Patent Publication Number: US-2017347689-A1

Title: A probiotic-fermented maca composition and a method for preparing the composition and use of the composition

Description:
TECHNICAL FIELD 
     The present invention relates to a maca composition fermented by one or more bowel probiotics and a method for preparing the composition, and belongs to the field of functional health drinks. 
     BACKGROUND TECHNIQUE 
     Maca ( Lepidium meyenii  Walp) is a plant of the  Lepidium  genus, crucifer family native to the Andes region with an altitude of 3500-4500 m in South America. Maca has been eaten as food in South America for 5800 years, and used traditionally as food and herbal medicine for improving sexual function, enhancing fertility, ameliorating fatigue and tumor, and treating depression, asthma and menopausal syndrome. Maca has also been planted in Tashkurgan County in Xinjiang Region and Lijiang area in Yunnan province in China. Maca powder was approved as New resource food on Announcement No. 13 issued by Ministry of Health of China in 2011. 
     Maca is rich in nutrients. The dried maca root contains 10.2% proteins, 9-12% sugars, 59% carbohydrates, and a remarkable amount of minerals such as potassium, calcium, iron, zinc, etc. The maca has an appropriate amino acid constitution which has a high amount of branched amino acids which have important physiological activities. The maca comprises a less amount of fatty acids, up to 52% of which is unsaturated ones such as linoleic acid, linolenic acid, etc. The proteins, amino acids, carbohydrates, minerals and so on contained in maca have important contributions to anti-anemia, anti-blood-sugar-decline-after exercise, anti-fatigue activities of maca. Maca is also relatively rich in maca polysaccharides which are active substances capable of clearing superoxide radicals and inhibiting hydroxyl radicals. 
     Maca alkaloids are important secondary metabolites of the maca. Macamide and macaene, which are alkaloids of amides and contain special amide bond formed between amino group and fatty acid, are active ingredients obtained from maca extract by a Chinese American botanist B. L. Zheng in 1999. Macamide is an ingredient uniquely found in maca, and macaene, which is an acyclic ketoacid with small polarity, is normally found in chloroform extract of maca and also can be dissolved in methanol. Both the macamide and macaene play important roles in regulation of human endocrine system, such as systemically regulate body function, balance endocrine system, and improve sexual function, just like hormones. In 1980, glucosinolates (β-thioglucoside-N-hydroxysulfates) and isocyanates having volatility were found in maca by Johns. It has been found that isothiocyanates include mainly p-methoxybenzyl isothiocyanate and benzyl isothiocyanate. Glucosinolates are hydrophilic anionic secondary metabolites containing sulfur and nitrogen in maca which have a variable side chain (R) and a thio-β-D-glucopyranose group in molecules. The glucosinolates can produce cyanide, oxazolidine thione, thiocyanate and isothiocyanate enzymatically by myrosinase or bacterial enzymes in gastrointestinal tract. It was predicted that the fertility and sexual function-improving activity of the maca are possibly attributed to the glucosinolate and benzyl isothiocyanate which were also reported to have activity against cancers, such as stomach, esophagus, and lung cancer and leukemia and so on. 
     Fructus lycii is a dried ripe fruit of  Lycium chinense  Mill. or  Lycium barbarum  L. of Solanaceae family, and grown mainly in Hebei and Ningxia provinces in China, and also in Japan, Korea, Europe and North America, etc. Fructus lycii is an important herbal drugs in Chinese traditional medicine from ancient, and written as enhancing kidney, generating sperm, protecting liver, sparkling eyes, strengthening bones, ameliorating fatigue, lighting up face, calming nerve, and lengthening life in the “Compendium of Materia Medica”, as strengthening muscles and bones along with long-term usage in “Shen Nong&#39;s Herbal Classic”, as invigorating essential qi in “Mingyi Bielu”, and as invigorating body and ameliorating fatigue in “Herbal Food Medicine”. As verified by modern immunologic experiments and clinical medical experiments,  Lycium barbarum  L. has activities like immunomodulatory function-improvement, anti-oxidation, anti-aging, anti-fatigue, anti-cancer, anti-tumor, anti-hypertension, anti-hyperlipidemia, anti-hyperglycemia, protection of liver, kidney and neural system, and acceleration of excretion of harmful substances, especially heavy metals and carcinogens. Fructus lycii was recognized officially as both food and drug by Ministry of Health of China in 1988. Fructus lycii comprises mainly active ingredients, such as alkaloids, flavonoids and glycosides thereof, organic acids, coumarins, lignans, polysaccharides, etc. 
     Fructus rubi (i.e.  Rubus chingii  Hu) is a plant of  Rubus  genus, Rosaceae family, and grown mainly in East China, such as Zhejiang, Fujian, Anhui, Jiangxi, Jiangsu, etc. The immature dried fruit of the  Rubus chingii  Hu is used as a medicine for strengthen kidney, arresting spontaneous emission, reducing excessive urination, and for treating liver deficiency, enuresis, frequent micturition, impotence, premature ejaculation, spermatorrhea, spontaneous emission, etc. As verified by modern pharmacological research, Fructus rubi warms kidney, reinforces yang, acts against mutagenesis, oxidation, improves memory, delays aging, enhances immune activity, and inhibits bacteria. It was also observed from vaginal smear and endometrial slice of rats and rabbits that the fructus rubi administered had estrogen-like activities. The chemical components reported so far to be found in the fructus rubi comprises Fupenzic acid, Ellagic acid, β-sitosterol, Labdane diterpene glycoside goshonoside F5, and flavonoids triterpene acid. 
     Human gastrointestinal tract is colonized by a huge amount of microflora in which probiotics play crucial role on human health. Probiotics can catabolize food components which cannot be catabolized by digestive system, produce short-chain fatty acids, and supply nutrients for gastrointestinal mucosal cells. Probiotics can also convert inactive precursors of drug components to its active forms by catabolizing the precursors, especially naturally occurring ones, ingested orally. Currently used Probiotic formulations are mainly ones comprising simply live probiotics or ones comprising simply probiotic cells. 
     Currently used maca-containing products include maca liquor, maca jam, maca gum, etc. The more frequently seen maca-containing products are health foods and medicines made from powder of dried maca root or from concentrated maca extract. Dozens of health products mainly for enhancing energy, increasing fertility, improving sexual function and treating menopausal syndrome, etc. have been developed from worldwide including United States, Japan, Australia, Spain, Peru, United Kingdom, Taiwan, Hong Kong, etc. The commercially available maca-containing products are mainly made by extracting the maca or grinding the maca directly in combination with other active components, and there is no a product made by fermenting the maca or a composition containing the maca with probiotics yet. 
     The Content of the Invention 
     The present invention provides a probiotic-fermented composition made of maca, fructus lycii and fructus rubi, more particularly a fermented composition obtained by enzymatically hydrolyzing an extract of maca, fructus lycii and fructus rubi, and fermenting the hydrolysate with multiple enteric probiotics, and a method for preparing the composition, as well as use of the composition in the manufacture of a health product, food or beverage for improving fatigue tolerance and sexual function. 
     One purpose of the present invention is to provide a probiotic-fermented maca composition for improving fatigue tolerance and sexual function, 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 with a high optimal temperature; and wherein the probiotics are  Bifidobacterium  and  Lactobacillus.    
     Preferably, said composition of the present invention comprises: 3.0% (g/ml) of maca, 3.0% (g/ml) of fructus lycii, and 3.0% (g/ml) of fructus rubi. 
     Preferably, said  Bifidobacterium  used in the present invention is selected from  Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium adolescentis  or  Bifidobacterium infantis ; and said  Lactobacillus  used in the present invention is selected from  Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus rhamnosus  or  Lactobacillus plantarum . The strains are obtained from China General Microbiological Culture Collection Center (CGMCC China, Beijing) or isolated from feces or entero-mucosal membrane tissue of a healthy human. 
     To improve the taste of the composition, it is further preferable to add sweeteners into the composition of the present invention, wherein sweeteners 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) respectively. 
     Another purpose of the present invention is to provide a method for preparing a probiotic-fermented maca composition for improving fatigue tolerance and sexual function, comprising 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 above-obtained powders together; 
     maca, fructus lycii and fructus rubi are ground by conventional means resulting increased specific surface areas which facilitate dilution of active ingredients and thus increase yield of extraction. 
     (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; 
     (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. 
     Preferably, in the method of present invention as mentioned above, 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, 
     Preferably, in the method of present invention as mentioned above, 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 4 .7H 2 O, 4.4 mg/100 ml of ZnSO 4 .7H 2 O, and 50 mg/100 ml of MgSO 4 .7H 2 O. 
     Preferably, in the method of present invention as mentioned above, to improve the taste of the composition, 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. 
     Preferably, in the method of present invention as mentioned above, 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. 
     In a preferred embodiment, the composition of the present invention has a total sugar-content of 20-100 mg/ml and has a total probiotics quantity of 2.0-8.0×10 8  cells/ml as counted under microscope using blood cell counter (see Preparation Example 1). 
     A further purpose of the present invention is to provide use of the composition according to the present invention in the manufacture of a health product, food or beverage for improving fatigue tolerance and sexual function in a subject. 
     The composition of the present invention can be formulated directly into food or health product suitable for use in a subject suffered from fatigue and sexual dysfunction. 
     In a preferred embodiment, the composition prepared by the method of the present invention can be used to prepare a health product or a beverage for improving fatigue tolerance and sexual function. 
     For example, additional natural or synthetic ingredient(s) having the same, similar or different biological activities and having facilitative or synergistic activity with the active ingredients of the present invention while having no antagonistic activity with each other can be added into the composition of the present invention or supernatant thereof to form a mist, emulsion and the like suitable for oral administration by conventional means known in the art. 
     The composition of the present invention can also be dried and formulated into a solid powder into which additional natural or synthetic ingredient(s) having the same, similar or different biological activities and having facilitative or synergistic activity with the active ingredients of the present invention while having no antagonistic activity with each other can be added to form a capsule, tablet, granule and the like suitable for oral administration by conventional means known in the art. 
     To test anti-fatigue and sexual function-improving activity of the composition of the present invention, the effects of the composition of the present invention on duration time of pole-climbing and swimming, and levels of serum urea nitrogen, blood lactate and liver glycogen in mice were investigated. The result showed that the composition of the present invention could remarkably prolong the duration time of pole-climbing and swimming, keep the levels of serum urea nitrogen and blood lactate remarkably below the control, and keep the level of liver glycogen above the control in mice which indicated that the composition of the present invention had an ability to significantly improve fatigue-tolerance in a mouse. At the same time, the effects of the composition of the present invention on mating latency and mating frequency in male mice were investigated. The result showed that the composition of the present invention can significantly reduce the mating latency and increase mating frequency in male mice which indicated that the composition of the present invention had an ability to significantly improve sexual function in mice. The experiments also compared effects of the compositions fermented or unfermented with probiotics on fatigue-tolerance and sexual function improvement in mice and proved that the fermented composition was superior to the unfermented one which indicated that the method of the present invention improved physiological activity of the composition made of mixture of maca, fructus lycii and fructus rubi. 
    
    
     EXAMPLES 
     Preparation Example 1: Preparation of a Probiotic-Fermented Composition A 
     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 4 .7H 2 O, 4.4 mg/100 ml of ZnSO 4 .7H 2 O, and 50 mg/100 ml of MgSO 4 .7H 2 O), 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 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 
     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 4 .7H 2 O, 4.4 mg/100 ml of ZnSO 4 .7H 2 O, and 50 mg/100 ml of MgSO 4 .7H 2 O), 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 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 
     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 4 .7H 2 O, 4.4 mg/100 ml of ZnSO 4 .7H 2 O, and 50 mg/100 ml of MgSO 4 .7H 2 O), 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 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 
     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. 
     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”. 
     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. 
     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. 
     Instrument: a pole-climbing shelf, a swimming tank, UV spectrophotometer 754, ES-200 electronic balance, centrifuge, a swimming tank, intragastric feeder and so on. 
     Reagents: A serum urea nitrogen detection Kit (diacetyl oxime method). 
     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: 
     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 rd  falling, and the mean of the durations tested 3 times was set as “pole-climbing duration”. 
     2. Loaded Swimming Test: 
     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: 
     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 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) 
               
               
                   
                   
               
            
           
         
       
     
     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 standard , A Sample , and A Blank , were measured and calibrated with those measured with distilled water. 
     4. Measurement of Blood Lactate 
     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 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 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 
               
               
                   
               
            
           
         
       
     
     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 
     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. 
     Blank Agent: 1 mL of distilled water was placed in a clean centrifuge tube and set as the Blank Agent. 
     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. 
     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 
     1. Pole-climbing Test with Mice 
     
       
         
           
               
             
               
                 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      
               
               
                 Fermented 
                 A 1  Unfermented 
                 10 
                 443.10 ± 49.49*      
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 A 2  Unfermented 
                 10 
                 442.70 ± 59.04*      
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 A 
                 A 3  Unfermented 
                 10 
                 375.50 ± 70.83      
               
               
                   
                 Low-Dose Group 
               
               
                   
                 A 4  Fermented 
                 10 
                 511.60 ± 50.92* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 A 5  Fermented 
                 10 
                 502.40 ± 35.16* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 A 6  Fermented 
                 10 
                 486.40 ± 67.20* ▴   
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 B 1  Unfermented 
                 10 
                 472.10 ± 32.26*      
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 B 2  Unfermented 
                 10 
                 455.50 ± 46.30*      
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 B 
                 B 3  Unfermented 
                 10 
                 378.60 ± 63.21      
               
               
                   
                 Low-Dose Group 
               
               
                   
                 B 4  Fermented 
                 10 
                 564.20 ± 54.31* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 B 5  Fermented 
                 10 
                 546.30 ± 36.64* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 B 6  Fermented 
                 10 
                 528.40 ± 48.02* ▴   
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 C 1  Unfermented 
                 10 
                 502.30 ± 42.16*      
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 C 2  Unfermented 
                 10 
                 494.50 ± 38.45*      
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 C 
                 C 3  Unfermented 
                 10 
                 437.60 ± 32.18*      
               
               
                   
                 Low-Dose Group 
               
               
                   
                 C 4  Fermented 
                 10 
                 598.30 ± 31.06* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 C 5  Fermented 
                 10 
                 576.40 ± 42.28* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 C 6  Fermented 
                 10 
                 554.20 ± 50.83* ▴   
               
               
                   
                 Low-Dose Group 
               
               
                   
               
               
                 *Significant difference compared with the control group (P &lt; 0.05). 
               
               
                   ▴ Significant difference between the fermented and unfermented compositions at the same concentrations (P &lt; 0.05). 
               
            
           
         
       
     
     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 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      
               
               
                 Fermented 
                 A 1  Unfermented 
                 10 
                 426.40 ± 62.24*      
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 A 2  Unfermented 
                 10 
                 401.18 ± 56.54      
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 A 
                 A 3  Unfermented 
                 10 
                 412.00 ± 44.19      
               
               
                   
                 Low-Dose Group 
               
               
                   
                 A 4  Fermented 
                 10 
                 503.40 ± 61.10* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 A 5  Fermented 
                 10 
                 491.60 ± 57.31* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 A 6  Fermented 
                 10 
                 483.10 ± 84.76* ▴   
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 B 1  Unfermented 
                 10 
                 462.60 ± 53.12*      
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 B 2  Unfermented 
                 10 
                 446.20 ± 48.62*      
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 B 
                 B 3  Unfermented 
                 10 
                 408.54 ± 39.76      
               
               
                   
                 Low-Dose Group 
               
               
                   
                 B 4  Fermented 
                 10 
                 561.30 ± 62.14* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 B 5  Fermented 
                 10 
                 547.60 ± 45.24* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 B 6  Fermented 
                 10 
                 506.82 ± 54.68* ▴   
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 C 1  Unfermented 
                 10 
                 503.62 ± 48.21*      
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 C 2  Unfermented 
                 10 
                 485.25 ± 52.13*      
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 C 
                 C 3  Unfermented 
                 10 
                 446.30 ± 32.18*      
               
               
                   
                 Low-Dose Group 
               
               
                   
                 C 4  Fermented 
                 10 
                 601.20 ± 59.35* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 C 5  Fermented 
                 10 
                 586.75 ± 46.12* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 C 6  Fermented 
                 10 
                 543.50 ± 61.20* ▴   
               
               
                   
                 Low-Dose Group 
               
               
                   
               
               
                 *Significant difference compared with the control group (P &lt; 0.05). 
               
               
                   ▴ Significant difference between the fermented and unfermented compositions at the same concentrations (P &lt; 0.05). 
               
            
           
         
       
     
     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 
       
     
       
         
           
               
             
               
                 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 1  Unfermented 
                 10 
                 28.81 ± 1.92  
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 A 2  Unfermented 
                 10 
                 29.07 ± 1.78  
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 A 
                 A 3  Unfermented 
                 10 
                 28.59 ± 2.28  
               
               
                   
                 Low-Dose Group 
               
               
                   
                 A 4  Fermented 
                 10 
                 26.97 ± 1.59* 
               
               
                   
                 High-Dose Group 
               
               
                   
                 A 5  Fermented 
                 10 
                 27.31 ± 1.44* 
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 A 6  Fermented 
                 10 
                 27.97 ± 2.63  
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 B 1  Unfermented 
                 10 
                 28.35 ± 1.65  
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 B 2  Unfermented 
                 10 
                 28.98 ± 1.38  
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 B 
                 B 3  Unfermented 
                 10 
                 29.12 ± 2.06  
               
               
                   
                 Low-Dose Group 
               
               
                   
                 B 4  Fermented 
                 10 
                 26.34 ± 1.24* 
               
               
                   
                 High-Dose Group 
               
               
                   
                 B 5  Fermented 
                 10 
                 26.83 ± 1.55* 
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 B 6  Fermented 
                 10 
                 27.25 ± 1.38* 
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 C 1  Unfermented 
                 10 
                 27.13 ± 1.23* 
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 C 2  Unfermented 
                 10 
                 27.64 ± 1.45* 
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 C 
                 C 3  Unfermented 
                 10 
                 28.26 ± 1.63  
               
               
                   
                 Low-Dose Group 
               
               
                   
                 C 4  Fermented 
                 10 
                 25.08 ± 1.26* 
               
               
                   
                 High-Dose Group 
               
               
                   
                 C 5  Fermented 
                 10 
                 26.42 ± 1.32* 
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 C 6  Fermented 
                 10 
                 26.89 ± 1.48* 
               
               
                   
                 Low-Dose Group 
               
               
                   
               
               
                 *Significant difference compared with the control group (P &lt; 0.05). 
               
               
                 ▴ Significant difference between the fermented and unfermented compositions at the same concentrations (P &lt; 0.05). 
               
            
           
         
       
     
     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 
       
     
       
         
           
               
             
               
                 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 1  Unfermented 
                 10 
                 2.41 ± 0.30 
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 A 2  Unfermented 
                 10 
                 2.21 ± 0.17 
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 A 
                 A 3  Unfermented 
                 10 
                 2.19 ± 0.16 
               
               
                   
                 Low-Dose Group 
               
               
                   
                 A 4  Fermented 
                 10 
                  2.79 ± 0.34* 
               
               
                   
                 High-Dose Group 
               
               
                   
                 A 5  Fermented 
                 10 
                 2.47 ± 0.37 
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 A 6  Fermented 
                 10 
                 2.42 ± 0.24 
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 B 1  Unfermented 
                 10 
                  2.69 ± 0.21* 
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 B 2  Unfermented 
                 10 
                 2.31 ± 0.15 
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 B 
                 B 3  Unfermented 
                 10 
                 2.27 ± 0.16 
               
               
                   
                 Low-Dose Group 
               
               
                   
                 B 4  Fermented 
                 10 
                     3.16 ± 0.35* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 B 5  Fermented 
                 10 
                     2.89 ± 0.23* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 B 6  Fermented 
                 10 
                 2.52 ± 0.14 
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 C 1  Unfermented 
                 10 
                  2.93 ± 0.32* 
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 C 2  Unfermented 
                 10 
                  2.79 ± 0.25* 
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 C 
                 C 3  Unfermented 
                 10 
                  2.71 ± 0.15* 
               
               
                   
                 Low-Dose Group 
               
               
                   
                 C 4  Fermented 
                 10 
                     3.52 ± 0.19* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 C 5  Fermented 
                 10 
                     3.36 ± 0.17* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 C 6  Fermented 
                 10 
                     3.18 ± 0.21* ▴   
               
               
                   
                 Low-Dose Group 
               
               
                   
               
               
                 *Significant difference compared with the control group (P &lt; 0.05). 
               
               
                   ▴ Significant difference between the fermented and unfermented compositions at the same concentrations (P &lt; 0.05). 
               
            
           
         
       
     
     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 
       
     
       
         
           
               
             
               
                 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 1  Unfermented 
                 10 
                 22.70 ± 2.95* 
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 A 2  Unfermented 
                 10 
                 27.87 ± 3.22* 
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 A 
                 A 3  Unfermented 
                 10 
                 27.20 ± 3.76* 
               
               
                   
                 Low-Dose Group 
               
               
                   
                 A 4  Fermented 
                 10 
                 20.47 ± 2.17* 
               
               
                   
                 High-Dose Group 
               
               
                   
                 A 5  Fermented 
                 10 
                     22.16 ± 2.44* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 A 6  Fermented 
                 10 
                 24.33 ± 4.28* 
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 B 1  Unfermented 
                 10 
                 20.65 ± 2.81* 
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 B 2  Unfermented 
                 10 
                 25.36 ± 2.65* 
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 B 
                 B 3  Unfermented 
                 10 
                 26.05 ± 2.13* 
               
               
                   
                 Low-Dose Group 
               
               
                   
                 B 4  Fermented 
                 10 
                 18.54 ± 3.15* 
               
               
                   
                 High-Dose Group 
               
               
                   
                 B 5  Fermented 
                 10 
                     19.35 ± 2.86* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 B 6  Fermented 
                 10 
                     20.48 ± 2.15* ▴   
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 C 1  Unfermented 
                 10 
                 19.65 ± 2.98* 
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 C 2  Unfermented 
                 10 
                 20.58 ± 3.16* 
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 C 
                 C 3  Unfermented 
                 10 
                 22.15 ± 2.65* 
               
               
                   
                 Low-Dose Group 
               
               
                   
                 C 4  Fermented 
                 10 
                     14.24 ± 2.46* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 C 5  Fermented 
                 10 
                 15.68 ± 2.14* 
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 C 6  Fermented 
                 10 
                     15.95 ± 3.02* ▴   
               
               
                   
                 Low-Dose Group 
               
               
                   
               
               
                 *Significant difference compared with the control group (P &lt; 0.05). 
               
               
                   ▴ Significant difference between the fermented and unfermented compositions at the same concentrations (P &lt; 0.05). 
               
            
           
         
       
     
     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 
     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 
     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. 
     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”. 
     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. 
     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. 
     Instrument: mice cage, timer. 
     Reagents: pregnant mare serum. 
     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 
     The effects of the compositions on the mating latency in mice were shown in Table 8. 
     
       
         
           
               
             
               
                 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 1  Unfermented 
                 10 
                 615.00 ± 42.32* 
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 A 2  Unfermented 
                 10 
                 627.11 ± 26.05* 
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 A 
                 A 3  Unfermented 
                 10 
                 608.50 ± 19.13* 
               
               
                   
                 Low-Dose Group 
               
               
                   
                 A 4  Fermented 
                 10 
                     570.30 ± 40.96* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 A 5  Fermented 
                 10 
                 598.60 ± 33.88* 
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 A 6  Fermented 
                 10 
                 601.10 ± 41.48* 
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 B 1  Unfermented 
                 10 
                 592.15 ± 36.54* 
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 B 2  Unfermented 
                 10 
                 604.50 ± 28.15* 
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 B 
                 B 3  Unfermented 
                 10 
                 618.36 ± 42.34* 
               
               
                   
                 Low-Dose Group 
               
               
                   
                 B 4  Fermented 
                 10 
                     516.60 ± 41.26* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 B 5  Fermented 
                 10 
                     531.53 ± 25.36* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 B 6  Fermented 
                 10 
                 593.62 ± 28.14* 
               
               
                   
                 Low-Dose Group 
               
               
                 Fermented 
                 C 1  Unfermented 
                 10 
                 564.30 ± 35.18* 
               
               
                 and 
                 High-Dose Group 
               
               
                 unfermented 
                 C 2  Unfermented 
                 10 
                 582.15 ± 42.85* 
               
               
                 Composition 
                 Middle-Dose Group 
               
               
                 C 
                 C 3  Unfermented 
                 10 
                 605.80 ± 28.19* 
               
               
                   
                 Low-Dose Group 
               
               
                   
                 C 4  Fermented 
                 10 
                     496.48 ± 26.72* ▴   
               
               
                   
                 High-Dose Group 
               
               
                   
                 C 5  Fermented 
                 10 
                     510.20 ± 36.86* ▴   
               
               
                   
                 Middle-Dose Group 
               
               
                   
                 C 6  Fermented 
                 10 
                     538.60 ± 42.18* ▴   
               
               
                   
                 Low-Dose Group 
               
               
                   
               
               
                 *Significant difference compared with the control group (P &lt; 0.05). 
               
               
                   ▴ Significant difference between the fermented and unfermented compositions at the same concentrations (P &lt; 0.05). 
               
            
           
         
       
     
     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 
       
     
       
         
           
               
             
               
                 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 1  Unfermented 
                 10 
                 4.18 ± 0.98* 
               
               
                   
                 and 
                 High-Dose Group 
               
               
                   
                 unfermented 
                 A 2  Unfermented 
                 10 
                 3.91 ± 1.38  
               
               
                   
                 Composition 
                 Middle-Dose Group 
               
               
                   
                 A 
                 A 3  Unfermented 
                 10 
                 3.42 ± 1.08  
               
               
                   
                   
                 Low-Dose Group 
               
               
                   
                   
                 A 4  Fermented 
                 10 
                 4.36 ± 1.21* 
               
               
                   
                   
                 High-Dose Group 
               
               
                   
                   
                 A 5  Fermented 
                 10 
                 4.27 ± 1.56* 
               
               
                   
                   
                 Middle-Dose Group 
               
               
                   
                   
                 A 6  Fermented 
                 10 
                 3.91 ± 1.14  
               
               
                   
                   
                 Low-Dose Group 
               
               
                   
                 Fermented 
                 B 1  Unfermented 
                 10 
                 4.52 ± 1.06* 
               
               
                   
                 and 
                 High-Dose Group 
               
               
                   
                 unfermented 
                 B 2  Unfermented 
                 10 
                 4.21 ± 1.21* 
               
               
                   
                 Composition 
                 Middle-Dose Group 
               
               
                   
                 B 
                 B 3  Unfermented 
                 10 
                 4.08 ± 1.35* 
               
               
                   
                   
                 Low-Dose Group 
               
               
                   
                   
                 B 4  Fermented 
                 10 
                 4.78 ± 1.42* 
               
               
                   
                   
                 High-Dose Group 
               
               
                   
                   
                 B 5  Fermented 
                 10 
                 4.56 ± 1.05* 
               
               
                   
                   
                 Middle-Dose Group 
               
               
                   
                   
                 B 6  Fermented 
                 10 
                 4.32 ± 1.25* 
               
               
                   
                   
                 Low-Dose Group 
               
               
                   
                 Fermented 
                 C 1  Unfermented 
                 10 
                 4.64 ± 0.96* 
               
               
                   
                 and 
                 High-Dose Group 
               
               
                   
                 unfermented 
                 C 2  Unfermented 
                 10 
                 4.46 ± 1.34* 
               
               
                   
                 Composition 
                 Middle-Dose Group 
               
               
                   
                 C 
                 C 3  Unfermented 
                 10 
                 4.25 ± 1.52* 
               
               
                   
                   
                 Low-Dose Group 
               
               
                   
                   
                 C 4  Fermented 
                 10 
                 4.96 ± 1.02* 
               
               
                   
                   
                 High-Dose Group 
               
               
                   
                   
                 C 5  Fermented 
                 10 
                 4.87 ± 1.41* 
               
               
                   
                   
                 Middle-Dose Group 
               
               
                   
                   
                 C 6  Fermented 
                 10 
                 4.65 ± 1.35* 
               
               
                   
                   
                 Low-Dose Group 
               
               
                   
                   
               
               
                   
                 *Significant difference compared with the control group (P &lt; 0.05). 
               
               
                   
                 ▴ Significant difference between the fermented and unfermented compositions at the same concentrations (P &lt; 0.05). 
               
            
           
         
       
     
     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 
     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.