Bifidobacterium longum subsp. infantis for relieving gastrointestinal discomfort of infants and its application

Abstract

This invention provides a subspecies of Bifidobacterium longum that can relieve gastrointestinal discomfort in infants and young children, and its application.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a subspecies of Bifidobacterium longum that can relieve gastrointestinal discomfort in infants and young children and its applications. Background Technology

[0002] The digestive system of infants aged 0-3 years is not yet fully mature, and their gut microbiota is not yet fully established. They are more susceptible to various factors, such as changes in diet, infection, and allergies, which can lead to gastrointestinal discomfort, such as nausea, vomiting, and rectal pain, which is detrimental to the health of infants.

[0003] In recent years, probiotics have received widespread attention due to their positive effects on gut health. However, there are currently limited probiotic products on the market for relieving gastrointestinal discomfort in infants and young children.

[0004] Therefore, developing a probiotic product that can effectively alleviate gastrointestinal discomfort in infants and young children can help meet the growing health needs. Summary of the Invention

[0005] This invention provides a subspecies of Bifidobacterium longum that can relieve gastrointestinal discomfort in infants and young children, and its application, which can effectively relieve gastrointestinal discomfort in infants and young children.

[0006] This invention provides the use of Bifidobacterium longum infantis subspecies YLGB-1496 strain in the preparation of products that relieve gastrointestinal discomfort.

[0007] For the purposes described above, the *Bifidobacterium longum* subspecies *YLGB-1496* strain is deposited at the China Center for Type Culture Collection (CCTCC) of Wuhan University, with accession number CCTCC NO: M2011122.

[0008] As described above, the gastrointestinal discomfort includes one or more of nausea, vomiting, and rectal pain.

[0009] As described above, the product comprises the *Bifidobacterium longum* subsp. *infantii* strain YLGB-1496.

[0010] As described above, in the product, the viable count of the *Bifidobacterium longum* subsp. *infantii* YLGB-1496 strain is 10. 6 CFU / g ~ 10 11 CFU / g.

[0011] As described above, the product is administered to the subject via the gastrointestinal route.

[0012] For the purposes described above, the subjects include infants aged 0 to 3 years.

[0013] As described above, the product includes one or more of the following: general food, health food, solid beverage, nutritional supplement, infant formula, food for special medical purposes, and pharmaceuticals.

[0014] The present invention also provides a composition comprising Bifidobacterium longum subsp. infantis YLGB-1496 strain.

[0015] The present invention also provides a method for preparing the composition as described above, comprising: mixing a raw material system including the Bifidobacterium longum subsp. infantis YLGB-1496 strain to obtain the composition.

[0016] This invention provides a *Bifidobacterium longum* subspecies *infantitidis* that can relieve gastrointestinal discomfort in infants and young children, and its application. The *Bifidobacterium longum* subspecies *infantitidis* (YLGB-1496) strain was cultured and isolated from the breast milk of a exclusively breastfed mother. Its gene sequencing results were compared with published *Bifidobacterium infantitidis* gene sequences, confirming it as a novel *Bifidobacterium infantitidis* strain. Experiments have demonstrated that this *Bifidobacterium longum* subspecies *infantitidis* strain YLGB-1496 has a good effect on relieving gastrointestinal discomfort in infants and young children, such as nausea, vomiting, and rectal pain. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a detailed technical roadmap for clinical trials;

[0019] Figure 2 A bar chart showing the number of days a subject experienced nausea in the past month;

[0020] Figure 3 A bar chart showing the number of days a subject vomited in the past month;

[0021] Figure 4 A bar chart showing the number of days a subject experienced rectal pain in the past month. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Gut microecology refers to the complex ecosystem formed by the interaction between microorganisms that live in the human gut and the human body.

[0024] The formation of the gut microbiota is a complex process. The initial microorganisms in a newborn mainly originate from the mother's birth canal, feces, and the surrounding environment. The period from 0 to 3 years of age is a critical window for the establishment and evolution of the gut microbiota, and breastfeeding is the most important factor determining the early gut microbiota of infants. Full-term, vaginally delivered newborns who are breastfed are more likely to develop a healthy gut microbiota, such as a gut microbiota with appropriate levels and types of Bifidobacteria and Lactobacilli.

[0025] Breast milk contains probiotics and prebiotics such as human milk oligosaccharides (HMOs). The first literature report in 2003 stated that breast milk contains bacteria that directly affect the gut microbiota of infants; subsequent studies have provided conclusive evidence that the gut microbiota of breastfed infants originates from breast milk. Numerous studies on probiotics in breast milk have been conducted, including their discovery, isolation, and research, and patents have been filed. For example, the University of Granada in Spain has filed patents globally and even in China for discovering three probiotic strains in breast milk (two Lactobacillus strains and one Bifidobacterium strain: Lactobacillus rhamnosus HERO 22A (CNCM I-4036), Lactobacillus paracasei HERO7 (CNCM I-4034), and Bifidobacterium breve HERO 15B (CNCM I-4035)).

[0026] Many countries have lists of bacterial strains that can be added to food, including Bifidobacterium, Lactobacillus, and Streptococcus, specifying the genus. The "List of Probiotic Strains that Can Be Used in Health Foods" and the "List of Bacterial Strains that Can Be Used in Foods" specify the edible probiotics down to the strain level. The "List of Bacterial Strains that Can Be Used in Infant Foods," published in 2011, further refines the list of probiotics that can be used in food down to the strain level. Lactobacillus and Bifidobacterium are widely recognized as probiotics.

[0027] Currently, only one strain of *Lactobacillus fermentum* CECT5716, originating from Spain, is listed as a suitable microbial strain for use in infant formula. All other strains are not derived from breast milk. Furthermore, the latest list of microbial strains suitable for use in food, updated in August 2022, includes *Bifidobacterium longum* subsp. infantum, and it is also included in the European Food Safety Authority's Qualified Professionals (QPS) list, indicating that this strain is safe for consumption. In recent years, numerous studies have confirmed the importance of *Bifidobacterium longum* subsp. infantum (Bifidobacterium infantis) for the gut health of infants.

[0028] Given that breast milk contains microorganisms and is crucial for the establishment of the gut microbiota in early life, especially the important role of *Bifidobacterium longum* subsp. infantis in infant milk for infant health, *Bifidobacterium longum* subsp. infantis in breast milk has become a research hotspot.

[0029] Based on this, embodiments of the present invention provide the use of Bifidobacterium longum infantis subsp. YLGB-1496 strain in the preparation of products that relieve gastrointestinal discomfort.

[0030] The aforementioned *Bifidobacterium longum subsp. Infantis* GB-1496 strain was deposited on April 10, 2011, at the China Center for Type Culture Collection (CCTCC) of Wuhan University, with accession number CCTCC NO: M2011122.

[0031] The Bifidobacterium longum subspecies in infant (Bifidobacterium infantis) YLGB-1496 strain was a Bifidobacterium infantis strain cultured and isolated from the breast milk of a exclusively breastfed mother. Its gene sequencing results were compared with the published gene sequences of Bifidobacterium infantis, confirming that it is a new Bifidobacterium infantis strain.

[0032] In some embodiments, the aforementioned gastrointestinal discomfort includes one or more of nausea, vomiting, and rectal pain.

[0033] Experiments have shown that the Bifidobacterium longum infant subspecies YLGB-1496 strain has a good effect in relieving at least one of gastrointestinal discomfort, such as nausea, vomiting, and rectal pain. Therefore, it can be used to prepare products for relieving gastrointestinal discomfort.

[0034] The embodiments of the present invention do not limit the specific types of the above-mentioned products. For example, the products may include one or more of the following: ordinary food, health food, solid beverage, nutritional supplement, infant formula, special medical purpose formula food, and medicine.

[0035] Understandably, the above products include the Bifidobacterium longum subsp. infantis strain YLGB-1496.

[0036] In the aforementioned products, the viable count of *Bifidobacterium longum* subsp. *infantitidis* YLGB-1496 can be greater than or equal to 10⁻⁶. 6 CFU / g.

[0037] Furthermore, in the aforementioned product, the viable count of the *Bifidobacterium longum* subsp. *infantii* YLGB-1496 strain can be 10-1. 6 CFU / g ~ 10 11 CFU / g.

[0038] In one specific implementation, the above product is administered to the subject via the gastrointestinal route to relieve the subject's gastrointestinal discomfort and effectively alleviate problems such as nausea, vomiting, and rectal pain.

[0039] Numerous studies have confirmed the crucial role of the gut microbiota in early life development during the maturation of the immune system, and the diversity and maturity of the infancy gut microbiota are closely related to health. Existing research data confirms that the aforementioned *Bifidobacterium longum* subsp. infantis YLGB-1496 strain is resistant to the strong acidity and bile salts of the artificial gastrointestinal tract, meaning it can cross the stomach and upper small intestine of the human digestive tract, ultimately reaching the lower small intestine and colon to exert its activity. For infants (e.g., those aged 0-3 years), because the acidity of gastric acid and the concentration of bile salts are lower than in adults, the aforementioned *Bifidobacterium longum* subsp. infantis YLGB-1496 strain, as a probiotic, is more likely to colonize the infant gut early.

[0040] In one specific implementation, the subjects included infants aged 0-3 years, such as children aged 1-3 years. The product can effectively relieve gastrointestinal discomfort in infants aged 0-3 years.

[0041] Clinical trial data confirms that the aforementioned Bifidobacterium longum infant subspecies YLGB-1496 strain can effectively alleviate nausea, vomiting, rectal pain, and other problems in infants aged 0-3 years.

[0042] This invention also provides a composition comprising *Bifidobacterium longum* subsp. infantis strain YLGB-1496. This composition can be used to relieve gastrointestinal discomfort and can effectively alleviate nausea, vomiting, rectal pain, and other problems in infants aged 0-3 years.

[0043] In addition, the composition may also include excipients such as maltodextrin.

[0044] Furthermore, in the above composition, the viable count of the *Bifidobacterium longum* subsp. *infantii* YLGB-1496 strain is greater than or equal to 10. 6 CFU / g.

[0045] Furthermore, in the above composition, the viable count of the *Bifidobacterium longum* subsp. *infantii* YLGB-1496 strain can be 10-1. 6 CFU / g ~ 10 11 CFU / g.

[0046] The present invention also provides a method for preparing the above composition, comprising: mixing a raw material system including Bifidobacterium longum subsp. infantis YLGB-1496 strain to obtain the composition.

[0047] In practice, a bacterial powder of Bifidobacterium longum subsp. infantis YLGB-1496 can be prepared first, and then the raw material system including the bacterial powder can be mixed evenly to obtain the above composition.

[0048] The above-mentioned Bifidobacterium longum infantis subspecies YLGB-1496 bacterial powder must meet the following requirement upon completion of production: viable lactic acid bacteria count ≥ 1.5 × 10⁻⁶. 11 CFU / g, with a water activity of 0.05 to 0.1, for example, 0.05, 0.07, 0.1 or any combination thereof.

[0049] In addition, the above-mentioned raw material system may also include excipients, such as maltodextrin.

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0051] Example 1

[0052] Clinical trials were conducted on infants aged 1-3 years, specifically targeting the *Bifidobacterium longum* subsp. *infantile* YLGB-1496 strain. The trials investigated the clinical manifestations of supplementation with this strain and its intervention in infants aged 1-3 years, and studied the alleviating effects of YLGB-1496 strain on nausea, vomiting, and rectal pain in this early-life population. The trial was a multicenter, randomized, double-blind, controlled study. The specific technical approach is as follows: Figure 1 As shown.

[0053] This study has been registered on ClinicalTrials.gov (identifier NCT05794815).

[0054] 1) Children aged 1-3 years were recruited as subjects. All parents of the subjects signed written informed consent before the study began. The inclusion and exclusion criteria for subjects are as follows:

[0055] Inclusion criteria include: current weight within the 20th-80th percentile of the children's scale, currently using infant formula without probiotics, not allowed to use prebiotics or probiotics, and willing to participate in the entire experiment;

[0056] Exclusion criteria include: any malformation, maternal metabolic and / or chronic disease, current or previous conditions that may interfere with the study or intervention, taking oral antibiotics within two weeks prior to the intervention, known probiotic allergy or possible food allergy, participation in other clinical studies within four weeks prior to the intervention, or reluctance to participate for any special reason.

[0057] 2) Preparation of probiotic products and placebos

[0058] The intervention used in this study was *Bifidobacterium longum* subsp. infantis YLGB-1496 bacterial powder, with maltodextrin as the excipient, in a 2g / bag specification; the placebo was maltodextrin, 2g / bag, with identical appearance, packaging, weight, and color to the intervention. Both the intervention and placebo were tested for *Escherichia coli*, *Salmonella*, *Staphylococcus aureus*, and mold by SGS-CSTC Standards Technical Services (Shanghai) Co., Ltd., and the results all met national standards. Additionally, heavy metals and additives such as lead (Pb), benzoic acid, sorbic acid, sodium saccharin cyclohexylsulfamate (cyclamate), and tartrazine were tested, and none were detected.

[0059] The intervention (bacterial powder) was frozen at -20 degrees Celsius; after the intervention was distributed to the subjects (study subjects), it was frozen at -18 degrees Celsius in a home refrigerator.

[0060] 3) Research and implementation methods

[0061] After enrollment, participants were randomly assigned to either the probiotic group or the placebo group based on inclusion and exclusion criteria. Eligible participants were randomly assigned in a 1:1 ratio to either group, identified by a unique code, from a computer-generated list. Randomization was performed by the research statistician, who did not have contact with the participants. No member of the research team received the assignment sequences until the study was completed.

[0062] Researchers distributed interventions to all enrolled infants and young children according to their group information.

[0063] Probiotic intervention group (intervention group): Once a day, one packet (sachet) each time (containing 1.5×10⁻⁶ live YLGB-1496 bacteria). 10 (CFU / packet), start using the probiotic product on the first day and continue for 3 months.

[0064] Placebo control group (placebo group or control group): 1 packet (maltodextrin, without Bifidobacterium infantis strain YLGB-1496) once a day, starting from the day of use of the placebo product, for 3 consecutive months.

[0065] Two groups of infants and their primary caregivers were visited at three follow-up points (0w, 6w, and 12w) to collect information on diet and health records. The relief of nausea, vomiting, and rectal pain in the subjects was recorded and tested at the three follow-up points.

[0066] 4) Data entry and statistical analysis

[0067] Data entry for the study was conducted using Epidata software with two people working on two computers. The database was then compared twice. Any issues discovered during the process were promptly communicated to the monitor, and the researchers were required to provide responses. A question form was used to record any questions raised during the data entry process, along with the answers and resolutions. This question form was kept for future reference.

[0068] The study data were analyzed using SPSS software version 20.0. Considering the skewed distribution and nonparametric nature of the data, the Mann-Whitney U test was used to compare differences between groups, while the chi-square test was used to compare routine data. Spearman rank correlation was used in nonparametric correlation analysis, with a correlation coefficient of rho(r). Negative binomial regression was also used to assess the relief of nausea, vomiting, and rectal pain caused by probiotic intervention. All tests were two-tailed, and *P < 0.05 was considered statistically significant. Unless otherwise stated, data are expressed as mean ± standard error. Specific results are shown in Table 1. Figure 2 , Figure 3 , Figure 4 .

[0069] Test results

[0070] Table 1. Number of days (days / weeks) during which subjects experienced nausea, vomiting, and rectal pain in the past month.

[0071]

[0072] Note: The past month refers to the month prior to data collection (follow-up point).

[0073] Combined with Table 1, Figures 2-4 It can be seen that the nausea, vomiting, and rectal pain in the probiotic intervention group (intervention group) were significantly relieved, which confirms that Bifidobacterium longum infant subsp. YLGB-1496 can effectively relieve nausea, vomiting, and rectal pain.

[0074] The preferred embodiments and experimental verifications of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of Bifidobacterium longum subsp. infantis YLGB-1496 strain in the preparation of products to relieve gastrointestinal discomfort.

2. Use according to claim 1, characterized in that, The *Bifidobacterium longum* subspecies *YLGB-1496* strain is deposited at the China Center for Type Culture Collection (CCTCC) of Wuhan University, with accession number CCTCC NO: M2011122.

3. Use according to claim 1 or 2, characterized in that, The gastrointestinal discomfort includes one or more of the following: nausea, vomiting, and rectal pain.

4. The use according to any one of claims 1-3, characterized in that, The product includes the *Bifidobacterium longum* infant subspecies YLGB-1496 strain.

5. The use according to claim 4, characterized in that, In the product, the viable cell count of the Bifidobacterium longum subsp. infantis YLGB-1496 strain is 10 6 CFU / g ~ 10 11 CFU / g.

6. The use according to any one of claims 1-5, characterized in that, The product is administered to the subject via the gastrointestinal route.

7. The use according to claim 6, characterized in that, The subjects included infants aged 0 to 3 years.

8. The use according to any one of claims 1-7, characterized in that, The products include one or more of the following: general food, health food, solid beverage, nutritional supplement, infant formula, special medical purpose formula food, and pharmaceuticals.

9. A composition, characterized in that, The composition comprises Bifidobacterium longum infantis subspecies YLGB-1496 strain.

10. A method for preparing the composition of claim 9, characterized in that, include: The raw material system including the Bifidobacterium longum infant subspecies YLGB-1496 strain was mixed to obtain the composition.

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