Ganoderma lucidum fermented beverage and preparation method thereof

Abstract

The invention relates to the technical field of microbial fermentation and food production, and particularly provides a ganoderma lucidum fermented beverage and a preparation method thereof. The ganoderma lucidum fermented beverage is sour and sweet in taste, has ganoderma lucidum fragrance, has the living bacteria quantity exceeding 10 < 11 > CFU / mL after leaving a factory, can still maintain the level of 10 < 10 > CFU / mL after being stored for 6 months at normal temperature, and is high in storage stability. The ganoderma lucidum fermented beverage is rich in functional components such as polysaccharides, triterpenes and sterols, has the effects of enhancing immunity, resisting oxidation and the like, and is wide in market prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation and food production, and specifically relates to a ganoderma fermented beverage and a preparation method thereof. Background Art

[0002] Ganoderma lucidum (Curtis) P.Karst.) is a fungus of the Ganoderma family and the genus Ganoderma. Most of the fruiting bodies of Ganoderma are annual, and a few are perennial. They have stalks, and the stalks are lateral. The cap is woody, corky, fan-shaped, with grooves, kidney-shaped, semicircular or nearly circular, with a brownish yellow or reddish brown surface, blood red to chestnut, and sometimes the edges gradually turn into light yellow brown to yellowish white, with a lacquer-like luster. There are concentric grooves on the cap surface, with sharp or slightly blunt edges, often involute. The flesh is white to light brown, often light brown near the tube, the tube is small, the tube pore surface is light white, white cinnamon, light brown to light yellow brown, the tube mouth is nearly circular, the stipe is lateral, eccentric or central, nearly cylindrical, and has a strong lacquer-like luster. The basidiospores are ovate or flat-topped, double-walled, the outer wall is transparent and smooth, the inner wall is brown or light brown, with small thorns, and an oil drop in the center.

[0004] Probiotics refer to living microorganisms that can have a beneficial effect on the host's health. They have the functions of promoting digestion, promoting nutrient absorption and maintaining intestinal health. Probiotics can produce a variety of organic acids, regulate the pH of the human intestine, and produce bacteriocins that can effectively inhibit the reproduction of a variety of harmful strains. They can also produce antioxidant enzymes, etc. Probiotic fermentation can further improve the nutritional value of Ganoderma lucidum and enhance its antioxidant effect, which is of great significance. Summary of the invention

[0005] The purpose of the present invention is to provide a fermented ganoderma drink and a preparation method thereof. The fermented ganoderma drink has a sweet and sour taste, has a ganoderma fragrance, is rich in effective ingredients such as polysaccharides, triterpenes and sterols, has the effects of enhancing immunity and anti-oxidation, and has broad market prospects.

[0006] In one aspect, the present invention provides a fermented ganoderma beverage comprising Lactobacillus rhamnosus and Bacillus coagulans.

[0007] The rhamnosus Lactobacillus is Lactobacillus rhamnosus YKSW, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on March 29, 2022, with a deposit number of CCTCC NO: M2022337.

[0008] The Bacillus coagulans is Bacillus coagulans KDB-18, which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on July 1, 2024, and its deposit number is CCTCC NO: M20241408.

[0009] The present invention also provides a method for preparing a fermented ganoderma beverage, comprising the following steps:

[0010] (1) mixing ganoderma lucidum powder and water in proportion, subjecting the mixture to ultrasonic cell wall breaking treatment, and adding protease for enzymolysis to obtain ganoderma lucidum liquid;

[0011] (2) preparing a fermentation medium containing Ganoderma lucidum liquid, sterilizing it, and cooling it;

[0012] (3) inoculating Lactobacillus rhamnosus seed liquid into the fermentation medium, culturing in a closed manner at 36-38° C. for 15-20 hours, then inoculating Bacillus coagulans seed liquid, and fermenting aerobically at 36-38° C. for 24-36 hours; after the fermentation is completed, cooling the fermentation liquid to below 10° C.;

[0013] (4) The fermented liquid is aseptically filled and packaged to obtain a Ganoderma lucidum fermented beverage.

[0014] Further preferably, the protease in step (1) is pepsin.

[0015] Further preferably, the fermentation medium in step (2) contains Ganoderma lucidum liquid, brown sugar, whey powder, glucose, MgSO4, NaCl, K2HPO4, and KH2PO4.

[0016] Further preferably, the components in the fermentation medium in step (2) and their mass volume ratios are: Ganoderma lucidum liquid 200-300 g / L, brown sugar 100-150 g / L, whey powder 20-50 g / L, glucose 20-45 g / L, MgSO4 0.2-0.5 g / L, NaCl 0.2-0.5 g / L, K2HPO4 0.2-0.4 g / L, KH2PO4 0.1-0.3 g / L.

[0017] Further preferably, the method for preparing the Ganoderma lucidum fermented beverage comprises the following steps:

[0018] (1) Ganoderma lucidum powder and water were mixed at a mass ratio of 1:50, subjected to ultrasonic cell wall breaking for 40 min, and the pH value was adjusted to 2.0±0.2; pepsin was added at a mass ratio of 5%, and after enzymatic hydrolysis at 37° C. for 3 h, the enzyme was inactivated to obtain Ganoderma lucidum liquid;

[0019] (2) Adding ganoderma lucidum liquid, brown sugar, whey powder, glucose, MgSO4, NaCl, K2HPO4, and KH2PO4 into a fermentation tank, stirring and dissolving, and then fixing the volume to obtain a fermentation medium, wherein the final mass volume ratio of each component is: ganoderma lucidum liquid 200-300 g / L, brown sugar 100-150 g / L, whey powder 20-50 g / L, glucose 20-45 g / L, MgSO4 0.2-0.5 g / L, NaCl 0.2-0.5 g / L, K2HPO4 0.2-0.4 g / L, KH2PO4 0.1-0.3 g / L; the initial pH value of the medium is 5.5-6.5; sterilizing by high temperature steam for 25 minutes, and cooling to 37-40°C after completion;

[0020] (3) inoculating Lactobacillus rhamnosus YKSW seed liquid into the fermentation medium at a volume ratio of 5-10%, culturing at 36-38° C. in a closed culture for 15-20 hours, then inoculating Bacillus coagulans KDB-18 seed liquid at a volume ratio of 5-10%, and fermenting aerobically at 36-38° C. for 24-36 hours; after the fermentation is completed, cooling the fermentation liquid to below 10° C.;

[0021] (4) The fermented liquid is aseptically filled and packaged to obtain a Ganoderma lucidum fermented beverage.

[0022] Further preferably, the method for preparing the Ganoderma lucidum fermented beverage comprises the following steps:

[0023] (1) Ganoderma lucidum powder and water were mixed at a mass ratio of 1:50, subjected to ultrasonic cell wall breaking for 40 minutes, and the pH value was adjusted to 2.0±0.2; pepsin was added at a mass ratio of 5%, and after enzymatic hydrolysis at 37°C for 3 hours, the enzyme was inactivated at 100°C for 10 minutes to obtain Ganoderma lucidum liquid;

[0024] (2) Adding ganoderma lucidum liquid, brown sugar, whey powder, glucose, MgSO4, NaCl, K2HPO4, and KH2PO4 into a fermentation tank, stirring and dissolving, and then fixing the volume to obtain a fermentation medium, wherein the final mass volume ratio of each component is: ganoderma lucidum liquid 300g / L, brown sugar 100g / L, whey powder 20g / L, glucose 45g / L, MgSO4 0.2g / L, NaCl 0.5g / L, K2HPO4 0.4g / L, KH2PO4 0.1g / L; the initial pH value of the medium is 5.5-6.5; sterilizing by high temperature steam for 25min, and cooling to 37-40°C after completion;

[0025] (3) inoculating Lactobacillus rhamnosus YKSW seed liquid into the fermentation medium at a volume ratio of 5%, culturing in a closed manner at 36-38° C. for 15 h, and then inoculating Bacillus coagulans KDB-18 seed liquid at a volume ratio of 5%, and fermenting aerobically at 36-38° C. for 36 h; after the fermentation is completed, cooling the fermentation liquid to below 10° C.;

[0026] (4) The fermented liquid is aseptically filled and packaged to obtain a Ganoderma lucidum fermented beverage.

[0027] Beneficial effects of the present invention:

[0028] The fermented ganoderma lucidum beverage prepared by the two-step fermentation method of enzymolysis and probiotics is light brown or dark brown, is a uniform liquid, has a pH value of 2.9-4.0, tastes sweet and sour, has a ganoderma lucidum flavor, and has a live bacteria count of more than 10 11 CFU / mL, and the number of viable bacteria can still maintain 10 after 6 months of storage at room temperature. 10 CFU / mL level, strong storage stability.

[0029] The fermented Ganoderma lucidum beverage is rich in effective ingredients such as polysaccharides, triterpenes and sterols, among which the polysaccharide content reaches 3.85-3.92g / L, and the triterpenes and sterols content reaches 455-604mg / L, achieving unexpected technical effects.

[0030] The fermented ganoderma drink has a significant antioxidant effect, with a scavenging rate of 90.21%-92.69% for DPPH free radicals and a scavenging rate of 83.31%-86.22% for superoxide anions; among them, the fermented ganoderma drink prepared in Example 6 has the highest antioxidant effect, with scavenging rates of 93.69% and 86.22% for DPPH free radicals and superoxide anion radicals, respectively, achieving unexpected technical effects.

[0031] In summary, the fermented Ganoderma lucidum beverage provided by the present invention has excellent taste and flavor, is rich in nutrition, has the effects of enhancing immunity and anti-oxidation, is beneficial to physical health, and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the colony morphology of Bacillus coagulans KDB-18. DETAILED DESCRIPTION

[0033] The following examples are provided to better illustrate the present invention, and those skilled in the art can better understand and grasp the present invention with the help of the examples. However, the protection and claims of the present invention are not limited to the examples provided.

[0034] The Lactobacillus rhamnosus YKSW selected in the embodiment of the present invention was screened from fresh feces of healthy people and was deposited in the China Center for Type Culture Collection of Wuhan University, Wuhan, China on March 29, 2022, with the deposit number of CCTCCNO:

[0035] M2022337.

[0036] The Bacillus coagulans KDB-18 selected in the embodiment of the present invention was screened from naturally fermented soybean paste and was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on July 1, 2024, with a preservation number of CCTCC NO: M20241408.

[0037] The pepsin described in the embodiments of the present invention can be purchased from Zhengzhou Jiuting Chemical Products Co., Ltd.

[0038] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods so that those skilled in the art can better understand the present invention, but the present invention is not limited thereto.

[0039] Example 1 Isolation, screening, identification and performance analysis of Bacillus coagulans KDB-18

[0040] The present invention isolates and screens a mucilage- and sugar-producing Bacillus from a naturally fermented soybean paste sample in Huacun Village, Dalian, and the extracellular crude polysaccharide yield in the fermentation broth reaches 3.64 g / L. Combined with the colony morphology, physiological and biochemical characteristics and molecular biological identification results of the Bacillus, it is determined to be a new type of coagulant Bacillus, named Bacillus coagulans KDB-18, which has been deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on July 1, 2024, and its deposit number is CCTCC NO: M20241408.

[0041] The colony morphology of the Bacillus coagulans KDB-18 is as follows Figure 1 As shown, the diameter of a single colony is 2-3mm, white and shiny, with a moist, flat surface and neat edges. Observation under a microscope shows that the strain is rod-shaped, arranged singly, in pairs or chains, and the spores are terminal. The strain is sensitive to common antibiotics such as erythromycin, gentamicin, tetracycline, and ampicillin, and has good biosafety.

[0042] The bacillus coagulans KDB-18 has a strong antioxidant capacity, and the scavenging rates of the supernatant thereof on DPPH free radicals, hydroxyl free radicals and superoxide anions reach 89.35%, 70.19% and 80.56% respectively.

[0043] The Bacillus coagulans KDB-18 can efficiently remove cholesterol, with an in vitro removal rate of up to 85.21%.

[0044] The inhibition rates of the Bacillus coagulans KDB-18 on alpha-amylase and glucosidase reach 82.28% and 34.06% respectively, and the in vitro hypoglycemic activity is high, so the invention has potential application prospects for hypoglycemic effect.

[0045] Example 2 Analysis of protease production ability of Bacillus coagulans KDB-18

[0046] The activated Bacillus coagulans KDB-18 was inoculated into LB liquid culture medium, and cultured at 37°C and 160 rpm for 24 hours; centrifuged at 4°C and 12000 rpm for 15 minutes, and the supernatant was collected.

[0047] The protease activity of the obtained fermentation supernatant was determined by referring to Appendix B "Determination of protease activity by Folin method" in the national standard GB / T 23527-2009 "Protease preparations".

[0048] The test results show that the protease activity in the fermentation supernatant of Bacillus coagulans KDB-18 provided by the present invention reaches 358.8 U / mL, achieving an unexpected technical effect.

[0049] Example 3 Analysis of antibacterial ability of Bacillus coagulans KDB-18

[0050] Staphylococcus aureus, Escherichia coli, Shigella, and Salmonella were inoculated into LB liquid culture medium at a volume ratio of 1%, and then cultured at 37°C for 12 h. The suspension of pathogenic bacteria was adjusted to 10% with sterile saline. 6 -10 7 CFU / mL.

[0051] The activated Bacillus coagulans KDB-18 was inoculated into LB liquid medium at a volume ratio of 1% and cultured at 37°C for 24 hours. 100 μL of pathogenic bacteria suspension was evenly spread on LB solid medium, a sterile Oxford cup was placed vertically into the solid medium and punched, then taken out, 200 μL of Bacillus coagulans KDB-18 fermentation liquid was added to the hole, and a commercially available Bacillus coagulans was used as a control, cultured at 37°C for 24 hours, and the diameter of the inhibition zone was measured. The experiment was repeated three times and the average value was taken.

[0052] The specific results are shown in Table 1.

[0053] Table 1 Bacillus coagulans KDB-18 antibacterial results

[0054]

[0055] It can be seen from the data in Table 1 that compared with commercially available Bacillus coagulans, the Bacillus coagulans KDB-18 provided by the present invention has a stronger inhibitory effect on four pathogenic bacteria, Staphylococcus aureus, Escherichia coli, Salmonella and Shigella, and the diameters of the inhibition zones for Escherichia coli and Salmonella are as high as 28.32 and 31.43 mm, respectively, achieving unexpected technical effects.

[0056] Example 4 Effect of Bacillus coagulans KDB-18 on intestinal health of mice

[0057] The experimental animals were Kunming male mice, 5 weeks old, weighing (22±3) g.

[0058] The mice used in this example were acclimatized to the environment for one week before the experiment. During this period, the temperature was 23±2°C and the humidity was 50%.

[0059] ±10%, 12h light-dark cycle, normal supply of drinking water and diet.

[0060] The experimental mice were randomly divided into 3 groups, namely normal control group, Escherichia coli group and Bacillus coagulans KDB-18 group, with 10 mice in each group. The mice were fasted for 24 hours before the experiment. The treatment methods of mice in each group are as follows:

[0061] The mice in the E. coli group and the Bacillus coagulans KDB-18 group were gavaged with 400 μL of an equal proportion of E. coli O157 and E. coli H7 suspension (10 9 After the inflammation model was successfully established, the mice in the E. coli group were gavaged with 400 μL of normal saline daily, and the mice in the Bacillus coagulans KDB-18 group were gavaged with 400 μL of Bacillus coagulans KDB-18 suspension daily (10 8 CFU / mL) for 5 consecutive days. The mice in the normal control group were gavaged with 400 μL of normal saline every day.

[0062] During the experiment, mice were free to eat and drink water, and their weight and food intake were measured every day.

[0063] After the experiment, the mice were fasted for 12 h and then killed by anesthesia.

[0064] Blood was collected from the eyeballs of all mice and centrifuged at 4°C and 3500 rpm for 15 minutes to obtain serum. The enzyme-linked immunosorbent assay (ELISA) method was used to detect the expression of biochemical indicators and inflammatory factors in the serum of mice in each group.

[0065] Colon tissues were collected from all mice, and feces in the cecal contents were collected. The feces and ether were mixed in a 1:1 ratio, vortexed for 2 minutes, ultrasonicated for 3 minutes to extract short-chain fatty acids, centrifuged for 10 minutes to remove the precipitate, and the supernatant was passed through a 0.22-micron organic filter membrane, wrapped with a sealing film and stored in a -20°C refrigerator for determination. A short-chain fatty acid standard curve was prepared using a gas chromatograph to determine the content of short-chain fatty acids in the cecal contents of mice in different groups.

[0066] 1. Effect of Bacillus coagulans KDB-18 on the body weight of mice

[0067] Compared with the normal control group, the body weight of mice in the Escherichia coli group was significantly reduced, and the body weight of mice showed a significant decline on the 4th day, while the body weight of mice in the Bacillus coagulans KDB-18 group was significantly higher than that in the Escherichia coli group.

[0068] 2. Regulatory effect of Bacillus coagulans KDB-18 on immune response in mice

[0069] Compared with the normal control group, the concentrations of pro-inflammatory factors IL-6, TNF-α, IL-1β, and C-reactive protein in the serum of mice in the E. coli group were significantly increased; while the concentrations of IL-1β, IL-6, and TNF-α in the serum of mice in the Bacillus coagulans KDB-18 group were significantly lower than those in the E. coli group. This shows that the Bacillus coagulans KDB-18 provided by the present invention can effectively reduce the expression of pro-inflammatory factors in the serum of mice, reduce the level of C-reactive protein, and effectively enhance the body immunity of mice.

[0070] 3. Effects of Bacillus coagulans KDB-18 on the level of short-chain fatty acids (SCFAs) in the cecum of mice

[0071] Compared with the normal control group, the contents of acetic acid, propionic acid and butyric acid in the cecal contents of the Escherichia coli group mice were significantly reduced, while the contents of three short-chain fatty acids, acetic acid, propionic acid and butyric acid, in the cecal contents of the Bacillus coagulans KDB-18 group were increased by 88.6%, 60.2% and 130.3% respectively compared with the Escherichia coli group. It is thus shown that the Bacillus coagulans KDB-18 provided by the present invention can significantly increase the content of short-chain fatty acids in the intestine of mice, which is beneficial to promote digestion and absorption, improve the body's anti-inflammatory level, maintain intestinal health, and achieve unexpected technical effects.

[0072] In summary, the Bacillus coagulans KDB-18 obtained by the present invention is sensitive to common antibiotics, has good biosafety, strong resistance to the gastrointestinal environment, can produce high amounts of extracellular polysaccharides and proteases, has strong antibacterial and antioxidant abilities, can efficiently degrade cholesterol, and lower blood sugar. Animal experimental results show that the strain can effectively reduce the expression of pro-inflammatory factors in mouse serum and significantly increase the content of short-chain fatty acids in the intestine, thereby promoting digestion and absorption, improving the body's anti-inflammatory level and immunity, and maintaining intestinal health.

[0073] Example 5 A Ganoderma lucidum fermented beverage and its preparation method

[0074] (1) Ganoderma lucidum powder and water were mixed at a mass ratio of 1:50, subjected to ultrasonic cell wall breaking for 40 minutes, and the pH value was adjusted to 2.0±0.2; pepsin was added at a mass ratio of 5%, and after enzymatic hydrolysis at 37°C for 3 hours, the enzyme was inactivated at 100°C for 10 minutes to obtain Ganoderma lucidum liquid;

[0075] (2) adding ganoderma lucidum liquid, brown sugar, whey powder, glucose, MgSO4, NaCl, K2HPO4, and KH2PO4 into a fermentation tank, stirring and dissolving, and then fixing the volume to obtain a fermentation medium, wherein the final mass volume ratio of each component is: ganoderma lucidum liquid 200 g / L, brown sugar 150 g / L, whey powder 50 g / L, glucose 20 g / L, MgSO4 0.5 g / L, NaCl 0.2 g / L, K2HPO4 0.2 g / L, KH2PO4 0.3% g / L; the initial pH value of the medium is 5.5-6.5; sterilizing by high temperature steam for 25 minutes, and cooling to 37-40° C. after completion;

[0076] (3) inoculating the Lactobacillus rhamnosus YKSW seed liquid into the fermentation medium at a volume ratio of 10%, and incubating it in a closed environment at 36-38° C. for 20 h, and then inoculating the Bacillus coagulans KDB-18 seed liquid at a volume ratio of 10%, and fermenting it aerobically at 36-38° C. for 24 h; after the fermentation is completed, cooling the fermentation liquid to below 10° C.;

[0077] (4) The fermented liquid is aseptically filled and packaged to obtain a Ganoderma lucidum fermented beverage.

[0078] Example 6 A Ganoderma lucidum fermented beverage and its preparation method

[0079] (1) Ganoderma lucidum powder and water were mixed at a mass ratio of 1:50, subjected to ultrasonic cell wall breaking for 40 minutes, and the pH value was adjusted to 2.0±0.2; pepsin was added at a mass ratio of 5%, and after enzymatic hydrolysis at 37°C for 3 hours, the enzyme was inactivated at 100°C for 10 minutes to obtain Ganoderma lucidum liquid;

[0080] (2) Adding ganoderma lucidum liquid, brown sugar, whey powder, glucose, MgSO4, NaCl, K2HPO4, and KH2PO4 into a fermentation tank, stirring and dissolving, and then fixing the volume to obtain a fermentation medium, wherein the final mass volume ratio of each component is: ganoderma lucidum liquid 300g / L, brown sugar 100g / L, whey powder 20g / L, glucose 45g / L, MgSO4 0.2g / L, NaCl 0.5g / L, K2HPO4 0.4g / L, KH2PO4 0.1g / L; the initial pH value of the medium is 5.5-6.5; sterilizing by high temperature steam for 25min, and cooling to 37-40°C after completion;

[0081] (3) inoculating Lactobacillus rhamnosus YKSW seed liquid into the fermentation medium at a volume ratio of 5%, culturing in a closed manner at 36-38° C. for 15 h, and then inoculating Bacillus coagulans KDB-18 seed liquid at a volume ratio of 5%, and fermenting aerobically at 36-38° C. for 36 h; after the fermentation is completed, cooling the fermentation liquid to below 10° C.;

[0082] (4) The fermented liquid is aseptically filled and packaged to obtain a Ganoderma lucidum fermented beverage.

[0083] Example 7 A fermented ganoderma beverage and its preparation method

[0084] (1) Ganoderma lucidum powder and water were mixed at a mass ratio of 1:50, subjected to ultrasonic cell wall breaking for 40 minutes, and the pH value was adjusted to 2.0±0.2; pepsin was added at a mass ratio of 5%, and after enzymatic hydrolysis at 37°C for 3 hours, the enzyme was inactivated at 100°C for 10 minutes to obtain Ganoderma lucidum liquid;

[0085] (2) Adding ganoderma lucidum liquid, brown sugar, whey powder, glucose, MgSO4, NaCl, K2HPO4, and KH2PO4 into a fermentation tank, stirring to dissolve and then fixing the volume to obtain a fermentation medium, wherein the final mass volume ratio of each component is: ganoderma lucidum liquid 250 g / L, brown sugar 120 g / L, whey powder 40 g / L, glucose 30 g / L, MgSO4 0.3 g / L, NaCl 0.3 g / L, K2HPO4 0.2 g / L, KH2PO4 0.2 g / L; the initial pH value of the medium is 5.5-6.5; sterilizing by high temperature steam for 25 min, and then cooling to 37-40° C.;

[0086] (3) inoculating the Lactobacillus rhamnosus YKSW seed liquid into the fermentation medium at a volume ratio of 6%, culturing in a closed manner at 36-38° C. for 18 h, and then inoculating the Bacillus coagulans KDB-18 seed liquid at a volume ratio of 8%, and fermenting aerobically at 36-38° C. for 30 h; after the fermentation is completed, cooling the fermentation liquid to below 10° C.;

[0087] (4) The fermented liquid is aseptically filled and packaged to obtain a Ganoderma lucidum fermented beverage.

[0088] Example 9 Performance Analysis of Ganoderma Lucidum Fermented Beverage

[0089] 1. Sensory evaluation

[0090] The fermented ganoderma beverages prepared in Examples 5-7 of the present invention are light brown or dark brown, are uniform liquids, taste sweet and sour, and have the aroma of ganoderma.

[0091] 2. pH value

[0092] The pH value of Ganoderma lucidum fermented beverage was measured using a handheld pH meter.

[0093] The results showed that the pH value of the fermented Ganoderma lucidum beverage provided by the present invention was 2.9-4.0.

[0094] 3. Live bacteria content

[0095] The plate count method was used to detect the number of viable bacteria (CFU / mL) in the Polygonatum sibiricum probiotic fermented drink.

[0096] The results showed that the live bacteria count of the Ganoderma lucidum fermented beverages provided by the present invention exceeded 10 11 CFU / mL, and the number of viable bacteria can still maintain 10 after 6 months of storage at room temperature. 10 The level of CFU / mL is stable.

[0097] 4. Triterpenoid and sterol content

[0098] The contents of triterpenes and sterols in Ganoderma lucidum fermented beverage were determined by HPLC.

[0099] The results showed that the content of triterpenes and sterols in the fermented Ganoderma lucidum beverage provided by the present invention reached 455-604 mg / L.

[0100] 5. Polysaccharide content

[0101] The fermented Ganoderma lucidum beverage prepared in Example 5-7 was centrifuged at 4°C and 12000 rpm for 15 min to obtain a supernatant. At least 3 volumes of anhydrous ethanol were added to the supernatant and the mixture was allowed to stand for centrifugation. The precipitate was collected to obtain the crude extracellular polysaccharide.

[0102] The crude extracellular polysaccharide was washed with 95% ethanol by volume, centrifuged at about 10,000 rpm and 4°C for 15 minutes, the supernatant was discarded, and the precipitate was washed again with 95% ethanol by volume, centrifuged at about 10,000 rpm and 4°C for 15 minutes, the supernatant was discarded, and the final precipitate was dissolved in 85°C distilled water. After removing the protein in the crude polysaccharide using the Sevage method, pure water was used as the dialysate in a dialysis bag with a molecular weight cutoff of 7000 for 48 hours, and the dialysate was replaced every 12 hours. The polysaccharide solution in the dialysis bag was freeze-dried using a freeze dryer. The freeze dryer had a condensation temperature of -40°C, a vacuum degree of 20 Pa, and a freezing time of about 20 hours to obtain dry powdered extracellular polysaccharides.

[0103] The results showed that the extracellular polysaccharide content in the Ganoderma lucidum fermented beverage provided by the present invention reached 3.85-3.92 g / L.

[0104] 4. Antioxidant capacity analysis

[0105] The fermented Ganoderma lucidum beverage prepared in Examples 5-7 of the present invention was centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was taken to measure the scavenging ability of the supernatant for DPPH free radicals and superoxide anions to indicate the comprehensive antioxidant capacity of the beverage.

[0106] (1) DPPH free radical scavenging rate

[0107] 2 mL of the supernatant of the fermented Ganoderma lucidum beverage was thoroughly mixed with 2 mL of 0.2 mmol / L DPPH 95% ethanol solution, respectively, and then placed at room temperature for 30 minutes, and the absorbance was measured at 517 nm.

[0108] DPPH free radical scavenging rate = (1-(A1-A2) / A3) × 100%.

[0109] Wherein: A1 is the absorbance value measured by the reaction of DPPH solution with sample; A2 is the absorbance value measured by the reaction of 95% ethanol with sample instead of DPPH solution; A3 is the absorbance value measured by the reaction of distilled water with sample instead of DPPH solution.

[0110] (2) Superoxide anion clearance rate:

[0111] The superoxide anion scavenging ability of the strain was determined by the pyrogallol auto-oxidation method. 1 mL of 150 mmol / L Tris-HCl (pH 8.2), 1.2 mmol / L pyrogallol and 3 mmol / L diethylenetriaminepentaacetic acid were added to the sample, kept at 25°C for 10 min, and the absorbance was measured at 325 nm.

[0112] The calculation formula is as follows:

[0113] Superoxide anion clearance rate (%) = [1-(A b -A c ) / (A d -A e )]×100%.

[0114] Where: A b is the absorbance value without sample and pyrogallol; A c A is the absorbance value without sample and containing pyrogallol; d A is the absorbance value of the sample without pyrogallol; e is the absorbance value of the sample and pyrogallol.

[0115] The above test results show that the scavenging rate of the Ganoderma lucidum fermented beverage provided by the present invention for DPPH free radicals reaches 90.21%-92.69%, and the scavenging rate for superoxide anions reaches 83.31%-86.22%. Among them, the Ganoderma lucidum fermented beverage prepared in Example 6 has the most significant antioxidant effect, and its scavenging rates for DPPH free radicals and superoxide anion radicals reach 93.69% and 86.22%, respectively, achieving unexpected technical effects.

[0116] In summary, the Ganoderma lucidum fermented beverage prepared by the two-step fermentation method of enzymatic hydrolysis and probiotics in the present invention has a sweet and sour taste, has a Ganoderma lucidum aroma, is rich in medicinal ingredients such as polysaccharides, triterpenes and sterols, has the effects of enhancing immunity and anti-oxidation, is beneficial to physical health, and has broad market prospects.

Claims

1. A fermented ganoderma beverage, characterized in that: The fermented Lingzhi beverage contains Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) and Bacillus coagulans ( Bacillus coagulans ).

2. The fermented ganoderma beverage according to claim 1, characterized in that: The preservation number of the Lactobacillus rhamnosus is CCTCC NO: M2022337.

3. The fermented ganoderma beverage according to claim 1 or 2, characterized in that: The deposit number of the Bacillus coagulans is CCTCC NO: M20241408.

4. The method for preparing the fermented ganoderma beverage according to claim 3, characterized in that: The preparation method comprises the following steps: (1) mixing ganoderma lucidum powder and water in appropriate proportions, subjecting the mixture to ultrasonic cell wall breaking treatment, and adding protease for enzymolysis to obtain ganoderma lucidum liquid; (2) preparing a fermentation medium containing Ganoderma lucidum liquid, sterilizing it, and cooling it; (3) Inoculate the Lactobacillus rhamnosus seed liquid into the fermentation medium, culture it in a closed environment at 36-38°C for 15-20 hours, then inoculate the Bacillus coagulans seed liquid, and ferment it aerobically at 36-38°C for 24-36 hours; after the fermentation is completed, cool the fermentation liquid to below 10°C; (4) The fermentation liquid is aseptically filled and packaged to obtain a Ganoderma lucidum fermented beverage.

5. The method for preparing the fermented ganoderma beverage according to claim 4, characterized in that: The protease in step (1) is pepsin.

6. The method for preparing the fermented ganoderma beverage according to claim 5, characterized in that: The fermentation medium in step (2) contains ganoderma lucidum liquid, brown sugar, whey powder, glucose, MgSO4, NaCl, K2HPO4, and KH2PO4.

7. The method for preparing the fermented ganoderma beverage according to claim 6, characterized in that: The components and their mass volume ratios in the fermentation medium in step (2) are: Ganoderma lucidum liquid 200-300 g / L, brown sugar 100-150 g / L, whey powder 20-50 g / L, glucose 20-45 g / L, MgSO4 0.2-0.5 g / L, NaCl 0.2-0.5 g / L, K2HPO4 0.2-0.4 g / L, KH2PO4 0.1-0.3 g / L.

8. The method for preparing the fermented ganoderma beverage according to claim 7, characterized in that: The preparation method comprises the following steps: (1) Ganoderma lucidum powder and water were mixed at a mass ratio of 1:50, and the mixture was subjected to ultrasonic cell wall breaking for 40 min. The pH value was adjusted to 2.0±0.

2. Pepsin was added at a mass ratio of 5%, and the mixture was enzymatically hydrolyzed at 37°C for 3 h, and the enzyme was inactivated to obtain Ganoderma lucidum liquid. (2) Adding ganoderma lucidum liquid, brown sugar, whey powder, glucose, MgSO4, NaCl, K2HPO4 and KH2PO4 into a fermentation tank, stirring to dissolve and then fixing the volume to obtain a fermentation medium, wherein the final mass volume ratio of each component is: ganoderma lucidum liquid 200-300 g / L, brown sugar 100-150 g / L, whey powder 20-50 g / L, glucose 20-45 g / L, MgSO4 0.2-0.5 g / L, NaCl 0.2-0.5 g / L, K2HPO4 0.2-0.4 g / L, KH2PO4 0.1-0.3 g / L; the initial pH value of the medium is 5.5-6.5; sterilizing by high temperature steam for 25 minutes, and then cooling to 37-40°C; (3) Inoculate the Lactobacillus rhamnosus seed liquid into the fermentation medium at a volume ratio of 5-10%, culture at 36-38°C in a closed environment for 15-20 hours, then inoculate the Bacillus coagulans seed liquid at a volume ratio of 5-10%, and ferment aerobically at 36-38°C for 24-36 hours; after the fermentation is completed, cool the fermentation liquid to below 10°C; (4) The fermentation liquid is aseptically filled and packaged to obtain a Ganoderma lucidum fermented beverage.

9. The method for preparing a fermented ganoderma beverage according to claim 8, characterized in that: The preparation method comprises the following steps: (1) Ganoderma lucidum powder and water were mixed at a mass ratio of 1:50, and the mixture was subjected to ultrasonic cell wall breaking for 40 min. The pH value was adjusted to 2.0±0.

2. Pepsin was added at a mass ratio of 5%, and the mixture was hydrolyzed at 37°C for 3 h. The mixture was then treated at 100°C for 10 min to inactivate the enzyme, thereby obtaining Ganoderma lucidum liquid. (2) Add Ganoderma lucidum liquid, brown sugar, whey powder, glucose, MgSO4, NaCl, K2HPO4, and KH2PO4 into a fermentation tank, stir to dissolve, and then adjust the volume to obtain a fermentation medium, in which the final mass volume ratios of the components are: Ganoderma lucidum liquid 300 g / L, brown sugar 100 g / L, whey powder 20 g / L, glucose 45 g / L, MgSO4 0.2 g / L, NaCl 0.5 g / L, K2HPO4 0.4 g / L, KH2PO4 0.1 g / L; the initial pH value of the medium is 5.5-6.5; sterilize with high-temperature steam for 25 minutes, and then cool to 37-40°C; (3) Inoculate the Lactobacillus rhamnosus seed liquid into the fermentation medium at a volume ratio of 5%, culture it in a closed environment at 36-38°C for 15 hours, then inoculate the Bacillus coagulans seed liquid at a volume ratio of 5%, and ferment it aerobically at 36-38°C for 36 hours; after the fermentation is completed, cool the fermentation liquid to below 10°C; (4) The fermentation liquid is aseptically filled and packaged to obtain a Ganoderma lucidum fermented beverage.

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