Patent Application
20240374660
The present disclosure relates to a B. longum CCFM1206 capable of producing sulforaphane and relieving inflammation, and belongs to the field of microbial technology.
Sulforaphane, with the molecular formula C6H11NOS2, is an isothiocyanate that is a secondary metabolite of thioglucoside (primarily glucoraphanin) found in cruciferous plants. However, the sulforaphane is not normally found in natural plants, but is stably found in the plant in the form of its precursor glucoraphanin. Only when the plant tissue is damaged does the hydrolysis of glucoraphanin with myrosinase result in the production of sulforaphane. The hydrolysis process is affected by a variety of factors such as pH, temperature, and moisture, which can lead to a decrease in yield. In addition, sulforaphane is unstable and extremely volatile, making it difficult to extract sulforaphane from natural plants.
Cruciferous vegetables such as broccoli, cabbage, and kale have been reported to be rich in glucoraphanin. However, after cooking treatments such as boiling and stir-frying, the plant-derived myrosinase will be inactivated due to heat and will no longer have the ability to hydrolyze glucoraphanin. Although the human intestinal microbiota also has the ability to convert glucoraphanin into sulforaphane, there are individual differences in the conversion of glucoraphanin. The results of a population-based experiment showed that the conversion rates of glucoraphanin in volunteers ranged from 1.1% to 40%, but the average conversion rate was only 10.4%-11.8%. Therefore, improving the metabolism of glucoraphanin by intestinal microbiota is conducive to the absorption and efficacy of sulforaphane.
Inflammation is often the cause of many diseases, and Sturm and Wagner have directly linked inflammatory states to the risk of cancer development. The sulforaphane is among the isothiocyanates with the most potent chemopreventive effect on inflammation and good anti-inflammatory properties. Studies have shown that the sulforaphane is an inducer of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which exerts antioxidant effects by upregulating antioxidant enzymes, such as quinone oxidoreductase-1 (NQO1) and superoxide dismutase (SOD), through activation of the Nrf2 signaling pathway. In addition, sulforaphane can inhibit the binding of redox-sensitive DNA and the trans-activation of NF-κB by interacting with sulfhydryl groups through the formation of dithiocarbamate, thus suppressing the inflammatory response.
Currently, there are no bacterial strains capable of effectively converting glucoraphanin to sulforaphane with full anti-inflammatory activity. Therefore, it is desirable to screen for a strain capable of bioconverting and producing sulforaphane and that the synergistic effect of sulforaphane and the strain is superior to the effect when they are present alone.
The technical problem to be solved by the present disclosure is to provide a strain of B. longum CCFM1206 capable of producing sulforaphane and to prevent and relive inflammation with B. longum CCFM1206 alone or in combination with glucoraphanin (or a composition containing glucoraphanin).
The present disclosure provides a B. longum CCFM1206, which was deposited at Guangdong Microbial Culture Collection Center on Dec. 15, 2021, with a deposit number of GDMCC NO: 62129.
The B. longum CCFM1206 has the following characteristics:
The present disclosure also provides a probiotic preparation including the B. longum CCFM1206.
In one embodiment, the B. longum CCFM1206 is present in the probiotic preparation in a level of ≥1×106 CFU/g or 1×106 CFU/mL.
In one embodiment, the probiotic preparation further contains glucoraphanin.
In one embodiment, the probiotic preparation further contains a broccoli seed aqueous extract, where the level of glucoraphanin is ≥40 mg/g.
In one embodiment, the probiotic preparation is a lyophilized powder prepared from a bacterial solution of the B. longum CCFM1206, where the active B. longum CCFM1206 is present in a level of 1.0×106 cfu/g or more.
In one embodiment, the probiotic preparation is prepared by inoculating the B. longum CCFM1206 at an inoculum size of 2%-4% into an MRS medium, anaerobically incubating at 37° C. for 24 h, and centrifuging to collect the bacterium, which is rinsed with a phosphate buffer at pH 7.0-7.2 for 2-4 times and resuspended with a protective agent to reach a concentration of 1010 cfu/mL; then incubating the suspension at 37° C. under an anaerobic condition for 1 h and lyophilizing to obtain the probiotic preparation.
In one embodiment, the protective agent contains 100 g/L skimmed milk powder, 30 mL/L glycerol, 100 g/L maltodextrin, 150 g/L trehalose, and 10 g/L sodium L-glutamate.
The present disclosure also provides a method for obtaining sulforaphane by biotransforming, including inoculating B. longum CCFM1206 into a fermentation medium and incubating for at least 24 h, where glucoraphanin is used as a carbon source in the fermentation medium.
In one embodiment, the fermentation medium contains: 10 g/L peptone, 10 g/L beef extract, 10 g/L broccoli seed aqueous extract, 2 g/L anhydrous sodium acetate, 5 g/L yeast powder, 2 g/L diammonium hydrogen citrate, 2.6 g/L K2HPO4·3H2O, 0.1 g/L MgSO4·7H2O, 0.05 g/L MnSO4, 1 ml/L Tween 80, and 0.5 g/L cysteine hydrochloride at pH=6.8.
In one embodiment, the level of the broccoli seed aqueous extract is 10 g/L.
The present disclosure also provides a method for preventing and/or ameliorating the symptoms of ulcerative colitis (UC) and/or systemic inflammation, including using the B. longum CCFM1206 alone or in combination with glucoraphanin as a medication.
In one embodiment, the medication further includes a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient means any diluent, adjuvant and/or carrier that can be used in the pharmaceutical field.
The present disclosure also provides a food product, nutraceutical, dietary supplement or medication containing the B. longum CCFM1206.
In one embodiment, B. longum CCFM1206 is present in the food product, nutraceutical and dietary supplement in a level of not less than 1.0×106 CFU/mL or 1.0×106 CFU/g.
In one embodiment, the food product, nutraceutical and dietary supplement further contains a composition containing glucoraphanin.
In one embodiment, the composition containing glucoraphanin means a vegetable or vegetable extract containing glucoraphanin; the vegetable includes, but is not limited to, one or a mixture of more than one of broccoli, cabbage, kale, and mustard.
In one embodiment, the fermented food product includes a fermented cow's milk and milk beverage as well as a fermented fruit and vegetable product; the fruit and vegetable product includes a juice beverage, a fruit and vegetable puree, and a kimchi made from broccoli, cabbage, and the like.
In one embodiment, the amelioration of the symptoms of ulcerative colitis includes, but is not limited to, the following:
In one embodiment, the pro-inflammatory factors in colon include TNF-α, IL-6 and IL-1β.
In one embodiment, the tight junction proteins in colon include Claudin-1, Occudin and ZO-1.
In one embodiment, the amelioration of the symptoms of LPS-induced systemic inflammation includes, but is not limited to, the following:
In one embodiment, the pro-inflammatory factors in serum include TNF-α, IL-6 and IL-1B.
In one embodiment, the pro-inflammatory factors in liver include TNF-α and IL-6 and the anti-inflammatory factor in liver includes IL-10.
In one embodiment, the short-chain fatty acids include acetic acid, propionic acid, and butyric acid.
Beneficial effects:
A B. longum CCFM1206 having the taxonomic name of B. longum was deposited at Guangdong Microbial Culture Collection Center on Dec. 15, 2021 with a deposit number of GDMCC NO: 62129, and the deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.
The 6-week-old SPF male C57BL/6J mice involved in the following examples were purchased from Vital River Laboratory Animal Technology Co., Ltd. The dextran sulfate sodium salt (DSS) and lipopolysaccharide (LPS) involved in the following examples were purchased from Sigma in Shanghai. The broccoli seed aqueous extract was purchased from Ganzhou Huahan Biotechnology Co., Ltd. and the level of glucoraphanin per gram of broccoli seed aqueous extract was 20% by mass. The ELISA kit involved in the following examples was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. Other reagents involved in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd.
MRS solid medium: 10 g/L peptone, 10 g/L beef extract, 20 g/L glucose, 2 g/L sodium acetate, 5 g/L yeast powder, 2 g/L diammonium hydrogen citrate, 2.6 g/L K2HPO4·3H2O, 0.1 g/L MgSO4·7 H2O, 0.05 g/L MnSO4, 1 mL/L Tween 80, 20 g/L agar, and 0.5 g/L cysteine hydrochloride.
MRS liquid medium: 10 g/L peptone, 10 g/L beef extract, 20 g/L glucose, 2 g/L sodium acetate, 5 g/L yeast powder, 2 g/L diammonium hydrogen citrate, 2.6 g/L K2HPO4·3H2O, 0.1 g/L MgSO4·7 H2O, 0.05 g/L MnSO4, 1 mL/L Tween 80, and 0.5 g/L cysteine hydrochloride.
Modified MRS solid medium: 10 g/L peptone, 10 g/L beef extract, 10 g/L broccoli seed aqueous extract, 2 g/L anhydrous sodium acetate, 5 g/L yeast powder, 2 g/L diammonium hydrogen citrate, 2.6 g/L K2HPO4·3H2O, 0.1 g/L MgSO4·7H2O, 0.05 g/L MnSO4, 1 ml/L Tween 80, 20 g/L agar, and 0.5 g/L cysteine hydrochloride.
Modified MRS liquid medium: 10 g/L peptone, 10 g/L beef extract, 10 g/L broccoli seed aqueous extract, 2 g/L anhydrous sodium acetate, 5 g/L yeast powder, 2 g/L diammonium hydrogen citrate, 2.6 g/L K2HPO4·3H2O, 0.1 g/L MgSO4·7H2O, 0.05 g/L MnSO4, 1 ml/L Tween 80, and 0.5 g/L cysteine hydrochloride.
Detection method of sulforaphane: qualitative and quantitative analyses were performed using UPLC-Q Exactive quadrupole-electrostatic field orbit trap high-resolution mass spectrometer (Thermo Fisher Scientific, Inc., USA) by parallel-reaction-monitoring (PRM). The chromatographic column was a Waters HSS T3 column (1.8 μm×2.1 mm×100 mm); the column temperature was 35° C.; the mobile phases were as follows: A-0.1% formic acid in water and B-acetonitrile; the flow rate was 0.3 mL/min; the injection volume was 2 μL; and the gradient elution was as follows: 0-3.0 min 5% B, 3-9 min 5%-30% B, 9-15 min 30%-100% B, 15-16 min 100% B, 16-16.5 min 100%-5% B, and 16.5-20 min 5% B. The ion source was a HESI source (heated ESI); the spray voltages were 3.5 kV (+) and 3.2 kV (−); the sheath gas volumetric flow rate was 35 μl min−1; the ion transfer tube temperature was 320° C.; the auxiliary gas flow rate was 15 μL-min−1; and the auxiliary gas temperature was 320° C. The scanning mode was PRM (100-500 m/z); the resolution was 35000; the acquisition polarity was positive; the AGC target was 5e 5; and the Maximum IT was 100 ms.
Determination of short-chain fatty acid level: Trace 1300 GC-MS (Thermo Fisher Scientific, Inc., USA) was used for the determination. The chromatographic column was a Rtx Wax column (30 m long, 25 μm inner diameter); the carrier gas was helium at a flow rate of 2 mL/min; the injection volume was 1 μL, and the sample was heated up to 140° C. at 7.5° C./min, and then heated up to 200° C. at 60° C./min for 3 min, with the ionization temperature of 20° C. The analysis was carried out in a full scan mode in order to make a standard curve by external standardization, thus calculating the concentration of each short-chain fatty acid.
0.5 g of fresh fecal sample from a healthy adult was added to a 4.5 ml of 0.9% normal saline for gradient dilution, an appropriate gradient dilution was selected and plated in a modified MRS solid medium with 0.2% bromocresol violet, and incubated under an anaerobic condition at 37° C. for 24-48 h. A single colony whose discoloration circle turned yellow obviously was selected and inoculated onto MRS plates for streaking purification. The single colony was picked and transferred to an MRS liquid medium for bacterial enrichment and preserved in 30% glycerol to obtain a strain of B. longum CCFM1206.
The whole genomic DNA of strain CCFM1206 was extracted for 16S rDNA amplification, and the amplified DNA fragments were collected for sequencing. The sequencing was accomplished by Suzhou GENEWIZ Biotechnology Co., Ltd. The sequence was subjected to a nucleic acid sequence alignment in NCBI, which showed that the strain was B. longum and was named as B. longum CCFM1206.
The bacterial solution of B. longum CCFM1206 was picked through dipping and streaked on an MRS solid medium, and anaerobically incubated at 37° C. for 48 h. After which, the colonies were observed and found to be round, white and smooth (
The single colony of B. longum CCFM1206 was picked and inoculated into an MRS liquid medium, and anaerobically incubated at 37° C. for 24 h to obtain the bacterial solution. 1 ml of bacterial solution was charged in a sterile centrifuge tube and centrifuged at 8000 r/min for 3 min, then the upper layer of medium was discarded, and the bacterial sludge was resuspended into a 30% glycerol solution and preserved at −80° C.
B. longum CCFM1206 preserved at −80° C. was streaked in an MRS solid medium, anaerobically incubated at 37° C. for 24-48 h, passaged with an MRS liquid medium for 2-3 times, then inoculated into a modified MRS liquid medium at an inoculum size of 2%-4% and anaerobically incubated at 37° C. for 24 h. The fermentation broth obtained was used for determining the sulforaphane level.
The results are shown in
40 healthy male C57BL/6J mice aged 6 weeks were acclimatized for one week, and then randomly divided into 5 groups, 8 mice per group. The 5 groups were as follows: a control group, a model group, a glucoraphanin-containing diet group (BSE), a B. longum CCFM1206 group (CCFM1206), and a glucoraphanin-containing diet in combination with B. longum CCFM1206 group (BSE+CCFM1206).
The 14-day gavage intervention period was from day 8 to day 21, the dosage for each gavage was 0.2 mL/mouse, and the time for daily gavage was the same. The control and model groups were gavaged with a normal saline, the BSE group was gavaged with a 40 mg/ml solution of broccoli seed aqueous extract, the CCFM1206 group was gavaged with a 5×109 CFU/ml bacterial suspension, and the BSE+CCFM1206 group was gavaged with a mixture containing a 40 mg/ml broccoli seed aqueous extract and a 5×109 CFU/mL bacterial suspension.
Day 15 to day 21 were the last 7 days of the intervention period for ulcerative colitis modeling. DSS was added to the drinking water at a concentration of 2.5% (w/v). Mice were sacrificed on day 22, and serum, tissues and the like were collected for relevant index measurements. The experimental animals were grouped and treated as shown in Table 1.
B. longum CCFM1206
In the modeling period (DSS treatment period), mice were weighed regularly every day and the percentage change in body weight was calculated. After sacrificing the mice, the length of the mouse colon was measured and the average colon length was calculated for each group. The experimental results are shown in
C57BL/6J mice were grouped, modeled and treated as in Example 3.
Mice were sacrificed on day 22, and mouse colon tissues were collected to make paraffin sections of mouse colon. The specific experimental steps were as follows: a 1 cm segment of distal colon 1 cm from the anus was taken and fixed with 4% paraformaldehyde for 48 h. The fixed colon tissues were rinsed with running water for 8 h, then sequentially added into 70%, 80%, and 90% ethanol solutions for dehydration, 30 min per dehydration, and then added into 95% and 100% ethanol solution, 20 min per addition. The colon samples were added in a 1:1 mixture of xylene and alcohol for 15 min, followed by xylene I and xylene II for 15 min, respectively. The colon tissues were transferred to a half-and-half mixture of xylene and paraffin for 15 min, and then added in paraffin I and paraffin II for permeation for 1 h, respectively, during which the temperature was kept at 60° C. The colon was embedded in the re-melted wax block using a Leica paraffin embedding machine, and the embedded tissues were sectioned with a tissue slicer at a thickness of 5 μm. The sections were adhered, then air-dried, and added in an oven at 62° C. for 1 h.
After the paraffin sections were made, HE staining was carried out. The specific experimental steps were as follows: paraffin sections were deparaffinized by xylene I and II for 5 min, respectively, then sequentially added into 100%, 95%, 90%, 80%, and 70% ethanol solution for 3-5 min, respectively, and finally added into distilled water for 3 min. The staining was carried out with hematoxylin for 20 s, and the unbound hematoxylin was washed away with distilled water. Then the sections were stained with eosin for 2 s, sequentially added into 95% ethanol I and II and 70% ethanol and removed quickly, then added into 80% ethanol for 50-55 s, and added into anhydrous ethanol for 2 min. Sections were added into a 1:1 mixture of xylene and alcohol for 1 min, followed by xylene I and II for 2-3 min, respectively, and then sealed with neutral gum.
The results are shown in
C57BL/6J mice were grouped, modeled and treated as in Example 3.
Mice were sacrificed on day 22 and mouse colon tissue was collected. Mouse colon tissues were added to a pre-cooled PBS buffer at a ratio of 1:9 for tissue grinding and centrifuged at 12,000 g for 15 min. The supernatant was removed and the levels of TNF-α, IL-1β and IL-6 in the colon were determined according to the detection method of TNF-α, IL-1β and IL-6 ELISA kit, respectively.
The experimental results are shown in
C57BL/6J mice were grouped, modeled and treated as in Example 3.
Mice were sacrificed on day 22, and mouse colon tissues were collected to determine the transcription levels of tight junction-associated proteins Claudin-1, Occudin, and ZO-1 in the colon.
The assay was performed as follows. The primer sequences for Claudin-1, Occudin, ZO-1 and GAPDH were synthesized, and the primer information is shown in Table 3. 1 cm of colon tissue from the same part of the mouse was taken, quickly added into liquid nitrogen, frozen and stored in the refrigerator at −80° C. The frozen colon tissue was then added into a 1.5 ml enzyme-free centrifugal tube with 1 mL of TRIzol and 3 zirconium beads, homogenized thoroughly by using a tissue homogenizer, and allowed to stand at room temperature for 5 min. 0.2 mL of chloroform was added, shaken vigorously for 30 s, and allowed to stand for 10 min. Centrifugation was then performed at 12000 g for 15 min at 4° C. The upper aqueous phase was carefully pipetted into a 1.5 mL new enzyme-free centrifuge tube, and an equal volume of isopropanol was added, which were mixed by gently inverting up and down and allowed to stand at room temperature for 10 min. Centrifugation was then performed at 12000 g for 15 min at 4° C. The supernatant was discarded, 1 mL of a pre-cooled 75% ethanol was added, and the precipitate was washed by flicking. Centrifugation was performed at 12000 g for 5 min at 4° C. The supernatant was carefully aspirated and discarded, and the precipitate was blown dry on an ultra-clean bench. 50 μl of enzyme-free ultrapure water was added to dissolve the RNA. The concentration of extracted RNA was determined using a micro-spectrophotometer, and OD260/OD280 ranged from 1.9 to 2.0 was of acceptable quality. The extracted total RNA of acceptable quality was used as a template, and cDNA was synthesized according to the steps of the instruction for the reverse transcription kit. cDNA obtained by reverse transcription was subjected to qRT-PCR, and the PCR system was as follows: 5 μL of SYBR Green Supermix, 3 μl of deionized water, 0.5 μL of upstream primer (10 μmol/L), 0.5 μl of downstream primer (10 μmol/L), and 1 μL of cDNA template (100 ng/μL). qPCR program was set as follows: 94° C. for 2 min, 39 cycles (94° C. for 30 s; 61° C. for 30 s; 72° C. for 20 s). The target genes were detected by real-time PCR, and then a relative gene expression analysis was performed using GAPDH as the internal reference gene by a 2−ΔΔCT method.
The experimental results are shown in
40 healthy male C57BL/6J mice aged 6 weeks were acclimatized for one week, and then randomly divided into 5 groups, 8 mice per group. The 5 groups were as follows: a control group, a model group, a glucoraphanin-containing diet group (BSE), a B. longum CCFM1206 group (CCFM1206), and a B. longum CCFM1206 in combination with glucoraphanin-containing diet composition group (BSE+CCFM1206).
The 14-day gavage intervention period was from day 8 to day 21, the dosage for each gavage was 0.2 mL/mouse, and the time for daily gavage was the same. Among which, the control and model groups were gavaged with a normal saline, the BSE group was gavaged with a 40 mg/ml solution of broccoli seed aqueous extract, the CCFM1206 group was gavaged with a 5×109 CFU/mL bacterial suspension, and the BSE+CCFM1206 group was gavaged with a mixture containing a 5×109 CFU/mL bacterial suspension and a 40 mg/ml broccoli seed aqueous extract.
The mice were injected intraperitoneally on day 22 with 0.9% normal saline in the control group and 6 mg/kg LPS in the other groups. The mice were weighed after 4 h. The mice were sacrificed, and serum, tissues and the like were collected for the determination of the relevant indexes. The experimental animal grouping and treatments are shown in Table 3.
LPS-induced systemic inflammation causes splenomegaly in mice. The spleens of mice were weighed and the spleen index was calculated as follows:
The experimental results are shown in
C57BL/6J mice were grouped, modeled and treated as in Example 7.
On day 22, the plasma was collected from eyeballs in mice and centrifuged at 3500 g for 15 min, and the supernatant was taken to obtain serum. The levels of pro-inflammatory factors in serum were determined according to the detection method of TNF-α, IL-1β and IL-6 ELISA kit, respectively.
The experimental results are shown in
C57BL/6J mice were grouped, modeled and treated as in Example 7.
On day 22, the mice were sacrificed and then dissected to obtain the livers. Mouse liver tissues were added to a pre-cooled PBS buffer at a ratio of 1:9 for tissue grinding and centrifuged at 12,000 g for 15 min. The supernatants were removed and the levels of TNF-α, IL-6 and IL-10 in the colon were determined according to the detection method of TNF-α, IL-6 and IL-10 ELISA kit, respectively.
The experimental results are shown in
C57BL/6J mice were grouped, modeled and treated as in Example 7.
The mice were sacrificed on day 22, and the contents of the mouse colon were collected, frozen and stored at −80° C. Firstly, the feces were lyophilized. 50 mg of feces was weighed, resuspended in 500 μL of saturated NaCl solution, and acidified by adding 20 μl of 10% H2SO4. 800 μL of anhydrous ether was added and shaken well. The fatty acids were extracted and then centrifuged at 13,000 g for 15 min. The upper ether phase was removed, dried by adding 0.25 g of anhydrous Na2SO4, shaken and mixed well for 30 s, and then centrifuged at 13,000 g for 10 min. The upper ether phase was removed, and the levels of short-chain fatty acids acetic acid, propionic acid, and butyric acid in the lyophilized feces of mice were determined by GC-MS.
The experimental results are shown in
C57BL/6J mice were grouped, modeled and treated as in Examples 3 and 7.
The mice were sacrificed on day 22, and then the contents of mouse colon were collected, frozen and stored at −80° C. 100 mg of the contents were weighed and 800 μl of methanol were added to precipitate the protein. 2-3 zirconium beads were added, ground at 60 Hz for 5 min, and centrifuged at 13,000 g for 15 min (4° C.). 400 μL of supernatant was removed, evaporated to dryness, then reconstituted by adding 200 μl of methanol-water (4:1), and centrifuged at 13,000 g for 15 min (4° C.). The supernatant was passed through a 0.22 μm filter membrane and then assayed.
The experimental results are shown in
24 healthy male C57BL/6J mice aged 6 weeks were acclimatized for one week, and then randomly divided into 3 groups, 8 mice per group. The 3 groups were as follows: a normal diet group, a glucoraphanin-containing diet feed group, and a glucoraphanin-containing diet feed plus gavage of B. longum CCFM1206 group (combination group).
The glucoraphanin-containing diet feed was a normal commercially available mouse feed additionally added with a lyophilized powder of glucoraphanin-containing vegetables or a glucoraphanin-containing vegetable extract with 0.6 mg of glucoraphanin per gram of feed. The vegetables include, but are not limited to, mixtures of vegetables such as broccoli, cabbage, and kale.
The 7-day gavage intervention period was from day 8 to day 14, the dosage for each gavage was 0.2 mL/mouse, the time for daily gavage was the same, and the feed intake in mice was about 3 g/day. Among which, the normal diet and glucoraphanin-containing diet feed groups were gavaged with a normal saline, and the combination group was gavaged with a bacterial suspension containing 5×109 CFU/mL B. longum CCFM1206. Mice were sacrificed on day 15, and the contents of the mouse colon were collected, frozen and stored at −80° C. 100 mg of the contents were weighed and 800 μl of methanol were added to precipitate the protein. 2-3 zirconium beads were added, ground at 60 Hz for 5 min, and centrifuged at 13,000 g for 15 min (4° C.). 400 μl of supernatant was removed, evaporated to dryness, then reconstituted by adding 200 μL of methanol-water (4:1), and centrifuged at 13,000 g for 15 min (4° C.). The supernatant was passed through a 0.22 μm filter membrane and then assayed for the levels of sulforaphane. The experimental animals were grouped and treated as shown in Table 4.
The experimental results are shown in
The medium was prepared as follows: 10 g/L peptone, 10 g/L beef extract, 20 g/L glucose, 2 g/L sodium acetate, 5 g/L yeast powder, 2 g/L diammonium hydrogen citrate, 2.6 g/L K2HPO4·3H2O, 0.1 g/L MgSO4·7H2O, 0.05 g/L MnSO4, 1 mL/L Tween 80, and 0.5 g/L cysteine hydrochloride at pH 6.8.
Preparation of lyophilized protective agent: a protective agent containing 100 g/L skimmed milk powder, 30 mL/L glycerol, 100 g/L maltodextrin, 150 g/L trehalose, and 10 g/L sodium L-glutamate was prepared by mixing water and the ingredients of protective agent.
B. longum CCFM1206 was inoculated at an inoculum size of 2%-4% into the MRS medium, anaerobically incubated at 37° C. for 24 h, and centrifuged to collect the bacterium, which was rinsed with a phosphate buffer at pH=7.0-7.2 for 2-4 times and resuspended with the protective agent to reach a concentration of 1010 cfu/mL. After which, the suspension was incubated at 37° C. under an anaerobic condition for 1 h and then lyophilized to obtain the preparation of B. longum CCFM1206.
Optionally, the preparation is mixed with broccoli seed aqueous extract to ensure that the number of live bacteria of B. longum CCFM1206 in the composition is not less than 1.0×106 CFU/mL or 1.0×106 CFU/g, and the level of broccoli seed aqueous extract is not less than 200 mg/g.
Optionally, a dietary supplement may be prepared by combining the preparation of B. longum CCFM1206 with a vegetable or vegetable extract containing glucoraphanin. The vegetable includes, but is not limited to, one or a mixture of more than one of broccoli, cabbage, kale, and mustard.
Optionally, a functional preparation, a fermented food, or a pharmaceutical composition can also be prepared using B. longum CCFM1206.
Although the present disclosure has been provided as above in preferred examples, they are not intended to limit the present disclosure. Various changes and modifications can be made by any person familiar with the technology without departing from the spirit and scope according to the present disclosure, and thus the protection scope according to the present disclosure should be based on that defined in the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022104047266 | Apr 2022 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2023/085418 | Mar 2023 | WO |
| Child | 18780491 | US |
