TW201210509A - Anti-allergy lactic acid bacteria - Google Patents

Abstract

The invention described relates to novel strains of lactic acid bacteria and to novel compositions containing a novel strain or strains not previously known to be useful in anti-allergy. The composition may be in the form of foodstuffs or in the form of pharmaceutical compositions. The invention described how to screen the strains of lactic acid bacteria can regulate the immune balance between Th1 type and Th2 type.

Description

201210509 VI. Description of the Invention: [Technical Field to Which the Invention Is Applicable] The present invention relates to an application to a food or medicine and its use in anti-allergic ability. Livure fungi, and [prior art] Allergic diseases have increased year by year in recent years, and from the occurrence of phlegm, allergic rhinitis and bronchial asthma ^; The progress of civilization, with a wide range of pollution and infection, may lead to an increase in allergies. At present, the treatment of asthma is mainly based on drug treatment. However, cockroaches, including anti-inflammatory and gas-swelling agents, can't really change the allergic immune response, so it can only be said to be treated by $. The only treatment that can be said to change the allergic immune constitution: desensitization therapy, but desensitization treatment usually takes a long time, and = some side effects will occur - so not all patients can use. The increase in allergic diseases is currently considered to be closely related to changes in the environment, especially in terms of the environment. The &intestines in the human gut stimulate the immune system, but if the food in daily life contains too much or too much steroid content, it will lead to a decrease in probiotics in the intestine, thus effectively stimulating immune cells. The production of a type 1 τ helper cell (Thl), which is closely related to the development of allergic diseases associated with type 2 T helper cells (Th2). Therefore, if we can use health foods to stimulate our immune system, and induce a Th2-type immune response caused by a balanced type of allergic immune response that can regulate allergic immune reactions, it will be able to achieve the effect of changing the body and sensitive body. In the history of human applied microorganisms, the application of lactic acid bacteria originated quite early. From the processing of early dairy products, it has been found that the consumption of foods fermented by lactic acid bacteria helps to improve the incidence of human gastrointestinal diseases and prolong the average life expectancy. From the beginning of 201210509, we will discuss the continuous fermentation of milk in the promotion of human health. In 1965, the concept of "Pr〇b1〇tlc-probiotics" was made by L% Guanlan, and became the generic name of functional lactic acid bacteria. . The effect of the production (10) has the health of the material effect: Although the number of lactic acid is 4 in the natural world, only a few strains have anti-allergic (four). A small number of these strains have the characteristics of acid resistance and '/ orphan force, ability to adsorb mucosal epithelial cells and ability to survive after passing through the gastrointestinal tract, and are important for screening and promoting the effect of spreading. There were only a few strains of Phytophthora strains that proved to have anti-allergic health effects on the 5th, and the function of lactic acid bacteria on body health lies in the specificity of the strain rather than the species. Such a strain that has a special effect on human health is called a functional probiotic (Guidelines f〇r the

Evaluation of probiotics in food ; Report of joint FAO/WHO woridng group on drafting guidelines for the evaluation of probiotics in food ; London Ontario, Canada April 30 and May 1 2002 : 1-7). Allergic diseases such as allergic eczema, urticaria, recurrent allergic rhinitis and allergic asthma have become serious social problems in Taiwan and other developed countries. There is a reason for the increase in the prevalence of allergic diseases. 'This well-known health hypothesis is: The less chance that infants and young children will be exposed to immune stimuli, the more likely they are to cause allergic-related diseases (Strachan, 1989) . When an allergic disease occurs, the immune response in the human body will decrease the number of Th1 cells, and continuously produce a variety of cytokines to promote the immune response toward the Th2 pathway, forming a humoral immune response, such as the production of IgE and excessive eosinophils (Romagnani, 1994; Holt , 1995). In the children of Estonia and Sweden, children with allergic diseases have fewer lactobacilli in the gut than those without allergies (Bjo “rksten et al., 1999”. Lactobacillus is thought to promote Thl The immunization against 201210509 should further improve allergy symptoms (Cross et al., 2001). Furthermore, in an animal model of albumin-induced mouse allergy, it was found that the mice were orally administered Lactobacillus casei strain Shitota after heat treatment. It can inhibit the content of IgE in mouse serum (Matsuzaki et al) 1998). In addition, the animal model of casein-induced mouse allergy was demonstrated in the intraperitoneal injection of heat-treated

Lactobacillus plantarum L-137 also inhibits the production of IgE (Murosaki et al., 1998). The allergic symptoms caused by hay fever pollen allergy, oral Enterococcus faecalis FK-23 extract, can reduce the amount of eosinophils accumulated in the peritoneum (81^11^(^61, 2003). In human clinical trials, Taking Lactobacillus rhamnosus strain GG during labor, it was found that two years after birth (Kallioma"ki et al., 2001) and after infancy (Kallioma..ki et al, 2003) can reduce allergic eczema in infants and young children. Risk and incidence.'

Lactobacillus rhamnosus 19070-2 and Lactobacillus reuteri DSM122460 can also moderately improve the allergic symptoms of severe eczema in children with atopic dermatitis (R0Senfeldt et al., 2003). SUMMARY OF THE INVENTION In one aspect, the present invention broadly includes any of the following biologically pure cultures, Lactobacillus acidophilus PM-A0002 strain, and food industry development research on February 23, 1995. Registered number BCRC 910308, Lactobacillus gasseri PM-A0005 strain, January 11, 1995 Food Industry Development Institute registration number BCRC 910304, Lactobacillus salivarius subsp salicinms PM-A0006 strain, the Food Industry Development Institute of the Republic of China on January 11, 1995, registered number BCRC 910305, Lactobacillus johnsonii PM-A0009 strain, October 17th, the Republic of China food industry development The Institute registered number BCRC 91〇336, Lactobacillus acidophilus PM-A0013 strain, and the Food Industry Development Institute registered number BCRC 910309 on February 23, 1995. A composition consisting of a physiologically acceptable excipient or diluent. 201210509 In a specific embodiment, the composition contains one or more such strains, such as a physiologically acceptable thief, which is a preferred form of food, which may be fermented milk, yogurt, Any of the cheese, the dairy drink, the milk powder, the tea beverage or the coffee is preferably, or alternatively, the composition is a pharmaceutical composition and the bismuth or diluent should be a pharmaceutically acceptable elixirs or Diluent 0 In the case of 遐贳 遐贳 , , , , , , , , , , , 生物 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强 增强5 strain, saliva milk ^ =: 06 strain, Yogurt lactic acid rod 3 ΡΜ · Α _9 strain or E. acidophilus ΡΜ - Α 0013 strain. In a specific embodiment, the present day and the month can broadly include inhibiting immunity by regulating the type III immune response by promoting the glass or interferon 7; g improving the allergic phenomenon of excessive immune reaction of the type G, and the pair is feeding the seed === ===; The diluent is preferably administered in the form of a composition and the release agent is a food, and the food is preferably _milk, yogurt, tea, or coffee. ^ 疋 疋 pharmaceutical composition 'and thief-shaped agent or turn on?, 'dix-shaped agent or thinner. I can accept the definition of the i ϊ ϊ 包括 包括 包括 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本 本Zhang, Shi-Complete things have been with the present hair == Jin, 201210509 [Embodiment] <The present invention can broadly include any of the following biological pure cultures: Pseudomonas PM-A0002 strain, Republic of China ninety-five On February 23, the food, exhibition research institute BCRC 9_8, mechanical milk transfer g ρΜ: 〇〇〇5 囷 ,, off the ninety-five years - liver-eclipse industry development institute registration number BCRC 910304, Lactobacillus saliva strain, the Food Industry Development Institute of the Republic of China on January 11, 1995, registered number BCRC 910305, Lactobacillus johnsonii PM-A00G9 strain, October 17th, 1995 Food Safety Development Institute The registration number BCRC 910336, Lactobacillus acidophilus ΡΜ_Α〇〇13 strain, registered in the Food Industry Development Institute of the Republic of China on January 12, BCRC 910309. Many literatures have pointed out that increasing the secretion of first-type T-cell cytokines including interleukin (IL-12) and interferon-gamma (iFN-gamma) can effectively improve allergy symptoms; further studies have pointed out specific lactic acid strains and trees. The binding of Toll-like receptors on the spheroid cells, the transfer of the transgenic proteins in the activated cells to the nucleus and the release of a large number of cytokines, is a part of innate immunity, so some specific lactic acid bacteria species have more cell walls. Sugar substances such as peptid〇glycan, lipopolysaccharide, polysaccharide, etc., through the innate immune system, can indeed activate the development of T cells. , strain name number Lactobacillus acidophilus PM-A0002 BCRC910308 February 23, 2006 Lactobacillus kawaii PM-A0005 BCRC910304 January 11, 2006 Lactobacillus saliva PM-A0006 BCRC910305 January 11, 2006 Yogurt Bacillus PM-A0009 BCRC910336 October 17, 2006 Lactobacillus acidophilus PM-A0013 BCRC910309 The frozen-dried culture of the five bacterial strains has been deposited at the Taiwan Food Industry Development Institute at the address of the Republic of China on February 23, 2006. No. 331, Food Road, Hsinchu City. The details of the deposit are shown in Table 1: 201210509 Table 1 The five strains of lactic acid bacteria deposited in the above listed patents have been found to have anti-allergic ability' including atopic dermatitis, urticaria, allergic rhinitis, food allergies and Symptoms of asthma have a function of slowing and treating. Example 1: Morphology and general properties of five anti-allergic lactic acid bacteria. According to the 16S rDNA sequence analysis and the results of the API bacterial identification system analysis, the taxonomic characteristics of the strain were confirmed. Found that Dongyu strain number? ]^_^〇〇〇2,, Lactobacillus acidophilus; Dongyu strain number PM-A0005 is Lactobacillus added; Dongyu strain number PM-A0006 is Lactobacillus saliva; Dongyu bacteria Tielu·09 is about lactic acid Bacillus and Dongyu strain No. PM-AGG13 are U. acidophilus. The morphological and general characteristics of the five strains are listed in detail. Table Lactobacillus acidophilus PM-A0002: is a successor, high-profile, usually single aerobic and occupational touch n, motility, in...±lc, belongs to Facultative Arr rt · Fen I touch 4 denier τ%,, . _ " -- Lactobacillus bergii PM-A0005 : Short rod-shaped, paired or short-chained when cultured in MRS culture. Long, usually single Gram staining rod g, does not produce spores, can grow in both aerobic and anaerobic environments, most suitable for ^_ and motility, in the facultative salivary bacillus ΡΜ-Α0006. ___ in MRS training When the liquid is cultured, the cells are short rod-shaped and paired in a short chain. - The code is rounded 'usually appears alone, Gram-positive bacilli, does not produce long diminidase and is motility'. No gas is produced in the saliva lactic acid rod! 201210509 Lactobacillus johnsonii PM-A0009: - The end is round, usually W-shaped or slightly longer, Gram-positive bacilli, can grow without augmentation of aerobic and anaerobic environment, the most suitable growth enzyme And exercise 'in money»^! Glucose metabolism does not produce gas: j 37 soil 1 C, belongs to facultative Lactobacillus acidophilus PM-A0013 when cultured in MRS medium, short or slightly longer - Mountain Gram Stain-positive bacilli, do not produce spores, do not form a square 'chain. Sub = and disgusting money _ can grow, the most suitable growth temperature is π when the gas is not produced. Table 2 ~ ~ ~ = 2: anti-allergic lactic acid bacteria to promote the secretion of interferon γ to increase the force of the immortal (to the peripheral blood Mononuclear cells were tested for in vitro efficacy. Five strains of anti-allergic lactic acid strains, Lactobacillus pneumoniae pM_A〇〇〇2 strain, 2 lactic acid ΡΜ·Α_5 g strain, Weiwei lake pM_A_6 strain, Lactobacillus johnii PM -A0009 strain or Lactobacillus acidophilus pM_A〇〇 strain enhances the immunity of TM type, and the amount of interferon γ is measured by co-cultivation of the blood and the anti-allergic lactic acid bacteria. The ability of the strain. Use the following experimental steps: I extract the appropriate human blood volume about 2 〇〇mi each time. 2. Take an equal ratio of Ficoll-paque and blood at 18_2 〇. 40 minutes. 3. Wash the cells 2-3 times with the Peripheral blood mononuclear cell (PBMC) layer buffer solution and suspend the cells in a suitable medium (eg RPMI-1640). 4. Activate the cells with activated cells. 3-day lactic acid strain 1 : 1 〇 ratio co-culture 48 201210509 hours. 5. Collect supernatant from cell culture. Use enzyme immunoassay (ELISA) to detect the content of interferon 7 (IFN-gamma) in the supernatant. § Ten analysis is shown in Table 3 and Figure 1, represented by Mean Shi SD. Figure 1 shows the strains of Lactobacillus acidophilus PM-A0002 at 1〇6 to 1〇8, respectively. , Lactobacillus gasseri PM-A0005 strain, Lactobacillus salivarius subsp. salicinius PM-A0006 strain, Lactobacillus johnsonii PM-A0009 strain or Lactobacillus acidophilus The PM-A0013 strain was co-cultured with 1〇5 to 1〇7 human peripheral blood leukocytes (PBMC) for 48 hours, and the cell supernatant was collected. The enzyme immunoassay was used to detect interferon gamma (IFN-gamma) in the supernatant. The content of the liquid, in which the anti-allergic lactic acid bacteria (Lactobacillus casd BCRC12249) was used as the negative control group 'PHA as the positive control group, the interferon γ was stimulated to detect the concentration. The results showed that the test group can significantly stimulate people. Peripheral blood leukocytes (PBMC) secreted interferon gamma and the negative control group were significantly different. Table 3 shows the secretion of interferon γ (pg/ml) by five anti-allergic strains and human peripheral blood mononuclear cells, respectively. The Lactobacillus acidophilus PM-A0002 19833±2767 j-mouth Bacteria PM-A0005 46625±3624 Saliva standard ΡΜ-ΑΟΟΟό 25850±2347 iiML Acid bacillus ΡΜ-Α0009 25725±2008 ΡΜ-ΡΜ0013 17416±2803 (L. casei BCRC 12249) 11±2.3 to control group (positive control) __ 44666+2488_^ Table 3 Example 3; Anti-allergic lactic acid bacteria promote the secretion of intercellular white pigment (IL_12) 201210509 The role of Thl-type immunity (to the dendritic cells such as Fuhua (2) 体外 for in vitro efficacy verification platform ). Five anti-allergic lactic acid strains, Lactobacillus acidophilus pM_A〇〇〇2 strain, Lactobacillus calyceii PM-A0005 strain, Lactobacillus salivarius PM_A〇〇〇6 strain, Lactobacillus johnsonii PM-A0009 strain or cola sour milk The effect of the strain PM-A0013 on the immunity of Th1 type is determined by measuring the secretion amount of intercellular melanin (IL_12) after co-culture of human dendritic cells with anti-allergic lactic acid bacteria to select strains having anti-allergic ability. Use the following experimental steps: 1. Draw appropriate human blood volume approximately 2 〇〇mi each time. 2. Take an equal ratio of blood cell separation solution (Fic〇u_paque) and blood at 18_2〇. Centrifuge at 400g for 30-40 minutes. 3. Take the human peripheral blood leukocyte (pbMC) layer, wash the cells with buffer solution 2-3 and then suspend the cells in a suitable medium (eg RPMj_164〇). 4. Human peripheral blood leukocyte (PBMC) cells were purified from CD14+ mononuclear cells of the cells by CD14+ microbeads (MiniMACS system). 5. The cells were differentiated into dendritic cells by cytokines (IL-4) and growth hormone GM-CSF, and the differentiated dendritic cells were collected at a culture time of 6-7 days. 6. The anti-allergic lactic acid strain was activated 3 days before co-cultivation, followed by 1 〇〇. 〇 Heat lethal lactic acid strain for 30 minutes. 7. The dendritic cells were co-cultured with the heat-killed lactic acid strain at a ratio of 1:10 for 48 hours. 8. Collect the supernatant from the cell culture. Enzyme immunoassay (ELISA) was used to detect the content of intercellular white pigment (IL-12) in the supernatant. The statistical analysis of the data is shown in Table 4 and Figure 2, and is represented by Mean Shi SD. Figure 2 shows the killing of 1〇6 to 1〇Lactobacillus acidophilus PM-A0002 strain, Lactobacillus gasseri PM-A0005 strain, Lactobacillus salivarum by heat. Lactobacillus salivarius subsp. salicinius) PM-A0006 strain, Yorkshire 201210509 Lactobacillus johnsonii PM-A0009 strain or Lactobacillus acidophilus PM-A0013 strain and 105 to 1〇7 human dendritic cells (dentritic Cell) was co-cultured for 48 hours, cell supernatant was collected, and the amount of interleukin (IL-12) in the supernatant was detected by enzyme immunoassay. Among them, Lactobacillus casei BCRC12249 was used as a negative control group, PHA was a positive control group, and the interleukin (IL_12) stimulated concentration was detected. The results showed that the experimental group significantly stimulated the secretion of intercellular white pigment (IL-12) from human dendritic cells and the negative control group was significantly different. Table 4 shows the secretion of intercellular white pigment (IL-12) in the name of intercellular white pigment (IL_12) strains (pg/ml) by five heat-killing anti-allergic strains and dendritic cells, respectively. Lactobacillus PM-A0002 15019±569 Lactobacillus lactis PM-A0005 19222士212 Salivary lactic acid bacteria PM-A0006 18625士365 Lactobacillus johnsonii PM-A0009 18291士39 Lactobacillus acidophilus PM-A0013 17836±168 Negative control Group (L. casei BCRC 12249, 80±15 positive control group 13786±341 Table 4 Example 4: Anti-allergic lactic acid bacteria resistant to gastric acid bile salt test. Examination of five anti-allergic lactic acid strains, Lactobacillus acidophilus pM_A〇 Can the 〇〇2 strain, Lactobacillus calyceii PM-A0005 strain, Lactobacillus salivarius pM_A〇〇〇6 strain, Lactobacillus johnsonii PM-A0009 strain or Lactobacillus acidophilus PM_A〇〇13 have the ability to pass the test of gastric acid bile salt Smoothly exert its anti-allergic function in the intestines. The test procedure is as follows: 1. Activate five anti-allergic lactic acid bacteria for 3 days. 2. Calculate the original bacteria number by taking 1 mL of bacterial liquid, and centrifuge the remaining lactic acid bacteria at 5 〇〇g. 12 201210509 : Knowing to add deionized water to clean lactic acid bacteria 2-3 times. Live fine, clean the water 2 butterfly 'calculation save 5 · After the centrifugation, the precipitate will be centrifuged to contain 37 cells, and the number of live cells will be 4 hours. 7. The growth rate of acid g' is calculated as the lactic acid bacteria in the sample of gastric biliary tract in the presence of gastric acid and bile salts, and the growth of the strain is analyzed by the analysis in Table 5 and Figure 3, the third After the activation of the medium, the total bacterial sensitivity of the acid lactic acid bacteria decreased significantly; the adjusted solution and the specific: =6, ^^ 趟Hi and the tolerance to gastric acid, but the strain passed through the gallbladder ^处=, Laiwu strain has a slight decline, but it is still tolerant to bile salts. Note that the five strains of lactic acid can pass the digestive system of the human body. The environment S is a hydrochloric acid and anti-(5) value of 2.5. Allergy lactic acid bacteria mixed test

Two = change, lay down sharply, - two 13 201210509 i yogurt to eat j__PM^0013j 1.78X109 1.83X109! l,86xl〇jl, 73xl〇9 Table 5 The results and analysis of the gallbladder salt in Table 6 and As shown in Figure 3, Table 6 tests the bile salt tolerance of 1.5% bile mixed with anti-allergic lactic acid bacteria. Strain name Original bacteria number 1.5%bile-lhr 1.5%bile-2hr 1.5%bile-3hr 1.5%bile-4hr Lactobacillus acidophilus PM-A0002 8.2〇xl〇8 4.3〇xl〇6 1.5〇xl〇6 5.45x10s 4.4 〇xl05 Plus Lactobacillus PM-A0005 2.65xl〇9 5.8〇xl〇4 5.55xl04 2.4〇xl04 1.89xl04 Lactobacillus saliva PM-A0006 2.55xl〇9 1.45x10s 9.2〇xl07 1.02xl08 8.3〇xl07 Lactobacillus faecalis PM -A0009 6.87xl〇8 8.65xl〇7 1.13xl08 5.7〇xl〇7 8.3〇xl06 Lactobacillus acidophilus PM-A0013 1.78xl〇9 6.72x10s 5.73x10s 8.0〇xl〇6 3.00xl05 Table 6 Example 5. Anti-allergy The adsorption capacity of lactic acid bacteria on human intestinal epithelial cells (CaC〇_2). Five different anti-allergic lactic acid bacteria strains were used to analyze the ability of human anti-allergic lactic acid bacteria to adsorb human-tall epidermal cells (CaCo 2 ) in vitro. Human intestinal epithelial cells (CaCo-2) monolayers were first grown on coverslips and plated into monolayer cells and placed in a multi-well culture dish. Then, the ratio of cells: lactic acid bacteria was 1:200, co-cultured, /-3 hours, washed with Ixpbs and fixed with sterol and the remaining number of bacteria, washed with IxPBS and fixed with methanol and remaining After adhering the number of bacteria, gram staining was performed and the number of attached bacteria was determined. Referring to the analysis method of Jacobsen et al. (1999), 20 fields of 201210509 were counted under a 1× microscope, and when the average number of bacteria per field of view was less than 4〇, it was judged as nonadhesive; When the average number of bacteria in the field of view is between 41 and 1 判定, it is judged as adherence; when the average number of bacteria per field of view is greater than 1 ,, it is judged to be strongly adhesive 〇 data of 5*1* The analysis is expressed in Mean ± SD. On average, the calculated values of forty-five fields of view are taken. The results are summarized in Tables 7 and 4. Figure 4 shows that according to the analysis method of Jacobsen et al. (1999), when the average number of bacteria per field of view is less than 40, it is judged as not attached (n〇nadhesive); when the average number of bacteria per field of view is 41 When it was 100, it was judged to be adherent; when the average number of bacteria per field of view was greater than 100 days, it was judged to be strongly attached (glyphs adhesive). Lactabidais acid〇philUS PM-A0002 strain, Lactobacillus calvus gaSSeri PM-A_5 strain, Lactobacillus salivarius (sp. SaliciniUS) PM-A0006 strain, Four anti-allergic strains such as Lactobacillus johnsonii j:hns〇nii) PM-A0009 strain were judged to be "strongly attached" and Lactobacillus acidophilus PM-A0013 strain was judged to be attached. Table 7 shows the adsorption capacity of five anti-allergic lactic acid bacteria on human intestinal epithelial cells (CaCo-2) cell line: strain name Lactobacillus acidophilus PM-A0002 MeansiSD --*--- 115.4±4.4 Lactobacillus lactis PM -A0005 100.1士2.6 Lactobacillus salivarius PM-A0006 108.3 Soil 1.4 Lactobacillus johnsonii PM-A0009 208.3 Soil 22.1 Lactobacillus acidophilus PM-A0013 02.8±5.0 Table 7 Example 6: Treatment with anti-allergic lactic acid bacteria as supplementary feed or The effect of soothing allergic asthma was evaluated by the following experiment using the following experiment with the administration of Lactobacillus sp. Lactobacillus ρμ_α〇〇〇6 to the protein (ovalbumin; OVA)-induced asthmatic mice. A. Experimental design Experimental animals Fine mice were used, 6-8 weeks old female rats, control group and experimental group each = 4 rats. The commonly used female ancestors were selected, 8 weeks old, and eat for six weeks, each number ^14. Purchased sPF six-week-old BALB/c 昧 'mother mice from the National Taiwan University Animal Center were housed in stainless steel cages at room temperature, controlled at 22±2〇c, and cycled coffee for 12 hours (shine in the morning) , 6 points dark, free drinking water and feed, the mice were randomly divided into groups at the age of eight weeks, began to give the test lactic acid by tube silver. 2. Establish an animal model of respiratory tract inflammation. The two-step procedure is as follows: · On the second day of the experiment, 5 ton of egg white egg (OVA) and adjuvant Aklm 4 mg were intraperitoneally injected into the mouse, in the experiment - In 30 and 44 days, mice were injected with 25 tons of albumin (〇VA) and 4 mg of Ahm^f. After sensitization, on the 54th and 55th day of the experiment, the mice were anesthetized, and intramary (mtra-naSal, in) drops of 100-way albumin (〇v hair respiratory inflammatory reaction. Ji 3. Experimental flow ^ as the first In the figure 5, the anti-allergic lactic acid g improves the design process of the allergy experiment. The stage of feeding the anti-allergic lactic acid bacteria is divided into four phases: the first phase is 〇~22 days; the second phase is 23~36 days; the third phase is 37~5〇天; the fourth period is Μ~幻天. In the meantime, mice were sensitized by intraperitoneal injection of egg protein (0VA), which was sensitized four times, the second time was the first day of apology; the second time was The 16th day of the start of the tube; the third day is the 30th day of the start of the tube; the fourth time is the first day of the tube: the intraperitoneal injection of the albumin (OVA) sensitized mice will be blood-tested every other week. Changes in the content of IgE in mice. In the first 4 days (day%) of the sacrificial mice, the mice were trained by nasal mucosa inhalation for 2 consecutive days, and the lungs of the mice were reacted with the force response. Sacrificial mice were tested for other biochemical values. Β·Experimental method 201210509 i. Animal sacrifice and experimental material collection and analysis a. Collection of serum samples = test When 23,37,51 Λ mice were sacrificed and blood samples were collected __bital). After cutting and anaesthetizing the rats, the blood is quickly collected from the orbital sinus, and about 2 〇〇25 blood is taken. The cells were allowed to stand for 2 to 4 hours, centrifuged at 12,000 rpm for 20 minutes, and the serum was collected and stored at -80 °C. VIII b. Determination of the protein (OVA)-specific antibody by enzyme immunoassay (ELISA) Dissolve 〇.5mg of antigen in lOOgL carb〇nate coating buffer ((λ 1 Μ

NaHC〇3) was added to a microplate and allowed to stand overnight at 4 °C. On the second day, the microplate was washed with IX PBS buffer containing 0.05% Tween 20. Then block for more than two hours with blocking solution (1% BSA in IX PBS buffer), after 3 washes, then add ι〇〇μ^^ The sample to be tested is allowed to stand at 4 ° C overnight, after 4 washes. Add lOOpL anti-mouse igE antibody to react at room temperature for one to two hours, and then wash 5 times. Add 1 〇〇pL to the appropriately diluted avidin-HRP (avidin-horseradish peroxidase conjugated) for one hour, wash 6 times, and finally add 100 μ]: ABTS ( 2.2,-Azino-bis-3- Ethylbenzthiazoline -6-Sulfonic Acid After the substrate was colored at room temperature for about 30 minutes, the reaction was terminated with 100 pL of 5% SDS, and the absorbance was read with a microplate reader. The results are expressed as follows by enzyme immunoassay (ELISA) unit: ELISA. Ulllt _ (Agample Ablank) / (Apositive control — Ablank)

c. Airway hyperresponsiveness (AHR) assay To investigate whether oral antiallergic lactic acid bacteria can alleviate allergic reactions in sensitized mice, and receive airway resistance tests the next day after two intratracheal allergen stimuli. The test system was Buxco system (Biosystem XA, Buxco Electronics Inc. Sharon, CT, USA). Values were calculated using a transducer (differential pressure transducer, Buxco) and preamplifier (MAX 17 201210509 II, Buxco) to collect information on changes in respiratory tract in mice. The Penh value is calculated as pause x PIF/PEF ; Pause = Listen _ Tr) / Tr, (PIF : peak inspiratory flow ; PEF : peak expiratory flow ; Te : expiratory time ; Tr : relaxation time). The mice were placed in the whole body plethysmograph chamber under conscious state, and the normal saline was introduced into the chamber for three minutes by ultrasonic vibration. Then, the average lung respiratory resistance value Penh was recorded for three minutes in the mouse, and then the concentration was increased.

Methacholine (6.25, 12·5, 25, 50 mg/mL) was spray-administered to the mice, and each concentration was stimulated for three minutes and three minutes of respiratory physiological changes were recorded. Record the average penh value for each minute in three minutes. d. Lung lavage fluid and lung cell analysis test lung respiratory resistance test the next day, the mouse sacrificed, cut the neck and flesh to expose the trachea, the venous catheter is inserted into the trachea, the first time I i sterile HBSS buffer Rinse the lungs, and in the second and third time, wash the lungs with HBSSbufFeM containing 2% BSA. The first obtained lung washing solution was centrifuged at 15 rpm for 5 minutes, and the supernatant was separated and stored at _8 (rc Subsequent analysis. The cell part was then inoculated into two lung lavage fluids, centrifuged at 15 〇〇 jpm for $ minutes, the supernatant was discarded, and the cells were floated with 1 mL of HBSS buffer containing 2% BSA, and trypan Blue staining count, take about! χ 1〇5 cells to centrifuge two centrifugation (Cyt〇Spin) 500, centrifuge for 3 minutes to make cell smear, after it is naturally dried, stained with LhiA and LiuB in sequence, with a small amount of gum arabic Capsules, use oil mirror (lGGG X) to read at least five fields of view cells or a total of more than 3 cells, calculate the proportion of lymphocytes and multinuclear spheres. e. Spleen cells culture under sterile conditions, open the small gas abdominal cavity to remove the spleen Placed in 3 this 2Gmmpetridish has been added, no The syringe was ground, and the lymphocytes were released. The cell suspension was inhaled for 15 minutes and then left to stand for 5 minutes. The supernatant was centrifuged at i5QQ rpm for 5 minutes. After removing the supernatant from 201210509, add 1 mL of RBC lysis. Buffer, let stand for 1 minute to dissolve red blood cells, add 5 mL HBSS buffer, centrifuge at 1500 φΐη for 5 minutes to 'absorb the supernatant; repeat twice with HBSS buffer, then add 5 mL of RPMI-1640 with 5% FBS Complete medium to suspend the cells. Calculate the total number of cells by trypan blue staining, adjust the number of cells to 1 X 1〇7 cells/mL medium, add 0.5 mL of cell solution to 48-well plate, add equal volume of culture medium, and split. The culture medium of the hormone or egg protein (OVA) is allowed to have a final concentration of 5 x 1 〇 6 cells/mL medium. The culture supernatant is placed in a strange temperature incubator at 37 ° C and 5% C 〇 2 for 48 hours. After storage, the amount of cytokine secretion in each cell was analyzed. f. Determination of cytokine The content of cytokines secreted by spleen cells was determined by sandwich enzyme immunoassay (ELISA). Briefly described as follows: NaHC〇3 buffer (pH 9.6 After dilution, a 96-well microplate was added and allowed to stand overnight at 4 ° C. The plate was washed 3 times the next day, and then blocked by reacting with PBS containing 1% BSA at room temperature for at least 1 hour. After adding the sample to be tested, it can be 37. (: reaction for 2 hours or at 4 ° C for overnight. After washing 4 times, add bi〇tin_c〇njugated anti-cytokine antibody diluted in 1% BSA-PBS buffer for 1 hour at room temperature 'wash 5 times, then Add qing (4) _ conjugated peroxidase and then react for 丨 hours. Finally, after washing, add 1 μμ of enzyme substrate Tβ substrate (Tetmmethylbenzidine substrate) to each well for about 2 〇 minutes at room temperature. Autoreader reads the 2_statistical method &m (four) ΐΐίΐΤ mean value 4 of the wavelength 450 nm (MeanS±S_. Each Ρ<= analysis, P<aG5 is considered to be significantly different (*, Ρ 0·05,, ρ&lt ;0·001). Further analysis of p<〇1 experimental results: β左兵19 201210509 ^Determination of white (OVA)-specific antibody enzyme immunoassay lisa) π > egg protein (OVA) specificity In the antibody titer portion, the control group of A0006 lactic acid bacteria, the protein (OVA:) specific antibody in the serum of ovarian (〇VA) sensitized mice showed a trend of decreasing. The results are shown in Table 8. Figure 8. Table 8 shows the specific antibody 姊 determination of the printed protein (〇VA) Results group A 1 A fly? A v aiuc-J control group IgE 0.66±0.05 0.44±0.03 (P=0.06)# ELISA unit = #:P<0.1 sample A blank) ! (A positive control ~ A blank Table 8 B. Airway hyperresponsiveness (AHR) As a result of lung respiratory resistance, compared with the control group, the feeding of PM-A0006 lactic acid bacteria significantly reduced the mice under the stimulation of high concentration of Methcholine. Penh value. The results are shown in Table 9, which is shown in Figure 7. Table 9 shows the results of airway hyperreactivity (Means ± SEMs, N = 14, P value = P) group (Mch : mg / mL) control group (Penh) experimental group (Penh) 0 1.41 ± 0.05 1.44 ± 0.08 ( P=0.86) 6.25 1.50±0.11 1.35±0.11 (P=0.62) 12.5 2.59 Soil 0.24 1.94±0.20 (P=0.28) 25 4.38±0.47 1.95±0.12 (P=〇.〇8)# 50 5.10±0.32 2.63± 0.13 (P=0.〇〇2)* 20 201210509

Mch=Methacholne formulated concentration

Penh=pause x PEF/PEF ; Pause=(Te - Tr) / Tr, (PIF : peak inspiratory flow; PEF : peak expiratory flow; Te : expiratory time; Tr : relaxation time) * : p < 0.05 # :P&lt ;0.1___ Table 9 C. Lung lavage fluid and lung cell analysis results The cellular components in the lung lavage fluid, compared to the control group, the experimental group fed with PM-A0006 lactic acid bacteria had significantly lower eosinophil infiltration. proportion. The results are summarized in Table 10, which is shown in Figure 8. Table 10 shows the lung washing solution group control group (%) Experimental group (%) eosinophil 27.5 ± 1.37 17.1 ± 1.66 (Ρ = 0.02) * % = In percentage 0 / 〇 * : P < 0.05 Table 10 D. Lung The cytokine analysis results of the rinsing liquid compared with the control group, the experimental group fed with PM-A0006 lactic acid bacteria can significantly reduce the amount of e〇taxin secretion in the lung rinsing and the tendency of pGE2& The results are summarized in Table 11, which is shown in Figure 9 and Figure 1 . Table η shows lung flushing cell stimulation [^ analysis results (Means ± Sg ^ [s. N = 14, P value = P) level: _ control χ, Α ^, Α ν χ / experimental group 38.8 ± 3.00 22.7 ± 1.54 (Ρ=0.02)* pge2 ---£__ 8.1±1.〇4 3.82±0.41(Ρ=0.08)# * : Ρ<0.05 21 201210509 Table 11 E. Spleen cell culture supernatant cytokine analysis results spleen After the cells were stimulated with ConA or egg protein (OVA) for 48 hours, IFN-γ secreted by the spleen's visceral cells of the experimental group fed with PM-A0006 lactic acid bacteria was significantly higher than that of the control group under ConA stimulation. The effect of stimulating substances on cytokine secretion of spleen cells (Means±SEMs, N=14, P value=P) group control group ConA 5185 558 9748±966(Ρ=〇·〇5,* * : PO.05 Table 12 Combining the above-mentioned 'electrochemical immunoassay test# of the invention_immunization reaction effectively overcomes the dry problem of electrochemical detection and improves the detection sensitivity In addition, the present invention can provide a single-step rapid test strip and quantitative detection, and further combines biotechnology and electronic secret processing to produce a low-cost technical test strip. The food composition of the present invention contains a summary Any combination of any one or more strains, such as milk powder, tea or coffee, can be stored in the composition / #(四) strain can live the dead bacteria (4) In addition to finding out that there is a specific function ϊ ίί = Wei Cai human stomach coffee (four) environment, ^ wants to have a good and allergic reaction to small intestinal mucosal epithelial cells with a strong anti-allergic lactic acid bacteria 匕 lh2 too rich. Invention of a 22 201210509 The goal is to provide the public with a new choice of steroids or anti-tissue ammonia in allergies. The present invention finds: = action and beneficial anti-allergic milk _ as the H-allergic system for allergy treatment The features of the present invention are described by way of examples, and the invention is intended to be understood by those skilled in the art of the invention. The modification or modification of the spirit of the disclosure should still be included in the scope of the patent application described below. [Simplified illustration of the schema] Figure 1 shows that when ίο6 to 108 cfil Lactobacillus acidophilus (Lactobacillus) Addophilus) PM-A0002 strain, Lactobacillus gasseri PM-A0005 strain, Lactobacillus salivarius subsp. salicinius PM-A0006 strain, Lactobacillus johnsonii PM-A0009 strain or acidophilus Lactobacillus acidophilus PM-A0013 strain was co-cultured with 1〇5 to 1 () 7 human peripheral white blood cells (PBMC) for 48 hours, and the cell supernatant was collected, ' Enzyme immunoassay was used to detect the interferon-gamma (IFN-gamma) in the supernatant. Among them, Lact〇bacillus casei BCRC12249 was used as the negative control group, and PHA was the positive control group. Interferon gamma is stimulated by concentration. The results showed that the experimental group significantly stimulated the secretion of interferon-gamma in human peripheral blood leukocytes (PBMC) and the negative control group. Figure 2 shows that 'when killing 1〇6 to 〇, respectively, cfU Lactobacillus acidophilus PM-A0002 strain, Lactobacillus gasseri PM-A0005 strain, salivary lactic acid Lactobacillus salivarius subsp. salicinius PM-A0006 strain, Lactobacillus johnsonii PM-A0009 strain or Lactobacillus acidophilus PM-A0013 strain and 1〇5 to 1〇7 human tree The dendritic cells were co-cultured for 48 hours, and the supernatant of the cells was collected, and the content of interleukin IL-12 in the supernatant was detected by an enzyme immunoassay. In 23 201210509, the lactic acid bacteria (Lact〇baciiius casei BCRC12249) was used as the control group, and PHA was used as the positive control group to detect the interleukin (IL_12) stimulated concentration. The test group can be based on the difference between the leucovorin (IW2) and the sputum (four) group (four). The force/graph shows that 'knot_the commercial anti-allergic lactic acid bacteria in the medium after the activation of the total number of bacteria via the acidic buffer solution and bile salt treatment, the number of bacteria decreased significantly; the patented lactic acid strain that regulates allergies after the medium was activated The strains, acidic buffer solution and bile salt, PM_A〇〇〇6, pM_A〇(), 9 and pM_AQQi3 were not significantly affected by changes in gastric choline, pM_A〇〇〇2 and ΡΜ·^ 0005 Although the number of bacteria affected by gastric acid proved to be resistant to gastric acid, when the strain was treated with bile salts, although the strain was slightly decreased, it was still tolerant to bile salts, demonstrating the five anti-allergic strains. Lactic acid strains can pass the test of the strict environment of the human body. Figure 4 shows that 'according to the analysis method of jac〇bsen et al. (1999), when the average g-number of visual fields is less than 40, it is judged to be unique (n_dhesive); when the average number of bacteria per field of view is 41 to When it was 1 ,, it was judged to be adherent; when the average number of bacteria per field of view was more than 1 ,, it was judged to be strongly adhesive. Lact〇baciUus acidophilus PM-A0002 strain, Lact〇baciUus gasseri PM-A0005 strain, Lactobacillus subtilis (Lact〇baciUussalivarius subsp. salicimus) PM-A0006 strain, Yogurt lactic acid Four strains of anti-allergic strains such as Lactobacillus johnsonii PM-A0009 strain were judged to be "strongly attached" and Lactobacillus acidophilus P]V [-A0013 strain was judged to be attached. 'Figure 5 shows that the anti-allergic lactic acid bacteria improve the design process of allergy experiments, the stage of feeding anti-allergic lactic acid bacteria is divided into four phases: the first phase is 〇~22 days; the second phase is 23~36 days; the third phase is 37 to 50 days; the fourth period is 51 to 53 days. In the meantime, mice were sensitized by intraperitoneal injection of ovalbumin (OVA), which was sensitized four times. The first time was 24 201210509 cm: 6 days; the third was started = 3 days, the fourth was: artificial tube The 44th day of Rao. Intraperitoneal injection of albumin (ovalbumm; 〇VA) sensitized mice will be bloody 4 weeks = sensitized mice for 2 consecutive days, _ mouse lung respiratory resistance response 'satually sacrificed mice every other day Detection of other biochemical values. =6 shows that the mice are supplemented with salivary lactic acid rod g ρΜ_Α〇_lactate protein-sensitized mouse serum egg protein (〇VA) specific anti-feeding fresh increase, prion protein (0VA) sensitized mice There is a tendency for the reduction of the [beta]-protein (OVA)-specific IgE antibody in serum. ^ 7 shows that the mouse is supplemented with salivary lactic acid rod g pM_A〇〇〇6 lactic acid strain = 夺 以, protein-sensitized mouse lung respiratory resistance, relative to the control = ' ^ can be in high concentration 醯 醯Low mouse Penh values stimulated by methach〇line. The #=8 image shows that the cells in the lung lavage fluid of the lactic acid-sensitized mouse larvae supplemented with Lactobacillus salivarius pM_A〇(8)6 lactate are relatively low in eosinophil infiltration ratio.

Figure 9 shows that feeding PM_A〇〇〇6 lactic acid bacteria significantly reduced the amount of e〇taxin secreted in the lung wash relative to the control group. Fig. 10 shows that, compared with the control group, feeding ρμ_Α0006 reduced the amount of Jane 2 secretion in lung washing. == Day 2 shows that spleen cells were stimulated by conA or egg protein (0VA). After 2 days, the spleen secreted by the PM_A_6 lactic acid bacteria group was significantly higher than that of the sputum. f main element symbol description] None 25

Claims (1)

201210509 VII. Patent application scope: L A biologically pure culture, wherein the culture is selected from the group consisting of Lactobacillus acid philus pMA〇〇〇2 (aka Pf°). -2). Food Industry Development Institute registration number BCRC 910308; and Lactobacillus acidophilus PM-A0013 (aka PRO-13): Food Industry Development Institute registration number BCRC 910309. 2. A composition for treating allergy comprising at least one strain of lactobacillus which promotes secretion of __丫 and _12 and reduces the number of IgE antibodies and eosinophils, wherein the strain of lactic acid is selected from an acidophilus Lactobacillus (Lact〇baciUus add〇PhilUS) PM-A0002 (aka PR0_2): Food Industry Development Institute registration number BCRC 910308 and Lactobacillus acidophilus (Lact〇bac s add〇Philus) PM-A0013 (aka PR 〇_l3): Group of Food Industry Development Institute registration number BCRC 910309 and its combination. 3. The composition of claim 2, wherein the strains are live or dead. 4. The composition of claim 2, wherein the composition is added to the food. 5. The composition of claim 4, wherein the food is selected from the group consisting of fermented milk, yogurt, cheese, dairy powder, tea and coffee. 6. The culture of claim 1, wherein the culture is Lactobacillus acidophilus PM-A0002 (aka PR〇_2): Food Industry Development Institute, registration number BCRC 910308. < 7. The culture of claim 1 wherein the culture refers to Lactobacillus acidophilus PM-A0013 (aka PR〇_i3): registration number BCRC 910309. 26