Use of Lactobacillus fermentum GKF3 in the preparation of oral formulations to reduce nicotine cravings

Abstract

This invention relates to the use of Lactobacillus fermentum GKF3 in the preparation of oral formulations that reduce nicotine cravings. After administration of Lactobacillus fermentum GKF3 to smoking subjects, the levels of nicotine metabolites in the subjects' urine decreased, indicating that Lactobacillus fermentum GKF3 can reduce nicotine cravings.

Description

[Technical Field]

[0001] This invention relates to the use of a Lactobacillus fermentum GKF3, and more particularly to the use of a Lactobacillus fermentum GKF3 in the preparation of an oral composition for reducing nicotine cravings. [Background Technology]

[0002] When tobacco is burned, it releases over 7,000 chemical substances, including more than 90 known carcinogens. These chemicals not only harm the smoker's body but also endanger the health of others. However, smokers find it difficult to quit because nicotine in tobacco is addictive. When a smoker inhales, nicotine is transported from the lungs to the brain via the bloodstream within seconds, giving the smoker a feeling of excitement and / or pleasure. Once the nicotine level in the blood decreases, smokers are prone to nicotine withdrawal symptoms, including anxiety, irritability, difficulty concentrating, depression, insomnia, coughing, and headaches. Furthermore, the brain can develop nicotine tolerance, requiring smokers to increase their smoking to alleviate withdrawal symptoms.

[0003] Currently available smoking cessation medications assist smokers in quitting by alleviating nicotine withdrawal symptoms. These medications can be divided into nicotine replacement therapy (NRT) and non-nicotine replacement therapy (NRT). Nicotine replacement therapy provides smokers with low doses of nicotine in various forms (lozenges, inhalers, nasal sprays, and / or patches), and gradually reduces nicotine dependence through step-up dosage adjustments. Non-nicotine replacement therapy not only alleviates nicotine withdrawal symptoms when smoking is reduced, but also reduces the euphoria and / or pleasure derived from smoking. However, these smoking cessation medications have many side effects.

[0004] In view of this, there is an urgent need for an oral formulation with fewer side effects and the ability to reduce nicotine addiction in order to solve the above problems. [Summary of the Invention]

[0005] Therefore, one aspect of the present invention is to provide the use of Lactobacillus fermentum GKF3 in the preparation of an oral composition for reducing nicotine addiction, thereby reducing the content of nicotine metabolites in the urine of smoking subjects.

[0006] Another aspect of the present invention is to provide the use of Lactobacillus fermentum GKF3 in the preparation of an oral composition for reducing nicotine addiction, wherein the oral composition contains an effective dose of Lactobacillus fermentum GKF3 as an active ingredient.

[0007] According to the above-described form of the present invention, a use is provided for Lactobacillus fermentum GKF3 in the preparation of an oral composition for reducing nicotine cravings, thereby reducing the content of nicotine metabolites in the urine of smoking subjects. The oral composition may contain, but is not limited to, an effective dose of Lactobacillus fermentum GKF3 as an active ingredient. This Lactobacillus fermentum GKF3 may, for example, be deposited on January 12, 2018, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing 100101, China), with accession number CGMCC 15203.

[0008] According to the above embodiments of the present invention, compared with the smoking subjects who did not receive the oral composition, the content of nicotine metabolites in the urine of the smoking subjects who received the oral composition was reduced by 10 ng / mL to 1100 ng / mL.

[0009] According to the above embodiments of the present invention, nicotine metabolites may include, but are not limited to, cotinine and / or trans-3-hydroxycotinine.

[0010] According to the above embodiments of the present invention, the effective dose may be, for example, from 700 mg / 60 kg body weight (bw) / day to 900 mg / 60 kg bw / day.

[0011] According to the above embodiments of the present invention, the oral composition is administered to smoking subjects for a duration of time, which may be, for example, 25 to 30 days.

[0012] According to another aspect of the present invention, a use is provided for Lactobacillus fermentum GKF3 in the preparation of an oral composition for reducing nicotine cravings, wherein the oral composition may contain, but is not limited to, an effective dose of Lactobacillus fermentum GKF3 (accession number: CGMCC15203) as an active ingredient, and the effective dose may be, for example, 700 mg / 60 kg bw / day to 900 mg / 60 kg bw / day.

[0013] According to the above embodiments of the present invention, the oral composition reduces the content of nicotine metabolites in the urine of smoking subjects.

[0014] According to the above embodiments of the present invention, after the oral composition was administered to smoking subjects, the content of nicotine metabolites in the urine of smoking subjects decreased by 10 ng / mL to 1100 ng / mL compared with the absence of oral composition.

[0015] According to another aspect of the present invention, a use is provided for Lactobacillus fermentum GKF3 in the preparation of an oral composition for reducing nicotine cravings, wherein the oral composition may contain, but is not limited to, an effective dose of Lactobacillus fermentum GKF3 (accession number: CGMCC15203) as an active ingredient, and the effective dose may be, for example, 10 10 Colony forming units (CFU) / 60 kg·bw / day to 10 12 CFU / 60kg·bw / day.

[0016] According to the above embodiments of the present invention, the oral composition is administered to smoking subjects for a period of time, which may be, for example, 25 to 30 days.

[0017] The use of Lactobacillus fermentum GKF3 of the present invention in the preparation of an oral composition for reducing nicotine addiction, wherein after the oral composition was administered to smoking subjects, the amount of smoking by the smoking subjects was reduced and no nicotine withdrawal symptoms were produced, indicating that Lactobacillus fermentum GKF3 has the effect of reducing nicotine addiction.

[0018] [Simplified Explanation of the Diagram]

[0019] To make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the detailed description of the accompanying drawings is as follows:

[0020] [ Figure 1 [ ] is an experimental flowchart illustrating the first group according to an embodiment of the present invention.

[0021] [ Figure 2 [ ] is an experimental flowchart illustrating the second group according to an embodiment of the present invention.

[0022] [ Figure 3 [This figure shows the percentage of people whose smoking addiction decreased in different treatment groups according to an embodiment of the present invention.]

[0023] [ Figure 4 The diagram illustrates the CES-D total score of a subject at different times according to an embodiment of the present invention.

Detailed Implementation Methods

[0024] The singular forms “a,” “an,” and “the” used in this invention include plural references unless the context clearly specifies otherwise. Numerical ranges (e.g., 10% to 11% of A) include upper and lower limits unless otherwise specified (i.e., 10% ≤ A ≤ 11%); if a numerical range does not define a lower limit (e.g., less than 0.2% of B, or B below 0.2%), then the lower limit may be 0 (i.e., 0% ≤ B ≤ 0.2%). The foregoing terms are for illustrative and understanding purposes only, and not for limiting the invention.

[0025] As previously stated, the present invention provides the use of Lactobacillus fermentum GKF3 in the preparation of an oral composition for reducing nicotine addiction, wherein the oral composition may contain, but is not limited to, an effective dose of Lactobacillus fermentum GKF3 as an active ingredient.

[0026] In one embodiment, this Lactobacillus fermentum GKF3 (also known as strain GKF3) was deposited on January 12, 2018 at the China General Microbiological Culture Collection Center (CGMCC, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing 100101, China) with accession number CGMCC 15203.

[0027] It should be noted that *Lactobacillus fermentum* is a Gram-positive bacterium in the genus *Lactobacillus*, commonly found in animal and / or plant fermentations, such as sourdough or pickles. Furthermore, certain specific *Lactobacillus fermentum* strains, such as *Lactobacillus fermentum* GKF3, can be used as probiotics for animals, and when administered at safe doses (e.g., orally), they have virtually no side effects.

[0028] In one embodiment, the effective dose of the above-mentioned oral composition may be, for example, 700 mg / 60 kg bw / day to 900 mg / 60 kg bw / day, equivalent to 10 10 CFU / 60kg·bw / day up to 10 12 CFU / 60 kg·bw / day. If the effective dose is less than 700 mg / 60 kg·bw / day and / or 10 10 The effect of CFU / 60kg·bw / day on reducing nicotine addiction is poor. If the effective dose is higher than 800mg / 60kg·bw / day and / or 10... 12 With CFU / 60kg·bw / day, the efficacy in reducing nicotine addiction did not significantly improve despite the increased manufacturing costs. In one embodiment, oral administration of the composition for a period of time can effectively reduce nicotine addiction, wherein the aforementioned administration time may be, for example, 25 to 30 days.

[0029] The aforementioned "smoking subjects" can be referenced from the international definition of a current smoker, which refers to someone who has smoked more than 100 cigarettes (5 packs) from the past to the present and has used tobacco products in the last 30 days.

[0030] One of the technical features of this invention is that it uses both objective physiological test values ​​and subjective cognitive scales to evaluate the efficacy of Lactobacillus fermentum GKF3 in reducing nicotine addiction. Specifically, objectively, the aforementioned "reducing nicotine addiction" refers to a decrease in the amount of smoking (e.g., a decrease in the frequency and / or quantity of smoking). Subjectively, the aforementioned "reducing nicotine addiction" refers to the absence of nicotine withdrawal symptoms when the decrease in the amount of smoking leads to a decrease in the nicotine content in the blood.

[0031] Specifically, nicotine metabolites can be detected in the urine of smoking subjects 7 to 10 days after smoking, and the level of nicotine metabolites is positively correlated with the amount of smoking. Therefore, when the level of nicotine metabolites in the urine of a smoking subject decreases to a preset concentration of 10 ng / mL, it is determined that the smoking amount of the smoking subject has decreased. The aforementioned nicotine metabolites may include, but are not limited to, cotinine and / or trans-3-hydroxycotinine.

[0032] Therefore, the efficacy of reducing nicotine addiction can be assessed by the decrease in urinary nicotine metabolite levels after administration of the oral formulation to smoking subjects. Secondly, the efficacy can also be assessed by the percentage of subjects whose nicotine addiction decreased. This percentage is the percentage of all smoking subjects whose urinary nicotine metabolite levels decreased by more than 10 ng / mL after administration of the oral formulation compared to those who did not.

[0033] In one embodiment, the aforementioned nicotine withdrawal symptoms can be assessed, for example, using the Center for Epidemiologic Studies Depression Scale (CES-D) from the National Institutes of Mental Health. The CES-D is a commonly used epidemiological depression scale, quantifying the frequency of subjectively assessed depressive moods in smoking subjects using the total CES-D score. If a smoking subject's smoking volume decreases and the total CES-D score increases, it is determined that nicotine withdrawal symptoms are occurring. Conversely, if the total CES-D score decreases, it is determined that nicotine withdrawal symptoms are not occurring.

[0034] Human trials have confirmed that, compared to smokers who did not receive Lactobacillus fermentum GKF3, those who received Lactobacillus fermentum GKF3 for 4 weeks showed a 10 ng / mL to 1100 ng / mL reduction in urinary nicotine metabolite levels. Furthermore, compared to smokers who did not receive Lactobacillus fermentum GKF3, the percentage of smokers who received Lactobacillus fermentum GKF3 showed a higher reduction in nicotine addiction, demonstrating that Lactobacillus fermentum GKF3 can objectively reduce nicotine addiction.

[0035] Secondly, according to the CES-D assessment, compared with those who did not receive Lactobacillus fermentum GKF3, the smoking subjects who received Lactobacillus fermentum GKF3 had lower CES-D scores, indicating a decrease in the frequency of depressive moods and the absence of nicotine withdrawal symptoms. This demonstrates that Lactobacillus fermentum GKF3 can subjectively reduce nicotine cravings. In conclusion, Lactobacillus fermentum GKF3 can reduce the amount of smoking in smoking subjects without producing nicotine withdrawal symptoms, thus having a nicotine craving-reducing effect.

[0036] The following examples illustrate the application of the present invention, but they are not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention.

[0037] Example 1: Source and Preparation of Strains

[0038] Fermented kimchi sold in Beipu City, Hsinchu, Taiwan was diluted with 10 times its volume of water and homogenized to obtain a homogenate. Then, 1 mL of the homogenate was inoculated into MRS medium containing 0.1% bromocresol green to screen for strains (including lactic acid bacteria) that prefer a weakly acidic environment (pH 6.5±0.2). After incubation at 37°C for 16 hours, a culture medium was obtained. The culture medium was then serially diluted and spread onto five solid media [Rogosa agar, bifidobacterium iodoacetate medium 25 (BIM-25), agar containing potato starch and yeast extract, bromocresol green MRS agar, and bromocresol purple MRS agar]. After incubation at 37°C for 40 hours, 15 to 20 single colonies were selected from each solid medium to obtain the first screening strain.

[0039] Next, RAW 264.7 macrophages treated with lipopolysaccharide (hereinafter referred to as model cells) were used as a cellular inflammatory response model to screen for strains with anti-inflammatory effects. If the secretion of nitric oxide and tumor necrosis factor-α (TNF-α) in model cells treated with the strain decreased by 80% compared to model cells not treated with the strain, then the strain was considered to have anti-inflammatory effects. Then, from these anti-inflammatory strains, strain GKF3, which possesses tryptophan decarboxylase activity, was screened out.

[0040] After purifying the genomic DNA (gDNA) of strain GKF3, polymerase chain reaction (PCR) was performed using the upstream and downstream primers shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, to obtain the DNA fragment of the recN gene with the nucleic acid sequence shown in SEQ ID NO: 3. Sequence alignment confirmed that strain GKF3 is *Lactobacillus fermentum*.

[0041] Lactobacillus fermentum strain GKF3 was deposited on January 12, 2018, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing 100101, China), with accession number CGMCC15203. Lactobacillus fermentum strain GKF3 was also deposited on February 12, 2018, at the Bioresource Center of the Food Industry Research and Development Institute, Taiwan (Address: No. 331, Food Road, East District, Hsinchu City, Taiwan), with accession number BCRC 910824.

[0042] Lactobacillus fermentum GKF3 was inoculated onto MRS agar medium for solid-state culture to obtain single colonies. These single colonies were then inoculated into a fermentation substrate and fermented for 18 hours to obtain fermented product. The solid-state culture was conducted at 37°C. The fermentation was carried out at 37°C with a carbon dioxide aeration rate of 0.5 vvm / vvm and a shaking rate of 500 rpm. The formulation of the fermentation substrate is shown in Table 1.

[0043] Table 1

[0044]

[0045] The above-mentioned fermented material was centrifuged at 10,000 rpm to obtain a bacterial slurry. Then, the bacterial slurry was mixed with 20 wt% skim milk powder and freeze-dried to obtain GKF3 freeze-dried powder. Freeze-drying is a known procedure and does not affect subsequent evaluations, so it will not be described further. Example 2: Evaluation of the efficacy of Lactobacillus fermentum GKF3 in reducing nicotine addiction using physiological test values.

[0046] The GKF3 lyophilized powder was encapsulated, so that each capsule contained 400mg of GKF3 lyophilized powder, equivalent to 5 × 10 9CFU of live Lactobacillus fermentum GKF3 was used to obtain Lactobacillus fermentum GKF3 experimental bacterial powder capsules. Secondly, microcrystalline α-cellulose was encapsulated in the capsules as a placebo.

[0047] Participants were recruited from healthy young men aged 20 to 50 who were smokers and had no serious medical conditions. It is noteworthy that participants meeting any of the following criteria were excluded:

[0048] (1) Those suffering from serious mental illness;

[0049] (2) Those taking antibiotics;

[0050] (3) Individuals with severe alcohol addiction or other behaviors that may affect the results of this test;

[0051] (4) Those currently taking psychotropic medications (e.g., antidepressants);

[0052] (5) Those who have received or are currently receiving nicotine-related replacement therapy;

[0053] (6) Those who have been using probiotics for the past three months or are currently using probiotics.

[0054] Secondly, if a subject experiences discomfort with the reagents (referring to Lactobacillus fermentum GKF3 powder capsules and / or placebo capsules) during the evaluation trial, fails to use the reagents as prescribed, does not follow the prescribed lifestyle, making it impossible to determine the efficacy of the reagents or affecting the determination of efficacy or safety due to incomplete data, or develops an illness or takes drugs or health foods that may affect the results of this trial during the evaluation trial, or fails to cooperate with urine collection and / or complete CES-D, the subject should withdraw from the trial.

[0055] The subjects (n=45) were randomly divided into group 1 (n=23) and group 2 (n=22). There were no statistically significant differences in any of the blood biochemical parameters between groups 1 and 2. Please refer to [link / reference]. Figure 1 and Figure 2 ,in Figure 1 This is a flowchart illustrating the first group of experiments according to an embodiment of the present invention, and Figure 2This is a flowchart illustrating the second group of experiments according to an embodiment of the present invention. In procedures 100 and 200 below, the urine test detects the content of nicotine metabolites in the subject's urine, wherein the nicotine metabolites include cintinib and trans-3-hydroxycintinib. The detection of cintinib and trans-3-hydroxycintinib is common knowledge in the technical field to which this invention pertains and does not affect efficacy, and will not be described further. The control treatment is the administration of two placebo capsules daily after breakfast. The experimental treatment is the administration of two Lactobacillus fermentum GKF3 powder capsules daily after breakfast. It is particularly noteworthy that during procedures 100 and 200 above, the subject may smoke according to their own wishes and / or needs.

[0056] Please see Figure 1 Procedure 100, as shown in step 110, involves testing urine. Next, a control treatment is administered for 4 weeks, followed by urine testing again, as described in steps 115 and 120. Then, according to step 130, the control treatment is stopped for 2 weeks. Next, as shown in step 140, urine is tested. Then, the experimental treatment is administered for 4 weeks, followed by urine testing again, as described in steps 145 and 150, for a total of 10 weeks.

[0057] Please see Figure 2 Procedure 200, as shown in step 210, involves testing urine. Next, the experimental treatment is performed for 4 weeks, and urine is tested again, as described in steps 215 and 220. Then, according to step 230, the experimental treatment is stopped for 2 weeks. Next, as shown in step 240, urine is tested. Then, a control treatment is performed for 4 weeks, and urine is tested again, as described in steps 245 and 250, for a total of 10 weeks.

[0058] Procedures 100 and 200 employed a crossover trial design, which has the advantage of providing evaluation results for each subject under both the control treatment (control group) and the experimental treatment (experimental group), thus reducing the required number of subjects. The average levels of nicotine metabolites in urine before and after different treatments are recorded in Table 2.

[0059] Table 2

[0060] Average nicotine metabolite content in urine (ng / mL) forward back Control treatment 3030.5 4441.8 Experimental treatment 3701.1 2653.4

[0061] As shown in Table 2, after the experimental treatment, the average content of nicotine metabolites in the subjects' urine was 1047.7 ng / mL lower than before the experimental treatment. However, the average content of nicotine metabolites in the urine after the control treatment was higher than before the control treatment. This indicates that the administration of Lactobacillus fermentum GKF3 reduced the content of nicotine metabolites in the subjects' urine, suggesting a reduction in their smoking, and proving that taking Lactobacillus fermentum GKF3 can reduce nicotine addiction.

[0062] The percentage of subjects whose urinary nicotine metabolite levels decreased by more than 10 ng / mL after experimental treatment can be used to determine the percentage of subjects with reduced nicotine addiction in the experimental treatment group. Similarly, the percentage of subjects whose urinary nicotine metabolite levels decreased by more than 10 ng / mL after control treatment can be used to determine the percentage of subjects with reduced nicotine addiction in the control treatment group.

[0063] Figure 3 This plot shows the percentage (%) of people whose smoking addiction decreased in different treatment groups according to an embodiment of the present invention, where the horizontal axis represents the control group and the experimental group from left to right, and the vertical axis represents the percentage of people whose smoking addiction decreased (unit: %). Figure 3 As shown, the percentage of people whose smoking addiction decreased in the experimental treatment group was 67% (30 / 45 people), which was 31% higher than the percentage of people whose smoking addiction decreased in the control treatment group (36%, 16 / 45 people), proving that taking Lactobacillus fermentum GKF3 has the effect of reducing smoking addiction.

[0064] Example 3: Using a cognitive scale to assess the efficacy of Lactobacillus fermentum GKF3 in reducing nicotine addiction.

[0065] Using the Cognitive Emotional Scale (CES-D), participants subjectively assessed the frequency of depressive symptoms within one week. Scores of 0, 1, 2, and 3 were assigned for frequencies of none, very few (less than one day per week), sometimes (one to two days per week), and frequently (three to seven days per week), respectively. The depressive symptoms included 20 items such as depressed mood, feelings of guilt and worthlessness, feelings of helplessness and hopelessness, psychomotor retardation, loss of appetite, and sleep disturbance. Therefore, the total CES-D score ranged from 0 to 60. A significant increase in the total CES-D score when smoking decreased indicated the onset of nicotine withdrawal symptoms.

[0066] The first group of subjects completed CES-D before step 115 (week 0 of the trial), after step 115 (week 4 of the trial), and after step 145 (week 10 of the trial) in procedure 100. The second group of subjects completed CES-D before step 215 (week 0 of the trial), after step 215 (week 4 of the trial), and after step 245 (week 10 of the trial) in procedure 200. The total CES-D scores of the first and second groups of subjects were calculated. Figure 4This diagram illustrates the CES-D total score of subjects at different time points according to an embodiment of the present invention. The horizontal axis represents the week number, the vertical axis represents the CES-D total score, bar 401 represents the control group, and bar 403 represents the experimental group. It should be noted that bar 401 represents the first group of subjects in weeks 0 and 4, but the second group in week 10. Conversely, bar 403 represents the second group of subjects in weeks 0 and 4, but the first group in week 10.

[0067] like Figure 4 As shown, the mean ± standard deviation of the total CES-D score for the first group of subjects was 12.4 ± 3 (bar 401) at week 0, 10.2 ± 4.6 (bar 401) at week 4 (after control treatment), and 9.4 ± 6 (bar 403) at week 10 (after experimental treatment). Similarly, the mean ± standard deviation of the total CES-D score for the second group of subjects was 12.4 ± 3 (bar 403) at week 0, 10.2 ± 4.6 (bar 403) at week 4 (after experimental treatment), and 11.6 ± 4.9 (bar 401) at week 10 (after control treatment).

[0068] Depend on Figure 4 It can be seen that, compared to the group without experimental treatment (bar 401 in week 4), the subjects in the first group who underwent experimental treatment (bar 403 in week 10) experienced a decrease in their total CES-D score. Secondly, compared to the group without experimental treatment (bar 403 in week 4), the subjects in the second group who underwent experimental treatment (bar 403 in week 10) also experienced a decrease in their total CES-D score. Furthermore, considering... Figure 3 and Figure 4 The results showed that after the experimental treatment, the subjects' smoking amount decreased, indicating that taking Lactobacillus fermentum GKF3 not only reduced the subjects' smoking amount but also did not cause nicotine withdrawal symptoms. It also tended to reduce the frequency of depressive symptoms, proving that Lactobacillus fermentum GKF3 has the effect of reducing nicotine addiction.

[0069] In summary, the specific processes, administration methods, subjects, and evaluation methods described above are merely illustrative of the use of *Lactobacillus fermentum* GKF3 in preparing oral compositions for reducing nicotine cravings. However, those skilled in the art will understand that other processes, administration methods, subjects, and evaluation methods can be used to illustrate the use of *Lactobacillus fermentum* GKF3 in preparing oral compositions for reducing nicotine cravings without departing from the spirit and scope of the invention, and are not limited to the above. For example, other processes, administration methods, subjects, and evaluation methods can be used without affecting the nicotine-reducing efficacy of *Lactobacillus fermentum* GKF3.

[0070] As can be seen from the above embodiments, the use of Lactobacillus fermentum GKF3 of the present invention in preparing oral components for reducing nicotine addiction has the advantage that, by using probiotics, nicotine addiction can be reduced without providing nicotine and with almost no side effects, and it can be applied as a food and / or medicine to aid in smoking cessation.

[0071] While the present invention has been disclosed above with reference to several specific embodiments, various modifications, alterations, and substitutions can be made to the foregoing disclosure. It should be understood that, without departing from the spirit and scope of the invention, certain features of the embodiments of the invention may be used in some cases, but other features may not be used accordingly. Therefore, the spirit and scope of the invention should not be limited to the embodiments described above.

[0072] [Preservation of Biological Materials]

[0073] The Lactobacillus fermentum GKF3 line is deposited at the China General Microbiological Culture Collection Center (CGMCC, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China) on January 12, 2018, with accession number CGMCC15203. sequence list <110> Grape King Biotechnology Co., Ltd. <120> Use of Lactobacillus fermentum GKF3 in the preparation of oral formulations to reduce nicotine cravings <160> 3 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <221> Upstream primer ★ <400> 1 atccaaggtc aaaatgagca 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <221> Downstream primer ★ <400> 2 cttcaacccg ttggttagtg 20 <210> 3 <211> 1071 <212> DNA <213> Lactobacillus fermentum GKF3 <400> 3 taacgctaag gttcagcccc tactagcgtc gtaccaagag cagtaccaag agtaccggcg 60 tttggaggcg gcggttaacc aaaagaaggc caacgagcaa cagtgggccc agcgcctcga 120 catgttgcgc taccaagtta aagaaattgg tgacgccgac ttgcgggccg acgaagaaga 180 tgaattaatc gccgaacgtg agcgactgga acacttccaa cagattgcca ccaccctcca 240 gcaggtggtt ggggtgttga acgatgacga agaggcgcct gtcttagacc aggtcgccac 300 catcatgaat gcagcccaag agattgcgcc cttcgatccc gaatacgatg acctggccca 360 gtccctttct gacgcctact attcactgca agacgtttcc aaccaagctg gccaccagtt 420 ggatagcctc gagtttgatg aggagcgact ggccacgatc aacgcccgct tagcgacgat 480 tgccgacttg gagcacaagt atggtgaaag cttagccgac gttttggcgt actacgacca 540 gatcaaagaa gagcttaccg atatggaggc cgcggccgat tcaggttccg atttggaaga 600 gcggttaaat gcggtccagg ccgatttgtt aaaccaaggg aacgccctga gccaagtgcg 660 gcaaacggcg gcccgcaaat tagccaagca ggttcacacc caactaaagg aactgtacat 720 ggataaggcg gtttttgaag tgaactttgc caaaaccaag aagccggtct tctctgccac 780 cgggatcgat caggttgaat tctacattca aaccaatcct ggtgaggcaa tgggaccctt 840 ggcccggatc gcctccgggg gggaactttc ccgggtgatg ctggccttaa agacgatctt 900 tgcccagggc gaaggggtta caagcatcat ctttgacgaa gtcgatactg gggtttccgg 960 gcgggtcgcc caagccattg ccgataagat tcgcttgatt gccgagggct cacaggtcct 1020 ttgcattact cacttaccac aggtggcagc ggttgcccaa caccacctct t 1071

Claims

1. Use of Lactobacillus fermentum GKF3 for the preparation of an oral composition for reducing the craving for tobacco, characterized in that, wherein the oral composition is composed of an effective dose of the Lactobacillus fermentum GKF3, which was deposited with the China General Microbiological Culture Collection Center on January 12, 2018, under Accession No. CGMCC 15203, to reduce the content of nicotine metabolites in urine of a smoking subject, and the smoking subject is excluded from having received or being receiving nicotine-related replacement therapy.

2. Use of Lactobacillus fermentum GKF3 according to claim 1 for the preparation of an oral composition for reducing the craving for tobacco, characterized in that, wherein the Lactobacillus fermentum GKF3 is a freeze-dried powder obtained by performing a solid culture step, a fermentation step, and freeze-drying.

3. Use of Lactobacillus fermentum GKF3 according to claim 2 for the preparation of an oral composition for reducing the craving for tobacco, characterized in that, wherein the solid culture step is performed at a temperature of 37°C.

4. Use of Lactobacillus fermentum GKF3 according to claim 2 for the preparation of an oral composition for reducing the craving for tobacco, characterized in that, wherein the solid culture step is performed using MRS agar medium.

5. Use of Lactobacillus fermentum GKF3 according to claim 2 for the preparation of an oral composition for reducing the craving for tobacco, characterized in that, wherein the fermentation step is performed at a temperature of 37°C, with a carbon dioxide aeration amount of 0.5vvm, and a shaking rate of 500rpm.

6. Use of Lactobacillus fermentum GKF3 according to claim 2 for the preparation of an oral composition for reducing the craving for tobacco, characterized in that, wherein the fermentation step is performed using a fermentation substrate, and the fermentation substrate comprises 1-10 wt% glucose, 0.1-5 wt% yeast extract, 0.1-5 wt% peptone, 0.01-2% trace elements, 0.01-0.1% cysteine, and 0.05-1% TWEEN-80.

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