DE2457303A1 - PROCESS FOR THE PRODUCTION OF YEAST CULTURES FOR BULGARIAN YOGURT - Google Patents

Description

(Priority: December 4, 1973 - Bulgaria - No. 25 150)

The invention relates to new, more active, better breeding properties such as yeast cultures with proteolytic activity and aromatic substances for real Bulgarian yogurt, which obtained as a result of the good symbiosis of the new strains Streptococcus thermophilus and Lactobacterium bulgaricum and isolated, purified and differentiated by the authors. '

It is known that for the production of Bulgarian yoghurt strains of Streptococcus thermophilus and Lactobacterium ' bulgaricum are used, which have a good mutual symbiotic Have relationship. Some are known from the Bulgarian copyright certificate No. 10 640, where a new technology for Production of Bulgarian yogurt is described.

It is the object of the present invention to provide the possibilities for producing more effective and much better operating properties owning yeast cultures and thereby the production of Bulgarian yoghurt with higher nutritional value and with a distinctive taste and aroma.

509824/0943

This task is to create new active strains of Streptococcus thermophilus and Lactobacterium bulgaricum isolated, purified and differentiated, which have a good symbiosis and thus more active yeast cultures with to be able to produce better operating properties.

The object of the invention is achieved by the fact that new strains of Streptococcus thermophilus and Lactobacterium bulgaricum isolates, purifies and differentiates, which as an advantage and compared to the previously known and too for this purpose, strains used a better biological and. morphological characteristics, better proteolitical properties (especially the strains Lactobacterium bulgaricum), good symbiosis, very good ability of aromatic substances to form, have and as a result of their symbiosis more active combinations of yeast cultures with significantly better operating properties for the production of real original) Bulgarian yoghurt from sheep's or cow's milk.

These strains are isolated from natural yeast cultures, raw milk, plants, and raw cream. After being cleaned three times, they are differentiated according to the characteristic values given by Sehleman, Berge, Rogosa and Charp, namely: Determination of the optimal development temperature and reduction of liquorice milk. Organoleptic estimation, coagulation moment and limit acidity. Morphological characteristics of the colonies and cells. Growth in milk with 0.1 % methylene blue. Growth in broth (hydrolyzed milk or MRS) at pH 9.2 and 9.6. Grow in broth (hydrolyzed milk or MRS) with 2, 4 and 6 % bile. Grown in broth (hydrolyzed milk or MRS) with 2, 4 and 6.5 % salt. Development in esculin broth. Catalase samples. Diacetyl formation. Fermentation of the sugars. Proteolitic activity indicated by the number and quantity of free amino acids. Development in bouillon (MRS) and milk with phenol. Thermoresistance. Determination of homo- and heterofermentativity. Aromatic substances.

S0982A / 0943

Tables: Streptococcus thermophilus No. 12, 14, 18,

Lactobacterium bulgarictun "5» 37, 26, 144.

These stems are as follows:

1. Streptococcus thermophilus No. 12, 14, 18, 22

Characteristic values No. 12 No. 14 origin Grastiza village /
Bulgaria Dalboki village /
Bulgaria morphology
a) Type of colony
Surface growth small, round around b) Microscopic picture. Diplococci and short chains Organoleptic estimation a) Consistency
bJ aroma
c) taste slimy
wed 1 chs
wed 1 chs slimy
aures
aures Optimal development
temperature 45 ° C 45 ° C reduction red Re d u k t i ο η Development at 10 ° C
it at 23 ° C
. »» 32 ° C
«« 37 ° c
it ti 5o ° c 3 days
14 hours
6.30 hours
12 hours 3 days
14 hours
7 hours
12 hours Limit acidity 124 ° T 123 ° T Growing in milk,
with 0.1 % methylene blue rr Coagulation moment 6-8 hours 6-8 hours

Development in bouillon

at pH 9.2 and 9.6 '-

Development in broth with 2, 4, 6% bile

Development in bouillon

with 2, 4, 6.5 % salt ■ - '

Developed in esculin broth

Formation of diacetyl
(Vogus-Proskauer). -

Fermentation of sugars ferment glycose,

Thermal resistance

63 - 30 minutes +

65 ° - 30 minutes +

75 - 30 minutes

509824/09

Sucrose and lactose

Proteolitic activity expressed in terms of type and quantity of free amino acids

8 hours 48 hours 15th day 8 hours 48 hours 15th day (D (2) (3) (4) (5) (6)

1. Cysteic acid _ + - + + - + - - - + 2. X 1, 2, 3, 4, 6 +++++ - +++++ traces +++++ traces +++++ +++++ +++++ - 3. Aspartic acid 0.026 - 0.020 traces - 0.018 traces 0.021 0.018 0.019 traces 4. Methionine sulfone - +++ - It +++ - +++ - - - ■ +++ 5. Threonine 0.008 traces 0.011 Il traces 0.009 traces 0.006 0.009 0.011 ■ + 6. Serine 0.015 traces 0.017 It traces 0.019 0.013 0.018 0.024 0.018 0.015 7. Glutamic acid 0.124 + 0.140 + 0.116 + 0.096 0.110 0.121 + 8. Proline 0.050 traces 0.045 _ 0.064 traces 0.056 0.051 0.061 _ 9. Glycine ' traces 0.014 0.010 0.010 0.015 0.011 10. Alanine 0.039 0.059 0.054 0.023 0.047 0.057 11. 1/2 cystine - ■ - -. - _- 12. X 7 + + + + + 13- valine traces 0.005 traces traces Ό, 006 14. X 8 traces traces Traces. traces traces 15. Methionine It It It It It 16. Isoleucine Il ti Il It dd 17. Leucine Il It ti Il It 18th X 9th , + + 19 · tyrosine - - 20. Phenylalanine - - 21.X 10, 11, 12 traces traces 22. Lysine traces + 23. Histidine traces traces 24. Ammonia + + 25. Arginine traces _

Note: The quantity of amino acids is in micromoles 1 ml of coagulated milk indicated

X = means unknown amino acids.

5 09824/0943

Characteristic values

No. 18

No. 22

Origin Dalboki village - Bulgaria Ireslavez village - Bulgaria

morphology

a) Type of colony

Surface growth kleinj b) Microscopic picture ι Organoleptic estimation

a) Consistency

b) aroma

c) taste

Optimal development temperature reduction

Development at 10 ⁰ C

round small, round

Diplococci and short chains

r a H m a r t i S. e S. m i 1 C. H S. a U r e S. m i 1 C. H S. a U r

red reduction

ti it 23 ⁰ C. .3 days .4 days η ti ₃₂ O ₀ 12 hours 15 hours 11 it 37O ₀ 6.30 hours 15 hours ti ti RQ ^ G 12 hours .15 hours Limit acidity 120 ⁰ T 144 ⁰ T Growing in milk with
0.1% methylene blue. Coagulation moment 6-8 hours 6-8 hours Development in bouillon
at pH 9.2 and 9 »6 - ■ Development in bouillon
with 2, 4 and 6 % bile - Development in bouillon
at 2.4 u, 6.5 % salt - - Development in esculin
bouillon Catalase sample mm

Formation of diazety!) (Vogeä-Eroskauer;

Formation of sugars'

Thermoresistance
63 ° - 30 minutes ..
65 ° - 30 minutes
- 75 ° - 30th minutes

ferment glycose, sucrose and lactose

609824 / .0 943

/ ■

Proteolitic activity expressed in free amino acids by type, quantity

8 hours 48th St.

15 days 8 hours 48 hours 15 days

1. Cysteic acid - - - 2. X 1, 2, 3, 4, 6 +++++ ++++ Sp. ++++ - ί- 3. Aspartic acid 0.023 0.023 0.017 5 “Threonine o, o11 0.008 0.011 6. Serine 0.108 0.021 o, o15 7 · glutamic acid. 0.060 0.061 0.031 8. Proline traces 0.035 0.055 9. Alanine 0.035 0.032 10. Glucine 0.018 0.013 0.011 11. 1/2 cystine - - - 12. X 7 + + + 13. Valine traces traces 0.008 14. Methionine traces traces +, 15 isoleucine - - 0.006 16. Leucine traces traces 0.005 17th X 9th traces + . + 18. Tyrosine - traces 19. Phenylalanine - - traces 20.X 10, 11, 12 traces traces +++ 21. Lysine traces . traces +. 22. Histidine traces traces 0.015 23 ammonia + + + 24 “Arginine + - -

traces

X s "means unknown amino acids.

5 09824/0943

dab

INSPECTED

~ ⁷ Λ

2 · Lactobacillus

Nu NS 5, J7, 26 etc.

Characteristic values

NS

NU 37

Dalboki village Bulgaria

Hristene village - Bulgari

R - colonies S - colonies

short rods with volutine grains 5- 10 jx 4- - 8 ^ i;

Origin .

Mophology

a) Type of colony • Microscopic picture

Cell size

örganoleptischo estimate

a) Consistency '. slightly grainy

b) Aroma · _ milksavS? es' specific

c) Taste · pronounced lactic acid taste. Optimal development temperature - '. -4-5 ⁰ C development at 15 ° C

tt «ap ^u n
"50 ° C 23 hours
11 hours
9.30 hours 20 hours
10 hours
12.30 hours Coagulation moment. 7-8 hours 7-8 hours Limiting acidity "'* 14-1 ⁰ I. 176 ⁰ I. Growing in milk with
0.1% methylene blue ■ "- ■ ■ Development in bouillon
pH 9.2 -.

Development in broth with 2.4 and 6% bile. Development in broth with 2.4 and 6.5 % salt

Development in esculin broth

Reaction for hetero- and homofermentativity

Formation of ammonia from arginine

Thermal resistance 63 ° - 30 minutes 65 ° - 30 minutes 70 ° - 30 minutes

Development in milk and '* bouillon with phenol

a): in MRS broth with 0.1% Hionol 0.2% "

"b) Milk with 0.1% Hienol

It

homofermentative

W-

509824/0943

CQPY

'ORIGINAL INSPECTED.

0.1+% phenol

Fermentation of the sugars ferment glycose and lactose

Catalase sample ■

509824/0943

Proteolitical activity
given in free Ami ~
nosacids of the type and
Quantity according to

8 St. 48 S, t. 15 days 8 hours 48 hours, 15 days «

“Cystoic acid ■ ι +++++ 0.134 - -. - 2.X1, 2, 3, 4, 5, 6 0.034 0.179 - +++++ +++++ +++++ 3. Aspartic acid - - 0.225 0.043 0.084 ¹ 0.137 4. Methionine} 3ulfone 0.087 0.339 0.353 - . _ _ 5. Threonine 0.188 0.522 0.719 0.077 0.102 0.150 6 serine 0.228 1.037 0.575 0.290 0.303 "0.447 7. Glutamic acid 0.385 0.772 0.121 0.41.6 0.563 0.745 8, proline. 0.069 0.183 0.147 0.680 0.669 0.959 9. Glycine 0.164 0.135 0.015sp .0.038 0.060 10. Alanine - .- · traces 0.164 0.099 0.284 11, 1/2 cystine traces traces 0.375 traces - - ' 12. X 7 .0.224 0.583 0.079 traces traces traces 13. Valine 0.049 0.113 - 0.246 0.258 0.269 O ₈ 382 14, methionine 0.089 0.321 0.353 traces 0.023 0.045 15 isoleucine | 138 ' 0.511 • · + 0.052 0.095 0.127 16. Leucine ο + ν + 0.106 0.083 0.147 ' 0.191 17th X 9th 0.054 0.145 0.125 traces traces traces 18. Tyrosine 0.028 0.178 traces 0.015 • 0.021 19 · phenylalanine ₊₊ , + Sp. 0.210 0.028 0.057 0.076 20th X 10, 11th 0.0173 0.268 0.074 + Sp. ++ ++ 21. Lyßin 0.080 0.125 · + 0.015 0.053 0.071 22. Histidine + t 0.120 0.058 0.065 0.084 23 ammonia o, o74 0.176 Is amino acids + + ₊ 24. Arginine omerkunp ;: The quantity more free 0.034 0.048 0.070 in micromoles in1 ml koagi

lated milk.

X β "means unknown amino acids,

509824709A 3

ORIGINAL INSPECTED

-ίο

Characteristic values

# 144

No.

Novo selo village - Bulgaria Grastiza village - Bulgaria

origin

morphology

a) Type of colony.

Microscopic picture cell size

Organoleptic estimation

a) Slightly grainy consistency

b) aroma. lactic acid ■ specific

c) Taste - ·. pronounced lactic acid taste

Optimal development temperature 6-7 hours. Coagulation

R - colonies S - colonies

short sticks with volutine grains

Development "at

32C 37 ° c ⁰ hours
hours
Stlinden. . 7. -30 hours x 7-8.40 hours

Coagulation moment

Limit acidity

Growth in milk with 0.1% methylene blue

Develops in broth at pH 9.2

Development in broth with 2, 4 and 6% bile

Development in broth with 2, 4, 6, S ^ salt

Development · in esculin broth

Reaction for homo- and heteroformativity

Formation of ammonia from

Arginine '. -

Thermoresistance

63 °, .- 30 minutes' + 65 ° - 30 minutes +

70 ° - 30 minutes

Development in milk and bouillon with "phenol:

a) in MRS broth with <

0.1% phenol. · · +

0.2% "

Milk with

0.1% phenol '+

0.2% "+

19.30 hours 19.30 hours 10 hours 8/30 hours

6-8 hours

2% +

for everyone homofermentative

0.4%

Fermentation of the sugars catalase probo ferment glucose and lactose -509 82 4/0943

ORIGINAL

Protoolitischo activity c anßeßobon in: Crolen amino acids doi ¹ type and quality according to

48 St. 15

8 hours 48 hours 15

1. Cystic acid - - ++++++ 2. X 1, 2, 3, 4, 5, 6 ++++++ ++++++ 0.200 3. Aspartic acid 0.066 .0.098 -. 4. Methionine sulfone - - 0.199 5 .. threonine 0.096 j 0.128 0.486 6. Serine 0.268 0.312 1,380 7 * - glutamic acid 0.835 0.825 • 1.320 8 · proline 0.768 0.757 0.082 .9. Wisteria 0.020 0.049 0.319 10. Alanine 0.041 0.134 - 11. 1/2 cystine - - traces 12. X 7 traces traces 0.576 13- valine 0.356 0.345 0.076 14. Methionine o, o41 0.041 0.304 15 isoleucine 0.168 0.173 0.383 16. Leucine 0.198 0.217 traces 17th X 9th traces traces 0.085. 18. Tyrosine 0.050 0.050 0.148 19 · phenylalanine 0.067 0.084 ■ ++ 20th X 10, 11th ++. ++. 0.31? 21. Lysine 0.185 .0.177 0.184 22. Histidine 0.161 0.104 + 23. Ammonia + + 0.153 24. Arginine 0.077 0.088

X - "does not mean" known amino acids.

509824/0943

BATH ORIGINAL

COPY

From the described strains Streptococcus thermophilus and Lactobacterium bulgaricum, 300 combinations of yeast cultures are produced in the following way: Flasks each with 100 ml of sterile cow's milk at a temperature of 45 ° C (-1 ° C) are mixed with 1 ml of each strain Streptococcus thermophilus and Lactobacterium bulgaricum. The acidified milk (in the flask) is thermostated at + 45 ° C. The coagulation time is observed. The two-hour coagulation combinations are secreted and examined microscopically. The combinations exhibiting normal cell morphology of both strains are re-secreted. Ratio ^1: Subsequently, 100 ml of sterile sheep's milk or cow's milk Geraisch of sheep and in is 1 with 1% each of the separate combinations yeast cultures inoculated. Vaccination in the same medium takes place every week for 4 months. After a further 2 months, sterile cow's milk is inoculated with 1% of the corresponding yeast culture every week. During these 6 months, the coagulation time, the microscopic picture and the relationship between Streptococcus thermophilus and Lactobacterium bulgaricum are observed for each vaccination. If the cells of the two microorganisms do not change morphologically during this period and the · relationship between the two in the examined combination yeast cultures shows a stability, they are separated for a test under operating conditions •. Before this, the yoghurt produced under laboratory conditions of the individual combinations according to the characteristics listed below will be tasted.

Of the 300 combinations, 7 are singled out. In the 7 selected combinations, the strains exhibit Streptococcus thermophilus and Lactobacterium bulgaricum have a very good symbiosis and a stable relationship.

The evaluation of the new yeast cultures for the production of yoghurt is based on the following parameters:

S09824 / Q943

1. Microscopic picture of the yeast culture that immediately followed the coagulation is placed in a refrigerator at + 4 ° C with a retention time of 24 hours. The observed morphological Characteristics of Streptococcus thermophilus must correspond to well-developed and well-colored diplococci and the of Lactobacterium bulgaricum - compact, well-defined rods, isolated, in pairs or as short chains. The relationship between the two microorganisms must vary from 1: 3-1: 10.

2. The coagulation time is observed at + 45 ° C (± -1 ° C) using 1 % yeast culture,

3. Organoleptic estimation - it is carried out on samples prepared from homogenized cow's milk by pasteurizing the milk at 95 ° C for 30 minutes. The samples are cooled and acidified at +45 C with 1% of the yeast cultures examined. The samples are thermostated at 45 ° C. After coagulation, the samples remain at room temperature until the acidity level is 75 ° T, after which they are placed in the refrigerator at a temperature of + 4 ° C. The tasting takes place in the 24th hour after the samples have been pre-tempered at room temperature. The organoleptic estimation is made according to the following parameters: Appearance of the coagulum, color, consistency and structure, breakage, taste and aroma. The volatile acids, degree of acidity in Törner degrees and pH, the free amino acids - in terms of quality and quantity, the aromatic substances and the activity of the yeast cultures in liquid and dry lyophilized form are determined: a) using the limiting dilution method and reading the number of Streptococcus thermophilus and Lactobacterium bulgaricum and their ratio ^ according to MacKredi and b) during coagulation - for dry lyophilized yeast culture (0.1 g in 100 ml of sterile cow's milk) in 3.30 hours and for liquid yeast culture (1 ml in 100 ml sterile cow's milk) - whole milk or skimmed milk) in 2 hours. The yeast cultures for yoghurt production No. 5 - 12, 5 - 22, 37 - 12, 37 - 18, 26 - 12, 144 - 12 and 144 - 14 are characterized by the following parameters:

50 9 8 24/09 43

2A57303

Connexions

5-ia

5-22

37-12

37-10

Mikrookopisches Diplococci, medium length, compact © rods, isolated, Picture riaren or alo short kotton

Coaculation time for every 2 hours at 45 ° C (1 ml for 100 inl)

'' for every 3.30 hours (0.1 g dry for 100 ml)

Appearance of the coagulum

tight for allo

with echinuiernder smoothness for allo specific, well pronounced for allo

a) Consistency

bi fracture

c) Gosc) icack u »Aroma Froio Amino Acids t Loucin

rhenylalanine

Triptophan

Valine Uethionln - ιi »«

Tyrosine ++

Adino oleic acid + ■

Proline ++++

Alfaalanine + * +

Threonine +

Glutanic acid ++ glycine

Serine

Asparcinoma

Asparcin 'histidine

traces

Ornithine

Cystidine '

Oxyproline

activity

a) liquid yeast culture

St. Thermophilus 575 mill / ml 550 mill / ml Lb.Bulgaricum 211 kill / ml 242 mill / ml

b) dry Hefok'jiltur (lyophill,)

St. Thermophilus 4,500 ΓΑϋΙ / β .. 4,500 Mill / a 4,500 Mill / π .. Lb.Bulgaricum 45 mill / g 45 mill / ß 9.5 mill / g

volatile acidity 15.60 15 »^ 0 18.20

Mill / ml
Mill / ml

JpUTCJl

• H +++

580 hill /: 250 liill /:

2.5ΟΟ mil! / J 9.5 mill / f,

15.60

B0982A / U943

COPY

ORIGINAL INSPECTED

Connexions 26-12 144-12 144-14 Appearance of the coagulum di! cht for Kotten +++. a) liquid yeast culture 1,983 mill / ml . 2,500 mill / g well pronounced for, 100 ml) ++ • Microscopic Diplococci, medium length compact rods, single, i a) Consistency ml for 100 ml) St Thermophilus 497 mill / ml '9.5 Mill / K '+ '783 mill / m: image Baaren or as Icur ^ o b) breakage (0.1 ^ dry ■ "++ Lb.Bulgaricum b) dry yeast culture (lyophill.) 15.60 +++ 383 mill / m; Coagulation ^ zoit for every 2 hours (1 ++. , St Thermophilus . + Everyone . +. 3.30 hours Everyone Lb.Bulgaricum + f-r-H- ' for all . ++ 4,5OO mill / m: with shimmering smoothness for ++++ volatile acids ++ - ++++ 45 mill / g c) taste and aroma specific, +++ . t ++++ "15.20 .Free amino acids: +++++ Leucine + +++ ++++ Phenylalanine + Valine methionine Tyrosine traces Amino oleic acid Proline Λν ^Γ alanine traces traces Threonine Glutamine acid GIyzin Serine Aspar gins aur e ¹ Asparagine Histidine Ly s in Ornithine 1.300 mill / ml Cystidine 213 mill / ml Oxyproline Triptophan 4,500 mill / g activity 9.5 mill / g 17.60

Comment:

The yeast cultures 144-12 and 144-14. Show "with special technological Relationships a slimy habit and become therefore "in the reservoir production of yoghurt type" Mladost " used.

509 824/0943

COPY ORIGINAL INSPECTED

On December 1, 1973 the strains are: Lactobacterium bulgaric um

tribe No. 5 under No. 1 51 It ti 26th It Il 1 52 ti It 37 It Il 1 53 ti It 144 It Il 1 54

and
Streptococcus thermophilus

tribe No. 12th under No. 155 ti It 1.4 It Il 156 Il Il 18th It It 157 Il II. 22nd It H 158

at the State Institute of Control of Medicinal Products at the Ministry of Health, Boul. Vladimir Zaimov No. 26 has been deposited.

509824/0943

Claims (7)

Claims nj Process for the production of yeast cultures for real Bulgarian yoghurt, characterized in that the following new strains: Streptococcus thermophilus No. 12, 14, 18, 22 and Lactobacterium bulgaricum No. 5, 26, 37 and 144 were used will. 2. The method according to claim 1, characterized in that g e k e η n.-records, that one in combination the strain Streptococcus thermophilus No. 12 and the strain Lactobacillus bulgaricum No. 5 is produced on v / iron yeast culture No. 5 - '12. 3. The method according to claim 1, characterized in that one of the strain Streptococcus thermophilus No. 22 and Lactobacterium bulgaricum No. 5 strain containing yeast culture No. 5-22 is prepared. 4. The method according to claim 1, characterized in that one of the strain Streptococcus thermophilus No. 18 and Lactobacterium bulgaricum No. 37 strain containing yeast culture No. 37-18 is prepared. 5. ' Process according to claim 1, characterized in that one of the strain Streptococcus thermophilus No. 12 ^ and the strain Lactobacterium bulgaricum No. 37 having yeast culture No. 37-12 is produced. 5Q9824 / 0943 6. The method according to claim 1, characterized in that one of the strain Streptococcus therrnophilus No. 12 and yeast culture No. 144-12 containing Lactobacterium bulgaricum No. 7. The method according to claim 1, characterized in that one of the strain Streptococcus thermophilus No. 14 and strain No. 144 - Lactobacterium bulgaricum containing yeast culture No. 144-14 is prepared. 5098 2 4/0943 Blank page