Note: Descriptions are shown in the official language in which they were submitted.
10484Z9
This invention relates to a process for the production
of sour milk products, in particular, but not e~xclusively,
Bulgarian sour milk (yoghurt).
Sour milk products are characteristically produced by
culturing, in combination, strains of Lactobacillus bulgaricus
and Streptococcus thermophilus in milk. The two strains co-exist
symbiotically. According to the common knowledge, Lactobacillus
bulgaricus and Streptococcus thermophilus belong to the order
Eubacteriales, of the Lactobacillaceae family.
The Bulgarian authorship certificate No. 10640 has
already described a new technology for producing Bulgarian
sour milk.
We have now found a number of novel strains of _.
bulgaricus and S. thermophilus which are particularly effective
when used in combination in the production of sour milk products,
especially yoghurt of the Bulgarian type. The strain combinations
possess high activity, a good symbiotic link, a stable relation-
ship, good proteolytic activity and give a high yield of aromatic
substances.
According to the present invention, there is provided
a process for the production of sour milk products which
comprises adding to a milk sample a starter combination
comprising at least one of the strains of Streptococcus thermophilus
herein given the Nos 12, 14, 18 and 22 having the cultural, morpho-
logical and biochemical characteristics set out herein and
at least one of the strains of Lactobacillus bulgaricus
herein given the Nos 5, 26, 37 and 144 having the cultural,
morphological and biochemical characteristics set out herein,
and cultivating the starter combination in the milk sample
until a sour milk product is obtained. Depending on the
type of product required, it is possible to use, for example,
whole cow milk or skimmed cow milk or even sheep milk.
-- 1 --
1048429
The description of the present invention which follows
is divided into two parts, the first one dealing with the morpho-
logical, cultural and biochemical properties of newly discovered
Streptococcus thermophilus and Lactobacillus bulgaricus strains
used in the starters of the invention and the second with the
characteristics of the starter-combination Nos. 5-12, 5-22,
37-18, 37-12, 26-12, 144-12, 144-14 obtained using the above
described strains of the two species. Seven of the eight
strains referred to herein were deposited in the State
Institute for Control of Medicine, Blvd. Vl. Zaimov 26,
Sofia, Bulgaria on 21st December, 1973 as follows:
1. L. bulgaricus No. 5 under No. 151
2. L. bulgaricus No. 26 under No. 152
- 3. L. bulqaricus No. 37 under No. 153
4. L. bulgaricus No. 144 under No. 154
5. S. thermophilus No. 12 under No. 155
6. S. thermophilus No. 14 under No. 156
7. S. thermophilus No. 18 under No. 157
The trePtococcus thermophilus and Lactobacillus
bulqaricus strains which coexist well were isolated from
unprocessed milk and cream and from vegetation originating from
different parts of Bulgaria.
After obtaining the strains from the abovesaid sources,
they were purified three times and examined for their morphological,
cultural and biochemical characteristics, which are: Defining the
optimal temperature of growth and reduction of litmus milk.
Organoleptic evaluation. Moment of coagulation and limit
acidity. Morphological characteristics of the colonies and the
cells. Gro~th in milk with 0,1% methylene blue. Growth in
broth (hydrolyzed milk
-- 2 --
Bl
1048429
c~ MRS) at pH 9,2 and 9,6. Growth in broth (hydrolyzed milk or
MRS) with 2,4 and 6% bladder. Growth in broth (hydrolyzed milk
or MRS) with 2%, 4% and 6,5~ galt. Development in liquid media. Cataly-
tical test. Formation of diacetyl. Sugar fermentation. Proteo-
lytic activity, expressed in the number and quantity of the free
amino acids. Development in broth (MRS) and milk with phenol.
Thermoresistance. Defining the homo and heterofermentability.
Aromatic substances.
Figures 1 to 8 of the accompanying drawings are the
spectral records of aromatic substances produced in culture media
containing respectively strains 12, 14, 18 and 22 of S. thermophilus
and strains 5, 37, 144 and 26 of L. bulgaricus.
The aromatic substances produced by each strain (or
combinations of strains - see hereinafter) were determined by
the head space method of Bassette et al using a gas chromatogram
employing a column filled with the material "Porapak n~, 80-100 mesh.
For general descriptions of the head space method, see:
1. Bassette R, Ozeris S. and Whitnah C.H. - "Gas Chromatographic
Analysis of head space gas of dilute aqueous solutions", Analytical
Chemistry, 34, p. 1541, 1962, and
2. Bassette R., Bawdon R. and Clayden T.J. - "Production of
volatile materials in milk by some species of bacteria", Journal
of Dairy Science 50, p. 167, 1967. For description of experimen-
tal method used, see hereinafter. The aromatic substances have
not been identified, but their chromatographs are reproduced.
The retention time in minutes of the individual components is
recorded on the abscissa and the number of millivolts is shown
on the ordinate.
In the foregoing description, reference is made to
MRS-broth. This is a culture medium attributed to:
a) M. Rogosa, National Institute of Dental Research,
National Institutes of Health, USA Public Health Service, Bethesda,
Maryland, U.S.A.; and
3 --
~0484Z9
b) Dr. Elizabeth Sharp, National Institute for Research
in Dairying, University of Reading, England.
The ingredients of the MRS-culture medium are:
trypton - 10.0 g
yeast extract 5,0 g
KH2PO4 6.0 g
NH4-citrate 2.0 g
Na-acetate 5.0 g
salt solution 5.0 ml
10 glucose 10.0 g
sucrose 10.0 g
arabinose 5.0 g
water
The strains are as follows:
1.S. thermophilus N, N 12, 14, 18, 22
Characteristics N 12 N 14
Origin v. Grashtiza - Bul. v. Dalboki - Bul.
Morphology
a) Colony type at
surface growth small, round round
b) Microscopic picture diplococci and diplococci and
short chains short chains
Organoleptic evaluation
a) Consistency mucous mucous
b) Aroma sour milk sour milk
c) Taste sour milk ~ sour milk
Optimal growth temperature 45C 45C
Reduction red reduction
Development at 10C
-"- 23C 3 days 3 days
-"- 32C 14 hours 14 hours
-"- 37 C 6,30 hours 7 hours
-"- 50C 12 hours 12 hours
.~
10484Z9
Characteristics N 12 N 14
. .
Limit acidity 124 T 128 T
Growth in milk with 0,1%
Methylene blue - -
Moment of coagulation 6-8 hours 6-8 hours
Development in broth at
pH 9,2 and 9,6
Development in broth with
2, 4, 6% bladder
Development in broth with
2, 4, 6.5~ salt
Development in liquid media
Catalytical test
Diacetyl formation
(Voges-Proskauer)
Carbohydrates fermentation ferment glucose,saccharose and lactose
Thermoresistance
63 - 30 min + +
65 - 30 min + +
75 - 30 min
---
activity 8 h. 48 h.15 days 8 h. 48 h. 15 d.
expressed in
the number and
quantity of the (1) (2) (3) (4) (5) (6)
free amino acids
1. Cysteic
acid
2. Xl,2,3,4,6+++++ +++++ +++++ +++++ +++++ +++++
3. Aspartic
acid 0.026 0.020 0.018 0.021 0.018 0.019
4. Methionine
sulfone - - - - - -
305. Threonine0.0080.011 0.009 0.006 0.009 0.011
6. Serine 0.015 0.017 0.019 0.018 0.024 0.018
7. Glutamine
acid 0.124 0.140 0.116 0.096 0.110 0.121
-- 5 --
-` 1048429
_
activity 8 h. 48 h.15 days 8 h. 48 h. 15 d.
expressed in
the number ans
free amono acids (1) (2) (3) (4) (5) (6)
8. Proline0.050 0.045 0.064 0.056 0.051 0.061
9. Glycinetraces0.014 0.010 0.010 0.015 0.011
10. Alanine0.039 0.059 0.054 0.023 0.047 0.057
11. 1/2 Cystine - - - - - -
12. X7 + + + + + +
13. Valinetracestraces 0.005 tracestraces 0.006
14. X8 traces traces traces traces traces traces
15. Methionine " " " " "
16. Isoleucine " " " " "
17. Leucine " " " " " '~
18. X9 + + + + + +
19. Tyrosine - - traces - - -
20. Phenylalanine - - " - - traces
21. X10,11,12 +++ +++ +++ tracestraces +++
22. Lysine traces traces traces traces + +
23. Histidine " " 0.013 ~ traces 0.015
24. Ammonia + + + + + +
25. Arginine traces - traces traces
Note: The amino acids quantity is in micromoles in 1 ml
coagulated milk
X = unknown amino acids
Characteristics N 18 N 22
Origin v. Dalbo~i-Bulg. v. Preslavez-Bulg.
30 Morphology
a) Type of colony at
surface growth small, round small, round
-- 6 --
. ~
1~4~3429
Characteristics N 18 N 22
b) Microscopic picture diplococci and short chains
Organic evaluation
- a) Consistency like cream of milk
b) Aroma sour milk- sour milk
c) Taste sour milk sour milk
Optimal growth temperature 45C 45C
10 Reduction red reduction
Development at 10 C - -
" 23C 3 days 4 days
" 32C 12 hours 15 hours
" 37 C 6.30 hours 15 hours
" 50C 12 hours 15 hours
Limit acidity 120 T 144 T
Growth in milk with 0,1%
Methylene blue
Moment of coagulation 6-8hours 6-8 hours
Development in broth at
2,4 and 6% bladder
Development in broth at
pH 9.2 and 9.6
Development in broth with
2~, 4~ and 6,5% salt
Development in liquid media
Catalytical test
Diacetyl formation
(Voges-Proskauer)
Carbohydrates fermentation ferment glucose, saccharose and lactose
Thermoresistance
63 - 30 min + +
65 - 30 min + +
75 - 30 min
- 7 -
, . ,,~
1~48429
_
Proteolytlc activity 8 hours48 h. 15 d. 8 h. 48 h. 15 d.
expressed in the klnd
and quantity of the
free amino acids
1 2 3 4 5 6
1. Cysteic acid
2. X 1,2,3,4,6 +++++ +~+++ +++++
3. Aspartic acid0.023 0.023 0.017 +
4. Methionine sulphone - - - -
5. Threonine 0.011 0.008 0.011 - - +
6. Serine 0.108 0.021 0,015 + ++ ++
7. Glutamine acid 0.060 0,061 0,031 + +
8. Prolinetraces 0.035 0,055 + ++
9. Alanine- 0.035 0.032 traces
10. Glycine0.018 0.013 0.011 + ++
11. 1/2 cystine - - - - - _
12. X7 + + +
13. Valine traces traces 0.008
14. Methionine traces traces +
15. Isoleucine - - 0.006 + +
16. Leucine traces traces 0.005
20 17. X9 traces + +
18. Tyrosine - - traces
19. Phenylalanine - - traces
20. X10,11,12traces traces +++
21. Lysinetraces traces +
22. Histidine traces traces 0.015
23. Ammonia + + +
24. Arginine +
-- 8 --
"` 1048429
2. L. bulgaricus N. N. 5,37, 26 and 144
_
Characteristics No. 5 No. 37
Origin v. Dalboki-Bulg. v. Hrishtene-Bulg.
Morphology
a) Type of colony R colonies S colonies
Microscopic picture short bacilli with short bacilli with
volutin grains volutin grains
Cell size 5-10~ 4-8
Organoleptic evaluation
a) Consistency slightly grainlike slightly grainlike
b) Aroma lactic acid specific
c) Taste characteristic lactic characteristic lactic
acid taste acid t~ste
Optimal T of growth - 45 C
Development at 15 C - -
" " 32C 23 hours 20 hours
" " 37C 11 " 10 "
- ~' 50C 9.30 hours 12.30 hours
Moment of coagulation 7-8 h. 7-8 h.
Limit acidity 141T 176T
Growth in milk with 0,1%
methylene blue
Development in broth
at pH 9,2
Development in broth
at 2.4 and 6% bladder
Development in broth
with 2%, 4% and 6,5% salt 2%+ 2%+
Development in
liquid media
Catalytical test
Reaction for homo and
heterofermentability homofermentative homofermentative
Ammonia formation from
argine
~ ~, _ g
~048429
~acteristics N 5 N 37
Thermoresistance
63 - 30 min ~ + +
65 - 30 min + +
70 - 30 min
Development in milk
and broth with phenol:
a) In MRS broth with
10 0,1~ phenol + +
0,2% phenol
b) milk with
0,1% phenol + +
0,2% phenol + +
0,3% phenol + +
0,4% phenol
Carbohydrates fermentation ferment glucose, ferment glucos ,
saccharose and saccharose and
Proteolytic activity expressed lactose lactose
in the type and quantity of
20 free amino acids 8 h. 48 h. 15 d. 8 h. 48 h. 15 d.
1. Cysteic acid
2. X1,2,3,4,5,6 +++++ +++++ +++++ +++++ +++++ +++++
3. Aspartic acid 0.034 0.179 0.134 0.043 0.084 0.137
4. Methionine sulphone
5~ Threonine 0.087 0.339 0.225 0.077 0.102 0.150
6. Serine 0.188 0.522 0.358 0.299 0.303 0.447
7. Glutamine acid 0.228 1.037 0.719 0.416 0.563 0.745
8- Proline 0.385 0.772 0.575 0.680 0.669 0.959
9~ Glycine 0.069 0.183 0.121 0.015 0.038 0.060
30 10. Alanine 0.164 0.135 0.147 0.164 0.099 0.284
11. 1/2 Cystine ~ ~ ~ traces
12. X7 traces traces tracestraces tracestraces
13. Valine 0.224 0.583 0.375 0.258 0.269 0.382
14. Methionine 0.049 0.113 0.079 traces0.023 0.045
15. Isoleucine 0.089 0.321 0.246 0.052 0.095 0.127
-- 10 --
lV48429
Characteristics N 5 N 37
Carbohydrates fermentation ferment glucose, ferment glucose,
saccharose and lactose saccharose and
Proteolytic activity expressed lactose
in the type and quantity of
free amino acids 8 h. 48 h. 15 d. 8 h. 48 h. 15 d.
16. Leucine 0.138 0.511 0.353 0.083 0.147 0.191
17. X9 + + + tracestracestraces
18. Tyrosine 0.054 0.145 0.106 tracesO.015 0.021
19. Phenylanine 0.028 0.178 0.125 0.028 0.057 0.076
20. X10,11 ++ +tr. ++ +tr. ++ ++
21. Lysine 0.01730.268 0.210 0.015 0.053 0.071
22. Histidine 0.080 0.125 0.074 0.058 0.065 0.084
23. Ammonia + + + + + +
24. Arginine 0.074 0.176 0.120 0.034 0.048 0.070
Note: The free amino acids quantity is in micromoles
in 1 ml coagulated milk
Characteristics N 144 N 26
Origin V. Novo selo-Bulg. v. Grashiza-Bulg.
Morphology
a) Colony type R colonies S colonies
Microscopic picture Short bacilli with Short bacilli with
volutin grains volutin grains
Cell size 5-8~ 5-10~
Organoleptic evaluation slightly grainlike slightly grainlike
a) Consistency
b) Aroma sour milk specific
c) Taste characteristic characteristic
lactic acid taste lactic acid taste
Optimal growth temperature 6-7 hours 6-7 hours
coagulation coagulation
""` 1048429
Characteristics N 144 N 26
Development at 15C 20 hours 19.30 h.
" " 32C 20 " 19.30 h.
" " 37C 11 " 10 h.
" " 50C 7.30 h. 8.30 h.
Moment of coagulation 7-8,40 h. 6-8 h.
Limit acidity 189T 225T
Development in milk with 0,1%
Methylene blue
Development in broth at
pH 9,2
Development in broth at
2.4 and 6% bladder
Development in broth with
2~, 4% or 6.5% salt 2%+ 2%+
Development in l-iquid media
Catalytical test
Reaction for homo and
20 heterofermentability homofermentative homofermentative
Ammonia formation
from arginine
Thermoresistance
63 - 30 min + +
65 - 30 min + +
70 - 30 min
Development in milk
and broth with phenol:
a) with MRS broth with
0,1% phenol + +
0,2%
- 12 -
1~48429
-
Characteristics N 144 N 26
~Development in milk
and broth with phenol:
b) milk with
0,1% phenol + +
0,2% " + +
0,3% " + +
0,4% _ _
Carbohydrates fermentation ferment glucose ferment glucose
and lactose and lactose
Proteolytic activity
expressed in the type
and quantity of free
amino acids 8 h. 48 h. 15 d.8 h. 48 h. 15 d.
1. Cysteic acid
2. Xl,2,3,4,5,6 ++++++ ++++++ ++++++
3. Aspartic acid 0.066 0.0980.200 ++ +
4. Methionine sulphone - - -
5. Threonine 0.096 0.1280.199 ++ +
6. Serine 0.268 0.3120.486 + +
7. Glutamine acid 0.835 0.8251.380 +++++ ++
8. Proline 0.768 0.7571.320 +++++ ++ ++++
9. Glycine 0.020 0.0490.082 + +
10. Alanine 0.041 0.1340.319 ++ ++
11. 1/2 Cystine - - -
12. X7 traces traces traces
13. Valine 0.356 0.345 0.576 ++++++ ++++++
14. Methionine 0.041 0.041 0.076 ++++++ ++++++
15. Isoleucine 0.168 0.173 0.304 +++ ++
16. Leucine 0.198 0.217 0.383
17. X9 traces traces traces
18. Tyrosine 0.050 0.050 0.085 +++ ++
19. Phenylalanine 0.067 0.084 0.148 ++
-13-
1~484Z9
P~oteolytic activity
expressed in the type
and quantity of free
amino acids 8 h. 48 h. 15 d. 8 h. 48 h. 15 d.
20. X10,11 ++ ++ ++
21. Lysine 0.185 0.177 0.312 +++
22. Histidine 0.161 0.104 0.184 ++
23. Ammonia + + +
24. Arginine 0.077 0.088 0.153 +++ +
The strains Streptococcus Thermophilus and Lactobacillus
Bulgaricus described above, have produced 300 yeast combinations
in the following way: retorts with 100 ml sterile cow milk at
45 C(-+ 1C) are being inoculated with 1 ml in each of them from
each strain Streptococcus Thermophilus and Lactobacillus Bulga-
ricus. The milks thus yeasted are being thermostated at + 45C.
The coagulation time should be observed. The combinations with
two-hours coagulation are separated and put under microscope.
When the cells morphology of both strains is normal, the combina-
tions are being separated. Immediately they are reinoculated
with 1% in 100 ml sterile sheep milk or in a mixture of sheep and
cow milk in a ratio 1:1. Throughout 4 months they are being
reinoculated in the same media every week. Then for 2 more months
they are being reinoculated every week in sterile cow milk with
1% of the respective yeast. Throughout these 6 months the coagu-
lation time at each reinoculation should be observed, as well as
the microscopic picture and the ratio between Streptococcus Ther-
mo~hilus and Lactobacillus Bulgaricus. If the cell of both mi-
croorganisms do not change morphologically throughout this period
and the ratio in the yeast combinations observed remains the same,
they are separated for production tests. This is preceded by de-
gustation evaluation of sour milk produced in laboratory condi-
tions out of the separate combinationsafter the characteristics
r 14
1~)48429
, ~.~
cl~ d below.
Out of the initial 300 combinations of bacterial strains,
7 passed the aforesaid tests particularly well. In each of these
combinations, a strain of S. thermophilus and a strain of _.
bulgaricus coexist well and keep a constant ratio. These novel
combinations of bacterial strains have been subjected to detailed
characterization as follows:
1. Microscopic examination of bacteria in a sample of yoghurt
stored for 24 hours at 4C. In each case, the morphological charac-
teristics of S. thermophilus were observed, namely well shaped singlediplococci which showed up well when stained. L. bulgaricus cells
could be seen to be dense rods existing singly, in pairs or in
short chains. The ratio between the two microorganisms in the
samples had been stabilized over the six month test period at values
in the range from 3:1 to 10:1.
2. The coagulation time was observed at 45C + 1C using 1% by
volume of starter added to sterile cows milk.
3. Organoleptic evaluation was effected using samples of whole cow
milk pasteurized at 95C and kept at that temperature for 30 minutes.
The samples were then cooled and inoculated when at 45C with 1%
by volume of the selected starter. The milk samples were thermostat-
ted at 45C until coagulation occurred and the milk samples were
then allowed to stand at the ambient temperature until their acidity
reaches 75T. The samples were then stored at 4C. After 24 hours,
the samples were allowed to warm up to ambient temperature (20C)
and subjected to organoleptic evaluation to determine the following
characteristics type of coagulum, colour, consistency and structure,
fracture, taste and aroma. In addition, there were determined the
acidity in degree t~rner (T~ (this is an alternative to determina-
tion of pH), free amino acids (qualitatively), the presence ofaromatic substances and volatile acids (quantitively).
- 15 -
.
~048429
By volatile ~cids is meant the ~uantity of volatile sub-
stances obtained by water-vapour distillation of 50 g of starter.
200 ml of the distillate produced in this way were titrated with
0.01 N NaOH/100 g starter. Furthermore, the activity of the starters
in liquid and dry (lyophilized) form was determined using (a) the end
dilution method and record of the number of cells of S. thermophilus
and L. bulgaricus, after McCready, and (b) coagulation time. Coagula-
tion when using 1 ml of a liquid starter should occur within 2 hours
and when using a dry liophilized starter, within 3~ hours. In the
end dilution method, the cell number of L.bulgaricus strains can be
seen to be very low when lyophilized samples were used. This is
because lyophilisation causes a high death rate among L. bulgaricus
cells.
The results of the aforesaid tests carried out on the
7 combination starters which were found to be most satisfactory for
use in the production of sour milk (yoghurt) are set out in Tables 5
and 6 which follow. The combination starters were as follows:
A. L. bulgaricus strain No. 5 and S. thermophilus strain
No. 12.
B. L. bulgaricus strain No. 5 and S. thermophilus strain
No. 22.
C. L. bulgaricus strain No. 37 and S. thermophilus strain
No. 12.
D. L. bulgaricus strain No. 37 and S. thermophilus strain
No. 18.
E. L. bulgaricus strain No. 26 and S. thermophilus strain
No. 12.
F. L. bulgaricus strain No. 144 and S. thermophilus strain
No. 12.
G. L. bulgaricus strain No. 144 and S. thermophilus strain
No. 14.
Spectra confirming the presence of aromatic substances in
coagulated milk samples produced using the combination starters are
shown in the appended chromatographs of the accompanying drawings.
- 16 -
1048429
Further to the above comments it was found that bacterial
combinations F and G produce sour milk products having a particu-
larly mucous consistency. This mucous consistency is not typical
of the usual forms of Bulgarian yoghurt. However, starter combina-
tions F and G are very suitable for the preparation of what is
termed "stirred" yoghurt of "reservoir" yoghurt. This type of
yoghurt is obtained by stirring immediately after coagulation and
can be produced in different variants with the addition of syrups,
fruit or juices for which it serves as a reservoir. A favourable
property of this product is that after canning it there occurs no
syneresis. In Bulgaria this type of sour milk is known as
"Mladost".
The sour milk yeasts N.N. 5-12, 5-22, 37-12, 37-18; 26-12,
144-12 and 144-12 are characterized by the following:
Characteristics 5-12 5-22 37-12 37-18
Microscopic picture Diplococci of S. thermophilus strains:
medium-long, dense rods of L. bulqaricus
in single, paired or small chains.
Coagulation time:
(a) Liq. inocculant 2 hours at + 45 C (1 ml for 100 ml) in
each case
(b) dry inocculant 32 hours (0.1 g dry for 100 ml) in each
case
Type of coagulum
a) Consistency dense for all
b) Fracture smooth-glittering for all
c) Taste and aroma specific, well expressed for all
Free amino acids:
Leucine +++ +++
Phenyl alanine
Valine methionine +++++++ +++++ traces
Tyrosine ++ +++ +
Aminobutyric acid + +
Proline ++++ ++++++ +++ +++++
- 17 -
1~48429
. _ . . ..
Characteristics 5-12 5-22 37-12 37-18
~lpha alanine +++ +++ +++ +++++
Threonine + +
Glutamine acid ++ ++++++ ++ +++
Glycine serine ++
Serine +
Aspartic acid
Aspargine traces
Histidine
Lysine ++ +
Ornithine +
Cystedine +++
Oxiproline +
Tryptophan
Activity (cells x 10 /ml)
a) Liquid yeasting
Streptococcus Thermophilus 575 550 962 580
Lactobacillus Bulgaricus 211 242 309 250
b) Dry yeast (~ioph)
(cells x 10 /g)
S. Thermophilus 4SOQ 4500 4500 2500
L. Bulqaricus 45 45 9.5 9.5
Volatile acids 1560 1540 1820 1560
Characteristics 26-12 144-12 144-14
. .
Microscopic picture Diplococci, medium-long, dense rods
single, in couples or like short chains
Time of coagulation For all 2 hours (1 ml for 100 ml)
3,30 h (0,1 g dry for 100 ml)
Type of coagulum
a) Consistency dense for all
b) Fracture Smooth - glittering for all
c) Taste and aroma Specific, well expressed for all
- 18 -
1t~484Z9
Characteristics26-12 144-12 144-14
Free amino acids:
Leucine +++ + ++
Phenyl alanine + +
Tryptophan
Valin methionine ++ ++++ +++
Tyrosine ++ ++ +
Aminobutyric acid + ++
10 Proline ++++ +++++ ++++
Alpha alanine +++ +++ ++++
Threonine
Glutamine acid + ++++ ++++
Glycine + +
Serine
Aspartic acid traces
Aspargine traces
Histidine
Lysine traces
20 Ornethine
Cystedine
Oxyproline
Activity (cells x 106/ml)
a) liquid yeasting
S. Thermophilus 1983 1300 783
.
L. Bulgaricus 497 213 383
b) dry yeast(l~oph)
(cells x 10 /g)
S. Thermophilus 2500 4500 1500
L. Bulgaricus 95 95 45
30 Volatile acids 15.60 17.60 15.20
-- 19 --
1~48429
The head space method for the determination of arGmatic
substances referred to hereinabove was carried out using the method
attributed to Bassette et al as follows:
In each determination, 8 ml. of a sour milk culture is
maintained at 45C for sufficient time to undergo coagulation. The
culture is then poured into a 50 ml. round bottomed flask which was
sealed-by means of a plastic plug. 4 grams of dry sodium sulphate
are placed in the flask and the flask is stored under refrigerating
conditions at -13C for a maximum time from three to -five hours.
The flask is then removed from the refrigerator employed, the con-
tents thereof are collodium-sealed and stirred and the flask is
then kept for 20 minutes at a temperature of 70C, undergoing stir-
ring at least twice.
2.5 cc of gas are then withdrawn from the volatiles-enri-
ched gas space within the flask using a gas syringe (for example
*
of the type Hamilton - 1005H). Before the chosen amount of gas
is withdrawn, the syringe is pre-operated without withdrawal of
the needle from the seal of the flask. The sample obtained is
analyzed by means of a gas chromatograph of the Fractovap , type D
manufactured by the Company Carlo Erba. For this purpose, a column
2 metres long having an inner diameter of 2 mm is filled with the
product Porapak Q80 - 100 US mesh (made in U.S.A.). Analysis is
carried out in a chamber kept at 155C. During the determination,
the carrying gas, nitrogen, is supplied at a flow rate of 25-30 ml/min.
The burning gas, hydrogen, is supplied at the same rate.
Under these conditions, the aromatograms of the strains
shown in the accompanying drawings were obtained.
* Trademark
- 20 -
~i
