Note: Descriptions are shown in the official language in which they were submitted.
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PROCESS OF IMPROVING THE STABILITY OF VITAMIN D
IN A NUTRITIONAL PRODUCT
CONTAINING HYDROLYZED PROTEIN AND PRODUCT PRODUCED THEREBY
i5
Technical Field
This invention relates to a method of improving the stability of Vitamin D in
a liquid nutritional product
that comprises hydrolyzed proteins and/or free amino acids. The process
comprises the addition of
methionine to the nutritional product to assure nutritional adequacy and the
reduced or eliminated
supplementation of cys6ne. The invention also relates to nutritional products
made through the
inventive process.
Background of the invention
Some adults and infants have food allergies that include sensitivity to intact
protein or protein
maldigestion. These individuals require a special nutritional formula, such as
a hypoallergenic
formula, to meet their dietary needs. Hypoallergenic formulas, that are also
sometimes referred to
as elemental formulas, are characterized in that they typically contain
hydrolysates such as soy
protein hydrolysate, casein hydrolysate, whey protein hydrolysate or a
combination of animal and
vegetable protein hydrolysates as the major source of amino nitrogen.
Hypoallergenic formulas may
also use only free amino acids as the source of amino nitrogen. The protein
hydrolysates comprise
short peptide fragments and/or free amino acids instead of the intact protein
found, for example, in
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cow's milk and soy protein isolate-based formulas. These short peptide
fragments and free amino
acids have been found to be less immunogenic and allergenic than intact
proteins. infants who
experience adverse reactions to intact protein often are able to tolerate
hydrolysate-based liquid
nutritional products. Typical sources of hydrolyzed protein include casein,
whey, rice, soy and beef
collagen.
Alimentum~ is one such hypoallergenic nutritional formula that is manufactured
by the Ross Products
Division of Abbott Laboratories, Columbus, Ohio. Alimentum~ is a nutritionally
complete formula
that contains an extensively hydrolyzed casein which is approximately
60°~ by weight free amino
acids, the remainder being small peptides. Such predigestion virtually
eliminates allergenicity.
During the manufacture of the protein hydrolysate, the concentration of a
number of amino acids,
such as tryptophan and tyrosine, is significantly reduced. Thus, fortification
of the hydrolysate with
these "lost" amino acids is needed to restore nutritional adequacy.
Although cystine is not an essential amino acid, products such as Alimentum~
have traditionally
been fortified with cystine in order to mimic the amino acid profile of human
breast milk. Thus, in
addition to the casein that has been enzymatically hydrolyzed and charcoal
treated, Alimentum has
added L-cystine dihydrochloride, L-tyrosine, L-tryptophan, taurine and L-
camitine. Pregestimil~ and
Nutramigen~, which are hypoallergenic infant nutritionals manufactured by the
Mead Johnson
Division of Bristol Meyers Squibb of Evansville, Indiana, are supplemented
with L-cystine;
L-tryptophan, L-tyrosine, taurine and L-camitine. All of these hypoallergenic
pediafic formula are
also fortified with Vitamin D at a level above 45 IU (label claim) per five
fluid ounce (142 ml) or 100
calories.
In addition to the protein hydrolysate, most nutritionally balanced
hypoallergenic formulas contain
carbohydrates, lipids, vitamins, and minerals. These hypoallergenic formulas
are utilized for feeding
infants, children and adults who have allergies or sensitivities to intact
protein, and an: often
medically used in the treatment of cys~c fibrosis, chronic diarrhea,
galactosemia, small bowel
resection, steatorrhea and protein-calorie malnutrition.
Advera~, a specialized, complete nutritional for the dietary management of
people with HIV infection
or AIDS; AlitraQ~, a specialized, elemental nutritional with glutamine for
metabolically stressed
patients; and Perative~, a specialized liquid nutritional for the dietary
management of metabolically
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stressed patients all contain hydrolyzed protein andlor free amino acids as
their source of amino
nitrogen. All of these products are manufactured and distributed by the Ross
Products Division of
Abbott Laboratories, Columbus, Ohio. All of these products also contain
Vitamin D and are
susceptible to Vitamin D degradation when in liquid form due to the presence
of protein hydrolysates
and free amino acids.
The beneficial qualities of Vitamin D are well documented and accepted. For
example, Utamin D
regulates the calcium level in the body and is responsible for depositing
calcium and phosphorous
into bone from the blood. Too little Vitamin D can cause soft bones, muscle
weakness, poor growth,
bone fractures and secondary hyperthyroidism. Although vitamin supplements in
tablet farm may be
taken, Vitamin D is generally easily obtainable from the diet, especially a
diet containing Vitamin D
enriched foods.
The most common biologically active forms of Vitamin D are previtamins D2 and
D3 and vitamins D2
and D3. Previtamin D2 and Vitamin D2 are produced from ergosterol and are
biologically active in
humans, cattle, swine and other mammals. Previtamin D3 and Vitamin D3 are
biologically active
and are produced in the skin of many animals following irradiation (i.e.,
exposure to the sun) of
7-dehydrocholesterol. The other isomeric forms of Vitamin D show no
significant biological activity.
The isomerization of bio-available Vitamin D to inactive forms occurs in
solution. The stability of
Vitamin D in a nutritional matrix is dependent upon a number of factors. For
example, light, high
temperatuna and iodine catalyze the conversion of the biologically active
forms of Vitamin D to
inactive forms.
One excellent source of Vitamin D is liquid nutritional products that an:
fortified with Vitamin D. With
respect to fortification with Vitamin D and infant formula, Vitamin D levels
in infant products are
federally regulated in the U.S.A. under the Infant Formula Act (IFA).
Nutritional products containing
levels above or below those set forth in the IFA may not legally be offered
for sale.
Hypoallergenic formulas encounter a major problem reganiing Vitamin D
stability. Vitamin D
undergoes significant degradation in protein hydrolysate formulas andlor
formulas containing free
amino acids. In fact, research has shown that the more hydrolyzed the protein,
the greater the
Vitamin D degradation. Specifically, the inherent and fortified sources of
free or combined cysteine
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and cystine are capable of forming the thiyl-free radical (in the presence of
a free radical initiator
such as oxygen or peroxides) that isomerizes Vitamin D into non-bioavailable
forms. These free
radical reactions also make Vitamin D more susceptible to oxidation that
converts the Vitamin D to
non-bioavailable forms. This ability to isomerize Vitamin D to non-
bioavailable forms by a free
~5 radical reaction involving the thiyl radical is true for all sulfur
compounds present as a thiol (such as
cysteine) or as a disulfide (such as cystine). The reaction also occurs with
cysteine-containing
peptides or disulfide-linked peptides but is not generally observed with
intact proteins due to steric
hindrances.
The reactions do not occur in sulfur-containing molecules in which the sulfur
atom is not bonded to
hydrogen or sulfur, due to the inability of these compounds to readily form
the thiyl free radical.
Therefore, sulfur compounds such as the salts and esters of methionine,
cystathionine,
S-methylcystine and cysteine sulfonate do not induce Vitamin D degradation.
In view of the problems with Vitamin D degradation in liquid nutritional
products containing
hydrolyzed protein andlor free amino acids, some products are over fortified
with Vitamin D. Over
fortification is used to meet the label claim for Vitamin D content over the
shelf life of the product.
However, as men~oned above, acceptable upper limits of Vitamin D concentration
exist, since too
much Vitamin D can cause hypercalcemia, hypercalciuria, urinary tract stones,
extraskeletal
calcifications and malfunction of the kidneys and other organs. In similar
fashion, if the Vitamin D
concentration drops below a certain limit due to degradation, the product must
be removed from sale
to the public. The costs associated with replacing, shipping and overall
monitoring of the Vitamin D
level in liquid nutrifional products are significant. A need presently exists
in the industry to enhance
the stability of Vitamin D in a liquid, hypoallergenic formula.
U.S. Patent No. 4,836,957 to Nemoto, et al., discloses a preparation
containing an active form of
Vitamin D3 that is stabilized by incorporation of an amino acid that is
neither containing a sulfur atom
nor an acid amino group in its structure, nor an acidic amino acid, nor a salt
of a basic amino acid.
This patent is not concerned with Vitamin D stability in liquid nutritional
formula that contain protein
hydrolysates andlor free amino acids.
U.S. Patents 5,382,439 and 5,456,926 to Hill, et al., disclose a method for
improving the stability of
Vitamin D in liquid nutritional products that contain hydrolyzed protein or
free amino acids. These
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patents teach that Vitamin C should be added at a concentration exceeding 300
mgs per liter and
that cystine be added to the liquid nutritional product after complefron of a
preliminary heat
treatment. While these patents disclose methods that address the problem of
Vitamin D stability in a
hypoallergenic product, they are less than totally satisfactory.
It is thus apparent that a need exists for a method to improve the stability
of Vitamin D in the
presence of protein hydrolysates andlor free amino acids without adversely
affecting the nufitional
quality of the formula.
Disclosure of the Invention
In general, this invention relates to the discovery that Vitamin D stability
in liquid nutritional products
can be dramatically improved by eliminating or limiting cystine fortification
without sacrificing
nutritional adequacy provided methionine is used to provide the requirements
for sulfur-containing
amino acids as assessed by animal growth are met. The present invention also
provides an
improved hypoallergenic formula that minimizes the need for over fort~cation
with Vitamin D and
provides excellent protein quality for normal growth in humans.
Thus, there is disclosed a method for improving the stability of Vitamin D in
a liquid nutritional
product wherein said nutritional product comprises Vitamin D and a source of
amino nitrogen
selected from free amino acids, hydrolyzed protein and mixtures thereof, said
method comprising the
addition of L-cystine to said product at a level not to exceed 7.0 mgs (in
addition to inherent levels)
per gram of protein and the addition of L-methionine to said product at a
level of at least 9.0 mgs (in
addition to inherent levels) per gram of protein.
In a more preferred embodiment, the liquid nutritional product is an infant
formula. In a yet more
preferred embodiment less than two mgs of cystine is added to the nutritional
product per gram of
total protein and at least 12 mgs of L-methionine is added per gram of total
protein. Most
preferably, no more than 0.5 mgs per gram of L-cystine is added and at least
14 mgs of
L-methionine is added per gram of total protein. In a yet most preferred
embodiment, no L-cystine is
added to the nutritional product and at least 15 grams of L-methionine is
added.
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There is also disclosed a liquid nutritional product comprising a source of
amino nitrogen selected
from the group consisting of hydrolyzed proteins, free amino acids and
mixtures thereof, Vitamin D
and L-methionine as an added substance at a concentration of at least 9.0 mgs
per gram of protein.
From another perspective the invention can be viewed and implemented in terms
of total content of
the sulfur-containing amino acids methionine, cystine and cysteine. The
protein hydrolysate used in
the hypoallergenic formula will contain inherent levels of methionine, cystine
and cysteine. For
example, intact casein inherently has 30 mgs of methionine per gram of protein
and four mgs of
cystine (cystine plus cysteine) per gram of protein. As used herein and in the
claims the levels
associated with cystine are to be understood as meaning the sum of cystine
plus cysteine. This is a
result of the analytical chemistry. In contrast, the casein hydrolysate used
in Alimentum has an
inherent level of methionine of about 26 mgs per gram of protein and about six
mgs of cystine per
gram of protein. To realize the benefits of improved Vitamin D stability and
adequate growth, the
inventors have determined that at least 45 mgs (320 micro-moles (NM)) of
sulfur-containing amino
acids (cystine, methionine and cysteine) per gram of protein be present in the
inventive formula from
the protein hydrolysate and amino acid supplementation. Further, at least 34
mgs of the 45 mgs of
the sulfur-containing amino acids should be methionine. Thus, a hydrolysate
containing 28 mgs of
methionine per gram of protein and seven mgs of cystine per gram of protein
(inherent levels) will be
fortified with at least six mgs of methionine (34-28=6) per gram of protein
and may, but not
necessarily, be additionally fortified with four mgs of cystine (6+28+7+4=45).
Thus, there is also disclosed a method for improving the stability of Vitamin
D in a liquid nutritional
product wherein said nutritional product comprises Vitamin D and a source of
amino nitrogen
selected from hydrolyzed protein, free amino acids and mixtures thereof, said
method comprising the
steps of: a) providing a liquid nutritional product containing Vitamin D; b)
adding L-methionine to
said product such that the total level (inherent plus supplemental} of L-
methionine is at least 34 mgs
per gram of protein and the addition of at least one amino acid selected from
the group consisting of
lysine, tryptophan, tyrosine, taurine and L-camitine.
There is further disclosed a method for improving the stability of Vitamin D
in a liquid nutritional
product wherein said nutritional product comprises Utamin D and a source of
amino nitrogen
selected from hydrolyzed protein, free amino acids and mixtures thereof, said
method comprising the
addition of L-methionine to achieve a total level of L-methionine of at least
34 mgs per gram of
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protein and the addition of at least one amino acid selected from the group
consisting of cys~ne,
cysteine and mixtures thereof to achieve a concentration of at least 45 mgs of
sulfur-containing
amino acids per gram of protein.
There is also disclosed an improved hypoallergenic enteral nutritional
comprising lipid, carbohydrate,
Vitamin D and a source of amino nitrogen, the improvement characterized in
that the source of
amino nitrogen is selected from hydrolyzed protein, free amino acids and
mixtures thereof wherein
the sum of all sulfur-containing amino acids is at least 50 mgs per gram of
protein and the level of
methionine is at least 40 mgs per gram of protein. In a more preferred
embodiment the level of
methionine is at least 42 mgs per gram of protein and the level of cystine is
less than 12 mgs per
gram of protein. In yet a more preferred embodiment, the formula comprises at
least 43 mgs of
methionine (inherent plus supplemented) per gram of protein and less than ten
mgs of cystine
(inherent plus supplemented) per gram of protein. In a most preferred
embodiment, the formula
comprises less than eight mgs of cystine per gram of protein and at least 42
mgs of methionine per
gram of protein.
This inven~on also relates to an enteral nutritional with an improved PER
comprising a source of
amino nitrogen, the improvement characterized in that the source of amino
nitrogen is selected from
hydrolyzed protein, free amino acids and mixtures thereof and wherein the
concentration of
L-methionine is at least 34 mgs per gram of protein.
In yet another view of the present invention, the inventors have determined
that an improved
hypoallergenic formula comprises Vitamin D and at least 34 mgs of total
(inherent plus fortification)
methionine per gram of protein and less than 12 mgs of total (inherent plus
fortification} cystine per
gram of protein. In yet a more preferred embodiment, the formula comprises at
least 40 mgs of
methionine per gram of protein and less than ten mgs of cystine. In a most
preferred embodiment
the formula comprises at least 42 mgs of methionine per gram of protein and
less than eight mgs of
cys~ne per gram of protein.
In similar fashion, a process for the production of an improved hypoallergenic
enteral formula
comprising protein hydrolysates is disclosed, said process comprising the
steps of supplementing
said formula with Vitamin D and free methionine to achieve a total (inherent
plus fortification)
concentration of methionine of at least 34 mgs per gram of protein, more
preferably at least 40 mgs
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per gram of protein, and most preferably at least 42 mgs per gram of protein.
The process may
additionally comprise the addition of free cys6ne to achieve a total (inherent
plus fortification)
concentration of cystine not to exceed 12 mgs per gram of protein.
The hydrolyzed protein that is used in the present invention can be any edible
source of protein such
as animal (i.e., meat and fish), cereal (i.e., rice and com) and vegetable
proteins (i.e., soy). More
specifically, the source of protein can be milk proteins such as casein and
whey. The most preferred
source of protein for hydrolysis is casein.
In addition to the hydrolyzed protein, the nutritional product of this
invention can additionally contain
free amino acids other than L-methionine. Any amino acid can be added to the
formula, however,
the addition of cystine andlor cysteine should be avoided or held to a
minimum. Thus, in addition to
L-methionine, the nutritional according to this invention is preferably
fortified with tryptophan,
tyrosine, taurine and L-camitine.
There is further disclosed an improved hypoallergenic enteral nutritional
comprising, based on total
calories of the nutritional, about 30-65°~ carbohydrates, about 30-60%
lipids, about 5-20°~ of a
source of amino nitrogen selected from hydrolyzed protein, free amino acids
and mixtures thereof;
and about 400 to 1,000 IU per liter of Vitamin D, the improvement
characterized in that said
nutritional comprises L-methionine as an added component at a concentration of
at least 9 mgs per
gram of protein. In a more preferred embodiment, L-methionine is at a
concentration of at least 12
mgs per gram of protein; and most preferably at a concentration of at least 14
mgs per gram of
protein. The hypoallergenic nutritional according to this invention also may
contain as an added
component an amino acid selected from lysine, tryptophan, tyrosine, taurine, L-
camitine and
mixtures thereof.
The present invention provides a method for improving the stability of Vitamin
D in liquid nutritional
products having hydrolyzed protein andlor free amino acids as the primary
source of amino nitrogen.
The method is extremely easy to practice, very reliable and does not
compromise the nutritional
quality of the formula. While the primary focus of the invention is directed
to infant formula, the
invention is also applicable to other liquid nutritional products that utilize
hydrolyzed proteins andlor
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amino acids as a source of amino nitrogen. The formula may take the form of a
ready-to-feed
product, a concentrate or a powder.
In yet another embodiment, this invention relates to a method of improving the
stability of Vitamin D
in a liquid nutritional product that contains hydrolyzed protein andlor free
amino acids as the source
of amino nitrogen, the method comprising the step of adding L-methionine to
said liquid nutritional
product at a concentration of added L-methionine of at least 9 mgs per gram of
protein.
There is also disclosed an improved liquid nutritional formula with enhanced
Vitamin D stability and a
source of amino nitrogen that provides for acceptable growth, made in
accordance with the methods
disclosed above. Other objects and advantages of this invention will be
apparent from the following
description and the appended claims.
As used herein and in the claims, the total protein upon which the level of
amino acid fortification is
based is determined through Kjeldahl's method for the determination of total
nitrogen. The principle
of ICjeldahl's method is the conversion of the nitrogen containing substances
to ammonium sulfate by
boiling with sulfuric acid in the presence of a catalyst, usually copper
sulfate. Potassium sulfate is
added to raise the boiling point. The mixture is then made alkaline and the
ammonia distilled off into
standard acid. Details of this technique can be found in numerous textbooks on
chemical analysis or
practical biochemistry. The nitrogen value is converted to grams of protein by
multiplying grams of
nitrogen by 6.25.
As used herein and in the claims the levels of added methionine or any other
amino acid are based
on total protein content including the added amino acids. For example; if a
formula contains ten
grams of protein per liter, the ten grams is the sum of all proteins, protein
fragments and free amino
acids and a fortification level of 15 mgs per gram of protein includes the 15
mgs in each gram of
protein.
Cystine or dicysteine (CsH,2Nz04Sz) has the chemical structure:
HzC-S-S-CHz
HzN -CH HC -NHZ
COOH COOH
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Cystine consists of two molecules of cysteine (cystine yields cysteine upon
reduction). Thus, due to
the two sulfur atoms, one mole of cystine provides two moles of sulfur-
containing amino acid
equivalents. Cystine is particularly abundant in the proteins of the skeletal,
connective tissues, hair
and wool of animals.
Methionine or 1-Amino-3-methylmercaptobutyric acid (C5H"N02S) has the chemical
structure:
CH3 - S - CHz - CHz - CH - COOH
~2
Methionine is one of the natural sulfur-containing amino acids and is present
in small quantities in
the hydrolysis products of proteins compared to most of the other amino acids.
It is an essential
constituent of the food of mammals and is particularly important in that it
and choline are the only
compounds in the diet known to take part in methylating reactions. As used
herein and in the
claims, the term °methionine" includes the salts and esters of
methionine; cystathionine,
s-methylcystine and cysteine sulfonate. Also as used herein and in the claims,
all of the recited
amino acids, including cystine and methionine, are understood to be in the L-
or laevorotatory form.
Detailed Descridtion of the Invention
The hypoallergenic formula according to the present invention is made by
blending carbohydrates,
lipids and a protein hydrolysate, homogenizing the mixture into a stable
emulsion, adding the
supplemental free amino acids and sterilizing the product in the pH range from
about 6 to about 7.
The protein hydrolysate of the invention may be any suitable protein
hydrolysate utilized in a
nutritional formula such as soy protein hydrolysate, cereal grain protein
hydrolysate, casein
hydrolysate, whey protein hydrolysate, animal and vegetable protein
hydrolysates and mixtures
thereof. The protein hydrolysate of the hypoallergenic formula of the
invention is preferably a soy
protein hydrolysate or a casein hydrotysate comprising short peptides and free
amino acids. The
immunogenicity of the formula of the present invention depends largely on the
extent of hydrolysis of
the selected protein. To insure hypoallergenicity of the fom~ula, the protein
hydrolysate should be
extensively hydrolyzed to yield very short peptides and free amino acids. This
is important since
free amino acids and di-and tri-peptides are known to be absorbed through the
small intestine
without any digestive breakdown. High molecular weight peptides are preferably
avoided because
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they are more allergenic and cause precipitation and emulsion destabilization
of the liquid nutritional.
In a preferred embodiment, the protein hydrolysate of the invention.contains a
high percentage of
low molecular weight peptide fragments.
In general, any known technique can be used to produce the protein
hydrolysate. Preferably, the
protein is hydrolyzed to such an extent that the ratio of amino nitrogen (AN)
from the free amino
acids in the hydrolysate to total nitrogen (TN) from intact protein, protein
fragments and free amino
acids ranges from about 0.3 AN to 1.0 TN to about 0.8 AN to 1.0 TN. Such
hydrolysis generally
yields hydrolysates with the following representative molecular weight
distribution.
Moiecular Wei ht Daitons Weight Percent in H drol
sate
77-95
500-1500 3-12
1500-5000
>5000 ~3
Hypoallergenic formula may also use only free amino acids as the source of
amino nitrogen or may
be more hydrolyzed than that given above.
One aspect of this invention resides in the discovery that the hydrolyzed
protein source be
supplemented with significant levels of methionine and only sparingly, if at
all, with cystine and still
be nutritionally adequate for growth. The formula may be fortified with other
free amino acids such
as L-tryptophan, L-tyrosine, L-arginine, L-taurine and L-camitine. The total
caloric value of the
protein hydrolysate and supplemented free amino acid mixtures in the
hypoallergenic formula may
range from about 8% to about 20°f° of the total calories of the
formula and is preferably in the range
of about 10% to about 14°~ of total calories.
While the presence of cystine in a hypoallergenic nutritional beverage is
known to increase the
degradation of the Vitamin D, a replacement is required to assure that proper
growth and protein
maintenance is achieved. One aspect of the invention resides in the discovery
that methionine can
be added to the nutritional formula without degrading Vitamin D while
promoting acceptable growth
of the animal.
"Protein Efficiency Ratio" (PER) is a measure of protein quality using
laboratory rats. The inventors
have discovered that the replacement of cystine with methionine in micro-moles
(NM) of
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sulfur-containing amino acid equivalents (e.g., two moles of methionine for
each mole of cystine),
results in a formula that provides adequate nutrition for growth (amino
nitrogen), has an excellent
PER and dramatically improves the stability of Vitamin D in liquid
nutritionals.
Analyrtical Method for Vitamin D
In order to determine whether the method of the inven~on is effective in
reducing Vitamin D
degradation, it was necessary to accurately determine the concentration of bio-
active Vitamin D in
the formula. The technique used by the inventors to quantitate Vitamin D was a
modfication of the
method published by Sertl and Molitor, Journal of the Association of 011fcial
Analytical Chemists,
Volume 68, Number 2,177-182 (1985). The method consists of saponif)ring the
sample, extracting
the saponified sample, subjecting the extract to preliminary liquid
chromatography, cleaning-up and
subjecfing the cleaned-up extract to quantitative liquid chromatography.
Details of this analytical
procedures can be found in U.S. Patent 5,456,926 to Hill, et al.
Under the Infant Formula Act (IFA) in the U.S.A. there are certain maximum and
minimum levels
assigned to Vitamin D in infant formula. For example, the upper limit for a 20
calorie per fl, oz.
formula is 676 IU per liter, or 100 IU per 100 calories. The liquid
nutritional product is typically
over-fortified such that by zero-time (the time when the container is n:ady
for sale) the Vitamin D
concentration has dropped into the acceptable range. During the shelf life of
the product, the
Vitamin D concentration can fall below the acceptable minimum and in that
event, the product would
have to be recalled or exchanged. According to the Infant Formula Act in the
U.S.A., the acceptable
minimum level of Vitamin D is 40 lU per 100 calories or 270 IU per liter for
an infant formula
containing 20 calories per fl. oz.
EXAMPLE I
Stu~yr of PER
This experiment was conducted to evaluate the PER of intact casein, casein
hydrolysates and
casein hydrolysates fortified with methionine at two levels, and ~rith lysine
andlor tryptophan. The
investigation was conducted in accordance with AOAC Method 960.48, AOAC
Official Methods of
Analysis,15th Edition,1990. There were two Control groups used in this
experiment; one was the
casein (intact protein) based diet having the composition given in the
Official AOAC method and the
second was based on an unfortifled casein hydrolysate that is used in
Alimentum~ (neither shown in
Table I). A total of six experimental diets were prepared that were based on
the casein hydrolysate
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with varied levels and types of fofificafion. Table I sets forth the
fortification of each diet based on
milligrams of added amino acid per 1.0 g of total protein (nitrogen times
6.25).
TABLEI
Experimental Diets
Casein Hvdrolvsate Base Plus Milligram of Amino Acid Per 1.0 Gram of Tcltai
Pmtpin
Diet 11 Dlet 22 Diet 3 filet 4 -filet 5 '
' .~ diet
6
5.0 g MET* 5.0 MET 5.0 MET 5.0 MET 10 MET 10
10 LYS* 3.0 TRP* 10 LYS MET
3.0 TRP 10
LYS
3.0
TRP
- not i = memionme
LYS = lysine hydrochloride
TRP = tryptophan
Each diet was virtually identical in caloric content, source and distribution
of calories from
carbohydrate and fat, and in macro- and micro-nutrients. The casein
hydrolysate Control diet
contained an inherent total of about 27-28 mgs of methionine per gram of
protein. Diets 1 through 4
contained 32-33 mgs of methionine as actually analyzed inherent plus
supplemental) per gram of
protein and Diets 5 and 6 contained 34 mgs of methionine per gram of protein
as actually analyzed.
Weaning male Sprague-Dawley rats between 21 and 23 days of age with an initial
weight between
40 and 60 grams were used in this experiment. The animals were placed in test
cages for an
acclimatization period of three to four days. During this time, the rats
received a standard rat chow
diet. After acclimatization,10 rats per group were assigned a Control or
Experimental diet. The rats
were fed their assigned diet and water ad libitum. The animals were weighed at
the beginning of the
study and every week during the four week study period. Consumption of the
diets was measured
and the PER was calculated by the method given in the AOAC PER procedure,15th
Ed., Method
960.48,1990.
Table II sets forth the data generated in this experiment. For each diet, the
average weight gain per
animal, the average amount of protein consumed and the PER for each diet is
reported.
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TABLE II
ER for Control and Exoerimen at ~i~tc
Control AOAC 82.9 29.2 2.50
Control 60.6 27.42 1.94
Diet 1 88.4 31.21 2.49
Diet 2 84.6 32.19 2,31
Diet 3 87.4 31.90 2.41
Diet 4 80.4 29.91 2.37
Diet 5 115.3 35.96 2,8~
Diet 6 109.2 35.18 2,75a
Values shanng the same superscnpt letters do not s~grnficanby differ (p values
> 0.05), Tukey's Muitiple Comparison
Procedure
The data indicate that supplementation of the base casein hydrolysate with
methionine at a total
level 34 mgs per gram protein level significantly improves the PER of the
protein system (Diets 5 and
6). Surprisingly, no response to lysine andlor tryptophan supplementation was
evident. It thus
appears that supplementation of a casein hydrolysate with methionine can
greatly enhance the
growth efficiency of the protein system. This interesting result forms an
aspect of the present
invention.
EXAMPLE II
Vitamin D Stability
Commercially available Alimentum~served as the Control in this experiment that
contains a casein
hydrolysate fortified with the amino acids L-cystine (11 mgs per gram of
protein), L-tryptophan and
L-tyrosine. The Experimental formula contained the same casein hydrolysate but
was supplemented
with 15 or 9.2 mgs of methionine (depending on the total protein concentration
in the formula} in
place of the 11 mgs of cystine per gram of total protein in the Control
product. Table III sets forth the
Control and Experimental products.
TABLE III
- meaian vane or ~u iors
The Vitamin D levels were determined during shelf life storage on product held
at room temperature.
The results are set forth in Table IV.
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TABLE N
Vitamin D Concentrations ~(IU~)i After Storage at Room Temp;
NT - Not Tested
From Table IV, it is evident that the Experimental products in accordance with
the invention
experienced less degradation of Vitamin D than the Control product. Table V
sets forth the Vitamin
D loss from processing and over the 12-month study period. Total loss in IU
per liter and % of
original fortification is also set forth in Table V.
TABLE V
Vitamin D Loss (IUILl
0 to 12 Months Loss
Control 207 31.0
Sam a I 95 14.5
Sam le II 113 18.0
Sam le III 98 16.5
It is quite evident from Table V that hypoallergenic products using the
present invention, (Samples
I-III) are significantly less likely to fall outside of Vitamin D acceptable
ranges during storage than the
Control product that used the techniques of the prior art.
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EXAMPLE III
A formula in accordance with this invention was analyzed for PER substantially
in the manner set
forth in Example I. Groups of 10 rats each, were fed a diet for 28 days
according to the invention or
a casein based formula using AOAC Method-960.48, AOAC D~cia! Methods of
Analysis,15th
Edition,1990. Every seven days, the rats were weighed and their food
consumption was recorded.
At the end of 28 days, the total weight gain and protein composition of the
two groups were
calculated. These values were used to calculate the PER. The composition of
each diet is set forth
in Table VI.
TABLE VI
Composition of Diets
- mgslg or iorai prorem
"' cystine addition prior to first heat treatment
*'"" cystine addition subsequent to first heat treatment
+ added as cystine dihydrochforide
The intact casein contained inherent levels of methionine ranging from 26-30
mgs per gram of
protein and cystine at about 4.0 mgs per gram of protein. The casein
hydrolysate contained about
26 mgs methionine per gram of protein and about 4.0 mgs of cystine per gram of
protein.
Diets 7 and 8 were identical except that the cystine was added prior to or
subsequent to a heat
treatment that was employed during the manufacture of the infant formula. The
Control diet and
Diets 7-10 contained the same blend of edible oils at about the same percent
of total calories (34.2
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to 39.8°~). The carbohydrate was the same in all the diets and was from
about 51.0 to 56.4% of
total calories. As all components of the diets were similar, except for the
source of amino nitrogen,
differences in growth of the animals can be attributed to the efficacy of the
protein system to support
growth. Thus, the higher the PER, theoretically, the better the protein
system. Table VII sets forth
the PER for each diet and the percent increase over the Control.
TABLE VII
Control 2.50 -
Diet 7 2.78 111
Diet 8 2.89 116
Diet 9 3.07 123
Diet 10 ~ 3.10 124
The results of this experiment indicate that the protein system in accordance
with the present
invention (Diets 9 and 10) has a significantly greater (Tukey's Studentized
Range Test for variable)
protein efficiency ratio than the conventional protein system (Control and
Diets 7 and 8) wherein
cystine fortification is utilized.
Diets 9 and 10, that were fo~ified with methionine (39.6 and 44 mgs of total
methionine per gram of
total protein) and decreased or no cystine (total cystine of 4.0 and 10.4 mgs
per gram of protein)
dihydrochloride fortification tested the highest in the study. The other diets
where no methionine
fortification occurred, tested significantly lower than the methionine
fortified diets. Diet 8 was very
similar to the presently available commercial Alimentum~ product (30 mgs of
total methionine and 15
mgs of total cystine per gram of protein) and had a PER of only 2.89. This
experiment supports one
aspect of the present invention; that being, increased fortification of sulfur-
containing amino acids,
other than cystine, has a beneficial effect on the growth of animals. This
aspect, in combination with
the reduction or elimination of cystine fortification, results in a
hypoallergenic nutritional product with
improved Vitamin D stability and a protein system for acceptable growth. As a
result of this work, an
improved hypoallergenic nutritional formula with acceptable protein quality
and Vitamin D stability
has been developed.
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EXAMPLE IV
Clinical Testing
As the protein quality and Vitamin D stability of a hypoallergenic nutritional
formula according to the
present invention had been demonstrated, the following clinical study was
undertaken to
demonstrate efficacy in human infants. The purpose of the study was to
investigate if substituting
methionine for cystine fortification or modifying the protein level and amino
acid fortification would
have any effect on the growth of healthy male, term infants.
The Control formula was commercially available Alimentum~. Diet 11 was
Alimentum~ with
increased levels of protein and methionine while cystine fortification was
reduced. Diet 12 was
Alimentum~ with methionine fortification and no cystine fortfication. Vitamin
D stability was also
measured in this study. Other biochemical responses of the infants such as
plasma albumin, urea
nitrogen and plasma amino acids were also evaluated.
This clinical trial was a controlled, blinded, randomized,16-week growth study
of male infants
enrolled within the first five days of life. Shortly after parturition,
parents of eligible infants were
contacted by the investigators. The study was explained and parents of infants
were requested to
sign an Informed Consent Form. On Day One of enrollment (SD1), the infants
were assigned to a
feeding group by an investigator, typically a pediatrician, and seen again by
the physician on weeks
four, eight and 16 (~ three days) after enrollment. Anthropometric
measurements were obtained on
entry into the study and at subsequent visits.
The Control and Experimental formula were prepared in similar fashion. Table
VI II sets forth the
target addition levels of cystine, methionine, tyrosine and tryptophan for the
Control and Diets 11
and 12.
TABLE VIII
Target Addiition Rates
Control'' 11.0 0.0 _ 6.0 ___ _3.5
Diet 11 0.0 15 6.0 ~ ~ 3.5
Diet 12 0.0 9.2 6.0 3.5
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Table IX sets forth the analyzed levels of protein, Vitamin D, Vitamin C,
cystine, methionine, tyrosine
and tryptophan for the Control and Experimental diets. Table IX also contains
data on the selected
amino acid content of representative human breast milk and a representative
intact casein.
TABLE IX
Level of Various Components
~Control* 18.8 664 v 418 14.5 27.0 20.5 16.0
Diet 11 * 18.7 641 399 5.8 43.5 22.0 14.0
Diet 12 21.6 595 350 5.0 38.0 21.0 14.0
Intact Casein - - - 4.0 30 52 9.6
Human - - - 21.9 19.0 43.8 20
Mitk**
* - average of two batches
** - values calculated from Peafiatric Nutrition Handbook, American Academy of
Pediatrics, 2nd Ed.,1985
The equipment and procedures used to prepare the Control and Experimental
formula are
conventional and well known to those skilled in the art. All study feedings
were supplied as
ready-to-feed (RTF) in 32 fluid ounce (907 ml) cans and provided 20 kcal per
fl. oz. All formulas met
or exceeded levels of nutrients as recommended by the Committee on Nutrition
of the American
Academy of Pediatrics (1985) and the Infant Formula Act (1980) and subsequent
amendments
(1986).
Formula volume intakes, incidence of spit-up, vomiting and stool patterns were
recorded on diaries
completed by the parents for three days immediately prior to the study visits
at four, eight and 16
weeks. Caloric and protein intakes were calculated based on formula volume
intakes and nutrient
composition of the formula fed.
Birth weight, length and head circumference were obtained from hospital
records. Weight, length
and head circumference were measured at SD1 and at each study visit (four,
eight and 16 weeks)
by the same examiner at each site, according to procedures in the Guide to
Growth Assessment of
Infants in Clinical Studies provided by Ross Products Division, Abbott
Laboratories. Nude body
weights were obtained using an electronic balance. Length boards were used to
obtain lengths,
measuring tapes were used to obtain the head and upper arm circumferences,
while calipers were
used to obtain triceps skin-fold thickness.
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Approximately 2 cc of blood was collected from the infanth by venipuncture by
a certified
technologist, nurse or physician at four and 16 weeks of age far determination
of plasma albumin,
urea nitrogen and plasma amino acids. The sample was drawn 150 to 180 minutes
after the
beginning of the last feeding. Plasma was frozen at -70° C unfit
analyzed.
The three feeding groups were evaluated for comparability at entry. Birth and
entrance
anthropometrics were analyzed using ANOVA. Gestational age and age at SD1 were
compared
using ANOVA on the ranked data. Ethnicity and five-minute Apgar scores were
analyzed using
Fisher's Exact test.
Evaluation of feeding effects was tamed out using Repeated Measures ANOVA.
These were also
analyzed separately at each visit using ANOVA. Ordinal variables were compared
separately at
each time point using ANOVA or Cochran Mantel-Haenszel tests: Categorical
variables were
analyzed at each time point using Fisher's Exact test.
Location of the study (site) was used as a blocking factor in the ANOVA
models. Ranking
transformations were used in the analyses of the percentage tolerance and
stool variables,
predominant stool consistency, volume intake and blood biochemistries. Mean
rank stool
consistency was also analyzed using the untransformed data. All tests were
carried out using a 5%
significance level. The primary analysis was done on the subset of completers
only. An
intent-to-treat analysis including available data on all subjects was done as
a secondary analysis.
Subject
A total of 73 infants successfully completed the study (23 on Control; 25 on
Diet 11; 25 on Diet 12).
A total of 27 infants were considered to be treatment failures due to
intolerance to the study feeding
(13 on Control; six on Diet 11; eight on Diet 12) and 22 infants were
considered to be protocol
failures.
No statistically significant differences were observed among groups for
gestational appropriateness.
One infant was considered small for gestation age, 88 infants were appropriate
for gestational age
and 33 infants were considered large for gestabonal age. No significant
differences were observed
among groups for age at SD1, gestational age or five minute Apgar score. The
mean age of infants
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at entry into the study ranged from 1.8 to 2.3 days (range 0 to five days of
age). The mean
gestationai age at birth ranged from 39.3 to 39.4 weeks.
Anthro~ometric Measurements
No statistically significant differences were observed in birth weight, length
or head circumference or
in their respective NCHS Z-scores. No statistically significant differences in
weight, length or head
circumference or their respective NCHS Z-scores were observed at SD1 or at
four, eight or 16
weeks. Most importantly, no statistically significant differences were
observed among groups in
weight gains, adjusted weight gains, length gains or head circumference gains
at four, eight or 16
weeks. In addition, no statistically significant differences were observed
among groups for total
upper arm area, upper arm muscle area, upper arm fat area or arm fat index at
SD1, five, eight or 16
weeks or in gains of these at four, eight or 16 weeks.
The study also revealed that there were no signficant differences among groups
in the mean
number of feedings per day, the mean volume of formula intake, mean adjusted
volume of formula
intake, adjusted caloric intake or caloric efficiency at four weeks or eight
weeks. Adjusted mean
protein intakes pooled across visits differed significantly (P<0.01) among the
feeding groups. Infants
fed Diet 12 had significantly greater mean adjusted protein intakes than
infants fed Diet 11 at four
weeks (P<0.01) and significan~y greater mean adjusted protein intakes than
infants fed Diet 11 and
Control at eight weeks. At 16 weeks, infants fed Diet 12 had significantly
greater volume of formula
intakes, greater adjusted volume of formula intakes, and adjusted caloric
intakes than those infants
fed Diet 11 (P<0.05). Also, at 16 weeks, infants fed Diet 12 had significantly
greater adjusted protein
intakes than infants fed Diet 11 or Control (P<0.01). Infants fed Diet 11 had
significantly greater
caloric efficiency at 16 weeks than infants fed Diet 12 (P<0.05). No
differences in the percent of
feedings with spit-up andlor vomiting were observed among groups during the
study.
No significant differences were observed among groups in the number of stools
per day during the
study. Also, no significant differences were observed among groups in the
percent of stools that
were watery, looselmushy or formed at each visit during the study. However,
infants fed Diet 12 had
significantly higher plasma urea nitrogen concentrations than infants fed Diet
11 and Control at four
weeks.
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For data pooled across visits, plasma methionine concentrations differed
significantly between
feeding groups (P<0.01). Infants fed Diet 12 had significantly higher plasma
concentrations of
asparagine, praline, valine, isoleucine, leucine and tyrosine than infants fed
Control at four weeks.
Infants fed Diet 11 and 12 had significan~y higher serum methionine
concentrations than infants fed
Control at four weeks. When only the data of successful completers were
analyzed, only plasma
methionine differences were statistically significant. At 16 weeks, the only
significant difference
observed was that infants fed Diet 11 had a significantly higher plasma
methionine level than infants
fed Control.
Conclusion
The inventors herein had theorized that the amino acid methionine could be
used to fortify a
hypoallergenic formula in place of cys~ne to insure that the requirements for
sulfur-containing amino
acids were met. This study was conducted to determine the.effects of
modiiycation of the protein
system of a hypoallergenic formula on the growth and biochemical response of
infants.
The results of this study confirmed that substituting methionine for cystine
resulted in no altera~ons
in growth of healthy term, male infants during the first four months compared
to infants fed the
Control formula, that was commercially available Alimentum~. The study results
also indicate that
there was no advantage in feeding infants an increased protein-containing
formula with methionine
fortification and no cystine fortification. All groups grew in a remarkably
similar fashion during the
study and when weights of these infants were compared to the weights of
infants fed Similac° With
Iron (an intact protein infant formula marketed by the Ross Products Division
of Abbott Laboratories,
Columbus, Ohio) in recent studies, the weights were also very similar. Most
importantly, no adverse
effects were observed in the group of infants fed a methionine fortified
product.
Industrial A~~alicabilitv
The results from these experiments demonstrate that the hypoallergenic enteral
formula of this
invention is effective in providing adequate growth without the need to over-
fortify Vitamin D. The
medical community is constantly searching for nutritional formulas that will
benefit the infantlpatient.
The present invention can clearly fill that need. The manufacture of the
formula utilizes conventional
equipment and may be readily accomplished. Further, the discoveries of the
invention have
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application to other liquid nutri~onal products that u~lize hydrolyzed
proteins and/or free amino acids
and need an increased level of Vitamin D stability.
While the formula and method of making said formula disclosed herein
constitute a preferred
embodiment of this invenfion, it is to be understood that the invention is not
limited to this precise
formulation or method and that changes may be made therein without departing
from the scope of
the invention that is defined in the appended claims.
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