity, TAG to FA ratio of 1:8 and reaction temperature of 56°C were ultimately established as the optimal conditions to achieve the target of the present study, and the OPO and OPL contents in the final products were 23.1% and 26.2%, respectively. Finally, molecular distillation was applied to purify above achieved breast milk fat substrates. Higher oil dropping speed, oil temperature, and agitation speed were favorable conditions, and the lowest acid value that achieved in the present study was 3.46 mgKOH/g. Overall, we established an efficient preliminary process for the simultaneous production of OPO and OPL, which could provide an important reference for commercial production of breast milk fat substrates for Chinese infants.