TWI401032B - Cordyceps sinensis solid-phase fermentation applied as feed additives to enhance poultry sexual maturity - Google Patents

Description

Application of Solid Fermented Cordyceps sinensis Feed Additive to Promote Sexual Maturity in Poultry

The present invention refers to a new application for adding a solid fermentation ferment of Cordyceps sinensis to poultry feed to promote sexual maturity of poultry. A solid fermentation of Cordyceps sinensis promotes weight gain, cockscomb color, testosterone production and StAR gene expression levels of 7%, 125%, 133%, and 155%, respectively (compared with Cordyceps 1.9 mg-2.85 mg/kg) A feed additive for the development of a fine tube, the additive comprising adenosine (Adenosine) comprising a ferment is 0.95 mg - 2.85 mg / kg. The invention relates to a feed additive for improving spermatogenesis and production of testosterone, mainly containing Cordyceps sinensis. Powders and water-soluble powders and premixes applicable to solid fermented feed additives

There are more than 400 species of Cordyceps sinensis known, the most representative of which are two species, one is Cordyceps sinensis and the other is Cordyceps militaris. The Compendium of Materia Medica records that Cordyceps sinensis has Baofeiyi. Kidney, hemostasis, phlegm and blood stasis; "Compendium of Materia Medica" also pointed out that it can benefit lung and kidney, replenish the essence, stop bleeding and phlegm; "Chinese Dictionary" shows that Cordyceps sinensis is sweet, flat, fragrant, into The lungs and kidneys can be strong, beneficial to the lungs and kidneys, tonic deficiency, benefit essence, detoxification, stop bleeding and phlegm.

Cordyceps extract has hypoglycemic effect, Lo et al. (2004) pointed out that Cordyceps sinensis fruit extract and artificially cultured Cordyceps sinensis mycelium extract can reduce hyperglycemia in streptozotocin (STZ)-induced diabetic rats. Symptoms, and the effect of artificial culture is the same as that of natural fruiting bodies, which can also alleviate the weight loss of male rats caused by diabetes. It has been proved by animal experiments that it can increase the pancreas of diabetic mice induced by alloxan-induced diabetes. Dirty insulin secretion and reduced insulin metabolism. The anti-oxidation power of the artificially cultivated Cordyceps sinensis is about 10-30 times higher than that of the wild Cordyceps sinensis. The Cordyceps polysaccharide has the ability to scavenge free radicals. Cordycepin is the main secondary generation of Cordyceps militaris Xie, can induce apoptotic bodies in canine and human breast cancer cells. Cordycepin can also effectively reduce the production of nitric oxide, prostaglandins, interleukin IL-1 and tumor necrosis factor, and inhibit the expression of mRN A and protein of inflammatory factors iNOS and COX-2.

Cordyceps 50% methanol extract stimulates human mononuclear cells, and 100% methanol extract inhibits IL-2 and IFN-γ secretion by human peripheral blood mononuclear cells. Oral administration of Cordyceps extract in mice with lymphoma increases the phagocytic capacity of macrophages and increases mouse immunity against Salmonella infection. Adenosine, uridine and guanosine contained in Cordyceps sinensis are one of the active constituents of Cordyceps sinensis. Adenosine has obvious pharmacological effects, can reduce the resistance of coronary artery, and can play a protective role on the myocardium in hypoxia. Cordyceps sinensis mycelium extract treated Ledi's cells in the primary culture of mice, which stimulated the synthesis of steroids by cells. Cordyceps sinensis was given to mice that had not been sexually mature and sexually mature, and the three-day and seven-day tests significantly increased the level of plasma testosterone.

The invention adopts poultry-chicken as a test animal, and studies the application of the solid fermentation product of Cordyceps sinensis to promote the sexual maturity of poultry in poultry, and the test results prove that the spermatogenesis and the production of testosterone can be promoted, and the invention is completed.

The invention focuses on (1) adding a solid fermented product of Cordyceps sinensis to the feed as a feed additive for cock. (B) using weight growth, cockscomb color, sputum pills to observe the development of fine tube, steroid formation and gene expression as indicators, to promote growth and sexual maturity, shorten the feeding process and improve the weight gain of chickens.

The invention further provides a solid fermentation product of Cordyceps sinensis to promote weight growth, color of cockscomb, cockroach formation and StAR gene expression of 7%, 125%, 133%, 155%, respectively (control group and Cordyceps 1.9 mg-2.85/kg) Comparing) and feed additives for the development of fine tubes, the additive is adenosine (Adenosine) including 0.95 mg to 2.85 mg/kg.

In a preferred embodiment of the invention, the adenosine is 1.9 mg/kg.

In this experiment, the solid fermentation technology was used. After culture, the adenosine content reached an average of 1.988 mg/g, which was about the same as the adenosine content of the natural Cordyceps fruit body 2.47 mg/g.

1. Cordyceps sinensis activation and enrichment

The present invention uses Cordyceps sinensis (Berk.) Sacc. strain, which is cultured by solid state fermentation to produce high amount of adenosine. The Cordyceps strain is subdivided with PDA (potato dextrose agar) plate at 22 ° C. After culturing for 14 days, PDB culture solution was placed in a 500 mL triangular flask, and after sterilization, sterile water was added to the plate medium, and the hyphae were scraped off, and the bacterial solution was inoculated into a triangular flask, and at 22 At a constant temperature of °C, the rotation speed was controlled at 150 rpm and cultured for 7 days to activate the bacteria.

Second, solid fermentation medium

The present invention uses 200 g of wheat as a fermentation substrate, and 10% yeast extract, 0.1% CaCO3, 0.05%, MgSO4.7H2O, 0.1% NaH2PO4, 50% H2O, 0.05% KH2PO4, and 1% glucose are added. The pre-activated bacterial solution was inoculated with 10 mL in a solid medium, cultured at 23 ° C, and cultured for 7 days.

Third, the fermentation product adenosine (adenosine) analysis

The adenosine standards were diluted with 15% methanol to 0,20,40,60,80 and 100 μ g / ml, and then analyzed by high-performance liquid chromatography and the standard curve. The sample of the fermentation product was collected and dried by hot air at 45 ° C for 12 hours. After the sample was homogenized into a powder by a homogenizer, 1 g of each sample was weighed into a 15 mL centrifuge tube, and then 10 mL of 15% methanol was added and placed in an oven at 100 ° C for extraction. 1 hour. The extract was centrifuged at 14,000 x g for 10 minutes, and the supernatant was collected, filtered through a 0.22 μm filter, and placed in a 1.5 mL microcentrifuge tube for HPLC analysis.

Fourth, HPLC analysis conditions

The mobile phase is 0.02M KH2PO4:methanol (85:15), the sample injection HPLC volume is 20 μl , the flow rate is 1ml/min, the UV detector detects the wavelength of 254nm, and the analytical column is a highly efficient Cartesian reverse phase chromatography column RP. -18 endcapped (5 μ m).

5. Chickens and feed:

From 4 weeks old black-feathered chicken, a total of 72, divided into four groups, each group of 3 repetitions, each repeated 6 chickens, with cages as repeating units. The solid fermentation product of Cordyceps sinensis (Berk.) Sacc. was added to the feed, and the adenosine contained in the ferment was used as the index, which was 0 mg/kg, 0.95 mg/kg, 1.9 mg/kg, respectively. 2.85mg/kg.

Six, the color difference measurement of the cockscomb

After the sacrifice of the chicken, the maximum length and the maximum height are measured with a vernier caliper, and the area is calculated. The cockscomb was cut with a scalpel along the chicken skull and placed on a glass plate, which was then read with a color difference meter.

Seven, testicular tissue biopsy (evaluation of spermatogenesis)

The sample of Formalin fixed testicles was trimmed to 2-4 mm3, dehydrated with 95% and 100% alcohol, and the tissue was immersed in xylene for transparency, paraffin embedding and section dewaxing, stained with hematoxylin and eosin. The method was stained with hematoxyline and eosin stain (HE), and the sections were observed under a microscope.

Eight, analysis of testosterone in the blood

Determination of testosterone by RIA radioimmunoassay. The anti-tanosterone-containing serum was added to the sample and 3H-testosterone, co-cultured for two hours at room temperature, and the reaction was stopped with ice. Add activated carbon, activated carbon is adsorbable 3H-testosterone/testosterone complex with antiserum, so the bound part was removed by activated carbon, and after reacting for 15 minutes at 4 ° C, the activated carbon was centrifuged to measure the remaining free 3H-testosterone, and the estimated content was The amount of steroids.

Nine, reverse transcription polymerase chain reaction analysis of StAR gene expression

RNA was added to 4 μl of RT buffer (5×Frist-Strand Buffer), 1 μl of 0.1 M DTT, 1 μl of RNA reverse transcriptase and 1 μl of RNAase inhibitor. After reacting at 50 ° C for one hour, the reaction was stopped at 70 ° C for 15 minutes. The product was 20 μl cDNA. 2 μl of cDNA was added to 0.4 μl of U/μl Taq DNA polymerase, 1 μl of 10 mM dNTP, 5 μl of 10X PCR buffer, and 2 μl of 20 μM primer was added, as shown in the following table: The product was analyzed for gene expression after electrophoresis.

Example 1. Effect of fermentation of fermented solids of Cordyceps sinensis on adenosine content during fermentation

After inoculation with Cordyceps sinensis using solid-state fermentation substrate, the change of adenosine content at 0, 7, 14, 21 days of fermentation was analyzed by HPLC. As a result, as shown in Fig. 1 and Fig. 2, the adenosine retention time was about 7.57 minutes, and the fermentation process reached a maximum of 1.9 mg/g on the 7th day, and then gradually decreased.

Example 2. Effect of solid feed of Cordyceps sinensis on chicken body weight, cockscomb red-green index and blood testosterone concentration

The solid fermentation product of Cordyceps sinensis was mixed with chicken feed. The amount of additive was divided into control group (no addition), 0.95, 1.9, 2.85 mg/kg based on the amount of adenosine contained in the fermentation product per kg of feed. The composition of the feed formula is as follows

The results are shown in Fig. 3. The chickens were weighed after 6 weeks of feeding, and the weight of adenosine 1.9 mg/kg was increased by 7% to 2.38 kg in the control group of 2.38 kg. The degree of rosy rosin is expressed by the red and green index of the cockscomb. The results are shown in Figure 4. With the addition of Cordyceps sinensis significantly increased the red-green index compared with the control group, and the highest in the 1.9 mg group 1.8 was 125% higher than the control group 0.8. The results of measuring the concentration of testosterone in chicken blood are shown in Figure 5. The concentration of testosterone in the Cordyceps group increased the concentration of ketone. The optimal 2.8 ng/mL increased by 133% compared with the control group of 1.2 ng/mL.

Example 3. Effect of chicken fed Cordyceps sinensis solid fermented material on spermatogenesis and StAR gene expression of testosterone synthesis in chickens

The chickens were fed with the fermentation of the Cordyceps sinensis to the tissue sections of the testicles, as shown in the results of Figure 6. In the control group, only spermatogonial cells (Spermatogonia) existed in the seminiferous tubules. In the 0.95 mg group of Cordyceps adenosine, a small number of spermatozoa (spermatozoa) were observed, while in the 1.9 mg group, all the seminiferous tubules were in the 1.9 mg group. There is a large amount of sperm, but there is no sperm and a few spermatozoa (spermatid) in the 2.85 mg group. The performance of StAR gene was analyzed by RT-PCR. The results are shown in Fig. 7. The performance of StAR gene in the fermentation of Cordyceps sinensis was 1.15 mg in the group with the added amount of 1.15 mg and 155% in the control group.

in conclusion:

The solid feed of Cordyceps sinensis in the chicken feed can promote weight growth, cockscomb color, sperm spermatogenesis in the fine tube of the sputum, and the formation of the sputum and StAR gene, thus promoting growth and sexual maturity, shortening the feeding process and improving the chicken. The application of weight gain only, the present invention discloses that a solid fermented product of Cordyceps sinensis can be used for the manufacture of a new application of a feed additive for promoting sexual maturity in poultry.

Fig. 1 and Fig. 2 show the HPLC chromatogram of the adenosine of the solid fermentation product of Cordyceps sinensis according to the present invention and the change of adenosine content during fermentation. Fig. 3 and Fig. 4 show the effect of the solid fermented product of Cordyceps sinensis on the weight of the chicken and the redness index of the chicken. Fig. 5, Fig. 6, and Fig. 7 are the effects of the solid fermentation product of Cordyceps sinensis on the concentration of testosterone, the spermatogenic effect of the testis tissue, and the StAR gene expression of the testis tissue.

Fig. 1 is a view showing the HPLC analysis of adenosine containing a solid fermentation product of Cordyceps sinensis according to the present invention.

Fig. 2 is a graph showing changes in adenosine content during fermentation of the solid fermentation product of Cordyceps sinensis according to the present invention.

Fig. 3 is a graph showing the effect of the solid fermented product of the chicken of the present invention on the weight of the chicken.

Fig. 4 is a graph showing the effect of the solid fermented product of the chicken of the present invention on the red-green index of the cockscomb.

Fig. 5 is a graph showing the effect of the solid fermentation of the Cordyceps sinensis on the concentration of testosterone in the blood of the chicken of the present invention.

Fig. 6 is a graph showing the effect of the fermented product of the chicken of the present invention on the tissue spermatogenesis of the testis.

Fig. 7 is a graph showing the effect of the solid fermented product of the chicken of the present invention on the StAR gene expression of the testis tissue.

Claims (10)

The invention relates to a method for producing a solid-state fermented Cordyceps sinensis feed additive, which comprises the following steps: (a) activation and enrichment of Cordyceps sinensis strain: Cordyceps sinensis strain is subcultured with PDA (potato dextrose agar) plate and cultured at 22 ° C for 14 days. The scraped hyphae were rinsed with sterile water, and the bacterial solution was inoculated into a triangular flask containing PDB culture solution, and cultured at 22 ° C, 150 rpm for 7 days to activate the bacteria; (b) solid fermentation: 200 g of wheat was taken For the solid fermentation substrate, 10 mL (a) activated bacterial solution was inoculated at 23 ° C for 7 days, dried at 45 ° C for 12 hours, and dried to form a powder; (c) Analysis of the fermentation product adenosine content: 1 g (b) Solid-state fermentation powder was added to 10 mL of 15% methanol and extracted in an oven at 100 ° C for 1 hour, centrifuged at 14,000 x g for 10 minutes, and the supernatant was collected, filtered and analyzed by HPLC; (d) added to the feed: in the fermentation product The content of adenosine contained in the feed is 0.95 mg/kg, 1.95 mg/kg and 2.85 mg/kg in the feed; the feed additive can promote the application of sexual maturity in poultry. The manufacturing method of claim 1, wherein promoting the sexual maturity of the poultry refers to the spermatogenesis of the poultry, the formation of testosterone, the growth of weight, and the redness of the cocks. For example, the manufacturing method of claim 2, wherein promoting the production of testosterone, means that (a) the content of adenosine contained in the fermentation product is 2.85 mg/kg, and the amount of StAR gene produced by poultry testosterone is compared with that of the control. The group increased by 155%; and (b) the addition of 1.9 mg/kg of adenosine in the fermentation product to the feed increased the concentration of testosterone in the blood of the poultry by 133% compared with the control group. For example, in the manufacturing method of claim 2, wherein the adenosine content of the fermentation product is 1.9 mg/kg, the body weight of the poultry can be increased by 7% compared with the control group. A feed additive for a solid fermented product of Cordyceps sinensis, which is obtained by the manufacturing method of any one of claims 1-5, characterized in that the content of adenosine contained in the solid fermentation product is added as an additive index, respectively, adding 0.95 Mg/kg, 1.95 mg/kg and 2.85 mg/kg in feed can be used to promote sexual maturity in poultry. For example, the feed additive of claim 5 can be applied to powders, water-soluble powders and premixes. A feed additive for promoting sexual maturity of poultry is obtained by the manufacturing method of any one of claims 1-5, which is characterized in that the content of adenosine contained in the solid fermentation product is added as an additive index, and 0.95 is added respectively. Mg/kg, 1.95 mg/kg and 2.85 mg/kg in feed can promote sexual maturity in poultry. For example, the feed additive of claim 7 of the patent scope, wherein promoting the sexual maturity of the poultry refers to the spermatogenesis of the poultry, the formation of testosterone, the growth of weight, and the redness of the crown. For example, the feed additive of claim 8 of the patent scope can promote the sexual maturity of poultry. (a) The addition of the adenosine content of the fermentation product to the feed is 2.85 mg/kg, which can make the StAR gene expression of the poultry testosterone The control group increased by 155%, and (b) the content of adenosine contained in the fermentation product was 1.9 mg/kg, which increased the concentration of testosterone in the blood of the poultry by 133% compared with the control group. For example, the feed additive of claim 8 of the patent scope, which can promote the sexual maturity of poultry, refers to the addition of 1.9 mg/kg of adenosine content in the fermentation product to the feed, which can increase the body weight of the poultry by 7% compared with the control group.