AU649424B2 - Method of preparing milk-fermented food - Google Patents

Description

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COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 Form COMPLETE SPECIFICATION FOR OFFICE USE Short Title: Int. Cl: Application Number: Lodged:

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c* C Complete Specification-Lodged: Accepted: Lapsed: Published: Priority: Related Art: TO BE COMPLETED BY APPLICANT Name of Applicant: KABUSHIKI KAISHA YAKULT HONSHA Address of Applicant: 1-19, Higashi Shinbashi 1-chome, Minato-ku, TOKYO, JAPAN Actual Inventor: Masako Yajima; Shinji Hashimoto; Taketsugu Saita and Kunio Matsuzaki Address for Service: GRIFFITH HACK CO 71 YORK STREET SYDNEY NSW 2000 Complete Specification for the invention entitled: METHOD OF PREPARING MILK-FERMENTED FOOD The following statement is a full description of this invention, including the best method of performing it known to us:- 10803-AO:GJH:RK 8542A:rk C I02 TITLE OF THE INVENTION METHOD OF PREPARING MILK-FERMENTED FOOD BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a method of producing milk-fermented food, and more particularly, to a method of producing milk-fermented food by fermenting milk with bifidobacterium and /or lactic acid bacteria, and also to milk-fermented food containing bifidobacteria and/or lactic O acid bacteria.

It "Milk" herein means whole milk or skimmed milk obtained from cows, goats or like animals, or reconstituted milk 9* prepared from powdered milk made from these two kinds of milk, or a mixture of all the kinds of milks mentioned I$ above. "Fermented food includes beverages such as lactic acid bacteria beverages and those beverages processed by heating and sterilization after fermentation. "Acidity" means volume(ml) of O.1N-NaOH per ml to neutralize 10 ml of sample.

DESCRIPTION OF THE PRIOR ART Bifidobacterium is one of the most dominant bacterial flora in the intestine of infants and most healthy adults 1A- S...i S

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0 and seems to contribute to the maintenance of a normal flora of intestine. It has been reported that the administration of these live bacteria to human or domestic animals suffering from diarrhea or patients infected with superinfection remarkably improves their symptom.

Accordingly, yogurt, milk beverages and sweets which contain live Bifidobacteria have been developed and also have been commercially available in order to improve people in their own health by allowing regular ingestion of such bifidobacteria.

l.fidobacterium is, however, originally anaerobic and thus readily affected by oxygen and easily destroyed in a milk-fermented product having high acidity. Lactic acid bacteria has a tendency to die out, but not so readily as bifidobacterium when pH1 falls during fermentation.

In milk-fermented food products, It is necessary to maintain the numbers of viable cell at a high level at least for two or more weeks, however, bifidobacteria and lactic acid bacteria will die out because of the decrease in pH caused by their own generation of organic acids. Such qualities limit their acidity to a low level in the milkfermented food obtained by use of the bacteria. Also, it is difficult to maintain high levels of viable cell count of the product for long period even at moderately suppressed acidic condition. Therefore, a more improved taste cannot 1'~ 2- I f be pursued by decreasing the pH of the food. High numbers of viable cells are also difficult to maintain for long periods when the acidity is suppressed in milk-fermented food.

In addition, bifidobacteria requires some growth promoters to obtain sufficient growth in pure milk-medium.

Conventionally, there have been growth promoters such as yeast extract, sulphur content amino acids (for example, cystein, methionine and the like soybean peptide (refer to Monthly Food Chemical, 8, 64, 1988) and so on. In many

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cases these substances cannot be used in sufficient amounts because of their own characteristic taste.

The growth accelerating effect of soybean milk is disclosed in Japanese Patent Publication Nos. 45-9822, 51- 14256 and 55-89390, however, soybean milk has its own ft e peculiar smell and thus changes for the worse in taste are unavoidable when sufficient amounts of soybean milk are used.

A method is disclosed in Japanese Patent Publication No. 59-179064, wherein soybean milk was neutralized by calcium hydroxide in the presence of calcium dichloride after the removal of its protein by the addition of phosphoric acid or phosphoric acid salts, and then it was precipitated by heating and desalting, thereby condensing the fraction of growth promoting substance for 3 bifidobacteria contained in the soybean milk. The fraction of the growth promoting substance for bifidobacterium thus obtained was compcied of 7.2% of crude protein, 77.3% of carbohydrate and 15.5% of crude ash, the carbohydrate was composed of 51.9% of sucrose, 27.0% of stachyose and 11.2% of raffinose. It has been also reported that a highly activated growth promoting substance for bifidobacterium was obtained b( purifying this fraction with the use of a membrane separation method and, which was made of 12.3% of raffinose, 84.5% of stachyose and 3.2% of verbacose (Japanese Patent Publication No. 60-66978). Japanese Patent Publication No. 62-155082 disclosed a growth promoting substance for bifidobacterium which was directly extracted from defatted soybean meal by using a 20 to 60 w/v% of alcoholic solution. The main component of the accelerator was an oligosaccharide which was composed of 82.5% to 85.1% of carbohydrates, 6.9% to 7.5% of protein, 1.0% to 2.8% of lipid and 6.3% to 6.4% of ash. These bifidobacterium accelerators mainly made up of oligosaccharides are so difficult to digest that they go down to a lower area of the intestines, thereby effectively stimulating the growth of bifidobacterium in an entero carbon source-deficient environment. These oligosaccharides are, however, not so effective for stimulating the growth of bifidobacterium in a culturing process of producing milk-fermented food -4because of the simultaneous presence of other readily available saccharides, lactose or glucose or so on.

Various efforts have been exerted to shorten the fermenting period when employing a lactic acid bacterium, accordingly, more effective means have been expected to emerge.

SUMMARY OF THE INVENTION In view of the foregoing, the present invention is directed to alleviating at least one of the above disadvantages of production of milk-fermented food.

More specifically, one embodiment of the present nvention provides a method of producing milk-fermented Mood, wherein the fermenting period for producing the milkt ermented food which is fermented by bifidobacteria and/or .:.actic acid bacteria is shortened to increase the numbers .of live bacteria in the product, increasing their survival .::-rate and maintaining the numbers thereof for a long period, :,-.hereby providing milk-fermented food with fresh flavor ased on high acidity.

In a further embodiment, the present invention provides a milk-fermented food manufactured by the above producing method wherein the survival rate of the bifidobacteria is improved so as to maintain the numbers of viable bacteria.

6 According to a first aspect of the present invention, there is provided a method of producing a fresh flavoured, high acidity milk-fermented food comprising the steps of: inoculating and culturing in milk bifidobacteria or lactic acid bacteria or a combination of said two bacteria, said milk having added thereto from 0.1 w/v% to w/v% of an isolated soybean protein with respect to the volume of said milk, wherein the method provides a shortened culturing period and increases the numbers and survival rate of live bacteria in the food.

According to a second aspect of the present invention, there is provided a method of producing a fresh-flavoured, high acidity milk-fermented food comprising the steps of: inoculating and culturing in milk bifidobacteria or lactic acid bacteria or a combination of said two bacteria, and then o 20 adding to said culture from 0.1 w/v% to 1.0 w/v% of an isolated soybean protein with respect to the volume of the culture obtained by culturing said bacteria to produce the milk-fermented food, 2wherein the method provides a shortened culturing 25 period and increases the numbers and survival rate of live bacteria in the food.

According to a third aspect of the present invention there is provided a milk-fermented food produced by a o. method according to the first or second aspects of the 30 invention, BRIEF DESCRIPTION OF THE DRAWINGS Fig.l is an illustration of a graph showing results of Experiment 1; and

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Fig.2 is an illustration of a graph showing the time course of numbers of viable bacteria in products during storage at 10° C, respectively as in Examples 4 to 6 and Comparative Examples 1 to 3.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE

INVENTION

The present invention is illustrated hereunder in more detail. The method of the present invention can be performed in a conventional way with the species of bacteria, milk culture medium and fermenting conditions, specified according to the kinds of milk-fermented food to Sproduced, except that bifidobacLeria or lactic acid acteria or a combination of these tw6 bacteria are 'jnoculaed into and cultured in the culture medium composed t inly of milk to which was added isolated soybean protein.

The isolated soybean protein in this invention is :prepared by the neutralization and drying of soybean ::protbein that was gathered after precipitation of protein soybean milk by adding an acid to the soybean milk ',.obtained from defatted soybean by extraction with water.

isolated soybean protein can be used since it is being mass-produced and is commercially available in these days.

The isolated soybean protein can be arbitrarily added to a milk culture medium for culturing the above-mentioned bacteria. It is, however, preferable to dissolve the 7 soybean protein in water together with a culture medium composed mainly of milk, for example, culture medium materials such as powdered milk, and to heat-sterilize the resulting mixture.

The amount of addition of the isolated soybean protein to the culture medium composed mainly milk is preferably approximately 0.1 w/v% to 7.0 and more suitably S approximately 0.1 w/v% to 5.0 If this protein is added to the culture medium in greater amounts, improvimfients in the growth accelerating effect and survival rate effect are not appreciable, but on the contrary, this will provide products with a characteristic soybean odor in certain kinds of products, so high addition rates of the soybean protein exceeding the above-mentioned 6eI@ range are not very meaningful except when trying to regulate a nutritive balance with the soybean protein.

The growth of the bacteria proceeds remarkably fast and I generation of acids is also accelerated by adding the isolated soybean protein, thereby cultivating time in reaching a pre-determined pH is greatly shortened.

Furthermore, the generated amount of organic acids such as acetic acid, lactic acid and the like, which are metabolites generated during the growth of bifidobacteria or lactic acid bacteria, increases when the isolated soybean protein is added to the cultured medium. The -8original acid and base buffering action by the isolated soybean protein itself, however, restrains a decrease in pH so that resultant pH of the culture with isolated soybean protein is not so low but the acidity thereof is higher than culture without isolated soybean protein. Such effect may provide a milk-fermented food with high acidity without decreasing the survival rate of the bacteria used in the fermentation.

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Oa* Other arbitrary growth accelerators may be usable together with an isolated soybean protein as long as the above-mentioned excellent characteristics of the soybean protein are not spoiled. Specifically, a yeast extract is suitably preferred when added to a medium in small amounts without exerting bad effects on the flavor of food. The growth of the bacteria is highly accelerated when the isolated protein is added to the medium together with the yeast extract. The amount of addition of the yeast extract to the milk culture medium is preferably approximately

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0.01w/v% to O.lw/v%.

Further, in the method of the present invention, the isolated soybean protein may be added to the bacterial culture after discontinuing cultivation when acidity of the inoculate reaches a pre-determined level. Protein is added to th,. same medium during processes such as homogenization, mixing with other milk-fermented substances, and addition 9 of sweetenings, seasonings or spices to the inoculate and dilution of the same according to the kind of final product.

An addition of the isolated soybean protein to the bacterial culture after cultivation can increase the survival rate of bacteria in milk-fermented food fermented with bifidobacterium or lactic acid bacteria, and also can produce products having a fresh tasty flavor.

0* In such a case, the addition of the isolated soybean protein to the culture is approximately O.lw/v% to and is preferably approximately O.lw/v% to l.Ow/v%.

If the isolated soybean protein is added to the culture medium in greater amounts, effects in improving the survival rate of bacteria are not appreciable, but on the **9e contrary, this provides some kinds of products with a characteristic soybean odor.

The product may be packed after processes such as homogenization, mixing with other milk-fermented substances, addition of sweetenings, seasonings or spices, dilution of the inoculate and sterilization by heating according to the kind of final product.

It is apparent from the foregoing that the method disclosed in this invention can shorten the time need to produce milkfermented food fermented with bifidobacterium or lactic acid bacteria, and also can improve the viable numbers of live bifidobacteria, 10 enabling its survival rate thereof toincrease. Since the bacterial culture with isolated soybean protein has a higher pH level than that of a conventional one with no isolated soybean proteiin added, at the same acidity, the pH at the completion of cultivation can be set at a high level without worrying about the taste becoming unsavory due to unsufficient acidity, thereby improving the survival rate of bifidobacteria and also providing a tasty milk-fermented food which maintains relatively higher numbers of living bacteria for long period.

The isolated soybean protein employed in the present invention does not have as strong a soybean odor as soybean milk, and the weak soybean odor is sufficiently removed during fermentation. Also it has ncne of bitter taste peculiar to peptides. Accordingly, sufficient amounts of the soybean protein can be used without fear of adversely effecting taste, thereby creating excellent effects and also contributing to an improvement of the nutritive 4 balance of the milk-fermented food.

EXAMPLES

Methodrs of the present invention are illustrated with reference to the following examples, but the invention is not intended to be limited only to these following examples.

11- The isolated soybean protein employed in each example was Pujipro CL made by Fujiseiyu Co., LTD.

Experiment 1 Bifidobacteriumn breve YIT4010 was inoculated in a milk culture medium (10w/v% of skim milk powder, 0.03w/v% of yeast extract) which was prepared anaerobically under flushing nitrogen gass, and cultured at 370 C overnight. The culture solution thus obtained was specified as a starter.

i 3.2 w/v% of isolated soybean protein and/or 0.05w/v% of yeast extract was added to a basic milk culture medium S (5w/v% of whole milk powder, 12w/v% of skim milk powder).

The culture medium thus obtained was inoculated with lv/v% of starter and cultured at 340 C. During cultivation, pH in the culture was measured, and the growth of the g bifidobacteria was compared with pH as an indication.

The results are illustrated in Fig.l. Further, none of the culture were found to have any deterioration of flavor.

As will be clear from Fig.l, the growth rate of bifidobacteria was remarkably promoted by addition of the isolated soybean protein and was further promoted by the simultaneous use of the yeast extract. It is impossible to compare the growth accelerating effect of the isolated soybean protein (ith that of the yeast extract at the 12 substance level since the adding concentrations of these two solutions are different.

However, further yeast extract is added to the culture medium, the flavor thereof is spoiled, and therefore, it is clear that the isolated soybean protein is superior in achievable growth promoting effect without adversely affecting flavor.

Experiment 2 S, The starters were prepared by using Bifidobacterium 0 breve YIT4010(I), Bifidobacterium bifidum YIT4007(II), 0 Bifidobacterium bifidum E319(III), Bifidobacterium longum 6* YIT4035(IV) and Bifidobacterium longum ATCC15707(V), respectively in a similar manner as in expe'iment 1.

The basic culture medium used in this experiment was composed of 13w/v% of whole milk powder. This basic culture i. 2 was employed as a control culture. Another culture medium with a growth promoting substance added was prepared by the

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addition of 0.2w/v% or 1.0w/v% of isolated soybean protein and 0.03w/v% of yeast extract. The culture media thus obtained were inoculated with 2v/v% of bifidobacteria starters, respectively and were cultured at 370 C for 16 hours. The growth in the culture media were compared using the pH of culture solution as an indication. The results were illustrated in Table 1.

13 Table 1. pH of culture solution after 16hours of cultivation at 37oC Growth accelerating Biidobacterium Bifidobacterium substance yeast Isolated soybean extract protein 0 0 5.48 5.06 5.77 6.21 5.74 0.03 0 4.88 4.75 5.44 6.03 5.10 0 0.2 5.34 4.73 5.60 6.05 5.62 0.03 0.2 4.75 4.67 5.41 5.24 5.43 0 1.0 4.67 4.61 5.41 5.64 5.04 0.03 1.0 4.36 4.57 5.27 5.01 5.08 ease *0 0 0 0 00 ak 0041

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The four out of the five sample strains show more or less similar tendencies, but Bifidobacterium longum ATCC15707(V) has shown a different tendency. Namely, bifidobacteria grew slowly in the basic culture but the growth of bifidobacteria was stimulated when the yeast extract or the isolated soybean protein was added thereto.

When the concentration of the isolated soybean protein was the growth rates of the bifidobacteria were the same or slightly inferior to those rates when the yeast extract alone was added thereto. When the isoliced soybean protein concentration was the growth rate of the bifidobacteria showed better results than in the former case. The growth rates were further stimulated when both 14 the isolated soybean protein and the yeast extract were added to the basic culture medium. Specifically, Bifidobacterium breve YIT4010(I) and Bifidobacterium longum YIT4035(IV) exhibited this tendency. Bifidobacterium longum ATCC15707(V) showed a sharp effect in growth rate when the yeast extract alone was added to the basic medium.

Simultaneous addition of the isolated soybean protein to the medium did not stimulate the growth of the bifidobacteria. However, the growth thereof was still stimulated by the addition of the isolated soybean protein

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alone.

Example 1 *0 Culture media were prepared by adding an isolated soybean protein to a basic culture medium which contained of whole milk powder and 12w/v% of skim milk powder.

The isolated soybean protein concentration of the medium attained 3.2w/v% or 4.8w/v% A control culture rrmdium was prepared by adding 0.03w/v% of yeast extract to the basic S culture medium without adding the isolated soybean protein.

Culture media were inoculated with lv/v% of a starter a' of Bifidobacterium breve YIT4010(B. breve) stock prepared in a similar manner to Experiment 1, and were cultured at 370 C until the pH of such resulting culture solution attained 4.6.(the yeast extract was added to the control culture medium because bifidobacteria showed a slow growth 15 rate in a culture medium having no growth accelerator, and also could not be fermented to attain a pH of 4.6 for comparing their survival rates at the same pH. The survival rate of bifidobacteria did not change for the worse by the addition of the yeast extract).

3 volumes of the bifidobacterium culture solution thus obtained was mixed with 2.3 volumes of the basic culture medium, 0.55w/v% of pectin and 0.4w/v% of gelatin were added to the mixture, and water was added for preparing a total of 10 volumes of yogurt-like milk-fermented food.

This product was preserved at 100 C for 15 days, and the

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results of the change in the viable cell number of bifidobacteria are shown in Table 2.

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16- Table 2 Decrease in viable cell number of B. breve by preservation Concentration of viable cell number of B.breve Isolated soybean Survival protein r in B.breve culture Immediately After 15 days ate after production 0 9.6 X 108 2.8X 107 2.9 3.2 2.3 X 109 3.9 X 108 17.0 4.8 2.5 X 109 5.9 X 108 23.6 *50S

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0 0 The food contained isolated soybean protein food had entirely no soybean odor when 3.2w/v% of isolated soybean protein (lw/v%of concentration in food product) was added, but rather there was an increase in acidic taste and in the feeling of freshness, therefore to excel in flavor. When the isolated soybean protein itself was dissolved in water, it had a soybean odor. However, such odor had been removed during fermentation due to the bifidobacterium. As the concentration of the isolated soybean protein increased, soybean odor had generated gradually, but smelled only slightly even when added with 4.8w/v% concentration in the food product).

Example 2 17 A culture medium was prepared by adding an isolated soybean protein to the basic culture medium which contained w/v% of whole milk powder and 8 w/v% of skim milk powder until the isolated soybean protein concentration of the medium attained to 2.18 The basic culture medium was employed as a control culture medium to which an isolated soybean protein was not added. Both culture media were inoculated with 0.5 v/v% of starter of a lactic acid bacterium (Streptococcus thermophilus YIT2021) prepared in a manner similar to Experiment 1, and then were cultivated at 370 c until the pH of the culture attained 4.55.

ft S 9 It took 23 hours to cultivate the culture medium to which an isolated soybean protein was not added, but took *see only 14 hours and 45 minutes to complete cultivation in a culture medium to which an isolated soybean protein was added, thereby the cultivation time to ferment could become greatly short. The viable cell number of B.breve in the culture were 1.3X 10 9 CFU/ml when the isolated soybean S* S protein was not added, and were 3.OX109CFU/ml when the isolated soybean protein was added.

0 S 2.3 volumes of the latic acid bacterial culture thus obtained was mixed with pectin and gelatin until 0.55w/v% and 0.4w/v% concentrations were attained, respectively in the medium. A yogurt-like milk-fermented food was prepared 18 by adding water to the resulting culture medium until this medium attained a total of 10 volumes.

The transient in the numbers of live bacteria by preservation was examined after this product was preserved at 100 c for 15days. There was almost no change in numbers of viable cells.

Example 3 A culture medium was prepared by adding an isolated soybean protein to a basic culture medium which contained 16w/v% of skimmed milk powder and 0.05w/v% of yeast extract O until the isolated soybean protein concentration of the S* Smedium attained 0.lw/v% or 0.2 A basic culture medium to which the isolated soybean protein was not added, was employed as a control medium. Both culture media were inoculated with 2v/v% of a starter of Bifidobacterium breve YIT4010 prepared in a similar manner to Experiment 1, and then were cultivated at 340 C until the pH of the culture attained 4.55.

0 0 The above-mentioned basic culture medium was inoculated with 0.5v/v% of starter of Streptococcus thermophilus YIT2021 prepared in a similar manner to Experiment 1, and

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Go" was cultured at 340 C until the pH of the culture solution attained 4.40.

1 5 volumes of a bifidobacterium culture solution obtained by the above-mentioned cultivation was mixed with 19 a 6OSS

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a S. 55 volumes of a lactic acid bacteria culture solution, pectin was added to the mixture until the pectin concentration attained 0.3w/v%, and then sucrose solution was added until the concentration of sucrose attained Latic fermenting beverage was prepared by adding water to the resulting solution until the total amount of food product attained 10 volumes.

The flavor of the product was good irrespective of the addition of the isolated soybean protein to the product.

The result of preservation test at 100 C is illustrated in Table 3.

Table 3. Decrease in numbers of live bacteria by preservation Concentration of Viable cell number of B.breve Isolated soybean Survival protein ate(% in B.breve culture Immediately After 15 days after production 0 5.2 X 108 2.3 X107 4.4 0.1 7.9 X 108 5.3 X 107 6.7 0.2 7.9 X 108 6.2 X107 7.9 Example 4 A culture medium was prepared by adding an isolated soybean protein to a basic culture medium which contained 13w/v% of whole milk powder and 0.03w/v% of yeast extract 20 until the soybean protein concentration of the medium attained Said culture medium was inoculated with 2v/v% of a starter of Bifidobacterium longum ATCC15707 stock prepared in a similar manner to Experiment 1, and then was cultivated at 34°C until the pH of the cultivation solution attained 4.65.

The culture medium which contained 7.5w/v% of whole milk powder, 21w/v% of skim milk and O.lw/v% of yeast extract was inoculated with 9.5v/v% of starter of Streptococcus thermophilus prepared in a similar manner to Experiment 1, and was cultured at 37°C until the pH of the culture solution attained 4.50.

1.5 volumes of a bifidobacterium culture solution obtained by the above-mentioned cultivation was mixed with 3.0 volumes of a latic acid bacteria culture solution, pectin was added to the mixture until the pectin concentration attained 0.3w/v%, and then sucrose solution was added until the degree of sweetness attained A yogurt was prepared by adding water to the resulting solution 00*0 until the total amount of food product attained 10 volumes.

The resultant yogurt had entirely no soybean odor and had a fresh tasty flavor.

Example SA culture medium was prepared by adding an isolated soybean protein to a basic culture medium which contained 13w/v% of whole milk powder and 0.03w/v% of yeast extract until the soybean protein concentration of the medium 21 attained 0.5w/v%. Said culture medium was inoculated with 2v/v% of a starter of Bifidobacterium infantis ATCC15697 stock prepared in a similar manner to Experiment 1, and then was cultivated at 340 C until the pH of the cultivation solution attained 4.65.

On the other hand, the cultured product was obtained by culturing Streptococcus thermophilus in a manner qimilar to Example 4.

volumes of a bifidobacterium culture solution obtained by the above-mentioned cultivation was mixed with 3.0 volumes of a lactic acid bacteria culture solution, 0 pectin was added to the mixture until the pectin concentration attained 0.3w/v%, and then sucrose solution was added until the degree of sweetness attained 60. A yogurt was prepared by adding water to the resulting solution until the total amount of food product attained volumes.

The resultant yogurt had entirely no soybean odor and had a fresh tasty flavor.

Experiments 3 to 6 An isolated soybean protein was added to a basic S culture medium which contained 13 w/v% of whole milk powder and 0.03 w/v% of yeast extract until the concentration of the isolated soybean protein attained 3.2 This medium was cultured with 1 v/v% of a starter of Bifidobacterium 22 breve prepared in a manner similar to Experiment 1, and then was cultured at 3'o C until the pH of the culture solution attained 4.65. Next, 0.5 v/v% of a starter of Streptococcus thermophilus prepared in a similar manner to Experiment 1 was inoculated into a culture medium which contained 7.5 w/v% of whole milk powder, 21 w/v% of skim milk powder and 0.1 w/v% of yeast extract, and then was cultured at 370 C until the pH of the culture solution attained 4.50.

1.5 volumes of the Bifidobacterium breve culture

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I solution thus obtained, was mixed with 3 volumes of Streptococuss thermophilus culture solution. A yogurt was prepared by adding pectin and syrup and water to the mixture until the total volume of the mixture attained volumes (Experiment 3).

o Another yogurt was prepared in a similar manner as described above except that an isolated soybean protein was not added to the Bifidobacterium breve culture medium 99 a (Experiment 4).

9 9 Furthermore, a yogurt was prepared in a manner similar to Experiment 3 except that Streptococcus thermophilus was 0 cultivated until the pH of the culture solution attained 4.35. (Experiment In addition, a yogurt was prepared in a manner similar to Experiment 4 except that Streptococuss 23 thermophilus was cultivated until the pH of the culture solution attained 4.35 (Experiment 6).

Table 4. shows characteristic values and a sensory evaluation of each product. on account of the use of the isolated soybean protein, we could produce a yogurt having both higher acidity and a higher pH value, thereby producing a yogurt having sourness, or suitabid acidity without being dim.

24 Table 4.

pH Acidity Sensory evaluation Experiment 3 4.46 8.85 Fresh and clean tasting Ex, eriment 4 4,47 8.04 No punch and weak Experiment 5 4.35 9.67 Delicious, but sour Experiment 6 4.34 8,95 Ordinary taste Examples 6 to 8 and Comparative Examples 1 to 3 l 1 v/v% of a starter of Bifidobacterium breve YIT4010 prepared in a man':er similar to Experiment 1 was inoculated into a milk culture medium (13 w/v% of skim milk powder and 0.03% w/v% of yeast extract), and then was cultured at 370 C until pH of the culture medium attained 4.65.

After sterilization of a culture medium, which was composed of w/v% of whole milk powder and 21 w/v% of skim milk powder, the medium was inoculated with a lactic 4. acid bacteria (Streptococcus thermophilus), and then was cultivated at 37u C until the pH of the culture medium attained 4.35.

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An isolated soybean protein to be added to the abovementioned bncterial culture media was a 2.7 w/v% of solution which was sterilized at 1210 C and then cooled to

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25 The incubates of the above bifidobacteria (hereinafter termed as B bacteria), the lactic acid bacteria (hereinafter termed as L bacteria) and the isolated soybean protein solution (hereinafter termed as SPI solution) were mixed together as shown in Table 5 (Examples 6 to 8 and Comparative Examples 1 to A syrup was further added to the resulting mixture to give a total amount of 10 w/v% of the yogurt-like milk-fermented food. Such product was sealed and packed, and then preserved at not more than *see *OO* 1 0 oC.

*6 The results of the measurements of pH, acidity and S* change in the numbers of the viable bacteria in each product are shown in Table 6 and Fig.2. It has been recognized that this table and figure show the improvements of buffering capacity of the product, and of survival rate of bacteria by the addition of the isolated soybean protein.

ee o 0 i 26 Table 5. Composition I B bacterium L bacterium SP souto culture culture Comparative 1.5volume 3 volume example 1 Example 6 1.5volume 3 volume 3.7volume Comparative example 2 Example 7 4.5volume 3 .7volume Comparative 4.5 volume example 3 Examcple 8 4.5 volume 3.7 volume 00 *0 0@ 0 0 03, Table 6. pHl and acidity o day of production 15days after production pH Acidity pH1 Acidity Comp. Exam. 1 4.34 9,20 4,34 9.57 Example 6 4.49 9.50 4.34 10.87 Comp.Exam.2 4.48 5,18 4.47 5.45 Example 7 4.68 5.40 4.66 5.71 Comp.Exam.3 4.32 11.26 4.31 11.63 L-Example 8 4.44 11,46 4.38 12.12 -27 When the flavor of the products was ex:amined, there was a faint pleasant odor when isolated soybea'i protein was added but there was no unsavory soybean odor such as a fishy smell or pungency.

Example 9 and Comparative Examples 4 and 1 w/v% of a starter of Bifidobacterium breve YIT4010 prepared in a similar manner to Example 1 was inoculated into a culture medium which contained 13 w/v% of whole milk powder and 0.05w/v% of yeast extract and cultured at 370C S**e a' until the pH of the culture attained 4.50 (hereinafter the culture thus obtained is .med as A).

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4 S, 0.5w/v of a starter of Streptococcus thermophilus YIT2021 prepared in a manner similar to Example 1 wa inoculated into a culture medium which contained 7.5 w/v% of whole milk powder, 21w/v% of skim powder milk and 0.lw/v% of yeast extract, and then was cultured at 370 c until the pH of the inoculate attained 4.50 (hereinafter S the culture thus obtained is termed as B).

volumes of culture A, 3.0 volumes of culture B and Ba 3.7 volumes of isolated soybean protein solution (that was a

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commercially available and was solubilized in water to become 2.68w/v%, then was sterilized) were mixed together, and pectin and syrup were added to such mixture to give a total amount of 10 volumes of yogurt (Example 9).

28volumes of culture A, 3.0 volumes of culture B and volumes of soybean milk (made of soybean in the conventional way and sterilized; 5% soybean protein content) were mixed together, and pectin and sterilized water were added to such mixture to such mixture to give a total amount of 10 volumes of yogurt (Comparative Example 4) The content of soybean protein in Example 9 and Comparative Example 4 were both 1.Ow/v%.

Further, 1.5 volumes of culture A and 3.0 volumes of culture B were mixed together, and pectin, syrup and sterilized water were added to such mixture to give a total amount of 10 volumes of yogurt without addition of isolated soybean protein (Comparative Example This yogurt was subjected a sensory test as a stewnard product.

The results of the sensory test on soybean odor (fishy I smell) and taste were examined by 0 well-trained panelists are illustrated in le 7. As can be seen from Table 7, this isolated soybean protein had almost no characteristic soybean odor or pungency. Evaluation standards were as follows: *b 1. Soybean odor 0:None l:Slight -29 2 :Ordinary 3 :Fairly 4 :High 2. Taste +2:Quite tasty +1 :Tasty o :Ordinary Unsavory -2:Quite unsavory ate In the product in Example 9, it was stated by the panelists that the product become tasty by the very weak and slight soybean flavor.

a *0 30 Soybean odor Taste D Significant RDSignificant difference difference Examn.9 0.8±0.23 1.2±0.37 Isolated soybean protein added Comp. 3.2± 0.35 *-0.8±0.23* Exam. 4 soybean milk added product Comp. 0.5 ±0.23 1.4± 0.35 Plain yogurt Fi g.7 (*Significant difference when risk ratio is lee.

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-31

Claims (9)

1. A method of producing a fresh flavoured, high acidity milk-feriiented food comprising the steps of: inoculating and culturing in milk bifidobacteria or lactic acid bacteria or a combination of said two bacteria, said milk having added thereto from 0.1 w/v% to w/v% of an isolated soybean protein with respect to the volume of said milk, wherein the method provides a shortened culturing period and increases the numbers and survival rate of live bacteria in the food.

2. A method of producing milk-fermented food as claimed in Claim 1, wherein a yeast extract is added together with said isolated protein to said milk.

3. A method of producing milk-fermented food as claimed in Claims 1 or 2 wherein 0.01 w/v% to 0.1 w/v% of said yeast extract is added to said milk. 8600

4. A method of producing milk-fermented food as claimed in any one of claims 1 to 3, wherein said bifidobacteria 20 are one or more species of bacteria selected from the group consisting of Bifidobacterium breve, Bifidobacterium lonum, Bifidobacterium bifidum, and Bifidobacterium infantis.

A method of producing a fresh-flav-ured, high 25 acidity milk-fermented food comprising the steps of: inoculating and culturing in milk bifidobacteria or lactic acid bacteria or a combination of said two bacteria, and then adding to said culture from 0,1 w/v% to 1.0 w/v% of an isolated soybean protein with respect to the volume of the culture obtained by culturing said bacteria to s s. produce the milk-fermented food, wherein the method provides a shortened culturing period and increases the numbers and survival rate of I %5.ziU live bacteria in the food. -33

6. A method of producing milk-fermented food as claimed in Claim 5, wherein said bifidobacteria are one or more species of bacteria selected from the group consisting of Bifidobacterium breve, Bifidobacterium lonqum, Bifidobacterium bifidum and Bifidobacterium infantis.

7. A milk-fermented food produced by a method as defined in any one of claims 1 to 6.

8. A method of producing milk-fermented food using isolated soybean protein substantially as hereinbefore described with reference to any one of Examples 1 to 9 and Experiments I to 3, excluding comparative Examples 1 to

9. A milk-fermented food produced with the use of isolated soybean protein substantially as hereinbefore described with reference to any one of Examples 1 to 9 and Experiments 1 to 3, excluding comparative Examples 1 to 9 DATED this lv.h day of February 1994 o. KABUSHIKI KAISHA YAKULT HONSHA 20 By their Patent Attorney 9" 9 GRIFFITH HACK CO. 99 l* e 0 I S:10803AO