**2. Benefits and analysis of human milk**

#### **2.1 Importance of human milk in newborn health**

Breastfeeding provides numerous health benefits, both short and long-term for breastfed newborn [10]. The short-term benefits include immune system development, reduction of gastrointestinal diseases (diarrhea), respiratory diseases (pneumonia), skin diseases (atopic dermatitis), allergies, leukemia, sudden death syndrome, diabetes and ear inflammation during childhood [10, 11]. Long-term evidence has shown various benefits to public health problems such as improved cognitive development [12] and reduction of chronic diseases, for example diabetes (type 1 and 2), obesity, hypertension, cardiovascular diseases, hyperlipidemia and selected categories of cancer in adult life [13].

#### *2.1.1 Importance of different lipid classes of human milk*

Milk TAG are formed in the endoplasmic reticulum from circulating FA or newly synthesized in the mammary epithelial cells of glucose. The initial step in the FA synthesis is the conversion of acetyl-CoA to malonyl-CoA, afterwards, the synthase enzyme catalyzes the sequence of fatty acid reactions, then each sequence adds twocarbon unit to the growing chain, resulting in the de novo synthesis of mediumchain and intermediate chain FA as well as explaining the elevated content of these FA in milk [14].

Long-chain TAG are digested by a lingual lipase, while the medium and short chain TAG undergoes the action of a stomach lipase and are absorbed in the stomach as FA and glycerol. In the intestine, TAG non-hydrolyzed, especially long chain triglycerides, undergo the action of bile salts and pancreatic enzyme, being reduced to MAGs, FA and glycerol, which are absorbed, distributed and utilized by the tissues [15].

Phospholipids contribute to 1–2% of the total lipids of human milk [16]. The major phospholipids of milk fat globule membrane are phosphatidylcholines, phosphatidylethanolamines and sphingomyelins, and each of it contributes to 20–40% of the total phospholipids [17]. The nutritional importance of these lipids is based on the variety of specific lipids provided, plus it also has particular bioactivities in the gastrointestinal.

**93**

*Lipids and Fatty Acids in Human Milk: Benefits and Analysis*

tion of cholesterol homeostasis in adult life [20].

anticarcinogenic agents and antidiabetic effects [23].

acids are linked to antimicrobial biological activities [26].

*2.1.2 Importance of fatty acids in human milk*

cry behavior [14].

The sphingomyelin demonstrates robust anti-tumor activity, may influence the cholesterol metabolism, and exhibits anti-infective activity [4]. The phosphatidylcholine and sphingomyelin contribute to approximately 10% of the total choline intake of infants [18]. Thus, in quantitative terms, water-soluble choline in milk is more significant, although there are good indications that the metabolization of free and esterified dietetic choline is distinctive and it may have specific effects on plasma cholesterol levels and even in the brain development of the baby [19].

Cholesterol content in human milk is low (0.5%), serving as structural component of the milk fat globule membrane, this characteristic is related to the provision of sufficient stabilization and fluidity, it is also essential in lipid metabolism [5]. Breastfed babies present higher plasma cholesterol levels in comparison to babies receiving infant formulas; however, early exposure may favor the metabolic regula-

Human milk fat accomplish an important position as energy source, structural and regulatory functions, [21] in which FA are essential for the development of the central nervous system [22] antiprotozoal activity (free fatty acid (FFA) produced during gastric and intestinal digestion of milk fat), increased immune response,

The principal saturated fatty acid (SFA) in human milk is the palmitic acid (16:0) [24]. It is located in the TAG sn-2 region, simplifying the pancreatic lipase action that specifically hydrolyzes the FA at the sn-1 and sn-3 positions converting the palmitic acid to sn-2 MAG, which is generally well absorbed resulting in improvement of intestinal discomfort, decreasing colic and crying of the newborn. [25] In addition, the palmitic acid position influences the n-acylentanolamides, including levels of anandamide, which presents analgesic effects contributing to the enlightenment of the association between the palmitic acid position and the baby

It is also noteworthy that the butyrate SFA (4:0) present functions as modulation of the gene expression regulation and reduction of inflammation processes in the intestine. The SFAs caproic (6:0), caprylic (8:0), capric (10:0) and lauric (12:0)

In particular, the most significant FA in human milk are the long-chain polyunsaturated fatty acids (LCPUFA) [22]. The homologs of linoleic acid (18:2n-6; LA) from n-6 series are precursors of arachidonic acid (20:4n-6; AA), while homologs of α-linolenic acid (18:3n-3; ALA) from n-3 series are precursors of eicosapentaenoic acid (20:5 n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA). Therefore, breast milk contains the indispensable FA precursors (LA and ALA) to produce AA and DHA, which present crucial function in visual, immune, cognitive and motor development in newborns. Besides, it present important function in allergy protection, asthma, improvement of lung function and reduction of childhood inflammation and obesity rates, plus an additional advantage is the increase of 4.5 IQ points

in infants breastfed in comparison to infants that did not received it [24].

**2.2 Nomenclature and terminology of main fatty acids in human milk**

The IUPAC nomenclature system is technically clear. The fatty acid names are excessively long, principally the long chain acids, therefore, for convenience, common or trivial name and abbreviated notations are often employed in scientific texts. Researchers working in different study areas on fatty acid composition are familiar with the chemical structure and commonly use of the notation C:D to

*DOI: http://dx.doi.org/10.5772/intechopen.80429*

#### *Lipids and Fatty Acids in Human Milk: Benefits and Analysis DOI: http://dx.doi.org/10.5772/intechopen.80429*

*Biochemistry and Health Benefits of Fatty Acids*

chemical properties of human milk fat [7].

**2. Benefits and analysis of human milk**

selected categories of cancer in adult life [13].

*2.1.1 Importance of different lipid classes of human milk*

**2.1 Importance of human milk in newborn health**

infant's body [9].

In human milk, the lipids are present as fat globules form, mainly constituted of TAG surrounded by a structural membrane composed of phospholipids, cholesterol, proteins and glycoproteins [5]. The fat from human milk is its main energy source, consisting 98% (m/m) of neutral lipids (TAG, DAG and MAG) [6]. Hence, the fatty acid composition of these constituents defines the nutritional and physico-

TAG are molecules of glycerol esterified to three fatty acids (FA), which may be located at the TAG sn-1, sn-2 and sn-3 positions. However, the FA position in TAG is also related to the human milk quality. In TAG from human milk, for example, palmitic acid is positioned normally on sn-2 (the central carbon atom) [8], which facilitates the action of pancreatic lipase. Besides, it leads to improved absorption of fat and calcium by newborns due to the subsequent metabolism of these TAG in the

Therefore, numerous analytical techniques are employed to attempt the verification of the FA composition as well as TAG structure present in human milk fat. This chapter will address the benefits associated with the consumption of human milk,

Breastfeeding provides numerous health benefits, both short and long-term for breastfed newborn [10]. The short-term benefits include immune system development, reduction of gastrointestinal diseases (diarrhea), respiratory diseases (pneumonia), skin diseases (atopic dermatitis), allergies, leukemia, sudden death syndrome, diabetes and ear inflammation during childhood [10, 11]. Long-term evidence has shown various benefits to public health problems such as improved cognitive development [12] and reduction of chronic diseases, for example diabetes (type 1 and 2), obesity, hypertension, cardiovascular diseases, hyperlipidemia and

Milk TAG are formed in the endoplasmic reticulum from circulating FA or newly synthesized in the mammary epithelial cells of glucose. The initial step in the FA synthesis is the conversion of acetyl-CoA to malonyl-CoA, afterwards, the synthase enzyme catalyzes the sequence of fatty acid reactions, then each sequence adds twocarbon unit to the growing chain, resulting in the de novo synthesis of mediumchain and intermediate chain FA as well as explaining the elevated content of these

Long-chain TAG are digested by a lingual lipase, while the medium and short chain TAG undergoes the action of a stomach lipase and are absorbed in the stomach as FA and glycerol. In the intestine, TAG non-hydrolyzed, especially long chain triglycerides, undergo the action of bile salts and pancreatic enzyme, being reduced to MAGs, FA and glycerol, which are absorbed, distributed and utilized by the tissues [15]. Phospholipids contribute to 1–2% of the total lipids of human milk [16]. The major phospholipids of milk fat globule membrane are phosphatidylcholines, phosphatidylethanolamines and sphingomyelins, and each of it contributes to 20–40% of the total phospholipids [17]. The nutritional importance of these lipids is based on the variety of specific lipids provided, plus it also has particular bioactivities in

as well as analytical techniques employed to assess its lipid quality.

**92**

FA in milk [14].

the gastrointestinal.

The sphingomyelin demonstrates robust anti-tumor activity, may influence the cholesterol metabolism, and exhibits anti-infective activity [4]. The phosphatidylcholine and sphingomyelin contribute to approximately 10% of the total choline intake of infants [18]. Thus, in quantitative terms, water-soluble choline in milk is more significant, although there are good indications that the metabolization of free and esterified dietetic choline is distinctive and it may have specific effects on plasma cholesterol levels and even in the brain development of the baby [19].

Cholesterol content in human milk is low (0.5%), serving as structural component of the milk fat globule membrane, this characteristic is related to the provision of sufficient stabilization and fluidity, it is also essential in lipid metabolism [5]. Breastfed babies present higher plasma cholesterol levels in comparison to babies receiving infant formulas; however, early exposure may favor the metabolic regulation of cholesterol homeostasis in adult life [20].

## *2.1.2 Importance of fatty acids in human milk*

Human milk fat accomplish an important position as energy source, structural and regulatory functions, [21] in which FA are essential for the development of the central nervous system [22] antiprotozoal activity (free fatty acid (FFA) produced during gastric and intestinal digestion of milk fat), increased immune response, anticarcinogenic agents and antidiabetic effects [23].

The principal saturated fatty acid (SFA) in human milk is the palmitic acid (16:0) [24]. It is located in the TAG sn-2 region, simplifying the pancreatic lipase action that specifically hydrolyzes the FA at the sn-1 and sn-3 positions converting the palmitic acid to sn-2 MAG, which is generally well absorbed resulting in improvement of intestinal discomfort, decreasing colic and crying of the newborn. [25] In addition, the palmitic acid position influences the n-acylentanolamides, including levels of anandamide, which presents analgesic effects contributing to the enlightenment of the association between the palmitic acid position and the baby cry behavior [14].

It is also noteworthy that the butyrate SFA (4:0) present functions as modulation of the gene expression regulation and reduction of inflammation processes in the intestine. The SFAs caproic (6:0), caprylic (8:0), capric (10:0) and lauric (12:0) acids are linked to antimicrobial biological activities [26].

In particular, the most significant FA in human milk are the long-chain polyunsaturated fatty acids (LCPUFA) [22]. The homologs of linoleic acid (18:2n-6; LA) from n-6 series are precursors of arachidonic acid (20:4n-6; AA), while homologs of α-linolenic acid (18:3n-3; ALA) from n-3 series are precursors of eicosapentaenoic acid (20:5 n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA). Therefore, breast milk contains the indispensable FA precursors (LA and ALA) to produce AA and DHA, which present crucial function in visual, immune, cognitive and motor development in newborns. Besides, it present important function in allergy protection, asthma, improvement of lung function and reduction of childhood inflammation and obesity rates, plus an additional advantage is the increase of 4.5 IQ points in infants breastfed in comparison to infants that did not received it [24].

### **2.2 Nomenclature and terminology of main fatty acids in human milk**

The IUPAC nomenclature system is technically clear. The fatty acid names are excessively long, principally the long chain acids, therefore, for convenience, common or trivial name and abbreviated notations are often employed in scientific texts. Researchers working in different study areas on fatty acid composition are familiar with the chemical structure and commonly use of the notation C:D to

represent the FA, being C the number of carbon atoms and D the number of double bonds in the carbon chain. Some researches frequently employ the "omega" system (n minus system), Shorthand Designation, as a notation to define the different series, such as n-3, n-6, n-9, n-12. This system is applicable to unsaturated fatty acids from natural sources (cis configuration). Unsaturated fatty acids have double bond in the carbon chain and, commonly, in the PUFA, the double bonds of carbon chain is interrupted by a methylene group (cis, cis-1,4-pentadiene group).

Thus, the term n minus refers to the position of the double bond closest to the methyl end carbon chain. However, in this system, the position of another double bond in PUFA acid carbon chain is not denoted and the configuration (cis or trans) is not specified. So, this system is not employed for FA with trans configuration and PUFA group, although it is widely used by researchers. Therefore, the IUPAC-IUB Commission does not recommend the 'omega' system [27].

### *2.2.1 Main fatty acids in human milk*

Cis fatty acid composition is one of the major components of woman's breast milk, and it is influenced by different factors, that can be grouped as follows: (i) variable: method of feeding, genetic factors, dietary habits, maternal diet composition, hormones, gestational age at birth, parity, seasonality, between lactation daily, caloric content of food and mutual proportions of particular dietary components (carbohydrate and fat contents), (ii) positive modulation: duration of the lactation period, adiposity, stage of lactation and maternal age, (iii) negative modulation: maternal malnutrition, infectious (mastitis), metabolic disorders (diabetes) and medications [28, 29].

In particular, trans fatty acids (TFA) in human milk have raised concerns because of the possible adverse effects on infant growth and development. The TFA have been associated with adverse effects on LCPUFA and essential fatty acids (LA, 18:2n-6 and ALA, 18:3n-3) metabolism, oxidative stress and low density lipoprotein cholesterol levels. These negative effects of TFA are predominantly associated with different isomers from hydrogenated vegetable shortening, such as 6/7/8/9/10 (trans) 18:1, and barely with TFA of natural sources such as ruminants fats as 11 (trans) 18:1 [30]. Composition of the TFA in human milk from Canadian and American woman has reduced since the mandatory TFA labeling was introduced in those countries [30, 31].

In this chapter, the group prepared a review concerning the composition of FA commonly encountered in studies of human milk from different countries, including five regions of China, Canada, Spain, Brazil, Poland, Germany, Hungary, Finland, Sweden Slovakia, United Kingdom, Denmark, Egypt, Uganda, and Tanzania. A list of FA is elaborated on **Table 1**, including: saturated FA, monounsaturated FA, polyunsaturated FA, branched chain FA, trans FA and conjugated linoleic [14, 26, 31–34].

#### **2.3 Contribution of omega-3 fatty acids from human milk on immunity**

Human milk contains numerous growth and antimicrobial factors, as well as cells and antibodies from mother, which are responsible by innate and acquired immune responses in the newborn [35]. The breastfeeding permits these components to cover the neonatal gastrointestinal tract, the main access of microorganisms in early life, and influences the maturation of immune system. It is assumed that human milk can perform in the induction of specific immune responses in the intestine, favoring a microbiota that competes with pathogenic bacteria [36].

**95**

*Lipids and Fatty Acids in Human Milk: Benefits and Analysis*

**SFA MUFA**

**designation**

Butiric 4:0 Lauroleic 12:1n-3 Caproic 6:0 Tsuzuic 14:1n-10 Enantic 7:0 Physeteric 14:1n-9 Caprylic 8:0 None 14:1n-7 Pelargonic 9:0 Myristoleic 14:1n-5 Capric 10:0 None 15:1n-5 Undecylic 11:0 Sapienic 16:1n-10 Lauric 12:0 None 16:1n-9 Tridecylic 13:0 Palmitoleic 16:1n-7 Myristic 14:0 None 17:1n-8 Pentadecylic 15:0 None 17:1n-7 Palmitic 16:0 Petroselinic 18:1n-12 Margaric 17:0 None 18:1n-10 Stearic 18:0 Oleic 18:1n-9 Arachidic 20:0 Vaccenic 18:1n-7 Heneicosylic 21:0 None 18:1n-6 Behenic 22:0 None 18:1n-5 Tricosylic 23:0 None 18:1n-4 Lignoceric 24:0 None 18:1n-3 **BCFA** None 18:1n-2 Isotridecylic 13:0 iso Gadoleic 20:1n-11 Anteisotridecylic 13:0 anteiso Gondoic 20:1n-9 Isomyristic 14:0 iso Paullinic 20:1n-7 Isopentadecylic 15:0 iso Cetoleic 22:1n-11 Anteisopentadecylic 15:0 anteiso Erucic 22:1n-9 Isopalmitic 16:0 iso Nervonic 24:1n-9

**Trivial name Shorthand designation**

*DOI: http://dx.doi.org/10.5772/intechopen.80429*

**Trivial name Shorthand** 

Isomargaric 17:0 iso Anteisomargaric 17:0 anteiso

**PUFA TFA**

Linoleic 18:2n-6 (LA) Myristelaidic t-14:1n-5 Gamma-linolenic 18:3n-6 None t-15:1n-5

Stearidonic 18:4n-3 Linolelaidic trans,trans 18:2n-6 Meadacid 20:3n-9 None cis-9, trans-12 18:2 Dihomo-γ-linolenic 20:3n-6 None trans-9,cis-12 18:2 Dihomo-ALA 20:3n-3 None trans-11,cis-15 18:2

Alpha-linolenic 18:3n-3 (ALA) Isomers 3/4/5/6/7/8/9/10/11/12/13 (trans) 16:1 None 20:2n-6 Isomers 6/7/8/9/10/11/12/13/14 (trans) 18:1


#### *Lipids and Fatty Acids in Human Milk: Benefits and Analysis DOI: http://dx.doi.org/10.5772/intechopen.80429*

*Biochemistry and Health Benefits of Fatty Acids*

*2.2.1 Main fatty acids in human milk*

medications [28, 29].

those countries [30, 31].

linoleic [14, 26, 31–34].

represent the FA, being C the number of carbon atoms and D the number of double bonds in the carbon chain. Some researches frequently employ the "omega" system (n minus system), Shorthand Designation, as a notation to define the different series, such as n-3, n-6, n-9, n-12. This system is applicable to unsaturated fatty acids from natural sources (cis configuration). Unsaturated fatty acids have double bond in the carbon chain and, commonly, in the PUFA, the double bonds of carbon

Thus, the term n minus refers to the position of the double bond closest to the methyl end carbon chain. However, in this system, the position of another double bond in PUFA acid carbon chain is not denoted and the configuration (cis or trans) is not specified. So, this system is not employed for FA with trans configuration and PUFA group, although it is widely used by researchers. Therefore, the IUPAC-IUB

Cis fatty acid composition is one of the major components of woman's breast milk, and it is influenced by different factors, that can be grouped as follows: (i) variable: method of feeding, genetic factors, dietary habits, maternal diet composition, hormones, gestational age at birth, parity, seasonality, between lactation daily, caloric content of food and mutual proportions of particular dietary components (carbohydrate and fat contents), (ii) positive modulation: duration of the lactation period, adiposity, stage of lactation and maternal age, (iii) negative modulation: maternal malnutrition, infectious (mastitis), metabolic disorders (diabetes) and

In particular, trans fatty acids (TFA) in human milk have raised concerns because of the possible adverse effects on infant growth and development. The TFA have been associated with adverse effects on LCPUFA and essential fatty acids (LA, 18:2n-6 and ALA, 18:3n-3) metabolism, oxidative stress and low density lipoprotein cholesterol levels. These negative effects of TFA are predominantly associated with different isomers from hydrogenated vegetable shortening, such as 6/7/8/9/10 (trans) 18:1, and barely with TFA of natural sources such as ruminants fats as 11 (trans) 18:1 [30]. Composition of the TFA in human milk from Canadian and American woman has reduced since the mandatory TFA labeling was introduced in

In this chapter, the group prepared a review concerning the composition of FA commonly encountered in studies of human milk from different countries, including five regions of China, Canada, Spain, Brazil, Poland, Germany, Hungary, Finland, Sweden Slovakia, United Kingdom, Denmark, Egypt, Uganda, and Tanzania. A list of FA is elaborated on **Table 1**, including: saturated FA, monounsaturated FA, polyunsaturated FA, branched chain FA, trans FA and conjugated

**2.3 Contribution of omega-3 fatty acids from human milk on immunity**

as cells and antibodies from mother, which are responsible by innate and acquired immune responses in the newborn [35]. The breastfeeding permits these components to cover the neonatal gastrointestinal tract, the main access of microorganisms in early life, and influences the maturation of immune system. It is assumed that human milk can perform in the induction of specific immune responses in the intestine, favoring a microbiota that competes with pathogenic

Human milk contains numerous growth and antimicrobial factors, as well

chain is interrupted by a methylene group (cis, cis-1,4-pentadiene group).

Commission does not recommend the 'omega' system [27].

**94**

bacteria [36].


*Fatty acids abbreviations: SFA—saturated fatty acid, MUFA—monounsaturated fatty acid, PUFA—polyunsaturated fatty acid, BCFA—branched chain fatty acid, TFA—trans fatty acid, CLA—conjugated linoleic acid.*

#### **Table 1.**

*Fatty acids commonly encountered in human milk.*

Other components constantly present in human milk are the LCPUFA DHA and AA [37], essentials as cell membranes components and also as immunomodulators, by production and regulation of inflammatory cytokines, leukotrienes, prostaglandins, and thromboxanes, recognized as eicosanoids [38].

LCPUFA in human milk can modulate immunological responses, affecting the balance between T-helper cell type-1 (Th1) and Th2 [39], and regulatory T and T helper 17 cells from the acquired immune response [40].

These subsets of CD4+ T cells, Th1, Th2 [41], Th17 [42], and regulatory T (Treg) cells [43] participle producing cytokines with the most diverse functions. Interferon-γ (IFN-γ), tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) are the products of Th1, and IL-4, IL-5, IL-9, IL-10, IL-13, and IL-25 are of Th2 cells. Th17 cells produce IL-17A, IL-17F, IL-21 and IL-22; while Treg cells produce IL-10 and TGF-β1.

Th1 cells present important functions in cellular immunity against intracellular bacteria and protozoa, while Th2 cells mediate the response against extracellular parasites, as helminths, and participate in allergies [44]. Th17 cells apparently perform against different classes of pathogens and autoimmune conditions [45], and Treg cells perform regulating the inflammation, autoimmunity, allergy, infection, and tumors.

In general, preterm infants have an immature immunoregulatory system, with potential for chronic inflammation [46], but an increase in Tregs and their function in early neonates has been observed [47], suggesting a transient increase of activated Treg in mature and full-term infants.

Although, generally, AA is considered proinflammatory and DHA immunoregulatory, the addition of it to infant formula has been indicated to increase the immunoregulatory system and to reduce inflammatory cytokines in infants, indicating an effect of LCPUFA on immune maturation [48].

However, according to [49], diets rich in DHA can reduce suppressive and migratory functions of regulatory T-cells [48]. Thereby, we must carefully examine the influence of FA during breastfeeding, since the knowledge from the DHA data on immune response in preterm infants, and the generation and maintenance of Tregs are still not well comprehended. Finally, the addition of AA and DHA in infant formulas should consider balancing its amounts, as DHA in excess may suppress the benefits provided by AA [50].

#### **2.4 Atopic disease and polyunsaturated fatty acids (PUFA)**

Atopic disease is defined as a set of disease such as atopic dermatitis, asthma and rhinitis, but it may differ according to the authors. The prevalence of childhood atopic

**97**

extended period [64].

*Lipids and Fatty Acids in Human Milk: Benefits and Analysis*

opment of IgE-associated with allergic disease in infancy [58].

200 mg DHA, to result in a milk with DHA content of 0.3% of FA [53].

effect on atopic sensitization in the primary 12 months of life [58].

which could explain the variability of the results [61].

A systematic review concluded that there is heterogeneity among studies in terms of presenting the association between PUFA and allergy, which could influence the results [59]. Some studies observed associations between n-3 and n-6 PUFAs and allergic disease [60], and the magnitude of this effect varied greatly. Otherwise it is known that breast milk contains different composition of PUFA,

A cohort study has shown the ratio of n-6: n-3 FA in milk is associated with the risk of non-atopic eczema at 6 months, and perhaps the high level of n-6 may increases the risk of rhinitis [62]. Other authors hypothesized that variations in the lipid composition of milk could, in part, explain some of the controversies regarding the protective effects of breastfeeding against allergy, and concluded that the fatty acid composition of human milk is disturbed in atopic mothers having an

**2.5 Processing, composition in antioxidants and lipid stability in human milk**

The methods of processing human milk employed in milk banks aim the preservation or inhibition of microbial growth, the prevention or delay of decomposition caused by the presence of enzymes, chemical reactions, and the preservation by grime, such as insects, hair, animals, etc. [63]. Generally, pasteurization and freezing techniques are employed. In addition, recent studies combine both processes with lyophilization in order to preserve the original characteristics of milk for an

However, food processing can cause nutritional loss and structural modifications. In milk banks, pasteurization is followed by freezing storage at −18°C for

disease has been increasing and studies suggested the possibility that breastfeeding may reduce allergic manifestations in high-risk individuals [51, 52]. This association is possible linked with the breast milk PUFA, which are essential for adequate growth and development. It is also recognized that an early ingestion of it may affect the growth as well as the neurological and immune functions in later life [53].

Part of the PUFA breast milk composition depends on the mother dietary, and it has provided support to the hypothesis that omega-3 (n-3) PUFA in breast milk possibly protect against atopic diseases. The ratio between n-3 and omega-6 (n-6) PUFA levels seems to influence the development of atopic disorders [54, 55]. The n-3 FA supplementation, during pregnancy, and lactation have been extensively studied. Pregnant mothers consuming n-3 FA may enhance levels of IgA (adaptative immune response) and soluble CD14 (innate immune response) in breast milk. This theory reinforces the importance of the PUFA immune modulation and the idea that it can improve the immune system directly and indirectly [56]. The composition of breast milk has been shown to reflect in the infant's serum, by increase of immunomodulatory cytokines, such as TGF-1 and TGF-β2, associated to the protection against atopic diseases [57]. Moreover, EPA and DHA levels in colostrum and early mature milk were related to the protective effect in the devel-

An Asian study revealed that n-3 FA supplementation during pregnancy could reduce the chance of preterm birth. According to this article the intake recommendation to pregnant women is minimum of 200 mg (DHA) per day over and above the intake level recommended for adult general health, resulting in a total DHA intake of at least 300 mg/day. Likewise, PUFA supply and fish ingestion may positively influence the development of immune responses involved in allergic reactions, and reduce the risk of allergic diseases (asthma and eczema). It is recommended to women who breastfeed to achieve a minimum average daily supply of

*DOI: http://dx.doi.org/10.5772/intechopen.80429*

#### *Lipids and Fatty Acids in Human Milk: Benefits and Analysis DOI: http://dx.doi.org/10.5772/intechopen.80429*

*Biochemistry and Health Benefits of Fatty Acids*

**Trivial name Shorthand** 

Cervonic 22:6n-3 (DHA)

*Fatty acids commonly encountered in human milk.*

**Table 1.**

None 20:4n-3

**designation**

Timnodonic 20:5n-3 (EPA) Rumenic cis-9, trans 11 18:2 Adrenic 22:4n-6 None trans-9, cis-11 18:2 Osbond 22:5n-6 None trans-11, cis-13 18:2 Clupadonic 22:5n-3 None trans-11,trans-13 18:2

*fatty acid, BCFA—branched chain fatty acid, TFA—trans fatty acid, CLA—conjugated linoleic acid.*

Aracdonic 20:4n-6 (AA) **CLA**

Other components constantly present in human milk are the LCPUFA DHA and AA [37], essentials as cell membranes components and also as immunomodulators, by production and regulation of inflammatory cytokines, leukotrienes, prostaglan-

*Fatty acids abbreviations: SFA—saturated fatty acid, MUFA—monounsaturated fatty acid, PUFA—polyunsaturated* 

**Trivial name Shorthand designation**

LCPUFA in human milk can modulate immunological responses, affecting the balance between T-helper cell type-1 (Th1) and Th2 [39], and regulatory T and T

These subsets of CD4+ T cells, Th1, Th2 [41], Th17 [42], and regulatory T (Treg) cells [43] participle producing cytokines with the most diverse functions. Interferon-γ (IFN-γ), tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) are the products of Th1, and IL-4, IL-5, IL-9, IL-10, IL-13, and IL-25 are of Th2 cells. Th17 cells produce IL-17A, IL-17F, IL-21 and IL-22; while Treg cells produce IL-10 and TGF-β1. Th1 cells present important functions in cellular immunity against intracellular bacteria and protozoa, while Th2 cells mediate the response against extracellular parasites, as helminths, and participate in allergies [44]. Th17 cells apparently perform against different classes of pathogens and autoimmune conditions [45], and Treg cells perform regulating the inflammation, autoimmunity, allergy, infection, and tumors. In general, preterm infants have an immature immunoregulatory system, with potential for chronic inflammation [46], but an increase in Tregs and their function in early neonates has been observed [47], suggesting a transient increase of acti-

Although, generally, AA is considered proinflammatory and DHA immunoregulatory, the addition of it to infant formula has been indicated to increase the immunoregulatory system and to reduce inflammatory cytokines in infants, indicating an

However, according to [49], diets rich in DHA can reduce suppressive and migratory functions of regulatory T-cells [48]. Thereby, we must carefully examine the influence of FA during breastfeeding, since the knowledge from the DHA data on immune response in preterm infants, and the generation and maintenance of Tregs are still not well comprehended. Finally, the addition of AA and DHA in infant formulas should consider balancing its amounts, as DHA in excess may suppress the benefits provided by AA [50].

Atopic disease is defined as a set of disease such as atopic dermatitis, asthma and rhinitis, but it may differ according to the authors. The prevalence of childhood atopic

dins, and thromboxanes, recognized as eicosanoids [38].

helper 17 cells from the acquired immune response [40].

vated Treg in mature and full-term infants.

effect of LCPUFA on immune maturation [48].

**2.4 Atopic disease and polyunsaturated fatty acids (PUFA)**

**96**

disease has been increasing and studies suggested the possibility that breastfeeding may reduce allergic manifestations in high-risk individuals [51, 52]. This association is possible linked with the breast milk PUFA, which are essential for adequate growth and development. It is also recognized that an early ingestion of it may affect the growth as well as the neurological and immune functions in later life [53].

Part of the PUFA breast milk composition depends on the mother dietary, and it has provided support to the hypothesis that omega-3 (n-3) PUFA in breast milk possibly protect against atopic diseases. The ratio between n-3 and omega-6 (n-6) PUFA levels seems to influence the development of atopic disorders [54, 55].

The n-3 FA supplementation, during pregnancy, and lactation have been extensively studied. Pregnant mothers consuming n-3 FA may enhance levels of IgA (adaptative immune response) and soluble CD14 (innate immune response) in breast milk. This theory reinforces the importance of the PUFA immune modulation and the idea that it can improve the immune system directly and indirectly [56].

The composition of breast milk has been shown to reflect in the infant's serum, by increase of immunomodulatory cytokines, such as TGF-1 and TGF-β2, associated to the protection against atopic diseases [57]. Moreover, EPA and DHA levels in colostrum and early mature milk were related to the protective effect in the development of IgE-associated with allergic disease in infancy [58].

An Asian study revealed that n-3 FA supplementation during pregnancy could reduce the chance of preterm birth. According to this article the intake recommendation to pregnant women is minimum of 200 mg (DHA) per day over and above the intake level recommended for adult general health, resulting in a total DHA intake of at least 300 mg/day. Likewise, PUFA supply and fish ingestion may positively influence the development of immune responses involved in allergic reactions, and reduce the risk of allergic diseases (asthma and eczema). It is recommended to women who breastfeed to achieve a minimum average daily supply of 200 mg DHA, to result in a milk with DHA content of 0.3% of FA [53].

A systematic review concluded that there is heterogeneity among studies in terms of presenting the association between PUFA and allergy, which could influence the results [59]. Some studies observed associations between n-3 and n-6 PUFAs and allergic disease [60], and the magnitude of this effect varied greatly. Otherwise it is known that breast milk contains different composition of PUFA, which could explain the variability of the results [61].

A cohort study has shown the ratio of n-6: n-3 FA in milk is associated with the risk of non-atopic eczema at 6 months, and perhaps the high level of n-6 may increases the risk of rhinitis [62]. Other authors hypothesized that variations in the lipid composition of milk could, in part, explain some of the controversies regarding the protective effects of breastfeeding against allergy, and concluded that the fatty acid composition of human milk is disturbed in atopic mothers having an effect on atopic sensitization in the primary 12 months of life [58].

#### **2.5 Processing, composition in antioxidants and lipid stability in human milk**

The methods of processing human milk employed in milk banks aim the preservation or inhibition of microbial growth, the prevention or delay of decomposition caused by the presence of enzymes, chemical reactions, and the preservation by grime, such as insects, hair, animals, etc. [63]. Generally, pasteurization and freezing techniques are employed. In addition, recent studies combine both processes with lyophilization in order to preserve the original characteristics of milk for an extended period [64].

However, food processing can cause nutritional loss and structural modifications. In milk banks, pasteurization is followed by freezing storage at −18°C for

up to 6 months. But, the thermal process causes modifications in the milk due to the inadequate intensity of the set time and temperature that has been applied. Consequently, proteins can be denatured, enzymes become inactive, lipids suffer oxidation and vitamins and minerals are unstructured [65].

The Holder pasteurization (30 minutes of heat at 62.5°C and frozen at −20°C) imposed by the global guideline was evaluated by studies demonstrating that the lipolytic activity increased, doubling the concentration of free fatty acids (FFA), while the low temperature reduced the lipolysis rate, even if it had been increased by the storage time under freezing [66].

The lyophilization process removes the water from food by sublimation, allowing its preservation at room temperature, with the addition of water the product returns to its original form without nutritional losses. This technique, applied in human milk, demonstrated to be effective, as it inhibits microbial contamination, preserves nutrients and oxidative markers, as well as ensures a prolonged conservation period in comparison to pasteurized human milk [67].

Lipids are the most compromised macronutrients present in milk during processing due to the autoxidation of the fatty acid. This degradation reaction can occur with or without oxygen, as well as be catalyzed by light, heat, irradiation and free radicals, forming toxic compounds, such as peroxides. According to the thermal processes, fatty acid may undergo structural isomers by generating trans molecules or losing their total or partially insaturations, damaging the product and causing nutritional loss. However, studies indicate that the PUFA is stable during pasteurization and it may be justified due to the high antioxidant activity of the human milk [68].

The milk naturally presents antioxidant compounds that delaying or preventing molecules from being affected by the oxidative processes [69]. These compounds operate according to diverse action mechanisms for cell protection, such as: (i) eliminate substances that initiate peroxidation, (ii) chelate metallic ions, turning it incapable of decomposing peroxides or forming free radicals, (iii) block the action of reactive species, (iv) interrupt the auto oxidizing chain reaction, and/or (v) reduce the local concentrations of O2 [70].

Regarding the antioxidant category, it can be classified chemically by enzymatic and non-enzymatic, both perform synergistic actions in free radicals elimination [71]. Among the antioxidant enzymes, the milk is composed by the superoxide dismutase and the catalase, and the glutathione peroxidase that contains selenium. There are also other enzymes that catalyze the synthesis or regeneration of non-enzymatic antioxidants, named support enzymes, among which are glucose-6-phosphate dehydrogenase and glutathione reductase [72].

Non-enzymatic antioxidants present in breast milk are glutathione, amino acids arginine, citrulline and taurine, creatine, metallic ions selenium and zinc, ascorbic acid (vitamin C), carotenoids, flavonoids, coenzyme Q10, vitamins E and lactoferrin. Among these antioxidants that are three distinct classes: (i) antioxidants that performs as free radical abductor in the lipid milk portion, such as vitamin E and A, carotenoids and coenzyme Q10, (ii) antioxidants that performs in the aqueous phase, such as ascorbic acid, and (iii) antioxidants that performs in both cases, such as flavonoids [73].

Among the lipophilic antioxidants, the principals are: carotenoids, vitamin A and α-tocopherol. A vitamin E constituent is present in greater amount in colostrum, first phase of breast milk, providing it a yellowish color due to the intense presence of the pigment carotenoid β-carotene, and decrease from the beginning of lactation, despite the increase of total lipids [74]. The average level of vitamin A

**99**

*Lipids and Fatty Acids in Human Milk: Benefits and Analysis*

isolated factor, determines its oxidative stability [76].

*2.6.1 Different extraction methods for total lipids (TL)*

Gerber method (butyrometer) [79], and gravimetric [80].

tion and not compromising the chemical structure of the lipids.

*2.6.2 Quantification of fatty acids in human milk by GC-FID*

FA (in mass) per volume or mass in human milk.

chromatography has been proposed [81].

on the third day of lactation comes to be three times superior than in mature milk. Similarly, the amount of vitamin E in colostrum can be the triple of that found in the mature milk and the carotenoids can present a level up to 10 times higher [75]. In this way, the precise balance of various antioxidants in breast milk, instead of any

The fatty acid composition of human milk has been extensively studied over the

Recent methods have been proposed in the direct quantification of FA in human milk by gas chromatography [81]. However, the gravimetric method is considered the gold standard for extracting the total lipid (TL) content in human milk, and Folch et al. [80] is one of the most recommended methods for it. This methodology extracts non polar, polar and neutral lipid using mixture of cold solvents for extrac-

Different methods of fat extraction have been proposed to determine the fat content in human milk by traditional method: crematocrit [77], esterified FA [78],

Prior to chromatographic analysis, a derivation step of the TL is required, converting the different lipid classes in fatty acids methyl esters (FAMEs). This step is necessary to enable the volatilization of the compounds of interest, and to allow

Normally, the quantitative determination of FA in the human milk is data generally normalized as g of FA per 100 g of FA or expressed as percentage of weight (area normalization) FA relative to all FA exposed in a chromatogram. In the normalization methods, all the FA of the sample must be considered and, in the case of omission of a component, the other components are affected. On the other hand, the results presented by the normalization present difficulties of interpretation and, therefore, in nutritional values of the human milk [81]. The main drawback about normalization methods is that the data set does give information on the amount of

Recent studies express the composition of FA in mass concentrations of FA by mass of liquid human milk, and direct quantification of FA in human milk by gas

The determination qualitative and quantitative of FAMEs by GC-FID is among the most commonplace analyses in lipid matrices. Quantification of FAMEs by GC-FID has been effectively performed whereas detection with GC tandem mass spectrometry (CG-MS) has been employed mainly for qualitative analysis of FA. Both detectors FID and MS, for chromatographic analysis, the derivation step of the lipids classes is required to conversion of TAG, DAG, MAG, phospholipids (transesterification process), FFA (esterification process) in FAMEs [83].

The American Oil Chemists Society (AOCS) and the Association of Analytical Chemists (AOAC) recommend parameters for accurate quantification of FA. Both sources indicate the use of internal standard (IS, methyl tricosanoate - 23:0) and

the determination by GC; there are several methods for this purpose [82].

**2.6 Analytical methods for determination of fatty acids in human milk by gas chromatography: flame ionization detector (GC-FID)**

last 25 years and almost all of the studies are obtained after lipid extraction.

*DOI: http://dx.doi.org/10.5772/intechopen.80429*

*Lipids and Fatty Acids in Human Milk: Benefits and Analysis DOI: http://dx.doi.org/10.5772/intechopen.80429*

*Biochemistry and Health Benefits of Fatty Acids*

by the storage time under freezing [66].

reduce the local concentrations of O2 [70].

6-phosphate dehydrogenase and glutathione reductase [72].

human milk [68].

as flavonoids [73].

up to 6 months. But, the thermal process causes modifications in the milk due to the inadequate intensity of the set time and temperature that has been applied. Consequently, proteins can be denatured, enzymes become inactive, lipids suffer

The Holder pasteurization (30 minutes of heat at 62.5°C and frozen at −20°C) imposed by the global guideline was evaluated by studies demonstrating that the lipolytic activity increased, doubling the concentration of free fatty acids (FFA), while the low temperature reduced the lipolysis rate, even if it had been increased

The lyophilization process removes the water from food by sublimation, allowing its preservation at room temperature, with the addition of water the product returns to its original form without nutritional losses. This technique, applied in human milk, demonstrated to be effective, as it inhibits microbial contamination, preserves nutrients and oxidative markers, as well as ensures a prolonged conserva-

Lipids are the most compromised macronutrients present in milk during processing due to the autoxidation of the fatty acid. This degradation reaction can occur with or without oxygen, as well as be catalyzed by light, heat, irradiation and free radicals, forming toxic compounds, such as peroxides. According to the thermal processes, fatty acid may undergo structural isomers by generating trans molecules or losing their total or partially insaturations, damaging the product and causing nutritional loss. However, studies indicate that the PUFA is stable during pasteurization and it may be justified due to the high antioxidant activity of the

The milk naturally presents antioxidant compounds that delaying or preventing molecules from being affected by the oxidative processes [69]. These compounds operate according to diverse action mechanisms for cell protection, such as: (i) eliminate substances that initiate peroxidation, (ii) chelate metallic ions, turning it incapable of decomposing peroxides or forming free radicals, (iii) block the action of reactive species, (iv) interrupt the auto oxidizing chain reaction, and/or (v)

Regarding the antioxidant category, it can be classified chemically by enzymatic and non-enzymatic, both perform synergistic actions in free radicals elimination [71]. Among the antioxidant enzymes, the milk is composed by the superoxide dismutase and the catalase, and the glutathione peroxidase that contains selenium. There are also other enzymes that catalyze the synthesis or regeneration of non-enzymatic antioxidants, named support enzymes, among which are glucose-

Non-enzymatic antioxidants present in breast milk are glutathione, amino acids arginine, citrulline and taurine, creatine, metallic ions selenium and zinc, ascorbic acid (vitamin C), carotenoids, flavonoids, coenzyme Q10, vitamins E and lactoferrin. Among these antioxidants that are three distinct classes: (i) antioxidants that performs as free radical abductor in the lipid milk portion, such as vitamin E and A, carotenoids and coenzyme Q10, (ii) antioxidants that performs in the aqueous phase, such as ascorbic acid, and (iii) antioxidants that performs in both cases, such

Among the lipophilic antioxidants, the principals are: carotenoids, vitamin A and α-tocopherol. A vitamin E constituent is present in greater amount in colostrum, first phase of breast milk, providing it a yellowish color due to the intense presence of the pigment carotenoid β-carotene, and decrease from the beginning of lactation, despite the increase of total lipids [74]. The average level of vitamin A

oxidation and vitamins and minerals are unstructured [65].

tion period in comparison to pasteurized human milk [67].

**98**

on the third day of lactation comes to be three times superior than in mature milk. Similarly, the amount of vitamin E in colostrum can be the triple of that found in the mature milk and the carotenoids can present a level up to 10 times higher [75]. In this way, the precise balance of various antioxidants in breast milk, instead of any isolated factor, determines its oxidative stability [76].
