JPH0859500A - Peptide mixture that prevents intestinal bacteria from entering the body, and composition containing the peptide mixture - Google Patents

Abstract

(57) [Summary] (Modified) [Structure] The lactoferrin hydrolyzate is isolated, and its physicochemical and biological properties prevent transfer of intestinal bacteria from the intestinal tract to the organ, and antibacterial activity in vitro. It has no action, and its antigenicity is 1/100 of that of undegraded lactoferrin.
0 or less, the average molecular weight measured by high performance liquid chromatography is 13,000 daltons or less, and the decomposition rate of lactoferrin is at least 6%.
And a composition containing the peptide mixture as an active ingredient. [Effect] It has the effect of preventing the intestinal bacteria from migrating from the intestinal tract to the body and preventing diseases such as opportunistic infections, anaerobic bacterial infections, and sepsis; patients with reduced digestive function or infection resistance. It is used as a component of oral or tube feeding nutritional composition with few side effects; it enables reduction of drug dose in the treatment of infectious diseases caused by intestinal bacteria; antibiotics, anticancer agents, etc. It has the effect of alleviating side effects that damage the intestinal tract.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a substance which exhibits an effect of inhibiting the intestinal bacterium from entering the body (Bacterial translocation, sometimes referred to as BT hereinafter), that is, a hydrolyzate of lactoferrin to form an antibacterial peptide. The present invention relates to a peptide mixture derived from a lactoferrin hydrolyzate obtained by removing antibacterial fractions such as, and a composition such as a food, a drug or a feed containing the peptide mixture as an active ingredient. More specifically, the present invention shows no antibacterial activity in vitro by itself, and inhibits the intestinal fine transfer from the intestinal tract to the organ by oral or tube administration to humans and animals. However, an opportunistic infection caused by migration of intestinal bacteria into the body, anaerobic bacterial infection, a peptide mixture having no antibacterial activity derived from lactoferrin hydrolyzate having the effect of preventing diseases such as sepsis, and this peptide mixture The present invention relates to a composition containing an active ingredient.

[0002]

2. Description of the Related Art In recent years, development of chemotherapy with antibiotics and improvement of nutritional and hygiene significantly reduce infections of pathogenic microorganisms, resulting in longevity of humans and animals, critically ill human patients and severely ill animals. The extension of survival period was realized. On the other hand, the number of people called so-called immunocompromised hosts, such as premature infants, children, elderly people, patients with surgery, burns, rheumatism, cancer, AIDS, etc., who have weak resistance to infection, increases, and opportunistic infections and anaerobic Morbidity such as bacterial infection and sepsis has become a serious problem.

In these infectious diseases, bacteria that are resident in the digestive tract are detected in the blood and lesions of patients [Journal
Journal of Inf
ectious Diseases), Vol. 152, pp. 99-103, 1985], it is considered that the transfer of intestinal bacteria from the intestinal tract to the internal organs is the cause. In addition, people suffering from these infectious diseases often have a deteriorated digestive tract function, and are mainly enteral nutrition or total parenteral nutrition (TPN) by high calorie infusion.
However, it is known that these nutritional methods further promote BT.

Since TPN does not use the digestive tract, the function of the digestive tract is deteriorated, and atrophy of the digestive tract mucosa and a decrease in the amount of mucus are observed (Transfusion and Nutrition Journal, Volume 15, Vol. 1).
311-1319, 1993). Originally, the gastrointestinal tract protects the host from the invasion of microorganisms by mucous membranes and mucus, but atrophy of the gastrointestinal mucosa and reduction of mucus in TPN increase the permeability of the gastrointestinal mucosa, and Causes BT which passes through the mucous membrane and migrates to the lymphatic system and portal vein. BT spreads to intestinal membrane lymph nodes, spleen, liver, kidney and other organs, blood, and systemic infections, and induces infections in immunocompromised hosts [Fuller, ed., “Probiotics The Scientific Basis "(Probiotics, The Scientific Basis), Chapman & Hall, London, 55th-5th
8 pages, 1992].

On the other hand, enteral nutrition has a lesser degree of morphological change in the gastrointestinal mucosa than TPN, and is also superior in nutritional or clinical aspects such as sugar, amino acid metabolism, and immune function. (Surgery, Vol. 51, pp. 24-31, 1989), it is recommended that nutritional supplementation should shift from TPN to enteral nutrition at an early stage. However, even in the case of enteral nutrition, differences in the structure and function of the digestive tract mucosa have been recognized depending on the component composition (Transfusion and Nutrition Journal, Vol. 13, 1043-1048, 1991).
Year). In animal experiments in which high-calorie infusion was orally administered, B
Because of the recognition of T, [Journ of Per-Enteral and Enteral Nutrition (Journ
al of Parenteral and Enteral Nutrition), 14th
Vol., Pp. 442-447, 1990], it has been pointed out that enteral nutrition may induce BT due to differences in nutritional components.

[0006] Further, in many cases, these patients in need of nutritional supplementation are simultaneously subjected to stress such as radiation therapy, administration of antibiotics, anticancer agents, etc. for the treatment of diseases or prevention of infectious diseases. There is a high risk that side effects such as damage to the gastrointestinal tract mucosa and changes in the intestinal tract flora will occur, and synergistically induce BT to worsen the infection. In experimental examples using animals, normal solid feed, enteral nutrition,
It is also known that BT frequently occurs by intraperitoneally administering an anticancer agent such as 5-fluorouracil or methotrexate or an immunosuppressive agent to a mouse bred with TPN or the like [Current Microbiology (Curren
Microbiology), Volume 8, pp. 285-292,
1983]. In addition, antibiotics (penicillin,
It is also known that BT frequently occurs when oral administration of clindamycin etc. [infection
Infection and immunity,
Vol. 33, pp. 854-861, 1984].

[0007] As described above, humans and animals ingesting only liquid or liquid foods are in a state in which BT is easily induced, and further BT is induced by drugs used for treatment, treatment methods and the like. In spite of the serious situation of illness, few effective treatment methods have been developed. On the other hand, there are some reports on the components effective in preventing BT. It has been reported that TPN for high calorie infusions containing glutamine or nucleic acid components is effective in animal tests [Infusion and nutrition journal, Volume 12, pages 1251-1255, 1990, Surgery, Volume 104, 917-923
Page, 1988, and the 47th Annual Meeting of the Japan Society for Nutrition and Food Science, 203, p. 1993], the effect of oral administration is unknown.

[0008] In addition, it has been reported that non-fermentable cellulose and kaolin, which increase the content in the digestive tract, are effective in animal tests as components effective in preventing BT in enteral nutrition [Journal Of the perennial
And General Nutrition (Journal of P
arenteral and Enteral Nutrition), Volume 14, Volume 44
Pages 2-447, 1990, and Nutrition, Volume 8, Pages 266-271, 1.
992], because these ingredients are not digested in the intestine,
The range of use for enteral nutrition that requires low residue is extremely limited.

As described above, an ingredient effective for preventing BT by ingesting orally or by tube administration and preventing infectious diseases, and pharmaceuticals, foods, feeds and the like containing this ingredient have hardly been developed. Recently, the efficacy of orally administered bovine lactoferrin has been reported for the endogenous sepsis of mice induced by the administration of the anticancer drug 5-fluorouracil (Abstracts of the 71st Annual Meeting of the Kanto Branch of the Japanese Society for Bacteriology, p. 35, 1994). It is presumed that BT is involved in this endogenous sepsis, but the inhibitory effect of lactoferrin was observed only when ingesting a solid feed, and the effect of ingesting a liquid enteral nutrient has not been examined at all. Absent. Furthermore, no consideration has been given to the effect of treating lactoferrin with a proteolytic enzyme or the like in order to improve digestive and absorbability and reduce antigenicity.

Lactoferrin is an iron-binding glycoprotein contained in tears, saliva, peripheral blood and milk.
It is known to show antibacterial action against harmful microorganisms such as Escherichia coli, Candida, Clostridium, etc. [Journal of Pediatric
s), Vol. 94, pp. 1-9, 1979]. As a physiologically active substance derived from lactoferrin, an antibacterial agent containing lactoferrin hydrolyzate as an active ingredient (JP-A-5-3200).
68), antibacterial peptides and antibacterial agents (Japanese Patent Laid-Open No. Hei 5
-92994), many application examples to foods, pharmaceuticals, etc. are reported. However, a fraction containing a mixture of peptides and glycopeptides obtained by removing antibacterial peptides from lactoferrin hydrolyzate as a main component (hereinafter referred to as a peptide mixture), that is, a physiological activity of a peptide mixture that does not exhibit antibacterial activity by itself. Is not known at all and there is no report on its use.

[0011]

As described above, a substance effective for preventing BT by ingesting orally or tube-wise and preventing infectious diseases caused by BT, pharmaceuticals and foods containing this substance In spite of the long-awaited development of feed, etc., no reports have been made on such substances and compositions, except for the limited reports mentioned above.

The present invention has been made in view of the above-mentioned circumstances, and humans and animals having reduced digestive tract functions and infection resistance can take enteral bacteria only by oral or tube feeding. It is an object of the present invention to provide a peptide mixture which is effective in blocking BT and preventing infectious diseases, is excellent in digestion and absorption, low antigenicity and low residue, and has no side effect.

Another object of the present invention is to provide a composition containing the above peptide mixture, that is, a food, a drug, a feed or the like.

[0014]

SUMMARY OF THE INVENTION The inventors of the present invention have
In order to solve the above-mentioned problems, protease hydrolysates of various proteins were prepared and screened for BT-blocking activity. Among them, a peptide mixture showing no antibacterial activity in vitro by itself showed BT. A peptide mixture derived from lactoferrin hydrolyzate, which has an inhibitory activity, that is, exhibits no antibacterial activity, is orally or tube-guided to humans and animals, so that B
The present invention has been completed by discovering that it has an effect of blocking T.

That is, the present invention provides a peptide mixture which is separated from lactoferrin hydrolyzate and has the following physicochemical and biological properties: a) inhibiting the transfer of intestinal bacteria from the intestinal tract to the organ, b ) It has no antibacterial activity in vitro, c) its antigenicity is 1/1000 or less of undegraded lactoferrin, d) its average molecular weight measured by high performance liquid chromatography is 13,000 daltons or less, e ) The degradation rate of lactoferrin is at least 6%.

The present invention also provides a composition containing as an active ingredient a peptide mixture having the physicochemical and biological properties as described above. Hereinafter, the present invention will be described in detail. The peptide mixture derived from the lactoferrin hydrolyzate that blocks BT of the present invention is, for example,
It can be produced according to the method described in JP-A-5-238948. Specifically, when fractionating and purifying an antimicrobial peptide by hydrophobic chromatography from a pepsin hydrolyzate of lactoferrin, each peptide is an antimicrobial peptide adsorbed on a chromatographic carrier,
It is fractionated into a peptide mixture other than the antibacterial peptide that is eluted without being adsorbed on the chromatographic carrier. The peptide mixture of the present invention is present in the latter fraction and contains peptides and glycopeptides as main components, and as is clear from the test examples described below, the peptide mixture itself has antibacterial activity in vitro. do not do.

The peptide mixture of the present invention comprises a residue obtained by separating an antibacterial peptide from a lactoferrin hydrolyzate,
A powder dried by a conventional method (for example, spray drying, vacuum drying, freeze drying, etc.), a concentrated solution concentrated by an ultrafiltration membrane or a powder dried by a conventional method can be produced at the lowest cost, If production cost is ignored, it can be obtained from a hydrolyzate of lactoferrin hydrolyzed by various proteases without producing antibacterial peptides.

Since the peptide mixture of the present invention undergoes the step of inactivating the enzyme by heating after the step of hydrolyzing lactoferrin, sterilization has already been achieved in this step of inactivating heat. You can also do it.
The peptide mixture of the present invention obtained as described above has the following physicochemical and biological properties a) to e). a) Prevent the transfer of intestinal bacteria from the intestinal tract to the organ. b) It has no antibacterial effect in vitro. c) The antigenicity is 1/1000 to 1/100000 of undegraded lactoferrin. d) The average molecular weight measured by high performance liquid chromatography is 300 to 13,000 daltons. e) The decomposition rate of lactoferrin is 6% to 50%.

In addition, since the peptide mixture of the present invention is mainly composed of a peptide obtained by hydrolyzing lactoferrin contained in milk with a protease to remove an antibacterial peptide, it is soluble in water. It has excellent digestive and absorption properties, low antigenicity and low residue, and has no side effects. The physicochemical and biological properties are based on the test by the following method, and the test methods in Test Examples and Examples described later are also based on the following method. 1) Antigenicity ELISA method using rabbit anti-lactoferrin serum (ELIS
A: Enzyme linked immunosorbent assay. Toho Medical Journal, Vol. 35, pp. 509-516, 1989).
It was measured by and expressed as a relative value to undegraded lactoferrin. 2) Average molecular weight Conventional method (edited by Nobuo Ui et al., “High Performance Liquid Chromatography of Proteins and Peptides”, Kagaku Special Issue No. 102, No. 24)
1-251 pages, Kagaku Dojin Co., Ltd., 1984), the molecular weight distribution was measured using a high performance liquid chromatograph system (manufactured by Shimadzu Corporation. LC-7A), and the obtained data was analyzed by an analysis program (manufactured by Shimadzu Corporation). CR-4
It is indicated by the weight average molecular weight calculated using A). 3) Decomposition rate The total nitrogen of the sample was measured by the Kjeldahl method, and the formol nitrogen of the sample was measured by the formol titration method, and the numerical values calculated from the following formulas were displayed from these values.

Decomposition rate (%) = (formol nitrogen / total nitrogen) × 100 4) In vitro antibacterial property In vitro antibacterial property was determined by the method described in Test Example 1. That is, 0 ml was added to 2 ml of a liquid medium consisting of 1% bactopeptone (manufactured by Difco Laboratory) and water.
Samples were added stepwise in a concentration range of 20 mg / ml, and Escherichia coli O111 in the logarithmic growth phase (sold from the Institute of Medical Science, University of Tokyo) was inoculated at a viable cell concentration of 10 ⁶ / ml,
When culturing at 37 ° C. for 16 to 20 hours, measuring the growth state of the test bacterium in the culture broth after culturing by the absorbance at 660 nm, and when the test bacterium grows even when the sample is added to any concentration It was judged that there was no antibacterial property. 5) BT blocking effect B produced by the method described in Test Example 2, 3 or 6
In T-induced mice, the BT incidence was measured when the mice were raised on a feed containing the sample, and when a decrease in the BT incidence was observed, it was determined to have a BT inhibitory effect. B
The method of measuring the T occurrence rate is as follows.

After the rearing, the mouse is dissected, mesenteric lymph nodes are aseptically collected, and a small amount of diluted solution (Mitsuoka, Tomohushi,
"The world of intestinal bacteria", page 322, Moubunsha, 1980.
, And the homogenized suspension with a Teflon homogenizer is divided into two equal parts, and BL agar plate medium and TS agar plate medium (Michioka Tomoaki, "World of Enterobacteriaceae", pages 319 and 327). , Koubunsha, 1980), and the BL agar plate medium is cultured under anaerobic conditions and the TS agar plate medium under aerobic conditions at 37 ° C. for 48 hours. It was evaluated that BT had occurred when bacterial colonies were formed on the agar medium after culturing. Thus, the ratio of the number of mice in which bacteria were detected in the mesenteric lymph node to the number of tested mice was expressed as the BT incidence.

Next, a composition containing the peptide mixture of the present invention as an active ingredient will be described. The composition of the present invention can be processed milk, whey beverage, baby milk, therapeutic milk, feed milk, food or feed in the form of liquid or liquid when enteral or tube feeding, etc. is there. As an enteral or tube feeding agent, normal liquid food, blender food, complete liquid food such as concentrated liquid food, low protein liquid food,
It may be a special liquid food such as a low-fat liquid food, a low-cholesterol liquid food, a low-sodium liquid food, a therapeutic milk, a component nutrition liquid food. Peptide mixture added to the composition of the present invention, food, pharmaceutical,
When it is added to feed or the like, it can be added at any stage of the production process.

The composition of this invention comprises at least 0.01
% (Weight; hereinafter the same unless otherwise specified), preferably containing 0.1 to 5% of the peptide mixture, the infection resistance,
Oral or tube administration to humans and animals with reduced gastrointestinal function has the effect of blocking BT and can prevent any infectious disease caused by BT.

The effective dose for oral or tube administration to humans and animals was confirmed using model mice that frequently induce BT, as shown in Test Examples 2 to 6 described later. . This model mouse is similar to humans and animals with reduced infection resistance and gastrointestinal function,
The mice were bred only on a liquid or liquid diet, and in some cases, the anticancer drug methotrexate was administered to induce BT. The site of BT occurring in humans and animals is the earliest and most frequently found in the mesenteric lymph nodes among the organs. The BT incidence in the mesenteric lymph nodes of this model mouse was 0.01% in the diet with the peptide mixture of the present invention.
It decreases when added and administered, especially 0.1 to 5%
There was a marked effect when the peptide mixture was added at the concentration of. From these results, it is clear that the peptide mixture of the present invention suppresses BT that develops in humans and animals with reduced infection resistance and gastrointestinal function.

The present invention will be described in more detail with reference to test examples. (Test Example 1) This test was conducted to examine the antibacterial activity of the peptide mixture of the present invention. Liquid medium 2 consisting of 1% bactopeptone (Difco Laboratory) water
The peptide mixture of the present invention (manufactured by the same method as in Example 1) was gradually added to ml in the concentration range of 0 to 20 mg / ml, and Escherichia coli O111 in the logarithmic growth phase was added.
(From the Institute of Medical Science, University of Tokyo) was inoculated at a viable cell concentration of 10 ⁶ / ml, and cultured at 37 ° C for 16 to 20 hours.
After culturing, the growth state of the test bacterium in the culture solution was measured by the absorbance at 660 nm. As a result, no growth inhibition of the test bacterium was observed at any concentration of the peptide mixture, and the peptide mixture showed antibacterial activity. Was found not to exist.

On the other hand, undegraded bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) used as a control completely inhibited the growth of the test bacteria at a concentration of 2 mg / ml or more, and showed antibacterial peptide (JP-A-5-238948). Was produced by the same method as in Example 1), and completely inhibited the growth of the test bacteria at a concentration of 6 μg / ml or more. The same test was performed on the peptide mixture of the present invention produced by another method, but almost the same results were obtained. (Test Example 2) This test was carried out in order to generate BT-induced mice necessary for testing the BT inhibitory effect of the peptide mixture of the present invention.

First, a model mouse that frequently induces BT when a liquid or liquid food used in this test was administered to the mouse was prepared as follows. That is, 9
Week old SPF mouse (purchased from Japan SLC), BALB
/ C, female, 20 animals were divided into 2 groups of 10 animals each, placed in a cage provided with a dung-preventing net, and fed for 2 weeks with normal solid feed and water. Then, one group of mice was orally allowed to freely ingest only a high-calorie infusion solution having the composition shown in Table 1 from a sterile pack, and kept for 2 weeks. The other group was similarly fed with chow and water as a control for 2 weeks.

[0028]

[Table 1]

After the rearing of the mice, the mice were dissected, mesenteric lymph nodes were aseptically collected, and a small amount of diluting solution (Mitsuoka Tomozu,
"The world of intestinal bacteria", page 322, Moubunsha, 1980.
) Was added and homogenized with a Teflon homogenizer. This homogenized suspension was divided into two equal parts, and BL agar plate medium and TS agar plate medium (by Tomohashi Mitsuoka, "World of Enterobacteriaceae", pages 319 and 327, Sanbunsha, 1
980) and BL agar plate medium under anaerobic conditions and TS agar plate medium under aerobic conditions at 37 ° C.
The cells were cultured for 48 hours. In the case of mice in which bacterial colonies were formed on the agar medium after culturing, that is, in the case of bacteria in which the bacteria were detected in the mesenteric lymph nodes, it was evaluated that BT had occurred, and the occurrence of BT in each group The rate was measured.

As a result, the BT occurrence rate of the group fed with the solid feed was 1/10 (indicating that BT occurred in 1 out of 10 animals. The same applies hereafter unless otherwise specified). The rate of BT in the group bred with high calorie infusion was 8/1
It was 0. Therefore, when the mice were fed with the solid feed, BT was hardly generated, but it was confirmed that the BT-induced mice were produced at a high frequency by feeding only with the high-calorie infusion. (Test Example 3) This test was conducted to examine another method for producing BT-induced mice necessary for testing the BT inhibitory effect of the peptide mixture of the present invention.

Four-week-old SPF mice (purchased from Japan SLC), BALB / c, 20 females, 20 each were divided into 2 groups and placed in cages provided with a fecal feed control net, and only commercially available milk was added. The animals were raised for 2 weeks by freely ingesting them from a sterile pack. The mouse after breeding was dissected, mesenteric lymph nodes were aseptically collected, and the BT incidence was measured by the same method as in Test Example 2.

As a result, it was confirmed that the BT incidence was 15/20, and that BT-inducing mice could be prepared with high frequency even by feeding only milk. (Test Example 4) This test was conducted to examine the BT inhibitory effect of the peptide mixture of the present invention. The peptide mixture of the present invention prepared by the same method as in Example 1 was added to
Test Example 2 at each concentration of 0.01, 0.1, 1 and 3%
Was added to the same high-calorie infusion solution as above and tested in the same manner as in Test Example 2 to compare the BT incidence. The total concentration of the amino acid and peptide mixture in the tested high calorie infusion solution was adjusted to 4.25%. The mice were 9-week-old SPF mice (purchased from Japan SLC), BAL.
B / c, 20 females in each group, 100 in total were used. The results of this test are shown in Table 2.

From the results shown in Table 2, it was confirmed that the peptide mixture of the present invention markedly inhibited the generation of BT at an addition concentration of 0.01% or more. On the other hand, as a control, milk casein enzyme hydrolyzate (Morinaga Milk Industry Co., Ltd.) was added to a high-calorie infusion at 3%.
Was added and tested in the same manner, but the BT incidence was 16
/ 20, and no BT inhibition effect was observed. A similar test was conducted using the peptide mixture of the present invention produced by another method, but almost the same BT blocking effect was obtained.

[0034]

[Table 2]

(Test Example 5) This test was conducted to examine the BT inhibitory effect of the peptide mixture of the present invention. The peptide mixture of the present invention prepared by the same method as in Example 1 was added to commercially available milk at each concentration of 0, 0.01, 0.1, 1 and 5% and tested by the same method as in Test Example 3. Was performed to compare the BT incidence. 4 mice were used
Week old SPF mouse (purchased from Japan SLC), BALB
/ C, 10 females in each group, a total of 50 females were used.

The results of this test are shown in Table 3. From the results in Table 3, it was confirmed that the peptide mixture markedly inhibited the generation of BT at the addition concentration of 0.01% or more. On the other hand, as a control, a casein enzyme hydrolyzate (manufactured by Morinaga Milk Industry Co., Ltd.) was added to milk at a concentration of 5% and the same test was conducted, but the BT occurrence rate was 8/10 and no BT inhibitory effect was observed.

A similar test was carried out using the peptide mixture of the present invention produced by another method, but almost the same BT blocking effect was obtained.

[0038]

[Table 3]

Test Example 6 This test was carried out to examine the BT inhibitory effect of the peptide mixture when a drug was administered to mice to induce BT at a high frequency. Commercial enteral nutritional supplement Ensure (available from Nippon Dynabot Co., Ltd.), and a peptide mixture of the present invention (Example 1)
(Manufactured by the same method as above) was used as a diet with two kinds of nutrients containing 0.5% of SPF mice (Japan SLC
(Purchased from Japan), BALB / c, females were allowed to freely ingest for 3 weeks. For each mouse, 10 mice per group were used, and 20 mice in total in 2 groups were used. Methotrexate (manufactured by Sigma) dissolved in physiological saline was used for 50 mg and 100 mg of mouse per 1 kg of body weight for 2 groups of mice fed with each feed for 1 week.
The mice were intraperitoneally administered once at a rate of. Two weeks after the administration, the mice were dissected after being fed with each feed, and the mesenteric lymph nodes were aseptically collected, and the BT incidence was measured by the same method as in Test Example 2. The results of this test are shown in Table 4.

From the results in Table 4, it was confirmed that the peptide mixture of the present invention markedly inhibits the drug-induced BT generation. In addition, the same test was performed for the peptides of the present invention obtained by other production methods,
Almost the same BT blocking effect was obtained.

[0041]

[Table 4]

(Test Example 7) This test was conducted on a peptide mixture.
The purpose of this study was to investigate the acute toxicity of. 1) Animal used 6-week-old CD (SD) strain rat (purchased from Japan SLC
Male and female at random in each of 3 groups
Each of them was divided into 6 groups (5 animals per group) for use. 2) Test method The peptide mixture produced by the same method as in Example 1 was weighed.
For injection at a rate of 1000 and 2000 mg per kg
Dissolve in water (Otsuka Pharmaceutical Co., Ltd.) at a concentration of 10%, and weigh 10
Needles with metal balls at a rate of 1 ml and 2 ml per 0 g
Acute toxicity after a single oral gavage with the drug
Was tested. As a control, each one group of male and female,
Water for injection was similarly added at a rate of 2 ml per 100 g of body weight.
Oral administration. 3) Test results The results of this test are shown in Table 5. From Table 5
As is apparent, this peptide mixture was
kg body weight and 2000 mg / kg body weight
There were no deaths in any of the groups. Therefore, this pepti
LD of mixture ₅₀Is 2000 mg / kg body weight or more
The toxicity was found to be extremely low.

A similar test was conducted on the peptide mixture of the present invention obtained by another production method.
Almost similar results were obtained.

[0044]

[Table 5]

Next, the present invention will be described in more detail and concretely by showing examples, but the present invention is not limited to the following examples.

[0046]

【Example】

Example 1 1 kg of commercially available lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) separated from milk was dissolved in 9 liters of purified water, and 1N hydrochloric acid was added to adjust the pH to 3.0. 30 g of commercially available pig pepsin (1: 10000; manufactured by Wako Pure Chemical Industries, Ltd.) was added and uniformly mixed, and the mixture was kept at 37 ° C. for 4 hours and then at 85 ° C. for 10 hours.
Heated for minutes to inactivate the enzyme. Next, 1N sodium hydroxide was added to adjust the pH to 7.0, insoluble materials were removed by filtration, and a lactoferrin degradation product solution was obtained. This lactoferrin degradation product solution was freeze-dried to obtain about 960 g of a powder of the lactoferrin degradation product.

The decomposition rate of this lactoferrin degradation product was measured by the same method as described above, and it was 11.3%. 800 g of the powder is dissolved in 20 liters of purified water,
Butyl Toyopearl (trademark, manufactured by Tosoh Corporation) 650M was passed through a column (36 cm in diameter × 15 cm in height) equilibrated with purified water in advance at a flow rate of 0.2 liter / min, and the absorbance of the effluent at 280 nm The purified water was passed through at a flow rate of 0.6 liter / minute until the water content was 0.01 or less. About 60 liters of the outflow fraction not adsorbed on the obtained butyl toyopearl was concentrated by a reverse osmosis membrane (manufactured by Asahi Kasei Corp.),
Further, it was freeze-dried to obtain about 720 g of a powder of the peptide mixture.

The obtained peptide mixture was tested by the same method as above. As a result, there was no antibacterial activity, the antigenicity was reduced to 1 / 10,000 of that of undegraded lactoferrin, and the degradation rate was 12%. , The average molecular weight is about 32
It was 00 Dalton. Example 2 100 liters of milk was heated to 70 ° C., and 150 kg / c
Homogenized under a pressure of m ² , sterilized at 90 ° C. for 10 minutes, and sterilized milk (49.5 liters) was added with 0.5 kg of the peptide mixture powder prepared by the same method as in Example 1 to uniformly dissolve, About 50 kg of a milk drink containing the peptide mixture of the invention in a final concentration of 1% was prepared.

This milk drink and commercially available milk were dispensed into a sterile pack, and in the same manner as in Test Example 5, 4-week-old SPF mice (purchased from Japan SLC), BALB / c, 10 females each were ingested freely for 2 weeks. Let The mouse after breeding was dissected, mesenteric lymph nodes were aseptically collected, and the BT incidence was measured by the same method as in Test Example 2. As a result, the BT incidence was 8/10 when the commercially available milk was administered, whereas it was 1/10 when the milk drink prepared by adding the peptide mixture described in Example 1 was administered. And the occurrence of BT was significantly suppressed. Example 3 4 kg of commercially available lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd.) separated from milk was dissolved in 36 liters of purified water, 120 g of commercially available pig trypsin (manufactured by Sigma) was added and mixed uniformly, and the mixture was kept at 37 ° C. for 4 hours. The mixture was held, heated at 85 ° C. for 10 minutes to inactivate the enzyme, insoluble matter was removed by filtration, and freeze-dried to obtain about 4 kg of a powder of lactoferrin degradation product.

The decomposition rate of this lactoferrin degradation product was measured by the same method as described above, and was 7.8%.

800 g of the above powder was dissolved in 20 liters of purified water, and butyl Toyopearl (trademark; manufactured by Tosoh Corporation) 650.
Column packed with M and pre-equilibrated with purified water (diameter 36
(cm x height 15 cm) at a flow rate of 0.2 l / min. The absorbance of the effluent at 280 nm is then 0.
Purified water was passed through at a flow rate of 0.6 liter / minute to wash the solution until it became 01 or less. About 65 liters of the outflow fraction not adsorbed on butyl toyopearl thus obtained was concentrated by a reverse osmosis membrane (manufactured by Asahi Kasei Corp.) and further freeze-dried to obtain about 760 g of a peptide mixture powder. This series of fractionation operations was repeated 4 times to obtain a total of about 3 kg of the powder of the peptide mixture.

The obtained peptide mixture was tested in the same manner as described above. As a result, it had no antibacterial activity, the antigenicity was reduced to 1/1000 of that of undegraded lactoferrin, and the degradation rate was 7.9%. And the average molecular weight is about 1
It was 3,000 daltons. Example 4 11.8 kg of enzymatically decomposed product of whey protein (manufactured by Morinaga Milk Industry Co., Ltd.),
36 kg of dextrin (manufactured by Showa Sangyo Co., Ltd.) and a small amount of water-soluble vitamins and minerals were dissolved in 200 kg of water to prepare an aqueous phase in a tank. On the other hand, soybean salad oil (Taiyo Yushi Co., Ltd.) 3 kg, palm oil (Taiyo Yushi Co., Ltd.) 8.5 kg
And safflower oil (Taiyo Yushi Co., Ltd.) 2.5 kg, lecithin (Ajinomoto Co., Inc.) 0.2 kg, fatty acid monoglyceride (Kao Co., Ltd.) 0.2 kg, and a small amount of fat-soluble vitamin were mixed and dissolved to prepare an oil phase. . The oil phase was added to the aqueous phase in the tank, mixed by stirring, heated to 70 ° C., homogenized with a homogenizer at a pressure of 150 kg / cm ² , and
Sterilized at ℃ for 10 minutes, concentrated and spray-dried to obtain about 60 kg of intermediate product powder.

To 50 kg of this intermediate product powder was added 6.7 kg of sucrose (made by Hokuren), 3 kg of the powder of the peptide mixture produced by the same method as in Example 3, and 0.3 kg of an amino acid mixed powder (produced by Ajinomoto Co., Inc.) to obtain a uniform mixture. Then, about 60 kg of powdery enteral nutritional supplement A was obtained. Separately, 5 kg of intermediate product powder, 0.67 kg of sucrose (made by Hokuren),
Whey protein enzymatic decomposition product (Morinaga Milk Industry Co., Ltd.) 0.3 kg and amino acid mixed powder (Ajinomoto Co., Inc.) 0.03 kg were added and uniformly mixed to obtain about 6 kg of powdery enteral nutrient B.

15% of these two types of enteral nutritional powders with water
Of 0.75% (hereinafter referred to as enteral nutritional supplement A) and 0% (hereinafter referred to as enteral nutritional supplement B) of the peptide mixture described in Example 3 respectively. Enteral nutrition was prepared. These two types of enteral nutrients were dispensed into a sterile pack, and in the same manner as in Test Example 6, 10-week-old SPF mice (SLC), BALB / c, and 10 females each were allowed to freely ingest for 3 weeks. One week after the breeding, methotrexate dissolved in physiological saline was intraperitoneally administered to each mouse at a rate of 100 mg per 1 kg of mouse body weight. After being fed with each feed for 2 weeks after the administration, the mice were dissected, mesenteric lymph nodes were aseptically collected, and the BT incidence was measured.
As a result, the BT incidence was 9 in the case of the mice to which the enteral nutritional supplement B containing no peptide mixture described in Example 3 was administered.
/ 10, whereas it was 1/10 when the enteral nutritional supplement A containing the peptide mixture of the present invention was administered,
The occurrence of BT was significantly suppressed.

[0055]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides a peptide mixture derived from a lactoferrin hydrolyzate having no antibacterial property, and a composition containing the peptide mixture as an active ingredient. It is effective. 1) It has the effect of preventing the intestinal bacteria from migrating from the intestinal tract of humans and animals to the organs, and preventing diseases such as opportunistic infections, anaerobic bacterial infections, and sepsis. 2) As a component of oral or tube feeding nutritional composition for humans with reduced digestive function and infection resistance in premature babies, newborns, infants, old people, patients with chronic digestive system diseases, postoperative patients, etc. Used. 3) It enables the reduction of drug dose in the treatment of infectious diseases caused by the transfer of intestinal bacteria into the body. 4) It has the effect of alleviating side effects such as antibiotics and anti-cancer agents that damage the intestinal tract of humans and animals.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shin Koichiro 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry Co., Ltd., Institute of Nutritional Sciences (72) Inventor Hidefumi Kuwata 5-1-183 Higashihara, Zama City, Kanagawa Prefecture Morinaga Dairy Co., Ltd. Nutritional Research Institute

Claims (2)

[Claims] 1. A peptide mixture which is separated from lactoferrin hydrolyzate and has the following physicochemical and biological properties: a) inhibiting the transfer of intestinal bacteria from the intestinal tract to the organ, b) in vitro. It has no antibacterial action, c) the antigenicity is 1/1000 or less of undegraded lactoferrin, d) the average molecular weight measured by high performance liquid chromatography is 13,000 daltons or less, and e) the lactoferrin Decomposition rate is at least 6%. 2. A composition, which is separated from lactoferrin hydrolyzate and contains, as an active ingredient, a peptide mixture having the following physicochemical and biological properties: a) Inhibition of intestinal bacterial transfer from the intestinal tract to an organ. B) has no in vitro antibacterial action, c) has an antigenicity of 1/1000 or less of undegraded lactoferrin, and d) has an average molecular weight of 13,000 daltons or less measured by high performance liquid chromatography. E) The decomposition rate of lactoferrin is at least 6%.