JPS6318457B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a food or drink containing a Bifidobacterium growth promoting factor. Various substances have been reported to have the effect of promoting the growth of Bifidobacterium in vivo or in vitro (hereinafter referred to as Bifidobacterium promoting factors), and their production methods or powdered milk containing them have been reported. Many manufacturing methods such as
45-6865, 45-21606, 49-40956, 45-21606, 49-40956, 45-21606, 49-40956,
49-40957 etc.). However, the effects of most of the bifidus growth factors that have been reported so far have been confirmed through in vitro culture experiments, and their in vivo activity is unknown or extremely insufficient. Bifidobacterium is a useful bacterium that grows in the human intestine, and it is too late to discuss in detail its physiological significance and the efforts being made to increase the proportion of this bacterium among intestinal bacteria. No need to do it. Therefore, Bifidus growth factors that are artificially included in foods and drinks, etc. are not only used for culturing Bifidobacterium in vitro, but can also be expected to have reliable and sufficient effects in vivo. From this point of view, there has been a strong desire to develop a better bifid growth factor. In order to meet the above-mentioned demands, the specification of Japanese Patent Application No. 54-12837 (Japanese Patent Publication No. 58-20266) describes an invention related to bifidus growth factors by the same inventors as the present inventors, that is, a general patent application. Formula Gal−
A Bifidobacterium growth promoter consisting of a transferred oligosaccharide represented by (Gal)n-Glc (where Gal is a galactose residue, Glc is a glucose residue, and n is an integer from 1 to 4). In addition, the invention of a method for producing the above-mentioned Bifidobacterium growth promoter by treating lactose with β-galactosidase produced by Aspergillus oryzae is also described. Most of the above oligosaccharides that are bifidus growth factors are
The present inventors discovered for the first time during their research on the transfer reaction that occurs together with the hydrolysis reaction when β-galactosidase acts on lactose.
Regarding the above rearrangement reaction, Wallen-fels in 1951
Although there have been many reports since the first report, the only transferred oligosaccharides that have been isolated and confirmed to date are Gal
-(β-1,2)-Glc, Gal-(β-1,3)-
Glc, Gal-(β-1,6)-Glc, Gal-(β-1,
3) -Gal, Gal-(β-1,6)-Gal disaccharide,
Gal-(β-1,6)-Gal-(β-1,4)-Glc,
It is a trisaccharide of Gal-(β-1,6)-Gal-(β-1,6)-Glc. (The brackets indicate the galactoside bond). The existence of tetrasaccharides has been confirmed by Huber et al., but their structures have not been clarified, and there has been no confirmation of the production of polysaccharides of pentasaccharides or higher. The contents of these reports are limited to the elucidation of the transfer mechanism and the structural study of the produced oligosaccharides, and none report on the relationship between transferred oligosaccharides and the growth of Bifidobacterium. In addition, US Patent No. 2,826,503 describes a method for producing oligosaccharides by treating lactose with lactase, but there is no description of the structure of the oligosaccharides obtained by this method, and there is no description of the use thereof. only mentions stabilizers for ice cream and thiokolate milk drinks, soft-centered thiokolate fillers, adhesives or raw materials for lacquers, films, coatings, etc. The present invention has been completed based on the above-mentioned inventions of the present inventors concerning bifidus growth factors and methods for producing the same, and is a delicious product containing both bifidus growth factors and bifidobacterium that are effective in vivo. Moreover, the method for producing foods and drinks useful for maintaining and promoting health is that by treating milk with β-galactosidase produced by Aspergillus oryzae, all or part of the lactose in milk is removed using the general formula Gal- (Gal)
n-Glc (where Gal is a galactose residue, Glc
is a glucose residue, and n is an integer from 1 to 4, respectively) into a transferred oligosaccharide (hereinafter, the term oligosaccharide refers to this specific oligosaccharide), and then heated to inactivate the enzyme. and then inoculating the milk after the above treatment with a starter of Bifidobacterium and culturing it, provides a method for producing a food or drink containing Bifidobacterium and its growth promoting factor. It is. In the production method of the present invention, as the raw material milk, whole milk, skim milk, or reconstituted milk from powdered milk of these milks can be used. The β-galactosidase used is one produced by Aspergillus oryzae, and as shown in Experimental Example 3 below, the β-galactosidase produced by Aspergillus oryzae is - Galactosidase is significantly superior to other β-galactosidases (of different origin). When performing β-galactosidase treatment, the milk solids concentration is approximately 15-30%, the pH is 3-8 (preferably 6-7), the enzyme concentration is 1-100 units/ml, and the temperature is 20%.
~50°C is suitable. Reaction time has a great influence on the yield of bifidus growth factors. As is clear from Figure 1, which shows the relationship between the reaction time and the amount of sugar produced in an example of a model enzyme treatment in which lactose is treated with β-galactosidase produced by Aspergillus oryzae, glucose and galactose are and oligosaccharides increase almost linearly, but thereafter they all draw somewhat complicated curves, and oligosaccharides tend to gradually decrease from a certain point. Since the time at which the yield of oligosaccharides reaches the maximum varies depending on other reaction conditions, it is desirable to confirm the optimal reaction time by experiment. Note that the oligosaccharide in the reaction mixture can be separated from other components by, for example, thin layer chromatography, and then quantified by the Anthrone method. For the enzyme reaction, the treated milk is heated to approximately 90℃ or higher for 5 to 10 minutes.
It can be stopped by heating for minutes to inactivate the enzyme. After the enzyme treatment, the milk is heated and sterilized.
This sterilization treatment may be performed simultaneously with the enzyme deactivation treatment. Then, transfer to a culture tank, inoculate with Bifidobacterium starter, and culture at around 37°C. The culture can be carried out by a conventional method of culturing Bifidobacterium using milk as a medium, or it may be cultured by inoculating lactic acid bacteria together with Bifidobacterium. During cultivation, Bifidobacterium preferentially utilizes easily assimilated monosaccharides and disaccharides, so that most of the bifid growth factors consisting of oligosaccharides remain in the milk after cultivation. The culture obtained as described above contains Bifidus growth factor and Bifidobacterium,
Since it has a flavor similar to normal fermented milk produced by Bifidobacterium, it can be eaten as is, but it may be diluted or homogenized as appropriate, or it may be processed with sweeteners, flavorings, etc. You may adjust the form and taste of the food or drink by, for example, adding . According to the present invention, the food and drink is highly effective in increasing the number of Bifidobacterium bacteria in the human intestine and has excellent nutritional balance, as it contains Bifidobacterium and Bifidobacterium growth factors that are effective in vivo. can be easily produced by using milk as a raw material and adding simple enzyme treatment and culture treatment to it as described above. Hereinafter, Experimental Examples 1 and 2 confirming the effect of the bifidus growth factor contained in the food and drink obtained by the production method of the present invention, Experimental Example 3, which confirmed the difference in the yield of bifidus growth factor depending on the type of β-galactosidase, As well as showing examples of the present invention,
The present invention will be explained. Experimental example 1 Dissolve 3.6 kg of lactose in about 6 kg of warm water,
Add 50 ml of 1M acetic acid buffer solution (PH4.6), 100,000 units of β-galactosidase and water to make 10, and make 37
The reaction was carried out at ℃ for 5 hours. The reaction solution was then heated to inactivate the enzyme, and the resulting denatured protein was filtered off, followed by passing through a cation exchange resin column. The flow-through was contacted with a 30 x 30 cm column packed with activated carbon overnight, after which the activated carbon was washed with deionized water 60 to elute monosaccharides, followed by 5% ethanol 60 and then 50% ethanol 60. This 50% ethanol elution fraction was concentrated to about 7%, sterile filtered using a membrane filter with a pore size of 0.2μ, and then ion-exchanged again to obtain a transparent sugar solution. Add this to about 2.5
The resulting viscous liquid was concentrated under reduced pressure to
TOS) obtained 500g. This TOS is approximately 55% trisaccharides and approximately 32% tetrasaccharides.
%, pentasaccharides or more are about 13%, and the molar ratio of glucose to galactol in these oligosaccharides is 1:2 for trisaccharides, 1:3 for tetrasaccharides, and 1 for pentasaccharides. :It was 4. Using this TOS, the following experiment was conducted on five healthy adults. The administered bacterial solution was Bifidobacterium breve in VL-G medium.
After 48 hours of culture, the cells were obtained by centrifugation and suspended in a milk medium. Experimental schedule 1st week: Administer only bacterial liquid 2nd week: Administer bacterial liquid and TOS (3 g/day) 3rd week: Administer bacterial liquid and TOS (10 g/day) Number of bacteria administered: 10 ⁹ / Daily TOS administration method: Dissolve in lukewarm water and drink after lunch Measurement: Measure the number of Bifidobacterium breves bacteria in each person's stool on the 3rd, 5th, and 7th day of each week, and average the number of Bifidobacterium breve bacteria for that week. I asked for a number. The results of the experiment are shown in Figure 2, and the TOS
It can be seen that the number of Bifidobacterium bacteria in the intestine increases significantly by administration of . Experimental Example 2 The following experiment was conducted on five healthy adults. Experimental schedule 1st week...no TOS administered 2nd week...TOS (3g/day) administered 3rd week...TOS (10g/day) administered TOS administration method: Same measurements as Experimental Example 1: Experimental Example 1 Measure the total number of Bifidobacterium bacteria in the stool in the same manner as above. The experimental results are shown in Figure 3. The figure shows that administration of TOS increases the number of Bifidobacterium bacteria resident in the human intestine. Experimental Example 3 The following six types of β-galactosidases of different origins were treated in the same manner as in Experimental Example 1 (however, the pH was adjusted to the optimum pH of each enzyme, and enzymes A, B, and F
(4.5 for enzymes C, D, and E, and 7.0 for enzymes C, D, and E), and changes in the sugar composition in the reaction mixture with the passage of treatment time when lactose was treated were investigated. Enzyme A: β-galactosidase produced using Aspergillus oryzae. Enzyme B: β produced by Aspergillus niger
- Galactosidase (Kyowa Hakko Co., Ltd. product). Enzyme C: β-galactosidase produced by Satucharomyces fragilis (Kyowa Hakko Co., Ltd. product). Enzyme D: β-galactosidase produced using Kryuiveomyces lactis. Enzyme E: β-galactosidase produced by Escherichia coli (product of Boehringer Mannheim). Enzyme F: β-galactosidase produced from Apricot emulsin (common name: apricot) The sugar content in the reaction mixture was determined by high performance liquid chromatography under the following conditions. Column: YMC-pack A-612 (NH ₂ ) Shimakyu Co., Ltd. products, 6 mmφ x 15 mm Mobile phase: Acetonitrile/water (70/30) Flow rate: 2 ml/min Detector: Differential refractometer Sugar in the reaction mixture in each example The economic changes in composition are shown in Figures 4 to 9, and the reaction mixture composition (in %) at the time when the oligosaccharide production reached its maximum was as shown in Table 1.

[Table] The experimental results show that the β-galactosidase (enzyme A) produced by Aspergillus oryzae gives a significantly better oligosaccharide yield than the others. In addition, when this enzyme is used, not only is the reaction yield high, but the rate of rearrangement reaction to the target oligosaccharide is high, and the amount of oligosaccharides produced is approximately 1 hour after the start of the reaction compared to the maximum yield. It reaches a level of 76% (in this respect it is superior to enzymes C, E, and F). After that, the maximum yield is reached in about 5 hours, but there is almost no tendency to convert the produced oligosaccharides into monosaccharides or transfer disaccharides, and therefore, there are some fluctuations in the conditions of the enzymatic reaction. They also have the advantage of always achieving stable yields and quality (enzymes C, D, E, and F have a remarkable increase and decrease in oligosaccharides and other saccharides as the reaction time progresses). From the above, it can be seen that β-galactosidase produced by Aspergillus oryzae has significantly excellent performance as an enzyme for producing oligosaccharides. Example 1 200 kg of skimmed milk was dissolved in water to make 1000, to which 10 million units of β-galactosidase produced by Aspergillus oryzae was added, and the mixture was heated at 40°C for 2 hours.
After being kept at a low temperature, the enzymes were deactivated and sterilized by heating. Next, it was transferred to a culture tank, inoculated with Bifidobacterium starter and cultured at 37°C for 24 hours. After the culture was completed, sweeteners were added and homogenized to produce 35g of oligosaccharide and Bifidobacterium. Fermented milk containing approximately 10 ⁹ bacteria/ml was obtained.

[Brief explanation of the drawing]

Figure 1: Graph showing the relationship between reaction time and saccharide yield when lactose is treated with β-galactosidase. FIG. 2: Graph showing the experimental results of Experimental Example 1. FIG. 3: Graph showing the experimental results of Experimental Example 2. Figures 4 to 9: Graphs showing the experimental results of Experimental Example 3.

Claims (1)

[Claims] 1. By treating milk with β-galactosidase produced by Aspergillus oryzae, all or part of the lactose in milk can be converted to the general formula Gal- (Gal).
n-Glc (where Gal is a galactose residue, Glc
is a glucose residue and n is an integer from 1 to 4, respectively), the enzyme is inactivated by heating, and then the milk after the above treatment is inoculated with Bifidobacterium. A method for producing a food or drink containing a Bifidobacterium growth promoting factor, which comprises inoculating and culturing a starter.