A PROBIOTIC-FERMENTED MACA COMPOSITION AND A METHOD FOR PREPARING THE COMPOSITION AND USE OF THE COMPOSITION

Abstract

Provided are a probiotic-fermented maca composition, a preparation method therefor, and use thereof. The composition is prepared from maca, fructus lycii and fructus rubi by milling, water extraction, enzymolysis and fermentation by Bifidobacterium and Lactobacillus, and can be used for preparing foods, healthcare products or beverages for improving fatigue tolerance and sexual function.

Claims

1. A probiotic-fermented maca composition, wherein the composition is obtained by extracting the maca, fructus lycii and fructus rubi with water, hydrolyzing the extracts enzymatically, and subjecting the hydrolysate to a probiotic-fermentation; wherein said composition comprises: 1.0-10% (g/ml) of maca, 2.0-8.0% (g/ml) of fructus lycii, and 2.0-8.0% (g/ml) of fructus rubi, wherein the enzyme used in the enzymatic hydrolysis is a high temperature amylase; and wherein the probiotics are Bifidobacterium and Lactobacillus.

2. The composition according to claim 1, wherein said composition comprises: 3.0% (g/ml) of maca, 3.0% (g/ml) of fructus lycii, and 3.0% (g/ml) of fructus rubi.

3. The composition according to claim 1, wherein said Bifidobacterium is selected from Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis or Bifidobacterium infantis; and said Lactobacillus is selected from Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus rhamnosus or Lactobacillus plantarum.

4. The composition according to claim 1, wherein the composition further comprises sweeteners which are sucrose which is present in an amount of 3.0% (g/ml), and sucralose which is present in an amount of 0.008% (g/ml).

5. A method for preparing the composition according to claim 1, wherein the method comprises the steps of: (1) obtaining a mixed powder of maca, fructus lycii and fructus rubi by grinding the maca to a powder with a fineness of 100 mesh or more, grinding the mixture of the fructus lycii and fructus rubi to a powder with a fineness of 10 mesh or more, and mixing the powders of the maca, fructus lycii and fructus rubi together; (2) obtaining a complex extract of the maca, fructus lycii and fructus rubi by immersing the mixed powder obtained in the above step (1) in water weighed 10-16 times greater than the mixed powder for 30 min to obtain a mixture, extracting the mixture at around 115° C. under high-pressure for 40 minutes to obtain an extract, hydrolyzing the extract with a high-temperature amylase after cooling to 85-90° C. and adjusting the pH to around 5.5, filtering and concentrating the hydrolysate under reduced pressure after sterilization to obtain a filtrate from which insoluble residues have been removed, and adding into the filtrate an amount of glucose and inorganic salts which is followed by adjustment of pH to 6.5-7.0 and sterilization; and (3) obtaining a probiotic-fermented maca composition by a probiotic-fermentation which comprises: seeding 0.5% (v/v) of one or two or more pre-cultured Bifidobacterium into the complex extract obtained in the above step (2) when it is cooled to about 39° C. and culturing for about 3 to 8 hours, seeding thereto further 0.5% (v/v) of one or two or more pre-cultured Lactobacillus and culturing for about 20 to 22 hours, decreasing the culture temperature to 28° C. when the pH is dropped to 4.2 or less, and continuing the fermentation until the pH is dropped to 4.0 and stabilizing the resultant ferment.

6. The method according to claim 5, wherein said composition comprises: 1.0-10% (g/ml) of maca, 2.0-8.0% (g/ml) of fructus lycii, and 2.0-8.0% (g/ml) of fructus rubi.

7. The method according to claim 5, wherein the ratio between the high-temperature amylase and the mixed powder in the step (2) is 30-40 U/g; the glucose is added into the filtrate in an amount of 0.5% (g/ml); and the inorganic salts are 5.0 mg/100 ml of FeSO.sub.4.7H.sub.2O, 4.4 mg/100 ml of ZnSO.sub.4.7H.sub.2O, and 50 mg/100 ml of MgSO.sub.4.7H.sub.2O.

8. The method according to claim 5, wherein the method further comprises, after the enzymatic hydrolysis and filtration in the step (2), adding sweeteners which are 3.0 g/100 ml of sucrose, and 0.008 g/100 ml of sucralose.

9. The method according to claim 5, wherein in the step (3), said Bifidobacterium is selected from Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis or Bifidobacterium infantis; and said Lactobacillus is selected from Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus rhamnosus or Lactobacillus plantarum.

10. Use of the composition according to claim 1, in the manufacture of a health product, food or beverage for improving fatigue tolerance and sexual function in a subject.

11. The composition according to claim 2, wherein the composition further comprises sweeteners which are a) sucrose which is present in an amount of 3.0% (g/ml), and b) sucralose which is present in an amount of 0.008% (g/ml).

12. The composition according to claim 3, wherein the composition further comprises sweeteners which are a) sucrose which is present in an amount of 3.0% (g/ml), and b) sucralose which is present in an amount of 0.008% (g/ml).

13. The method according to claim 6, wherein in the step (3), a) said Bifidobacterium is selected from Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis or Bifidobacterium infantis; and b) said Lactobacillus is selected from Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus rhamnosus or Lactobacillus plantarum.

14. The method according to claim 7, wherein in the step (3), a) said Bifidobacterium is selected from Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis or Bifidobacterium infantis; and b) said Lactobacillus is selected from Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus rhamnosus or Lactobacillus plantarum.

15. The method according to claim 8, wherein in the step (3), a) said Bifidobacterium is selected from Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis or Bifidobacterium infantis; and b) said Lactobacillus is selected from Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus rhamnosus or Lactobacillus plantarum.

16. Use of the composition according to claim 2, in the manufacture of a health product, food or beverage for improving fatigue tolerance and sexual function in a subject.

17. Use of the composition according to claim 3, in the manufacture of a health product, food or beverage for improving fatigue tolerance and sexual function in a subject.

18. Use of the composition according to claim 4, in the manufacture of a health product, food or beverage for improving fatigue tolerance and sexual function in a subject.

19. The composition according to claim 1, wherein the composition is obtained by a) extracting the maca, fructus lycii and fructus rubi with water, b) hydrolyzing the extracts enzymatically, and c) subjecting the hydrolysate to a probiotic-fermentation; wherein said composition of maca, fructus lycii and fructus rubi is comprised of: i) 3.0% (g/ml) of maca, ii) 3.0% (g/ml) of fructus lycii, and iii) 3.0% (g/ml) of fructus rubi, wherein the enzyme used in the enzymatic hydrolysis is a high temperature amylase; and the probiotics are Bifidobacterium and Lactobacillus and wherein: a) said Bifidobacterium is selected from Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis or Bifidobacterium infantis; and b) said Lactobacillus is selected from Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus rhamnosus or Lactobacillus plantarum; wherein the composition further comprises sweeteners which are a) sucrose which is present in an amount of 3.0% (g/ml), and b) sucralose which is present in an amount of 0.008% (g/ml).

20. A method according to claim 5 for preparing the composition of claim 1, wherein the method comprises the steps of: (1) obtaining a mixed powder of maca, fructus lycii and fructus rubi by a) grinding the maca to a powder with a fineness of 100 mesh or more, grinding the mixture of the fructus lycii and fructus rubi to a powder with a fineness of 10 mesh or more, and b) mixing the powders of the maca, fructus lycii and fructus rubi together; wherein said powder are in the following percents: i) 1.0-10% (g/ml) of maca, ii) 2.0-8.0% (g/ml) of fructus lycii, and iii) 2.0-8.0% (g/ml) of fructus rubi (2) obtaining a complex extract of the maca, fructus lycii and fructus rubi by a) immersing the mixed powder obtained in the above step (1) in water weighed 10-16 times greater than the mixed powder for 30 min to obtain a mixture, b) extracting the mixture at around 115° C. under high-pressure for 40 minutes to obtain an extract, c) hydrolyzing the extract with a high-temperature amylase after cooling to 85-90° C. and adjusting the pH to around 5.5, d) filtering and concentrating the hydrolysate under reduced pressure after sterilization to obtain a filtrate from which insoluble residues have been removed, and e) adding into the filtrate an amount of glucose and inorganic salts which is followed by adjustment of pH to 6.5-7.0 and sterilization; and wherein; i) the ratio between the high-temperature amylase and the mixed powder in the step (2) is 30-40 U/g; ii) the glucose is added into the filtrate in an amount of 0.5% (g/ml); and iii) the inorganic salts are 1) 5.0 mg/100 ml of FeSO.sub.4.7H.sub.2O, 2) 4.4 mg/100 ml of ZnSO.sub.4.7H.sub.2O, and 3) 50 mg/100 ml of MgSO.sub.4.7H.sub.2O wherein after the enzymatic hydrolysis and filtration in the step (2), adding sweeteners which are: a) 3.0 g/100 ml of sucrose, and b) 0.008 g/100 ml of sucralose, followed by; (3) obtaining a probiotic-fermented maca composition by a probiotic-fermentation which comprises: a) seeding 0.5% (v/v) of one or two or more pre-cultured Bifidobacterium into the complex extract obtained in the above step (2) when it is cooled to about 39° C. and culturing for about 3 to 8 hours, b) seeding thereto further 0.5% (v/v) of one or two or more pre-cultured Lactobacillus and culturing for about 20 to 22 hours, c) decreasing the culture temperature to 28° C. when the pH is dropped to 4.2 or less, and d) continuing the fermentation until the pH is dropped to 4.0 and stabilizing the resultant ferment, wherein; i) said Bifidobacterium is selected from Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis or Bifidobacterium infantis; and ii) said Lactobacillus is selected from Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus rhamnosus or Lactobacillus plantarum.

Description

EXAMPLES

Preparation Example 1: Preparation of a Probiotic-Fermented Composition A

[0031] 30 g of dried maca (Lepidium meyenii Walp), which had been ground and sieved through a sieve of 100 mesh, and 30 g of dried fructus lycii (Lycium barbarum L) and 30 g of dried fructus rubi (Rubus chingii Hu), which had been mixed, ground and sieved through a sieve of 10 mesh, were mixed together to obtain a mixed powder of maca, fructus lycii and fructus rubi. The mixed powder was immersed in water weighed 12 times greater than the mixed powder for 30 minutes to obtain a mixture. The mixture was extracted at around 115° C. under high-pressure for 40 minutes to obtain an extract. After the extract had been cooled to 85-90° C. and the pH of the extract had been adjusted to around 5.5 with sodium hydroxide, a 2700 units (30 U/g mixed powder) of high-temperature amylase which had been diluted with water was added into the extract and stirred well, and the enzymolysis was allowed to occur with the temperature being maintained at 85-90° C. The enzymolysis was allowed to end when the indicator of iodine was not colored blue remarkably as compared to the control which was not hydrolyzed enzymatically. The enzyme was inactivated at 100° C. for 10 minutes, and the hydrolysate was filtered though a filter of 200 mesh, centrifuged and recovered for the filtrate. The filtrate was concentrated to a concentration of 9.0% (g/ml) under reduced pressure. 0.5% (g/ml) of glucose and inorganic salts (5.0 mg/100 ml of FeSO.sub.4.7H.sub.2O, 4.4 mg/100 ml of ZnSO.sub.4.7H.sub.2O, and 50 mg/100 ml of MgSO.sub.4.7H.sub.2O), 3.0 g/100 ml of sucrose, and 0.008 g/100 ml of sucralose were added into the concentrated filtrate which was followed by adjustment of pH to about 7.0 and sterilization at 115° C. for 40 minutes to obtain a complex extract of the maca, fructus lycii and fructus rubi. A 0.5% (v/v) of pre-cultured Bifidobacterium bifidum (isolated from normal human body and identified by the Chinese Academy of Sciences Institute of Microbiology) and Bifidobacterium breve (AS 1.2213) were seeded into the complex extract when it had been cooled to about 39° C., and allowed to ferment for 6 hours. A 0.5% (v/v) of pre-cultured Lactobacillus delbrueckii (AS 1.1480) and Lactobacillus plantarum (AS 1.19) were further seeded thereto, and allowed to ferment at 37° C. for 20-22 hours. The temperature of fermentation was decreased when the pH was dropped to 4.2 or less and the fermentation was continued until the pH was dropped to 4.0. The ferment was stabilized in a water bath of 60° C. for 4 hours after the fermentation was stopped to obtain the Composition A which had a total probiotics quantity of 3.2×10.sup.8 cells/ml as counted under microscope using blood cell counter and had a total sugar-content of 35 mg/ml.

Preparation Example 2: Preparation of a Probiotic-Fermented Composition B

[0032] 50 g of dried maca (Lepidium meyenii Walp), which had been ground and sieved through a sieve of 100 mesh, and 40 g of dried fructus lycii (Lycium barbarum L) and 20 g of dried fructus rubi (Rubus chingii Hu), which had been mixed, ground and sieved through a sieve of 10 mesh, were mixed together to obtain a mixed powder of maca, fructus lycii and fructus rubi. The mixed powder was immersed in water weighed 14 times greater than the mixed powder for 30 minutes to obtain a mixture. The mixture was extracted at around 115° C. under high-pressure for 40 minutes to obtain an extract. After the extract had been cooled to 85-90° C. and the pH of the extract had been adjusted to around 5.5 with sodium hydroxide, a 3850 units (35 U/g mixed powder) of high-temperature amylase which had been diluted with water was added into the extract and stirred well, and the enzymolysis was allowed to occur with the temperature being maintained at 85-90° C. The enzymolysis was allowed to end when the indicator of iodine was not colored blue remarkably as compared to the control which was not hydrolyzed enzymatically. The enzyme was inactivated at 100° C. for 10 minutes, and the hydrolysate was filtered though a filter of 200 mesh, centrifuged and recovered for the filtrate. The filtrate was concentrated to a concentration of 11.0% (g/ml) under reduced pressure. 0.5% (g/ml) of glucose and inorganic salts (5.0 mg/100 ml of FeSO.sub.4.7H.sub.2O, 4.4 mg/100 ml of ZnSO.sub.4.7H.sub.2O, and 50 mg/100 ml of MgSO.sub.4.7H.sub.2O), 3.0 g/100 ml of sucrose, and 0.008 g/100 ml of sucralose were added into the concentrated filtrate which was followed by adjustment of pH to about 7.0 and sterilization at 115° C. for 40 minutes to obtain a complex extract of the maca, fructus lycii and fructus rubi. A 0.5% (v/v) of pre-cultured Bifidobacterium bifidum (isolated from normal human body and identified by the Chinese Academy of Sciences Institute of Microbiology) and Bifidobacterium adolescentis (AS1.2190) were seeded into the complex extract when it had been cooled to about 39° C., and allowed to ferment for 6 hours. A 0.5% (v/v) of pre-cultured Lactobacillus acidophilus (AS 1.1854) and Lactobacillus plantarum (AS 1.19) were further seeded thereto, and allowed to ferment at 37° C. for 20-22 hours. The temperature of fermentation was decreased when the pH was dropped to 4.2 or less and the fermentation was continued until the pH was dropped to 4.0. The ferment was stabilized in a water bath of 60° C. for 4 hours after the fermentation was stopped to obtain the Composition B which had a total probiotics quantity of 4.2×10.sup.8 cells/ml as counted under microscope using blood cell counter and had a total sugar-content of 65 mg/ml.

Preparation Example 3: Preparation of a Probiotic-Fermented Composition C

[0033] 80 g of dried maca (Lepidium meyenii Walp), which had been ground and sieved through a sieve of 100 mesh, and 50 g of dried fructus lycii (Lycium barbarum L) and 20 g of dried fructus rubi (Rubus chingii Hu), which had been mixed, ground and sieved through a sieve of 10 mesh, were mixed together to obtain a mixed powder of maca, fructus lycii and fructus rubi. The mixed powder was immersed in water weighed 16 times greater than the mixed powder for 30 minutes to obtain a mixture. The mixture was extracted at around 115° C. under high-pressure for 40 minutes to obtain an extract. After the extract had been cooled to 85-90° C. and the pH of the extract had been adjusted to around 5.5 with sodium hydroxide, a 6000 units (40 U/g mixed powder) of high-temperature amylase which had been diluted with water was added into the extract and stirred well, and the enzymolysis was allowed to occur with the temperature being maintained at 85-90° C. The enzymolysis was allowed to end when the indicator of iodine was not colored blue remarkably as compared to the control which was not hydrolyzed enzymatically. The enzyme was inactivated at 100° C. for 10 minutes, and the hydrolysate was filtered though a filter of 200 mesh, centrifuged and recovered for the filtrate. The filtrate was concentrated to a concentration of 15.0% (g/ml) under reduced pressure. 0.5% (g/ml) of glucose and inorganic salts (5.0 mg/100 ml of FeSO.sub.4.7H.sub.2O, 4.4 mg/100 ml of ZnSO.sub.4.7H.sub.2O, and 50 mg/100 ml of MgSO.sub.4.7H.sub.2O), 3.0 g/100 ml of sucrose, and 0.008 g/100 ml of sucralose were added into the concentrated filtrate which was followed by adjustment of pH to about 7.0 and sterilization at 115° C. for 40 minutes to obtain a complex extract of the maca, fructus lycii and fructus rubi. A 0.5% (v/v) of pre-cultured Bifidobacterium infantis (AS1.853) and Bifidobacterium breve (AS 1.2213) were seeded into the complex extract when it had been cooled to about 39° C., and allowed to ferment for 6 hours. A 0.5% (v/v) of pre-cultured Lactobacillus delbrueckii (AS 1.1480) and Lactobacillus rhamnosus (AS 1.22) were further seeded thereto, and allowed to ferment at 37° C. for 20-22 hours. The temperature of fermentation was decreased when the pH was dropped to 4.2 or less and the fermentation was continued until the pH was dropped to 4.0. The ferment was stabilized in a water bath of 60° C. for 4 hours after the fermentation was stopped to obtain the Composition C which had a total probiotics quantity of 5.8×10.sup.8 cells/ml as counted under microscope using blood cell counter and had a total sugar-content of 84 mg/ml.

Experimental Example 1: Investigation of Effect of the Fermented Composition of the Present Invention on Fatigue-Tolerance

[0034] The present experimental example aimed to investigate effect of the composition comprising maca, fructus lycii and fructus rubi fermented by several probiotics on fatigue-tolerance in mice by pole-climbing test, loaded swimming test, blood lactate measurement, serum urea nitrogen measurement, and liver glycogen measurement.

[0035] The Compositions A, B and C obtained in the Preparation Examples 1, 2 and 3 were served as “fermented samples”, while the compositions of the same components without subjecting to fermentation with Bifidobacterium and Lactobacillus were served as “unfermented samples”.

[0036] Experimental animals: in this experiment, 1-month-old healthy SPF male Kunming mice with body-weight of 20±3 g provided by Research Center for Experimental Animal in Shandong University of Traditional Chinese Medicine were used and randomized.

[0037] Doses: The recommended dose for human was 100 mL/day/60 kg body weight. The equivalent dose for mouse was 10 times more than that of human. An oral dosage form of 16.7 mL/day/kg body weight which is mouse dose equivalent to recommended human dose was set as middle dose, while the 8.3 mL/Day/kg body weight which is 0.5 times of the middle dose and 33.3 mL/day/kg body weight which is 2 times of the middle dose were set as low dose and high dose respectively. The doses were diluted with distilled water and administered to the mice intragastrically at 1.2 mL/day for 25 days continuously.

[0038] Instrument: a pole-climbing shelf, a swimming tank, UV spectrophotometer 754, ES-200 electronic balance, centrifuge, a swimming tank, intragastric feeder and so on.

[0039] Reagents: A serum urea nitrogen detection Kit (diacetyl oxime method).

[0040] 5% of Trichloroacetic acid (TCA), glucose standard solution, concentrated sulfuric acid, anthrone reagent, 4% copper sulfate solution, 1% sodium fluoride solution, protein precipitant, 1.5% para-hydroxybiphenyl solution, the standard solution of lactic acid.

Experimental Method

1. Pole-Climbing Test:

[0041] The mice were placed on a rod of plexiglass in a pole-climbing shelf with their muscles being on a state of static tension, 30 min after the last administration of the test substance. The duration time of the mice retaining on the rod before they falling down due to fatigue was measured. The test was stopped upon the 3.sup.rd falling, and the mean of the durations tested 3 times was set as “pole-climbing duration”.

2. Loaded Swimming Test:

[0042] The mice were loaded with lead wires of 5% of their body-weight, 30 min after the last administration of the test substance. The mice were placed in a swimming tank with a water depth of 30 cm or more and a water temperature of 25° C., and allowed to swim. The duration from beginning to swim to entire head being immersed in the water while being unable to float over the surface of the water for 8 seconds was set as “swimming duration”.

3. Measurement of Serum Urea Nitrogen:

[0043] The mice were placed in water at a temperature of 25° C. and allowed to swim for 90 min, 30 min after the last administration of the test substance. Serum was taken from blood sampled from eyes removed from the mice, and tested with a kit according to the procedure as shown in the Table 1.

TABLE-US-00001 TABLE 1 Reagents Blank Standard Test BUN20 mg/dL Standard 0.02 Solution (mL) Sample (mL) 0.02 Diacetyl oxime Solution 3.0 3.0 3.0 (mL) Ferric chloride-phosphate 2.5 2.5 2.5 Solution (mL)

[0044] The serum were mixed with the agents thoroughly, placed in a boiling water bath for 10 minutes, and then cooled in cold water. Absorbances at 520 nm, i.e. A.sub.standard, A.sub.Sample, and A.sub.Blank, were measured and calibrated with those measured with distilled water.

4. Measurement of Blood Lactate

[0045] The mice were placed in water at a temperature of 25° C. and allowed to swim for 60 min, 30 min after the last administration of the test substance. Blood was sampled from eyes removed from the mice after leaving arrested for 15 min. 0.48 mL of 1% NaF solution was added into a 5 ml centrifuge tube, into bottom of which 20 μL of whole blood was then accurately added with the pipette being washed with the supernatant by multiple-pipetting. 1.5 mL protein precipitant was further added into the tube, and the tube was vibrated thoroughly and centrifuged at 3000 r/min for 10 min, and the supernatant was subjected to operation in accordance with Table 2.

TABLE-US-00002 TABLE 2 Blank Tube Standard Tube Test Tube (mL) (mL) (mL) Precipitating Agent-NaF 0.5 — — Mix-Solution Lactic acid Standard Solution — 0.5 — Sample — — 0.5 4%CuSO.sub.4 0.1 0.1 0.1 Concentrated sulfuric acid 3 3   3 Mixed thoroughly, placed in a boiling water bath for 5 min, and cooled in icy water for 10 min 1.5% p-Hydroxydiphenyl 0.1 0.1 0.1

[0046] The resultant was shaken thoroughly and placed in a 30° C. water bath for 30 min with shaking at every 10 min after completion of the foregoing steps. The resultant was then placed in a boiling water bath for 90 s, cooled to room-temperature, and then subjected to colorimetry at 560 nm and calibrated with blank tube.

5. Measurement of Liver Glycogen with Anthrone Method

[0047] The mice were sacrificed, 30 min after the last administration of the test substance. Livers were taken from the mice, rinsed with a saline, and dried with a filter paper. An accurately weighed 200 mg of liver piece was placed in a tube into which 4 mL of TCA was added, and then homogenized for 1 min. The homogenate was poured into a centrifuge tube, and centrifuged at 3000 r/min for 15 min, and the supernatant was transferred into another tube. 4 mL of TCA was added to the precipitating agent, homogenized for 1 min, and further centrifuged for 15 min, and the supernatant was pooled with the previous supernatant and mixed thoroughly. 1 mL of the pooled supernatant was placed into 10 ml centrifuge tubes into which 4 mL of 95% ethanol was added, and mixed thoroughly until there was no interface between the two liquids. The tube was plugged with a clean plug, and left at room temperature overnight. The tube was centrifuged at 3000 r/min for 15 min after precipitation was completed. The supernatant was discarded carefully and the tube was left upside-down for 10 min. The glycogen was dissolved in 2 mL distilled water with the glycogen on the wall of the tube being washed down by the distilled water, and shaken for thorough dissolution. 0.1 ml of the solution was diluted in 0.9 mL of distilled water.

[0048] Blank Agent: 1 mL of distilled water was placed in a clean centrifuge tube and set as the Blank Agent.

[0049] Standard tube: 0.1 mL of standard solution containing 100 mg/dL glucose was placed into a tube into which 1.5 ml of distilled water was added.

[0050] Assay: 5 mL of anthrone reagent was injected directly into center of each tube which is in a cold water bath, and mixed thoroughly. Upon the temperature of the mixture was decreased to that of the cold water bath, the tube was transferred to a boiling water bath for 15 min. The tube was then transferred to an icy water bath until it cooled to room-temperature. The content of the tube was transferred to a colorimetric tube, and absorbance at 620 nm was measured and calibrated with that measured with the Blank tube. The absorbance of the standard tube was also measured. The measured absorbances were converted to content of liver glycogen against weight of the liver (expressed at g/100 g of liver).

Results

[0051] 1. Pole-climbing Test with Mice

TABLE-US-00003 TABLE 3 Effects of the fermented Compositions A, B and C and the corresponding compositions without fermentation on pole-climbing durations in mice (X ± SD) Number of Pole-Climbing Groups Animals Durations (sec) 1 Control 10 370.00 ± 37.82 .sup.  Fermented A.sub.1 Unfermented 10 443.10 ± 49.49* .sup.  and High-Dose Group unfermented A.sub.2 Unfermented 10 442.70 ± 59.04* .sup.  Composition Middle-Dose Group A A.sub.3 Unfermented 10 375.50 ± 70.83 .sup.  Low-Dose Group A.sub.4 Fermented 10 511.60 ± 50.92*.sup..box-tangle-solidup. High-Dose Group A.sub.5 Fermented 10 502.40 ± 35.16*.sup..box-tangle-solidup. Middle-Dose Group A.sub.6 Fermented 10 486.40 ± 67.20*.sup..box-tangle-solidup. Low-Dose Group Fermented B.sub.1 Unfermented 10 472.10 ± 32.26* .sup.  and High-Dose Group unfermented B.sub.2 Unfermented 10 455.50 ± 46.30* .sup.  Composition Middle-Dose Group B B.sub.3 Unfermented 10 378.60 ± 63.21 .sup.  Low-Dose Group B.sub.4 Fermented 10 564.20 ± 54.31*.sup..box-tangle-solidup. High-Dose Group B.sub.5 Fermented 10 546.30 ± 36.64*.sup..box-tangle-solidup. Middle-Dose Group B.sub.6 Fermented 10 528.40 ± 48.02*.sup..box-tangle-solidup. Low-Dose Group Fermented C.sub.1 Unfermented 10 502.30 ± 42.16* .sup.  and High-Dose Group unfermented C.sub.2 Unfermented 10 494.50 ± 38.45* .sup.  Composition Middle-Dose Group C C.sub.3 Unfermented 10 437.60 ± 32.18* .sup.  Low-Dose Group C.sub.4 Fermented 10 598.30 ± 31.06*.sup..box-tangle-solidup. High-Dose Group C.sub.5 Fermented 10 576.40 ± 42.28*.sup..box-tangle-solidup. Middle-Dose Group C.sub.6 Fermented 10 554.20 ± 50.83*.sup..box-tangle-solidup. Low-Dose Group *Significant difference compared with the control group (P < 0.05). .sup..box-tangle-solidup.Significant difference between the fermented and unfermented compositions at the same concentrations (P < 0.05).

[0052] The results (Table 3) showed that the pole-climbing durations of mice were increased by all of the fermented Composition A, B, and C at all doses, and the unfermented Compositions A and B at high and middle doses, and the unfermented Composition C at high, middle and low doses, with the fermented Compositions A, B and C at all doses acting significantly superior to the corresponding compositions at the corresponding doses without fermentation, which indicated that the extracts of maca, fructus lycii and fructus rubi fermented by probiotics were able to increase the pole-climbing duration of mice.

2. Swimming Test with Mice

TABLE-US-00004 TABLE 4 Effects of the fermented Compositions A, B and C and the corresponding compositions without fermentation on swimming duration in mice (X ± SD) Number of Swimming Groups Animals durations (sec) Control 10 362.31 ± 69.87 .sup.  Fermented A.sub.1 Unfermented 10 426.40 ± 62.24* .sup.  and High-Dose Group unfermented A.sub.2 Unfermented 10 401.18 ± 56.54 .sup.  Composition Middle-Dose Group A A.sub.3 Unfermented 10 412.00 ± 44.19 .sup.  Low-Dose Group A.sub.4 Fermented 10 503.40 ± 61.10*.sup..box-tangle-solidup. High-Dose Group A.sub.5 Fermented 10 491.60 ± 57.31*.sup..box-tangle-solidup. Middle-Dose Group A.sub.6 Fermented 10 483.10 ± 84.76*.sup..box-tangle-solidup. Low-Dose Group Fermented B.sub.1 Unfermented 10 462.60 ± 53.12* .sup.  and High-Dose Group unfermented B.sub.2 Unfermented 10 446.20 ± 48.62* .sup.  Composition Middle-Dose Group B B.sub.3 Unfermented 10 408.54 ± 39.76 .sup.  Low-Dose Group B.sub.4 Fermented 10 561.30 ± 62.14*.sup..box-tangle-solidup. High-Dose Group B.sub.5 Fermented 10 547.60 ± 45.24*.sup..box-tangle-solidup. Middle-Dose Group B.sub.6 Fermented 10 506.82 ± 54.68*.sup..box-tangle-solidup. Low-Dose Group Fermented C.sub.1 Unfermented 10 503.62 ± 48.21* .sup.  and High-Dose Group unfermented C.sub.2 Unfermented 10 485.25 ± 52.13* .sup.  Composition Middle-Dose Group C C.sub.3 Unfermented 10 446.30 ± 32.18* .sup.  Low-Dose Group C.sub.4 Fermented 10 601.20 ± 59.35*.sup..box-tangle-solidup. High-Dose Group C.sub.5 Fermented 10 586.75 ± 46.12*.sup..box-tangle-solidup. Middle-Dose Group C.sub.6 Fermented 10 543.50 ± 61.20*.sup..box-tangle-solidup. Low-Dose Group *Significant difference compared with the control group (P < 0.05). .sup..box-tangle-solidup.Significant difference between the fermented and unfermented compositions at the same concentrations (P < 0.05).

[0053] The results (Table 4) show that the swimming durations of mice were increased by all of the fermented Compositions A, B and C at all doses and the unfermented Composition A at high dose, the unfermented Composition B at high dose, the unfermented Composition C at high, middle and low doses, with the fermented Compositions A, B and C at all doses acting significantly superior to the corresponding compositions at the corresponding doses without fermentation, which indicated that the extracts of maca, fructus lycii and fructus rubi fermented by probiotics were able to increase swimming duration in mice.

3. Level of Serum Urea Nitrogen in Mice

[0054]

TABLE-US-00005 TABLE 5 Effect of fermented Compositions A, B and C and the corresponding compositions without fermentation on level of serum urea nitrogen in mice (X ± SD) Number of Level of serum Groups Animals urea nitrogen Control 10 31.29 ± 2.94  Fermented A.sub.1 Unfermented 10 28.81 ± 1.92  and High-Dose Group unfermented A.sub.2 Unfermented 10 29.07 ± 1.78  Composition Middle-Dose Group A A.sub.3 Unfermented 10 28.59 ± 2.28  Low-Dose Group A.sub.4 Fermented 10 26.97 ± 1.59* High-Dose Group A.sub.5 Fermented 10 27.31 ± 1.44* Middle-Dose Group A.sub.6 Fermented 10 27.97 ± 2.63  Low-Dose Group Fermented B.sub.1 Unfermented 10 28.35 ± 1.65  and High-Dose Group unfermented B.sub.2 Unfermented 10 28.98 ± 1.38  Composition Middle-Dose Group B B.sub.3 Unfermented 10 29.12 ± 2.06  Low-Dose Group B.sub.4 Fermented 10 26.34 ± 1.24* High-Dose Group B.sub.5 Fermented 10 26.83 ± 1.55* Middle-Dose Group B.sub.6 Fermented 10 27.25 ± 1.38* Low-Dose Group Fermented C.sub.1 Unfermented 10 27.13 ± 1.23* and High-Dose Group unfermented C.sub.2 Unfermented 10 27.64 ± 1.45* Composition Middle-Dose Group C C.sub.3 Unfermented 10 28.26 ± 1.63  Low-Dose Group C.sub.4 Fermented 10 25.08 ± 1.26* High-Dose Group C.sub.5 Fermented 10 26.42 ± 1.32* Middle-Dose Group C.sub.6 Fermented 10 26.89 ± 1.48* Low-Dose Group *Significant difference compared with the control group (P < 0.05). .box-tangle-solidup. Significant difference between the fermented and unfermented compositions at the same concentrations (P < 0.05).

[0055] The results (Table 5) show that the level of serum urea nitrogen in mice was decreased by all of the fermented Composition A at high dose, the fermented Compositions B and C at high, middle and low doses and the unfermented Composition C at high dose.

4. Level of Liver Glycogen in Mice

[0056]

TABLE-US-00006 TABLE 6 Effects of the fermented Compositions A, B and C and the corresponding compositions without fermentation on the level of liver glycogen in mice (X ± SD) Number Level of of Liver Groups Animals Glycogen Control 10 2.21 ± 0.24 Fermented A.sub.1 Unfermented 10 2.41 ± 0.30 and High-Dose Group unfermented A.sub.2 Unfermented 10 2.21 ± 0.17 Composition Middle-Dose Group A A.sub.3 Unfermented 10 2.19 ± 0.16 Low-Dose Group A.sub.4 Fermented 10  2.79 ± 0.34* High-Dose Group A.sub.5 Fermented 10 2.47 ± 0.37 Middle-Dose Group A.sub.6 Fermented 10 2.42 ± 0.24 Low-Dose Group Fermented B.sub.1 Unfermented 10  2.69 ± 0.21* and High-Dose Group unfermented B.sub.2 Unfermented 10 2.31 ± 0.15 Composition Middle-Dose Group B B.sub.3 Unfermented 10 2.27 ± 0.16 Low-Dose Group B.sub.4 Fermented 10  .sup. 3.16 ± 0.35*.sup..box-tangle-solidup. High-Dose Group B.sub.5 Fermented 10  .sup. 2.89 ± 0.23*.sup..box-tangle-solidup. Middle-Dose Group B.sub.6 Fermented 10 2.52 ± 0.14 Low-Dose Group Fermented C.sub.1 Unfermented 10  2.93 ± 0.32* and High-Dose Group unfermented C.sub.2 Unfermented 10  2.79 ± 0.25* Composition Middle-Dose Group C C.sub.3 Unfermented 10  2.71 ± 0.15* Low-Dose Group C.sub.4 Fermented 10  .sup. 3.52 ± 0.19*.sup..box-tangle-solidup. High-Dose Group C.sub.5 Fermented 10  .sup. 3.36 ± 0.17*.sup..box-tangle-solidup. Middle-Dose Group C.sub.6 Fermented 10  .sup. 3.18 ± 0.21*.sup..box-tangle-solidup. Low-Dose Group *Significant difference compared with the control group (P < 0.05). .sup..box-tangle-solidup.Significant difference between the fermented and unfermented compositions at the same concentrations (P < 0.05).

[0057] The results (Table 6) show that the levels of liver glycogen in mice were increased by all of the fermented Composition A at high dose, the fermented Composition B at high dose, the fermented Composition C at high, middle and low doses and the unfermented Composition B at high dose, the unfermented Composition C at high, middle and low doses, with the fermented Composition B at high and middle doses and the fermented Composition C at all doses acting significantly superior to the corresponding compositions at the corresponding doses without fermentation.

5. Level of Blood Lactate

[0058]

TABLE-US-00007 TABLE 7 Effects of the fermented Compositions A, B and C and the corresponding compositions without fermentation on level of blood lactate in mice (X ± SD) Number of Level of blood Groups Animals lactate Control 10 33.89 ± 3.98  Fermented A.sub.1 Unfermented 10 22.70 ± 2.95* and High-Dose Group unfermented A.sub.2 Unfermented 10 27.87 ± 3.22* Composition Middle-Dose Group A A.sub.3 Unfermented 10 27.20 ± 3.76* Low-Dose Group A.sub.4 Fermented 10 20.47 ± 2.17* High-Dose Group A.sub.5 Fermented 10  .sup. 22.16 ± 2.44*.sup..box-tangle-solidup. Middle-Dose Group A.sub.6 Fermented 10 24.33 ± 4.28* Low-Dose Group Fermented B.sub.1 Unfermented 10 20.65 ± 2.81* and High-Dose Group unfermented B.sub.2 Unfermented 10 25.36 ± 2.65* Composition Middle-Dose Group B B.sub.3 Unfermented 10 26.05 ± 2.13* Low-Dose Group B.sub.4 Fermented 10 18.54 ± 3.15* High-Dose Group B.sub.5 Fermented 10  .sup. 19.35 ± 2.86*.sup..box-tangle-solidup. Middle-Dose Group B.sub.6 Fermented 10  .sup. 20.48 ± 2.15*.sup..box-tangle-solidup. Low-Dose Group Fermented C.sub.1 Unfermented 10 19.65 ± 2.98* and High-Dose Group unfermented C.sub.2 Unfermented 10 20.58 ± 3.16* Composition Middle-Dose Group C C.sub.3 Unfermented 10 22.15 ± 2.65* Low-Dose Group C.sub.4 Fermented 10  .sup. 14.24 ± 2.46*.sup..box-tangle-solidup. High-Dose Group C.sub.5 Fermented 10 15.68 ± 2.14* Middle-Dose Group C.sub.6 Fermented 10  .sup. 15.95 ± 3.02*.sup..box-tangle-solidup. Low-Dose Group *Significant difference compared with the control group (P < 0.05). .sup..box-tangle-solidup.Significant difference between the fermented and unfermented compositions at the same concentrations (P < 0.05).

[0059] The results (Table 7) show that the levels of blood lactate were decreased by the fermented Compositions A, B and C and the unfermented Compositions A, B and C at all doses in mice after exercise, with the fermented Composition A at middle dose, the fermented Composition B at middle and low doses, the fermented Composition C at high and low doses acting superior to the corresponding compositions at the corresponding doses without fermentation.

Conclusions

[0060] The results of pole-Climbing test, swimming test, and measurement of levels of the serum urea nitrogen, liver glycogen, and blood lactate in mice showed that the compositions made of maca, fructus lycii and fructus rubi fermented by probiotics were able to ameliorate fatigue, and the probiotic-fermented compositions acted significantly superior to the corresponding compositions without fermentation, which indicated that the fermentation with probiotics was able to increase the anti-fatigue activities of the compositions made of maca, fructus lycii and fructus rubi.

Experimental Example 2: Investigation of Effects of the Fermented Composition of the Present Invention on Improvement of Sexual Function

[0061] The present experimental example aimed to investigate effects of the composition comprising maca, fructus lycii and fructus rubi fermented by several probiotics on sexual function in mice by measuring mating latency and mating frequency within 20 min in male mice.

[0062] The fermented Compositions A, B and C obtained in the Preparation Examples were served as “fermented samples”, while the compositions of the same components without subjecting to fermentation were served as “unfermented samples”.

[0063] Experimental animals: in this experiment, 1-month-old healthy SPF Kunming mice (male:female=1:3) with body-weight of 20±3 g provided by Research Center for Experimental Animal in Shandong University of Traditional Chinese Medicine were used and randomized.

[0064] Doses: The recommended dose for human was 100 mL/day/60 kg body weight. The equivalent dose for mouse was 10 times more than that of human. An oral dosage form of 16.7 mL/day/kg body weight which is mouse dose equivalent to recommended human dose was set as middle dose, while the 8.3 mL/Day/kg body weight which is 0.5 times of the middle dose and 33.3 mL/day/kg body weight which is 2 times of the middle dose were set as low dose and high dose respectively. The doses were administered to the mice intragastrically at 1.2 mL/day for 32 days continuously and their effects were measured.

[0065] Instrument: mice cage, timer.

[0066] Reagents: pregnant mare serum.

[0067] Detection methods: the test substances were administered to male mice once/day for continuous 32 days. The male mice were injected with 0.2 ml (10 IU) of 50 IU/ml pregnant mare serum intramuscularly 48 hours before the test for synchronizing their sexual cycle. The male mice were housed with female mice in same cages 30 min after the last administration of test substance, and their behaviors were observed in dark. The mating latency which was calculated as time-period from housing to the first mating and mating frequency within 20 min were recorded.

Results

1. Mating Latency in Mice

[0068] The effects of the compositions on the mating latency in mice were shown in Table 8.

TABLE-US-00008 TABLE 8 Effects of the fermented Compositions A, B and C and the corresponding compositions without fermentation on the mating latency in mice (X ± SD) Number of Mating Groups Animals latency (s) Control 10 688.42 ± 60.39  Fermented A.sub.1 Unfermented 10 615.00 ± 42.32* and High-Dose Group unfermented A.sub.2 Unfermented 10 627.11 ± 26.05* Composition Middle-Dose Group A A.sub.3 Unfermented 10 608.50 ± 19.13* Low-Dose Group A.sub.4 Fermented 10  .sup. 570.30 ± 40.96*.sup..box-tangle-solidup. High-Dose Group A.sub.5 Fermented 10 598.60 ± 33.88* Middle-Dose Group A.sub.6 Fermented 10 601.10 ± 41.48* Low-Dose Group Fermented B.sub.1 Unfermented 10 592.15 ± 36.54* and High-Dose Group unfermented B.sub.2 Unfermented 10 604.50 ± 28.15* Composition Middle-Dose Group B B.sub.3 Unfermented 10 618.36 ± 42.34* Low-Dose Group B.sub.4 Fermented 10  .sup. 516.60 ± 41.26*.sup..box-tangle-solidup. High-Dose Group B.sub.5 Fermented 10  .sup. 531.53 ± 25.36*.sup..box-tangle-solidup. Middle-Dose Group B.sub.6 Fermented 10 593.62 ± 28.14* Low-Dose Group Fermented C.sub.1 Unfermented 10 564.30 ± 35.18* and High-Dose Group unfermented C.sub.2 Unfermented 10 582.15 ± 42.85* Composition Middle-Dose Group C C.sub.3 Unfermented 10 605.80 ± 28.19* Low-Dose Group C.sub.4 Fermented 10  .sup. 496.48 ± 26.72*.sup..box-tangle-solidup. High-Dose Group C.sub.5 Fermented 10  .sup. 510.20 ± 36.86*.sup..box-tangle-solidup. Middle-Dose Group C.sub.6 Fermented 10  .sup. 538.60 ± 42.18*.sup..box-tangle-solidup. Low-Dose Group *Significant difference compared with the control group (P < 0.05). .sup..box-tangle-solidup.Significant difference between the fermented and unfermented compositions at the same concentrations (P < 0.05).

[0069] The results (Table 8) show that the mating latency was shortened by all of the fermented Compositions A, B and C and the unfermented Compositions A, B and C at all doses, with the fermented Composition A at high dose, the fermented Composition B at high dose, and the fermented Composition C at high, middle and low doses acting superior to the corresponding compositions at the corresponding doses without fermentation.

2. Mating Frequency in Mice

[0070]

TABLE-US-00009 TABLE 9 Effects of the fermented Compositions A, B and C and the corresponding compositions without fermentation on mating frequency in mice (X ± SD) Number of Mating Groups Animals frequency Control 10 2.92 ± 1.17  Fermented A.sub.1 Unfermented 10 4.18 ± 0.98* and High-Dose Group unfermented A.sub.2 Unfermented 10 3.91 ± 1.38  Composition Middle-Dose Group A A.sub.3 Unfermented 10 3.42 ± 1.08  Low-Dose Group A.sub.4 Fermented 10 4.36 ± 1.21* High-Dose Group A.sub.5 Fermented 10 4.27 ± 1.56* Middle-Dose Group A.sub.6 Fermented 10 3.91 ± 1.14  Low-Dose Group Fermented B.sub.1 Unfermented 10 4.52 ± 1.06* and High-Dose Group unfermented B.sub.2 Unfermented 10 4.21 ± 1.21* Composition Middle-Dose Group B B.sub.3 Unfermented 10 4.08 ± 1.35* Low-Dose Group B.sub.4 Fermented 10 4.78 ± 1.42* High-Dose Group B.sub.5 Fermented 10 4.56 ± 1.05* Middle-Dose Group B.sub.6 Fermented 10 4.32 ± 1.25* Low-Dose Group Fermented C.sub.1 Unfermented 10 4.64 ± 0.96* and High-Dose Group unfermented C.sub.2 Unfermented 10 4.46 ± 1.34* Composition Middle-Dose Group C C.sub.3 Unfermented 10 4.25 ± 1.52* Low-Dose Group C.sub.4 Fermented 10 4.96 ± 1.02* High-Dose Group C.sub.5 Fermented 10 4.87 ± 1.41* Middle-Dose Group C.sub.6 Fermented 10 4.65 ± 1.35* Low-Dose Group *Significant difference compared with the control group (P < 0.05). .box-tangle-solidup. Significant difference between the fermented and unfermented compositions at the same concentrations (P < 0.05).

[0071] The results (Table 9) shows that the mating frequency was increased by all of the fermented Composition A at high and middle doses, the fermented Compositions B and C at all doses, the unfermented Composition A at high dose, the unfermented Compositions B and C at all doses.

CONCLUSION

[0072] The compositions made of maca, fructus lycii and fructus rubi fermented by probiotics were able to improve sexual function, and the fermentation with probiotics was able to enhance the sexual function-improving activities of the compositions made of maca, fructus lycii and fructus rubi.