Formula of preparations for oral and/or enteral feeding of children

Abstract

The subject of the invention is a new and improved formula of preparations for the oral and enteral feeding of children and offspring of mammals, which cannot or should not be fed by the mother, characterized by the fact, that the preparations contain kynurenic acid or its salt at a concentration adapted to its content in the mother's natural milk at different times from delivery.

Claims

1. A nutritional composition for the oral and enteral feeding of children comprising kynurenic acid or its salt designed for a child 4-12 weeks, wherein kynurenic acid or a salt thereof is present, in an amount of 3.0-3.9 μg/100 ml.

2. A nutritional composition for the oral and enteral feeding of children comprising kynurenic acid or its salt designed for a child aged 4-6 months, wherein kynurenic acid or a salt thereof is present, in an amount of 4.0-5.0 μg/100 ml.

3. A nutritional composition for the oral and enteral feeding of children comprising kynurenic acid or its salt designed for a child aged 7-12 months, wherein kynurenic acid or a salt thereof is present, in the amount of 5.1-14.0 μg/100 ml.

4. A nutritional composition for the oral and enteral feeding of children comprising kynurenic acid or its salt designed for a child aged 3 weeks, wherein kynurenic acid or a salt thereof is present in an amount of 1.7-2.9 μg/100 ml.

Description

EXAMPLES

(1) Material Human Breast Milk

(2) Milk from 25 breastfeeding mothers was used for testing. Each mother provided a sample of 10 ml of fresh milk independently drawn by the nursing mother into a sterile test tube. Milk samples were taken from each mother 6 times on the third day, after one week, two weeks, one month, three months and six months after physiological labour.

(3) Formulas Intended for the Oral Feeding of Infants

(4) Standard formulas intended for oral feeding of infants up to 6 months of age were obtained commercially. Three packages of a commercial preparation were used for the study from each of the three analysed different series specified by the manufacturer on the packaging.

(5) Methods

(6) The 0.1 ml of 2 M perchloric acid was added to every 0.5 ml of human breast milk samples and centrifuged for 15 minutes at 6000 rpm. The obtained supernatant was stored at −20° C. to determine the concentration of kynurenic acid.

(7) Samples of formulas intended for feeding infants up to 6 months of age were prepared in accordance with the recipe provided by the manufacturer. Perchloric acid was added to liquid formula samples and the mixture was centrifuged like milk from nursing mothers. The obtained supernatant was used for further determinations.

(8) The supernatant was acidified with 2 M trichloric acid. The denatured proteins were separated by centrifugation and the obtained deproteinized supernatant was suspended in 1 M hydrochloric acid.

(9) The obtained deproteinized supernatant was applied to ion-exchange columns (Dowex 50W+, 200-400 mesh) filled with 0.1 M hydrochloric acid. The columns were washed with 1 ml 0.1 M HCl and 1 ml of deionized water. The obtained eluate was chromatographed (HPLC). The quantitative determination of kynurenic acid was carried out using the metric fluoride technique. The kynurenic acid standard was obtained from Sigma-Aldrich (St. Louis, USA). All reagents used to determine the kynurenic acid concentration came from the producers providing the highest degree of purity.

(10) Results

Example 1

(11) The Concentration of Kynurenic Acid in the Human Breast Milk

(12) As a result of the research, an increasing concentration of kynurenic acid in the human breast milk was found depending on the time elapsed from delivery. The average concentration of kynurenic acid in breast milk on the third day after delivery was 14 times lower than the average concentration after 6 months of breastfeeding (Table 1). The observed differences were statistically significant from the 14th day after delivery compared to the content on the 3rd day.

(13) TABLE-US-00001 TABLE 1 The concentration of kynurenic acid (μg/100 ml) in the human breast milk depending on the time passed since the delivery. Time passed since the delivery 3 × 24 6 × 24 2 4 3 6 hours hours weeks weeks months months arithmetic 0.39 1.07 2.11 3.74 4.15 5.66 mean SD 0.34 1.26 1.79 2.28 2.68 3.34 SEM 0.06 0.23 0.34 0.45 0.55 0.86 Median 0.30 0.59 1.70 2.99 3.16 5.18 Min 0.00 0.00 0.34 0.45 0.37 0.83 Max 1.53 4.88 9.24 10.36 11.03 13.78 ANOVA NS p <0.01 p <0.001 p <0.001 p <0.001 Dunn post-hoc test SD - standard deviation; SEM - standard error of the mean; min - lowest; max - the highest measurement value

Example 2

(14) The concentration of kynurenic acid in commercial preparations of milk intended for feeding newborns, infants and older children

(15) No producer specifies the content of kynurenic acid in the formulations they produce. The conducted tests revealed the presence of kynurenic acid in all tested milk preparations intended for feeding infants (Table 2). As a result of the analyses performed, no differences were found in kynurenic acid content in the tested preparations depending on the packaging from the same manufacturer's series. There were significant differences between the preparations of different manufacturers. The largest difference concerned NAN AR (0.03 μg/100 ml) and Bebilon 1 immuno fortis (1.08 μg/100 ml), indicating a 36-fold difference in the amount of kynurenic acid (Table 2).

(16) TABLE-US-00002 TABLE 2 The average content of kynurenic acid (μg/100 ml) in commercial preparations of milk intended for feeding to children depending on the feeding period recommended by the producer. since 4 since 6 since 9-10 since 12 since 24 Feeding period since birth months months months months months NAN AR 0.03 NAN pro HA 1 liquid 0.07 NAN HA 1 0.09 Bebilon 1 HA immuno fortis 0.21 NAN Active 1 0.24 Bebiko HA 1 0.25 NAN 1 0.25 NAN Pro 1 liquid 0.31 Bebilon Pepti liquid 0.33 Bebilon 1 immuno fortis 0.33 comfort Bebilon pepti 1 0.35 NAN AR 0.37 Bebilon 1 liquid 0.44 Bebilon AR 1 0.63 Nutramigen 1 0.66 Enfamil Premium 1 0.67 Bebiko Nenatal Premium 0.85 Enfamil AR 1 0.89 Bebiko 1 0.93 HIPP BIO 1 1.03 Bebilon 1 immuno fortis 1.08 Bebilon PEPTI 2 0.38 Nutramigen 2 0.58 Bebilon HA 2 0.29 NAN HA 2 0.31 Bebiko HA 2 0.33 Bebilon comfort 2 0.47 NAN 2 0.54 Bebiko 2R 0.70 Bebiko 2 0.71 NAN 2 R 0.71 NAN 2 Active 0.74 Enfamil premium 2 0.80 NAN pro d.noc B 0.95 Bebilon 2 immuno fortis 0.99 Enfamil AR 2 1.19 HIPP BIO 2 1.42 NAN HA 3 0.28 NAN PRO 3 0.98 NAN 3R 1.08 HIPP BIO 3 1.52 Bebiko Junior 3 0.65 Bebilon Junior 3 1.18 Bebiko Junior 4 0.67 Bebilon Junior 4 1.03

(17) The measurements were made in five milk packages and three different producer's series were compared (15 measurements for each tested preparation).

(18) A comparison of series of the same commercial preparations of milk intended for child nutrition showed significant differences in the content of kynurenic acid. The discrepancy between the series reached twice the amount of kynurenic acid calculated per 100 ml of the preparation intended for children (Table 3).

(19) TABLE-US-00003 TABLE 3 The concentration of kynurenic acid (μg/100 ml) in the tested milk preparations intended for feeding children with the indication of the series with the lowest and highest kynurenic acid content. Loose preparations Average SD SEM Min. Max. Bebilon 1 Immuno 0.33 0.12 0.07 0.20 0.45 fortis comfort Bebilon AR 1 0.64 0.12 0.07 0.51 0.77 Bebilon Junior 3 1.18 0.04 0.02 1.1 1.22 Bebilon Junior 4 1.03 0.02 0.01 1.01 1.05 Bebilon Pepti 2 0.38 0.08 0.05 0.33 0.47 Bebilon 1 Immuno fortis 1.08 0.16 0.09 0.98 1.27 Bebilon 2 Immuno fortis 0.99 0.36 0.21 0.7 1.4 Bebilon 1 HA 0.21 0.05 0.03 0.18 0.28 Immuno fortis Bebilon HA 2 0.29 0.02 0.01 0.28 0.31 Bebilon Comfort 2 0.47 0.27 0.16 0.3 0.78 Bebilon Pepti 1 0.35 0.05 0.03 0.31 0.41 Bebilon Nenatal 0.85 0.29 0.16 0.64 1.18 premium Bebiko 1 0.93 0.13 0.08 0.83 1.08 Bebiko 2 0.71 0.02 0.01 0.69 0.73 Bebiko 2R 0.7 0.07 0.04 0.63 0.77 Bebiko HA 1 0.25 0.01 0.01 0.23 0.27 Bebiko HA 2 0.32 0.03 0.02 0.29 0.36 Bebiko Junior 3 0.65 0.05 0.03 0.58 0.69 Bebiko Junior 4 0.66 0.02 0.01 0.65 0.68 Enfamil Premium 1 0.67 0.09 0.05 0.60 0.77 Enfamil Premium 2 0.80 0.07 0.04 0.72 0.84 Enfamil AR 1 0.89 0.07 0.04 0.83 0.96 Enfamil AR 2 1.19 0.31 0.18 0.95 1.54 HIPP Bio 1 1.03 0.05 0.03 0.99 1.08 HIPP Bio 2 1.42 0.03 0.02 1.38 1.45 HIPP Bio 3 1.52 0.23 0.13 1.28 1.74 NAN 1 0.25 0.03 0.02 0.22 0.27 NAN 2 0.54 0.10 0.06 0.43 0.64 NAN HA 1 0.09 0.02 0.01 0.07 0.12 NAN HA 2 0.31 0.10 0.06 0.19 0.40 NAN HA 3 0.28 0.08 0.04 0.21 0.36 NAN AR 0.37 0.14 0.08 0.24 0.53 NAN Active 1 0.24 0.05 0.03 0.20 0.30 NAN Active 2 0.73 0.07 0.05 0.68 0.78 NAN PRO d.night B 0.95 0.14 0.08 0.77 1.05 NAN PRO 3 0.98 0.05 0.03 0.93 1.03 NAN 2 R 0.71 0.37 0.21 0.33 1.06 NAN 3 R 1.08 0.11 0.02 1.08 1.08 Nutramigen 1 0.66 0.36 0.20 0.42 1.07 hypoallergenic Nutramigen 2 0.58 0.16 0.09 0.43 0.76 hypoallergenic Liquid preparations Average SD SEM Min. Max. Bebilon Nenatal 0.74 0.02 0.01 0.72 0.75 premium Bebilon Pepti liquid 0.33 0.03 0.02 0.29 0.35 Bebilon 1 0.44 0.08 0.05 0.39 0.53 NAN PRO 1 0.31 0.04 0.02 0.29 0.36 NAN PRO HA 1 0.07 0.01 0.01 0.06 0.07 Enfamil Premium 1 0.51 0.02 0.01 0.49 0.53 SD-standard deviation; SEM-standard error of the mean; Min-average minimum value in the series; Max-average maximum value in the series

Example 3

(20) Comparison of Kynurenic Acid Concentration in Commercial Preparations of Milk Intended for Child Nutrition with the Concentration in Human Breast Milk at the Same Time After Delivery

(21) In commercial preparations of milk intended for child nutrition in the period from birth, the average kynurenic acid content is very diverse and ranges from 0.03 to 1.08 μg/100 ml depending on the type of preparation, while the average content of kynurenic acid in breast milk on the third day after delivery is 0.39 μg/100 ml, on day 6, 1.07 μg/100 ml, at week 2, 2.11 μg/100 ml and week 4, 3.74 μg/100 ml. These amounts are significantly different from the amount of kynurenic acid in commercial preparations. There are also significant differences in the content of kynurenic acid between preparations from different production series. In addition, commercial preparations intended for children of this age contain a constant amount of kynurenic acid, whereas the content of kynurenic acid in breast milk increases almost 4 times in 4 weeks, from 0.39 μg/100 ml to 3.74 μg/100 ml. None of the products reflects the dynamics of changes in kynurenic acid content during this period.

(22) In commercial preparations of milk intended for children from 4 months of age, the average content of kynurenic acid is 0.38 to 0.58 μg/100 ml depending on the type of preparation, while the average content of kynurenic acid in breast milk at 3 months after delivery is 4.15 μg/100 ml. This amount exceeds the amount of kynurenic acid in commercial preparations more than 10 times.

(23) In commercial preparations of milk intended for child nutrition in the period from 6 months of age, the average kynurenic acid content is very diverse and ranges from 0.29 to 1.42 μg/100 ml depending on the type of preparation, while the average content of kynurenic acid in breast milk at 6 months after delivery is 5.66 μg/100 ml. This amount is much higher than the amount of kynurenic acid in commercial preparations appropriate to the age (4-20 times).

(24) In commercial preparations of milk intended for child nutrition in the period from 9-24 months of age, the average kynurenic acid content is very diverse and ranges from 0.28 to 1.52 μg/100 ml depending on the type of preparation. These amounts are significantly lower than the average kynurenic acid content in mother's milk at 6 months after delivery, which is 5.66 μg/100 ml. This amount is much higher than the amount of kynurenic acid in commercial preparations appropriate to the age (4-20 times).

Example 4

(25) The Preparation Method of Formula for Oral Feeding of Children

(26) The solubility of kynurenic acid in water is 0.95 mg/ml (http://www.hmdb.ca/metabolites/HMDB00715).

(27) Kynurenic acid is readily soluble in alkaline conditions; in 1.0 M NaOH, its solubility is 50 mg/ml (http://www4.mpbio.com/ecom/docs/proddata.nsf/440121766f8ee75e8525645d0068b043/2f736b869 41e7f61852569cb00688812? OpenDocument).

(28) There are salts of kynurenic acid which are well soluble in water, e.g. a sodium salt, with the solubility of 100 mM (http://www.abcam.com/kynurenic-acid-sodium-salt-ab120256.html), which corresponds to a solubility of 21 mg/ml.

(29) The results of our own research in the light of data from the literature indicate that the solubility of kynurenic acid in water is sufficient to obtain the highest recommended concentration of this substance which results from the determination of the physiological content of kynurenic acid in the milk of breast-feeding women. The solubility of kynurenic acid of 0.95 mg/ml is 6900 times higher than the highest content of kynurenic acid in the mother's milk tested in the study, which is 13.78 μg/100 ml (Table 1), which allows the production of a preparation with an increased content of kynurenic acid in comparison to physiological contents. For this purpose, a well-soluble kynurenic acid salt or kynurenic acid dissolved in alkali may also be used.

(30) Because kynurenic acid is soluble in water, kynurenic acid in powder form was added to the children's powder formula—Bebilon Nenatal in an amount corresponding to 10 times the highest content of kynurenic acid found in breast milk of 13.78 μg/100 ml (Table 1), that is 137.8 μg/100 ml converted into the volume recommended by the manufacturer. Then water was added in the amount recommended by the manufacturer and the whole was mixed well.

(31) Macroscopic examination proved that the addition of kynurenic acid did not change the colour, consistency and pH of the mixture, compared to the control mixture, without the addition of kynurenic acid.

(32) As nutritional preparations are available in ready liquid form, kynurenic acid dissolved in water was added to the finished liquid preparation Bebilon 1. The added amount of kynurenic acid corresponded to 10 times the highest content of kynurenic acid found in breast milk of 13.78 μg/ml (Table 1), that is 137.8 μg/100 ml.

(33) Macroscopic examination proved that the addition of kynurenic acid did not change the colour, consistency and pH of the mixture, compared to the control mixture, without the addition of kynurenic acid. Conclusions: The preparation for oral feeding of children is prepared by means known in the food industry, provided that the kynurenic acid content should preferably be 0.01-0.7 μg/100 ml for children 1-5 days; 0.8-1.6 μg/100 ml for children aged 6-14 days; 1.7-2.9 μg/100 ml for children aged 2-3 weeks; 3.0-3.9 μg/100 ml for children aged 4-12 weeks; 4.0-5.0 μg/100 ml for children aged 4-6 months; 5.1-14.0 μg/100 ml for children aged 7-12 months.

SUMMARY

(34) 1. Human breast milk contains kynurenic acid in an increasing amount with breastfeeding time. Concentrations of kynurenic acid in milk of breastfeeding women are 14 times higher in the 6th month of feeding compared to 3 days after delivery. 2. The content of kynurenic acid in the majority of preparations for children's nutrition is significantly lower compared to the content of kynurenic acid in the milk of breast-feeding women. Only commercial preparations with the highest content of kynurenic acid contain kynurenic acid in the amount similar to the content in milk of women on day 6 of breastfeeding. 3. The content of kynurenic acid in infant preparations is accidental, i.e. it is not deliberately planned by the manufacturer and is not controlled. 4. The research results argue in favour of the development of a preparation refined formula intended for feeding children and offspring of mammals taking into account natural changes in the content of kynurenic acid in breast milk of women and lactating animals. 5. In order to obtain the desired effect of kynurenic acid, the content of kynurenic acid in infant preparations should preferably be 0.01-0.7 μg/100 ml for children aged 1-5 days; 0.8-1.6 μg/100 ml for children aged 6-14 days; 1.7-2.9 μg/100 ml for children aged 2-3 weeks; 3.0-3.9 μg/100 ml for children aged 4-12 weeks; 4.0-5.0 μg/100 ml for children aged 4-6 months; 5.1-14.0 μg/100 ml for children aged 7-12 months. 6. Due to the sufficiently good solubility of kynurenic acid in water ensuring its content at the physiological level, kynurenic acid or its soluble salt may be used to prepare the formula for feeding children and offspring of mammals in a loose form, or a solution of kynurenic acid or a salt thereof for preparation in a liquid form.