Bifidobacterium longum B14 with efficient lactose degradation capability and low gas production characteristic and application of bifidobacterium longum B14

Abstract

The invention discloses bifidobacterium longum B14 with efficient lactose degradation capability and low gas production characteristic and application of the bifidobacterium longum B14, and belongs to the technical field of microorganisms. The preservation number of the bifidobacterium longum B14 is GDMCC No: 67276. The bifidobacterium longum B14 strain provided by the invention can effectively decompose lactose in intestinal tracts, meanwhile, uncomfortable symptoms such as abdominal distension caused by gas production are reduced to the maximum extent, and the technical problem that in the prior art, an efficient lactose degradation strain and the low gas production characteristic are difficult to consider at the same time is successfully solved. The invention provides a core strain resource for developing probiotic products capable of relieving main symptoms of lactose intolerance and remarkably reducing side effects. The strain has a definite application prospect and a remarkable industrial value in development of health care products and medicines.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Bifidobacterium longum B14 with high lactose degradation capacity and low gas production characteristics and its applications. Background Technology

[0002] Lactose intolerance is a common digestive disorder primarily caused by a deficiency or insufficient activity of lactase in the small intestine. This prevents the adequate hydrolysis of ingested lactose, leading to symptoms such as bloating, diarrhea, and abdominal pain. Currently, utilizing probiotics, particularly Lactobacillus and Bifidobacterium, to aid lactose digestion has become a popular nutritional intervention strategy. The theoretical basis for this is that these probiotics can express their own β-galactosidase (lactase) in the gut, thereby helping the host break down lactose and alleviate symptoms.

[0003] However, current technologies and practical applications show that not all probiotics are effective and suitable for alleviating lactose intolerance. First, different strains exhibit significant differences in lactose degradation efficiency, with many strains showing limited degradation capacity in vitro or in vivo, resulting in poor efficacy. Second, even if some strains demonstrate high lactose degradation activity in vitro, they may not be able to tolerate the harsh environment of the human digestive tract (such as stomach acid and bile salts), leading to low survival rates and insufficient reach of the colon to function. A more significant technical challenge is that many strains capable of efficiently degrading lactose produce large amounts of hydrogen and carbon dioxide during their metabolism, particularly during colonic fermentation. This vigorous gas production not only fails to alleviate symptoms in lactose-intolerant individuals but can also directly exacerbate bloating and abdominal pain, severely impacting the user experience and effectiveness of the product.

[0004] "Highly efficient degradation" and "low gas production" are often difficult to achieve simultaneously in microbial physiology, as rapid lactose glycolysis is often accompanied by vigorous gas production. Most publicly available strains only focus on one aspect, lacking ideal strains that can simultaneously meet both stringent criteria. This directly limits the development of highly efficient and low-side-effect probiotic products. Therefore, isolating and obtaining a novel probiotic strain that combines excellent lactose degradation efficiency with unique low-gas-producing metabolic characteristics is of paramount importance for advancing technology and upgrading products in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bifidobacterium longum B14 with high lactose degradation capacity and low gas production, and its applications, to solve the problems existing in the prior art. The Bifidobacterium longum B14 provided by this invention combines the dual advantages of high lactose degradation capacity and low gas production, effectively alleviating lactose intolerance symptoms while minimizing bloating side effects, thus providing an excellent strain resource for developing highly effective, low-side-effect probiotic products.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a strain of Bifidobacterium longum with high lactose degradation capacity and low gas production characteristics. Bifidobacterium longum Bifidobacterium longum B14 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 11, 2025, with accession number GDMCC No: 67276.

[0007] The present invention also provides the application of the aforementioned Bifidobacterium longum B14 in the preparation of probiotic formulations.

[0008] The present invention also provides a probiotic preparation, the active ingredient of which includes the aforementioned Bifidobacterium longum B14.

[0009] The present invention also provides the use of the aforementioned Bifidobacterium longum B14 or the aforementioned probiotic preparation in the preparation of products for improving lactose intolerance.

[0010] The present invention also provides a product for improving lactose intolerance, wherein the active ingredient comprises the aforementioned Bifidobacterium longum B14 or the aforementioned probiotic preparation.

[0011] Optionally, the products include health supplements and medicines.

[0012] Optionally, the drug may also include pharmaceutically acceptable excipients and / or carriers.

[0013] Optionally, the carrier includes at least one of a diluent, a dispersant, an excipient, a stabilizer, a lubricant, and a disintegrant.

[0014] Optionally, the dosage form of the drug includes tablets, capsules, powders, and granules.

[0015] Optionally, the capsules include spherical capsules using spherical encapsulation technology; the powders or granules include microcapsule particles made using microencapsulation technology.

[0016] The present invention discloses the following technical effects: The *Bifidobacterium longum* strain B14 provided by this invention successfully solves the technical challenge of simultaneously achieving high-efficiency lactose degradation and low gas production in existing technologies. Experiments demonstrate that this strain not only achieves 98.5% high-efficiency degradation in a culture medium with lactose as the sole carbon source, but also maintains a viable count of 6.08 log CFU / mL and a lactose degradation rate of 92.28% after being subjected to a simulated human digestive tract environment. Particularly in simulated colonic fermentation experiments, while completely degrading lactose, the B14 strain exhibits a significantly lower 24-hour cumulative gas production (5.6 ± 0.5 mL) compared to other control strains, demonstrating unique low-gas metabolic characteristics.

[0017] Based on the above characteristics, the strain of this invention can effectively break down lactose in the intestines while minimizing discomfort symptoms such as bloating caused by gas production. This provides a core strain resource for developing probiotic products that can both alleviate the main symptoms of lactose intolerance and significantly reduce side effects. This strain has clear application prospects and significant industrial value in the development of health products and drugs. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a morphological identification diagram of strain B14; Figure 2 This is a phylogenetic tree constructed based on the amplified sequence of strain B14. Detailed Implementation

[0020] This invention provides a strain of Bifidobacterium longum with high lactose degradation capacity and low gas production characteristics. Bifidobacterium longum B14, with accession number GDMCC No:67276.

[0021] The *Bifidobacterium longum* B14 strain was isolated from traditional fermented yogurt kefir and screened by the inventors from a previously established probiotic strain library. Experiments have confirmed that this *Bifidobacterium longum* possesses highly efficient lactose degradation capabilities. In a simulated human digestive environment, both its viable bacterial concentration and lactose degradation capacity can be maintained at high levels; under simulated colon conditions, this strain can rapidly utilize lactose for proliferation and exhibits low gas production characteristics.

[0022] The present invention also provides the application of the aforementioned Bifidobacterium longum B14 in the preparation of probiotic formulations.

[0023] Optionally, the dosage form of the probiotic preparation includes solid dosage form, semi-solid dosage form and liquid dosage form.

[0024] Further optionally, the solid dosage form includes bacterial powder, granules, capsules, and tablets; the capsules include spherical capsules using spherical encapsulation technology; the bacterial powder or granules include microcapsule particles made using microencapsulation technology.

[0025] The present invention also provides the use of the aforementioned Bifidobacterium longum B14 or the aforementioned probiotic preparation in the preparation of health products or drugs for improving lactose intolerance.

[0026] Optionally, the drug may also include pharmaceutically acceptable excipients and / or carriers.

[0027] Further optionally, the carrier includes at least one of a diluent, a dispersant, an excipient, a stabilizer, a lubricant, and a disintegrant.

[0028] Optionally, the dosage form of the drug includes tablets, capsules, powders, and granules.

[0029] Further optionally, the capsules include spherical capsules using spherical encapsulation technology; the powders or granules include microcapsule particles made using microencapsulation technology.

[0030] Optionally, the Bifidobacterium longum B14 in the application or product described in this invention exists in the form of live or dead or intermittently sterilized, or in the form of lysate and / or extract, or in the form of bacterial product, or in the form of supernatant or derivative, wherein the derivative form is preferably selected from: metabolites, metabolobioproducts, exosomes, probiotics, cell walls and their components, extracellular polysaccharides and compounds containing immunogenic components, preferably selected from: supernatant or inactivated bacterial cells.

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Example 1: Preliminary screening of highly efficient lactose-degrading probiotics Forty probiotic strains were selected from the inventor's previously established strain bank, including 20 strains of Lactobacillus (named L01-L20) and 20 strains of Bifidobacterium (named B01-B20). The 40 strains, stored at -80℃, were continuously streaked onto MRS plates for three generations to activate the strains. Single colonies were then picked and inoculated onto MRS plates. The plates were then incubated at 37℃ for 24 hours in an anaerobic environment.

[0037] Forty activated bacterial strains were used to prepare seed culture, which was then inoculated at a 1% (v / v) in MRS broth medium with lactose as the sole carbon source. The medium composition was: lactose 20.0 g / L, peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate heptahydrate 0.20 g / L, manganese sulfate tetrahydrate 0.05 g / L, dipotassium hydrogen phosphate 2.0 g / L, and Tween-80 1.0 g / L. The inoculated medium was incubated at 37°C in an anaerobic workstation. Samples were taken at 12 and 24 hours, and the residual lactose concentration in the medium was determined by high-performance liquid chromatography (HPLC). The lactose degradation rate was calculated using the following formula:

[0038] The lactose degradation capacity of 40 probiotic strains was evaluated, and the results are shown in Table 1.

[0039] Table 1. Lactose degradation rate of 40 candidate bacteria at different culture times in MRS medium with lactose as the sole carbon source.

[0040] Table 1 shows that the degradation abilities of different strains varied significantly. After 24 hours of cultivation, L10 ( L. plants L18 L. plantarum B04 B. bifidum B14 B. bifidum ) and B15 ( B. bifidumAll five strains exhibited excellent performance, with lactose degradation rates exceeding 96%. Specifically, strain L10 achieved the highest degradation rate at 98.8%, followed by B14 (98.5%), L18 (98.4%), B15 (97.7%), and B04 (96.9%). These five strains ranked among the top five in lactose degradation efficiency among all 40 candidate strains. Therefore, these five strains were selected for in vitro simulated human digestion experiments.

[0041] Example 2: Secondary screening based on an in vitro simulated human digestive model To further evaluate the survival ability and functional stability of the five probiotic strains (L10, L18, B04, B14 and B15) initially screened in Example 1 in the human digestive tract environment, an in vitro static digestion model was constructed in this example.

[0042] Commercially available whole milk was used as the substrate for the digestion experiment, with a lactose content of 4.92% (w / v). The initial concentration was 1×10⁻⁶. 9 Each bacterial suspension at CFU / mL was mixed with 5 mL of milk to obtain bacterial-containing milk. Subsequently, the system underwent treatment simulating three stages: oral cavity, stomach, and small intestine.

[0043] The in vitro simulated digestive fluid and its components are as follows: (1) The components of the simulated saliva (pH 7) are: potassium chloride 1.125 g / L, potassium dihydrogen phosphate 0.504 g / L, sodium bicarbonate 1.142 g / L, magnesium chloride hexahydrate 0.0305 g / L, ammonium carbonate 0.006 g / L, hydrogen chloride 0.040 g / L and calcium chloride dihydrate 0.221 g / L.

[0044] (2) The components of the simulated gastric juice (pH 3) are: potassium chloride 0.514 g / L, potassium dihydrogen phosphate 0.122 g / L, sodium bicarbonate 2.100 g / L, sodium chloride 2.759 g / L, magnesium chloride hexahydrate 0.024 g / L, ammonium carbonate 0.048 g / L, hydrogen chloride 0.569 g / L and calcium chloride dihydrate 0.022 g / L.

[0045] (3) The components of the simulated small intestinal fluid (pH 7) are: potassium chloride 0.507 g / L, potassium dihydrogen phosphate 0.109 g / L, sodium bicarbonate 7.141 g / L, sodium chloride 2.244 g / L, magnesium chloride hexahydrate 0.067 g / L, hydrogen chloride 0.306 g / L and calcium chloride dihydrate 0.088 g / L.

[0046] The in vitro simulated digestion process is as follows: (1) Oral stage: Mix the bacteria-containing milk with simulated saliva and incubate for 2 minutes; (2) Stomach stage: Add simulated gastric juice and adjust the pH to 3.0, incubate for 2 hours; (3) Small intestine stage: Add simulated intestinal fluid and adjust the pH to 7.0, and continue incubation for 2 hours.

[0047] Samples were taken at key time points in each stage, and the viable bacterial concentration was determined by plate counting. The lactose content was detected by high-performance liquid chromatography (HPLC) to calculate the degradation rate. The results are shown in Table 2.

[0048]

[0049] Table 2 shows that the viable cell counts of all strains decreased to varying degrees after treatment in a simulated digestive tract, as expected. Strain L18 exhibited the best tolerance, maintaining a viable cell count of 6.38 log CFU / mL after 2 hours of small intestinal digestion, while strain B04 showed the worst tolerance, with a significantly reduced viable cell count to 4.95 log CFU / mL. Significant differences were observed among the strains in the crucial lactose degradation function. After the complete digestion process, strain B14 achieved a lactose degradation rate of 92.28%, the best among all strains, and its viable cell count remained at a high level of 6.08 log CFU / mL. Furthermore, strains L10 and B15 also demonstrated strong lactose degradation capabilities, with degradation rates of 83.3% and 81.7%, respectively. Considering both the survival rate and lactose degradation efficiency throughout the digestion process, strains L10, B14, and B15 showed the most outstanding overall performance and were therefore selected for the next round of screening. Example 3: Screening for low gas production characteristics based on an in vitro simulated human colonic fermentation model

[0050] To ultimately determine the optimal strain that combines efficient lactose degradation with low gas production, this example places the three probiotic strains (L10, B14, and B15) that emerged victorious from the secondary screening stage in Example 2 into an in vitro simulated human colon fermentation system for evaluation.

[0051] The in vitro simulated human colon fermentation system consisted of lactose (0.5% w / v), 9.5 mL of basal solution, 0.125 mL of vitamin phosphate solution, 0.5 mL of carbonate buffer, and 0.125 mL of reducing agent solution. The specific components are as follows: (1) Basic solution: potassium chloride 0.6 g / L, sodium chloride 0.6 g / L, calcium chloride dihydrate 0.2 g / L, magnesium sulfate heptahydrate 0.5 g / L, ammonium chloride 0.54 g / L, peptone 1.0 g / L, resazurin 1.0 mg / L, manganese chloride tetrahydrate 0.25 mg / L, ferrous sulfate heptahydrate 0.2 mg / L, zinc chloride 0.25 mg / L, cuprous chloride dihydrate 0.2 mg / L, cobalt chloride hexahydrate 0.50 mg / L, selenium oxide 0.50 mg / L, boric acid 2.5 mg / L, heme 1.000 mg / L, acetic acid 68.5 mg / L, propionic acid 30.0 mg / L, butyric acid 18.4 mg / L and isobutyric acid 4.7 mg / L.

[0052] (2) Vitamin phosphate solution: Biotin 20.4 mg / L, folic acid 20.5 mg / L, calcium dextropantothenate 164.0 mg / L, nicotinamide 164.0 mg / L, riboflavin 164.0 mg / L, thiamine hydrochloride 164.0 mg / L, pyridoxine 164.0 mg / L, para-aminobenzoic acid 20.4 mg / L, cyanocobalamin 20.4 mg / L and potassium dihydrogen phosphate 54.2 g / L.

[0053] (3) The reducing agent solution contains 20.5 g / L of cysteine ​​hydrochloric acid.

[0054] The bacterial suspensions of each strain were diluted with 1×10⁻⁶. 7 An initial concentration of CFU / mL was inoculated into the fermentation system and cultured at 37℃. Samples were taken at 0, 12, and 24 h to detect the total viable cell count and lactose degradation rate, and the cumulative gas production volume was measured using a precision barometer to evaluate the gas production kinetics of the strain during fermentation. The results are shown in Table 3.

[0055]

[0056] Table 3 shows that all three strains exhibited strong proliferation capacity and lactose utilization efficiency in a simulated colonic environment, completely degrading lactose within 24 hours. However, they showed significant differences in gas production characteristics. After 24 hours of cultivation, strain L10 had the largest cumulative gas production volume, reaching 10.5 ± 0.7 mL; strain B15 was second, at 8.2 ± 0.6 mL. In contrast, strain B14 had a total gas production volume of only 5.6 ± 0.5 mL, significantly lower than the other two strains (p<0.05). This result indicates that although all three strains could efficiently ferment lactose, strain B14 produced the least gas during metabolism. Given that intestinal gas production is a major cause of bloating, abdominal pain, and other discomfort symptoms in lactose-intolerant individuals, the "efficient degradation, low gas production" metabolic characteristic of strain B14 is crucial for developing probiotic products that can effectively alleviate lactose intolerance symptoms while minimizing side effects. Therefore, B14 was ultimately selected as the target strain. Example 4: Identification and biopreservation of strain B14

[0057] 1. Morphological identification Strawberry strain B14 was streaked onto MRS agar plates and incubated anaerobically at 37°C for 48 hours before observation. The colonies were round, smooth, with regular, slightly convex edges, milky white, and opaque, with a diameter of approximately 1-2 mm.

[0058] Single colonies were picked and Gram-stained for observation under a light microscope. The results showed that strain B14 was a Gram-positive bacillus with pleomorphic cell structure, exhibiting various morphologies such as curved, forked, or rod-shaped cells. It was non-spore-forming and arranged singly, in pairs, or in short chains. Figure 1 The above morphological characteristics are similar to those of the genus Bifidobacterium ( ). Bifidobacterium It matches the typical characteristics of ).

[0059] 2. Molecular biological identification Take 1.5 mL of overnight cultured B14 bacterial culture and extract total genomic DNA of the strain using a bacterial genomic DNA extraction kit (Tiangen Biotech Co., Ltd.) according to its instructions.

[0060] Using the extracted DNA as a template, PCR amplification was performed using the universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') for the bacterial 16S rRNA gene.

[0061] PCR reaction system (50 μL): 10×PCR Buffer 5 μL, dNTP Mixture 4 μL, forward and reverse primers (10 μM) 2 μL each, template DNA 1 μL, Taq DNA polymerase (5 U / μL) 0.5 μL, add sterile ultrapure water to 50 μL.

[0062] PCR reaction procedure: 94℃ pre-denaturation for 5 minutes; followed by 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 1.5 minutes, for a total of 30 cycles; and finally 72℃ final extension for 10 minutes.

[0063] After the PCR products were detected by agarose gel electrophoresis, they were sent to a professional biotechnology company for purification and bidirectional sequencing.

[0064] The obtained 16S rRNA gene sequence was compared for homology in the NCBI database using the BLAST program. The results showed that the 16S rRNA gene sequence of strain B14 shared over 99.8% homology with the sequences of multiple strains of *Bifidobacterium longum* subsp. *longum*. Figure 2 The phylogenetic tree constructed based on this sequence also showed that strain B14 and *Bifidobacterium longum* belong to the same evolutionary branch. Combined with its morphological characteristics, this strain was ultimately identified as *Bifidobacterium longum*. Bifidobacterium longum ).

[0065] 3. Preservation of bacterial strains Strain B14 was deposited on November 11, 2025, at the Guangdong Microbial Culture Collection Center (GDMCC) in Guangzhou, China, with accession number GDMCC No:67276.

[0066] The address of the collection center is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China, Institute of Microbiology, Guangdong Academy of Sciences.

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Bifidobacterium longum with high lactose degradation capacity and low gas production characteristics ( Bifidobacterium longum B14, characterized in that, The Bifidobacterium longum B14 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 11, 2025, with accession number GDMCC No: 67276.

2. The use of Bifidobacterium longum B14 as described in claim 1 in the preparation of probiotic formulations.

3. A probiotic preparation, characterized in that, The active ingredient includes Bifidobacterium longum B14 as described in claim 1.

4. The use of the Bifidobacterium longum B14 of claim 1 or the probiotic preparation of claim 3 in the preparation of a product for improving lactose intolerance.

5. A product for improving lactose intolerance, characterized in that, The active ingredients include Bifidobacterium longum B14 as described in claim 1 or the probiotic preparation as described in claim 3.

6. The product according to claim 5, characterized in that, The products include health supplements and medicines.

7. The product according to claim 6, characterized in that, The drug also includes pharmaceutically acceptable excipients and / or carriers.

8. The product according to claim 7, characterized in that, The carrier includes at least one of diluent, dispersant, excipient, stabilizer, lubricant and disintegrant.

9. The product according to claim 6, characterized in that, The dosage forms of the drug include tablets, capsules, powders, and granules.

10. The product according to claim 9, characterized in that, The capsules include spherical capsules using spherical encapsulation technology; the powders or granules include microcapsule particles made using microencapsulation technology.

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