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Available lysine, protein digestibility and lactulose in commercial infant formulas
International Dairy Journal 13 (2003) 95–99
Available lysine, protein digestibility and lactulose in commercial infant formulas Adriana S. Pereyra Gonza! les, Gabriela B. Naranjo, Laura S. Malec, Mar!ıa S. Vigo* ! Departamento de Qu!ımica Organica, Area Bromatolog!ıa. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. 1428 Buenos Aires, Argentina Received 21 May 2002; accepted 3 November 2002
Abstract Available lysine, in vitro protein digestibility and lactulose values were determined in 23 commercial infant formulas. The mean available lysine content of the formulas based on dairy proteins was 66.779.5 mg g 1 protein, similar to that of human milk, while that of soy based formulas was considerably lower (45.078.3 mg g 1 protein). In vitro protein digestibility values ranged 85.5– 88.9% for soy-based formulas and 90.5–98.3% for formulas based on dairy proteins. Formulas based on milk enriched with whey had higher lactulose content than those based on cow’s milk. However, all values were below the limit of 600 mg L 1 recommended for UHT milk. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Infant formula; Available lysine; Protein digestibility; Lactulose
1. Introduction Human milk is considered the best food for infants. It meets all their nutritional requirements and promotes infant health and development. However, in some circumstances, e.g. insufficient milk syndrome, breastfeeding failure, social factors (wage-earners mothers) or for premature and low-birth-weight infants, it is necessary to substitute or fortify breast-feeding with specially designed formulas (Gurr, 1981; Guo, Hendricks, & Kindstedt, 1998). Theoretically, infant formulas can be based on any appropriate blend of proteins, carbohydrates, fats, minerals and vitamins. However, infant milk products are based predominantly on cow’s milk on the account of special properties of its proteins and lactose and its availability in Western countries. One of the major differences between cow’s milk and human milk is the protein composition and content. Casein constitutes 80% of the protein fraction in cow’s milk and not more than 40% in human milk (O’Connor, *Corresponding author. Tel.: +54-11-4576-3346; fax: +54-11-45763346. E-mail address: [email protected] (M.S. Vigo).
Masor, Paule, & Benson, 1997). Thus, in most infant formulas cow’s milk is enriched with whey or whey protein concentrate to attain usually a 40:60 casein/ whey protein ratio. The other major difference is the higher lactose content of human milk. In cow’s milk based infant formulas the lactose content is increased by the addition of whey and/or lactose alone. In some cases, substitution or fortification of human milk with cow’s milk based formulas is not a good choice as it can cause severe disturbances. Cow’s milk allergy prevalence has been reported from o1% to as high as 8%, as all the major protein fractions of cow’s milk are potentially antigenic and allergenic (Gurr, 1981; Lo & Kleinman, 1996). In some cases, formulas not based on cow’s milk but made with soy protein or hydrolyzates of milk proteins are available. On the other hand, though lactose intolerance is not very frequent in infants (American Academy of Pediatrics, Committee on Nutrition, 1978; Heubi et al., 2000), infant formulas in which lactose is replaced by other sugars, e.g. glucose, fructose, corn syrup or maltodextrin, are available, the protein source being soy protein or casein. Infant formulas combine a set of factors that make them highly sensitive to Maillard reaction, that is, high carbohydrate content, lysine-rich proteins, relatively
0958-6946/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0958-6946(02)00173-5
96
! et al. / International Dairy Journal 13 (2003) 95–99 A.S. Pereyra Gonzales
high temperature applied during the manufacturing process, and storage for long periods of time. The loss of lysine availability and the decrease of protein digestibility are the main nutritional consequences of Maillard reaction during heat treatment and storage of foods (Hurrell, 1990; Swaisgood & Catignani, 1991). The nutritional damage is of particular interest in infant formulas because frequently they are the only protein source during early infancy. Available lysine content is widely used as marker for nutritional loss. During heat treatment, lactose undergoes the Lobry de Bruyn-Alberda van Eckenstein rearrangement, which gives rise initially to isomeric disaccharides, mainly lactulose. Lactulose is not known to occur naturally in milk: it is formed in heated dairy products (Andrews, ! * & Olano, 1999). So, lactulose is 1986; Lopez-Fandi no also a useful indicator of heat damage in milk. Due to the wide difference in composition among formulas and the variability of time/temperature conditions during its processing, their sensitivity to Maillard reaction and lactose isomerization may be quite different. The aim of this work was to measure the available lysine, protein digestibility and lactulose content in commercial powder infant formulas available in Argentina and to determine if there is any variability of these values among the different formulas related to their protein and carbohydrate composition that may result in inadequate nutrient availability.
2. Materials and methods 2.1. Materials Twenty three different commercial powder infant formulas, manufactured by Arinco (Denmark), John Wyeth Laboratorios S.A. (Argentina), Kasdorf S.A. (Argentina), M & R Laboratoria B.V. (Netherlands), Mead Johnson (Canada), Mead Johnson (Me! xico), Milupa AG (Germany), N.V. Nutricia (Netherlands), Nestle! Argentina (Argentina), Nestle! Netherlands (Netherlands), Wyeth Nutritionals Inc. (USA) and Wyeth S.A. (Me! xico) and a sample of powdered cow’s milk were purchased from the local market. Four formulas were based on soy protein and the others on dairy proteins: four on casein, seven on cow’s milk and eight on milk enriched with whey. The last formulas contained a casein:whey proteins ratio 40:60 (as indicated on the label). Table 1 shows the protein and carbohydrate composition of the formulas. Before chemical analysis, the samples were defatted by extraction in a Soxhlet-type extractor with ether for 24 h. All chemicals used were of analytical grade.
2.2. Methods Nitrogen and available lysine determinations were performed after dialysis to remove free amino acids commonly added to infant formulas. The defatted samples were dialyzed against distilled water through dialysis tubing, 12 000 MW, with constant stirring at 41C for 72 h. Total nitrogen was determined in duplicate by the Kjeldahl method using a digestor Buchi . 430 and a N2 distillation unit Buchi . 320 (Buchi . LaboratoriumsTechnik AG, Flawil, Switzerland). Available lysine was measured using duplicate samples by a spectrophotometric method with o-phthaldial! dehyde (OPA) (Vigo, Malec, Gomez, & Llosa, 1992). Samples were dissolved in 10% sodium dodecylsulfate solution; 50 mL of sample solution were added to 2 mL OPA reagent (Goodno, Swaisgood, & Catignani, 1981) and the absorbance was measured at 340 nm with a Hewlett Packard spectrophotometer HP 8453 (Hewlett Packard, Palo Alto, USA). Six replicate measurements of each duplicate sample were carried out. The available lysine content was obtained from a standard curve prepared with casein dissolved in pH 9.0 sodium tetraborate buffer solution in the range 1.0– 10.0 mg mL 1. The interference of free amino groups of amino acids, small peptides and amines was checked according to Goodno et al. (1981) in the supernatant of samples dissolved in pH 9.0 sodium tetraborate buffer solution after treatment with 10% trichloroacetic acid solution. This interference was always negligible. An in vitro method using trypsin, chymotrypsin and porcine intestinal peptidase was used to evaluate protein digestibility in triplicate. The pH was adjusted to 8.0 and maintained for 5–10 min before adding 1 mL of the enzyme solution. Enzyme activity was estimated from the amount of NaOH 0.1 n required to maintain the pH at 7.98 for exactly 10 min. Digestibility was calculated using the equation for animal proteins (Pedersen & Eggum, 1983). For estimation of lactulose content in samples containing lactose, formulas and powdered cow’s milk were dissolved in distilled water (50% p/v) and 10 mL of the solution clarified with Carrez I and Carrez II solutions. The mixture was filtered and lactulose determined in a 5 mL aliquot of the filtrate by the enzymatic method of Geier and Klostermeyer (1980). Each sample was analyzed by duplicate.
2.3. Statistical analysis Statistical analyses were conducted using one-way analysis of variance (ANOVA) and the Student’s t test. Multiple comparisons were made using a multiple range test.
! et al. / International Dairy Journal 13 (2003) 95–99 A.S. Pereyra Gonzales
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Table 1 Protein and carbohydrate composition (according to information on label), available lysine, protein digestibility and lactulose values of infant formulas Formula basis and identification
Proteins
Carbohydrates
Available lysine (mg g 1protein)
Digestibility (%)
Lactulose (mg L 1 as fed)
Corn Corn Corn Corn
33.2 45.9 48.9 52.0
87.0 85.5 88.9 87.4
— — — —
Soy protein isolate 1 2 3 4
Soy Soy Soy Soy
Casein 5 6 7 8
Calcium caseinate Casein Acid casein Sodium caseinate
Glucose, maltodextrin Corn syrup Maltodextrin Maltodextrin
41.3 65.3 68.8 70.0
93.0 97.5 93.5 90.5
— — — —
Milk 9 10 11 12
Skim milk Whole milk Whole milk Skim milk
62.1 66.0 66.1 68.7
90.9 96.1 96.7 93.4
68 29 108 59
13 14 15
Skim milk Skim milk Skim milk
Lactose, sucrose Lactose Lactose Lactose, maltodextrin, sucrose Lactose Lactose Lactose, corn syrup
72.0 75.3 81.1
98.3 93.0 92.1
79 93 78
Lactose, maltodextrin
53.8
90.7
186
Lactose
55.9
87.3
199
Lactose, maltodextrin
57.8
89.5
175
Lactose, maltodextrin
64.3
88.7
312
Lactose
72.6
97.1
98
Lactose
73.9
91.8
97
Lactose
74.7
89.9
148
Lactose
77.8
88.5
113
Lactose
80.4
93.1
0
protein protein protein protein
isolate isolate isolate isolate
Milk enriched with wheya 16 Demineralized whey, skim milk 17 Demineralized whey, skim milk 18 Whey protein concentrate, skim milk 19 Demineralized whey, skim milk 20 Demineralized whey, skim milk 21 Demineralized whey concentrate, skim milk 22 Demineralized whey protein, skim milk, whole milk 23 Demineralized whey, skim milk Cow’s milk a
syrup, sucrose syrup syrup syrup, sucrose
Casein:whey proteins 40:60 (as indicated on the label).
3. Results and discussion 3.1. Available lysine Table 1 summarizes all the data obtained including the available lysine content, protein digestibility and lactulose. The ANOVA of the available lysine values among the three groups of dairy proteins based formulas (casein, milk and milk enriched with whey) showed no statistically significant differences at the 5% level. However,
available lysine content of sample 5, the only formula containing glucose, was about 40% lower than those of the other formulas with casein. This is consistent with Lewis and Lea (1950), Baxter (1995) and Naranjo, Malec, and Vigo (1998) who reported that glucose is considerably more reactive than the other sugars and could cause a greater loss of available lysine by Maillard reaction. The average available lysine value in dairy proteins based formulas was 66.779.5 mg g 1 of protein, quite similar to the mean value accepted for human milk
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(66 mg g 1; WHO, 2000). Available lysine contents of these formulas were lower than that for powdered milk obtained in our laboratory (80.4 mg g 1) or those reported in the literature (76.6–85.4 mg g 1 protein; Hurrell, Finot, & Ford, 1983; Erbersdobler & Hupe, 1991; van Mil & Jans, 1991; Vigo et al., 1992). Available lysine contents in soy based formulas were significantly lower than those found in dairy protein based formulas (po0:05) averaging 45.078.3 mg g 1 protein. The available lysine contents in dairy proteins based formulas were similar to those reported by Mitchell and Grundel (1986) and Ferrer, Alegr!ıa, Farre! , Abella! n, and Romero (2000). The corresponding values of soy based formulas were lower than those found by Mitchell and Grundel (1986). In contrast to Evangelisti, Calcagno, and Zunin (1994), no significant differences in available lysine content were observed between formulas with and without the partial replacement of lactose by corn syrup or maltodextrin. Most infant formulas had lower contents of available lysine than those reported for the corresponding protein sources, i.e. soy protein (63.4–64.1 mg g 1 protein; Steinke, Prescher, & Hopkins, 1980; Friedman & Brandon, 2001); casein (79.8–85.0 mg g 1 protein; Steinke et al., 1980; Smith & Friedman, 1984; Friedman & Brandon, 2001); milk (76.6–85.4 mg g 1 protein; Hurrell et al., 1983; Erbersdobler & Hupe, 1991; van Mil & Jans, 1991; Vigo et al., 1992); and whey protein (80–97.5 mg g 1 protein; Desrosiers, Savoie, Bergeron, & Parent, 1989, Lindemann-Schneider & Fennema, 1989; Erbersdobler & Hupe, 1991). The losses were probably a consequence of the combined effects of the type of protein, the treatments during the manufacture and the different time and conditions of storage. The recommended dietary allowance available lysine for infants below the age of 6 months is 103 mg kg 1 body weight day 1 (Harper, 1981; National Research Council, 1989; WHO, 2000). The daily intake of available lysine with each formula, prepared and consumed according to the manufacturer’s instructions was calculated. It was found that only sample 1 could not provide the recommended daily intake of available lysine for infants weighing more than 6 kg, while available lysine content in sample 2 was close to the limit and could be marginally deficient. Furthermore, the risk may be increased by losses during storage. 3.2. Protein digestibility Digestibilities of soy based formulas were significantly lower (po0:05) than those of formulas based on dairy proteins. However, all values were quite satisfactory from the nutritional standpoint. The values observed were consistent with those found by Mitchell and Grundel (1986) and Sarwar, Botting, and Peace . (1989). Rudloff and Lonnerdal (1992) reported values
lower than those of the present work, probably because they used a different method to evaluate protein digestibility. 3.3. Lactulose Lactulose, which is absent in raw milk, is considered a useful indicator to evaluate the thermal treatment applied to milk, as its content increases with the intensity of the treatment (Andrews, 1986; Ferrer, ! Alegr!ıa, Farre! , Abella! n, & Romero, 1999; Lopez* & Olano, 1999). The few reports about Fandino lactulose content in infant formulas showed usually higher values than those found in commercial milk (Bernhart, Gagliardi, Tomarelli, & Stribley, 1965; Beach & Menzies, 1986; Corzo, Delgado, Troyano, & Olano, 1994; Ferrer et al., 1999). Table 1 shows that lactulose contents of infant formulas containing lactose (formula 9 to 23) were between 29 and 108 mg L 1 of reconstituted formulas based on milk and 97 and 312 mg L 1 of reconstituted formulas based on milk enriched with whey, the difference between both groups being statistically significant (po0:05). Lactulose values observed in this study were quite low compared with those found by Bernhart et al. (1965) (500–2500 mg L 1 in sterilized infant formulas and not more than 290 mg L 1 in reconstituted spray-dried infant formulas), and similar to those reported by Beach and Menzies (1986) in UHT infant formulas (55–350 mg L 1). Although no limit to lactulose content in infant formulas has been established, the International Dairy Federation (1992) and the European Communities Commission (EC Commission, 1992) proposed 600 mg L 1 for commercial UHT milk. Lactulose contents in formulas included here were well below this limit. 4. Conclusions The type of dairy protein used in the infant formulas obtained from Argentina markets had no significant effect on the available lysine and protein digestibility values. Though the corresponding values in formulas based on soy protein were significantly lower than those of dairy proteins based formulas, they were, except for one sample, quite satisfactory considering the recommended values. Lactulose contents showed a great variability with a trend to higher values in those formulas containing milk enriched with whey. Acknowledgements This work was supported by a grant UBACYT No. TW08 from Secretar!ıa de Ciencia y Te! cnica de la Universidad de Buenos Aires.
! et al. / International Dairy Journal 13 (2003) 95–99 A.S. Pereyra Gonzales
References American Academy of Pediatrics, Committee on Nutrition (1978). The practical significance of lactose intolerance in children (RE0006). Pediatrics, 62, 240–245. Andrews, G. R. (1986). Formation and occurrence of lactulose in heated milk. Journal of Dairy Research, 53, 665–680. Baxter, J. R (1995). Free amino acid stability in reducing sugar systems. Journal of Food Science, 60, 405–408. Beach, R. C., & Menzies, I. (1986). Determination of lactulose and soya oligosaccharides in infant formula milk feeds. Journal of Dairy Research, 53, 293–299. Bernhart, F. W., Gagliardi, E. D., Tomarelli, R. M., & Stribley, R. C. (1965). Lactulose in modified milk products for infant nutrition. Journal of Dairy Science, 48, 399–400. Corzo, N., Delgado, T., Troyano, E., & Olano, A. (1994). Ratio of lactulose to furosine as indicator of quality of commercial milks. Journal of Food Protection, 57, 737–739. Desrosiers, T., Savoie, L., Bergeron, G., & Parent, G. (1989). Estimation of lysine damage in heated whey proteins by furosine determinations in conjunction with the digestion cell technique. Journal of Agricultural and Food Chemistry, 37, 1385–1391. EC Commission (1992). Proposed draft Commission decision laying down the limits and the methods for distinguishing between the different types of heat treated drinking milk. Dairy Chemist’s Group, Doc.VI/5726/92. Erbersdobler, H., & Hupe, A. (1991). Determination of lysine damage and calculation of lysine bio-availability in several processed foods. Zeitschrift fur 30, 46–49. . Ernahrungswissenschaft, . Evangelisti, F., Calcagno, C., & Zunin, P. (1994). Relationship between blocked lysine and carbohydrate composition of infant formulas. Journal of Food Science, 59, 335–337. Ferrer, E., Alegr!ıa, A., Farr!e, R., Abell!an, P., & Romero, F. (1999). Review: Selected indicators of the quality of thermal processed milk. Food Science and Technology International, 5, 447–461. Ferrer, E., Alegr!ıa, A., Farr!e, R., Abell!an, P., & Romero, F. (2000). Effects of thermal processing and storage on available lysine and furfural compounds contents of infant formulas. Journal of Agricultural and Food Chemistry, 48, 1817–1822. Friedman, M., & Brandon, D. L. (2001). Nutritional and health benefits of soy proteins. Journal of Agricultural and Food Chemistry, 49, 1069–1086. Geier, H., & Klostermeyer, H. (1980). Enzymatische Bestimmung von Lactulose. Zeitschrift fur . Lebensmittel-Unterschung und-Forschungwissenschaft, 171, 443–445. Goodno, C. C., Swaisgood, H. E., & Catignani, G. L. (1981). A fluorimetric assay for available lysine in proteins. Analytical Biochemistry, 115, 203–211. Guo, M. R., Hendricks, G. M., & Kindstedt, P. S. (1998). Component distribution and interactions in powdered infant formula. International Dairy Journal, 8, 333–339. Gurr, M. I. (1981). Review on the progress of dairy science: Human and artificial milks for infant feeding. Journal of Dairy Research, 48, 519–554. Harper, A. (1981). Amino acid scoring patterns. FAO/WHO/UNU. Rome. Heubi, J., Karasov, R., Reisinger, K., Blatter, M., Rosenberg, L., Vanderhood, J., Darden, P. M., Safier, J., Martin, T., & Euler, A. R. (2000). Randomized multicenter trial documenting the efficacy and safety of a lactose-free and a lactose-containing formula for term infants. Journal of the American Dietetic Association, 100, 212–217. Hurrell, R. F. (1990). Influence of the Maillard reaction on the nutritional value of foods. In: P. A. Finot, H. U. Aeschbacher, R. F. Hurrell, & R. Liardon (Eds.), The Maillard reaction in food
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processing, human nutrition and physiology (pp. 245–258). Basel: Birkh.auser Verlag. Hurrell, R. F., Finot, P. A., & Ford, J. E. (1983). Storage of milk powders under adverse conditions. 1. Losses of lysine and other essential amino acids as determined by chemical and microbiological methods. British Journal of Nutrition, 49, 343–354. International Dairy Federation (1992). Influence of technology on the quality of heat treated milk and fluid milk products. B-Doc.222, IDF, Brussels, Belgium. Lewis, V. M., & Lea, C. H. (1950). A note on the relative rates of reaction of several reductive sugars and sugar derivatives with casein. Biochimica et Biophysica Acta, 4, 532–534. Lindemann-Schneider, U., & Fennema, O. (1989). Stability of lysine, methionine, and tryptophan in dried whey concentrate during storage. Journal of Dairy Science, 72, 1740–1747. Lo, C. W. y Kleinman, R. E. (1996). Infant formula, past and future: Opportunities for improvement. American Journal of Clinical Nutrition, 63, 646S–650S. ! * R., & Olano, A. (1999). Review: Selected indicators of Lopez-Fandi no, the quality of thermal processed milk. Food Science and Technology International, 5, 121–137. Mitchell, G. V., & Grundel, E. (1986). Nutritional value of proteins in powdered infant formula: In vitro and in vivo methods. Journal of Agricultural and Food Chemistry, 34, 650–653. Naranjo, G. B., Malec, L. S., & Vigo, M. S. (1998). Reducing sugars effect on available lysine loss of casein by moderate heat treatment. Food Chemistry, 62, 309–313. National Research Council, Food and Nutrition Board (1989). Recommended Dietary Allowances (10th ed.). Washington, DC: National Academy Press. O’Connor, D. L., Masor, M. L., Paule, Ch., & Benson, J. (1997). Amino acid composition of cow’s milk and human requirements. In: R. A. S. Welch, D. J. W. Burns, S. R. Davis, A. I. Popay & C. G. Prosser (Eds.), Milk composition, production and biotechnology. Biotechnology and agricultural series Vol. 18. (pp. 203–213). Hamilton, New Zealand: CABI Publishing. Pedersen, B., & Eggum, B. O. (1983). Prediction of protein digestibility by an in vitro enzymatic pH-stat procedure. Zeitschrift fur und Futtermittelkunde, 49, . Tierphysiologie, Tierernahrung . 265–277. . Rudloff, S., & Lonnerdal, B. (1992). Solubility and digestibility of milk proteins in infant formulas exposed to different heat treatments. Journal of Pediatric Gastroenterology and Nutrition, 15, 25–33. Sarwar, G., Botting, H. G., & Peace, R. W. (1989). Amino acid rating methods for evaluating protein adequacy of infant formulas. Journal of the Association of Official Analytical Chemists, 72, 622–627. Smith, G.A., & Friedman, M. (1984). Effect of carbohydrates and heat on the amino acid composition and chemically available lysine content of casein. Journal of Food Science, 49, 817–20, 843. Steinke, F. H., Prescher, E. E., & Hopkins, D. T. (1980). Nutritional evaluation (PER) of isolated soybean protein and combinations of food proteins. Journal of Food Science, 45, 323–327. Swaisgood, H. E., & Catignani, G. L. (1991). Protein digestibility: In vitro methods of assessment. Advances in Food and Nutrition Research, 35, 185–236. van Mil, P. J. J. M., & Jans, J. A. (1991). Storage stability of whole milk powder: Effects of process and storage conditions on product properties. Netherland Milk and Dairy Journal, 45, 145–167. ! Vigo, M. S., Malec, L. S., Gomez, R. G., & Llosa, R. A. (1992). Spectrophotometric assay using o-phthaldialhyde for determination of reactive lysine in dairy products. Food Chemistry, 44, 363–365. WHO (2000). Energy and protein requirements. World Health Organization Technical Report Series 724. Geneva, Switzerland.
Available lysine, protein digestibility and lactulose in commercial infant formulas Adriana S. Pereyra Gonza! les, Gabriela B. Naranjo, Laura S. Malec, Mar!ıa S. Vigo* ! Departamento de Qu!ımica Organica, Area Bromatolog!ıa. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. 1428 Buenos Aires, Argentina Received 21 May 2002; accepted 3 November 2002
Abstract Available lysine, in vitro protein digestibility and lactulose values were determined in 23 commercial infant formulas. The mean available lysine content of the formulas based on dairy proteins was 66.779.5 mg g 1 protein, similar to that of human milk, while that of soy based formulas was considerably lower (45.078.3 mg g 1 protein). In vitro protein digestibility values ranged 85.5– 88.9% for soy-based formulas and 90.5–98.3% for formulas based on dairy proteins. Formulas based on milk enriched with whey had higher lactulose content than those based on cow’s milk. However, all values were below the limit of 600 mg L 1 recommended for UHT milk. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Infant formula; Available lysine; Protein digestibility; Lactulose
1. Introduction Human milk is considered the best food for infants. It meets all their nutritional requirements and promotes infant health and development. However, in some circumstances, e.g. insufficient milk syndrome, breastfeeding failure, social factors (wage-earners mothers) or for premature and low-birth-weight infants, it is necessary to substitute or fortify breast-feeding with specially designed formulas (Gurr, 1981; Guo, Hendricks, & Kindstedt, 1998). Theoretically, infant formulas can be based on any appropriate blend of proteins, carbohydrates, fats, minerals and vitamins. However, infant milk products are based predominantly on cow’s milk on the account of special properties of its proteins and lactose and its availability in Western countries. One of the major differences between cow’s milk and human milk is the protein composition and content. Casein constitutes 80% of the protein fraction in cow’s milk and not more than 40% in human milk (O’Connor, *Corresponding author. Tel.: +54-11-4576-3346; fax: +54-11-45763346. E-mail address: [email protected] (M.S. Vigo).
Masor, Paule, & Benson, 1997). Thus, in most infant formulas cow’s milk is enriched with whey or whey protein concentrate to attain usually a 40:60 casein/ whey protein ratio. The other major difference is the higher lactose content of human milk. In cow’s milk based infant formulas the lactose content is increased by the addition of whey and/or lactose alone. In some cases, substitution or fortification of human milk with cow’s milk based formulas is not a good choice as it can cause severe disturbances. Cow’s milk allergy prevalence has been reported from o1% to as high as 8%, as all the major protein fractions of cow’s milk are potentially antigenic and allergenic (Gurr, 1981; Lo & Kleinman, 1996). In some cases, formulas not based on cow’s milk but made with soy protein or hydrolyzates of milk proteins are available. On the other hand, though lactose intolerance is not very frequent in infants (American Academy of Pediatrics, Committee on Nutrition, 1978; Heubi et al., 2000), infant formulas in which lactose is replaced by other sugars, e.g. glucose, fructose, corn syrup or maltodextrin, are available, the protein source being soy protein or casein. Infant formulas combine a set of factors that make them highly sensitive to Maillard reaction, that is, high carbohydrate content, lysine-rich proteins, relatively
0958-6946/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0958-6946(02)00173-5
96
! et al. / International Dairy Journal 13 (2003) 95–99 A.S. Pereyra Gonzales
high temperature applied during the manufacturing process, and storage for long periods of time. The loss of lysine availability and the decrease of protein digestibility are the main nutritional consequences of Maillard reaction during heat treatment and storage of foods (Hurrell, 1990; Swaisgood & Catignani, 1991). The nutritional damage is of particular interest in infant formulas because frequently they are the only protein source during early infancy. Available lysine content is widely used as marker for nutritional loss. During heat treatment, lactose undergoes the Lobry de Bruyn-Alberda van Eckenstein rearrangement, which gives rise initially to isomeric disaccharides, mainly lactulose. Lactulose is not known to occur naturally in milk: it is formed in heated dairy products (Andrews, ! * & Olano, 1999). So, lactulose is 1986; Lopez-Fandi no also a useful indicator of heat damage in milk. Due to the wide difference in composition among formulas and the variability of time/temperature conditions during its processing, their sensitivity to Maillard reaction and lactose isomerization may be quite different. The aim of this work was to measure the available lysine, protein digestibility and lactulose content in commercial powder infant formulas available in Argentina and to determine if there is any variability of these values among the different formulas related to their protein and carbohydrate composition that may result in inadequate nutrient availability.
2. Materials and methods 2.1. Materials Twenty three different commercial powder infant formulas, manufactured by Arinco (Denmark), John Wyeth Laboratorios S.A. (Argentina), Kasdorf S.A. (Argentina), M & R Laboratoria B.V. (Netherlands), Mead Johnson (Canada), Mead Johnson (Me! xico), Milupa AG (Germany), N.V. Nutricia (Netherlands), Nestle! Argentina (Argentina), Nestle! Netherlands (Netherlands), Wyeth Nutritionals Inc. (USA) and Wyeth S.A. (Me! xico) and a sample of powdered cow’s milk were purchased from the local market. Four formulas were based on soy protein and the others on dairy proteins: four on casein, seven on cow’s milk and eight on milk enriched with whey. The last formulas contained a casein:whey proteins ratio 40:60 (as indicated on the label). Table 1 shows the protein and carbohydrate composition of the formulas. Before chemical analysis, the samples were defatted by extraction in a Soxhlet-type extractor with ether for 24 h. All chemicals used were of analytical grade.
2.2. Methods Nitrogen and available lysine determinations were performed after dialysis to remove free amino acids commonly added to infant formulas. The defatted samples were dialyzed against distilled water through dialysis tubing, 12 000 MW, with constant stirring at 41C for 72 h. Total nitrogen was determined in duplicate by the Kjeldahl method using a digestor Buchi . 430 and a N2 distillation unit Buchi . 320 (Buchi . LaboratoriumsTechnik AG, Flawil, Switzerland). Available lysine was measured using duplicate samples by a spectrophotometric method with o-phthaldial! dehyde (OPA) (Vigo, Malec, Gomez, & Llosa, 1992). Samples were dissolved in 10% sodium dodecylsulfate solution; 50 mL of sample solution were added to 2 mL OPA reagent (Goodno, Swaisgood, & Catignani, 1981) and the absorbance was measured at 340 nm with a Hewlett Packard spectrophotometer HP 8453 (Hewlett Packard, Palo Alto, USA). Six replicate measurements of each duplicate sample were carried out. The available lysine content was obtained from a standard curve prepared with casein dissolved in pH 9.0 sodium tetraborate buffer solution in the range 1.0– 10.0 mg mL 1. The interference of free amino groups of amino acids, small peptides and amines was checked according to Goodno et al. (1981) in the supernatant of samples dissolved in pH 9.0 sodium tetraborate buffer solution after treatment with 10% trichloroacetic acid solution. This interference was always negligible. An in vitro method using trypsin, chymotrypsin and porcine intestinal peptidase was used to evaluate protein digestibility in triplicate. The pH was adjusted to 8.0 and maintained for 5–10 min before adding 1 mL of the enzyme solution. Enzyme activity was estimated from the amount of NaOH 0.1 n required to maintain the pH at 7.98 for exactly 10 min. Digestibility was calculated using the equation for animal proteins (Pedersen & Eggum, 1983). For estimation of lactulose content in samples containing lactose, formulas and powdered cow’s milk were dissolved in distilled water (50% p/v) and 10 mL of the solution clarified with Carrez I and Carrez II solutions. The mixture was filtered and lactulose determined in a 5 mL aliquot of the filtrate by the enzymatic method of Geier and Klostermeyer (1980). Each sample was analyzed by duplicate.
2.3. Statistical analysis Statistical analyses were conducted using one-way analysis of variance (ANOVA) and the Student’s t test. Multiple comparisons were made using a multiple range test.
! et al. / International Dairy Journal 13 (2003) 95–99 A.S. Pereyra Gonzales
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Table 1 Protein and carbohydrate composition (according to information on label), available lysine, protein digestibility and lactulose values of infant formulas Formula basis and identification
Proteins
Carbohydrates
Available lysine (mg g 1protein)
Digestibility (%)
Lactulose (mg L 1 as fed)
Corn Corn Corn Corn
33.2 45.9 48.9 52.0
87.0 85.5 88.9 87.4
— — — —
Soy protein isolate 1 2 3 4
Soy Soy Soy Soy
Casein 5 6 7 8
Calcium caseinate Casein Acid casein Sodium caseinate
Glucose, maltodextrin Corn syrup Maltodextrin Maltodextrin
41.3 65.3 68.8 70.0
93.0 97.5 93.5 90.5
— — — —
Milk 9 10 11 12
Skim milk Whole milk Whole milk Skim milk
62.1 66.0 66.1 68.7
90.9 96.1 96.7 93.4
68 29 108 59
13 14 15
Skim milk Skim milk Skim milk
Lactose, sucrose Lactose Lactose Lactose, maltodextrin, sucrose Lactose Lactose Lactose, corn syrup
72.0 75.3 81.1
98.3 93.0 92.1
79 93 78
Lactose, maltodextrin
53.8
90.7
186
Lactose
55.9
87.3
199
Lactose, maltodextrin
57.8
89.5
175
Lactose, maltodextrin
64.3
88.7
312
Lactose
72.6
97.1
98
Lactose
73.9
91.8
97
Lactose
74.7
89.9
148
Lactose
77.8
88.5
113
Lactose
80.4
93.1
0
protein protein protein protein
isolate isolate isolate isolate
Milk enriched with wheya 16 Demineralized whey, skim milk 17 Demineralized whey, skim milk 18 Whey protein concentrate, skim milk 19 Demineralized whey, skim milk 20 Demineralized whey, skim milk 21 Demineralized whey concentrate, skim milk 22 Demineralized whey protein, skim milk, whole milk 23 Demineralized whey, skim milk Cow’s milk a
syrup, sucrose syrup syrup syrup, sucrose
Casein:whey proteins 40:60 (as indicated on the label).
3. Results and discussion 3.1. Available lysine Table 1 summarizes all the data obtained including the available lysine content, protein digestibility and lactulose. The ANOVA of the available lysine values among the three groups of dairy proteins based formulas (casein, milk and milk enriched with whey) showed no statistically significant differences at the 5% level. However,
available lysine content of sample 5, the only formula containing glucose, was about 40% lower than those of the other formulas with casein. This is consistent with Lewis and Lea (1950), Baxter (1995) and Naranjo, Malec, and Vigo (1998) who reported that glucose is considerably more reactive than the other sugars and could cause a greater loss of available lysine by Maillard reaction. The average available lysine value in dairy proteins based formulas was 66.779.5 mg g 1 of protein, quite similar to the mean value accepted for human milk
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! et al. / International Dairy Journal 13 (2003) 95–99 A.S. Pereyra Gonzales
(66 mg g 1; WHO, 2000). Available lysine contents of these formulas were lower than that for powdered milk obtained in our laboratory (80.4 mg g 1) or those reported in the literature (76.6–85.4 mg g 1 protein; Hurrell, Finot, & Ford, 1983; Erbersdobler & Hupe, 1991; van Mil & Jans, 1991; Vigo et al., 1992). Available lysine contents in soy based formulas were significantly lower than those found in dairy protein based formulas (po0:05) averaging 45.078.3 mg g 1 protein. The available lysine contents in dairy proteins based formulas were similar to those reported by Mitchell and Grundel (1986) and Ferrer, Alegr!ıa, Farre! , Abella! n, and Romero (2000). The corresponding values of soy based formulas were lower than those found by Mitchell and Grundel (1986). In contrast to Evangelisti, Calcagno, and Zunin (1994), no significant differences in available lysine content were observed between formulas with and without the partial replacement of lactose by corn syrup or maltodextrin. Most infant formulas had lower contents of available lysine than those reported for the corresponding protein sources, i.e. soy protein (63.4–64.1 mg g 1 protein; Steinke, Prescher, & Hopkins, 1980; Friedman & Brandon, 2001); casein (79.8–85.0 mg g 1 protein; Steinke et al., 1980; Smith & Friedman, 1984; Friedman & Brandon, 2001); milk (76.6–85.4 mg g 1 protein; Hurrell et al., 1983; Erbersdobler & Hupe, 1991; van Mil & Jans, 1991; Vigo et al., 1992); and whey protein (80–97.5 mg g 1 protein; Desrosiers, Savoie, Bergeron, & Parent, 1989, Lindemann-Schneider & Fennema, 1989; Erbersdobler & Hupe, 1991). The losses were probably a consequence of the combined effects of the type of protein, the treatments during the manufacture and the different time and conditions of storage. The recommended dietary allowance available lysine for infants below the age of 6 months is 103 mg kg 1 body weight day 1 (Harper, 1981; National Research Council, 1989; WHO, 2000). The daily intake of available lysine with each formula, prepared and consumed according to the manufacturer’s instructions was calculated. It was found that only sample 1 could not provide the recommended daily intake of available lysine for infants weighing more than 6 kg, while available lysine content in sample 2 was close to the limit and could be marginally deficient. Furthermore, the risk may be increased by losses during storage. 3.2. Protein digestibility Digestibilities of soy based formulas were significantly lower (po0:05) than those of formulas based on dairy proteins. However, all values were quite satisfactory from the nutritional standpoint. The values observed were consistent with those found by Mitchell and Grundel (1986) and Sarwar, Botting, and Peace . (1989). Rudloff and Lonnerdal (1992) reported values
lower than those of the present work, probably because they used a different method to evaluate protein digestibility. 3.3. Lactulose Lactulose, which is absent in raw milk, is considered a useful indicator to evaluate the thermal treatment applied to milk, as its content increases with the intensity of the treatment (Andrews, 1986; Ferrer, ! Alegr!ıa, Farre! , Abella! n, & Romero, 1999; Lopez* & Olano, 1999). The few reports about Fandino lactulose content in infant formulas showed usually higher values than those found in commercial milk (Bernhart, Gagliardi, Tomarelli, & Stribley, 1965; Beach & Menzies, 1986; Corzo, Delgado, Troyano, & Olano, 1994; Ferrer et al., 1999). Table 1 shows that lactulose contents of infant formulas containing lactose (formula 9 to 23) were between 29 and 108 mg L 1 of reconstituted formulas based on milk and 97 and 312 mg L 1 of reconstituted formulas based on milk enriched with whey, the difference between both groups being statistically significant (po0:05). Lactulose values observed in this study were quite low compared with those found by Bernhart et al. (1965) (500–2500 mg L 1 in sterilized infant formulas and not more than 290 mg L 1 in reconstituted spray-dried infant formulas), and similar to those reported by Beach and Menzies (1986) in UHT infant formulas (55–350 mg L 1). Although no limit to lactulose content in infant formulas has been established, the International Dairy Federation (1992) and the European Communities Commission (EC Commission, 1992) proposed 600 mg L 1 for commercial UHT milk. Lactulose contents in formulas included here were well below this limit. 4. Conclusions The type of dairy protein used in the infant formulas obtained from Argentina markets had no significant effect on the available lysine and protein digestibility values. Though the corresponding values in formulas based on soy protein were significantly lower than those of dairy proteins based formulas, they were, except for one sample, quite satisfactory considering the recommended values. Lactulose contents showed a great variability with a trend to higher values in those formulas containing milk enriched with whey. Acknowledgements This work was supported by a grant UBACYT No. TW08 from Secretar!ıa de Ciencia y Te! cnica de la Universidad de Buenos Aires.
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