TWI351962B - Use of selected lactic acid bacteria for reducing - Google Patents

Description

1351962 IX. Description of the Invention: [Technical Field to Which the Invention Is Applicable] The present invention provides specific Lactobacillus strains, and a method for selecting such strains and products comprising such strains; wherein these strains have an increased cytokine IL- A level of ability to be selected for pre-amp; and/or treatment of abdominal pain. [Prior Art] Although abdominal pain is common and painful, its characteristics and causes are still unclear. A mother described the situation as a baby who was very happy during the day, began to frown, her face turned red, her legs began to scream, and she continued to cry for 2 2 minutes, and then the attack suddenly stopped. Even the terminology used to describe this situation is controversial. These terms include “baby abdominal pain” and “evening abdominal pain” because the pain is mainly confined to the evening, and “three months of abdominal pain, which is caused by the symptoms three months after the baby is born (IUingw〇rth Rs.

Difficulties in breastfeeding. InRonaldS. Illingworth, ed. The Normal Child. 10th edn. Harcourt (India) Pvt. Ltd 1997; 39-44). Different authors use different definitions. Wessels' definition is that the abdominal pain is three or more hours of paroxysmal crying every day for three or more days in each week of at least three weeks, and is most widely accepted in the literature (SondergaardC, SkajaaE, Henriksen TB. Fetal growth and infantile colic. Arch Dis Child Fetal-Neonatal Ed 2000; 83 (1): F44-47). Currently, the main possible predisposing factors are divided into three groups: psychological and social, gastrointestinal and neurodevelopmental diseases. Psychological and social factors include: variants of normal crying, effects of atypical parenting behaviors, and performance of communication problems between parents and babies. Gastrointestinal diseases are involved in abdominal pain because of the position of the baby's legs and the distortion of the face during crying. Gastrointestinal factors mainly include the following: Incorrect feeding methods such as bottle feeding, horizontal position support, and lack of snoring after feeding are considered to be the cause of induction. In the first six months of the mother 5 1351962 colostrum was considered the only protective factor. In infants who are not breast-feeding, the risk of abdominal pain in infants is increased by 1.86 times. (Saavedra MA, Dacosta JS, Garcias G, Horta BL, Tomasi E, Mendoca R. Infantile colic incidence and associated risk factors: a cohort study.

Pediatr (Rio J) 2003; 79(2): 115-122) Lothe et al. believe that abdominal pain is sensitive to whey protein in milk (LotheL, LindbergT. Cow's milk whey protein elicits symptoms of infantile colic in colicky formula -fed infants : A double -blind cross over study. Pediatrics 1989; 83: 262).

In a recent study, it was concluded that food allergies are only a cause of abdominal pain in a small number of infants (Hill DJ, Hosking CS. Infantile colic and food hypersensitivity. J Pediatr

Gastrenterol Nutr 2000; 30 (Suppl): S67-76). recently

Buchanan has shown that hypoallergenic milk has not been tested in infants with abdominal pain.

Buchanan P · Effectiveness of treatment for infantile colic. Trial of hypoallergenic milk is not supported by strong fundamental evidence. BMJ 1998; 317 (7170): 1451-1452). Infants with abdominal pain in infants are considered to have a higher risk of allergic disease. However, in a recent study, markers of atopy, allergic rhinitis, asthma, wheezing, and changes in respiratory rate were comparable in infants with and without abdominal pain (Castro-Rodriguez JA, Stern DA, Halonen M, Wright AL, Holberg CJ et al. Relationship between infantile colic and Asthma/ atopy: A prospective study in an unselected population. Pediatrics 2001; 108(4): 878-882). Lactose absorption disorders have been reported by the authors based on respiratory hydrogen detection (Hyams J, Geerstama M, Etienne N, Treem W. Colonichy drogen production in infants with colic. J Pediatr 1989; 115: 592). Hydrogen concentration in feces was not found in infants with and without abdominal pain in infants (s 6 degrees difference. However, infants with higher levels of smoldering were found to have reduced abdominal pain, indicating that decane production can Relief (BelsonA, ShettyAK, YorginPD, Bujanover Y, PeledY, DarMH, Riaf S. Colonichy drogen elimination and methane production in infants with and without infantile colic syndrome. Dig Dis Sci 2003; 48(9): 1762-1766). (GIT) hormones such as activin, vasoactive intestinal peptides have been shown to be abnormally high in infants with abdominal pain. Lothe et al. found that infants developing abdominal pain in the future showed an increase in GIT levels from the first day of birth, as indicated by Abnormal GIT in patients with abdominal abdominal pain (Lothe L, Ivassson SA, Ekman R, Lindberg T. Motilin and infantile colic: A prospective study· Acta Pediatr Scand 1990; 79(4): 410-416). Disorders indicate that abdominal cramps and abdominal pain may be the result of excessive peristalsis. The evidence from this theory supports the use of anticholinergic agents to reduce the abdomen Symptoms (Gupta SK. Is colic a gastro intestinal disorder? Curr Opin Pediatr 2002; 14: 588-92). The fact that most babies get rid of abdominal pain in about 4 months supports the idea that neurodevelopmental factors induce abdominal pain (BaiT RG) · c〇lic and crying syndromes in infants. Pediatrics 1998; 102 (5 suppl E): 1282-6). According to the generally accepted definition, 'probiotics' are a living microbial feed additive that improves microbes in the host's intestines. Balancing and effectively affecting the host's movements. Although it is the most suitable additive for the farm's secret feed, it is easy to accept the definition of human beings. The main probiotics ingested by humans are lactic acid rod 8 and double secret gg lion milk. Food in the form of a product. It suggests that the T-prevalence of the probiotics affects the composition of the microbial community in the intestines. It has been suggested that the effects of probiotics on the intestinal ecosystem are beneficial to the ingestors. The use of probiotics to alter intestinal microbes has been There are many possibilities, including: improving the resistance of infectious diseases, especially the intestines, reducing the duration of the abdominal 1351962 diarrhea, reducing the liquid Force, lower blood cholesterol concentration, desensitization, stimulation of phagocytosis of peripheral blood lymphocytes, gene regulation of cytokine, adjuvant effects, tumor regression, and reduction of carcinogens or carcinogens: The first person reported probiotics The immunomodulatory effect of Lactobacillus bacterium, which is the Th1/Th2/Th3/Trl/Treg-copy of the dendritic cells (DCs). They showed that t-murine cells DCs exposed 』 = ^ nutrients, including Lod Lactic Acid (IV), which produced

, a 12, and the ability of TNF-a and the ability to label the surface of the == steroid and c:D86 in a concentration-dependent manner were adjusted to varying degrees. 3 5 rods ^ adjusted surface MHC11 class and (10) 6 label - showed 1224j in these studies, especially to note that L. lactis (strain m rod culture, it can inhibit proinflammatory cells in different degrees), iL_6 and TNF_a is produced, and these substances can be stimulated by the latter two types. The production of IL-10 remains unchanged under these conditions. The conclusion of the introduction is that "L. lactis may have a tree in the intestine =, shape, , the field cell produces a kind of environmental regulation, so that it produces no good tolerance for "dangerous lUi, benefit" while maintaining a good ability to respond to the risk signal, and "Lod Lactic Acid" The potential fine-grained treatment of Bacillus species can effectively regulate the production of 1L-i2 and IL-6) while inducing the anti-inflammatory factor IL_1〇, thus providing a method to counteract the intestinal inflammatory factor microbial community, and thus point Thl Intestinal stimulating effect of /Th2/Th3 'According to the group of intestinal microbiota, the intestinal microbiota includes ingested probiotics.,, (Christe

Diffe ^ , T H,PeStkB JJ (2〇〇2) Lact〇bacilli matu Γ+η m Y m〇dulate exPressi〇n ofcytokiness and juration surface markers in murine dendritic cells J

Immunol 168 171-178). J

Snuts et al. extended these studies and found that Lord's lactic acid rod g was able to open 8 1351962

DCs are stimulated by T-regulated (TR) cells. They were co-cultured with human monocyte-derived DCs in vitro using three different lactobacilli. Two of the Lactobacillus strains, a human L. lactis strain (ATCC 53609) and Lactobacillus casei, but not a Lactobacillus plantarum strain, activate these DCs to stimulate the development of TR cells. These TR cells have been shown to produce increased levels of IL-10 and are capable of inhibiting proliferation of bypass T cells in an IL~10-dependent manner (Smits HH, Engering A, van der Kleij D, de Jong EC, Schipper K, van Capel TMM, Zaat BAJ. Yazdanbakhsh M, Wierenga EA, van Kooyk Y, Kapsenberg L (2005) Selective probiotic bacteria induce IL-10-producing regulatory T cells in votro by modulating dendritic cell function through dendritic cell -specific intercellular adhesion Molecule 3-grabbing nonintegrin. J Allergy Clin Immunol 115 1260-1267). The first evidence of the effect of oral L. lactis on the host itself is the observed increase in the proportion of CD4+/CD8+ T cells in the chicken ileum (Walter J.

Dobrogosz, NUTRAfoods 2005 4(2/3) 15-28). Valeur et al

This research has recently been extended to the human ileum. They obtained in situ evidence in human subjects that (a) human-specific L. lactis ATCC 55730 individual cells are distributed throughout the human intestine and (b) L. lactis ATCC 55730 participates in the ileum that is specifically present in the intestine Recruitment and/or proliferation of regional CD4+ T cells. They concluded that the administration of L. lactis induces the recruitment of CD4+ T-helper cells to the human epithelium. This recruitment can be used as a factor in explaining the probiotic effect of this L. lactis strain in humans (ValeurN, Engel P) , Carbajal N, Connolly E, Ladefoged K (2004) Colonization and iramunomodulation by Lactobacillus reuteri ATCC 55730 in the human gastrointestinal tract, Appl Environ Microbiol 70 1176-1181).

Toll-like receptors (TLRs) recognize the structural motifs of microorganisms and initiate a line of genes that are produced by the genus 9 1351962. Usually, TLRs are considered to be microbial sensitivities Tlp?^ and our role is to induce an inflammatory response. However, the structural motifs that can be used in the box are not unique to the pathogen, but rather the general structural motifs used by all microorganisms' and how the immune system and the pathogens through the TLRs are not fully understood. Recently, data show that TLRs, although /, are gamma in the induction of inflammation, but also play a role in maintaining intestinal flora balance by recognizing symbiotic microbiota (Rak〇ff_驰_s, Paglin〇

J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell. 2004 Jul 23;118(2):229-41). + Various lactic acid bacteria species including L. lactic acid bacteria have been used to formulate probiotic preparations. Lactobacillus lordii is a naturally occurring resident bacterium in the gastrointestinal tract of animals and is commonly found in the intestines of healthy animals, including humans. It is known to have anti-microbial activity. See, for example, U.S. Patents 5, 439, 678, 5, 458, 875, 5' 534, 253, 5' 837, 238, and 5, 849, 289. When L. laudiohas cells are grown under anaerobic conditions in which glycerol is present, they produce an antimicrobial substance known as propionic acid (3-ΗΡΑ).

There is a clear and complex relationship between the immune system and the commensal flora in the intestine. Recently, it has been demonstrated that the endogenous flora can induce endogenous and adaptive host response processes in the intestinal epithelial cells induced by t〇11-like receptors (TLRs) and NOD-receptor initiation (Haller D) (Haller D , Jobin C. Interaction between resident liminal bacteria and the host: can a healthy relationship turn sour? J Pediatr Gastroenterol Nutr 2004; 38: 123-36. Rakoff-Naholm S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R Recognition of comm, ensal microflora by Toll-like receptors is required for intestinal homeostasis. Cell 2004; 118: 229-241). In animal models, cytokines are able to initiate an intra-arterial neuromuscular overreflex response via the God 10 1351962 trans- and muscle-immunological interactions (Mi 11a PJ. Inflammatory cells and the regulation of gut motility. J Pediatr Gastroenterol Nutr 2004; 39: S750.).

There is a clear and complex relationship between the immune system and the commensal flora in the intestine. Recently, it has been demonstrated that endophytic flora can initiate a key process of bacterial-induced natural and acquired host responses by initiating toll-like receptors (TLRs) and NOD-receptors on intestinal epithelial cells (Haller D, Jobin C) Interaction between resident liminal bacteria and the host: can a healthy relationship turn sour? J Pediatr Gastroenterol Nutr 2004; 38: 123-36. Rakoff-Naholm S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of Comm.ensal microflora by Toll-like receptors is required for intestinal homeostasis. Cell 2004; 118: 229-241). In animal models, cytokines are capable of initiating a hyperreflex response in the intestinal neuromuscular via neuro- and muscle-immunological interactions (Mi 11a PJ. Inflammatory cells and the regulation of gut motility J Pediatr Gastroenterol Nutr 2004; 39: S750).

It has been demonstrated in experimental and clinical studies that specific probiotic strains inhibit T cell proliferation and reduce the secretion of both Th1 and Th2 cytokines, while preferentially producing inhibitory cytokines such as IL-10 and TGF-β (Rautava S, Kalliomaki) M, Isolauri E. Probiotics during pregnancy and breast-feeding might adjunctive immunomodulatory protection against atopic disease in the infants. J

Allergy Clin Immunol 2002; 109: 119-121). Moreover, in human volunteers, L. lacticis clusters in the human gastrointestinal tract and is capable of exerting immunomodulatory activity 'including recruitment of CD4+ T-helper cells in human ileum epithelium (ValeurN, Engel P, Carbajal N, Connolly E, Ladefoged K. Colonization and imraunomodulation by Lactobacillus 1351962 reuteri ATCC 55730 in the human gastrointestinal tract.

Appl Environ Microbiol 2004; 70: 1176-81). The maturation of DCs is the process of transforming immature DCs into mature antigen-presenting cells and migrating lymph nodes. This process leads to the loss of immature DCs, a powerful loss of antigen uptake capacity, and upregulates the expression of various cytokines and costimulatory molecules (Mellman I, Steinman RM: Dendritic cells: specialized and regulated antigen processing machines. Cell 2001 , 106:255-8. Banchereau J, Briere F, Caux C, Davoust J, LebecqueS, LiuYJ, PulendranB, PaluckaK: Immunobiology of Dendritic cells. Annu Rev Immunol 2000, 18:767-811) 〇 Known maturity conditions are Based on the environment in which DCs meet during or after exposure to an antigen. The preferred embodiment of this is the use of monocyte-conditioned medium (MCM). MCM is produced in vitro by cultured monocytes and is subsequently used as a source of maturation factors for the supernatant of the culture. The main components responsible for maturation are the pro-inflammatory cytokines interleukin-1 beta (IL-1 β), IL-6 and TNF-ot (Reddy A, Sapp M, Feldman M,

Subklewe M, Bhardwaj N: A monocyte conditioned medium is more effective than defined cytokines in mediating the terminal maturation of human dendritic cells. Blood 1997, 90:3640-6.). Mature DCs produce a variety of cytokines that stimulate and mediate tau cell responses. Two of these cytokines are IL_10* IL_12. These cytokines have opposite effects in terms of induced T cell responses: il-12 induces a Th1 type response and IL-10 inhibits such a response. Thus the activation state of APCs (antigen-presenting cells), including DCs, determines the type and size of CD4+ T cell responses. Static APCs (including epithelial cells in the thymus) can promote the development of CD4+CD25+TR cells. Identification of microbial molecules by TLRs during pathogen infection leads to activation of APCs. The APCs then produce IL-6 and other solubility factors that together invalidate the TR cell inhibitory effect to allow efficient production of TE (T effector) cells against pathogens. 12 1351962 The dynamic balance between resting and activated APCs will also be affected by both TR and TE cell activity (Figure 1).

Pessi et al. (2000) disclose the production of il-10 after oral administration of L. rhamnosus in atopic children. However, unlike the present invention, this study did not disclose a specific strain capable of producing a high amount of IL-10, which was selected for its effectiveness against dysmotility and abdominal pain (pessiT, SutasY,

Isolauri E. Interleukin-10 generation in atopic children following oral Lactobacillus rhamnosus. GG. Clin Exp Allergy. 2000 Dec;30(12):1804-8).

A study by Hermelijn et al. (2005) showed that L. lactis and Lactobacillus casei, but not Lactobacillus plantarum, induced monocyte-derived fs to drive the development of TR cells. The resulting TR cells produced levels of IL-10. Unlike the invention herein, the author did not link the improvement of il_i〇 to intestinal motility or abdominal pain. Even when referring to strains, the authors describe two different bacterial species that are effective in increasing IL-i〇 levels. This is in contrast to the present invention, which shows that probiotics that most effectively reduce abdominal pain must be identified and selected at the strain level because different strains of the same species have different abilities to effectively increase IL-10 levels. There is currently no effective treatment for abdominal pain. The current paradigm for treating abdominal pain consists of pharmacological = and / or non-pharmacological methods to minimize symptoms. Therapeutic interventions for abdominal pain that are usually provided to parents include four strategies including dietary, physical, behavioral, and pharmacological. Dietary practices include professional advice on various feeding techniques' either using hypoallergenic milk, soy milk or lactose-free formula, and knowing: early introduction of solid foods (L〇the, L·., et al. cow, s milk Formula as a cause of infantile colic: a double-blind study. Pediatrics 1982; 70:7-10; Forsyth BW C. Colic and the effect 〇f changing formulas: a double- blind multiple-crossover study. J Pediatr 1989;115,521 - 6;

Treem, W R, et al. Evaluation of the effect of a 13 1351962 fiber-enriched formula on infant colic. J Pediatr 1991; 119695-701). However, whether using soy milk formula or changing feeding techniques is not effective for all kinds of abdominal pain. Studies of these proposed statistics indicate that hypoallergenic formulas, such as partially hydrolyzed or amino acid based, may be beneficial for only about 25% of infants (Lucassen, PLBJ, et al. infantile colic: crying time reduction with a whey hydrolysate: a double - blind , randomized placebo-controlled trial. Pediatrics 2000;106:1349-54;

Estep, D C, et al. Treatment of infant colic with amino acid-based infant formula: a preliminary study. Acta Paediatr 2000;89:22-7). Fi Physical strategies for treating abdominal pain include physically moving the body position to reduce gas/countercurrent flow, holding the baby, wrapping, pressing the abdomen, or massaging the baby. Other methods include distracting the baby to reduce its alertness to abdominal pain such as taking a baby by car, using a car simulator, using a crib oscillation or cradle (Lipton E L. Swaddling and child care practice: historical, cultural and experimental observations. Pediatrics 1965; 35:521-67; Byrne JM, Horowitz F D. Rocking as a soothing intervention: the influence of direction and type of movement. Infant Behav Dev 1981; 4:207-18). Another way is to play some recordings that are said to make the baby relax. However, there is evidence in the medical literature that these methods do not work (Parkin PC, Schwartz CJ, Manuel B A. Randomized controlled trial of three interventions in the management of persistent crying of infancy. Pediatrics 1993; 92(2): 197- 201). These strategies, in the best case, are only helpful in alleviating the symptoms of abdominal pain. It is suggested that behavioral intervention to treat abdominal pain is currently the most contradictory therapy. Some authors advocate an increase in sensory stimuli, while others advocate a reduction of such stimuli 14 1351962 (Balon A J. Management of infantile colic. Amer Pham. Physician 1997; 55: 235-242; Lucassen PLBJ, Assendelft WJJ, Gubbels JW, van Eijk TM, van Geldrop WJ,

• Effectiveness of treatments for infantile colic: systematic review. BMJ 1998;316(5): 1563-9; and Carey W • B' "Colic"—primary excessive crying as an - infant-environmental interaction. Pediatr Clin North Am 1984 ;31:993-1005). Other suggestions include responding to crying early, or allowing the baby to cry, providing a rubber nipple, performing a fixed feeding schedule, and using eye contact and interactive games. Pharmacological interventions for the treatment of abdominal pain lead to the use of prescription or over-the-counter medications. Prescription drugs currently in use include the belladonna domain and sedatives (analgesics), which can alleviate pain, but the accompanying risks include extrapyramidal symptoms, respiratory depression, and constipation. For example, anti-cholinergic drugs, which act like atropine, for example, LEVISINE® or GASTROSED® and bicyclic amine effervescent tablets, can speed up heartbeat and reduce saliva production 'eliminating the gastrointestinal and urethra and bronchi Hey. Although anti-cholinergic drugs are the only prescription drugs on the US market that have been shown to be effective for abdominal pain in infants, unfortunately, more than 5% of treated infants have side effects, including dyspnea, apnea, seizures, Syncope, sputum, coma, and muscle tone are too low (Williams J, Watkin- Jones R.

Dicyclomine: worrying symptoms associated with its use • in some small babies (BMJ 1984; 288:901; Myers JH, Moro Sutherland D, Shook J E. Anticholinergic poisoning in colicky infants treated with hyoscyamine sulfate. Am J Emerg Med 1997; 532-5) In addition, there are several reports of deaths among infants taking bicyclic amines (Garri〇tt JC, Rodriguez R, Norton L E. Two cases of death involving dicyclomine in infants.

Clinical Toxicol 1984; 22(5): 455-462). Over-the-counter medications that have been reported to be effective in treating abdominal pain in infants include several 15 sedative or hypnotic medications, including hyperpharmacological (high dose) diphenhydramine (BENADRYL®), stupid barbital, chloral hydrate, and even alcohol. . However, these substances may have serious side effects in children with respiratory diseases, thus limiting their widespread use in the treatment of abdominal pain (Balon A J. Management of infantile colic· Amer Pham Physician 1997; 55: 235-242;

Gurry D. infantile colic. Australian Pham Phys 1994;23(3):337-34632). More safe over-the-counter medications for abdominal pain include the administration of jsimethicone or polydimethyloxane, a non-absorbable non-reagent that reduces the size of intestinal air bubbles. Xiqiao oil is very safe and often recommended, but there are still several studies showing that the effect of guanidine oil on infants' abdominal pain is no better than placebo (Metcalf, TJ, et al., Pediatfies 1994 July; 94(1):29 -34. Sferra,TJ, etal^Pe^at^

North Am 1996 April; 43(2): 489-510. Danielson, B. et al., Acta Paediatr Scand 1985 May; 74(3): 446-50. Colon, AR, et al., Am Fam Physician 1989 December 40(6): 122-4). Therefore, the most common abdominal pain treatment today is just waiting for the baby to grow up to no longer suffer from abdominal pain. Therefore, there is a need for an effective and safe treatment for abdominal pain in infants and young children. The compositions and methods of the present invention meet this need by providing a product that is safe and effective in treating the symptoms associated with abdominal pain in infants. Patent application Ep1364483A10 discloses the treatment of intestinal-neuromuscular rejuvenation such as infant abdominal pain (IV). The applicant introduced several different bacterial species that can be used as probiotics. In contrast to the present invention, the probiotics of the present invention which are most effective in reducing abdominal pain are a specific lactobacillus which is selected because of an effective increase in the level of -1 ,, rather than an entire bacterial species, the present invention The inventors have demonstrated a significant difference in stimulation yield between different strains of the same species. In 2005, 'Savino proved that the addition of Lord's lactate rod 1351962 bacteria ATCC 55730 in 7 days of treatment can significantly change the symptoms of abdominal pain in breast-fed infants compared with the standard treatment with Xiqiaoshi oil. The effective rate of treatment with L. lactis A叩0/two infants with only lycopene oil was only 7%. He presented the results to the European Society for Pediatric Research (ESPR) at the meeting held in Siena, Italy from 2,5 years, 931° to September 3 (Pediatr Res. 2005;58(2): 411 ). Although the results of Savino’s showed beneficial effects, he did not know the relationship between specific strains that increased a-production and reduced intestinal motility and thus reduced abdominal pain. The invention herein provides a method of selecting such an optimal strain. • c〇llins discloses a series of symptoms of motor dysfunction that increase inflammation and immune activation in various areas of the intestine and nearby organs, such as edema, gastritis, and spontaneous enteritis (IBD). These studies indicate that the motility of the intestine is also affected by the immune system. In this case, the motor system may play an important role in the intestinal resistance to the harmful stimuli present in the lumen (c〇uins s., The Iramunomodulation of Enteric Neuromuscular Function: Implications for Motility and Inflammatory Disorders.

Gastroentrology 1996; 111: 1683-1699). This view is reflected, for example, by Vantrappen et al., which shows that bacterial overgrowth in the small intestine is accompanied by collapse of the normal loop pattern of digestive movement in the small intestine (Vantrappen G, Jannssens J, Hellemans J, Ghoos Y. The heron , interdigestive motor complex of normal subjects and patients with bacterial overgrowth of the small • intestine. J Clin Invest 1977; 9:1158-1168). It has been known for many years that increasing the level of IL-10 inhibits over-activated immune systems. It has also previously been demonstrated that intestinal motility is controlled by neural signals associated with the intestinal immune system and abdominal pain is the result of increased intestinal motility, for example, due to excessive bacterial growth. In industrialized countries, health measures have been taken as early as the delivery process, which disrupts the ability of newborns to obtain the mother's intestinal microflora. As a result, different microbial flora is established in infants. Instead of being invented by, for example, E. coli and lactic acid rods (S > 17 1351962 ί! Γίί'. The body of the bribe is more golden, and the other skin is fine, the inventors unexpectedly found that when the skin is finely overgrown When the 2m immune system 'causes excessive activation of intestinal motility and leads 】, has improved yield of ^1Q _ cooked: proliferation of P CD4+CD25+TR cells. The upper part of CD4+CD25+ cells leads to intestinal energy, low From (four) <t, Weilan baby production benefits.

The squatter was found to have a characteristic strain of this strain by the non-pathogenic 88 strain _ improve the characteristics of IL-10 and reduce abdominal pain in infants. The present invention relates to other strains selected by the method of using Lactobacillus lordum var. 17938 for the preparation of a medicament for preventing and/or treating abdominal pain. Further, it is a further object of the invention to provide a product comprising the strain and administered to the ’' including humans. Other objects and advantages will be apparent from the following disclosure and the appended claims. SUMMARY OF THE INVENTION The present invention provides a specific Lactobacillus strain, a method of selecting such a strain, and a product comprising such a strain. Among them, the Lactobacillus strain is selected for preventing and/or treating abdominal pain due to its ability to produce the yield of CD4+CD25+ TR cells and to promote the proliferation of CD4+CD25+ TR cells. Other objects and features will be apparent from the following description and claims. [Embodiment] The clustering of skin bacteria in the intestine and the lack of TR cells cause the neonatal immune system to be overactivated, resulting in excessive activation of intestinal motility and causing abdominal pain. A large number of special enterobacteria, such as L. lactis DSM 17938, have the ability to increase IL-10 production, leading to the maturation of the TR system, i.e., CD4+CD25+TR cell proliferation. Other Lactobacillus strains previously reported by many researchers have been able to induce 1351962, et al. (described above). (3) Peach (IV) on the cells and thus have a beneficial effect on abdominal pain. The strain having an increased level of 1 L-10 was the same strain as the halogen strain capable of reducing the time of + average time (Example 2).

Ben, Ming, including selected Lactobacillus strains, including Lord's lactate rod reuteri) DSM 17938 J ϊ: ΐίϊΐ force was chosen. Contains the entire fine from these strains

It is prepared by a method well known in the art, and generally includes an absorbable support of the strain, or a component obtained therefrom. Mu Miaoyi ^ in vitro research method was used as a method for selecting Lactobacillus strains. Because of the ability to stimulate IL-10 from monocyte-derived cells, it can be used to develop cells. Example 1). f shows that IL-10 is strongly stimulated by the specific strain L. lactis ATCC 5573 Lactobacillus sp. DSM 17938, and this regulation is one of the two specific g strains released into the medium during log/stationary growth. ,, qualitatively mediated. Conversely, the two other L. lactis bacteria correspond to IL-10 production. Further studies have been conducted to confirm that the selected strain is used to prevent or treat abdominal pain in clinically related breast-fed infants with abdominal pain. The features of the present invention will become more apparent from the following description of the embodiments. Example 1 Study of the ability of probiotic strains to increase IL-i 〇 production by DCs smashed by a single nucleus: Immature DCs produced from peripheral blood mononuclear cells (Hilkens, CMU, Ρ. Kal inski , Μ. deBoer, and Kapsenberg. 1997. (s 19 1351962

Human dendritic cells require exogenous interleukin-12-inducing factors to direct the development of naive T-helper cells toward the Thl phenotype. Blood 90: 1920) cultured in IMDM (Life Technologies, Paisley, UK), the medium containing 10% fetal Bovine serum (HyClone, Logan, UT, USA), recombinant human granulocyte macrophage colony (Recombinant Human rh GM-CSF) (500 U/ml; Dutch company Schering-Plough), and recombination Human IL-4 (250 U/ml; • Pharma Biotechnology, Hannover, Germany) (Kalinski, P., J. Η. Ν. Schuitemaker, C. Μ. U. Hilkens, EA Wierenga, and 9 ML Kapsenberg. 1999). The final mature dendritic cells are associated with decreased responsiveness to π?Ν-γ and IL-12 elicitors to bacilli: mature dendritic cells have reduced ability to produce IL-12 during interaction with Th cells (j. Immunol. 162:3231).

The strain detected in this example is L. lactis ATCC

55730, L. lactis DSM 17938, L. lactis ATCC PTA 4660 and L. lactis ATCC PTA 4964, all available from Atcc (Manassas, VA), and DSMZ (Brunswick, Germany) . The strain was cultured in Columbia agar containing 6.25% sheep blood (British, Basingstoke, Oxoid). Lactobacillus was incubated at 37 ° C in a 5% CO 2 atmosphere. After 3 days, the number of bacteria was determined by measuring the optical density at 620 nm (〇D620)_. Generally, the OD62 〇·35 for all strains to be tested corresponds to the ixi〇8 clone forming unit (cfu). On day 6, immature DCs were induced to mature by LpS (E. coli, Sigma-Aldrich, St. Louis, USA), containing cytokines (25 = /mL) and TNF-cx (50 ng / mL) Lactobacillus was tested in the presence of the composition and all used as a maturity factor (cell factor purchased from Peprotech, Inc., Rocky Mountain, NJ, USA). Soil As described by Vieira PL et al., 虬12, 20 1351962 IL-10, and IL-6 cytokines produced by mature DCs were determined by CD40 ligand-expressing mouse plasmacytoma cells for 24 hours. Vieira PL, de Jong EC, Wierenga EA, Kapsenberg ML, Kalinski P. Development of Thl-inducing capacity in myeloid dendritic cells requires environmental instruction. J Immunol

2000; 164: 4507-12). After 24 hours, the supernatant was collected and the concentration of IL-10 was measured by ELISA. The results are shown in Figure 2. As can be seen from the results, the ability of different Lactobacillus strains to increase the production of IL-10 by DCs is significantly different. Example 2 Comparison of selected probiotic strains of guanidine methyl ester in the treatment of abdominal pain in infants The Department of Pediatrics and Adolescent Science recruited breast-fed infants diagnosed with infant abdominal pain (Drottning Silvias University Hospital, G0teborg). The patient's age ranged from 21 to 90 days, term delivery, birth weight 2500 to 4000 g, abdominal pain symptoms consistent with Wessel's criteria, and almost 6 ± 1 day before enrollment, considered to be useful in this study (wessei ma, Cobb JC, Jackson EB, Harris GS, Detwiler AC. Paroxismal fussing in infancy, sometimes called “colic,, trics 1954; 14: 421-35.” All registered infants are strictly breastfed to reduce intestinal microflora due to dietary changes. Changes that may in turn affect the response to probiotics. If there is clinical evidence that the baby has a chronic or gastrointestinal illness or has received antibiotics or probiotics a week before enrollment, they will be excluded. In the study, infants with abdominal pain were randomly randomized to receive probiotics or cyprin oil in the following treatments: (P1) L. lactis strain ATCC 55730, also known as SD2112, (P2) L. lactis strain DSM 17938, according to the Budapest Agreement in 2006 Deposited at the DSMZ Microorganisms Collection in Germany on March 06 (Mascheroder Weg lb, D-38124 Braunschweig), 21 1351962 (Ρ3) Lactobacillus laudios strain ATCC ΡΤΑ 466 〇 and (S) psittac oil. 乳酸 L. lactic acid bacteria is added in increments of 108 clone forming units (CFU) at 5, commercially available MCT-oil (medium chain glycerol) The triester is suspended in 3 minutes and fed once a day for 28 days. MCT oil is naturally occurring, the most abundant source is coconut oil. Most MCT oils are refined from coconut oil. MCT oil is Clear, odorless, low-viscosity liquid. This oil suspension is stable for 21 months at 2~8 C (according to the manufacturer's literature, BioGaia AB, Stockholm, Sweden) (alternative MCT oil source is Swedish card ^Sahamen, BioGaia AB). During the study, parents were instructed to put the product in the refrigerator when not using the product. The dosage of the guanidine oil (s) was 6 days, to 3mL commercial solution (My 1 icon infants Gas Rel ief Dr〇ps, Shame 7050 Camp Hill Rd., Fort Washington, Pennsylvania =034-2210, USA) 'Apply after feeding, twice a day for 28 days. In ▲ » 己, all The mother was asked to abide by the reported no milk Food: Avoid milk, yogurt, fresh and dried cheese, cream, butter, biscuits. Persistence of recipes during all treatments is monitored through a diet diary. On the 7th, 14th, 2nd and 28th days, the investigator will check the persistence of this recipe.

~ The ethics committee of the Institute agreed to the conditions of the study and only the parent's written consent was used to participate in the study. & Follow-up Pediatrician's first time to see the baby is set to the first day (Day_, the baby is medically examined and the parents are interviewed to obtain the relevant points = age of birth weight, family history of gastrointestinal fistula and hereditary allergies #景数摅. In particular, if the baby's one or more family members, the father and/or the older brother and sister, suffer from allergic wet treatment and allergies, 'the last is positive. Moreover, the number and symptoms of any allergic disease during the study period were recorded. Parents were also asked to record the average number of crying and abdominal pain per day after recruiting 22 1351962 Day 0 (Day 0). The doctor randomly assigned the baby to any research group. Study production qQ 1 (Day 1) was started. Brother-big parents were asked to use a fixed notebook from the day of study to the 28th study and were asked to record the daily sequel to the sad and crying plot, Da Nadu and Xian and any control _ _ _ π 吐, infection reaction, etc.). In order to ensure that all parents are crying and ensuring that the baby is properly given the medicine, the researcher keeps in touch with the parents to help the parents. Heart ~ 疋 again = patients on the first 7, 14, 21 and 28 days of the 12G mother-in-law abdominal pain infants registered by the same pediatrician results, 3 () treated with L. lactis ATCC55730 (Ρ1) , 3 〇 德 Lactobacillus DSM Π 938 (Ρ 2) treatment, 3G randomized _ (10) treatment and 3 ° random mining S West; no infant withdrawal, because there is no experimental related sub = use. The age, birth weight, sex, type of childbirth, history of the disease, the history of the disease, and passive smoking were similar. 〆 Α ίί〇 and Day 1 The average daily crying time of each treatment group was similar. Infants receiving L. lactis ATCC 55730 (Ρ1) and biliary 17938 (Ρ2) had a significant reduction in daily aging on day 7 compared with infants receiving 甲石夕油(8). The crying time was significantly different in the 14th and 21st treatment groups. In 28 days, (10) and (8) ^ Menping = day crying __ difference from the beginning to the end of the study is 82 minutes 曰 and (the average daily crying time difference between S from the beginning to the study knot) υ致ϊ结 f It was seen that the four groups were essentially convinced before receiving the therapeutic product. After 7 days, the ferulic acid rods _ strains 5573 〇 and 23 1 351 962 DSM 17938 days later had a clear difference between the other two addition classes.

It is clear that the side is to be _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ [Simple diagram of the diagram] The role of the specific stalks selected by 〇τ in promoting the development of itO)4+C:D25+TR 24 1351962. Figure 2a shows a bar graph of IL-10 production by DC cells; Figure 2b shows a table of IL-10 production by DC cells. [Main component symbol description] None 25

Claims (1)

1351962 丨 Announcement 10, Application for profit-seeking range ··· 1. A biologically pure culture of Lactobacillus, wherein the Lactobacillus includes Lactobacillus reuteri DSM 17938, the Lo, Lactobacillus DSM Π 938 Development of Hsinchu Food Industry in Taiwan, Republic of China, = deposit number BCRC910384. π ι~ 2, a composition comprising a biologically pure culture of Lactobacillus, wherein the bacillus comprises Lactobacillus reuteri DSMng^8, and the Lod lactic acid rod _ DSM 17938 is deposited in Hsinchu, Taiwan, Republic of China Food Hall Development Research Institute, the registration number is BCRC910384. , α ° concentration 3, one, reducing the baby's abdomen, j method, the method includes selecting a lactobacillus strain that promotes human body than -10 expression and applying the strain to the infant diagnosed with infant pain; The lactic acid rod g includes a Lod lactic acid rod g (Lact 〇 bacill reuteri) DSM 17938, which is deposited in the Hsinchu Food Industry Development Research Institute of Taiwan, Republic of China, with a registration number of 84. 4. A product for reducing abdominal pain in a baby, the product comprising: a lactate rod 8 B strain capable of increasing IL_1 〇 water = and wherein the lactic acid rod g comprises a Lactobacillus reuteri DSM 17938, The L. lactis DSM is in the Hsinchu Food Development Research Institute of Taiwan, China. The registration number is bCKLyiLM84 〇5. If you apply for the product described in item 4, the towel, the medium-chain triglyceride- oil.