Ganoderma lucidum spores capable of carrying lactic acid bacteria and a preparation method thereof

Abstract

The present invention relates to Ganoderma lucidum spores capable of loading lactic acid bacteria and a preparation method thereof. It can efficiently load lactic acid bacteria. The method of the present invention includes Step 1: adding lipopeptide iturin A into deionized water, stirring until completely dissolved, and adding the uniformly dissolved iturin A into Ganoderma lucidum spores, mixing uniformly to obtain suspension A; Step 2: placing the suspension A obtained in Step 1 in a shaker and continuously shaking; Step 3: centrifuging the shaken suspension A obtained in Step 2, washing it three times alternately with ethanol and deionized water, removing the supernatant, and then freeze-drying to obtain Ganoderma lucidum spores treated with iturin A; Step 4: placing the Ganoderma lucidum spores treated with iturin A obtained in Step 3 in an HCl solution, stirring evenly, and placing it in a shaker and continuously shaking to obtain suspension B; Step 5: centrifuging the suspension B obtained in Step 4, washing it three times alternately with ethanol and deionized water, removing the supernatant, and then freeze-drying to obtain Ganoderma lucidum spores jointly treated with iturin A and HCl.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of carriers loaded with probiotics by Ganoderma lucidum spores, and particularly relates to a Ganoderma lucidum spore capable of loading lactic acid bacteria and a preparation method thereof. Background Art

[0002] Probiotics play an important role in maintaining human health and even assisting in the treatment of diseases. Lactic acid bacteria are a type of probiotics with significant efficacy and wide use. However, whether the lactic acid bacteria entering the body can exert their efficacy depends to a large extent on how many live lactic acid bacteria can colonize and survive in the intestine. Although methods such as encapsulation and capsule shell coating have been developed to reduce the loss of lactic acid bacteria activity when passing through the gastric juice, there are few related studies on transporting lactic acid bacteria to the intestinal mucosal surface and enabling them to effectively colonize.

[0003] Carriers are an important means for people to target the transport of drugs. However, there are very few reports on carriers for loading lactic acid bacteria. This is mainly because most carriers load drugs through the adsorption of porous structures, but the bacterial cells are much larger than these pore sizes and cannot effectively enter the interior of the carrier, thus unable to achieve effective loading.

[0004] With the increasing understanding of the active ingredients in Ganoderma lucidum spores, more and more Ganoderma lucidum spores are used to extract their fat-soluble substances or water-soluble polysaccharides. A large amount of the remaining, broken spore shells are not fully utilized, resulting in waste.

[0005] Although some researchers use modified Ganoderma lucidum spores to adsorb pollutants (more commonly dye molecules) in sewage, the main mechanism is that these pollutant molecules can enter the small pores of Ganoderma lucidum spores. However, the volume of lactic acid bacteria is much larger than these molecules and cannot enter the pore diameter. Therefore, no one has studied how to use Ganoderma lucidum spores to load lactic acid bacteria. Summary of the Invention

[0006] In view of this, the present invention provides a Ganoderma lucidum spore capable of loading lactic acid bacteria and a preparation method thereof, which can efficiently load lactic acid bacteria.

[0007] To solve the problems existing in the prior art, the technical solution of the present invention is: a preparation method of a Ganoderma lucidum spore capable of loading lactic acid bacteria, characterized in that:

[0008] The method comprises the following steps:

[0009] Step 1: Add lipopeptide iturin A to deionized water, stir until completely dissolved, and add the uniformly dissolved iturin A to Ganoderma lucidum spores (bGLS), mix evenly to obtain suspension A;

[0010] Step 2: Place the suspension A obtained in Step 1 in a shaker and shake continuously.

[0011] Step 3: Centrifuge the shaken suspension A obtained in Step 2, wash it three times alternately with ethanol and deionized water, remove the supernatant, and then freeze-dry to obtain Ganoderma lucidum spores treated with iturin A.

[0012] Step 4: Place the Ganoderma lucidum spores treated with iturin A obtained in Step 3 in an HCl solution, stir evenly, and place it in a shaker and shake continuously to obtain suspension B.

[0013] Step 5: Centrifuge the suspension B obtained in Step 4, wash it three times alternately with ethanol and deionized water, remove the supernatant, and then freeze-dry to obtain Ganoderma lucidum spores treated with a combination of iturin A and HCl, which are the Ganoderma lucidum spores that can effectively load lactic acid bacteria.

[0014] Furthermore, in Step 2, the shaking temperature of the shaker is 4 - 50 °C, and it is shaken at 50 - 300 rpm for 4 - 24 hours.

[0015] Furthermore, in Step 3, the suspension A is centrifuged at a rotational speed of 4000 - 10000 × g for 1 - 20 minutes, and the freeze-drying is carried out in a vacuum freeze-dryer.

[0016] Furthermore, in Step 4, the HCl concentration is 4 - 8 M, the shaker temperature is 4 - 50 °C, and it is shaken at 50 - 300 rpm for 4 - 24 hours.

[0017] Furthermore, in Step 5, the suspension B is centrifuged at a rotational speed of 4000 - 10000 × g for 1 - 20 minutes, and the freeze-drying is carried out in a vacuum freeze-dryer.

[0018] The Ganoderma lucidum spores prepared by the above preparation method.

[0019] Compared with the prior art, the advantages of the present invention are as follows:

[0020] 1) Based on the natural pore size of the broken Ganoderma lucidum spores, the present invention increases the number and pore size of the small holes on its surface and changes its surface hydrophilicity through technical treatment, so that it can effectively adsorb lactic acid bacteria.

[0021] 2) The present invention utilizes the broken Ganoderma lucidum spore shells from the waste materials, improving the comprehensive utilization of the waste materials and conforming to the concept of sustainable development.

[0022] 3) The materials used in the treatment method selected by the present invention are safe, the operation method is simple, and the steps are few.

[0023] 4) The carrier prepared by the present invention is safe and non-toxic.

[0024] 5) The carrier prepared by the present invention has a very high loading capacity for Lactobacillus rhamnosus.

[0025] 6) The carrier of the present invention can promote intestinal mucus secretion, which helps to target the delivery of lactic acid bacteria to the intestine and promote their intestinal colonization. Description of the Drawings

[0026] Figure 1 Hydrophilicity and hydrophobicity detection of Ganoderma lucidum spores obtained after treatment (represented by water contact angle); where: (a) untreated bGLS; (b) bGLS after treatment.

[0027] Figure 2 Weight changes in the 14-day acute oral toxicity evaluation of Ganoderma lucidum spores after treatment; where: (a) female mice; (b) male mice.

[0028] Figure 3 Weight changes in the 28-day oral toxicity evaluation of Ganoderma lucidum spores after treatment; where: (a) female mice; (b) male mice.

[0029] Figure 4 Results of PAS mucus staining and Alcian blue mucus staining of the gastric, small intestine, and colon tissues of mice after intragastric administration of Ganoderma lucidum spores after treatment.

[0030] Figure 5 Adsorption efficiency of Ganoderma lucidum spores after treatment on Lactobacillus rhamnosus.

[0031] Figure 6 Scanning electron micrographs of Ganoderma lucidum spores loaded with Lactobacillus rhamnosus after treatment; where: (a) untreated bGLS; (b) bGLS after treatment.

[0032] Figure 7 Retention time and distribution of Ganoderma lucidum spores labeled with FITC fluorescence in the intestine after intragastric administration to mice; where: (a) retention in the gastrointestinal tract at different time points; (b) fluorescence intensity of Ganoderma lucidum spores in the colon tissue; (c) statistical calculation of the fluorescence intensity of Ganoderma lucidum spores retained on the surface of the colon tissue.

[0033] Figure 8 Adsorption of Ganoderma lucidum spores after treatment in the mucus of gastrointestinal tissues; where: (a) adsorption of Ganoderma lucidum spores after treatment in the mucus of the stomach, small intestine, and colon; (b) adhesion of Ganoderma lucidum spores in gastrointestinal tissues indicated by rhodamine B fluorescence labeling. Detailed Embodiments

[0034] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Example:

[0036] The present invention relates to a method for preparing Ganoderma lucidum spores capable of loading lactic acid bacteria, comprising the following steps:

[0037] Step 1: 400 μg of lipopeptide iturin A is added to 20 mL of deionized water to make the final mass concentration of the lipopeptide iturin A solution 20 μg / mL, stirred until completely dissolved, and then 1 g of Ganoderma lucidum spores (bGLS) is added and mixed evenly. The final mass concentration of the Ganoderma lucidum spores is 50 mg / mL.

[0038] In Step 2, the evenly mixed iturin A and Ganoderma lucidum spore suspension is placed in a shaker and continuously shaken; the shaking temperature is 25°C, and shaken at 180 rpm for 12 hours.

[0039] In Step 3, the suspension obtained in Step 2 is centrifuged at a speed of 8000×g for 5 minutes at 4°C, the supernatant is removed to leave the precipitate, and the precipitate is washed three times alternately with ethanol and deionized water. Centrifuged in the same way to remove the supernatant and leave the precipitate. The freeze-drying operation is pre-frozen at -80°C for 2 hours and then freeze-dried in a vacuum freeze-dryer to obtain Ganoderma lucidum spores treated with iturin A.

[0040] In Step 4, 1 g of Ganoderma lucidum spores treated with iturin A obtained in Step 3 is added to 10 mL of HCl solution. The final mass concentration of the Ganoderma lucidum spores is 100 mg / mL, and the HCl concentration is 6 M. It is placed in a shaker and continuously shaken. The shaker temperature is 25°C, and shaken at 180 rpm for 12 hours.

[0041] In Step 5, the suspension obtained in Step 4 is centrifuged at a speed of 8000×g for 5 minutes at 4°C, the supernatant is removed to leave the precipitate, and the precipitate is washed three times alternately with ethanol and deionized water. Centrifuged in the same way to remove the supernatant and leave the precipitate. The freeze-drying operation is pre-frozen at -80°C for 2 hours and then freeze-dried in a vacuum freeze-dryer to obtain crushed Ganoderma lucidum spores jointly treated with iturin A and HCl, that is, a kind of Ganoderma lucidum spores capable of loading lactic acid bacteria.

[0042] The Ganoderma lucidum spores are purchased from the market in Changbai Mountain, Jilin Province, China. Iturin is prepared and purified from the culture supernatant of Bacillus subtilis CCTCC M207209. Hydrochloric acid (HCl, liquid) is purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

[0043] The Ganoderma lucidum spores obtained in the above example are subjected to performance testing as follows:

[0044] 1. Analysis of the hydrophilicity and hydrophobicity of Ganoderma lucidum spores

[0045] Measure the water contact angle of the treated Ganoderma lucidum spores, and the obtained results are asFigure 1 As shown. It can be seen that compared with the untreated Ganoderma lucidum spores ( Figure 1 a), the water contact angle of the treated Ganoderma lucidum spores ( Figure 1 b) increases significantly, indicating that its hydrophobicity increases and its hydrophilicity weakens.

[0046] 2. Edible safety evaluation experiment

[0047] According to the relevant experimental methods of GB 15193.3-2014 and GB15193.22-2014, acute oral toxicity test and 28-day oral toxicity test were carried out on mice. The breeding temperature was 20-23 °C, the management standard was clean grade, and the light / dark cycle was 12 h. The mice were fed with SPF-grade mouse food, with free drinking water and food intake. After 3 days of adaptive feeding, the mice were randomly divided into a control group and a treated bGLS group, and each group was divided into two cages according to gender (10 mice in each group).

[0048] Among them, the experimental method for acute oral safety evaluation was as follows: 5-week-old healthy Kunming mice with a body weight of 18-20 g were selected. One dose group of 15000 mg / (kg·bw) was set for the acute oral toxicity test. Oral gavage was performed at 20 mL / (kg·bw). The animals were fasted for 16 h before gavage, and the body weight was recorded after 14 days of exposure. The results are as Figure 2 shown. It can be seen that no individual death occurred in the mice during the 14-day observation period, and the body weight increased normally. After the experiment, the animals were sacrificed for dissection, and no visible tissue or organ abnormalities were found. This shows that the treated Ganoderma lucidum spores have an acute oral LD50 > 5000 mg / (kg·bw) for mice. According to the acute toxicity classification, the treated Ganoderma lucidum spores belong to the actual non-toxic level, that is, the food safety level.

[0049] The experimental method for 28-day oral safety evaluation was as follows: 6-week-old healthy Kunming mice were selected. Three dose groups of 500, 1000, and 2000 mg / (kg·bw) (equivalent to 25, 50, and 100 times the human recommended intake, respectively) and a control group (the control group was given an equal amount of sterile water by gavage to each mouse every day) were set for the 28-day feeding test. The body weight was recorded after 28 days of exposure. The results are as Figure 3 shown. The results show that after the mice were gavaged with Ganoderma lucidum spores for 28 days, their physical conditions were good, no individual death occurred at medium, high, and low doses, no poisoning phenomenon was found, and the body weight increased normally. After the experiment, the animals were sacrificed for dissection, and no visible abnormal damage was found in the tissues and organs, indicating that it has no chronic toxicity.

[0050] 3. Promote mucus secretion in gastrointestinal tissues

[0051] After 28 days of gavage, mice in the 500 mg / (kg·bw) group were sacrificed. After dissection, their gastric, small intestine, and colon tissues were taken to prepare sections, which were stained with PAS and Alcian blue respectively to evaluate the effect of Ganoderma lucidum spores after treatment on the changes in mucus in mouse tissues. The results are as follows Figure 4 shown. The figure shows that compared with the control group, after mice were fed Ganoderma lucidum spores after treatment, the PAS and Alcian blue staining of gastric, small intestine, and colon tissue sections was deeper, indicating a larger amount of mucus, suggesting that Ganoderma lucidum spores after treatment can promote mucus secretion in gastrointestinal tissues.

[0052] 4. Analysis of the Bacterial Loading Efficiency and Microscopic Structure Observation of Ganoderma lucidum Spores after Treatment

[0053] Using Lactobacillus rhamnosus CCTCC M2017839 as the lactic acid bacterium to be loaded, 100 μL of the strain preservation solution was taken, spread on MRS solid medium, and placed in an inverted position in a constant temperature incubator at 37 °C for 48 h. Single colonies of probiotics growing on MRS solid medium were picked and inoculated into 5 mL of MRS liquid medium, and then continued to be statically cultured in a constant temperature incubator at 37 °C for 48 h. According to the standard curve of the plate counting method counting results and the absorbance of the bacterial suspension at OD600nm, the cell concentration was calculated from the OD value. When the cell concentration reached 1×10 9 CFU / mL, the culture was stopped.

[0054] The above-mentioned bacterial suspension that reached the concentration requirement was centrifuged at 8000 r / min at 4 °C for 5 min, the cell precipitate was collected, washed three times with physiological saline, and then resuspended with physiological saline to a concentration of 1×10 9 CFU / mL for standby.

[0055] Take 5 mg of Ganoderma lucidum spore carriers in the control group, bGLS group, and bGLS group after treatment respectively, add them to 1 mL of 1×10 9 CFU / mL probiotic suspension respectively. After mixing evenly, let it stand and load at room temperature for 30 minutes. Then the solution was centrifuged at 1000×g for 1 minute, and the Ganoderma lucidum spore carriers loaded with bacteria were collected from the sediment. At the same time, the OD600nm value of the supernatant was measured, and according to the standard curve, the concentration of the unloaded bacteria remaining was measured.

[0056] Calculate the probiotic loading efficiency of the corresponding carrier according to the change in the cell concentration before and after the loading process. Use equation (1) to calculate the bacterial loading rate (log CFU / g) of Ganoderma lucidum spores:

[0057] Loading rate (log CFU / g) = (Initial concentration of the bacterial suspension - Concentration of the bacterial suspension after loading) / Mass of Ganoderma lucidum spores Equation (1)

[0058] After the experiment, the results obtained are as follows Figure 5 shown. It can be seen from this that the loading efficiency of the treated Ganoderma lucidum spores on Lactobacillus rhamnosus can reach 10 11 CFU / g, and the loading amount is increased by 10 times compared with the untreated spores.

[0059] When observing the Ganoderma lucidum spores loaded with Lactobacillus rhamnosus by scanning electron microscope, the results obtained are as follows Figure 6 shown. It can be seen from this that for the treated Ganoderma lucidum spores, the number and pore diameter of the surface pores increase, and the loaded bacterial cells are densely embedded in the Ganoderma lucidum spores, proving that the bacterial cells are indeed loaded by the Ganoderma lucidum spores.

[0060] 5. Retention time of the treated Ganoderma lucidum spores in the gastrointestinal tract of mice

[0061] Take 6-week-old healthy adult male Kunming mice, and intragastrically administer the FITC-labeled treated Ganoderma lucidum spores to the mice at a dose of 500 mg / (kg·bw). At 2, 4, 6, 8, 10, 12, 24, 30, 36, 48, 72, and 96 hours after intragastric administration, dissect the mice, take out the parts from the stomach to the anus, and observe the fluorescence distribution of the Ganoderma lucidum spores through a small animal in vivo imager. Take out the colon tissues at different time points and further dissect them to measure and calculate the fluorescence intensity of the treated bGLS adhering to the colon tissues.

[0062] The results obtained are as follows Figure 7 shown. It can be seen from this that the treated Ganoderma lucidum spores can remain in the body for more than 96 hours after intragastric administration, reach the colon at 6 hours after intragastric administration, and the treated Ganoderma lucidum spores can remain in the colon from 6 hours to 96 hours. Dissect the colon and measure the fluorescence intensity, and it is found that the most spores are aggregated in the colon at 48 hours after intragastric administration, and a strong fluorescence is still observed on the inner surface of the colon tissue at 96 hours, fully indicating that the treated Ganoderma lucidum spores can adhere and remain in the digestive tract of mice for a long time, thus contributing to the long-term colonization of probiotics.

[0063] 6. Adhesion of the treated Ganoderma lucidum spores to the gastrointestinal mucus layer

[0064] Evaluation of the adsorption of gastrointestinal mucus on processed Ganoderma lucidum spores: Six-week-old male healthy adult Kunming mice were sacrificed, and the gastric, small intestinal, and colonic tissues (2.5 cm long for small intestinal and colonic tissues) were removed. The tissues were placed on an ice plate, cut along the side of the tissues, and gently washed with PBS to remove the contents. The mucus substances on the inner surface of the tissues were scraped off with a blade. The scrapings were aspirated with a micro syringe, and after adding 0.1 mL of the scrapings to 5 mL of PBS and thoroughly mixing, they were centrifuged at 5000 r / min at low temperature for 30 min. Then, 1 mL of the supernatant was taken from each, and 5 mg of untreated bGLS and processed bGLS were added to each respectively. After incubation at 37 °C for 3 h, they were centrifuged at 1000 r / min at low temperature for 1 min to separate the spores adsorbed with mucus. The supernatant was left to incubate with 0.4 mg / mL of Alcian blue solution at 20 °C for 12 h, and then centrifuged at 2500 r / min for 10 min. The supernatant was taken, and the absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader at 615 nm to determine the adsorption of the spores themselves to mucus.

[0065] The adsorption rate of the corresponding spores to the mucus of different tissue sites was calculated based on the change in the amount of mucus before and after the adsorption process. It was calculated using equation (2):

[0066] Mucus adsorption rate (%) = (total mucus amount - unadsorbed mucus amount) / total mucus amount Equation (2)

[0067] Quantitative analysis of the adsorption of processed Ganoderma lucidum spores in gastrointestinal mucus: Six-week-old male healthy adult Kunming mice were sacrificed, and the gastric, small intestinal, and colonic tissues (2.5 cm long for small intestinal and colonic tissues) were removed. The gastric tissue was cut along the greater curvature of the stomach, and the ports of the small intestinal and colonic tissues were cut, and they were gently washed with PBS to remove the contents. 10 μL of Alexa Fluor 488-conjugated wheat germ agglutinin (WGA) was added to the gastric tissue, small intestinal tissue, and colon respectively to stain the mucin fibers. The gastric tissue, small intestine, and colon were sutured with surgical sutures. After incubation in PBS (pH 7.4) at 37 °C for 30 min, processed bGLS pre-stained with rhodamine B was injected. The tissues were incubated for 60 min and then collected and observed by confocal microscopy (Alexa Fluor 488-conjugated WGA excitation wavelength / emission wavelength = 488 / 520 nm; rhodamine B excitation wavelength / emission wavelength = 546 / 568 nm).

[0068] The results obtained were as Figure 8 shown. As can be seen from Figure 8 a, compared with the untreated spores, the adsorption rate of the processed Ganoderma lucidum spores in the mucus at the gastrointestinal tissue sites was greatly increased; Figure 8 b shows that the 3D layer scan results of the gastrointestinal mucus layer show that the processed Ganoderma lucidum spores can deeply adhere and embed into the mucus layers of the gastric, small intestinal, and colonic tissues, demonstrating the application potential of carrying and colonizing lactic acid bacteria here.

[0069] The above are only the preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements made thereto shall be regarded as within the protection scope of the present invention.

Claims

1. A preparation method of Ganoderma lucidum spores capable of loading lactic acid bacteria, characterized in that: the method comprises the following steps: Step 1: Add lipopeptide iturin A to deionized water, stir until completely dissolved, and add the uniformly dissolved iturin A to Ganoderma lucidum spores, mix evenly to obtain suspension A; Step 2: Place the suspension A obtained in Step 1 in a shaker and continuously shake; Step 3: Centrifuge the shaken suspension A obtained in Step 2, wash it three times alternately with ethanol and deionized water, remove the supernatant, and then freeze-dry to obtain Ganoderma lucidum spores treated with iturin A; Step 4: Place the Ganoderma lucidum spores treated with iturin A obtained in Step 3 in an HCl solution, stir evenly, and place it in a shaker and continuously shake to obtain suspension B; Step 5: Centrifuge the suspension B obtained in Step 4, wash it three times alternately with ethanol and deionized water, remove the supernatant, and then freeze-dry to obtain Ganoderma lucidum spores broken by combined treatment of iturin A and HCl, which are Ganoderma lucidum spores capable of effectively loading lactic acid bacteria; in Step 2, the shaker shaking temperature is 4 - 50 °C, and shake at 50 - 300 rpm for 4 - 24 hours; in Step 3, centrifuge the suspension A at a rotation speed of 4000 - 10000 × g for 1 - 20 minutes, and freeze-dry in a vacuum freeze dryer.

2. The preparation method of Ganoderma lucidum spores capable of loading lactic acid bacteria according to claim 1, characterized in that: in Step 4, the HCl concentration is 4 - 8 M, the shaker temperature is 4 - 50 °C, and shake at 50 - 300 rpm for 4 - 24 hours.

3. The preparation method of Ganoderma lucidum spores capable of loading lactic acid bacteria according to claim 1 or 2, characterized in that: in Step 5, centrifuge the suspension B at a rotation speed of 4000 - 10000 × g for 1 - 20 minutes, and freeze-dry in a vacuum freeze dryer.

4. Ganoderma lucidum spores prepared by the preparation method according to claim 1.

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