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Патент USA US3086873

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3,086,866
Patented Apr. 23, 1963
2
CGIAGULATION 0F MILK
3,086,866
addition to the sources of carbon and nitrogen, a small
amount of a purine such as guanine, adenine, xanthine
and the like. In general, it is found that an amount of
Thomas W. Humphreys, Plain?eld, and Dudley S. Titus,
West?eld, N.J., assignors to Merck 8: Co., Inc., Rahway,
purine of about 0.5 to 2 mg. per 100 ml. of milk or
more will accelerate the formation of lactic acid and there
NJ, a corporation of New Jersey
No Drawing. Filed Mar. 3, 1960, Ser. No. 12,519
1 Claim. (CI. 99-59)
This invention relates to an improved method for the
fore, the coagulation of the milk. Usually, it is found that
when the milk is supplemented with sources of carbon and
nitrogen and a small amount of purine an incubation
period of from about 16-24 hours at about 37° C.
coagulation of milk. More particularly, it is concerned 10 will bring about complete coagulation of the milk. These
purines are present in various yeast products, and hence
with a process for the coagulation of milk by the action
of Pediococcus cerevisiae.
when such materials are used as the source of assimilable
nitrogen, it is not necessary to add additional purines.
In carrying out the processes of the present invention
15 for the coagulation of milk, the formation of the lactic
known in the art.
'
acid can be allowed to continue until complete coagulation
It is an object of the present invention to provide an
occurs or the coagulation can be effected earlier when a
improved means for effecting the coagulation of milk.
particular desired level of titratable acidity is reached
Another object is to shorten the time required for the co
be the addition of rennet extract in accordance with
agulation of milk. A further object is to provide a method
which eliminates the time consumed in preparing large 20 procedures well known in this art.
The following examples which are presented to illus
volumes of inoculum by subculture techniques heretofore
trate the processes of the present invention were carried
used in coagulating milk. An additional object is to
out in ‘general as follows:
.
eliminate the interference by bacteriaphage frequently en
The milk samples employed in the examples were pre
countered with the presently used microorganisms used
for the coagulation of milk. These and other objects 25 pared by reconstituting commercial non-fat dry milk
solids using 10 g. of the milk solids in 90 ml. of distilled
of this invention will be readily apparent from the de
Water. The milk was dispensed in 10* ml. quantities in
tailed description of this invention hereinafter provided.
cotton-plugged pyrex test tubes, sterilized by autoclaving
In accordance ‘with the present invention, it is now
at 250° F. for 10 minutes, cooled quickly by immersion in
found that the time required for the coagulation of milk
can be shortened considerably by including in the milk 30 ‘tap water, inoculated with the Pediococcus cerevisiae cells
and incubated at 37° C. for 20-24 hours. At the end
Pediococcus cerevisiae together with suitable sources of
of the incubation period, the relative amount of lactic
carbon and nitrogen for the microorganism. Sources of
acid formed was determined by titrating the incubated
carbon that are suitable for this purpose are carbohy
tubes directly with N/ 10 sodium hydroxide to the phenol
drates which are assimilable by said microorganism.
phthalein end point. The nutrient source of carbon em
Thus, sugars such as dextrose, fructose, sucrose, mannose,
ployed in all of the examples unless otherwise indicated
galactose and maltose are satisfactory sources of carbon
was 1% by weight of dextrose. The dextrose and the.
forPediococcus cerevisiae. Pursuant to a preferred em-_
nutrient source of nitrogen, which is indicated in the ex
bodiment of the present invention, dextrose is preferably
amples as the additive, were added either from stock solu
used since this sugar is inexpensive and readily available.
The process of bringing about the coagulation of milk
by the addition of lactic acid-producing bacterium is well
If desired, the sugars can be added to the milk in the 40 tions or as solids and the pH of the milk was brought
to 6.6-6.8 by the addition of sodium hydroxide or hy
form of solutions thereof such as corn syrup and the like;
drochloric acid before adjusting the ?nal volume.
Alternatively, and in accordance with a further em
bodiment of the present invention, the milk itself can be
The non-fat dry milk solids employed in these examples
used as a source of'carbon for ‘the Pediococcus cerevisiae
were utilized as a convenience since it avoided any vari
by adding a suitable enzyme capable of converting the 45 ation which might occur in natural milk. However, it is
understood that in commercial practice of this inven
lactose of the milk to dextrose and galactose. Thus,
tion whole or skimmed milk either alone or enriched with
lactase can be added to the milk for this purpose.
non-fat dry milk solids would normally be used. The
Among the various sources of nitrogen Which can be
inoculum of Pediococcus cerevisiae was prepared by
used in carrying out the processes of the present inven
tion that might be mentioned are suitable sources of pro 50 growing this microorganism at 37° C. for 20-24 hours
in the following medium.
tein, amino acids and the like. Thus, yeast products
A two liter solution of the following composition was
such as yeast extract, yeast autolysate, solubilized yeast,
formed:
‘
food yeast and the like and amino acids such as hydro
lyzed proteins, for example, enzymatic digest of casein,
and proteins such as peptone and the like are suitable for
use in the present invention.
The amount of the source of carbon used in the process
of the present invention is not critical, but in general, it
Try-ptone _________________________ __grams_..
2'0
Yeast extract ______________________ ___do___..
Sodium citrate _____________________ .._do____ ,
10 '
10
K2HPO4.
Dextrose __________________________
(anhydrous) __________ __’____do____
__.do__..._
20
is preferred to use an amount not in excess of about 2%
Tween 80 ________________________ __,___ml.__
by weight of the milk. When dextrose is employed as 60 N-aNOz _
____
grams“
the source of carbon, it is preferred to use it in an amount
between about 0.5 and 2.0%. Usually, about 1% of
dextrose is su?icient and ‘gives good results in the im
2
-
0.2
Salt solution _________________________ __ml_.. 1,000
Distilled Water, q.s ___________________ __ml__ 2,000
proved process of the present invention. The quantity of
vThe salt solution employed was of the following com
the source of nitrogen used is likewise not critical and 65 position:
'
.
can vary depending in part upon the particular source
1.4
MnCl2.4H2O '_ _____________________ __grams__
being utilized. In general, it is preferred to use an amount
FeSo4.7H-2O _______________________ .__do___..
.4
of the source of nitrogen equivalent to about 0.5% to
MgSO4.7H2O ______________________ __do____
8.0v
about 2.0% by weight of the milk.
Distilled water _______________________ __ml__ 1,000
In accordance with a further embodiment of the present 70
invention, it is found that the coagulation of the milk
can be accelerated further by including in the milk, in
After preparing the above-described medium, the pH
iwas adjusted electrometrically to 7.12 with concentrated ,
3,080,800
3
4
hydrochloric acid and 50 g. of sodium chloride were
dissolved in the medium. The pH was then 6.8. Prior
to inoculation with the microorganism, the medium was
sterilized by autocl-aving at 120° C. ‘for 30 minutes.
E. L-histidine, DL-tryptophane, L-proline, hydroxy-L
proline.
F. L-arginine, L-lysine.
G. L-tyrosine, DL-phenyl-alanine.
. The broth culture at the time of use contained about 5 H. No amino acids added and no guanine added.
108-409 cells per ml. and 0.1 ml. of this broth culture
The incubation was 16 hours at 370 C_
was used to inoculate the 10 ml. milk samples.
‘
In these examples the nutrient source of nitrogen and
the source of the purine and the amounts thereof are
indicated as additives to the dextrose-containing milk. 10
M1. M10 NaOH/lo
mi'ofsample
Deletion
Inocu-
Example 1
lated
In this example di?erent enzymatic digests of casein
'
obtainable commercially and known under the trade
%- $360 $51132 ?giig?a?ygfggggg)65-m- 19-13
Con-
trol
Di?er
ence
g-gg
33
name N-Z-amines were tested to determine their s'uita- 15 CI smntainjng a4cids(3)__4______ ______ “Q1:
bllity as sources" of nitrogen for the Pediococcus cerevis~
g
glnggrsbggyhc aclds-
6100
4:46
1154
{Ii-g8
:
me.
F, Diamm-Q mopocarboxyllc acids (2)
s. 10
4.24
1.86
5%
7-25
4-16
3-09
3.57
2.60
0.97
.
.
.
.
.
.
These N-Z amine preparations were added in the
amount of 0.5% by‘ ‘weight and the results compared with
a test'_w1th 0.5% ‘by weight of yeast extract.
In this test
c
'
....
_____ _.
flcfslgfidzgé --n01) ___________________________ _.
the milk samples were incubated ‘for 20 hours at 37° C.
The results ‘were as follows:
-
20
- e
-
e
Mll-nll‘r/éggggg/m
Con-
Diller-
lated
n01
ence
‘
‘
"
teins and purines indicated in- the table below on the
termined.
.
~
I
25
Amounts. ,. of constituents
are expressed ‘as
. .
.
weight/100 ml. of milk.
‘
The incubation was 24 hours
at 37° C.
e. 10
3.50
2.60
4. e0
3. 50
1: 10
Ml. N/lO NaOH/IO
ago
- 5
~ 0'
2- 95
__
.
Inocu-
g
2- 60
Additive
m1‘ of Sample
‘1.90
30
-25
Inocu-
Oon-
Differ
0- 35
lated
trol
enee
10.00
3.97
6.93
3'50
1'75
Example 2
The ability of 0.5% yeast autolysate, solublllzed yeast, 35
and _f0od yeast natural, respectively to support acid pro-
A. Casarnino acids, 1.0 gm; L-cysteine nol,
égcalgzPgéterg’igte‘lpgggiég mcgsitésfgg
uracil and thymine; and_8 i3 vitamins’
was
ductlon
compared?wi-tl'i
of P. ce’revisiaa
0.5%in yeast
milk containing
extract alone
1% and
dextrose
with
manganese added (MnCl2: 0003 gm/mi. or milk). The
i<geb°§3$§?1m§gébg1é?§
ben'z'oiq
acid, and pyridoxlne hydro
glglogfeoligilgi8533356313333;gfofigg
table shows that these various yeast products were suitable 40
at the level of 11 per ml. of milk.)
sources of nitrogen for the microorganism. ’These reB- Aié?téxecfxcept purines and pyrimldmes
sults are as follows:
Incubation was 20 21 hours at 37
_
"
-
'
°
f
t
0 m0
r
ml. of sample
.
838
6‘ 89
g0
3_ 72
5
3.17
I. Yeast‘ extract, 0.5'g'ii1.
_
J. Control, no additive .................... _.
11.33
3.09
3.88
2.60
7. 45
0.49
45
-
I
Autolysate _________________________________ __
9.55
320
.
-
7j50
3j20
'
'
‘ V
merit of Pediococcus cerevisiae was determined.
Milk
635 50 containing per 100 ml; Casamino acids (Difco), 1.0
gxgtagt plus M11012‘ .............. -. -------- -
'
-
.
The effect of purines and pyrimidines on the develop
-
igugilized.
~
.
Example 5
211;?
O
Peptone, 0.5 gm ___________ __~ ______ __
y
Exct’rgc'tjjjjj _______ __
‘
5'25
8. AsinAeiEcept amino acids omitted ..... __
H. Tryptone' 05
.-
ML Nllo NaOH/m
F
>
development of Pedioc'occus cere‘vr'siae in milk was de
‘
l
.
The effect of the combinations of amino acids, pro
Additive
'
I
.
Example 4
‘
'
.
__
‘i.
gin; L-cysteine HCl, 10 mgm.; and DL-tryptophane, 1O
430
m-gm., was further supplemented as indicated in the
3.83
table with purine and/or pyrimidine. The amount of
f
each purine and each pyrimidine employed was 1 mgm./
Example 3
m 55 100 ml. of milk. The incubation was 18 hours at 37° C.
These tests were made to determine the effectiveness
Ml. N[10 NaOH/lO
of various groups of amino acids on the development of
ml. of sample
Additive
Pediococc'l'ls cereyisiaé in milk. In Test A all of the
amino acids and guanine were added, but in Tests B-G
the amino acids indicated were deleted. In the tabulated 60
results of Tests‘ B-G, the‘ nature and the‘ number of
amino acids deleted are indicated.
A. DL~valine, 160 mg.; L-leucine, 50 mg.; DL-isoleucine,
100 mg.; L-hydroxyproline, 5 mg; DL-phenylalanine,
100- mg.; L-_tyrosine, 35 Hmg.;p1.-cysti_ne, 5.0_ mg; L
arginine, HCl, 40 mg.; L-histidine, HCl, 20 mg; L
A. Cytoslne, uracil and thymine ........... ._
B. Adenine and guanine ................... ..
F. Guanine onl __
1. 64
6. 39
3.85
6.33
6. 94
6. 49
3. 85
3. 83
3.09
2. 66
4. 05
3. 65
5.32
4. 00
.._
5. 47
4. 10
1.37
Thymine only ______ ...‘ ......... _.____ ____ -.,.
5. 19
3. 72
1. 47
or purines added) _____________________ _-
3. 46'
2. 74'
0.72
J. Control milk (no amino acids, pyrimidines
I.
4. 00
4. 00
10.18
8. 70
G. Cytosine only..
DL-aspartic acid, 50 mg; glycine, 5 mg; DL-alanine,
40 mg; L-proline,"5 mg; L-cysteine, HCI 10, DL 70
V DL-serine, glycine, DL-alanine.
once
.
thymine"-.. 1
6-0 mg.; DL-serine, 20 mg; L-glutamic acid, 125 mg;
C. DL-methionin‘e, L-cystinefL-cysteine HCl.
D. L-aspartic acid, L-glut'amic acid.
Differ
trol
5. 64
10. 39
1);. Like 0 minus'c
E. Adenine only__
H. Uracil only_____
tryptophane 10, and guanine 2 mg. per 100 ml. of milk.
Con-
lated
C. Adenine, guanine, cytosine uracil and
lysine, 100mg; DL-methionine, 80mg; DL-threonine,
B. DL-valine, L-l'eucine, DL-isoleucine, DL-threonine,
Inocu-
Example 6
1.32
I
In this example, lactas'e was included in the milk
and the dextrose was not. The stimulating agent em
ployed, was yeast autolysate and the amount used was
75 0.5 wt. percent‘.
3,086,866
5
6
Various ch-anges and modi?cations of the invention
A commercial form of puri?ed lactase was used in
these tests. It was dissolved in water and sterilized by
passage through a membrane ?lter; subsequently, two
fold serial dilutions were made in milk. In the work
can be made and, to the extent that such variations in
corporate the spirit of this invention, they are intended
to be included within the scope of the appended claim.
What is claimed is:
‘In the process of coagulating milk the improvement
which comprises adding lactase in an amount su?icient to
with lactase, there were certain modi?cations of pro
cedures employed in the previous examples, as follows:
(a) modi?ed inoculum: 75 mgm. of P. cerevisiae cells
(cells lyophilized with non~fat dry milk solids—no dex
convert the lactose of the milk to dextrose and a source
trose added) were diluted to *25 ml. with distilled water and
of nitrogen in an amount between about 0.5% and 2%
used at the rate of 0.1 ml. per 5 m1. of milk sample. 1O by weight to milk and incubating said milk with Pedia
(b) Titrations were not carried out. Instead, tubes were
coccus cerevisiae until substantial amounts of lactic acid
observed for coagulation of the milk.
are produced.
The table shows that coagulation of the milk occurs
at an enzyme level as low as 0.0625 mgm./ml. (app.
006%) at 37° C. The level of enzyme required for 15
References Cited in the ?le of this patent
coagul-ation will vary with its activity. ‘In general, the
lactase used herein will yield 70-80% of the monoses
UNITED STATES PATENTS
2,681,858
of lactose with an initial substrate concentration of 10
15%, an incubation temperature of 40° C., and an incu
bation time of four hours at pH 6-7. The E/S ratio is 20
1/40.
Coagulation + or - in
Concentra
tion of
lactase
(mgmJmL)
16 hours, 37° 0., 24hours, 37° 0., 48hours, 37° 0., 25
inoculated
control
inoculated
control
inoculated
control
+
+
—
+
+
-
+
+
—
—
+
—
+
-
+
—
+
'
i
_
i
'
i
_
_
_
.+
_
Sti-mpson ____________ _... June 22, 1954
OTHER REFERENCES
Dacre: Journal of Dairy Research, October 1958, vol.
25, No. 3, pp. 409-417.
“Bergey’s Manual of Detenminative Bacteriology,”
published by Williams and Wilkins Co., Baltimore, Md.,
1957, pp. 530-531.
“The Genus Pediococcus,” by Pederson, C. S., 1949,
Bact. Rev., vol. 13, pp. 228-229.
30
1 Very soft coagulum.
NOTE.——+ equals solld coagulum. - equals no evidence of coagulation. 35
“Milk and Milk Processing,” by Herrington, B. L.,
1948, published by McGraw-Hill, pp. 21, 82, 90, 116.
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