U.S. patent application number 13/946020 was filed with the patent office on 2014-04-03 for human milk compositions and methods of making and using same.
This patent application is currently assigned to Prolacta Bioscience, Inc.. The applicant listed for this patent is Prolacta Bioscience, Inc.. Invention is credited to Joseph Fournell, Martin L. Lee, Elena M. Medo, David J. Rechtman.
Application Number | 20140093634 13/946020 |
Document ID | / |
Family ID | 39464002 |
Filed Date | 2014-04-03 |
United States Patent
Application |
20140093634 |
Kind Code |
A1 |
Medo; Elena M. ; et
al. |
April 3, 2014 |
HUMAN MILK COMPOSITIONS AND METHODS OF MAKING AND USING SAME
Abstract
The disclosure features human milk fortifier compositions,
standardized human milk, and methods of making and using same. In
one embodiment, a pasteurized human milk composition includes a
human protein constituent of about 35-85 mg/mL; a human fat
constituent of about 60-110 mg/mL; and a human carbohydrate
constituent of about 60-140 mg/mL.
Inventors: |
Medo; Elena M.; (Murrieta,
CA) ; Lee; Martin L.; (Studio City, CA) ;
Rechtman; David J.; (Hermosa Beach, CA) ; Fournell;
Joseph; (Newbury Park, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Prolacta Bioscience, Inc. |
City of Industry |
CA |
US |
|
|
Assignee: |
Prolacta Bioscience, Inc.
City of Industry
CA
|
Family ID: |
39464002 |
Appl. No.: |
13/946020 |
Filed: |
July 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11947580 |
Nov 29, 2007 |
8545920 |
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13946020 |
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PCT/US07/19234 |
Aug 30, 2007 |
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11947580 |
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60867748 |
Nov 29, 2006 |
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60841371 |
Aug 30, 2006 |
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Current U.S.
Class: |
426/586 |
Current CPC
Class: |
A23L 33/15 20160801;
A23L 33/16 20160801; A23L 33/19 20160801; A23L 33/40 20160801; A23C
9/206 20130101; A23L 2/66 20130101; A23L 33/10 20160801; A23L 2/52
20130101 |
Class at
Publication: |
426/586 |
International
Class: |
A23C 9/20 20060101
A23C009/20 |
Claims
1-65. (canceled)
66. A method for obtaining a pasteurized human milk fortifier, the
method comprising: (a) genetically screening pooled human milk for
one or more viruses selected from the group consisting of HIV-1,
HBV and HCV; (b) filtering the pooled human milk; (c) heat-treating
the pooled human milk: (d) separating the pooled human milk into
cream and skim; (e) adding a portion of the cream to skim such that
the resulting composition comprises: (i) 35-85 mg/mL of protein
(ii) 60-110 mg/mL of fat (iii) 60-140 mg/ml, of carbohydrate; and
(f) pasteurizing the resulting composition from (e) thereby
obtaining a pasteurized human milk fortifier.
67. The method of claim 66, wherein the heat-treating comprises
treating the pooled human milk at about 63.degree. C. or greater
for about 30 min.
68. The method of claim 66, further comprising ultrafiltration of
the skim after step (d).
69. The method of claim 66, wherein the genetic screening in step
(a) comprises utilizing the polymerase chain reaction.
70. The method of claim 66, wherein the pooled human milk is
filtered through a 200 micron filter in step (b).
71. The method of claim 66, further comprising running the cream
through a separator following step (d).
72. The method of claim 68, wherein ultrafiltration the skim
comprises filtering permeate out of the skim.
73. The method of claim 68, further comprising washing the
ultrafilters to obtain post wash solution.
74. The method of claim 73, further comprising combining the post
wash solution to skim.
75. The method of claim 66, further comprising carrying out mineral
analysis of the composition obtained after step (e).
76. The method of claim 66, further comprising adding to the
resulting composition obtained after step (e) one or more minerals
selected from the group consisting of calcium, chloride, copper,
iron, magnesium, manganese, phosphorus, potassium, sodium, and
zinc.
77. The method of claim 66, further comprising carrying out
biological testing of a portion of the pasteurized human milk
fortifier after step (f).
78. The method of claim 66, further comprising carrying out
nutritional testing of a portion of the composition after step
(f).
79. The method of claim 66 wherein adding a portion of the cream to
skim in step (e) is done such that the resulting composition
comprises: (i) 55-65 mg/mL of protein (ii) 60-110 mg/mL of fat
(iii) 60-140 mg/mL of carbohydrate.
80. A method for obtaining a pasteurized human milk fortifier, the
method comprising: (a) genetically screening pooled human milk by
polymerase chain reaction for one or more viruses selected from the
group consisting of HIV-1, HBV and HCV; (b) filtering the pooled
human milk through a 200 micron screen; (c) heat-treating the
pooled human milk at about 63.degree. C. or greater for about 30
minutes; (d) separating the pooled human milk into cream and skim;
(e) ultrafiltering the skim obtained in step (d) to remove permeate
(f) adding a portion of the cream obtained in step (d) to the
ultrafiltering skim obtained in step (e) such that the resulting
composition comprises: (i) 55-85 mg/mL of protein (ii) 60-110 mg/mL
of fat (iii) 60-140 mg/mL of carbohydrate; (g) carrying out mineral
analysis on the composition; (h) adding one or more of the minerals
to the composition wherein the one or more minerals are selected
from the group consisting of: calcium, chloride, copper, iron,
magnesium, manganese, phosphorus, potassium, sodium, and zinc; and
(i) pasteurizing the resulting composition from (h) thereby
obtaining at pasteurizing human milk fortifier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to provisional application
60/841,371 filed Aug. 30, 2006; provisional application 60/867,748,
filed Nov. 29, 2006; and PCT application PCT/US07/19234, filed Aug.
30, 2007. The contents of these applications are incorporated
herein by reference in their entireties.
TECHNICAL FIELD
[0002] The disclosure relates to human milk compositions, e.g.,
human milk fortifiers and standardized human milk formulations, and
methods of making and using such compositions.
BACKGROUND
[0003] Human milk is generally the food of choice for preterm and
term infants because of its nutritional composition and immunologic
benefits. The source of human milk can be, e.g., a donor or the
infant's mother. The nutritional value of raw or
conventionally-processed donor milk, however, varies and, in most
instances, is not sufficient to meet the needs of preterm infants.
In addition, a possibility of bacterial, viral and other
contamination of raw donor milk exists. For these and other
reasons, use of milk from the infant's own mother has become the
preferred nutritional approach in the modern neonatal intensive
care units (NICUs). Even the mother's own milk, however, is not
nutritionally sufficient for the premature infant. It is often
desirable to feed preterm infants milk that is fortified with
various supplements, e.g., proteins, added energy (calories) and/or
minerals.
SUMMARY
[0004] This disclosure features human milk compositions, e.g.,
pasteurized human milk compositions, and methods of making and
using such compositions. The compositions include human milk
fortifiers (e.g., PROLACTPLUS.TM. Human Milk Fortifiers, e.g.,
PROLACT+4.TM., PROLACT+6.TM., PROLACT+8.TM., and/or
PROLACT+10.TM.), which are produced from human milk and contain
various concentrations of nutritional components, e.g., protein,
fat, carbohydrates, vitamins, and/or minerals. These fortifiers can
be added to the milk of a nursing mother to provide an optimal
nutritional content of the milk for, e.g., a preterm infant.
Depending on the content of mother's own milk, various
concentrations of the fortifiers can be added to mother's milk. For
example, the protein concentration of the mother's milk can be
increased with the use of the fortifier. As mentioned above, the
fortifiers of the present disclosure are generated from human milk
and, therefore, provide infants with human-derived nutrients.
[0005] The disclosure also features standardized human milk
formulations (exemplified by PROLACT20.TM., NEO20.TM., and/or
PROLACT24), which are produced from human milk. Methods of making
and using such compositions are also described herein. These
standardized human milk formulations can be used to feed, e.g.,
preterm infants, without mixing them with other fortifiers or milk.
They provide a nutritional human-derived formulation and can
substitute for mother's milk. Human milk formulations can contain
various caloric contents, for example, PROLACT24.TM. (a full-feed
whole milk product) can contain about 24 Cal/oz or about 81 Cal/100
mL.
[0006] The methods featured herein are used to process large
volumes of donor milk, e.g., about 75-2,000 liters/lot of starting
material.
[0007] In one aspect, the disclosure features a pasteurized human
milk composition that includes: a human protein constituent of
about 35-85 mg/mL; a human fat constituent of about 60-110 mg/mL;
and a human carbohydrate constituent of about 60-140 mg/mL. The
carbohydrate constituent can include lactose. The composition can
further include IgA and/or one or more constituents selected from
the group consisting of: calcium, chloride, copper, iron,
magnesium, manganese, phosphorus, potassium, sodium, and zinc. In
one embodiment, the composition can be mixed with raw human milk to
provide a nutritional composition, wherein the raw human milk
comprises about 80%, about 70%, about 60%, or about 50% of the
nutritional composition.
[0008] Embodiments can include one or more of the following
features.
[0009] In one embodiment, the composition can include the protein
constituent of about 55-65 mg/mL; the fat constituent of about
85-95 mg/mL; and the carbohydrate constituent of about 70-120
mg/mL. The carbohydrate constituent can include lactose. The
composition can further include IgA and/or one or more constituents
selected from the group consisting of: calcium (e.g., at a
concentration of about 4.0-5.5 mg/mL or at 2.00-2.90 mg/mL);
chloride (e.g., at a concentration of about 0.35-0.95 mg/mL or at
about 0.175-0.475 mg/mL); copper (e.g., at a concentration of about
0.0005-0.0021 mg/mL or at about 0.00025-0.001 mg/mL); iron (e.g.,
at a concentration of about 0.001-0.007 mg/mL or at about
0.0005-0.0025 mg/mL); magnesium (e.g., at a concentration of about
0.180-0.292 mg/mL or at about 0.090-0.170 mg/mL); manganese (e.g.,
at a concentration of about 0.010-0.092 mcg/mL or at about
0.005-0.046 mcg/mL;); phosphorus (e.g., at a concentration of about
2.00-3.05 mg/mL or at about 1.00-2.90 mg/mL, e.g., at about
1.00-1.50 mg/mL); potassium (e.g., at a concentration of about
1.90-2.18 mg/mL or at about 0.95-1.41 mg/mL); sodium (e.g., at a
concentration of about 0.75-0.96 mg/mL or at about 0.375-0.608
mg/mL); and zinc (e.g., at a concentration of about 0.0200-0.0369
mg/mL or at about 0.010-0.0198 mg/mL). In one embodiment, the
composition can be mixed with raw human milk to provide a
nutritional composition, wherein the raw human milk comprises about
80%, about 70%, about 60%, or about 50% of the nutritional
composition.
[0010] In another aspect, the disclosure features a pasteurized
human milk composition that includes: a human protein constituent
of about 11-20 mg/mL, e.g., about 11-13 mg/mL; a human fat
constituent of about 35-55 mg/mL; and a human carbohydrate
constituent of about 70-120 mg/mL, e.g., about 80-105 mg/mL. The
carbohydrate constituent can include lactose. The caloric content
of the composition can be about 0.64 to about 1.10 Cal/mL.
[0011] Embodiments can include one or more of the following
features.
[0012] In one embodiment, the pasteurized human milk composition
can further include one or more of the following components:
calcium (e.g., at a concentration of about 0.40-1.50 mg/mL);
chloride (e.g., at a concentration of about 0.30-0.80 mg/mL);
copper (e.g., at a concentration of about 0.0005-0.0021 mg/mL);
iron (e.g., at a concentration of about 0.001-0.005 mg/mL);
magnesium (e.g., at a concentration of about 0.03-0.13 mg/mL);
manganese (e.g., at a concentration of about 0.01-0.092 mcg/mL);
phosphorus (e.g., at a concentration of about 0.15-0.631 mg/mL);
potassium (e.g., at a concentration of about 0.60-1.20 mg/mL);
sodium (e.g., at a concentration of about 0.20-0.60 mg/mL); and/or
zinc (e.g., at a concentration of about 0.0025-0.0120 mg/mL).
[0013] The disclosure also features method of making various human
milk compositions.
[0014] In one aspect, the disclosure features a method for
obtaining a pasteurized human milk composition. The method
includes: (a) genetically screening human milk for one or more
viruses; (b) filtering the milk; (c) heat-treating the milk, e.g.,
at about 63.degree. C. or greater for about 30 minutes; (d)
separating the milk into cream and skim; (e) adding a portion of
the cream to the skim; and (f) pasteurizing.
[0015] Embodiments include one or more of the following
features.
[0016] In one embodiment, the method can further include filtering
the skim through filters after step (d), e.g., to filter the water
out of the skim. After filtering the skim after step (d), the
filters used in the filtering can be washed to obtain a post wash
solution. The post wash solution can be added to the skim.
[0017] The genetic screening in step (a) can be polymerase chain
reaction and/or can include screening for one or more viruses,
e.g., HIV-1, HBV, and/or HCV. The milk can be filtered through an
about 200 micron screen in step (b). The method can further include
running cream, e.g., about 30-50% of cream, through a separator
following step (d). The composition of post wash and skim can
include about 7.0-7.2% of protein.
[0018] The method can further include carrying out mineral analysis
of the portion of the composition obtained after step (e). The
method can also include adding to the composition obtained after
step (e) one or more minerals selected from the group consisting
of: calcium, chloride, copper, iron, magnesium, manganese,
phosphorus, potassium, sodium, and zinc. Adding of the one or more
minerals can include heating the composition.
[0019] The method can also include cooling the composition after
step (f), carrying out biological testing of a portion of the
composition after step (f), and/or carrying out nutritional testing
of a portion of the composition after step (f).
[0020] The human milk of step (a) can be pooled human milk. The
methods featured herein can be carried out with large volumes of
the starting material, e.g., human milk, e.g., pooled human milk.
The volumes can be in the range of about 75-2,000 liters/lot of
starting material.
[0021] The composition obtained after step (f) can include about
8.5-9.5% fat, about 6.3-7.0% protein, and about 8.0-10.5%
lactose.
[0022] In another aspect, the disclosure features a method for
obtaining a pasteurized human milk composition. The method
includes: (a) genetically screening human milk for one or more
viruses; (b) filtering the milk; (c) adding cream; and (d)
pasteurizing.
[0023] Embodiments can include one or more of the following
features.
[0024] In one embodiment, the genetic screening in step (a) can be
polymerase chain reaction. The genetic screening can include
screening for one or more viruses, e.g., HIV-1, HBV, and/or
HCV.
[0025] The milk can be filtered through an about 200 micron screen
in step (b). The method can further include ultra filtering the
whole milk after step (b) through filters. The filters used during
ultra filtering can be post washed.
[0026] The composition can be cooled after step (d). Biological
and/or nutritional testing of the composition can be carried out
after step (d).
[0027] Human milk of step (a) can be pooled human milk. The methods
featured herein can be carried out with large volumes of the
starting material, e.g., human milk, e.g., pooled human milk. The
volumes can be in the range of about 75-2,000 liters/lot of
starting material.
[0028] The method can also include adding to the composition
obtained after step (c) one or more minerals selected from the
group consisting of: calcium, chloride, copper, iron, magnesium,
manganese, phosphorus, potassium, sodium, and zinc. In one
embodiment, the composition obtained after step (d) can include
about 11-20 mg/mL protein, about 35-55 mg/mL fat, and about 70-120
mg/mL carbohydrates.
[0029] In another aspect, the disclosure features a kit that
includes the pasteurized human milk compositions featured herein
(e.g., a fortifier) and a graduated container (e.g., a bottle, a
syringe, and a can) for mixing the featured compositions with raw
human milk.
[0030] In yet another aspect, the disclosure features a method of
obtaining a nutritional milk composition. The method includes
adding the pasteurized human milk compositions featured herein
(e.g., fortifiers) to raw human milk, thereby increasing the
nutritional concentration of the raw human milk. The caloric
composition of the raw human milk can be increased by about 2-10
Cal/oz.
[0031] In another aspect, the disclosure features a method of
providing supplemental nutrients to a premature human infant, the
method comprising adding the compositions (fortifiers) featured
herein to raw human milk to obtain a mixture and administering the
mixture to the premature infant.
[0032] The terms "premature", "preterm" and "low-birth-weight
(LBW)" infants are used interchangeably and refer to infants born
less than 37 weeks gestational age and/or with birth weights less
than 2500 gm.
[0033] By "whole milk" is meant milk from which no fat has been
removed.
[0034] By "bioburden" is meant microbiological contaminants and
pathogens (generally living) that can be present in milk, e.g.,
viruses, bacteria, mold, fungus and the like.
[0035] All patents, patent applications, and references cited
herein are incorporated in their entireties by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 is a chart of an embodiment of a method of making a
human milk fortifier.
[0037] FIG. 2 is a chart of an embodiment of a method of making
standardized human milk.
DETAILED DESCRIPTION
[0038] This disclosure features human milk compositions, e.g.,
pasteurized human milk compositions, and methods of making and
using such compositions. The compositions include human milk
fortifiers (e.g., PROLACTPLUS.TM. Human Milk Fortifiers, e.g.,
PROLACT+4.TM., PROLACT+6.TM., PROLACT+8.TM., and/or
PROLACT+10.TM.), which are produced from human milk and contain
various concentrations of nutritional components, e.g., vitamins,
and/or minerals. These fortifiers can be added to the milk of a
nursing mother to provide an optimal nutritional content of the
milk for, e.g., a preterm infant. Depending on the content of
mother's own milk, various concentrations of the fortifiers can be
added to mother's milk. For example, the protein concentration
and/or caloric content of the mother's milk can be increased with
the use of the fortifier. In one embodiment, the featured
fortifiers can deliver from about 3.3 to about 5.5 g of
protein/kg/day to the infant receiving a full feed of 150 mL and/or
3.2 to 4.1 grams of protein per 120 Cal/kg/day.
[0039] The disclosure also features standardized human milk
formulations (exemplified by PROLACT20.TM., NEO20.TM., and/or
PROLACT24.TM.), which are produced from human milk. Methods of
making and using such compositions are also described. These
standardized human milk formulations can be used to feed, e.g.,
preterm infants, without mixing them with other fortifiers or milk.
Thus, the compositions provide an infant with a human-derived
nutritional formulation that can substitute for mother's milk.
Human milk formulations can contain various caloric contents, for
example, PROLACT24 (a full feed whole milk formulation) can contain
about 24 Cal/oz or about 81 Cal/100 mL.
[0040] The compositions of the present disclosure are generated
from human donor milk, e.g., pooled milk, which undergoes rigorous
genetic screening, processing (e.g., to concentrate nutrients in
the fortifier compositions, and/or to reduce bioburden), and
pasteurization. The milk can be supplemented with various minerals
and/or vitamins. Thus, the disclosure also features methods of
obtaining and processing milk from human donors.
[0041] The methods of the present disclosure can be used to process
large volumes of donor milk, e.g., about 75-2,000 liters/lot of
starting material.
Nutritional Requirements of Premature Infants
[0042] There are many factors that can affect the clinical outcome
of a newborn, e.g., prematurely born, infant. Preterm infants have
vulnerable immune systems, immature digestive systems, and
increased total caloric and specific nutrient needs (when generally
compared with term infants). Thus, nutrition provided to such
infants is an important factor in their growth and development.
Human milk has long been recognized as the ideal food for preterm
and term infants because of its nutritional composition and
immunologic benefits. Not every mother, however, can or will
breastfeed her baby (or use a breast pump and store her milk). For
example, mothers who have certain diseases, e.g., active
tuberculosis, or are being administered radioisotopes,
antimetabolites, or chemotherapy may not breast feed their infants.
In addition, mother's own milk may not contain sufficient
nutritional content to sustain a preterm infant. Use of donor milk
can also be problematic, as such milk may not contain adequate
nutrition for a preterm infant.
[0043] The present disclosure features human milk compositions and
methods of making and using such compositions for feeding and/or
increasing the nutritional value of milk fed to infants, e.g.,
premature infants. The fortifiers described herein (e.g.,
PROLACTPLUS.TM.) can deliver a high level of, e.g., protein and/or
calories to the milk and therefore to the infant. The standardized
human milk formulations (e.g., PROLACT20.TM. and NEO20.TM.) can be
used instead of mother's own milk. The compositions can be
supplemented with various vitamins and/or minerals. The
compositions can also contain IgA (e.g., secretory IgA) and various
components described herein.
Obtaining Human Milk from Qualified and Selected Donors
[0044] The compositions of the present disclosure are generated
from donated human milk. Various techniques are used to identify
and qualify suitable donors. A potential donor must obtain a
release from her physician and her child's pediatrician as part of
the qualification process. This helps to insure, inter alia, that
the donor is not chronically ill and that her child will not suffer
as a result of the donation(s). Methods and systems for qualifying
and monitoring milk collection and distribution are described,
e.g., in U.S. patent application Ser. No. 11/526,127 (U.S.
2007/0098863), which is incorporated herein by reference in its
entirety.
[0045] Generally, the donor screening process includes both
interviews and biological sample processing. Any blood sample found
positive for, e.g., viral contamination, on screening removes the
donor from the qualification process.
[0046] Once a donor qualifies and begins sending milk, milk from
each of her shipments is tested for, e.g., B. cereus and drugs of
abuse (e.g., cocaine, opiates, methamphetamines, benzodiazepine,
amphetamines, and THC). Any positive finding results in the
deferral of the donor and destruction of all previously-collected
milk.
[0047] Donors may be periodically requalified. For example, a donor
is required to undergo screening by the protocol used in their
initial qualification every four months, if the donor wishes to
continue to donate. A donor who does not requalify or fails
qualification is deferred until such time as they do, or
permanently deferred if warranted by the results of requalification
screening. In the event of the latter situation, all remaining milk
provided by that donor is removed from inventory and destroyed.
[0048] A qualified donor may donate at a designated facility (e.g.,
a milk bank office) or, typically, expresses milk at home. The
qualified donor can be provided with supplies by a milk bank or
directly from a milk processor (the milk bank and processor may be
the same or different entities) to take home. The supplies will
typically comprise a computer readable code (e.g., a barcode-label)
on containers and may further include a breast pump. The donor can
then pump and freeze the milk at home at a temperature of about
-20.degree. C. or colder. The donor milk is accepted, provided that
the donor is a qualified donor; if such results are satisfactory,
an appointment is made for the donor to drop off the milk at the
center, or to have it collected from home. A donor can also ship
the milk directly to the milk bank center or milk processor in
insulated containers provided by the milk bank or milk processor.
The milk and container are examined for their condition and the
barcode information checked against the database. If satisfactory,
the units are placed in the donor milk center or processing center
freezer (-20.degree. C. or colder) until ready for further testing
and processing.
[0049] Because in some embodiments of the present methods the milk
is expressed by the donor at her home and not collected at the milk
banking facility, each donor's milk is sampled for genetic markers,
e.g., DNA markers, to guarantee that the milk is truly from the
registered donor. Such subject identification techniques are known
in the art (see, e.g., International Application Serial No.
PCT/US2006/36827, which is incorporated herein by reference in its
entirety). The milk may be stored (e.g., at -20.degree. C. or
colder) and quarantined until the test results are received.
Throughout the above process, any non-complying milk specimens are
discarded, and the donor is disqualified. Access to all
confidential information about the donor, including blood test
data, is carefully controlled and meets Health Insurance
Portability and Accountability Act (HIPAA) requirements.
Processing Donated Human Milk
[0050] Collected donor human milk is processed to obtain, e.g., a
human milk fortifier, and/or standardized human milk
formulation.
[0051] Methods of Obtaining Human Milk Fortifiers
[0052] FIG. 1 is a chart showing an embodiment of generating a
human milk fortifier (e.g., PROLACTPLUS.TM.). A detailed embodiment
is discussed in Example 1 below. As discussed above, donor milk is
carefully analyzed for both identification purposes and to avoid
contamination. The donor milk is frozen and, when desired, thawed
and pooled. It is then screened (step 1 of FIG. 1), e.g.,
genetically screened, e.g., by polymerase chain reaction (PCR).
Genetic screening is done to identify any contaminants, e.g.,
viral, e.g., HIV-1, HBV, and/or HCV. The milk then undergoes
filtering, e.g., through about a 200 micron filter (step 2), and
heat treatment (step 3). For example, the composition can be
treated at about 63.degree. C. or greater for about 30 minutes or
more. In step 4, the milk is transferred to a separator, e.g., a
centrifuge, to separate the cream from the skim. The skim can be
transferred into a second processing tank where it remains at about
2 to 8.degree. C. until a filtration step (step 5).
[0053] Optionally, the cream separated from the skim in step 4, can
undergo separation again to yield more skim.
[0054] Following separation of cream and skim (step 4), a desired
amount of cream is added to the skim, and the composition undergoes
further filtration (step 5), e.g., ultrafiltration. This process
concentrates the nutrients in the skim milk by filtering out the
water. The water obtained during the concentration is referred to
as the permeate. Filters used during the ultrafiltration can be
postwashed and the resulting solution added to the skim to maximize
the amount of nutrients obtained, e.g., obtaining a protein
concentration of about 7% to 7.2%. The skim is then blended with
the cream (step 6) and samples taken for analysis. At this point
during the process, the composition generally contains: about 8.5%
to 9.5% of fat; about 6.3% to 7.0% of protein; and about 8% to
10.5% of carbohydrates, e.g., lactose.
[0055] After the separation of cream and skim in step 4, the cream
flows into a holding tank, e.g., a stainless steel container. The
cream can be analyzed for its caloric, protein and fat content.
When the nutritional content of cream is known, a portion of the
cream can be added to the skim milk that has undergone filtration,
e.g., ultrafiltration, (step 5) to achieve the caloric, protein and
fat content required for the specific product being made. Minerals
can be added to the milk prior to pasteurization.
[0056] At this point, in one embodiment, the processed composition
can be frozen prior to the addition of minerals and thawed at a
later point for further processing. Any extra cream that was not
used can also be stored, e.g., frozen. Optionally, before the
processed composition is frozen, samples are taken for mineral
analysis. Once the mineral content of the processed milk is known,
the composition can be thawed (if it were frozen) and a desired
amount of minerals can be added to achieve target values.
[0057] After step 6 and/or the optional freezing and/or mineral
addition, the composition undergoes pasteurization (step 7). For
example, the composition can be placed in a process tank that is
connected to the high-temperature, short-time (HTST) pasteurizer
via platinum-cured silastic tubing. After pasteurization, the milk
can be collected into a second process tank and cooled. Other
methods of pasteurization known in the art can be used. For
example, in vat pasteurization the milk in the tank is heated to a
minimum of 63.degree. C. and held at that temperature for a minimum
of thirty minutes. The air above the milk is steam heated to at
least three degrees Celsius above the milk temperature. In one
embodiment, the product temperature is about 66.degree. C. or
greater, the air temperature above the product is about 69.degree.
C. or greater, and the product is pasteurized for about 30 minutes
or longer. In another embodiment, both HTST and vat pasteurization
are performed.
[0058] The resulting fortifier composition is generally processed
aseptically. After cooling to about 2 to 8.degree. C., the product
is filled into containers of desired volumes, and various samples
of the fortifier are taken for nutritional and bioburden analysis.
The nutritional analysis ensures proper content of the composition.
A label that reflects the nutritional analysis is generated for
each container. The bioburden analysis tests for presence of
contaminants, e.g., total aerobic count, B. cereus, E. coli,
Coliform, Pseudomonas, Salmonella, Staphylococcus, yeast, and/or
mold. Bioburden testing can be genetic testing. The product is
packaged and shipped once the analysis is complete and desired
results are obtained.
[0059] Methods of Obtaining Standardized Human Milk
Formulations
[0060] FIG. 2 and Examples 3 and 4 below show some embodiments of
the methods for obtaining standardized human milk formulations
(e.g., PROLACT20.TM. and NEO20.TM.)
[0061] As discussed above, donor milk is screened to ensure the
identity of the donors and reduce the possibility of contamination.
Donor milk is pooled and further screened (step 1 of FIG. 2), e.g.,
genetically screened (e.g., by PCR). The screening can identify,
e.g., viruses, e.g., HIV-1, HBV, and/or HCV. Milk that tests
positive is discarded. After the screening, the composition
undergoes filtering (step 2). The milk is filtered through about a
200 micron screen and then ultrafiltered. During ultrafiltration,
water is filtered out of the milk (and is referred to as permeate)
and the filters are postwashed using the permeate. Post wash
solution is added to the milk to recover any lost protein and
increase the concentration of the protein to, e.g., about 1.2% to
about 1.5%. Referring to FIG. 2, cream from another lot (e.g.,
excess cream from a previous fortifier lot) is added in step 3 to
increase the caloric content. At this stage of the process, the
composition generally contains: about 3.5% to 5.5% of fat; about
1.1% to 1.3% of protein; and about 8% to 10.5% of carbohydrates,
e.g., lactose.
[0062] At this stage, the composition can be frozen and thawed out
for further processing later.
[0063] Optionally, if the human milk formulation is to be fortified
with minerals, a mineral analysis of the composition is carried out
after step 3. Once the mineral content is known, a desired amount
of minerals can be added to achieve target values.
[0064] In step 4, the composition is pasteurized. Pasteurization
methods are known in the art. For example, the product can be
pasteurized in a tank that is jacketed. Hot glycol can be use to
heat up the tank. The product temperature can be about 63.degree.
C. or greater and the air temperature above the product about
66.degree. C. or greater. The product is pasteurized for a minimum
of about 30 minutes. Other pasteurizing techniques are known in the
art.
[0065] After cooling to about 2 to 8.degree. C., the product is
filled into containers of desired volumes and various samples of
the human milk formulation are taken for nutritional and bioburden
analysis. The nutritional analysis ensures proper content of the
composition. A label generated for each container reflects the
nutritional analysis. The bioburden analysis tests for presence of
contaminants, e.g., total aerobic count, B. cereus, E. coli,
Coliform, Pseudomonas, Salmonella, Staphylococcus, yeast, and/or
mold. The product is packaged and shipped once the analysis is
complete and desired results are achieved.
Effects of the Featured Methods on Components and Bioburden of
Human Milk
[0066] Human milk comprises about 100,000 different molecular
entities--proteins, lipids, carbohydrates, vitamins, and trace
minerals. Some specific components include the monomeric
immunoglobulin A (IgA) and the dimeric secretory IgA
(s[IgA].sub.2), lysozyme, and lactoferrin. Accordingly, the methods
featured herein (which include pasteurization), while helping to
ensure the safety of the milk product, should also preserve
adequate quantities of those components, which are potentially heat
labile. As described in Example 5 below and Table 3, pasteurization
methods featured herein can preserve a large amount of IgA,
lysozyme, and Vitamin B6 in the processed milk. As described in
Example 5 below and Table 2, the featured methods are able to
reduce any bioburden of human milk.
Human Milk Compositions
[0067] The compositions featured herein contain various amounts of
nutrients, e.g., protein, carbohydrates, fat, vitamins, and
minerals, as well as other milk components, such as IgA and
lysozyme. Human milk fortifiers provide a concentrated amount of
nutrients and calories that can be added to, e.g., mother's milk.
Standardized human milk formulations can be supplemented with
vitamins and/or minerals if desired and can be fed directly to an
infant, e.g., a premature infant. The methods of generating these
compositions are designed to optimize the amount of nutrients and
calories in the compositions. For example, the compositions
featured herein can deliver from about 3.3 to about 5.5 grams of
protein/kg/day to an infant receiving a full feeding of 150 mL per
day and/or 3.2 to 4.1 grams of protein/kg/day to infants receiving
120 Cal/kg/day.
[0068] Human Milk Fortifiers
[0069] The human milk fortifiers featured herein contain
concentrated amounts of nutrients. In one embodiment, the fortifier
can contain a human protein constituent of about 35-85 mg/mL; a
human fat constituent of about 60-110 mg/mL; and a human
carbohydrate constituent of about 60-140 mg/mL. Total caloric
content of the fortifier can be, e.g., about 0.92 to about 1.89
Cal/mL. In particular, one embodiment can include the protein
constituent of about 55-65 mg/mL; the fat constituent of about
85-95 mg/mL; and the carbohydrate constituent of about 70-120
mg/mL.
[0070] The fortifier can be supplemented with minerals, if desired.
In one embodiment, the minerals can include: calcium concentration
of about 4.0-5.5 mg/mL; chloride concentration of about 0.35-0.95
mg/mL; copper concentration of about 0.0005-0.0021 mg/mL; iron
concentration of about 0.001-0.700 mg/mL; magnesium concentration
of about 0.180-0.292 mg/mL; manganese concentration of about
0.010-0.092 micrograms/mL; phosphorus concentration of about
2.00-3.05 mg/mL; potassium concentration of about 1.90-2.18 mg/mL;
sodium concentration of about 0.75-0.96 mg/mL; and zinc
concentration of about 0.0200-0.0396 mg/mL. In another embodiment,
the minerals can include calcium concentration of about 2.00-2.9
mg/mL; chloride concentration of about 0.175-0.475 mg/mL; copper
concentration of about 0.00025-0.001 mg/mL; iron concentration of
about 0.0005-0.0025 mg/mL; magnesium concentration of about
0.090-0.170 mg/mL; manganese concentration of about 0.005-0.046
micrograms/mL; phosphorus concentration of about 1.00-1.50 mg/mL;
potassium concentration of about 0.95-1.41 mg/mL; sodium
concentration of about 0.375-0.608 mg/mL; and zinc concentration of
about 0.010-0.0198 mg/mL.
[0071] An exemplary fortifier (e.g., PROLACT+4.TM. and
PROLACT+6.TM.) can include the following components: human milk,
calcium carbonate, potassium phosphate, calcium phosphate, calcium
glycerophosphate, calcium gluconate, sodium citrate, magnesium
chloride, calcium chloride, magnesium phosphate, zinc sulphate,
cupric sulfate, and manganese sulfate. An exemplary fortifier can
have the following characteristics per 100 mL: about 135-155 Cal;
about 8.5-9.5 g of total fat; about 75-96 mg of sodium; about
190-218 mg of potassium; about 7.0-12.0 g of total carbohydrates;
about 5.5-10.0 g of sugars; about 5.5-6.5 g of protein; about
1000-5000 IU of Vitamin A; less than about 1 mg of Vitamin C; about
400-550 mg of calcium; about 0.1-0.5 mg of iron; about 200-305 mg
of phosphorus; about 18-29.2 mg of magnesium; about 35-95 mg of
chloride; about 2.0-4.0 mg of zinc; about 0.05-0.21 mcg of copper;
and less than about 9.2 mcg of manganese; with the osmolality of
.about.343 mOsm/Kg H.sub.2O (when mixed with raw human milk at the
ratio of about 80% raw milk to 20% fortifier).
[0072] Fortifiers with other constituents and constituents of
different concentrations are encompassed by this disclosure.
[0073] The fortifiers featured herein can be mixed with mother's
milk at various concentrations, depending on the nutritional
content of the mother's milk and the needs of the infant. For
example, a mother's raw milk can be tested to determine the raw
milk's nutritional value. Typical raw milk comprises, on average,
1.1 g protein, 3.2 g fat, 7.7 g carbohydrates (primarily lactose),
and supplies about 64 kcal of energy per 100 mL. After testing, the
mother's milk can be adjusted by adding a fortifier composition
discussed herein. For example, the fortifiers can add from about 2
to about 10 Cal/oz and/or from 3.3 to 5.5 g/protein/kg/day when
considering a full feeding of 150 mL/day and/or 3.2 to 4.1 grams of
protein/kg/day to infants receiving 120 Cal/kg/day. If a lower dose
of the fortifier and the nutrients is needed, the mixture can
include about 20% of the fortifier and about 80% of raw human milk
(adding about 4 Cal/oz) (e.g., the mixture with PROLACT+4.TM.). If
a higher dose of the nutrients is needed, the mixture can include
about 50% of raw human milk and about 50% of the fortifier (e.g.,
the mixture with PROLACT+10.TM.. Other ratios of the mixtures are
encompassed by this disclosure, including (but not limited to):
about 30% fortifier with about 70% raw human milk (e.g., the
mixture with PROLACT+6.TM.) and about 40% of fortifier with about
60% of raw human milk (e.g., the mixture with PROLACT+8.TM.).
Example 2 below and Table 2 show some embodiments of the mixtures
and their nutritional information.
[0074] Standardized Human Milk Formulations
[0075] The standardized human milk formulations featured herein can
be used in lieu of mother's own milk to feed the infants, e.g.,
premature infants. They include various nutritional components for
infant growth and development.
[0076] In one embodiment, the standardized human milk formulation
can include: a human protein constituent of about 11-20 mg/mL; a
human fat constituent of about 35-55 mg/mL; and a human
carbohydrate constituent of about 70-120 mg/mL. In a particular
embodiment, the formulation can contain: a human protein
constituent of about 11-13 mg/mL; a human fat constituent of about
35-55 mg/mL; and a human carbohydrate constituent of about 80-105
mg/mL. The total caloric content of the formulations can be, e.g.,
from about 0.68 Cal/mL to about 0.96 Cal/mL.
[0077] The milk formulation can be supplemented with vitamins
and/or minerals. In one embodiment, the composition can include:
calcium concentration of about 0.40-1.50 mg/mL; chloride
concentration of about 0.30-0.80 mg/mL; copper concentration of
about 0.0005-0.0021 mg/mL; iron concentration of about 0.001-0.005
mg/mL; magnesium concentration of about 0.03-0.13 mg/mL; manganese
concentration of about 0.01-0.092 mg/mL; phosphorus concentration
of about 0.15-0.631 mg/mL (e.g., about 0.15-0.60 mg/mL); potassium
concentration of about 0.60-1.20 mg/mL; sodium concentration of
about 0.20-0.60 mg/mL; and zinc concentration of about
0.0025-0.0120 mg/mL.
[0078] The human milk formulations can contain various caloric
content, e.g., 67 Kcal/dL (20 Calorie per ounce), and 81 Kcal/dL
(24 Calorie per ounce). An exemplary human milk formulation (e.g.,
PROLACT24N can include the following constituents: human milk,
calcium glycerophosphate, potassium citrate, calcium gluconate,
calcium carbonate, magnesium phosphate, sodium chloride, sodium
citrate, zinc sulfate, cupric sulfate, and manganese sulfate. This
exemplary composition can have the following characteristics per
100 ml: about 81 Cal; about 4.4 g of total fat; about 20.3 mg of
sodium; about 60.3 mg of potassium; about 8 g total carbohydrates;
about 5-9 g of sugars; about 2.3 g of protein; about 180-250 IU of
Vitamin A; less than about 1.0 mg of Vitamin C; about 40.0-150.0 mg
of calcium; about 100-150 mcg of iron; about 15-50 mg of
phosphorus; about 3-10 mg of magnesium; about 25-75.0 mg of
chloride; about 1.2 mcg of zinc; about 140-190 mcg of copper; less
than about 60.2 mcg of manganese; and Osmolarity of about 322
mOsm/Kg H.sub.2O. Milk formulations with other constituents and
constituents of different concentrations are encompassed by this
disclosure.
[0079] Specific Components of the Featured Compositions
[0080] One component of the milk compositions and fortifiers
featured herein is protein. In the body, protein is needed for
growth, synthesis of enzymes and hormones, and replacement of
protein lost from the skin, urine and feces. These metabolic
processes determine the need for both the total amount of protein
in a feeding and the relative amounts of specific amino acids. The
adequacy of the amount and type of protein in a feeding for infants
is determined by measuring growth, nitrogen absorption and
retention, plasma amino acids, certain blood analytes and metabolic
responses. Some proteins present in the featured compositions
beneficial for other than purely nutritional reasons include human
IgA, lysozyme, and lactoferrin.
[0081] Another constituent of the milk compositions described
herein is fat. Fat is generally a source of energy for LBW infants,
not only because of its high caloric density but also because of
its low osmotic activity in solution. Thus, optionally, the milk
compositions of the invention can be supplemented with fat
constituents. Such heterologous fat constituents include specific
fatty acids such as docosahexaenoic acid (DHA) and arachidonic
acid.
[0082] Vitamins and minerals are important to proper nutrition and
development of an infant. A premature infant or LBW infant requires
electrolytes, e.g., sodium, potassium and chloride for growth and
for acid-base balance. Sufficient intakes of these electrolytes are
also needed for replacement of losses in the urine and stool and
from the skin. Calcium, phosphorus and magnesium are needed for
proper bone mineralization and growth.
[0083] Trace minerals are associated with cell division, immune
function and growth. Consequently, sufficient amounts of trace
minerals are needed for infant growth and development. Some trace
minerals that are important include, e.g., copper, magnesium and
iron (which is important, e.g., for the synthesis of hemoglobin,
myoglobin and iron-containing enzymes). Zinc is needed, e.g., for
growth, for the activity of numerous enzymes, and for DNA, RNA and
protein synthesis. Copper is necessary for, e.g., the activity of
several important enzymes. Manganese is needed, e.g., for the
development of bone and cartilage and is important in the synthesis
of polysaccharides and glyoproteins. Accordingly, the human milk
formulations and fortifier compositions of the invention can be
supplemented with vitamins and minerals as described herein.
[0084] Vitamin A is a fat-soluble vitamin essential for, e.g.,
growth, cell differentiation, vision and proper functioning of the
immune system. Vitamin D is important, e.g., for absorption of
calcium and to a lesser extent, phosphorus, and for the development
of bone. Vitamin E (tocopherol) prevents peroxidation of
polyunsaturated fatty acids in the cell, thus preventing tissue
damage. Folic acid plays a role in, e.g., amino acid and nucleotide
metabolism. Serum folate concentrations have been shown to fall
below normal after 2 weeks of age in LBW infants with low folic
acid intakes. Additionally, several B vitamins are present at low
concentrations in preterm milk.
[0085] As described above, the variability of human milk vitamin
and mineral concentrations and the increased needs of the preterm
infant often require some fortification to insure that a developing
infant is receiving adequate amounts of vitamins and minerals.
Examples of vitamins and minerals that can be added to the human
milk compositions featured herein include: vitamin A, vitamin B1,
vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin
E, vitamin K, biotin, folic acid, pantothenic acid, niacin,
m-inositol, calcium, phosphorus, magnesium, zinc, manganese,
copper, sodium, potassium, chloride, iron and selenium. The
compositions can also be supplemented with: chromium, molybdenum,
iodine, taurine, carnitine and choline may also require
supplementation.
[0086] The osmolality of human milk fortifiers and standardized
milk formulations featured herein can affect adsorption,
absorption, and digestion of the compositions. High osmolality,
e.g., above about 400 mOsm/Kg H.sub.2O, has been associated with
increased rates of necrotizing enterocolitis (NEC), a
gastrointestinal disease that affects neonates (see, e.g.,
Srinivasan et al., Arch. Dis. Child Fetal Neonatal Ed. 89:514-17,
2004). The osmolality of the human milk composition and fortifier
(once mixed with raw milk) of the disclosure is typically less than
about 400 mOsm/Kg H.sub.2O. Typically the osmolality is from about
310 mOsm/Kg of water to about 380 mOsm/Kg of water. The osmolality
can be adjusted by methods known in the art.
Kits
[0087] The present disclosure also features kits that include the
human milk fortifiers described herein and containers for mixing
the fortifiers with raw human milk. The containers can include
bottles, e.g., graduated bottles to assist in proper dilution,
syringes, cans, and other containers known in the art.
Processing of the Featured Compositions in NICU
[0088] Preparation of the fortifiers and standardized milk products
in, e.g., NICUs, is adjusted depending on the needs of the patients
and the policies of various hospitals. Thus, the amount of milk
prepared, e.g., with the fortifiers, will be determined on
site.
[0089] The embodiments of the disclosure may, of course, be carried
out in other ways than those set forth herein without departing
from the spirit and scope of the disclosure. The embodiments are,
therefore, to be considered in all respects as illustrative and not
restrictive.
Example 1
Production and Compositions of PROLACTA.TM. Bioscience Human Milk
Fortifier (HMF)
[0090] The PROLACTA.TM. Bioscience Human Milk Fortifier (HMF)
(PROLACTPLUS.TM.) was produced using the following steps.
Processing was done in a class 100,000 clean room (ISO class 8),
and filling was done in a class 10,000 clean room (ISO class
7):
[0091] 1. Donor milk was pooled.
[0092] 2. A sample was taken for Polymerase Chain Reaction (PCR)
for the following viruses:
[0093] a. HIV-1
[0094] b. HBV--Hepatitis B
[0095] c. HCV--Hepatitis C
[0096] 3. Donor milk was filtered through a 200 micron screen.
[0097] 4. Donor milk was heat-treated at 63.degree. C. or greater
for 30 minutes.
[0098] 5. Milk was separated into in skim milk and cream, which had
the following concentrations:
[0099] a. Skim: 0.3%-0.69% Fat, 0.9%-1.2% Protein, 6%-10%
Lactose
[0100] b. Cream: 25%-46% Fat, 0.5%-2% Protein, 8-10% Lactose
[0101] 6. The Cream can be, if desired, taken and run through the
separator:
[0102] a. This step produces more skim milk, which increases the
yield because the more skim is produced, the more HMF can be
produced.
[0103] b. The product that comes out the non-skim side of the
separator is currently considered waste.
[0104] 7. Cream was added to skim for better ultra filtration
during concentration step. The fat in the skim was from 55% to 65%
of the protein content.
[0105] 8. Skim milk underwent ultra filtration. This step filtered
water out of the skim milk. The water was referred to as permeate.
The final concentration during this step was 7%-10% of protein.
[0106] 9. Post wash of the filters using permeate was carried out
to recover any protein that was trapped on the filters during the
ultra filtration process. The post wash was added to the
concentrated skim milk until protein concentration reached
7.0%-7.2%.
[0107] 10. During the Bulk formulation, cream was added to the
concentrated skim milk, after post wash was added. This step
increased calories to the correct target. At this point, the
product was at: [0108] a. Fat--8.5%-9.5%; [0109] b.
Protein--6.3%-7.0%; and [0110] c. Lactose--8%-10.5%.
[0111] 11. A sample of the Bulk was sent for mineral analysis. This
analysis of the initial mineral content allowed for later
determination of the amount of minerals that needed to be added to
the product (see, e.g., step 14).
[0112] 12. The Bulk was frozen at -20.degree. C. or colder.
[0113] 13. When the mineral results were returned from the lab, the
Bulk was thawed.
[0114] 14. Based on the mineral concentration in the starting
material (determined in step 11 above), it was calculated how much
more of the minerals needed to be added.
[0115] The final targets are set out below. PROLACT+4.TM.,
PROLACT+6.TM., PROLACT+8.TM., and PROLACT+10.TM. fortifiers are
used in mixtures with human milk and are further discussed in
Example 2 below.
[0116] a. PROLACT+4.TM. and PROLACT+6.TM. [0117] i. Calcium--400.0
mg/dL to 550.0 mg/dL [0118] ii. Chloride--35 mg/dL to 95.0 mg/dL
[0119] iii. Copper--0.05 mg/dL to 0.21 mg/dL [0120] iv. Iron--0.1
mg/dL to 0.7 mg/dL [0121] v. Magnesium--18.0 mg/dL to 29.2 mg/dL
[0122] vi. Manganese--1.0 micrograms/dL to 9.2 micrograms/dL [0123]
vii. Phosphorus--200.0 mg/dL to 305.0 mg/dL [0124] viii.
Potassium--190.0 mg/dL to 218.0 mg/dL [0125] ix. Sodium--75 mg/dL
to 96 mg/dL [0126] x. Zinc--2.0 mg/dL to 3.96 mg/dL
[0127] b. PROLACT+8.TM. and PROLACT+10.TM. [0128] i. Calcium--200.0
mg/dL to 290 mg/dL [0129] ii. Chloride--17.5 mg/dL to 47.5 mg/dL
[0130] iii. Copper--0.025 mg/dL to 0.1 mg/dL [0131] iv. Iron--0.05
mg/dL to 0.25 mg/dL [0132] v. Magnesium--9.0 mg/dL to 14.6 mg/dL
[0133] vi. Manganese--0.5 micrograms/dL to 4.6 micrograms/dL [0134]
vii. Phosphorus--100.0 mg/dL to 150.0 mg/dL [0135] viii.
Potassium--95.0 mg/dL to 141.0 mg/dL [0136] ix. Sodium--37.5 mg/dL
to 60.8 mg/dL [0137] x. Zinc--1.0 mg/dL to 1.98 mg/dL
[0138] c. PROLACT+6.TM. will also be made with the following
formulation: [0139] i. Calcium--250.0 mg/dL to 425.0 mg/dL [0140]
ii. Chloride--15 mg/dL to 75.0 mg/dL [0141] iii. Copper--0.05 mg/dL
to 0.21 mg/dL [0142] iv. Iron--0.1 mg/dL to 0.5 mg/dL [0143] v.
Magnesium--10.0 mg/dL to 25.0 mg/dL [0144] vi. Manganese--1.0
micrograms/dL to 9.2 micrograms/dL [0145] vii. Phosphorus--125.0
mg/dL to 225.0 mg/dL [0146] viii. Potassium--60.0 mg/dL to 105.0
mg/dL [0147] ix. Sodium--50.0 mg/dL to 90 mg/dL [0148] x. Zinc--2.0
mg/dL to 3.96 mg/dL
[0149] 15. The Final Bulk was heated to 50.degree. C.-55.degree. C.
and the minerals were mixed into the Final Bulk.
[0150] 16. After the minerals have been mixed, the product was
pasteurized in a tank. The tank was jacketed and hot glycol was
used to heat up the tank. The following parameters were
followed:
[0151] a. The product temperature was 66.degree. C. or greater;
[0152] b. The air temperature above the product was 69.degree. C.
or greater; and
[0153] c. The product was pasteurized for a minimum of 30
minutes.
[0154] 17. The product temperature was brought down to 2.degree.
C.-8.degree. C. using cold glycol in the jacketed tank.
[0155] 18. The product was filled into bottles using a
Watson-Marlow filling machine in the class 10,000 clean room. The
fill sizes were as follows:
[0156] a. PROLACT+4.TM. 10 mL
[0157] b. PROLACT+4.TM. 20 mL
[0158] c. PROLACT+4.TM. 100 mL
[0159] d. PROLACT+6.TM. 15 mL
[0160] e. PROLACT+6.TM. 30 mL
[0161] f. PROLACT+8.TM. 20 mL
[0162] g. PROLACT+8.TM. 40 mL
[0163] h. PROLACT+10.TM. 25 mL
[0164] i. PROLACT+10.TM. 50 mL.
[0165] 19. When the fill was finished, the bottles were used as
follows:
[0166] a. BioBurden samples--2 bottles from the beginning, middle
and end of the fill run, for a total of 6 bottles were taken for
tests;
[0167] b. Nutritional samples--1 bottle from the beginning, middle,
and end of the fill run, for a total of 3 bottles were taken for
tests; and
[0168] c. Retention samples--120 mL of product was retained.
[0169] 20. The analysis was done on the BioBurden samples for the
following:
[0170] a. Total Aerobic Count--<100 Colony Forming Unit/mL;
[0171] b. Bacillus cereus--<10 Colony Forming Unit/mL;
[0172] c. E. coli--<1 Colony Forming Unit/mL;
[0173] d. Coliform--<1 Colony Forming Unit/mL;
[0174] e. Pseudomonas--<1 Colony Forming Unit/mL;
[0175] f. Salmonella--<1 Colony Forming Unit/mL;
[0176] g. Staphylococcus--<1 Colony Forming Unit/mL;
[0177] h. Yeast--<100 Colony Forming Unit/mL; and
[0178] i. Mold--<100 Colony Forming Unit/mL.
[0179] 21. The following nutritional analysis was conducted.
[0180] a. PROLACT+4.TM. or PROLACT+6.TM. [0181] i. Total
Calories--1.35 Cal/mL to 1.55 Cal/mL [0182] ii. Protein--5.5 g/dL
to 6.5 g/dL [0183] iii. Fat--8.5 g/dL to 9.5 g/dL [0184] iv.
Lactose--7.0 g/dL to 12.0 g/dL [0185] v. Calcium--400.0 mg/dL to
550.0 mg/dL [0186] vi. Chloride--35 mg/dL to 95.0 mg/dL [0187] vii.
Copper--0.05 mg/dL to 0.21 mg/dL [0188] viii. Iron--0.1 mg/dL to
0.7 mg/dL [0189] ix. Magnesium--18.0 mg/dL to 29.2 mg/dL [0190] x.
Manganese--1.0 micrograms/dL to 9.2 micrograms/dL [0191] xi.
Phosphorus--200.0 mg/dL to 305.0 mg/dL [0192] xii. Potassium--190.0
mg/dL to 218.0 mg/dL [0193] xiii. Sodium--75 mg/dL to 96 mg/dL
[0194] xiv. Zinc--2.0 mg/dL to 3.96 mg/dL
[0195] b. PROLACT+8.TM. or PROLACT+10.TM. [0196] i. Total
Calories--1.35 Cal/mL to 1.55 Cal/mL [0197] ii. Protein--5.5 g/dL
to 6.5 g/dL [0198] iii. Fat--8.5 g/dL to 9.5 g/dL [0199] iv.
Lactose--7.0 g/dL to 12.0 g/dL [0200] v. Calcium--200.0 mg/dL to
275 mg/dL [0201] vi. Chloride--17.5 mg/dL to 47.5 mg/dL [0202] vii.
Copper--0.025 mg/dL to 0.1 mg/dL [0203] viii. Iron--0.05 mg/dL to
0.25 mg/dL [0204] ix. Magnesium--9.0 mg/dL to 14.6 mg/dL [0205] x.
Manganese--0.5 micrograms/dL to 4.6 micrograms/dL [0206] xi.
Phosphorus--100.0 mg/dL to 150.0 mg/dL [0207] xii. Potassium--95.0
mg/dL to 141.0 mg/dL [0208] xiii. Sodium--37.5 mg/dL to 60.8 mg/dL
[0209] xiv. Zinc--1.0 mg/dL to 1.98 mg/dL
[0210] c. PROLACT+6.TM. will also be made with the following
formulation: [0211] i. Total Calories--1.35 Cal/mL to 1.55 Cal/mL
[0212] ii. Protein--5.5 g/dL to 6.5 g/dL [0213] iii. Fat--8.5 g/dL
to 9.5 g/dL [0214] iv. Lactose--7.0 g/dL to 12.0 g/dL [0215] v.
Calcium--250.0 mg/dL to 425.0 mg/dL [0216] vi. Chloride--35 mg/dL
to 75.0 mg/dL [0217] vii. Copper--0.05 mg/dL to 0.21 mg/dL [0218]
viii. Iron--0.1 mg/dL to 0.5 mg/dL [0219] ix. Magnesium--10.0 mg/dL
to 25.0 mg/dL [0220] x. Manganese--1.0 micrograms/dL to 9.2
micrograms/dL [0221] xi. Phosphorus--125.0 mg/dL to 225.0 mg/dL
[0222] xii. Potassium--190.0 mg/dL to 218.0 mg/dL [0223] xiii.
Sodium--50 mg/dL to 90 mg/dL [0224] xiv. Zinc--2.0 mg/dL to 3.96
mg/dL
[0225] 22. Once the nutritional analysis was complete, a label was
generated with the actual values from the lab. Generally, a normal
procedure for foods is to put an average on the label. These
Prolact products, however, show what was specifically in each
bottle.
[0226] 23. The product was shipped to customers in insulated
coolers on dry ice. Cool blocks can also be used in lieu of dry ice
for shipping.
Example 2
Production and Compositions of Various Types of PROLACTA.TM. Human
Milk Products Based on Human Milk Fortifier
[0227] The human milk fortifiers (HMFs) produced, e.g., as
described in Example 1 supra, were mixed at various concentrations
with raw human milk (e.g., the milk from the mother of the
premature infant). Depending on the nutritional content of mother's
own milk and the needs of the infant, HMF can be mixed at various
ratios. PROLACT+4.TM. is a composition meant to be mixed at a
ration of about 80% of raw milk with 20% of HMF. PROLACT+6.TM. is a
composition meant to be mixed at a ratio of about 70% raw milk and
30% HMF. PROLACT+8.TM. is a composition meant to be mixed at a
ratio of about 60% raw milk and 40% HMF. PROLACT+10.TM. is meant to
be mixed at a ratio of about 50% of raw milk and 50% of HMF. The
mixing was done by the customers, not at the PROLACTA.TM.
facilities. The table below shows an exemplary nutritional
comparison of nutrients in raw milk, and the four PROLACTPLUS.TM.
compositions mixed at various ratios with raw milk.
TABLE-US-00001 TABLE 1 Nutritional Comparison of Raw Human Milk and
Four PROLACTPLUS Human Milk Fortifier Compositions Per 100 mL* Pre-
term Nutrient Unit Milk+ PROLACT + 4 .TM. PROLACT + 6 .TM. PROLACT
+ 8 .TM. PROLACT + 10 .TM. mixing ratios: 100% 80:20 70:30 60:40
50:50 mother to fortifier Volume mL 100 100 100 100 100 Energy cal
68 84 91 99 107 protein g 1.3 2.2 2.7 3.2 3.7 (human) carbohydrates
g 7.0 7.6 7.9 8.2 8.5 fat (human)++ g 3.9 4.9 5.4 5.9 6.5
Minerals** Sodium mg 46 54 58 54 56 Potassium mg 66.3 71.1 73.5
71.1 72.3 Calcium mg 28 128 178 128 153 Phosphorus mg 14 63.1 87.7
63.1 75.4 Magnesium mg 3.3 8 10.4 8 9.2 Chloride mg 78.1 83.4 86.1
83.4 84.7 Manganese mcg 0.4 2.36 3.2 2.36 2.79 Copper mg 0.08 0.1
0.1 0.1 0.1 Zinc mg 0.5 0.7 1.1 0.7 0.9 Iron mg 0.1 0.2 0.2 0.2 0.2
Osmolality mOsm/ .apprxeq.290 <335 <360 <325 <350
kgH.sub.2O +calculated based on various published studies on file
at Prolacta Bioscience ++data for pre-term milk is not available;
fat is calculated based upon total calories *nutritional values are
based upon minimum values from Product Manufacturing Specification
for PROLACTPLUS .TM. line of fortifiers **based upon average of
lots produced
Example 3
Production and Compositions of Standardized Human Milk
Formulations
[0228] The PROLACTA.TM. Bioscience NEO20.TM. is a standardized
human milk formulation that was produced with the following steps.
Processing was done in a class 100,000 clean room (ISO class 8),
and filling was done in a class 10,000 clean room (ISO class
7):
[0229] 1. Donor milk was pooled.
[0230] 2. A sample was taken for Polymerase Chain Reaction (PCR) to
test for the following viruses:
[0231] a. HIV-1
[0232] b. HBV--Hepatitis B
[0233] c. HCV--Hepatitis C
[0234] 3. Donor milk was filtered through 200 micron screen.
[0235] 4. Whole milk underwent ultra filtration. This step filtered
water out of the whole milk. The water was referred to as permeate.
The final concentration of protein during this step was
1.2%-1.3%.
[0236] 5. The filters were post-washed using permeate to recover
any protein that was trapped on the filters during the ultra
filtration process. The post wash was added to the concentrated
whole milk until the protein concentration was 1.2%-1.5%.
[0237] 6. During the Bulk formulation, cream from previous
fortifier lots was added to the concentrated whole milk, after
adding post wash to increase calories to the correct target. At
this point, the product was at:
[0238] a. Fat--3.5%-5.5%;
[0239] b. Protein--1.1%-1.3%; and
[0240] c. Lactose--8%-10.5%.
[0241] 7. Next, the product was pasteurized in a tank. The tank was
jacketed and hot glycol was used to heat up the tank. The following
parameters were followed:
[0242] a. The product temperature was 63.degree. C. or greater;
[0243] b. The air temperature above the product was 66.degree. C.
or greater; and
[0244] c. The product was pasteurized for a minimum of 30
minutes.
[0245] 8. The product temperature was brought down to 2.degree.
C.-8.degree. C. using cold glycol in the jacketed tank.
[0246] 9. The product was filled into bottles using a Watson-Marlow
filling machine in the class 10,000 clean room. The fill sizes were
as follows:
[0247] a. NEO20.TM. 30 mL;
[0248] b. NEO20.TM. 40 mL;
[0249] c. NEO20.TM. 50 mL;
[0250] d. NEO20.TM. 148 mL; and
[0251] e. NEO20.TM. 200 mL.
[0252] 10. When the fill was finished, the bottles were taken for
the following tests:
[0253] a. BioBurden samples--2 bottles from the beginning, middle
and end of the fill run were taken, for a total of 6 bottles;
[0254] b. Nutritional samples--1 bottle from the beginning, middle,
and end of the fill run was taken, for a total of 3 bottles;
and
[0255] c. Retention samples--120 mL of product was retained.
[0256] 11. The following parameters were tested in the BioBurden
samples:
[0257] a. Total Aerobic Count--<100 Colony Forming Unit/mL;
[0258] b. Bacillus cereus--<10 Colony Forming Unit/mL;
[0259] c. E. coli--<1 Colony Forming Unit/mL;
[0260] d. Coliform--<1 Colony Forming Unit/mL;
[0261] e. Pseudomonas--<1 Colony Forming Unit/mL;
[0262] f. Salmonella--<1 Colony Forming Unit/mL;
[0263] g. Staphylococcus--<1 Colony Forming Unit/mL;
[0264] h. Yeast--<100 Colony Forming Unit/mL; and
[0265] i. Mold--<100 Colony Forming Unit/mL.
[0266] 12. Nutritional analysis was conducted for all fill sizes of
NEO20.TM.. The concentration values were in the following
ranges:
[0267] i. Total Calories--0.69 Cal/mL to 0.74 Cal/mL;
[0268] ii. Protein--1.1 g/dL to 1.3 g/dL;
[0269] iii. Fat--3.5 g/dL to 5.5 g/dL; and
[0270] iv. Lactose--8.0 g/dL to 10.5 g/dL.
[0271] 13. Once the nutritional analysis was complete, a label was
generated with the actual values from the lab. Generally, a normal
procedure for foods is to put an average on the label. These
PROLACTA.TM. products, however, show what was specifically in each
bottle.
[0272] 14. The product was shipped to customers in insulated
coolers on dry ice. Cool blocks can be used for shipping in lieu of
dry ice.
Example 4
Production and Compositions of Standardized Human Milk Fortified
with Minerals
[0273] The PROLACTA.TM. Bioscience PROLACT20.TM. is a standardized
human milk product fortified with minerals. It was produced using
the following steps in a class 100,000 clean room (ISO 8). Filling
was done in a 10,000 clean room (ISO 7).
[0274] 1. Donor milk was pooled.
[0275] 2. A sample was taken for Polymerase Chain Reaction (PCR)
tests for the following viruses:
[0276] a. HIV-1
[0277] b. HBV--Hepatitis B
[0278] c. HCV--Hepatitis C
[0279] 3. Donor milk was filtered through 200 micron screen.
[0280] 4. Whole milk was ultra filtered. In this step water was
filtered out of the whole milk. The water was referred to as
permeate. The final concentration of protein during this step was
1.2%-1.3%.
[0281] 5. The filters were post washed using permeate to recover
any protein that was trapped on the filters during the ultra
filtration process. The post wash was added to the concentrated
whole milk until the protein was in the range of 1.2%-1.5%.
[0282] 6. During the Bulk formulation, cream from previous
fortifier lots was added to the concentrated whole milk after
adding post wash, to increase calories to the correct target. At
this point, the product was:
[0283] a. Fat--3.5%-5.5%;
[0284] b. Protein-1.1-1.3%; and
[0285] c. Lactose--8%-10.5%.
[0286] 7. At this stage, the product can be frozen and later thawed
for further processing.
[0287] 8. Based on the analysis of the starting minerals, it was
calculated how much more of minerals needed to be added. The final
targets were:
[0288] a. Calcium--40 mg/dL to 150 mg/dL;
[0289] b. Chloride--30 mg/dL to 80 mg/dL;
[0290] c. Copper--0.05 mg/dL to 0.21 mg/dL;
[0291] d. Iron--0.1 mg/dL to 0.5 mg/dL;
[0292] e. Magnesium--3.0 mg/dL to 13 mg/dL;
[0293] f. Manganese--1.0 micrograms/dL to 9.2 micrograms/dL;
[0294] g. Phosphorus--15 mg/dL to 60 mg/dL;
[0295] h. Potassium--60 mg/dL to 120 mg/dL;
[0296] i. Sodium--20 mg/dL to 60 mg/dL; and
[0297] j. Zinc--0.25 mg/dL to 1.2 mg/dL.
[0298] 9. After mineral addition (and thawing, if the product was
frozen), the product was pasteurized in a tank. The tank was
jacketed and hot glycol was used to heat up the tank. The following
parameters were followed:
[0299] a. The product temperature was 63.degree. C. or greater;
[0300] b. The air temperature above the product was 66.degree. C.
or greater; and
[0301] c. The product was pasteurized for a minimum of 30
minutes.
[0302] 10. The product temperature was brought down to 2.degree.
C.-8.degree. C. using cold glycol in the jacketed tank.
[0303] 11. The product was filled into bottles using a
Watson-Marlow filling machine in the class 10,000 clean room. The
fill sizes were as follows:
[0304] a. PROLACT20.TM. 30 mL
[0305] b. PROLACT20.TM. 40 mL
[0306] c. PROLACT20.TM. 50 mL
[0307] 12. When the fill was done, the bottles were taken for the
following:
[0308] a. BioBurden samples--2 bottles from the beginning, middle
and end of the fill run were taken, for a total of 6 bottles;
[0309] b. Nutritional samples--1 bottle from the beginning, middle,
and end of the fill run was taken, for a total of 3 bottles;
and
[0310] c. Retention samples--120 mL of product was retained.
[0311] 13. The following were tested in the BioBurden samples:
[0312] a. Total Aerobic Count--<100 Colony Forming Unit/mL;
[0313] b. Bacillus cereus--<10 Colony Forming Unit/mL;
[0314] c. E. coli--<1 Colony Forming Unit/mL;
[0315] d. Coliform--<1 Colony Forming Unit/mL;
[0316] e. Pseudomonas--<1 Colony Forming Unit/mL;
[0317] f. Salmonella--<1 Colony Forming Unit/mL;
[0318] g. Staphylococcus--<1 Colony Forming Unit/mL;
[0319] h. Yeast--<100 Colony Forming Unit/mL; and
[0320] i. Mold--<100 Colony Forming Unit/mL.
[0321] 14. Nutritional analysis was conducted for all fill sizes of
PROLACT20.TM. after minerals were added. The ranges included:
[0322] i. Total Calories--0.69 Cal/mL to 0.74 Cal/mL;
[0323] ii. Protein--1.1 g/dL to 1.3 g/dL;
[0324] iii. Fat--3.5 g/dL to 5.5 g/dL;
[0325] iv. Lactose--8.0 g/dL to 10.5 g/dL;
[0326] v. Calcium--40 mg/dL to 150 mg/dL;
[0327] vi. Chloride--30 mg/dL to 80 mg/dL;
[0328] vii. Copper--0.05 mg/dL to 0.21 mg/dL;
[0329] viii. Iron--0.1 mg/dL to 0.5 mg/dL;
[0330] ix. Magnesium--3.0 mg/dL to 13 mg/dL;
[0331] x. Manganese--1.0 micrograms/dL to 9.2 micrograms/dL;
[0332] xi. Phosphorus--15 mg/dL to 60 mg/dL;
[0333] xii. Potassium--60 mg/dL to 120 mg/dL;
[0334] xiii. Sodium--20 mg/dL to 60 mg/dL; and
[0335] xiv. Zinc--0.25 mg/dL to 1.2 mg/dL.
[0336] 15. Once the nutritional analysis was complete, a label was
generated with the actual values from the lab. In general food
procedures, an average is placed on the label. These PROLACTA.TM.
products, however, show what was specifically in each bottle.
[0337] 16. The product was shipped to customers in insulated
coolers on dry ice. Cool blocks can also be used for shipping in
lieu of dry ice.
Example 5
Validation of the Pasteurization Methods
[0338] The methods and compositions of the disclosure maintain
desirable activities of important proteins and vitamins in the
described compositions and eliminate bioburden (see, e.g., Terpstra
at al., Breastfeeding Med. 2(1):27-33, 2007).
[0339] A. Bioburden Validation
[0340] A validation of the effects of high-temperature short-time
(HTST) pasteurization and vat pasteurization on the bioburden of
human milk was carried out. The test organisms used in the
validation studies were the following bacteria and viruses: E.
coli, S. aureus, and S. agalactiae, human immunodeficiency virus
(HIV), hepatitis A virus (HAV), bovine viral diarrhea virus (BVDV)
and pseudorabies virus (PSR). HIV and HAV are known to be potential
contaminants of human milk and were, therefore, selected as
relevant viruses. Hepatitis C virus (HCV) is also known to be a
potential contaminant of human milk. However, this virus cannot be
cultured effectively in laboratory cell line systems so the
specific model virus BVDV is used. For the same technical reason,
hepatitis B virus (HBV), also a known potential contaminant of
human milk, is substituted by the general model pseudorabies virus
which, like HBV, is a lipid-enveloped virus. Using the methods
described herein, the following results were obtained:
TABLE-US-00002 TABLE 2 Log.sub.10 Reduction Values for Test
Organisms Log.sub.10 Pathogen Log.sub.10 Reduction (HTST) Reduction
(vat) E. coli >32 Not done S. aureus 15 Not done S. agalactiae
>26 Not done BVDVa >5.84 >6.1 HIV >7.27 >6.7 PSR
>7.68 >6.8 HAV ~2 ~1.7
[0341] These viral log reduction values do not represent the
maximum reduction that can be achieved by the methods of the
disclosure. Although S. aureus is fairly resistant to heat
treatment, this organism showed a 15 log reduction with the process
of the disclosure.
[0342] B. Validation of Milk Components
[0343] A validation of the level of various human milk components
after pasteurization was also carried out.
[0344] Immunoglobulin A (IgA) and secretory IgA (s[IgA.sub.2]) were
quantitated in human milk samples using sandwich ELISA procedures.
Following pasteurization using the HTST process featured herein,
IgA concentration declined about 27% (e.g., a range of about 7% to
about 47%), on average, and secretory IgA levels declined 17%
(e.g., a range of about 7% to about 27%), on average, compared to
the corresponding values in untreated human milk samples.
[0345] Lysozyme activity was determined by a microtiter assay using
a Micrococcus lysodeikticus suspension as a substrate. The lysozyme
activity in human milk after pasteurization was about 22,000 IU/mL,
57% (e.g., a range of about 47% to about 67% or more) of the
initial activity (39,000 IU/mL) in raw human milk.
[0346] Lactoferrin concentration was determined by an ELISA
technique. The lactoferrin content of human milk after
pasteurization using the methods of the disclosure was about 0.033
g/100 mL, about 14% (e.g., a range of about 4-24%) of the initial
concentration (0.24 g/100 mL) in raw human milk.
[0347] Vitamin analyses were performed by validated HPLC
procedures. Vitamin A, vitamin C, and .alpha.-, .gamma.-, and
.delta.-tocopherol levels remained unchanged following
pasteurization. The vitamin B6 content of human milk slightly
decreased to about 7.8 .mu.g/100 mL, about 89% of the initial
concentration of 8.8 .mu.g/100 mL. These results are presented in
Table 3.
TABLE-US-00003 TABLE 3 Effect of Pasteurization on Human Milk
Constituents Untreated Pasteurized % Constituent Milk Milk
Remaining Immunoglobulin A (mg/mL) 315 230 73 Secretory IgA (mg/mL)
462 379 82 Lysozyme (IU/mL) 39,000 22,000 57 Lactoferrin (g/100 mL)
0.24 0.033 14 Vitamin B6 (.mu.g/100 mL) 8.8 7.8 89
[0348] Other variations and embodiments of the invention described
herein will now be apparent to those of ordinary skill in the art
without departing from the scope of the invention or the spirit of
the claims below.
* * * * *