DE3048438A1 - FERMENTED MILK AND METHOD FOR THEIR PRODUCTION - Google Patents

Description

The invention relates to a milk fermented by Bifidobacterium and a method for producing this fermented milk Milk.

Bifodobacteria are useful microorganisms that can be found in the colonize and grow in the human intestine. As for its physiological importance, it was confirmed that Bifidobacteria have the ability to inhibit the growth of pathogenic microorganisms by reducing them of the pH in the intestine caused by the appearance of lactic acid, acetic acid, formic acid etc. and that they are other have valuable skills. Due to widespread recognition of the valuable physiological abilities of Bifodobacteria have made up a portion of the dairy market for numerous products that contain living cells from Contain bifidobacteria.

In terms of bacteriological properties, however, bifidobacteria are different from those normally used Milk-fermenting lactic acid bacteria that were previously used to produce fermented milk. Unlike a culture of lactic acid bacteria for the dairy industry, as is the case in the usual production of fermented When milk is applied, the culture of Bifodobacteria is extremely difficult to conduct and the obtained culture product often has bad taste for the following reasons:

(1) Bifidobacteria show complicated and strict auxotrophy and grow only very poorly in a milk medium which does not contain a growth factor such as yeast extract.

(2) Although bifidobacteria show growth in a milk medium containing a growth factor

this growth factor generally becomes expensive and uneconomical and, moreover, becomes in culture receive a product with a bad taste. (3) While lactic acid bacteria for dairy needs if facultative anaerobic microorganisms belong, bifidobacteria belong to the obligatory ones Anaerobes that show no growth in the presence of oxygen.

Fermented milk containing bifidobacteria is therefore hitherto for example have been manufactured using the following methods:

(A) Production of fermented milk using common dairy lactic acid bacteria, separate Obtaining cultured cells from Bifidobacteria and adding the obtained cells to the fermented milk, and

(B) Adding some suitable growth factors for Bifidobacteria to the milk to facilitate the fermentation of the To enable milk through Bifidobacterium.

However, all of the above methods have disadvantages such as

(A) additional costs, tenting and labor that is caused by the separate culture and

(B) the impossibility, the occurrence of bad taste to prevent completely in the fermentation products.

The attempt to produce a milk fermented with the help of bifidobacteria in a simple, large-scale process, addresses the conflicting problems that bifidobacteria are difficult to grow in a pure milk medium is to be accomplished, however, that the fermented milk produced using a milk medium which contains some growth factors contains, is formed, such a bad sex

taste that it is not used as food can be. The invention is based on investigations with the aim of solving the difficulties described above to eliminate and to provide an economical method for fermenting milk with Bifidobacterium, which can be carried out on a large scale and results in a great tasting product.

The invention thus relates to a process for the production of milk fermented with the aid of bifidobacteria, which is characterized in that bifidobacteria and yeast are inoculated into a milk medium and in the form of a mixed culture grown under aerobic conditions.

With the help of the method according to the invention, a milk fermented with Bifidobacteria with good taste and Aroma produced by a process which can be easily carried out, which cannot be achieved with any of the known methods could be.

Other objects and features as well as advantages of the invention will be apparent from the detailed description below. . . '-..- ..

According to the invention, fermentation is carried out with the help of bifidobacteria Milk with good taste produced using a process according to which a milk medium with bifidobacteria and yeast is inoculated and mixed culture is carried out under aerobic conditions. By this invention As a result, the following effects are achieved:

(1) Bifidobacteria achieve vigorous growth in the milk medium, in which Bifidobacteria when they do being present alone will only achieve very poor growth.

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(2) The mixed culture gives the milk fermented with Bifidobacteria an aroma that none of the usual methods could be achieved.

(3) The culture of Bifidobacteria can easily be carried out under aerobic conditions in a pure milk medium which does not contain generally expensive growth factors.

(4) To carry out the method according to the invention, all devices can be used that were previously used for production of fermented milk with the help of lactic acid bacteria has been used for dairy needs are.

The invention thus with the help of bifidobacteria fermented milk with good taste accessible that with With the help of a method which can be easily carried out, which is not possible with any of the methods previously used reached v / earth. For the consumer who consumes fermented milk containing the bifidobacteria, arise beneficial physiological effects.

The species of the genus Bifidobacterium which can be used for the purposes of the invention are not subject to any particular restriction. Various newly isolated species of the genus Bifidobacterium, their resistant strains and mutant strains, as well long-known strains such as Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum etc. can be used for the purposes of the invention.

kwCh the species of yeast to be used are not subject to any special restriction. For example, species of the genus Saccharomyces, the genus Candida, the genus Debaryomyces, of the genus Hansenula, the genus Pichia, the genus Torulopsis and the genus Trichosporon can be used. Special!

yeasts which are capable of producing a good aroma in the course of the culture are desirable. A preferred one Example of yeasts which cause extraordinarily good effects when the method according to the invention is carried out, is the genus Saccharomyces.

Milk is used as the culture medium for carrying out the mixed culture of bifidobacteria and yeasts, which generally is used for the culture of lactic acid bacteria for dairy needs. In this milk medium, Bifidobacterium, when used alone, it will not achieve complete growth under aerobic conditions. It's for invention characteristic that vigorous growth of Bifidobacterium can be achieved in this milk medium. For examples for the type of milk that can be used for the purposes of the invention include whole milk, skimmed milk, concentrated milk Milk and reconstituted milk that can be made from dry milk or concentrated milk. Albeit the concentration of milk solids in this one milk to be used according to the invention is not subject to any particular restriction, it is generally desirable that a milk solids range of 3 to 30% is maintained. The milk medium according to the invention can numerous additives can be added, suitably in amounts small enough to maintain the properties the milk as a medium not to interfere with it. According to the invention, additives are those that have the purpose of To improve the aroma or taste, to impart color or taste, to increase the nutritional value, to increase the stability improve etc. Of course, the use of other additives that has nothing to do with growth and behavior also changes of Bifidobacteria have nothing to do with the method according to the invention.

The term "mixed culture" used in the invention refers to the condition of the culture under which the growth of Bifidobacteria progresses in the milk medium while yeasts are present in the milk medium. The mixed culture can therefore can be carried out by growing bifidobacteria and yeast in the form of a mixture and the milk medium this mixed starter is added or by culturing pure Bifidobacterium and pure yeast separately and adding each culture in predetermined proportions to the milk medium, or by adding to the milk medium first Starter of Bifidobacterium or a starter of yeast is added to start the culture and then, after expiration at an appropriate time, the other starter is added to the milk medium to continue the culture. The constant supply of the starters in the large-scale mass production of the fermented milk can be facilitated by conducting a transfer culture of the starters in mixed form.

Any of the microorganisms used as a starter, Bifidobacterium and yeast, is generally in an amount of 0.5 to 10% by volume, based on the culture medium, in this inoculated. The number of species of Bifidobacterium and yeast that are used does not have to be a single one be limited. It is possible to appropriately use each different species in combination.

As used herein, the term "aerobic conditions" refers to culture in the atmosphere without gas exchange or the like which serves to remove oxygen. For example, meet the culture conditions under which plants for the production of yogurt and other milk beverages using Lactic acid bacteria for general dairy use

are operated, the conditions according to the invention.

The cultivation temperature is suitably selected in the range of 20 to 4O ⁰ C. Normally, the cultivation is carried out at a temperature in the region of 37 ^° C., which favors the growth of bifidobacteria, and this culture is continued for a suitable period, which depends on the intended use of the fermented milk to be produced. If the taste originating from the yeasts is to be emphasized specifically, the cultivation or culture can be ^{carried out at a temperature in the range from 20 to 30 °} C., which is particularly suitable for the formation of aroma by yeasts. Optionally, the culture can first be carried out at a lower temperature which is suitable for the yeast and can then be continued at a higher temperature which is better matched to the growth of bifidobacteria, or according to a further embodiment the culture can first be carried out at a higher temperature and then done at a lower temperature.

It is possible to use the fermented milk prepared in the manner described above as a starter for the next Use fermentation cycles.

The fermented milk obtained using the procedure described above can either be used directly in unchanged form or after the addition of sweeteners, fruit juice and flavorings, as are generally recognized for foods such as yoghurt, or as starting material for the production of desserts, cakes , tablets or various other edible preparations. The fermented milk can of course also be converted into powdered products and used for the same purposes.

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The invention is explained in more detail below with reference to the experiments and examples described.

In all of the experiments and examples given below, the number of living cells is given as the value that per ml of the culture liquid was determined and the acidity is given by the number of ml of a 0.1 η aqueous sodium hydroxide solution, that for the neutralization of 10 ml of the culture liquid are required.

Attempt 1

The cultivation took place statically at 37 ^° C. under aerobic conditions for 24 hours. A 10% strength reconstituted skimmed milk, which had been treated in an autoclave ^{at 121 ° C. for 15 minutes, was used as the culture medium.} Bifidobacteria and yeasts which had previously been separately subjected to a pure culture were each inoculated into the medium in an amount of 10%. The culture liquid obtained by repeating the same inoculation in an amount of 10% twice in order to eliminate possible effects of the pure culture medium was used as a starter for the main culture. This starter was added to the culture medium in an amount of 10%, and the main culture was then carried out. The culture medium was sampled before and after the cultivation and examined to determine the changes in the number of living cells and the acidity. The change in the living cell number is shown in Table 1, and that in acidity is shown in Table 2.

TABLE 1

Change in the number of living cells (number of living cells / 1 ml of culture fluid)

* -—— ■ incubation time (h)
Tribe ' - . 5687 1 2365 0 24 B. bifidum C-1762, FERM-P 8.2 x 10 ⁶ 6.2 x 10 ⁶ B. breve, ATCC 15701 2365 9.4 x 10 ³ 1, OxIO ⁴ B. lonqum, ATCC 15707 5687 5, 4xlO ³ 6, OxIO ³ ATCC 2365 7, 8x10 ⁶ 2, IxIO ⁸ B. bifidum C-1762, FERM-P 1, 2x10 ⁵ 2, OxIO ⁶ Saccharomyces cerevisiae, ATCC 7, OxIO ⁵ 4.6xlO ⁷ 1.7x10 ⁵ 3.7x10 ⁶ B. breve, ATCC 15701 ATCC 1, OxIO ⁵ 3.2 x 10 ⁶ Saccharomyces cerevisiae, 5.6 x 10 ⁵ 8.6 x 10 ⁶ B. lonqum, ATCC 15707 Saccharomyces cerevisiae,

Annotation :

FERM: Fermentation Research Institute, Agency of Industrial Science and Technology

ATCC: American Type Culture Collection

TABLE 2 Change in Acidity (ml)

~~~ -—----_________ Incubation time (h) '
Stem "" —— ~ ________ ^ 0 24 B. bifidum C-1762, FERM-P 5687 2.0
1.9
1/8 2.5
1.9
1.8 B. breve, ATCC 15701
B. lonqum, ATCC 15707 2.5 6.7 2.1 4.6 B. bifidum C-1762, FERM-P 5687 2.1 4.1 Saccharomyces cerevisiae, ATCC 2365 B. breve, ATCC 15701
Saccharomyces cerevisiae, ATCC 2365 B. lonqam, ATCC 15707 Saccharomyces cerevisiae, ATCC 2365

It is clear from Tables 1 and 2 that there is practically no change in the number of living cells and the acidity observed when bifidobacteria were grown on their own. It can also be seen that in the mixed culture from Bifidobacterium and yeasts in comparison with the pure culture the number of living cells of Bifidobacterium noticeably was increased and the acidity was increased. In all approaches to mixed culture, a pleasant, well-balanced taste was achieved educated.

Attempt 2

In a culture medium, which had been treated at 121 ⁰ C in an autoclave for 15 minutes washed Zellsüspensionen were inoculated by separately cultured different species of Bifidobacteria and yeasts in an amount of 2% as a starter and then under aerobic conditions of a static culture at 37 ⁰ C subject. During the course of the culture, samples of the culture were taken at fixed time intervals to determine the change in living cell counts and acidity with the lapse of time.

In some trial runs, 10% reconstituted skimmed milk was used as the culture medium. In other experiments, 10% reconstituted skimmed milk was used, to which an amount of yeast extract extracted from the yeast used as a starter by a conventional method was used
had been added.

an amount corresponding to 10 yeast cells per ml of the medium

The change in the number of living cells is shown in Table and that in acidity is shown in Table 4. The results des.organoleptic tests of taste, fermented Milk are listed in Table 5.

TABLE 3

Change in the number of living cells (number of living cells / 1 ml

Culture fluid)

Culture medium Il Tribes Incubation time (h) 17th 24 Skimmed milk Il B. bifidum C-1762, FERM-P 5687 0 2.9 x 10 ⁷ 3, IxIO ⁷ Il 1, OxIO ⁷ 9, IxIO ⁵ l, 3xlO ⁵ ' Il B-. infantis, ATCC 15697 7.3 x 10 ⁶ 6.3xlO ³ 3.2xlO ³ Il • 1.3x10 ⁴ 5.5 x 10 ⁸
2, OxIO ⁷ 4.9 x 10 ⁸
1.3x10 ⁷ Il B. longum, ATCC 15707 9, IxIO ⁶
1. 9x10 ⁷ 4, IxIO ⁸
2, OxIO ⁶ 4, OxIO ⁸
4.5 x 10 ⁶ Il 9.8 x 10 ⁶
8.3 x 10 ⁵ 1, IxIO ⁷
2.9 x 10 ⁶ 4.2 x 10 ⁷
3.5 x 10 ⁶ Il B. bifidum C-1762, FERM-P'5687 6.6xlO ⁶
2.2 x 10 ⁶ 8.9 x 10 ⁵
3.6xl0 ⁶ 1.3x10 ⁸
l / 5xlO ⁷ Hansenula anomala, IFO 0145 1.3x10 ⁴
8f4xlO ⁵ 2, OxIO ⁸
2.2 x 10 ⁴ 3.8 x 10 ⁸
5.4 x 10 ⁴ 8.4 x 10 ⁵
1.6x10 ⁴ 5, 9x10 ⁵
5, 3xlO ⁵ 2.8 x 10 ⁷
7.6 x 10 ⁵ B. bifidum C-1762, FERM-P 5687 3, OxIO ³
8, OxIO ⁴ Pichia. farinosa / IFO 0193 B. infantis, ATCC 15697 Sacchafomyces cerevisiae, ATCC 2365 B. lonqum, ATCC 15707 Kluyve.romyces fraqilis, IFO 0288 B. bifidum C-1762, FERM-P '5687 Schizosaccharomyces pombe, ATCC 2476 B. lonqum, ATCC 15707 Saccharomyces heteroqenicus CW-27,
FERM-P 568 6 . ·

TABLE 3 (continued)

Skimmed milk with additive.
set of yeast
extract
(Hansenula anomala. B. bifidun »C-1762, FERM-P 5687 t
ι, ΐχίο ⁷ 2.9 x 10 ⁷ 3, IxIO ⁷ IPO 0145) l, 2xlO ⁷ 3.3 x 10 ⁷ 3.3 x 10 ⁷ Skimmed milk with zu
set of yeast B. bifidum C-1762, FERM-P 5687 7.9 x 10 ⁶ 1, OxIO ⁵ extract (Pichia fari
nosa, IFO 0193) 1, IxIO ⁴ 8.6xlO ⁵ 2.4xlO ³ Skimmed milk with zu
set of yeast extract
(Saccharomyces cere- B. infantis. ATCC 15697 7.9 x 10 ³ visiae, ATCC 2365) Skimmed milk with zu
set of yeast extract
(Kluyveromvces
fragilis, ~ IFO B. lonqum, ATCC 15707 0288)

Note :

Skimmed milk: 10% reconstituted skimmed milk

IFO: Institute for Fermentation, Osaka, Japan

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TABLE 4

Change in acidity (ml)

Culture medium Tribes Incubation time (h) 17th 24 Skimmed milk B. bifidum C-1762, FERM-P 5687 0 1.8 2.6 Il B. infantis, ATCC 15697 1.6 1.9 1.9 Il B. lonqum, ATCC 15707 1.9 1.7 1.7 Il B. bifidum C-1762, FERM-P 5687 1.7 2.5 3.3 η Hansenula anomala, IFO 0145 1.6 2.4 3.3 Il B. bifidum C-1762, FERM-P 5687 1.6 2.1 2.7 Il Pichia farinosa, IFO 0193 1.9 3.4 4.3 Il B. infantis, ATCC 15697 1.8 2'8 4.3 Il Saccharomyces cerevisiae, ATCC 2365 1.9 2.0 2.5 B. longum, ATCC 15707 1.9 Kluyveromyces fragilis, IFO 0288 B. bifidum C-1762, FERM-P 5687 Schizosaccharomyces pombe, ATCC 2476 B. lonqum, ATCC 15707 Saccharomyces heterogenicus CW-27, FERM-P 5686

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TABLE 4 (continued)

Skimmed milk with zu
set of yeast extract
(Hansenula anomala,
IFO 0145) B. bifidum C-1762, FERM-P 5687 1.6 2, 0 3.2 Skimmed milk with zu
set of yeast extract
(Pichia farinosa,
IFO 0193) 1.6 1.8 2.6 Skimmed milk with zu
set of yeast extract
(Saccharomyces cere-
visiae, ATCC 2365) B. bifidum C-1762, FERM-P 5687 1.9 1.9 1.9 Skimmed milk with zu
set of yeast extract
(Kluyveromyces fra-
gilis, IFO 0288) 1.7 1.7 1.7 B. infantis, ATCC 15697 B. longum, ATCC 15707

Note : Skimmed milk: 10% reconstituted skimmed milk

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TABLE 5
Organoleptic test of the taste of fermented milk

Culture medium Il Tribes Taste of the
mentation product Total be
evaluation Skimmed milk Il B. bifidum C-1762, FERM-P 5687 weak fermenta
tion smell, no aroma
ma bad » B. infantis, ATCC 15697 Il Il Il B. lonqum, ATCC 15707 Il Il Il B. bifidum C-1762, FERM-P 5687
Hansenula anomala, IFO 0145 aromatic aroma
the fermentation Well H B. bifidum C-1762, FERM-P 5687
Pichia farinosa, IFO 0193 Il Il H B. infantis, ATCC 15697 Il Il n Saccharomyces cerevisiae, ATCC 2365 Il Il Il Il B. longum, ATCC 15707
Kluyveromyces fraqilis, IFO 0288 Il Il B. bifidum C-1762, FERM-P 5687
Schizosaccharomyces pombe, ATCC 2476 B. lonqum, ATCC 15707
Saccharomyces heterogenicus CW-27,
FERM-P 5686

TABLE 5 (continued)

Skimmed milk with zu
set of yeast extract
(Hansenula anomala,
IFO 0145) B. bifidum C-1762, FERM-P 5687 weak fer
mentationsge
smell, no
Aroma bad Skimmed milk with zu
set of yeast extract
(Pichia farinosa,
IFO 0193) M. Il Skimmed milk with zu
set of yeast extract
(Saccharomyces cere-
visiäe, ATCC 2365) B. bifidum C-1762. FERM-P 5687 Il Il Skimmed milk with zu
set of yeast extract
(Kluyveromyces fra-
gilis, IFO 0288) Il Il B. infantis. ATCC 15697. B. lonqum, ATCC 15707

Note :

The skimmed milk used was 10% reconstituted skimmed milk. (The organoleptic test was repeated three times and by 12 members one Expert group carried out.)

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As is clear from Tables 3, 4 and 5, the number of living cells of Bifidobacteria decreased rapidly or increased only very slightly over the course of the incubation period and the acidity changed only very much slightly or very slightly increased when bifidobacteria were bred for themselves.

As for the taste and aroma of the fermented milk, the fermentation odor was faint and substantial no flavor was formed.

When an extract obtained from the yeast used as a starter was added to the medium, the tendency was as follows Change in the number of living cells of Bifidobacterium and the acidity, as well as the aroma and taste of the fermented ones Milk similar to the case where the medium did not contain yeast extract.

When the same strains of Bifidobacterium and yeast one Were subjected to mixed culture, the number of living cells of Bifidobacterium was greatly increased and, moreover, the Acidity increased. In all approaches to mixed culture, the fermentation products had an aromatic taste and aroma educated.

example 1

An Erlenmeyer flask with a capacity of 5 l, which contained 3 l of a 10% strength reconstituted skimmed milk, was sealed with aluminum foil and treated in an autoclave ^{at 121 ° C. for 15 minutes.} Then it was cooled to 37 ⁰ C. According to the procedure of Experiment 2, Bifidobacterium bifidum C-1762, FERM-P 5687 and Saccharomyces cerevisiae, ATCC 2365, which had been cultured separately, were inoculated into the medium and subjected to static culture for 24 hours

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subjected to aerobic conditions to produce fermented milk containing both of the above Contained microorganism strains. In the fermented milk formed, the number of living cells of B. bifidum was

8 6

2.9 10 cells / ml and that of S. cerevisiae 1.6 χ 10 cells / ml and the acidity was 7.1 ml. The fermented milk showed an aromatic taste.

Example 2

A mixed culture of the strain Bifidobacterium longum, ATCC 15707 and the strain Kluyveromyces fragilis, IPO 0288, was in the The procedure of Example 1 was carried out with the formation of a fermented milk.

In the fermented milk formed, the living cell counts of B. longum were 1.3 × 10 cells / ml and of K. fragilis 1.2 10 cells / ml and the acidity corresponded 4.4 ml.

The fermented milk had an aromatic taste. By freeze-drying 1 l of this fermented milk were made about 90 g of dry milk.

Claims (2)

r ε, -, .- · .. · SHIP ν. FÜNER STREHL SCHÜBEL-HOPF EBBINGHAUS FINCK MARIAHILFPLATZ 2 & 3, MÖNCHEN 9O POST ADDRESS: POST BOX 95Ο16Ο, D-8OOO MÖNCHEN 95 AS PROFESSIONAL REPRESENTATIVE BEFORE THE EUROPEAN PATENT OFFICE (1964-1978) KARL LUDWlQ SCHIFF478. CHEM. DR. ALEXANDER V. FÖNER DIPL. ΙΝΘ. PETER STREHL DIPL. CHEM. DR. URSULA SCHÜBEL-HOPF DIPL. INTO THE. DIETER EaBINQHAUS DR. INQ. DIETER FINCK TELEFON (OSO) 48 2004 TELEX B-a3SSS AURO D TCLBORAMME AUROMARCPAt MUNICH CALPIS SHOKUHIN KOGYO DEA-13 494 KABUSHIKI KAISHA December 22, 1980 Fermented milk and its production process. PATENT CLAIMS 1. Fermented milk obtained by fermenting a Milk medium with a mixed culture of a strain of the genus Bifidobacterium and a yeast. Process for the production of fermented milk with the aid of Bifodobacterium, characterized in that a milk medium with a strain of the genus Bifodobacterium and © in © r Haf © is inoculated and subjected to a salmon culture under aerobic conditions.