JP3061316B2 - Fermented milk and lactic acid bacteria beverages in which lactic acid bacteria easily colonize the intestinal tract - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to fermented milk and lactic acid bacteria beverages in which lactic acid bacteria can reach and colonize the intestine and exert their physiological activities.

[0002]

2. Description of the Related Art Intestinal useful lactic acid bacteria such as Bifidobacterium and Lactobacillus inhabit the human intestinal tract and have various physiological activities such as protection against infection, prevention of intestinal rot, and promotion of intestinal motility. It is thought that it has demonstrated.
However, it is said that the flora in the intestinal tract is susceptible to stress, illness, aging and the like, and that useful bacteria are easily reduced and harmful bacteria become dominant. In addition, for people who consume a large amount of fermented milk containing such useful bacteria on a daily basis,
Epidemiological studies also report that many people live long. From such a viewpoint, the custom of ingesting fermented milk and lactic acid bacteria beverages as health foods has recently become common, and consumption of fermented milk such as plain yogurt has been increasing. However, there are many doubts as to whether these useful lactic acid bacteria taken orally pass through the stomach alive and colonize the intestinal tract. It is believed that the reason is that lactic acid bacteria taken orally are killed by stomach acid before reaching the intestinal tract, which is the original growth environment. For this purpose, an attempt has been made to produce fermented milk by screening for acid-resistant lactic acid bacteria capable of growing even in a medium having a high acidity (Japanese Patent Application Laid-Open No. 58-224685). However, such bacteria produce acetic acid. In addition, the acidity and fermentation smell become strong, and the flavor is remarkably reduced. In addition, the structure of the yogurt becomes coarse during storage and the smoothness of the mouth is lost. In order to solve these, fermented milk is used in which several types of lactic acid bacteria are combined and fermented under certain conditions, and acetic acid does not affect the taste, but fermentation conditions and the like must be strictly controlled. There is a new problem.

[0003]

DISCLOSURE OF THE INVENTION The present invention has been made to solve such a problem, and it is an object of the present invention to provide a fermented milk or a lactic acid bacteria beverage with a peptide fraction having an inhibitory effect on gastric acid secretion isolated from milk. By adding and containing stomach, the stomach acid secretion action in the stomach at the time of ingestion is suppressed, lactic acid bacteria reach the intestine while maintaining their activity without being killed in the stomach, and exert their physiological activities.

[0004] That is, an object of the present invention is to provide fermented milk and a lactic acid bacteria beverage capable of exerting its physiological activity by allowing useful lactic acid bacteria to reach and colonize the intestine alive. Another object of the present invention is to use a peptide fraction derived from milk, which is also a raw material for fermented milk, to slowly suppress gastric acid secretion, so that fermented milk and lactic acid bacteria capable of exhibiting physiological activity without causing side effects. It is to provide a beverage. Furthermore, an object of the present invention is to provide a fermented milk and a lactic acid bacteria beverage having a good taste and mouthfeel using a commonly used lactic acid bacterium without intentionally selecting a strain having excellent acid resistance having a problem in flavor. It is in.

[0005]

The present invention relates to a fermented milk or a lactic acid bacteria beverage containing lactic acid bacteria, which contains a peptide fraction having a gastric acid secretion inhibitory action isolated from human or mammalian milk, so that the lactic acid bacteria can be added to the stomach. The present invention relates to a fermented milk and a lactic acid bacteria beverage which reach the intestine and are easily fixed while maintaining the activity without dying.

The peptides according to the present invention include Japanese Patent Publication No. 59-27.
No. 358, JP-A-2-276542, Japanese Patent Application No. 2-9568
There is a crude fraction of glycomacropeptide (GMP) produced based on the method described in JP-A-6-284199 or JP-A-63-284199. It has long been known that GMP is released into whey during cheese production. As this raw material, cheese whey, protein-free cheese whey, whey protein concentrate (WPC) produced by ultrafiltration of cheese whey, lactose mother liquor and the like can be used. That is, the peptide in the present invention is human, cow, sheep, raw milk such as goat, skim milk, whey,
It is prepared using whey protein concentrate or lactose mother liquor as a raw material. As an example of this preparation method, for example, as disclosed in JP-A-2-276542, cheese whey, WPC and protein-free cheese whey obtained from these raw materials are obtained and adjusted to a pH of less than 4. After GMP is converted into a monomer, the filtrate is subjected to ultrafiltration using a membrane having a molecular weight cut-off of 10,000 to 50,000 to obtain a permeate. Then, after adjusting the permeate to pH 4.0 or higher to form GMP into an aggregate, the molecular weight cut off of 10,000 to
GMP crude fraction is obtained by concentration using 50,000 membranes. Alternatively, as described in Japanese Patent Application No. 3-19112, the adsorbed portion is adsorbed on an anion exchange resin, the adsorbed portion is eluted, and the obtained eluted fraction is subjected to membrane treatment as described above. You may. Further, the crude peptide fraction thus obtained may be a crude peptide fraction adsorbed on an anion exchange resin and then eluted.
Such adsorption to an anion exchange resin may be carried out by dissolving the crude GMP fraction in a buffer solution containing 0.2 M sodium chloride at pH 8.7 and adsorbing this at pH 8.7 to the anion exchange resin. . Then, the non-adsorbed portion is eluted using this buffer, and the adsorbed fraction is then eluted with a buffer containing 0.25 M or more, preferably 1M sodium chloride, at pH 8.7 to obtain a crude peptide fraction. At this time, Q-Sepharose, DEAE-Sepharose, QAE-Sephadex, QAE-Toyopearl, etc. are used as the anion exchange resin, and Tris-HCl buffer, ethanolamine buffer, phosphate buffer, etc. are used as the buffer. Used.

When producing fermented milk and lactic acid bacteria beverages containing these peptide fractions, these peptide fractions may be added at an appropriate time during the production process. For example, fermented milk or raw milk that is a raw material for lactic acid bacteria beverages, raw milk, skim milk or the like, these peptide fractions may remain in solution, or may be spray-dried, or freeze-dried and added together with sweeteners and stabilizers in the form of powder. Alternatively, it may be added when a starter or a flavor is added after sterilization of the raw material mix.

[0008] These peptide fractions in the present invention are peptides derived from milk components, and when taken orally, have no adverse effect on the human body, and the amount of intake is not particularly limited. . However, in order to actually establish the lactic acid bacteria in the intestinal tract, it is appropriate that the dose is 2 mg / kg body weight or more by oral ingestion. Since there is an individual difference in the amount of fermented milk, the upper limit of the amount of fermented milk is not intentionally limited, but a sufficient effect can be obtained with an amount of 2 to 20 mg / kg body weight. Therefore, assuming that the product weight is 100 to 250 g / piece, the compounding amount in the fermented milk may be about 0.05 to 2 g per 100 g of the product.

The effects of the present invention are summarized as follows. 1) Lactobacillus such as Bifidobacterium genus, which is easily killed by stomach acid, or Lactobacillus genus, which is said to be difficult to colonize the intestinal tract, is established in the intestinal tract, thereby preventing the infection of lactic acid bacteria and preventing intestinal decay. An effect, an intestinal motility promoting effect, an immunostimulating effect, an antitumor effect, and the like can be exerted. 2) A product can be produced using a common lactic acid bacterium commonly used in the production of fermented milk and lactic acid bacterium beverages having a good taste and mouthfeel without intentionally screening for acid-resistant lactic acid bacterium. 3) Milk-derived peptides that are usually taken as foods have a gastric acid secretion inhibitory effect, so that by taking them in, there is no need to worry about side effects and the like, and the physiological activity of lactic acid bacteria can be exerted.

Hereinafter, the present invention will be specifically described based on examples.

Example 1 This example shows a method for preparing a peptide fraction having an inhibitory effect on gastric acid secretion in the present invention. Whey protein concentrate (Taiyo Chemical, trade name Sunlac N-
2) Dissolve 1 kg in 50 liters of water at 50 ° C, and add concentrated hydrochloric acid to pH.
After adjusting to 3.5, ultrafiltration membrane (D
Ultrafiltration using DS, GR61pp) (pressure 0.4M)
Pa, average permeation flow rate 52.4 l / m ² · h). Permeate volume
When the volume reached 40 liters, 40 liters of water at 50 ° C. was added to the concentrated solution, followed by continuous ultrafiltration to obtain 160 liters of permeate. Add 25% sodium hydroxide solution to this permeate
The pH was adjusted to 7.0, and ultrafiltration was again performed using the same ultrafiltration membrane until the concentration of the concentrate became 5 liters, followed by desalting and concentration. Then 50 ℃
Of diafiltration was carried out under the same conditions as before while constantly maintaining the amount of the concentrated solution at 10 liters by adding water. When the amount of the permeate reached 80 liters, the addition of the concentrate to the concentrate was stopped, and the concentrate was concentrated to 2 liters, and then freeze-dried to obtain 54 g of a crude GMP fraction.
This fraction was found to be 51% pure as analyzed by urea-SDS electrophoresis.

[0011]

Example 2 1.0 g of crude GMP fraction obtained in Example 1
Was dissolved in 20 ml of 20 mM ethanolamine buffer (pH 8.7) containing 0.2 M sodium chloride, passed through 200 ml of Q-Sepharose Fast Flow Anion Exchange Resin, and the resin was washed with 600 ml of the same buffer to remove non-adsorbed resin. Elute fractions. Next, the eluted fraction is collected by passing through 300 ml of 20 mM ethanolamine buffer at pH 8.7 containing 0.3 M salt. This eluted fraction is freeze-dried and the crude peptide fraction of the present invention 50 mg
Get.

[0012]

Example 3 This example illustrates a method for producing fermented milk according to the present invention. A skim milk medium consisting of 11.5% non-fat milk solids and 0.5% yeast extract.
Longum SBT 2933R (Microtechnical Lab. No. 8743), Streptococcus thermophilus SBT 1021A (Microtechnical Lab. No. 106)
No. 58), and Lactobacillus acidophilus SBT 20
62 (Microtechnical Laboratories No. 10730) was cultured at 31 ° C. for 20 hours to prepare a composite bacteria starter. Lactic acidity after culture is 1.35
%, PH 4.35, and the number of bacteria was 6 × 10 ⁸ cfu / bacteria by Bifidobacterium longum SBT 2933R (Microtechnical Laboratory No. 8743).
g, 3 × 10 ⁸ cfu / g of Streptococcus thermophilus SBT 1021A (Microcosms No. 10658) and Lactobacillus acidophilus SBT 2062 (Microcosms No. 1073)
No. 0) was 2.5 × 10 ⁸ cfu / g. 4.4 g of milk and 50 g of skim milk powder and the GMP crude fraction 50 obtained in Example 1
g and stirred, then homogenized at 60-70 ° C (homogeneous pressure:
100-270 kg / cm ² ). The mixture was sterilized by keeping it at 90 to 95 ° C. for 5 to 10 minutes, cooled to 38 ° C., and 500 ml of the complex starter was added. After filling in a container and fermenting at 38 ° C. for 5 hours, it was cooled to 10 ° C. or lower to obtain fermented milk containing a GMP fraction of 1%. In addition, skim milk powder was added to 4.4 liters of raw milk.
95 g, 5 g of the GMP crude fraction obtained in Example 1, and 99.5 g of skim milk powder and the GMP crude fraction obtained in Example 1
0.5 g was added and the GMP crude fraction 0.1
% And a fermented milk containing 0.01% of GMP crude fraction. GMP-free fermented milk (control used in Example 3 and below) was similarly produced by adding 50 g of skim milk powder to 4.45 liters of raw milk.

[0013]

Example 4 This example shows an experimental example in which fermented milk to which the crude GMP fraction of the example was added was administered to a germ-free mouse, and the fixation of lactic acid bacteria was examined. Six-week-old germ-free mouse (ICR, female) receiving sterilized feed and tap water 16
Group, 4% fermented milk administration group with 1% GMP crude fraction,
GMP crude fraction 0.1% fermented milk added group (4 animals), GM
Fermented milk supplemented with P0.01% (four animals) and fermented milk without GMP (four animals) were separately administered, and the fermented milk was orally administered 0.1 ml each using a sonde. Two weeks after the administration, the small intestine and large intestine were removed, and the small intestine was divided into upper and lower parts, and each was incised. After thoroughly washing the intestinal tract with sterile physiological saline, each site was homogenized in 10 volumes of PBS (0.01 M phosphate buffer (pH 7.2) containing 0.15 M sodium chloride). This solution was spread on an agar plate medium, and cultured at 37 ° C. for 20 hours under anaerobic and aerobic conditions. After counting the number of colonies, as shown in Table 1, 1% of a crude GMP fraction was added. Fermented milk administration group and GMP crude fraction 0.1
The number of bacteria in the group to which the fermented milk was added was significantly (p <0.05) higher. Therefore, it was considered that the lactic acid bacteria in the fermented milk became significantly easier to settle by adding the crude GMP fraction to the fermented milk at 0.1% or more.

[0014]

[Table 1] Number of bacteria established in the intestine 管 Upper small intestine Lower small intestine Large intestine 発 酵 Anaerobic fermented milk group containing 1% GMP crude fraction Bacteria 5.84 ± 0.40 6.72 ± 0.52 7.21 ± 0.33 Aerobic bacteria 4.98 ± 0.23 3.87 ± 0.45 5.23 ± 0.43 ────────────────────────────発 酵 Fermented milk group with 0.1% GMP crude fraction added anaerobic bacteria 6.12 ± 0.65 6.32 ± 0.41 6.41 ± 0.66 Aerobic bacteria 4.34 ± 0.54 4.78 ± 0.23 5.89 ± 0.77 ────────発 酵 Anaerobic bacteria with fermented milk group supplemented with 0.01% GMP crude fraction 3.42 ± 0.56 3.12 ± 0.56 5.11 ± 0.12 Aerobic bacteria 2.13 ± 0.23 2.78 ± 0.24 3.11 ± 0.32 ───────────────────────────発 酵 GMP-free fermented milk group Anaerobic bacteria 2.35 ± 0.35 2.62 ± 0.42 4.77 ± 0.21 Aerobic bacteria 2.41 ± 0.27 2.11 ± 0.34 2.87 ± 0.31 ─────────── ───────────────────────── Calculate the common logarithm of the number of viable bacteria / intestinal weight (g) and calculate the mean ± standard deviation ( n = 6).

[0015]

Example 5 This example illustrates a method for producing a lactic acid bacteria beverage according to the present invention. The lactic acid bacteria beverage containing 1% of the crude GMP fraction according to Example 1 was manufactured as follows. milk
Add 4.4 g of skim milk powder and 60 g of GMP fraction to 4.4 liters and stir, then homogenize at 60-70 ° C (homogeneous pressure: 100-270k)
g / cm ² ). The mixture was sterilized by holding at 90 to 95 ° C. for 5 to 10 minutes, cooled to 38 ° C., and 500 ml of the composite strain starter prepared in Example 3 was added. After fermenting at 38 ° C. for 5 hours and cooling to 10 ° C. or less, 1 liter of a sugar solution mixture obtained by mixing and dissolving 100 g of sugar, 5 g of xanthan gum, and 5 g of orange flavor was added and stirred, followed by homogenization treatment (homogeneous pressure: 100 to 270 k).
g / cm ² ) to obtain a lactic acid bacteria drink containing 1% of a crude GMP fraction. GMP-free lactic acid bacterium drink is made from raw milk 4.
50 g of skim milk powder was added to 45 liters to produce in the same manner.

[0016]

Example 6 This example shows an experimental example in which a lactic acid bacterium drink to which a GMP crude fraction according to the example was added was administered to a germ-free mouse, and the fixation of lactic acid bacteria was examined. Six-week-old germ-free mice (ICR strain,
Female) 8 mice were divided into a group receiving lactic acid bacteria drink with 1% GMP crude fraction (4 animals) and a group receiving lactic acid bacteria drink without GMP (4 animals), and 0.1 ml of each lactic acid bacteria drink was orally administered using a sonde. Was administered. Two weeks after the administration, the small intestine and large intestine were removed, and the small intestine was divided into upper and lower parts, and each was incised. After thoroughly cleaning the intestinal tract with sterile physiological saline,
The mixture was homogenized in 10 volumes of PBS (0.01 M phosphate buffer (pH 7.2) containing 0.15 M sodium chloride). This solution was spread on an agar plate medium, cultured at 37 ° C. for 20 hours under anaerobic and aerobic conditions, and then the number of colonies was measured. As shown in Table 2, the lactic acid bacteria beverage containing 1% GMP was administered. The number of bacteria in the group was significantly (p <0.05) higher. Therefore, it was considered that the addition of the crude GMP fraction to the lactic acid bacteria beverage at 0.1% or more significantly facilitated the establishment of the lactic acid bacteria in the beverage.

[0017]

[Table 2] Number of bacteria colonized in the intestine ─────────────────────────────────── Upper small intestine Lower small intestine Large intestine乳酸 GMP 1% added lactic acid bacteria beverage group Anaerobic bacteria 5.15 ± 0.53 6.61 ± 0.84 7.83 ± 0.64 Aerobic bacteria 4.26 ± 0.72 3.68 ± 0.16 5.98 ± 0.75 ─────────────────────────────────── GMP-free lactic acid bacteria drink group Anaerobic bacteria 2.53 ± 0.85 2.88 ± 0.23 4.28 ± 0.44 Aerobic bacteria 2.18 ± 0.65 2.21 ± 0.84 2.65 ± 0.64 ──────────────────── ─────────────── values were calculated by calculating the common logarithm of the number of viable bacteria / intestinal weight (g), and expressed as mean ± standard deviation (n = 6).

[0018]

Example 7 A crude peptide fraction obtained by the method of Example 2 was used in place of the crude GMP fraction of Examples 5 and 6, and a similar method was carried out. The same result was obtained.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) A23C 9/00-9/146 A23J 1/20

Claims (3)

(57) [Claims] 1. A fermented milk or a lactic acid bacteria beverage containing lactic acid bacteria, which contains a peptide fraction having an inhibitory effect on gastric acid secretion isolated from human or mammalian milk to maintain the activity without killing the lactic acid bacteria in the stomach. A fermented milk and a lactic acid bacteria beverage characterized in that the fermented milk and the lactic acid bacterium have reached the intestine as they are and are easily established. 2. The molecular weight cut off of the peptide fraction at a pH below 4
The fermented milk and lactic acid bacteria beverage according to claim 1, which is a fraction that permeates an ultrafiltration membrane of 10,000 to 50,000 and is obtained by desalting and concentrating using an ultrafiltration membrane having a pH of 4 or more and a molecular weight cut off of 50,000 or less. 3. The molecular weight cut off of the peptide fraction at a pH below 4
Permeate through 10,000-50,000 ultrafiltration membrane, desalted and concentrated by ultrafiltration membrane with pH ≥4 and molecular weight cut off 50,000,
The fermented milk and lactic acid bacteria beverage according to claim 1, which is a fraction obtained by further adsorbing and eluting on an anion exchange resin.