CA1048429A - Method for obtaining yeasts for bulgarian yoghurt - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The present invention relates to a method for the production of a sour milk product, which comprises adding a starter combination comprising at least one of the strains of Streptococcus thermophilus Nos 12, 14, 18 and 22 and at least one of the strains of Lactobacillus bulgaricus Nos 5, 26, 37 and 144 to a milk sample and culturing the starter combination until a sour milk product is obtained.
The strains combinations ensure obtaining of original Bulgarian sour milk with higher nutritive qualities, characteristic taste and aroma.

Description

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.

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 Bl 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 ~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 .~

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 -` 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 . ~

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 , . ,,~

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. 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 +

"` 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 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%

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 ++

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).

.

~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.

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 ++++ ++++++ +++ +++++

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 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 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 ~i

Claims (6)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for the production of a sour milk product, which comprises adding a starter combination comprising at least one of the strains of Streptococcus thermophilus Nos 12, 14, 18 and 22 and at least one of the strains of Lactobacillus bulgaricus Nos 5, 26, 37 and 144 to a milk sample and culturing the starter combination until a sour milk product is obtained.

2. A method as claimed in claim 1, wherein the milk is whole cow milk or skimmed cow milk.

3. A method as claimed in claim 1, wherein the milk is sheep milk.

4. A method as claimed in any one of claims 1 to 3, wherein the combination of the strains: Streptococcus thermophi-lus No. 12 and Lactobacillus bulgaricus No.5; Streptococcus thermophilus No. 22 and Lactobacillus bulgaricus No. 5; Strep-tococcus thermophilus No. 18 and Lactobacillus bulgaricus No. 37;
Streptococcus thermophilus No. 12 and Lactobacillus bulgaricus No. 37 or Streptococcus thermophilus No. 12 and Lactobacillus bulgaricus No. 26 is used to produce yoghurt by a procedure which does not involve addition of dry skimmed milk.

5. A method as claimed in any one of claims 1 to 3, wherein the combination of the strains Streptococcus thermophilus No. 12 and Lactobacillus bulgaricus No. 144 or the strains Strep-tococcus thermophilus No. 14 and Lactobacillus bulgaricus No. 144 is used to produce "reservoir" sour milk of the "Mladost" type.

6. A sour milk product which has been produced by the method claimed in claim 1.