Research On The Extraction Process Of Total Isochlorogenic Acid From Stevia And Its Development As Feed Additives

As the main source of stevia sugar,stevia has the largest planting area and export volume in China.However,the traditional stevia extraction process produces a large amount of stevia waste residue.The direct disposal of waste residues not only pollutes the environment,but also causes a waste of functional components such as isochlorogenic acid.At present,feed additives developed with chlorogenic acid have been widely used in the livestock and poultry breeding industry,but there are still few breeding inputs developed with isochlorogenic acid.Isochlorogenic acid is similar in structure to chlorogenic acid,and their biological activities are also similar,such as antioxidant,antibacterial,antiviral,etc.,which is a potential resource for the development of feed additives.This article mainly explores the feasibility of stevia total isochlorogenic acid as a feed additive from three aspects of raw material source,quality control and function evaluation,and the specific content is as follows:(1)The extraction process of simultaneously extracting steviol glycosides and total isochlorogenic acid from Stevia has been developed,which provided a guarantee for the source of raw materials for stevia total isochlorogenic acid.Refering to the patent method,the dry leaf powder of stevia is extracted with 75-95% C1-C3 short-chain alcohol aqueous solution at 60℃ hot reflux for 2 times,and the ratio of solid to liquid was 1:8 and 1:7,and the extraction time was 3 h and 2 h.Combine the filtrate and use a rotary evaporator to dry it into an extract in low-temperature.The extract adopts ethyl acetate: water=1:1,material-liquid ratio 1:8.5,normal temperature,liquid-liquid extraction twice.The ester-soluble layer solutions are combined and concentrated to extract by rotary evaporation at a temperature of 60°C and a vacuum of-0.1±0.01 Mpa.The extract is powdered and then 10-30% C1-C3 short-chain alcohol aqueous solutionl is used,the ratio of material to liquid is 1:8.35,55℃,and solid-liquid extraction is performed once,and7.65 g of stevia total isochlorogenic acid can be obtained.The yield is 3.83%.The content of total isochlorogenic acid in the product extract measured by HPLC external standard method is 53.28%,and the total phenolic acid content was 64.49%.The verification results show that the process can realize the simultaneous extraction of steviol glycosides and total isochlorogenic acid in stevia,and concentration of total isochlorogenic acid in the product is high.(2)The HPLC characteristic chromatogram of stevia total isochlorogenic acid product was established to ensure the controllability and standardization of its quality.Refer to "Chinese Veterinary Pharmacopoeia" High Performance Liquid Chromatography(General Principle 0512),using Phenomenex Kinetex C18 chromatographic column,using 0.1%trifluoroacetic acid water and acetonitrile as mobile phases for gradient elution,column temperature 25℃,detection wavelength 330 nm,flow rate 1.5m L/min,injection volume3μL,successfully established 20 batches of stevia total isochlorogenic acid product HPLC characteristic chromatogram,identified 7 characteristic peaks,designated isochlorogenic acid A as the reference peak,calculated the characteristic peaks The relative retention time,the results are all within ±0.1% of the specified value,and the relative retention time RSD is less than ±0.3%,all of which meet the specified requirements,and can be used to quickly and effectively control the quality of stevia total isochlorogenic acid products of different production batches.(3)The in vitro antioxidant activity,the anti-oxidation effect of oil and the anti-mildew effect of feed of stevia total isochlorogenic acid were evaluated.The DPPH,FRAP and ABTS methods were used to evaluate the antioxidant activity of stevia total isochlorogenic acid in vitro.The Schaal oven heat test method and the detection of acid value and peroxide value in oil were used to evaluate the anti-oxidation effect of oil of stevia total isochlorogenic acid.The feed routine test and the national standard method were used to count the total number of molds in the feed to evaluate the anti-mold effect of stevia total isochlorogenic acid.The results show that: 0.1-1.0 mg/m L stevia total isochlorogenic acid has a scavenging rate of about 95.2% for DPPH free radicals,which is equivalent to VC but significantly better than propyl gallate;its FRAP and ABTS total antioxidant capacity are respectively It is 7.39 mmol/g and 1.42 mmol/g,which are better than Trolox;the best addition amount of stevia total isochlorogenic acid has better antioxidant effect on oil than the national standard addition amount of 0.2‰ BHA;0.5‰ of stevia total isochlorogenic acid of can effectively reduce the total number of molds in the feed.This result shows that stevia total isochlorogenic acid has strong in vitro antioxidant activity,oil anti-oxidation effect and feed anti-mold effect,and has broad development and utilization prospects in the field of feed antioxidants and anti-mold agents.