A kind of yacon hydrolyzate and its preparation method and application

Abstract

The invention belongs to the field of microbial fermentation, discloses a yacon hydrolyzate and a preparation method and application thereof, selects yacon, washes with clean water, cuts into 3-5 mm thin slices, dries at 60-70°C for 14-16h, grinds into powder with a pulverizer, passes through an 80-100 mesh sieve, takes a certain amount of yacon dry powder, adds it to deionized water, stirs evenly, adds inulin exonuclease for enzymatic hydrolysis or inorganic acid for acid hydrolysis, prepares yacon hydrolyzate, and utilizes yacon hydrolyzate to culture schizochytrium to produce DHA oil. The invention provides a cheap, safe and efficient novel fermentation substrate raw material, which can be used for culturing schizochytrium to produce high-yield DHA, utilizes cheap, abundant, easy to obtain and difficult to store yacon as raw material, adopts a microbial fermentation method, produces high value-added DHA, not only greatly improves the economic value of yacon, but also effectively reduces the production cost of algae oil DHA, ensures a higher algae oil DHA quality, and is very beneficial to promote the industrialization development of schizochytrium fermentation to produce DHA.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and relates to a yacon hydrolyzate and a preparation method and application thereof, and more specifically, relates to a preparation method of the yacon hydrolyzate and a method for culturing schizochytrium to produce DHA oil. Background Art

[0002] Docosahexaenoic acid (DHA) is an ω-3 polyunsaturated fatty acid that is very beneficial to human health. The DHA content in human cerebral cortical nerve tissue, retina, egg cells and sperm is particularly rich. It is one of the indispensable components of fatty acids in the structure of human brain and visual cells. DHA has multiple physiological functions, including promoting the development of infant brain and vision, preventing or alleviating atherosclerosis and coronary heart disease, anti-tumor, preventing Alzheimer's disease and delaying aging. The latest research shows that DHA can also help reduce the incidence and mortality of novel coronavirus pneumonia virus (COVID-19) infection. As a new generation of functional health factor, DHA is widely used in the food and pharmaceutical industries. For example, it is currently added to infant formula as a food nutrient enhancer. Due to the wide range of uses of DHA, its global market demand has also greatly increased. Traditionally, DHA is extracted from deep-sea fish oil, but it is difficult to meet market demand due to its strong fishy smell, low DHA content, high cholesterol and other unsaturated fatty acid content in fish oil, difficulty in separation and purification, limited fish resources, and increased marine pollution. With the continuous maturity of microbial fermentation technology and the gradual deepening of microbial oil research, the production of DHA by microbial fermentation can overcome the shortcomings of traditional fish oil extraction, easily realize large-scale industrial production, and has broad application prospects.

[0003] The microorganisms that can synthesize DHA in nature are mainly lower marine fungi and microalgae, such as Crythecodinium, Thraustochytrium, Schizochytrium, etc. Among them, Schizochytrium, also known as Schizochytrium, is a heterotrophic single-cell marine fungus with the advantages of fast growth, easy cultivation, high DHA content in oil and reliable food safety. It is considered to be one of the ideal microbial strains for producing DHA and is currently widely used in the industrial production of DHA. The food rights agencies in the United States and Europe have approved the addition of DHA from Schizochytrium to related foods. In 2010, the Ministry of Health of my country approved Schizochytrium DHA oil as a new resource food.

[0004] Schizochytrium is a heterotrophic microorganism. The current production cost of cultivating DHA oil produced by Schizochytrium is relatively high, which greatly limits the industrial production of DHA produced by microbial fermentation. In order to reduce production costs, many researchers use some industrial and agricultural wastes such as crude glycerol (biodiesel byproduct), sugarcane bagasse, sweet sorghum juice, potato scraps, etc. as raw materials, and after certain treatment, cultivate Schizochytrium to produce DHA oil. However, the DHA yield obtained after fermentation is too low to be applied to industrial production, and the use of these organic wastes as raw materials will also cause people to worry about the edible safety of the product. Therefore, it is very necessary to find a safe, cheap and efficient substrate as a raw material to cultivate Schizochytrium to ferment and produce DHA.

[0005] Yacon, also known as Jerusalem artichoke, snow lotus potato, and ground ginseng fruit, is a cheap non-food crop that can adapt to different altitudes, climates, and soils. It is widely planted around the world and is cultivated in Yunnan, Guizhou, Sichuan, Fujian, Hunan, Hubei, and other provinces in my country. The crop has a high yield, with a single plant producing 3-5 kg ​​of tubers and an acreage yield of 5-7 tons of tubers. Yacon tubers are currently mostly used for fresh food, but their water content is high, so they are not easy to store. The rotting of a large amount of yacon causes a huge waste of resources. A large amount of carbohydrates are stored in yacon tubers, mainly oligofructose, and also include a certain amount of inulin, fructose, and a small amount of glucose. Among them, oligofructose and inulin are both fructans with a linear structure, which are formed by the dehydration polymerization of D-fructose through β-(2→1) glycosidic bonds, and the reducing end is a glucose residue connected by α-(2→1) glycosidic bonds. In addition, yacon also contains polyphenols, flavonoids, vitamins, amino acids, inorganic salts and other substances, which are necessary for the growth and metabolism of microorganisms. Therefore, yacon is expected to be used as an excellent raw material for microbial cultivation and has great potential for cultivating industrial fermentation microorganisms. Summary of the invention

[0006] In order to make up for the shortcomings of the prior art, the present invention provides a safe, cheap and efficient substrate as a raw material to culture Schizochytrium to ferment and produce DHA, which is achieved by the following technical scheme:

[0007] The invention discloses a method for preparing a yacon hydrolyzate. The method comprises the following steps: selecting yacon without pests, mildew or decay, washing the yacon with clean water, cutting the yacon into 3-5 mm slices, drying the slices at 60-70 DEG C for 14-16 hours, grinding the slices into powder with a pulverizer, filtering the slices through an 80-100 mesh sieve, taking a certain amount of yacon dry powder, adding the powder into a certain volume of deionized water, stirring the mixture evenly, adding a certain amount of inulin exoenzyme for enzymatic hydrolysis or a certain amount of inorganic acid for acid hydrolysis, and preparing the yacon hydrolyzate.

[0008] Further, the enzymatic hydrolysis uses inulin exonuclease produced by a microorganism, the enzyme-producing microorganism is Kluyveromyces marxianus, the substrate concentration of yacon dry powder is 50-300 g / L, the pH is adjusted to 6.0-7.5 with 1 mol / L hydrochloric acid or sodium hydroxide solution, the enzyme addition amount is 30-60 U / g substrate, the reaction temperature is 40-50° C., the reaction time is 3-5 h, and then centrifuged to collect the supernatant to obtain yacon enzyme hydrolyzate;

[0009] Furthermore, the acid hydrolysis uses dilute hydrochloric acid or dilute sulfuric acid, the substrate concentration is 50-300g / L, the concentration of dilute hydrochloric acid or dilute sulfuric acid is 2-3% (m / v), the reaction temperature is 80-100°C, the reaction time is 50-80min, and then the pH is adjusted to 6.0-7.0 with 1mol / LNaOH solution, followed by centrifugation, and the supernatant is collected to obtain yacon acid hydrolyzate.

[0010] A yacon hydrolyzate is prepared according to the above enzymatic hydrolysis or acid hydrolysis method.

[0011] The invention discloses an application of yacon hydrolyzate, which utilizes the yacon hydrolyzate to culture Schizochytrium to produce DHA oil.

[0012] The specific steps include:

[0013] (1) Activation of strains: Take out the Schizochytrium glycerol tube strain stored at -80°C, transfer it to a slant culture medium, and culture it in a 25°C incubator for 48-72 hours;

[0014] (2) Seed solution preparation: Use an inoculation loop to pick up 3-4 loops of activated slant culture, inoculate into a 250-mL Erlenmeyer flask containing 30 mL of seed culture medium, and culture in a shaker at 25°C and 200 rpm for 48-60 h.

[0015] (3) Fermentation of yacon hydrolyzate: The fermentation medium was prepared using yacon enzymatic hydrolyzate or acid hydrolyzate. The seed liquid was inoculated at a rate of 5-10% into a 250-mL Erlenmeyer flask containing 30 mL of fermentation medium. The culture was shaken in a shaker at 25°C and 200 rpm for 96-120 h. The cells were centrifuged and the cell precipitate was collected. The cells were lysed by the acid-heat method and the DHA oil in the cells was extracted using a chloroform-methanol solution (2:1, v / v).

[0016] The specific process is as follows: add 4 mol / L HCl solution to the bacterial cells, shake evenly, place at room temperature for 30 minutes, place in a boiling water bath for 10 minutes, then cool at -20°C for 10 minutes, take out and add 2 times the volume of chloroform-methanol (v / v2:1) mixed solvent, mix completely, centrifuge at 4500r / min for 15 minutes, aspirate the lower chloroform phase into a dry test tube, evaporate and remove chloroform to obtain oil, place in a 70°C drying constant temperature box until constant weight, and weigh the oil amount.

[0017] Furthermore, the components of the slant culture medium are, in g / L, 30% glucose, 10% yeast extract powder, 2% peptone, 15% sea crystal, 15% agar powder, and pH 6.0.

[0018] Furthermore, the components of the seed culture medium are, in g / L, 30% glucose, 10% yeast extract powder, 2% peptone, 15% sea crystal, and pH 6.0.

[0019] Furthermore, the composition of the fermentation medium is, in g / L, 7.5% yeast extract, 7.5% sodium glutamate, 1% MgSO4·7H2O2, 1% KH2PO4·H2O, 15% sea crystal, and pH 6.0.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The yacon hydrolyzate is prepared by the present invention. The selected raw material yacon is a cheap, abundant and easily available non-grain crop biomass. Compared with the current fermentation industry which is mainly based on grain starch raw materials, it has the unique advantage of "not competing with others for grain". The present invention selects inulin exoenase or inorganic acid produced by Kluyveromyces marxianus as a catalyst. The hydrolysis process is simple, the reaction time is short, the energy consumption is low, the polysaccharide in the raw material is completely hydrolyzed, the hydrolysis yield is high, the hydrolyzate contains fermentable sugars that can be easily metabolized and utilized by microorganisms, and the hydrolyzate also contains polyphenols, flavonoids, vitamins, amino acids, inorganic salts and other nutrients required for the growth and metabolism of microorganisms. Therefore, the prepared yacon hydrolyzate is very beneficial to the cultivation of microorganisms.

[0022] The yacon hydrolyzate prepared by the present invention is used to culture Schizochytrium to produce DHA oil. The fermentation results show that compared with glucose or fructose, which are commonly used commercial carbon sources in the fermentation industry, the total antioxidant capacity of Schizochytrium cells cultured with the yacon hydrolyzate is increased by 85.3% and 96.2%, respectively, the content of active oxygen in the cells that causes cell damage and lipid peroxidation is reduced by 68.8% and 67.9%, respectively, the cell biomass is increased by 15.4% and 21.1%, respectively, the oil yield is increased by 16.4% and 12.6%, respectively, and the DHA yield is increased by 28.7% and 32.1%. The results fully illustrate that, compared with glucose or fructose, which are commonly used carbon sources in the fermentation industry, using the yacon hydrolyzate as a substrate is more conducive to improving the antioxidant capacity of Schizochytrium cells, reducing the harmful active oxygen content in the cells, and more effectively promoting the growth of Schizochytrium cells, oil accumulation and DHA production, showing a result that is better than that of commercial glucose or fructose in culturing Schizochytrium to produce DHA oil.

[0023] In summary, the present invention provides a cheap, safe and efficient new type of fermentation substrate raw material - yacon hydrolyzate, which can be used to cultivate Schizochytrium to produce high-yield DHA. The cheap, abundant, easily available and difficult to store yacon is used as a raw material, and a microbial fermentation method is adopted to produce high-value-added DHA, which not only greatly improves the economic value of yacon, but also effectively reduces the production cost of algae oil DHA, ensures a high quality of algae oil DHA, and is very beneficial to promote the industrialization development of Schizochytrium fermentation to produce DHA. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention in any way.

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0026] Example 1 Preparation of Yacon Hydrolyzate by Enzymatic Hydrolysis

[0027] 1. Select 5kg of yacon tubers that are free of pests, mildew, and rot, wash them with clean water, and cut them into 3-5mm slices;

[0028] 2. Place in a 65°C oven and bake for 16-18 hours, dry, grind into powder with a pulverizer, and pass through an 80-mesh sieve to obtain yacon dry powder;

[0029] 3. Add 20g of yacon powder to 100mL of deionized water and adjust the pH to 7.0 with 1mol / L hydrochloric acid or sodium hydroxide solution;

[0030] 4. Add 0.04 g of inulin exonuclease, which was purchased from Wuhan Jinke Tiancheng Technology Co., Ltd. and produced from Kluyveromyces marxianus, with an enzyme activity of 20,000 U / g. Stir evenly, place in a 40°C water bath, and let react for 4 hours;

[0031] 5. Centrifuge the reaction solution at 10,000 rpm for 10 min, collect the supernatant, and obtain yacon hydrolyzate.

[0032] Example 2 Preparation of Yacon Hydrolyzate by Acid Hydrolysis

[0033] 1. Select 5kg of yacon tubers that are free of pests, mildew, and rot, wash them with clean water, and cut them into 3-5mm slices;

[0034] 2. Place in a 65°C oven and bake for 16-18 hours, dry, grind into powder with a pulverizer, and pass through an 80-mesh sieve to obtain yacon dry powder;

[0035] 3. Add 20 g of yacon powder into 100 mL of 2% (m / v) dilute hydrochloric acid solution;

[0036] 4. React in 85℃ water bath for 4h;

[0037] 5. Adjust the pH value of the reaction solution to 7.0 with 1 mol / L sodium hydroxide solution, centrifuge the reaction solution at 10,000 rpm for 10 min, collect the supernatant, and obtain yacon hydrolyzate.

[0038] Example 3 Using Yacon Hydrolyzate to Cultivate Schizochytrium to Produce DHA Oil

[0039] 1. Activation of strains: Take out the Schizochytrium glycerol tube strain stored at -80℃, transfer it to a slant culture medium, the composition of which is (g / L): 30% glucose, 10% yeast extract powder, 2% peptone, 15% sea crystal, 15% agar powder, pH 6.0, and culture it in a 25℃ incubator for 72h;

[0040] 2. Seed solution preparation: Use an inoculation loop to pick 3-4 loops of activated slant bacteria and inoculate them into a 250mL Erlenmeyer flask containing 30mL of seed culture medium. The composition of the seed culture medium is (g / L): 30% glucose, 10% yeast extract powder, 2% peptone, 15% sea crystal, pH 6.0. Shake and culture in a shaker at 25°C and 200rpm for 48h.

[0041] 3. Shake flask fermentation: Use yacon hydrolyzate to prepare fermentation medium, add nitrogen source and inorganic salt, its composition is (g / L): yeast extract powder 7.5, sodium glutamate 7.5, MgSO4·7H2O2, KH2PO4·H2O 1, sea crystal 15, pH 6.0, according to the inoculation amount of 10%, the seed liquid is added to a 250mL triangular flask containing 30mL fermentation medium, shaken and cultured in a shaker at 25℃ and 200rpm for 96h, and samples are taken to determine cell biomass, oil yield and DHA yield. Biomass determination method: Take 2mL of fermentation liquid, centrifuge at 10000r / min for 20min, discard the supernatant, place the precipitate at 80℃ to dry to constant weight, and weigh; the oil yield determination method uses the acid-heat method described above to lyse the cells and then use chloroform-methanol solution (2:1, v / v) to extract DHA oil in the cells; the DHA yield determination method uses methyl esterification of oil and then uses GC-MS to determine the fatty acid composition content;

[0042] 4. After 96 hours of fermentation of yacon hydrolyzate, the cell biomass reached 48.1 g / L, the oil yield reached 23.9 g / L, and the DHA yield reached 10.8 g / L. Compared with the control experiment results using commercial glucose as the carbon source, the biomass, oil yield and DHA yield increased by 15.4%, 16.4% and 28.7%, respectively. Compared with the control experiment results using commercial fructose as the carbon source, the biomass, oil yield and DHA yield increased by 21.1%, 12.6% and 32.1%, respectively.

[0043] The embodiments of the present invention are not limited thereto. Based on the above contents of the present invention, according to the common technical knowledge and common methods in the art, without departing from the above basic technical ideas of the present invention, the present invention may also have other embodiments. The present invention may also be modified, replaced or changed in other forms, all of which fall within the scope of protection of the present invention.

Claims

1. A method for culturing Schizochytrium to produce DHA oil using yacon hydrolyzate, characterized in that: The method comprises the following steps of: selecting yacon without pests, mildew or decay, washing with clean water, cutting into 3-5 mm slices, drying at 60-70° C. for 14-16 h, grinding into powder with a pulverizer, passing through an 80-100 mesh sieve, adding the yacon dry powder into deionized water, stirring evenly, adding inulin exo-acid to perform enzymatic hydrolysis or inorganic acid to perform acid hydrolysis, and preparing yacon hydrolyzate; Select inulin exonuclease produced by microorganisms, and the enzyme-producing microorganism is Kluyveromyces marxianus Kluyveromyces marxianus , the substrate concentration of yacon dry powder is 50-300 g / L, the pH is adjusted to 6.0-7.5 with 1 mol / L hydrochloric acid or sodium hydroxide solution, the enzyme amount is 30-60 U / g substrate, the reaction temperature is 40-50 ℃, the reaction time is 3-5 h, and then centrifuged to collect the supernatant to obtain yacon enzyme hydrolyzate; The specific steps of using the above-mentioned yacon hydrolyzate to cultivate Schizochytrium to produce DHA oil include: (1) Activation of strains: Take out the glycerol tube strain of Schizochytrium stored at -80 ℃, transfer it to the slant culture medium, and culture it in a 25 ℃ incubator for 48-72 h; (2) Seed solution preparation: Use an inoculation loop to pick up 3-4 loops of activated slant culture medium, inoculate into a 250 mL Erlenmeyer flask containing 30 mL of seed culture medium, and culture in a shaker at 25 °C and 200 rpm for 48-60 h. (3) Fermentation of yacon hydrolyzate: Use yacon enzymatic hydrolyzate or acid hydrolyzate to prepare the fermentation medium. Inoculate the seed liquid into a 250 mL Erlenmeyer flask containing 30 mL of fermentation medium at an inoculation rate of 5-10%. Incubate the culture in a shaker at 25 °C and 200 rpm for 96-120 h. Centrifuge and collect the cell precipitate. Lyse the cells using the acid-heat method and extract the DHA oil from the cells using a chloroform-methanol solution (2:1, v / v).

2. The method for culturing Schizochytrium to produce DHA oil using yacon hydrolyzate as claimed in claim 1, characterized in that: The acid hydrolysis uses dilute hydrochloric acid or dilute sulfuric acid, the substrate concentration is 50-300 g / L, the concentration of dilute hydrochloric acid or dilute sulfuric acid is 2-3% (m / v), the reaction temperature is 80-100°C, the reaction time is 50-80 min, and then the pH is adjusted to 6.0-7.0 with 1 mol / L NaOH solution, followed by centrifugation, and the supernatant is collected to obtain yacon acid hydrolyzate.

3. The method for culturing Schizochytrium to produce DHA oil using yacon hydrolyzate as claimed in claim 1, characterized in that: The components of the slant culture medium are, in g / L, 30% glucose, 10% yeast extract powder, 2% peptone, 15% sea crystal, 15% agar powder, and pH 6.

0.

4. The method for culturing Schizochytrium to produce DHA oil using yacon hydrolyzate as claimed in claim 1, characterized in that: The components of the seed culture medium are, in g / L, 30% glucose, 10% yeast extract powder, 2% peptone, 15% sea crystal, and pH 6.

0.

5. The method for culturing Schizochytrium to produce DHA oil using yacon hydrolyzate as claimed in claim 1, characterized in that: The composition of the fermentation medium is calculated in g / L as follows: 7.5% yeast extract, 7.5% sodium glutamate, 2% MgSO4·7H2O, 1% KH2PO4·H2O, 15% sea crystal, pH 6.0.