Note: Descriptions are shown in the official language in which they were submitted.
:1218~3~5
-- 1 --
Milk and milk products depleted or not depleted in
calcium and enriched in magnesium.
The developed countries of the Northern
Hemisphere, especially Europe, underwent a very sign-
ficant change in their eating habits shortly after the second world war.
After the hardships of the years 1940-1947,
there was literally an obsessive reaction in favor of
rich foods and highly refined foods (like white flour,
white sugar and white salt) at the expense of bran,
flour having a lower extraction rate, unrefined sugar,
sea salt and all foodstuffs containing useful or even
essential mineral elements such as magnesium.
A second cause of the deficiency of magnesium
intake arises from the fact that the magnesium-rich
foods are nearly always excluded from our meals because
of their high caloric content. However, magnesium is
one of the most important of the vital elements. It
activates more than 300 enzymes and is involved in
2C regulating cell permeability and neuromuscular excite-
ability Jo Olin. Comma Olin. Become., volume 18,
1980, pages 257-270).
Thus, we are witnessing the beginning of a very
uncomfortable functional pathology whose classification
25 has been specified since the years 1959-1969 and is
designated as magnesium deficiency (M.D.).
The etiology of this disease is nutritional and
affects both animals and man. Confirmation it pro-
voided by veterinary surgeons, in the U.S.A., England
and Frowns describe grass Titan. Supplementing
the ration with magnesium eliminates attacks of Titan
in cattle.
In man, magnesium therapy, which has been
practiced for 15 years with acknowledged success, also
establishes the merits of the theory of nutritional
lo 395
-- 2
magnesium deficiency. The studies of D. AMITY et at.,
Journal de Médecine de Besanc,on, Thea year, no. 5
September-October 1969, and of Y. RAYSSIGUIER et at.,
Ann. Blot. aim. Block. Buffs. 1974, 14 (1), 145-156,
may be mentioned in this respect.
In addition to the existing therapies, the
object of the invention is to propose supplementing the
food ration of the population. Magnesium-enriched food
products already exist. Such products are marketed in
particular in the form of magnesium-enriched biscuits,
pancakes or bread.
The applicant has found that it is
possible to use milk and its derivatives as a vehicle
for magnesium. In fact, few people avoid the ingestion
of milk products in a raw or converted form (milk or
yogurt); yogurts are in the main group of milk-based
foods. Moreover, these two products are capable of
being kept for 3 weeks (yogurts) to several months
(long-life milk). These two reasons alone would amply
justify this choice of ford. There is at least one
other argument in favor of this choice: the Bible-
graphical studies of the prior art report that certain
sugars, and especially lactose, favor the assimilation
of magnesium. It will be recalled that the Cam ratio
in milk is approximately equal to 10. In fact, one
liter of cow's milk contains about 1.30 g of calcium
and 0.13 g of magnesium.
The invention thus relates to a magnesium-
enriched milk, the said milk being depleted or not
depleted in calcium. The invention also relates to the
milk products obtained from the milk according to the
invention, especially yogurts.
The magnesium-enriched milk according to the
invention can be obtained both from raw whole milk and
from semi-skimmed milk and milk pasteurized, for
A
3895
-- 3 --
example, by the HUT technique, by the addition of an
appropriate quantity of magnesium.
The quantity of magnesium Jo be added must
satisfy the dietetic criteria and be such as not to
interfere with the stability of the milk. To satisfy
this latter condition, it has been found that the
quantity of magnesium added can range up to 1 g/liter
of My++, whereby the Cam ratio drops from 10 to 1 or
even 0.6.
The milk according to the invention can also
be enriched in lactose or another sugar (for example
muons) in order to permit increased assimilation of
the magnesium.
In a preferred variant of the invention, the
magnesium-enriched milk is a milk previously depleted
in calcium by removal of 25 to 50% of the initial
calcium content, and enriched with magnesium up to
an My++ content capable of reaching 1 g/liter.
The milk according to the invention is obtained
by the addition of a magnesium salt, if appropriate
after calcium removal effected, for example, by passage
over an ion exchange resin or mixing with a resin of
this type. The magnesium can be added in the form of a
water-so~ub]e or even liposoluble salt or complex.
Magnesium lactate, chloride or pyrrolidonecarboxylate
are magnesium salts which are assimilated well and may
be mentioned as examples of magnesium salts which are
suitable for the purposes of the invention.
It is known that milk can be demineralized by
treatment with certain ion exchange resins (ARM. GUT-
HERMAN, "~echnologie dyes fails industries" ("Tech-
neology of industrial milk"); Labo-Pharma, no. 261
January 1977, page 25). A milk containing less than
60 milligrams of calcium per liter is even said to exist
on the foreign market (ARM. GUYOT-HERMANN, "lo fail
~;218895
-- 4 --
maternal et lies fails modifies" ("Mother's milk and
modified milk"), Sock Forum. de Lisle, no. 2, 1970,
page 95).
To remove calcium from the milk according to the
invention, an ion exchange resin of the sulfonic acid
type, trademark AMBERLITE IRE, marketed by the Room
and baas Company, was used in its sodium form. This
operation of calcium removal can be carried out:
- either by passage over a column of resin
. at a constant rate or
. at a variable rate,
- or by mixing the resin with the milk.
The milk or the milk-based products can also
contain conventional nutrients such as mineral elements
lo and vitamins, preferably vitamin By, in proportions of
10 to 40 mg/liter.
The present invention will now be described in
greater detail by the non-limiting illustrative examples
below.0 Example 1: removal of calcium from milk by passage at
a constant rate over a column containing
resin
A column of "AMBERLITE IT 120" resin and past
terraced whole milk were used. The height of the resin
bed was 80 cm and loosening was carried out by raising
the resin 50% by means of pressurized water in counter-
current.
2 liters of milk were passed over the column
in 45 minutes, i.e. a flow rate of 2.5 liters/hour.
The diameter of the column was 3 cm and the volume of
the resin bed was 565 ml. The equivalent flow rate was
therefore 5 liters/hour/liter of resin.
The first 120 ml were discarded and samples
were taken every 3 minutes 30 seconds, the taking of
each 14 ml sample lasting 20 seconds. The calcium in
. .
~Z:18~395
-- 5 --
the samples was determined by atomic absorption spectra-
photometry 24 hours after they had been taken, the
samples being stored at ambient temperature. For the
standard, the calcium content was 10320 g/liter. The
results of the calcium determinations on the successive
samples were as follows:
TABLE I
Sample calcium content of the Percentage of calcium
Numbereluate in mg/liter retained by the resin
10 1 80 93.94
2 82 93.78
3 91 93.10
4 98 92.57
101 92.34
15 6 106 91.77
7 113 91.44
8 123 90.68
9 138 89.54
151 88.56
2011 154 88.33
12 172 86.97
13 176 86.66
14 194 85.30
The percentage of calcium retained decreases
very distinctly but non-uniformly. None of the
samples stored at ambient temperature shows any phase
separation.
Example 2
The experiment o f Example 1 was repeated using
the same resin and the same column, but passing a
larger quantity of milk (4 liters in 1 hour 50 minutes),
i.e. a flow rate of 2.20 liters/hour. The volume of
the resin was the same as before, i.e. 565 ml, the
equivalent flow rate therefore being 4.40 liters/hour/
1.;2~389~
-- 6 --
liter of resin.
The first 100 milliliters were discarded and
samples were then taken every 500 milliliters. The
calcium in these samples was determined under the same
conditions as in the previous example. The results are
given in the table below, the calcium content of the
milk used being 1.243 g/liter at the start.
TABLE II
Sample Calcium content of the Percentage of calcium
Number equate in miter __ retained by the resin
1 255 79.48
2 505 59.37
3 560 54.94
4 600 51.73
640 48.51
6 690 44.48
7 710 42.80
8 715 42.47
The percentage of calcium retained by the resin
again decreases with time and it has an average value
of approximately 50%, taken over all the samples.
Example 3: removal of calcium from milk by passage at
a variable flow rate over a column con
twining ryes n
The same resin and the same column as in
Examples 1 and 2 were used.
1 liter of pasteurized whole milk was passed in
1 hour. The flow rate was:
- 2 liters/hour for samples 1 to 6
- 1.33 liters/hour for samples 7 and 8.
Samples were taken every 200 ml and the
determinations carried out as previously.
The results are as follows, the calcium content
of the standard milk being 1.350 g/liter.
~Z~3895
-- 7 --
TABLE III
Sample Calcium content of the Percentage of calcium
Number equate in mg/liter retained by the resin
1 228 83.11
2 294 78.22
3 304 77.4
4 320 76.29
74.81
6 358 73.48
7 338 74.96
8 332 75.40
From these results, it is seen that the flow
rate has an important influence on the percentage of
calcium retained.
The standard and the various samples were left
at ambient temperature for 48 hours. Whereas, after
this time, the standard was curdled on the surface and
had a fairly strong odor, the samples did not show any
change to the naked eye.0 Example 4: removal of calcium from milk by mixing with
the resin
It is also possible to remove some of the
calcium from milk by mixing it with an ion exchange
resin of the same type as that used in the previous
examples.
One liter of milk was mixed with 200 ml of
resin in a beaker by means of a magnetic stirrer. The
experiment lasted I hours and the samples were taken
in a first stage every half an hour and filtered on
filter paper.
The results are as follows, the initial calcium
content of the milk being 1.350 g/liter.
~2~8895
-- 8 --
TABLE IV
Calcium in Percentage of
mg/liter calcium retained
of milk by the resin
After 1/2 hour of mixing 368 72.74
" 1 hour of mixing 328 75.70
" I hours of mixing 315 76.66
" 2 hours of mixing 295 78.14
" I hours of mixing 2~4 78.96
In this case, the "equivalent flow rate" after
30 minutes was 5 liters/hour/liter of resin. This
method of calcium removal is preferred for the purposes
of the invention because of its speed.
The same experiment as above was repeated with
much shorter mixing times (3 to 15 minutes) and the
resulting percentage of calcium in the milk was of the
order of 50%.
The same experiment as above was repeated, but
only 100 milliliters of resin (instead of 200 ml as
above) were mixed in a beaker by means of a magnetic
stirrer.
Determination of the calcium in the milk after
mixing with the resin was carried out by atomic
absorption spectrophotometry at A a 422.7 no.
The results obtained as a function of mixing
time are indicated in Table V below, the calcium Cull-
tent of the standard being 1.210 g/liter.
~Z1~ 35
_ 9 _
TABLE V
Percentage of calcium
Calcium retained by remaining
in my the resin in the milk
After 3 mix of mixing 858 29.1 70.9
"6 mix " " 737 OWE 60.9
"9 mix " " 671 44.5 55.5
"15 mix " " 649 46.4 53.6
"20 mix " " 539 55.4 44.6
"30 mix " " 506 58.2 41.8
"40 mix " " 473 60.9 39.1
"50 mix " " 462 61.8 38.2
"60 mix " " 429 64.5 35.5
The above examples show that the use of ion
exchange resins makes it possible to remove up to 90%
of the calcium from milk, especially by passing the
milk over a column filled with a resin of this type.
It will be noted that, to achieve the calcium
removal preferred according to the invention (40-50%),
it therefore suffices to choose the volume of the resin
and the flow rate of passage over the column so as to
obtain a milk from which only 40-50% of the calcium has
been removed.
Example 5: preparation of the milk according to the
invention
The second stage of the process for the pro-
parathion of the milk according to the invention consists
in adding a known quantity of magnesium to the milk.
An operation for the removal of calcium from
milk according to Examples 1 and 2 was repeated: a
quantity of 1 g/liter of My + in the form of the
chloride, i.e. 4 glitter of magnesium chloride, was
added to each sample of milk leaving the column.
5.5 liters of pasteurized milk were passed in
~21889~;
-- 10 --
1 hour over a column of diameter 4 cm, filled with the
same resin as in Example 1, the volume of the resin bed
being 1 liter. The equivalent flow rate was therefore
5.5 liters/hour/liter of resin.
The first 250 milliliters were discarded and
isle milliliter samples A to I were taken every 500
milliliters.
Each sample A to I was divided up into five
22.5 ml flasks.
- The first flask is the standard used for the
calcium determination.
- The second flask was stored at ambient temperature.
- The third flask was stored in a refrigerator.
- The fourth flask, to which 4 g/liter of McCauley were
added, was stored at ambient temperature.
- The fifth flask, to which 4 g/liter of McCauley were
added, was stored in a refrigerator.
The calcium in the various samples was
determined in the same way as in Example 1.
The results were as follows, the calcium content
of the standard being 1.24 g/liter.
TABLE VI
Sample Calcium content of the Percentage of calcium
__ equate in mg/liter retained by the resin
A 67 94.59
B 73 94.11
C 79 93.63
D 84 93.22
E 100 91.93
F 123 90.~3
G 152 87.74
H 192 84.51
I 218 82.92
Lowe
-- 11
The various samples were also observed with
the naked eye.
- After standing for 24 hours at ambient temperature:
. Those flasks of samples A to E without
McCoy showed two very distinct phases, and the same
applied to those of samples F to I.
. Those flasks of samples A to E with McCoy
did not show any phase separation and those of samples
F to I only showed a very slight separation into two
phases.
- After standing for 24 hours in a refrigerator:
. Those flasks of samples A to E without
McCoy showed two distinct phases and those of samples
F to I showed two very distinct phases.
. Those flasks of all the samples with McCoy
showed two poorly visible phases.
After standing for 72 hours, all the flasks,
whether stored at ambient temperature or in a refrigera-
ion and whether containing McCoy or not, showed a
separation into two very distinct phases.
This example shows that magnesium-supplemented
calcium-depleted milk is more stable than non-
supplemented calcium-depleted milk and can be stored
for 24 hours virtually without phase separation.
The milk according to the invention can be
used for the production of yogurts, as shown in the
example below.
Example 6: yogurts produced with magnesium-supplemented
calcium-depleted milk according to the
invention
Two liters of "long-life, VHT-sterilized" milk
were passed over resin under the same conditions as in
Example 2. This gave milk from which about 46% of the
calcium which it contained had been removed. One series
of yogurts was produced with this calcium-depleted
~2~8~39S
- 12 -
milk without the addition of McCauley and another series
of yogurts was produced after the addition of variable
quantities of McCauley to this milk. The ferment used
for these various products consisted of milk powder
containing sucrose, lactose and Strep. thermophilus
LOB. Bul~aricus (blue ferment sold by Yalacta), in a
proportion of 4 g of ferment per liter of milk.
The standard sample, without added McCoy, made
it possible to obtain stable yogurts having good
keeping properties. The same applied to samples to
which 2 g/liter and 4 g/liter of McCoy had been added.
However, for an McCauley supplement of more than 4 g/liter?
the yogurts obtained showed rapid sedimentation.
It is therefore possible to produce yogurts
with the milk according to the invention, depleted in
calcium and supplemented with a maximum of 4 g/liter
of McCoy.
Example 7: effect of oral supplementation achieved
with the milk-based products according to
the invention in the case of magnesium
deficiency
a) Test protocol
Experiments on rats made it possible to assess
the effect of the oral administration of a magnesium-
enriched milk-based preparation according to the
invention in the case of magnesium deficiency.
Male rats of the Wisteria strain were divided
randomly into 3 groups. One of these groups received
ad lobotomy a purified diet having a normal magnesium
content (1 g/kg of solids) and the other two groups
received a magnesium-deficient diet (0.050 g/kg of
solids) for 16 days.
From day 13, the diet of one of the deprived
groups was supplemented with magnesium by administering
S ml of milk-based product according to the invention
sly
- 13 -
(approximately 50% calcium-depleted milk with 4 g/
liter of added McCoy) to the rats by means of a
stomach tube, twice a day for 4 days. A blood sample
was taken from each animal of the 3 groups on day 13
of the experiment start of the supplementation period)
and on day 16 (end of the experimental period). The
animals were sacrificed about 3 hours after the last
stomach tube had been inserted, the blood being
collected by cardiac puncture under ether anesthesia.
The magnesium was determined in the plasma and the red
blood corpuscles by absorption spectrophotometry
(Perking Elmer 420).
b) Results
The results are given in the table which
follows:
Level of My in the plasma
(average for each group of animals)
Control Deprived Deprived
groups groups (1) groups (2)
Samples on day 13 19.15 + 0.27 3.56 + 0.48 4.50 + 0.22
Supplemented
deprived
. groups
Samples on day 16 17.55 + 0.29 4.31 + 0.19 15.42 + 0.42
12~8895
- 14 -
Level of My in the red blood corpuscles
(average for each group)
Control I Deprived Deprived
groups groups (1) groups (2)
Samples on day 13 59.27 + 1.38 35.82 + 1.88 35.02 + 2.27
Supplemented
deprived
groups _
Samples on day 16 61.60 0.93 38.39 - 1.83 56.76 + 1.65
The following comments can be made on the
results:
On day 13 of deprivation, the classical signs
of magnesium deficiency appeared in an identical way
in the two deprived groups: a reduction in growth,
hyperemia of the ears and a significant reduction in
the concentrations of magnesium in the serum and red
blood corpuscles, compared with the control group.
Continuation of the deprivation in the non-
supplemented group did not modify the above parameters.
The supplementation of a deprived group with
the milk-based preparation according to the invention
resulted in a highly significant increase in the My
values of the plasma and red blood corpuscles, compared
with the deprived group. However, these values, which
are very close to the normal values, are significantly
lower than those of the control groups. The hyperemia
of the ears disappeared.
In conclusion, the preparation according to
the invention makes it possible, after administration
for 4 days, to correct a severe experimental magnesium
deficiency almost completely.
~Lzla~s~
- 15 -
having different Contents
The test protocol described in Example 7 was
repeated using magnesium-supplemented milk-based
preparation according to the invention, under the
conditions below.
5 groups of rats were used sod divided up as
follows:
. the first group (group A) served as the
control group while the other 4 were deprived by means
of a suitable diet.
. the second group (group B) was the deprived
control group and was continuously fed with the same
diet up to stout day 16.
. the third group (group C) was supplemented
from day 13 with a milk-based preparation containing
400 my of My++ cation per liter.
. the fourth group (group D) was supplemented
from day 13 with a milk-based preparation containing
700 my of My++ cation per liter.
. the fifth group (group E) was supplemented
from day 13 with a milk-based preparation containing
400 my of My++ cation plus 10 my of vitamin By per
liter.
The milk-based preparations used in the above
tests were yogurts prepared from an approximately 25%
calcium-depleted milk and having the following
characteristics:
Group C: 90 ml yogurts containing 400 my of My per
liter;
Group D: 90 ml yogurts containing 700 my of My per
liter;
Group E: 90 ml yogurts containing 400 my of My++ plus
10 my of vitamin By per liter.
lZ1889S
- 16 -
Results
On day 13 of deprivation, the classical signs
of magnesium deficiency appeared in an identical way
in the two deprived groups: a slight reduction in
growth, hyperemia of the ears and a significant reduce
lion in the concentrations of magnesium in the serum
and red blood corpuscles, compared with the control
group.
Continuation of the deprivation in the non-
supplemented group did not modify the above parameters.
Supplementation with different milk-based
preparations resulted in a highly significant increase
in the My values of the plasma and red blood corpuscles,
compared with the deprived group. However, these
values are lower than those of the control groups. The
hyperemia of the ears disappeared.
The different types of supplementation had
different effects on the serum magnesium.
The serum magnesium of group D is significantly
higher than than of groups C and E.
The serum magnesium of group E is significantly
higher than that of group C.
The various milk-based preparations according
to the invention make it possible rapidly to correct
an experimental magnesium deficiency.
The concentration of magnesium in the plasma
and red blood corpuscles and the concentration of
calcium in the first and second blood samples (on day
13 and day 16) have been indicated in Tables VII, VIII
and IX below. The number of animals used per group is
indicated in brackets in each table.
~218895
-- 17 --
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- 20 -
Example 9: effect of the calcium level on the no-
plenishing of deprived animals with
magnesium using the milk-based products
according to the invention
The test protocol of Example 7 was repeated
using male Wisteria rats of initial weight 200 go divided
randomly into 5 groups (T-0-F-G-H), These rats
received ad lobotomy a purified diet having a normal
magnesium content (group T) or a deficient magnesium
content (0, F, G, H) (1 g or 0.050 g/kg of solids) for
16 days.
From day 13 of the experimental period, the
supplementation of the deprived groups with My was
carried out by the administration of 5 ml of the milk-
based products by means of a stomach tube, twice a day
for 4 days (groups F, G, Ho A blood sample was taken
on day 13, before the start of supplementation, from 8
control groups and 8 magnesium-deprived groups, and on
day 16, at the end of the supplementation period, from
all the groups.
The animals were sacrificed about 3 hours after
the insertion of the last tube, and the blood was
collected by cardiac puncture under ether anesthesia.
The concentrations of magnesium and calcium in
I the plasma and of magnesium in the red blood corpuscles
were determined by absorption spectrometer (Perking
Elmer 420). The results obtained are shown in Tables
X to XIV below. The number of rats used in each group
is indicated in brackets in the various tables.
The milk-based products used in the above tests
were yogurts having the following characteristics:
Group F: 90 ml yogurts produced from non calcium-
depleted milk containing 1000 my of My + per liter;
Group G: 90 ml yogurts produced from approximately
35 25% calcium-depleted milk containing 1000 my of My
f~Q~ k
~8895
- 21 -
per liter;
Group H: 90 ml yogurts produced from approximately
50% calcium-depleted milk containing 1000 my of My++
per liter.
As in the above tests, on day 13 of deprivation,
the classical signs of magnesium deficiency appeared in
an identical way in the two deprived groups: a slight
reduction in growth, hyperemia of the ears, a signify-
cant reduction in the concentrations of magnesium in
the serum and red blood corpuscles, compared with the
control group, and hypercalcemia.
Supplementation with the various milk-based
preparations (F, I, H) resulted in a resumption of
growth in the deprived groups and a highly significant
increase in the My values of the plasma and red blood
corpuscles.
The different types of supplementation did not
have different effects on the serum magnesium, but the
concentration of magnesium in the red blood corpuscles
of group H is significantly higher than that of group
F (6.36 + 0.07 us 6.07 0.09; t = 2.28; P C 0.05).
In conclusion, the various preparations
studied make it possible to correct an experimental
magnesium deficiency rapidly. Although it was not
possible to demonstrate a difference in the effect of
the different preparations on the plasma magnesium,
preparation H is significantly more effective than
preparation F in raising the concentration of magnesium
in the red blood corpuscles.
~2~8~395
-- 22 --
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