COMPARISON OF IMMUNOGLOBULIN LEVELS IN HUMAN MILK, COW MILK AND INFANT FORMULA MILK USING FTIR SPECTROSCOPY Dr. R. RAZIYA SULTANA Asst.Professor, Department of Physics, Justice Basheer Ahmed Sayeed College for Women, Chennai-600018,Tamilnadu, India

Immunoglobulin (IgA, IgG, IgD and IgM) in human milk, cow milk and infant formula milk has been analysed by FTIR spectroscopy. This technique has been successfully carried out and the role of FTIR spectroscopy in analyzing the immunoglobulin of human milk, cow milk and infant formula milk are clearly demonstrated both qualitatively and quantitatively. Among the milk of human, cow and infant formula, the vibrational peaks of the immunoglobulins increased in human milk, whereas in infant formula milk decreases and there was a further decrease in cow milk. Therefore it is concluded that the immunoglobulin levels in human milk, cow milk and infant formula milk have been detected and marked spectral difference is observed by FTIR techniques. It paints the intestinal epithelium and protects the mucosal surfaces against entry of pathogenic bacteria and enteroviruses. The immunoglobulin , a membrane protective is missing unless the newborn is breast-fed, it is absent from the respiratory and urinary tracts as KIJSET/OCT-DEC2015/VOL-2/Iss-4/A4 ISSN:2348-5477 IMPACT FACTOR(2015) 3.8071 Copyright © 2015 Published by kaav publications. All rights reserved www.kaavpublications.org 26 well as in the gastrointestinal tract. The immune system is in great part suppressed, and it is a mechanism for preventing maternal recognition of paternal antigens with subsequent reject of the fetus. Immunoglobulin plays a very important role in infant and suggest that the infant should be breastfed instead of cow milk and infant formula milk. The identity of absorption peak ascribed to Immunoglobulins was established by FTIR spectroscopy. Introduction Infrared spectroscopy is the subset of spectroscopy that deals with the infrared region of the electromagnetic spectrum. It covers a range of techniques, the most common being a form of absorption spectroscopy. Fourier-transform infrared spectroscopy is a vibrational spectroscopic technique, meaning it takes advantage of asymmetric molecular stretching, vibration, and rotation of chemical bonds as they are exposed to designated wavelengths of light. Fourier transform is to transform the signal from the time domain to its representation in the frequency domain FT – IR can take wavelength readings across the whole IR region simultaneously and smoothly, making this a very rapid technique. The technique is non-invasive and non-destructive. Its resolution of 0.125 cm is not spectacular in comparison to other vibrational techniques.FTIR salivary signature may be applied to indicate different stage of pregnancy of different age group in which, in the case of normal pregnant women of age between 25 to 35 has a greater absorption value than the normal pregnant women of age 18 to 25. This may be attributed to the salivary IgA and IgM concentrations higher in ageing people (Raziya Sultana etal., 2011). Breastfeeding is the feeding of an infant of young child with breast milk directly fromfemale human breasts notfrom a baby bottle or other container. Infant suckling stimulates the nerve endings in the nipple and areola, which signal the pituitary gland in the brain to release two hormones, prolactin and oxytocin. Prolactin causes alveoli to take nutrients such as proteins, sugars from blood supply and turn them into breast milk. Oxytocin causes the cells around the alveoli to contract and eject milk down the milk ducts. This passing of the milk down the ducts is called the let-down milk ejection reflex (Talwar G P etal., 2003). KIJSET/OCT-DEC2015/VOL-2/Iss-4/A4 ISSN:2348-5477 IMPACT FACTOR(2015) 3.8071 Copyright © 2015 Published by kaav publications. All rights reserved www.kaavpublications.org 27 Human milk contains two types of proteins, whey and casein. Approximately 60% is whey, while 40% is casein. Lactoferrin inhibits the growth of iron-dependent bacteria in the gastrointestinal tract. Secretory IgA also works to protect the infant from viruses and bacteria, specifically those that the baby, mom, and family are exposed to. It also helps to protect against E. Coli and possibly allergies. Other immunoglobulins, including IgG and IgM, in breast milk also help protect against bacterial and viral infectionsLysozyme is an enzyme that protects the infant against E. Coli and Salmonella. It also promotes the growth of healthy intestinal flora and has anti-inflammatory functions. Bifidus factor supports the growth of lactobacillus that protects the baby against harmful bacteria by creating an acidic environment where it cannot survive. It is necessary for brain development, absorption of fat-soluble vitamins, and is a primary calorie source. Long chain fatty acids are needed for brain, retina, and nervous system development. They are deposited in the brain during the last trimester of pregnancy and are also found in breast milk. The amount and types of vitamins in breast milk is directly related to the mother’s vitamin intake. Fatsoluble vitamins, including vitamins A, D, E, and K, are all vital to the infant’s health. Water-soluble vitamins such as vitamin C, riboflavin, niacin, and panthothenic acid are also essential.Lactose is the primary carbohydrate found in human milk. Lactose helps to decrease the amount of unhealthy bacteria in the stomach, which improves the absorption of calcium, phosphorus, and magnesium. It helps to fight disease and promotes the growth of healthy bacteria in the stomach. Milk appears entirely opaque while in thin layers it is transparent, Milk as essentially an emulsion of fat in a watery solution of sugar and mineral salts and with protein in a colloidal suspension. Milk fat exists in milk in the form of minute globules in a true emulsion of the oil-in-water type the fat globules being in the dispersed phase. Milk fat is not a single chemical compound but a variable mixture of several different glycerides. Each glyceride is the result of the union of glycerol and one or more organic acids. The organic acids contained in milk fat and fats from other sources other than milk fat are commonly termed as Fatty acids. KIJSET/OCT-DEC2015/VOL-2/Iss-4/A4 ISSN:2348-5477 IMPACT FACTOR(2015) 3.8071 Copyright © 2015 Published by kaav publications. All rights reserved www.kaavpublications.org 28 Proteins of milk Proteins are the most complex of organic substances. They contain carbon,, hydrogen, oxygen, nitrogen, sulfur and phosphorus. Protein is an indispensable part of the food of animals as it is the chief constituent of the protoplasm which forms the vital part of every living cell. In milk of average composition, a fat content of about 3.8 percent. Nearly all samples of normal milk would come between 2.8 and 4.00 percent. The protein of milk is not a single compound but includes two major proteins and small quantities of others. Casein constitutes about 80 percent of the total and lactalbumin 18%. A third protein recognized as present in milk is lactoglobulin. It is present in very small amounts, probably about 0.05 to 0.07 %. The constituents of human milk are used as a reference in developing infant formula. The composition of human milk is continually changing within a feed,during a 24-hour period and with the age of the infant. Human milk contains antibodies secretory IgA, IgG and IgM, live cells such as macrophages, lymphocytes, neutrophils and epithelial cells and other bioactive substances. Human milk contains human protein.Long Chain Polyunsaturated Fatty Acids (LCPUFAs) LCPUFAs are nutritive substances added to infant formula.LCPUFAs play an important role in the development of an infant’s visual and central nervous system. Breast milk contains pre-formed LCPUFAs, namely DHA (docosahexaenoic acid) and AA (arachidonic acid), and their precursors, ALA (alphalinolenic)/omega-3, and LA (linoleic acid)/omega-6, meeting all of the essential fatty acid requirements of the growing infant.Rapid brain accumulation of DHA and AA is dependent prenatally on placental transfer and postnatally on dietary sources and limited endogenous synthesis Research evidence suggeststhat formulafed infants require dietary pre-formed DHA and AA to match the tissue accretion rates of infants receiving DHA and AA from breast milk. It has been suggested that low levels of LCPUFAs found in formula milk may contribute to lower cognitionand vision skills in term infants. Recent systematic reviews from the Cochrane Library concluded that feeding term infants with milk formula enriched with LCPUFA had no proven benefit regarding vision, cognition or physical growth. KIJSET/OCT-DEC2015/VOL-2/Iss-4/A4 ISSN:2348-5477 IMPACT FACTOR(2015) 3.8071 Copyright © 2015 Published by kaav publications. All rights reserved www.kaavpublications.org 29 Probiotics and Prebiotics Probiotics are dietary supplements containing potentially beneficial bacteria and yeast. The rationale for the addition of probiotics to infant formula is to simulate breast milk, which favours the growth of bifidobacteria and represents the growth of unfavourable gastrointestinal bacteria. These effects are both likely to contribute to the protection that breast milk provides against intestinal infection. Probiotics are also proposed as a practicalapproach to the management of gastrointestinal and systemic conditions. In infants, including antibiotic-associated diarrhoea, viral gastroenteritis and allergy. Prebiotics are non-digestible components of foods, usually a fibre, that are used by beneficial gastrointestinal bacteria as food, boosting their growth in the large bowel. Breast milk contains galacto-oligosaccharides (GOS) which stimulate the growth of bifidobacteria in the infant gastrointestinal tract. The present study reveals that the human milk is very essential for the infants and the secretary immnoglobulins is more in human milk compared to cow milk and infant formula milk. Milk is a biological package of nutrients harmonized together in adequate proportion. Due to the high biological values of proteins, presence of a number of short chain and unsaturated fatty acids and the unique carbohydrate lactose in milk, our food habits should contain milk and milk products (Clarence Henry Ecklesetal., 1957). Milk as a source of infant food occupies a pivotal position in the nutritional market. Every biological species synthesizing milk, tailors at the genetic level, the amount and nature of milk constituents, to suit the nutritional requirements of their offspring. Likewise, human milk possesses additional virtues compared to cow and buffalo milk as an infant food. Immunoglobulins The antibodies are globular proteins. Antibodies were all gamma globulins. There are five classes of immunoglobulins. These are IgG, IgA, IgM, IgD and IgE. IgA in milk is principally in the form of secretory IgA, serves as a first line of mucosal defense. Maternal supply of secretary IgA is important as infant intestinal Immunoglobulin A(IgA) production does not begin until several months of age. KIJSET/OCT-DEC2015/VOL-2/Iss-4/A4 ISSN:2348-5477 IMPACT FACTOR(2015) 3.8071 Copyright © 2015 Published by kaav publications. All rights reserved www.kaavpublications.org 30 IgG is a 4-peptide unit composed of two light chains and two heavy chains. The words light and heavy denote the relative size of the polypeptide chains. The light chains have approximately 214 amino acids whereas the heavy chain is made up of 440 amino acids. IgM,macro-globulin is composed of five 4-peptide units linked to each other through -S-Sbond and J-chain. It is the major immunoglobulin involved in defence in the initial stages of infections.IgD is a four-peptide unit as other immunoglobulins, in which twolight and two heavy chains. The heavy chain, characteristic of this class of immunoglobulin, as delta and the class of IgD. IgGis a myeloma proteins, it has become evident that there are subclasses of this immunoglobulin. Four subclasses have been recognized: IgG1, IgG2, IgG3 and IgG4. The corresponding heavy chains would be y1, y2, y3, and y4 (Talwar G P etal., 2003) The differences are mainly in amino acid sequence here and there and in disulphide bridging. These differences give rise to some variations in their biological behavior.Breast milk is important for brain and retinal development, it protects against pathogens & allergens, Kills pathogenic organisms or modifies their growth and it stimulates epithelial maturation for future defense and moreover it is a first immunization and it protects against a common respiratory and intestinal diseases, storing and processing lots of information Results and discussion The spectrum of infrared is the essence of reflection of the infrared colour characteristics pattern of the sample (Liu et al., 2002). A FTIR absorption spectrum of milk sample of breast milk, cow milk and infant formula milk is shown in (figure1-3). FT-IR spectroscopy has been employed to monitor the significant differences in comparison of immunoglobulin of milk sample.A vibrational band assignment is analysed with the idea of the group frequencies of the various analytes present in the sample. FTIR spectroscopy as a bio-diagonostic tool to predict changes in the milk sample of breast milk, cow milk and infant formula milk. An increase in the secretion of immunoglobulin in the breast milk compared to the cow and infant formula milk. The vibrational peaks are mainly dominated by the protein constituents of the sample (Petibois et al., 2001; Deleris&Petibois, 2003). In this KIJSET/OCT-DEC2015/VOL-2/Iss-4/A4 ISSN:2348-5477 IMPACT FACTOR(2015) 3.8071 Copyright © 2015 Published by kaav publications. All rights reserved www.kaavpublications.org 31 study, The FTIR analysis of human milk, cow milk and infant formula milk was observed qualitatively and quantitatively and it is observed that the corresponding changes in their specific regions and there is a marked spectral differences in which the absorbance is directly proportional to the concentration and the analysis is carried out by calculating the intensity ratio among the absorption peaks. The spectral region 1762cm is due to C=O stretching vibration (Chandra S etal.,2009) and it is mainly due to AlbuminThe absorption band at 1652cm is mainly occupied by Amide I (Mordechai, S.,etal., 2004) C2=O cytosine (Dovbeshko GI etal., 1997). Immunoglobulin IgG2 and the absorption band in the region 1600-1800 cm is mainly occupied by C=O stretching and the band at 1628cm is due to the immunoglobulin IgG3 and the peak value at 1622cm is due to IgG2. The absorption peak in which the group rise to a medium intensity band around 1505 cm-1 not observed in the spectra of other amides and can be assigned as a C-N stretch C-N (CH3)2 stretch and it is of immunoglobulin IgG4. The spectral region (1500–1300 cm) is mainly dominated by the deformations of the methyl,methylene and C-H groups. For the methyl substituted benzene derivatives the asymmetric and symmetric deformation vibrations(Bright A etal., 2010)of methyl group normally appear in the region 1465-1440 cmand 1390-1370 cm. The band at 1479cm-1and 1464cm-1 is due to the immunoglobulin IgD, The absorption peak at 1419 cm is (COO) (polysaccharides, pectin), (Shetty G etal., 2006) and it has immunoglobulin of IgG1. The region around 1375-1300cm ofsulfones,, sulfonyl chlorides, and the peak at 1372cm is due to immunoglobulin IgA and the band at 1361cm is IgG1. The vibrational band at 1285cm is due to C-C types of vibration Alcohols, ethers, esters, carboxylic acids and and the peak at 1237cm is due to anhydrides which is around the region 1300-1000 cm and it is due to immunoglobulin IgA and the band at 1187cmis due C – C – H asymmetric bending of IgG and prominent peak at 1160cm is due to CO stretching (Wang HP etal., 1997) of IgM. The prominent absorption at 1150 cm-1is due to C-O stretching vibration (Wood BR etal., 1996) C-O stretching mode of the carbohydrates (Fung M etal., 1996)CH8, CH”8 deformations (Schulz H etal., 2007) n(C-O-C), ring (polysaccharides, pectin) (Shetty G etal., 2006) of immunoglobulin IgA. The peak around 1137 cmis Oligosaccharide C-OH stretching band (Yoshida S etal., 1997) 2-Methylmannoside and it is due to IgA KIJSET/OCT-DEC2015/VOL-2/Iss-4/A4 ISSN:2348-5477 IMPACT FACTOR(2015) 3.8071 Copyright © 2015 Published by kaav publications. All rights reserved www.kaavpublications.org 32 and the band at 1028cm is the Glycogen absorption due to C-O and C-Cstretching and C-O-H deformation motions (Huleihel M etal., 2002). FTIR spectroscopy is used to study the spectral difference and exhibits vibrational band characteristics of the various group frequencies, the spectrum of a breast milk, cow milk, and formula milk samples in which there is a change in the positions of the peaks but different in terms of the absorption levels of the peaks. Fig. 1, 2, 3 shows the FTIR spectrum of breast milk, cow milk and infant formula milk respectively. It is observed that the amount of absorption increases in breast milk, followed by decrease in infant formula milk and further decrease in the cow milk. This decrease in absorption is due to the changes in the milk sample, an increase in the immunoglobulin levels (IgA, IgG, IgD and IgM) in breast milk sample is more than the cow milk and infant formula milk. In order to quantify the results, intensity ratio parameters of 5 characteristics absorption bands of milk samples are R1 (I1257/I1572), R2 (I1157/I1150), R3 (I1157/I1572) R4 (I1150/I1572) R5 (I1285/I1572) are calculated. It is observed that these values of immunoglobulin levels of IgA increase in breast milk, decreases in infant formula milk and further decreases in cow milk.Immunoglobulins IgA directly improve the immune status of the new born baby. It acts as antibodies against intestinal bacteria and viruses are found in human breast milk. Breast milk reduces the incidence and severity of diseases. Table 1 verifies the same absorption by intensity ratio calculation. The analysis is carried out in finding the immunoglobulin levels of IgMfor breast milk, infant formula milk and cow milk. Breast milk has a greater absorption value than infant formula milk and cow milk. Immunoglobulin IgM do not cross the placental barrier but it can be manufactured by the fetus. In order to quantify the results, 8 intensity ratio parameters R1 ( I1160/I1762 ) , R2 (I1160/I1372 ), R3 (I1028/I1464 ), R4 (I1160/I1479), R5 ( I1160/I1505), R6 (I1028/I1622), R7 (I1160/I1652), R8 (I1028/I1419 ) are calculated. The absorption levels of the peaks increases in breast milk and decreases in the infant formula milk than that of the breast milk and further decreases in the cow milk than that of the infant formula milk. Table-2 verifies the same absorption by intensity ratio calculation. Immunoglobulin IgDis the antibodies in breast milk, combat diseases without causing inflammation. There are six intensity ratio parameters have been calculated such as R1 ( I1464/I1628), R2 ( I1028/I1464), R3 (I1762/I1479 ), R4 (I1479/I1505), R5 ( I1464/I1652), R6 ( I1479/I1652 ) and foundthat the KIJSET/OCT-DEC2015/VOL-2/Iss-4/A4 ISSN:2348-5477 IMPACT FACTOR(2015) 3.8071 Copyright © 2015 Published by kaav publications. All rights reserved www.kaavpublications.org 33 immunoglobulin levels of IgD increases in breast milk compared to infant formula milk and cow milk. Table 3 verifies the same absorption value by the intensity ratio calculation. IgG antibodies to bacterial toxins and virusesprovide transient protection to thenewborn. ImmunoglobulinIgG is the primary immunoglobulin class found in milk and bovine colostrum. To quantify the results, 8 intensity ratio parameter of IgG2, are R1 ( I1187/I1622), R2 ( I1479/I1622), R3 ( I1237/I1652)R4 ( I1372/I1652), R5 ( I1505/I1652), R6 ( I1762/I1652) and immunoglobulin IgG3in which there are 7 intensity ratio parameters are R1 ( I1028/I1628), R2 ( I1150/I1628), R3 ( I1419/I1628), R4 ( I1237/I1628), R5 ( I1361/I1628), R6 ( I1505/I1628), R6 ( I1200/I1628) and immunoglobulin of IgG4 in which 5 intensity ratio parameters are taken as R1 ( I1028/I1505), R2 ( I1285/I1505), R3 ( I1137/I1505), R4 ( I1160/I1505), R5 ( I1200/I1505) have been calculated and observed that the immunoglobulin levels increases in breast milk compared to infant formula milk and cow milk. Table (4a, 4b, and 4c) verifies the absorption value of the intensity ratio calculation. Analysis with histogram From the FTIR spectra obtained a bar diagram as shown in Figure(4-9) shows a striking spectral difference between the immunoglobulin levels (IgA, IgM, IgD, IgG2, IgG3, IgG4) of breast milk, infant formula milk and cows has a greater abosorbance value of immunoglobulin levels of breast milk compared to infant formula milk and cow milk.