Abstract:
Specific strains of the micro-organisms Lactobacillus bulgaricum and Streptococcus thermophilus are isolated, identified and purified and used for the preparation of starting materials for production of Bulgarian sour milk. The use of these micro-organisms is described together with procedures in the present invention for the production of Bulgarian sour milk having improved nutrient value and improved characteristic taste and flavor. Specified mixtures of the above micro-organisms can also be advantageously employed for this purpose.

Description:
Thisis a continuation of application Ser. No. 727,040, filed Sept. 27, 1976, which is a continuation-in-part of application Ser. No. 529,475, filed Dec. 4, 1974, both now abandoned. 
    
    
     The present invention relates to new starting materials having greater activity and better production qualities, such as proteolytic activity and content of aromatic substances for the production of original Bulgarian sour milk. These starting materials are obtained with the newly isolated, purified and differentiated strains of Str. thermophilus and Lb. bulgaricus. 
     BACKGROUND OF THE INVENTION 
     It is known to produce Bulgarian sour milk using strains of St. thermophilus and Lactobacillus bulgaricus which establish a good symbiotic bond. Some of these micro-organisms have been reported in Bulgarian authorship certificate N10640, where a new technology for producing Bulgarian sour milk is described. 
     The present invention deals with new procedures for obtaining starting materials for sour milk, which is more active and having far better production qualities, for the production of original Bulgarian sour milk with higher nutritive qualities and characteristic taste and flavor. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The principal feature of the present invention is to isolate, purify and differentiate new active strains of St. thermophilus and Lactobacillus bulgaricus which coexist well in order to obtain more active starting materials with better production qualities for the production of original Bulgarian sour milk. These procedures are carried out by isolation, purification and differentiation of the new strains of St. thermophilus and Lactobacillus bulgaricus, which can be distinguished by their biological and morphological characteristics, better proteolytic qualities (especially the strains of Lactobacillus bulgaricus), good coexistence and very good ability to form aromatic substances, as compared to known strains used for the same purpose, thereby resulting in more active combinations of starters having better production qualities for production of original Bulgarian sour milk from sheep or cow milk. 
     The new strains were deposited in the Bulgarian State Institute of Drug Control on Dec. 21, 1973, and in the Japanese Fermentation Research Institute of Industrial Technology with the following depository designations: 
     
         ______________________________________            Bulgarian                     Japanese            Collection                     Collection______________________________________1) Lb. Bulgaricus         #5       151        30132) Lb. Bulgaricus         #26      152        30143) Lb. Bulgaricus         #37      153        30154) Lb. Bulgaricus          #144    154        30165) St. Thermophilus         #12      155        30096) St. Thermolphilus         #14      156        30107) St. Thermophilus         #18      157        30118) St. Thermolphilus         #22      158        3012______________________________________ 
    
     The above strains are isolated from natural starters, milk, herbs and raw cream. After being threefold purified, they are differentiated according to the following criteria defined by Zehleman, Berge, Rogasa, and Sharp: (1) defining the optimum temperature of growth and reduction of litmus milk, (2) organoleptic evaluation, (3) moment of coagulation and limit acidity, (4) morphologic characteristics of colonies and cells, (5) growth in milk with 0.1% methylene blue, (6) growth in broth (hydrolyzed milk or MRS) at pH 9.2 and 9.6, (7) growth in broth (hydrolyzed milk or MRS) with 2.4 and 6% bile, (8) growth in broth (hydrolyzed milk or MRS) with 2.4 and 6.5 salt, (9) development in esculin broth, (10) catalytical test, (11) formation of diacetyl, (12) carbohydrate fermentation, (13) proteolytic activity, expressed by number and quantity of free amino acids, (14) development in broth (MRS) and milk with phenol, (15) thermoresistance, (16) defining homo-and heterofermentability, (17 ) content of aromatic substances. 
     The strains are as follows: 
     1. St. thermophilus N, N12, 14, 18, 22 
     
                       TABLE I______________________________________Characteristics  N12         N14______________________________________ Origin          v. Grashtizo-                        v. Dalboki-Bul.            Bul.Morphologya) Colony type atsurface growth   small, round                        roundb) Microscopic pattern            diplococci and short chainsOrganoleptic evaluation:a) consistence   mucous      mucousb) flavor        milksourc) taste         milksourOptimal growth temperature            45° C.                        45° C.Reduction        red reduction                        --Development at 10° C.            -           -Development at 23° C.            3 days      3 daysDevelopment at 32° C.            14 hours    14 hoursDevelopment at 37° C.            6.5 hours   7 hoursDevelopment at 50° C.            12 hours    12 hoursLimit Acidity    124° T. (L+)                        123° T. (L+)Growth in milk with 0.1%methylene blue   -           -Moment of coagulation            6-8 hours   6-8 hoursDevelopment in broth atpH 9.2 and 9.6   -           -Development in broth with2.4 and 6% bile  -           -Development in broth with2.4 and 6.5% salt            -           -Development in esculin broth            -           -Catalytical test -           -Diacetyl formation(Foges-Proscauer)            -           -Carbohydrate fermentation            ferment glucose, saccharose and            lactosThermoresistance30 minree.       +           +30 minree.       +           +30 minree.       -           -______________________________________ 
    
     
                       TABLE I______________________________________Proleolytic activityexpressed by num-          N12ber and quantity of          8 h.      48 h.     15 d.free amino acids          (1)       (2)       (3)______________________________________1. Cysteic acid          -         -         -2. X1, 2, 3, 4, 5, 6          +++       +++       +++3. Asparginic acid          .026      .020      .0184. Methionine sulphone          -         -         -5. Threonine   .008      .011      .0096. Serine      .015      .017      .0197. Glutamic acid          .124      .140      .1168. Proline     .050      .045      .0649. Glycine     traces    .014      .01010. Alanine    .039      .059      .05411. 1/2 Cystine          -         -         -12. X7         +         +         +13. Valine     traces    traces    .00514. X8         traces    traces    traces15. Methionine traces    traces    traces16. Isoleucine traces    traces    traces17. Leucine    traces    traces    traces18. X9         +         +         +19. Tyrosine   -         -         traces20. Phenylalanine          -         -         traces21. X10, 11, 12          +++       +++       +++22. Lysine     traces    traces    traces23. Histidine  traces    traces    0.01324. Ammonia    +         +         +25. Arginine   traces    -         traces______________________________________ Note: The amino acids quantity is in micromoles in 1 ml coagulated milk X = unknown amino acids 
    
     
                       TABLE I______________________________________Proteolytic activityexpressed by num-          N14ber and quantity of          8 h.      48 h.     15 d.free amino acids          (4)       (5)       (6)______________________________________L. Cysteic acid          -         -         -2. X1, 2, 3, 4, 6          +++       +++       +++3. Asparginic acid          .021      .018      .0194. Methionine sulphone5. Threonine   .006      .009      .0116. Serine      .018      .024      .0187. Glutamic acid          .096      .110      .1218. Proline     .056      .051      .0619. Glycine     .010      .015      .01110. Alanine    .023      .047      .05711. 1/2 Cystine          -         -         -12. X7         +         +         +13. Valine     traces    traces    0.00614. X8         traces    traces    traces15. Methionine traces    traces    traces16. Isoleucine traces    traces    traces17. Leucine    traces    traces    traces18. X9         +         +         +19. Tyrosine   -         -         -20. Phenylalanine          -         -         traces21. X10, 11, 12          traces    traces    +++22. Lysine     traces    +         +23. Histidine  traces    traces    .01524. Ammonia    +         +         +25. Arginine   traces    -         -______________________________________ Note: The amino acids quantity is in micromoles in 1 ml coagulated milk X = unknown amino acids 
    
     
                       TABLE II______________________________________Characteristics  N18         N22______________________________________Origin           v. Dalboki-Bul.                        v. Preslavez-                        Bul.Morphologya) Type of colony at            small, round                        small, roundsurface growth   diplococci andb) Microscopical pattern            short chainsOrganoleptic evaluationa) consistence   like cream or            milkb) flavor        milk sourc) taste         milk sourOptimal growth temperature            45° C.                        45° C. -Reduction red reductionDevelopment at 10° C.            -           -Development at 23° C.            3 days      4 daysDevelopment at 32° C.            12 hours    15 hoursDevelopment at 37° C.            6.30 hours  15 hoursDevelopment at 50° C.            12 hours    15 hoursLimit Acidity    120°T. (L+)                        144° T. (L+)Growth in milk with 0.1%methylene blue   -           -Moment of coagulation            6-8 hours   6-8 hoursDevelopment in broth with2.4 and 6% bile  -           -Development in broth atpH 9.2 and 9.6   -           -Development in broth with2.4 and 6.5% salt            -           -Development in esculin broth            -           -Catalytical test -           -Diacetyl formation(Foges-Proscauer)            -           -Carbohydrates fermentation            ferment glucose, saccharose and            lactoseThermoresistance30 minree.       +           +30 minree.       +           +30 minree.       -           -______________________________________ 
    
     
                       TABLE II______________________________________Proteolytic activityexpressed by type          N18and quantity of          8 h.      48 h.     15 d.free amino acids          (1)       (2)       (3)______________________________________1. Cysteic acid          -         -         -2. X1, 2, 3, 4, 6,          +++++     +++++     traces3. Asparginic acid          .023      .023      .0174. Methionine sulphon          -         -         -5. Threonine   .011      .008      .0116. Serine      .108      .021      .0157. Glutamic acid          .060      .061      .0318. Proline     traces    .035      .0559. Alanine     -         .035      .03210. Glycine    .018      .013      .01111. 1/2 Cystine          -         -         -12. X 7        +         +         +13. Valine     traces    traces    .00814. Methionine traces    traces    +15. Isoleucine -         -         .00616. Leucine    traces    traces    .00517. X9         traces    +         +18. Tyrosine   -         -         traces19. Phenylalanine          -         -         traces20. X10, 11, 12          traces    traces    +++21. Lysine     traces    traces    +22. Histidine  traces    traces    .01523. Ammonia    +         +         +24. Arginine   +         -         -______________________________________ 
    
     
                       TABLE II______________________________________Proteolytic activityexpressed by type          N22and quantity of          8 h.      48 h.     15 d.free amino acids          (4)       (5)       (6)______________________________________1. Cysteic acid          -         -         -2. X1, 2, 3, 4, 63. Asparginic acid          +         -         -4. Methionine sulphone          -         -         -5. Threonine   -         -         +6. Serine      +         ++        ++7. Glutamic acid          +                   +8. Proline     +                   ++9. Alanine                         traces10. Glycine    +                   ++11. 1/2 Cystine          -         -         -12. X713. Valine     -         -         -14. Methionine -         -         -15. Isoleucine +                   +16. Leucine17. X 918. Tyrosine   -         -         -19. Phenylalanine          -         -         -20. X10, 11, 1221. Lysine     -         -         -22. HIstidine  -         -         -23. Ammonia24. Arginine______________________________________ 
    
     2. Lactobacillus bulgaricum N, N5, 37, 26 and 144 
     
                       TABLE III______________________________________Characteristics  N5          N37______________________________________Origin           v. Dalboki-Bul                       v. Hrishtene-BulMorphologya) Type of colony            R colonies S coloniesMicroscopical picture            short rods with volutine grainsCell size        5-10 μ   4-8 μOrganoleptic evaluation: -a) consistence            slightly    slightly            grainlike   grainlikeb) flavor        lactic acid specificc) taste         characteristic lactic acid tasteOptimal temperature growth            45° C.                       45° C.Development at 15° C.            -          -Development at 32° C.            23 hours   20 hoursDevelopment at 37° C.            11 hours   10 hoursDevelopment at 50° C.            9.30 hours 12.30 hoursMoment of coagulation            7-8 hours  7-8 hoursLimit acidity    141° T. (D-)                       176° T. (D-)Growth in milk with 0.1%methylene blue   -          -Development in brothat pH 9.2        -          -Development in broth with2.4 and 6% bile  -          -Development in broth with2.4 and 6.5% salt            2%+        2% +Development in esculin brothCatalytical test -          -Reaction for homo- andheterofermentability            homofermentativeAmmonia formation fromargine           -          -Thermoresistance30 minree.       +          +30 minree.       +          +30 minree.       -          -Development in milkand broth with phenol: -a) In MRS broth with0.1% phenol      +          +0.2% phenol      -          -b) milk with0.1% phenol      +          +0.2% phenol      +          +0.3% phenol      +          +0.4% phenol      -          -Carbonhydratesfermentation:    ferment glucose, saccharose            lactose______________________________________ 
    
     
                       TABLE III______________________________________Proteolytic activityexpressed by type        N5and quantity of free        8 h.      48 h.      15 d.amino acids  (1)       (2)        (3)______________________________________1. Cysteic acid2. X1, 2, 3, 4, 5, 6        +++++     +++++      +++++3. Asparginic acid        .034      .179       .1344. Methionine sulphon        -         -          -5. Threonine .087      .339       .2256. Serine    .188      .522       .3587. Glutamine acid        .228      1.037      .7198. Proline   .385      .772       .5759. Glycine   .069      .183       .121.10. Alanine .164      .135       .14711. 1/2 Cystine        -         -          -12. X7       traces    traces     traces13. Valine   .224      .583       .37514. Methionine        .049      .113       .07915. Isoleucine        .089      .321       .24616. Leucine  .138      .511       .35317. X9       +         +          +18. Tyrosine .054      .145       .10619. Phenylanine        .028      .178       .12510. X10, 11  ++        +tr.       ++21. Lysine   .173      .268       .21022. Histidine        .080      .125       .07423. Ammonia  +         +          +24. Arginine .074      .176       .120______________________________________ Note: The free amino acids quantity is in micromoles in 1 ml coagulated milk 
    
     
                       TABLE III______________________________________Proteolytic activityexpressed by type        N37and quantity of free        8 h.      48 h.      15 d.amino acids  (4)       (5)        (6)______________________________________1. Cysteic acid        -         -           -2. X1, 2, 3, 4, 5, 6        +++++     +++++      +++++3. Asparginic acid        .043      .084       .1374. Methionine sulphon        -         -          -5. Threonine .077      .102       .1506. Serine    .290      .303       .4477. Glutamine acid        .416      .563       .7458. Proline   .680      .669       .9599. Glycine   .015      .038       .06010. Alanine  .026      .099       .28411. 1/2 Cystine        traces    -          -12. X7       traces    traces     traces13. Valine   .258      .269       .38214. Methionine        traces    .023       .04515. Isoleucine        .052      .095       .12716. Leucine  .083      .147       .19117. X9       traces    traces     traces18. Tyrosine traces    .015       .02119. Phenylanine        .028      .057       .07620. X10, 11  +tr.      ++         ++21. Lysine   .015      .053       .07122. Histidine        .058      .065       .08423. Ammonia  +         +          +24. Arginine .034      .048       .070______________________________________ Note: The free amino acids quantity is in micromoles in 1 ml coagulated milk 
    
     
                       TABLE IV______________________________________Characteristics  N144        N26______________________________________Origin           v.Novoselo-Bul                        v. Grashiza-                        BulMorphologya)Colony type    R colonies  S coloniesMicroscopical pattern            short rods with volutine grainsCell size        5-8 μ    5-10 μOrganoleptic evaulation            slightly granlikea) consistenceb) flavor        milk sour   specificc) taste         characteristic lactic acid tasteOptimal growth temperature            45° C.                        45° C.Development at 15° C.Development at 32° C.            20 hours    19.30 hoursDevelopment at 37° C.            11 hours    10 hoursDevelopment at 50° C.            7-8 40 hours                        6-8 hoursLimit acidity    189° T. (D-)                        225° T. (D-)Development in milk with0.1% methylene blue            -           -Development in brothat pH 9.2        -           -Development in broth with2.4 and 6% bile  -           -Development in broth with2.4 and 6.5% salt            2%+         2%+Development in esculin broth            -           -Catalytical test --          --Reaction for homo- andheterofermentability            homofermentative for allAmmonia formationfrom arginine    -           -Thermoresistance63° C. - 30 min            +           +65° C. - 30 min            +           +70° C. - 30 min            -           -Development in milk and brothwith phenol:a) with MRS broth with0.1% phenol      +           +0.2% phenol      +           +b) milk with0.1% phenol      +           +0.2% phenol      +           +0.3% phenol      +           +0.4% phenol      -           -Carbohydrates fermentation:            ferment glucose and lactose______________________________________ 
    
     
                       TABLE IV______________________________________Proteolytic activityexpressed by type        N144and quantity of free        8 h.      48 h.      15 d.amino acids  (1)       (2)        (3)______________________________________1. Cysteic acid        -         -2. X1, 2, 3, 4, 5, 6        +++++     +++++      +++++3. Asparginic acid        .066      .098       .2004. Methlonone sulphon        -         -          -5. Threonine .096      .128       .1996. Serine    .268      .312       .4867. Glutamic acid        .835      .825       1.3808. Proline   .768      .757       1.3209. Glycine   .020      .049       .08210. Alanine  .041      .134       .31911. 1/2 Cystine        -         -          -12. X7       traces traces traces13. Valine   .356      .345 +     .57614. Methionine        .041      .041       .07615. Isoleucine        .168      .173       .30416. Leucine  .198      .217       .38317. X9       traces traces traces18. Tyrosine .050      .050       .08519. Phenylalanine        .067      .084       .14820. X10, 11  ++        ++         ++21. Lysine   .185      .177       .31222. Histidine        .161      .104       .18423. Ammonia  +         +          +24. Arginine .077      .088       .153______________________________________ 
    
     
                       TABLE IV______________________________________Proteolytic activityexpressed by type         N26and quantity of free         8 h.       48 h.    15 d.amino acids   (4)        (5)      (6)______________________________________1. Cysteic acid2. X1, 2, 3, 4, 5, 63. Asparginic acid         ++                  +4. Methlonone sulphon         -          -        -5. Threonine  ++                  +6. Serine     +                   +7. Glutamic acid         +++++               ++8. Proline    +++++               ++++9. Glycine    +                   +10. Alanine   ++                  ++11. 1/2 Cystine12. X713. Valine    ++++++              ++++++14. Methionine         ++++++              ++++++15. Isoleucine         +++                 ++16. Loucine17. X918. Tyrosine  +++                 ++19. Phenylalanine         ++20. X10, 1121. Lysine    +++                 +++22. Histidine ++23. Ammonia24. Arginine  +++                 +______________________________________ 
    
     Combination of Two Microorganisms 
     
                                           TABLE V__________________________________________________________________________Characteristics      5-12     5-22   37-12  37-18__________________________________________________________________________Microscopical      Diplococci Medium-long, dense rods, inpattern    single pairs or small chainsTime of coagu-      for all 2 hours at +45° C. (1 ml for 100 ml)lation:    for all 3.30 h. (0.1 g dry for 100 ml)Type of coagulum:a) consistence      dense for allb) fracture      smooth-flittering for allc) taste andflavor     specific, well expressed for allFree amino acids:Leucine                    +++    +++PhenylalanineValine meth-ionine     +++++++  +++++         tracesTyrosine   ++       +++           +Aminobutyricacid       +        +Proline    ++++     +++++  +++    +++++Alfa al-anine      +++      +++    +++    +++++Tronine    +        +Glutamine acid      ++       +++++  ++     +++Glycine serine             ++Serine                            +Asparginic acidAspargine                  tracesHistidineLysine     ++       +Ornithine           +Cystedine                  +++Oxiproline                        +TryptophanActivity:a) Liquid combin-ation starterSt. Thermo-philus     575 mln/ml               550 mln/ml                      962 mln/ml                             580 mln/mlLb. Bulgaricum      211 mln/ml               242 mln/ml                      309 mln/ml                             250 mln/mlb) Dry combina-tion starter(lioph) St. Therm-philus     4,500mln/g               4.500 mln/g                      4.500 mln/g                             2.500 mln/gLb. Bulgaricum      45 mln/g 45 mln/g                      9.5 mln/g                             9.5 mln/gVolatile acids      15.60    15.40  18.20  15.60__________________________________________________________________________ mln=million 
    
     
                       TABLE VI______________________________________Characteristics       26-12      144-12     144-14______________________________________Microscopic pattern       Diplococci, medium-long, dense rods,       single, in pairs or short chainsTime of Coagulation       For all 2 hours (1 ml for 100 ml)       3.30 h. (0.1 g dry for 10 ml)Type of coagulum:a) consistency       dense for allb) fracture smooth-glittering for allc) taste and flavor       specific, well expressed for allfree amino acids:Leucine     +++        +          ++Phenyl alanine         +          +TryptophanValin methionine       ++         +++++      +++Tyrosine    ++         ++         +Aminobutyric acid      +          ++Proline     ++++       +++++      ++++Alfa alanine       +++        +++        ++++TrionineGlutamine acid       +          ++++       ++++Glycine     +                     +SerineAsparginic acid       tracesAspargine              tracesHistidineLysine      tracesOrnethineCystedineOxyprolineActivity:a) LiquidcombinationStarterSt. Thermophilus       1.983mln/ml                  1.300mln/ml                             783mln/mlLb. Bulgaricum         497mln/ml                    213mln/ml                             383mln/mlb) dry combinationstarter (lioph) St.Thermophilus       2.500mln/g 4.500mln/g 4.500mln/gLb. Bulgaricum       9.5 mln/g  9.5 mln/g  10  45 mln/gVolatile acids       15.60      17.60      15.20______________________________________ Note: Combination starters 144-12 and 144-14 are able to produce mucous cosistency under special technologic circumstances and therefore they are used for producing reservoir sour milk 
    
     The strains of Streptococcus thermophilus and Lactobacterium bulgaricum described above, produced 300 combination starters in the following way: each of a number of retorts containing 100 ml sterile cow milk at 45° C. (±1° C.) were inoculated with a combination starter consisting of 1 ml of a strain of Str. thermophilus and 1 ml of a strain of Lb. bulg. The inoculated milk was thermostated at +45° C. and the coagulation time observed. The combination starters causing coagulation in two-hours time were examined under a microscope for normal cell morphology of both strains. Immediately after examination, 100 ml sterile sheep milk or a mixture of sheep and cow milk at a ratio 1:1 were inoculated each with 1% of an approved combination starter. The latter were subcultured in the same medium in the course of four months every day and in the course of another two--every week. The inoculation time of each reinoculation as well as the microscopic pattern and ratio between the strains of Str. therm. and Lb. bulg. were regularly observed throughout the whole six month period. If the cells of both micro-organisms did not change morphologically throughout this period and the ration of the combination starters observed remained the same, they were used for production tests. This was preceded by degustation evaluation of sour milk prepared with the approved combination starters under laboratory conditions after the criteria further cited. 
     Seven combination starters out of 300 combination starters were selected on the basis of a satisfactory coexistence and a stable ratio. 
     Evaluation of the new combination starters for sour milk is carried out according to the following criteria: 
     (1) microscopic pattern of the combination starters which immediately after coagulation were placed in a refrigerator at +4° C. for 24 hours. (a) Streptococcus thermophilus well shaped and well dyed diplococci, (b) Lactobacillus bulgaricus dense, clear cut small rods, single, in couples or small chains. The ratio between the two micro-organisms should vary from 1:3-1:10; 
     (2) the coagulation time was observed at +45° C. (±1° C.) using 1% combination starter--2 hours. 
     (3) organoleptic evaluation of sour milk samples prepared from homogenized whole cow milk pasteurised at 95° C. with holding time of 30 minutes. The samples were then cooled and inoculated at 45° C. each with 1% of an approved combination-starter followed by thermostating them at 45° C. After coagulation, the samples were left at indoors temperature until the acidity reached 75° T, whereafter they were put in a refrigerator at +4° C. They were tasted in 24 hours time after tempering them to indoors temperature. The organoloptic evaluation disclosed the characteristics below: type of coagulum, color, consistence and structure, fracture, taste and flavor. In addition, the volatile acids were defined, the acidity in grades Torner, the free amino acids-qualitatively or quantitatively, the aromatic substances and the activity of the combination starters in liquid and dry lyophilized form. The activity is determined on the basis of: (a) through the method of limiting dilution and recording the number of Streptococcus thermophilus and Lactobacillus bulgaricus as well as their ratio after McKredy and (b) time of coagulation lyophilized combination starter (0.1 g in 100 ml sterile cow milk-whole milk or skimmed) for 31/2 hours, liquid combination starter (1 ml in 100 ml of sterile whole or skimmed cow milk)--2 hours. 
     The sour milk combination starters NN5-12, 5-22, 37-12, 37-18, 26-12, 144-12 and 144-14 were characterized by the criteria given in the tables.