CN108812538A - A kind of preparation method of the nutritional egg with sight protectio and enhancing - Google Patents

A kind of preparation method of the nutritional egg with sight protectio and enhancing Download PDF

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CN108812538A
CN108812538A CN201810639292.1A CN201810639292A CN108812538A CN 108812538 A CN108812538 A CN 108812538A CN 201810639292 A CN201810639292 A CN 201810639292A CN 108812538 A CN108812538 A CN 108812538A
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egg
lutein
zeaxanthin
dha
feed
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张卫文
陈磊
刘璐
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Zao Neng Bio Tech Ltd Kunming
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Zao Neng Bio Tech Ltd Kunming
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/02—Breeding vertebrates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A23K10/22—Animal feeding-stuffs from material of animal origin from fish
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry

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  • Biomedical Technology (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Fodder In General (AREA)
  • Feed For Specific Animals (AREA)

Abstract

The present invention is a kind of preparation method of nutritional egg with sight protectio and enhancing.Step is：Laying hen several for selecting the health of laying period；In the daily feed of above-mentioned laying hen, while adding any one of each group in following three groups of constituents, first group：Lutein, marigold, other frequently seen plants rich in lutein；It is 0.0005%~0.01% that the quality of contained lutein, which accounts for the ratio of feed for nursing gross mass, in lutein quality or marigold or other plants rich in lutein；Second group：Zeaxanthin, corn, other frequently seen plants rich in zeaxanthin；It is 0.0003%~0.012% that the quality of contained zeaxanthin, which accounts for the ratio of feed for nursing gross mass, in zeaxanthin quality or corn or other plants rich in zeaxanthin；Third group：Fish meal, linseed, schizochytrium powder, schizochytrium mud, DHA fish oil, DHA algal oil, the quality of DHA contained therein are the 0.1~1.0% of feed for nursing gross mass；It is continuous to feed 1-6 weeks；Obtain egg.

Description

A kind of preparation method of the nutritional egg with sight protectio and enhancing
Technical field
The invention belongs to field of biotechnology, it is related to poultry farming, it is specifically a kind of to have effects that sight protectio and enhancing The preparation method of nutritional egg.
Background technique
The nutriments such as necessary amino acid, fatty acid, vitamin, minerals in egg rich in needed by human body, are consumption The daily necessity of person, while being also the good carrier of lutein, zeaxanthin and DHA.By nutrition regulation means, in egg Lutein is added in chicken feed, and perhaps DHA etc. can produce enrichment lutein or the egg of DHA.However, not having by changing also Become the feed ingredient that poultry is fed, produces the report containing high lutein, zeaxanthin and the egg of DHA three simultaneously.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, and for modern's work and life pressure, big, eyesight is asked The feature that topic takes place frequently, sight protectio and enhancing are in great demand, on the nutritive value of this traditional food of egg, the present invention is logical The feed addictive rich in lutein, zeaxanthin and DHA is crossed while adding into egg feedstuff, to produce while rich in leaf Huang The egg of element, zeaxanthin and DHA.
To solve the above-mentioned problems, a kind of system of the nutritional egg rich in lutein, zeaxanthin and DHA simultaneously of the present invention Preparation Method the technical solution adopted is that：
(1) laying hen several for selecting the health of laying period；
(2) in the daily feed of above-mentioned laying hen, each group in following three groups of constituents is added simultaneously according to mass percent Any one of, first group：Lutein, marigold, other frequently seen plants rich in lutein；Second group：Zeaxanthin, jade Rice, other frequently seen plants rich in zeaxanthin；Third group：Fish meal, linseed, schizochytrium powder, schizochytrium mud, DHA fish Oil, DHA algal oil；It is continuous to feed 1-6 weeks；
(3) the three kinds of additives added in above-mentioned egg feedstuff, lutein quality (or marigold or other yellow rich in leaf Element plant in contained lutein quality) account for feed for nursing gross mass ratio be 0.0005%~0.01%；Zeaxanthin The quality quality of contained zeaxanthin (or in corn or other plants rich in zeaxanthin) accounts for feed for nursing gross mass Ratio is 0.0003%~0.012%；Fish meal, linseed, schizochytrium powder, schizochytrium mud, DHA fish oil, institute in DHA algal oil Quality containing DHA is the 0.1~1.0% of feed for nursing gross mass；
The egg that laying hen produces, uses the content of its Lutein of high effective liquid chromatography for measuring for 0.5~5.0mg/100g Egg, using rp-hplc determination, wherein zeaxanthin is 0.3~6.0mg/100g egg, using efficient The content that gas chromatography measures wherein DHA is 50~500mg/100g egg；
Lutein (Lutein, 3 ' 3- dihydroxy-alpha-carotene) is that one kind is widely present in the plant such as vegetables, flowers, fruit Natural materials in object belong to " carotenoids class " race's substance, it is known that in nature there is more than 600 kinds of carotenoid, In only about 20 kinds be present in the blood and tissue of people.Medical experiment proves that natural carotenol contained in plant is a kind of property The excellent antioxidant of energy, a certain amount of lutein is added in food can prevent cell ageing and aging of body organs, simultaneously Visual impairment caused by senile eyeball retina macular degeneration and blindness can also be prevented, pass through a series of medical research, class Carrotene has been proposed as cancer preventive agent, life extender, ulcer resistant preparation, heart attack and coronary artery disease Inhibitor.Especially in terms of sight protectio and enhancing, lutein has a series of function, including：For the master of retina Pigment composition is wanted, the eyes of the mankind contain the lutein of a large amount, and this element is that human body can not manufacture, it is necessary to yellow by intake leaf It usually supplements, if lacking this element, eyes will blind；Phototoxis is protected the eyes from, the aging of eyes is delayed and is prevented Only lesion；Eyesight is protected, lutein can promote visual purple in retina cell as antioxidant and photoprotection (Rhodopsin) regeneration can prevent laser high myopia and detached retina, and can promote eyesight, protection eyesight, be particularly suitable for Student, driver et al. are edible；Alleviate visual fatigue symptom：(blurred vision, eye are dry and astringent, eye is swollen, ophthalmodynia, photophobia etc.)；Improve macula lutea Pigment density protects macula lutea, promotes macula lutea development；Prevent macular degeneration and retinal pigment degeneration etc..
Zeaxanthin (Zeaxanthin, 3,3 '-dihydroxy-beta carotene) is a kind of novel oil-soluble natural pigment, It is widely present in green leafy vegetable, flowers, fruit, fructus lycii and yellow maize.In nature usually with lutein, β-Hu Luo Bu Su, kryptoxanthin etc. coexist, and form carotenoid mixture.In human body, zeaxanthin be mainly distributed on eye, liver, pancreas, In the histoorgans such as kidney, spleen, ovary, play an important role to health.In eyes, zeaxanthin is concentrated mainly on view The macula lutea district center of film.Human body and animal itself can not synthesize zeaxanthin, it is necessary to pass through food or replenishers obtain.In human body Interior, zeaxanthin is a kind of powerful antioxidant, can also be protected by Anti-oxidation behaviors such as quenching singlet oxygen, removing free radicals Body tissue cell, thus protect biosystem from some since excessive oxidation reacts generated potential illeffects, Therefore there is antitumaous effect, prevention cardiovascular disease, prevention and other effects.If the not macular area of normal function, people Main visual function can gradually damage, or even have the danger of blindness.In macular area central point, incident light is most strong, the work of generation Property oxygen is also most.Epidemiological studies result also indicates that there is zeaxanthin specificity absorption most to damage to retina The effect of the blue ray of property, to protect the cone cell of fovea centralis.Many studies have shown that increasing maize in short term Matter intake can be such that macular pigment increases, to enhance the ability of macular area confrontation harmful substance and light ray damage, prevention With slow down senile macular degeneration.In addition, zeaxanthin itself has very high nutritive value, it can be in human liver after eating It is converted to biologically active vitamin A, it is disease-resistant to growth and development, protection eyesight and the epithelial cell, the raising that promote human body Ability extends the service life etc. with special effect.
DHA (Docosahexaenoic Acid, DHA, docosahexaenoic acid) is the important substance of human brain development, growth One of, it is the important composition ingredient of brain and retina, content is up to 20% in human brain cortex, in eye retina Proportion is maximum, accounts for about 50%.Lacking DHA intake will lead to DHA content decline in retinal tissue, and cause retina electric Scheme not normal.In addition, DHA plays the regeneration of visual pigment rhodopsin vital, and rhodopsin is in light It plays a crucial role in the system converted to visual pattern.Meanwhile it is newest research shows that the unsaturated fatty acids such as DHA also have drop Health benefits in terms of the eyes such as low intraocular pressure, crystalline lens pressure and glaucoma.Some researches show that contain in infant's diet and fill Sufficient DHA can make infant quick-eyed, and visual performance development is very fast.Since DHA concentration is relatively low in premature's body, spy is needed Not Bu Chong DHA to guarantee the normal development of premature's optic cell.Pregnant woman is rich in the nutriment of DHA in pregnancy period intake, leads to It crosses and is transported to fetal brain and retina, the nerve cell maturity of fetus can be made to improve.The U.S.《Clinical nutriology magazine》? At 2002 just it is reported that, baby feed rich in DHA 52 week of food after, vision obviously than in control group not into The healthy babies for eating DHA are sensitive.
Above-mentioned three kinds substances relevant to human body visual acuity and protection, cannot need to pass through meals synthesized by human body Food additionally takes the intake such as capsule.Wherein, the nutrient being can absorb when lutein is mankind's daily consumption fruit and vegetables, But absorption rate is generally lower, has been incorporated as dietary supplements early in lutein in 1996.Green pot-herb is leaf Huang The main dietary source of element, but wherein zeaxanthin is less.The United Nations FAO/WHO, the United Nations food additives expert committee Member can 1977 the 21st time report regulation：All to separate from known food, the unconverted pigment of chemical structure, use is dense When degree meets the natural concentration in former food again, it is considered as food, does not need toxicological information, and zeaxanthin is known food One of ingredient in object corn belongs to carotenoid, considers in conjunction with its physico-chemical analysis result, it is believed that it is safe and non-toxic, Ke Yizhi It scoops out in food, medicine and cosmetic industry, maximum usage amount to be 5.0g/Kg.It is deep-sea fish, linseed, rich in micro- algae powder Containing DHA, but it is difficult in the ordinary meal of most crowds comprising these foods, it is normal far from meeting by food intake The demand of human body.It is suitable for intake that each crowd DHA recommends in United States Medicine research institute (IOM) daily, is -18 years old 4 years old daily respectively 90-160 milligrams, daily 160 milligrams of adult, daily 200 milligrams of pregnant woman.Egg is universal as a kind of indispensable food of high nutrition It in people's lives, is a kind of very nutritious, relative low price common food, edible object is quite extensive, from 4 ~5 months babies are until old man, is suitable for edible egg.
While the present invention is by adjusting the optimal content of lutein, zeaxanthin, DHA three in egg, it ensure that Three together the effect of promote mutually, it is edible to safeguard eyes and nervous centralis health, be optimal to eyesight Effect is protected and enhanced, this has far reaching significance for the eye health level for improving China resident.Meanwhile feeding the present invention specially The feed of addition lutein, zeaxanthin and DHA, can significantly improve the immunity of laying hen, thus subtract significantly while benefit proposes The egg nutrient equilibrium of the use of few antibiotic, output is abundant, and yolk tinctorial yield is high, and antibiotic-free residual, mouthfeel is more aromatic, Also it is relatively beneficial to human health.
Bring benefit of the present invention is based on the following：One) lutein and zeaxanthin are only two types in eyes Carrotene, however, ratio collocation can not in most cases be optimal, such as green leaf in the common food of intake It is the main dietary source of lutein with vegetables, but wherein zeaxanthin is less.Two) lutein is with zeaxanthin The patron saint of DHA, DHA and lutein and zeaxanthin can more play the role of bringing out the best in each other with benefit, and retina is rich in DHA unsaturated fatty acid, DHA are easily oxidised, and such as lack lutein and zeaxanthin, mend DHA more again to retinal development With health-care effect and have a greatly reduced quality, and the anti-oxidation protection effect of lutein abundant contained by macular area can allow view Film is protected from the injury of fat oxidation, while can also prevent the generation of DHA fishlike smell.Three) some researches show that lutein, jade Cream-coloured matter and DHA (when such as being boiled, fried to egg, decocting cooking operation), can still retain major part under certain high temperature Activity.
Remarkable result of the present invention is, the egg rich in lutein, zeaxanthin and DHA while production, lutein, corn Yellow matter and the collocation of the content of DHA are rationally, edible to meet the needs of human body is for lutein, zeaxanthin and DHA simultaneously, and And play the role of effect and bring out the best in each other, can safeguard eyes and nervous centralis health, be optimal to the protection of eyesight and Reinforcing effect.Meanwhile laying hen is raised with the method for offer of the invention, lutein, zeaxanthin and DHA in feed can not only The nutritional ingredient of feed is protected, and the immunity of laying hen can be improved, thus greatly reduces the use of antibiotic, the egg of output Nutrient equilibrium is abundant, and yolk tinctorial yield is high, and antibiotic-free residual, mouthfeel is more aromatic, is also relatively beneficial to human health.
Specific embodiment
Below by embodiment, invention is further described in detail.
Embodiment 1：The laying hen for choosing 60 20 week old, is randomly divided into two groups, it is desirable that the laying hen between each group grows and lays eggs Situation no significant difference, one group of laying hen eats no added basal feed respectively during experiment, two groups of laying hens eat feed addition 0.01% lutein sterling, 0.002% zeaxanthin sterling with 2% algae oil (DHA content 40%), experimental period 42 It, daily observe laying hen growing state, situation of laying eggs, and in 42 days every group take 10 pieces of eggs to be detected.
The results show：Two groups of laying hens are without death in breeding process, and one group of laying hen dead 1, egg production and average egg Weight is without significant difference, but the produced egg yolk Lutein of two groups of laying hens, zeaxanthin and DHA content are high (table 1), yolk color It is deeper.
Remarkable result of the invention is, the egg rich in lutein, zeaxanthin and DHA while production, lutein, jade Cream-coloured matter and the collocation of the amount of DHA are rationally, edible to meet the needs of human body is for lutein, zeaxanthin and DHA simultaneously, and And play the role of effect and bring out the best in each other, can safeguard eyes and nervous centralis health, be optimal to the protection of eyesight and Reinforcing effect.Meanwhile laying hen is raised with the method for offer of the invention, lutein, zeaxanthin and DHA in feed can not only The nutritional ingredient of feed is protected, and the immunity of laying hen can be improved, thus greatly reduces the use of antibiotic, the egg of output Nutrient equilibrium is abundant, and yolk tinctorial yield is high, and antibiotic-free residual, mouthfeel is more aromatic, is also relatively beneficial to human health.
Embodiment 2：Same as Example 1, only two groups of egg feedstuffs are added to 0.005% lutein sterling, 0.001% Zeaxanthin sterling with 1% algae oil (DHA content 40%).
The results show：Two groups of laying hens are without death in breeding process, and one group of laying hen sick 1, egg production and average egg Weight is without significant difference, but the produced egg yolk Lutein of two groups of laying hens, zeaxanthin and DHA amount are high (table 2), and yolk color is more It is deep.
In conclusion the contents of the present invention are not limited in the above embodiments, the knowledgeable people in same area can Can propose other embodiments easily within technological guidance's thought of the invention, but this embodiment is included in this hair Within the scope of bright.
1. being measured using high performance liquid chromatography to the lutein content in egg
1.1 method summaries
After lutein acetone extraction in egg yolk, detected at 446nm in high performance liquid chromatograph, with external standard method It is quantitative.
1.2 instrument
High performance liquid chromatograph, UV detector, ultrasonic extractor, eddy mixer, centrifuge, uv-spectrophotometric Meter.
1.3 reagent
Lutein mark product：Lutein (purity > 99%, Fluka company of the U.S.).Acetone, methylene chloride, methanol (chromatography It is pure, J.T.Baker company of the U.S.)；2,6-di-tert-butyl p-cresol (BHT), methyl tertiary butyl ether(MTBE) (MTBE) (purity > 99%, Sigma Co., USA)；Egg to be measured
1.4 determination step
1.4.1 sample preparation
Raw egg takes yolk, and freeze-drying is pulverized afterwards for 24 hours.1.0g (being accurate to 0.001g) sample is accurately weighed, is used 5mL warm water sufficiently dissolves, and 10mL acetone soln (containing 0.12%BHT) and 5.0g NaCl is added, and shakes 15min, low-temperature centrifugation 5min (4000r/min), 4 DEG C), take supernatant into 10mL centrifuge tube, with acetone soln constant volume, after -40 DEG C of freezing 1h, again (4 DEG C) centrifugation 5min of low temperature.Supernatant is taken to cross 0.22 μm of filter membrane to be analyzed.It meets light due to lutein to be extremely easy in decomposition, Gu Yangpinchu Reason overall process should be protected from light as far as possible.
1.4.2 the preparation of lutein standard solution
1.0mg (being accurate to 0.1mg) lutein standard items are accurately weighed, 10mL is dissolved and be settled to methylene chloride, is matched 100mg/L standard reserving solution is made；Standard reserving solution accurately is pipetted, with methanol dilution, is configured to 20mg/L standard intermediate solution, It is kept in dark place after being distributed into several bottles in -80 DEG C of refrigerators.Standard intermediate solution is taken out from -80 DEG C of refrigerators before use, is put to room Wen Houyong acetone soln (containing 0.12% (mass fraction, similarly hereinafter) BHT) is diluted to the standard working solution of 20~500 μ g/L.
1.4.3 chromatographic condition
Chromatographic column：YMC Carotenoid C30 column (150mm × 4.6mm, 3 μm)；Mobile phase：Methanol-methyl tertbutyl Ether (70: 30, v/v), isocratic elution；Column temperature：25℃；Flow velocity：0.5mL/min；Sample volume：5μL；Detection wavelength：446nm.
1.4.4 measurement：Different standard solutions is taken respectively, is injected in high performance liquid chromatography and is separated, it is molten with standard The retention time of liquid appearance is qualitative, records corresponding peak area, canonical plotting is drawn, with quantified by external standard method.
1.5 results calculate
X=(c*V1*100)/(m*V2*1000)
X-sample Lutein content (mg/100g) in formula
The concentration (μ g/mL) for the sample liquid Lutein that c-is looked into from mark product curve
M-sample quality (g)
E-sample constant volume (mL)
K-sample measurement liquid product (mL)
The conversion coefficient of 1000-μ g conversion mg.
The content that egg Lutein can be obtained is 0.5~5.0mg/100g egg.
2. being measured using reversed-phased high performace liquid chromatographic to the zeaxanthin in egg
2.1 method summaries
The reversed-phased high performace liquid chromatographic of zeaxanthin, using C30 column, with acetonitrile-methanol-triethylamine (75:25: 0.05, V/V) and methyl tertiary butyl ether(MTBE)-triethylamine (100:0.05, V/V) gradient elution is carried out for mobile phase, UV detector exists It is detected at 450nm, quantified by external standard method.
2.2 instrument
High performance liquid chromatograph, UV detector, ultrasonic extractor, eddy mixer, centrifuge, uv-spectrophotometric Meter.
2.3 reagent
Lutern (95.8%), zeaxanthin (99.8%) Sigma Co., USA；Acetonitrile, methanol, methyl tertiary butyl ether(MTBE) Fisher company of (chromatographically pure) U.S.；Triethylamine, petroleum ether (30~60 DEG C of boiling range), dehydrated alcohol, anhydrous sodium sulfate, hydroxide Potassium, DBPC 2,6 ditertiary butyl p cresol (BHT), ascorbic acid (being that analysis is pure) Chinese medicines group chemical reagents corporation；Water is Milli-Q Gradient ultrapure water.
2.4 determination step
2.4.1 sample preparation
(1) it is saponified
Raw egg takes yolk, and freeze-drying is pulverized afterwards for 24 hours.It accurately weighs sample and weighs 5g, accurately to 0.0001g, It is placed in 250mL round-bottomed flask, adds 50mL ascorbic acid ethanol solution, be completely dispersed sample with homogenizer homogeneous, be passed through nitrogen Gas adds 10mL 500g/L potassium hydroxide solution, is uniformly mixed, and is placed in 60 DEG C of water-bath reflux 40min, oscillation prevents sample frequently It is adhered in bottle wall, saponification terminates, and uses 5mL ethyl alcohol respectively, and 5mL water autocondensation tube top end is rinsed inside it, and it is cooling to take out flask To about 40 DEG C.
(2) it extracts
It is quantitative to shift whole saponification liquors in the 500mL separatory funnel for filling 60mL petroleum ether, with 30~50mL distilled water Divide 2~3 flushing round-bottomed flasks to be incorporated to separatory funnel, fierceness oscillation 2min, deflates, then mix, stratification.Shift water phase In the 2nd separatory funnel, repeats to extract twice with 40mL petroleum ether by several times, discard water phase, merge 3 petroleum ether phases.Every time With 60mL distillation water washing petroleum ether extract 2~3 times to neutral, gently shaking by swirling when first washing prevents from emulsifying.Petroleum ether mentions Take liquid by anhydrous sodium sulfate be dehydrated, after be transferred in 250mL chicken heart bottle, add 100mg BHT to be allowed to dissolve.The above operation exists It is protected from light in vent cabinet and carries out.
(3) it is concentrated
Petroleum ether extract is placed on Rotary Evaporators, it is dry in 40 DEG C of partial vacuum, bath temperature condition evaporations.Residue It is dissolved with 2mL methanol, 0.22 μm of membrane filtration, is measured to high performance liquid chromatograph.
2.4.2 the preparation of zeaxanthin standard solution
Zeaxanthin standard reserving solution：With a small amount of acetone solution, it is respectively 100 that again with methanol, which is made into mass concentration for solvent, μ g/mL and 40 μ g/mL standard items stock solutions.It can be diluted to series standard solution as needed.
2.4.3 chromatographic condition
Chromatographic column：Ultimate XB-C30 chromatographic column (250mm × 4.6mm, 5 μm)；Flow velocity：1.0mL/min；Column temperature：25 ℃；Detector：UV detector, wavelength 450nm；Sampling volume：20μL.Mobile phase A：Acetonitrile-methanol-triethylamine (75: 25:0.05, V/V), Mobile phase B：Methyl tertiary butyl ether(MTBE)-triethylamine (100:0.05, V/V).
2.4.4 measurement：Different standard solutions is taken respectively, is injected in high performance liquid chromatography and is separated, it is molten with standard The retention time of liquid appearance is qualitative, records corresponding peak area, canonical plotting is drawn, with quantified by external standard method.
2.5 results calculate
X=(c*V1*100)/(m*V2*1000)
In formula in X-sample zeaxanthin content (mg/100g)
The concentration (μ g/mL) of c-zeaxanthin from the sample liquid that mark product curve is looked into
M-sample quality (g)
E-sample constant volume (mL)
K-sample measurement liquid product (mL)
The conversion coefficient of 1000-μ g conversion mg.
It is 0.3~6.0mg/100g egg that zeaxanthin in egg, which can be obtained,.
3. being measured using high resolution gas chromatography method to the DHA content in egg
3.1 mention oil：Raw egg takes yolk, and freeze-drying is pulverized afterwards for 24 hours.Powder sample obtained by about 25mg is weighed to be put into 15mL centrifuge tube records the weight.750 μ L chloroforms and 300 μ L ultrapure waters are added into the test tube, test tube is then placed in 37 DEG C Lower 180rpm/min shakes 1 hour.2mL fat-extraction liquid (chloroform is added into the test tube again：Methanol=2:1, v/v, 0.1% BHT), 180rpm/min at 37 DEG C is placed in shake 1 hour.Test tube is centrifuged 5min under 5500rpm room temperature, extracts subnatant Body is to another clean tube.Residue is added after 2mL fat-extraction liquid is placed in 37 DEG C of 180rpm/min concussions 1 hour and 1mL is added Ultrapure shaking water mixes, and is centrifuged 5min under 5500rpm room temperature again, extracts lower liquid and gained lower liquid is mixed for the first time It closes.1mL KCl (1mol/L) solution is added to mixed liquid and rocks mixing, 5min is centrifuged under 5500rpm room temperature, extracts Lower liquid is to another clean tube.1mL ultrapure water is added into gained lower liquid again, mixing is rocked, in 5500rpm room temperature Lower centrifugation 5min extracts 1mL lower liquid to a prior weighed (m1) clean 2mL centrifuge tube in, and in traditional vacuum It is 1 hour dry in spinner, it extracts again dried in traditional vacuum spinner in remaining lower liquid to the 2mL centrifuge tube Night.It is weighed again (m to 2mL centrifuge tube2) obtain grease obtained weight (m2-m1)。
The measurement of 3.2 DHA contents：Grease obtained middle addition 1mL n-hexane and 10 μ L KOH-CH into above-mentioned steps3OH (2mol/L) solution, 65 DEG C placement 4-6 hours, 12000rpm room temperature be centrifuged 30s, extract supernatant liquid body be settled to 1mL DHA content is carried out using gas chromatograph to measure to obtain DHA sample concentration.
3.2.1 chromatographic condition
HP-5 capillary chromatographic column (30m × 0.25mm × 0.25 μm), 280 DEG C of injector temperature, hydrogen flame (FID) detection 240 DEG C of device temperature, column temperature：200 DEG C of initial temperature, 30 DEG C/min of heating rate rises to 280 DEG C of holding 5min, flow rate of carrier gas 1.0mL/min, 1.0 μ L of sample volume, split ratio 100:1.It is qualitative with retention time, peak area quantified by external standard method, in this experiment item Under part, the retention time of DHA is 16.457min.
3.2.2 standard curve
3.2.2.1 the preparation of Standard Stock solutions
It accurately weighs DHA mark product 450mg and is diluted to graticule with n-hexane in 10mL volumetric flask, and immediately, standard is stored up at this time The concentration of standby solution, DHA 45mg/mL.
3.2.2.2 the production of standard curve
Standard Stock solutions carbon disulfide is diluted to the standard series (unit for being configured to following mass concentration step by step：mg/ mL).DHA：5.0.0,10.0,15.0,20.0,25.0.1.0 μ L of sample introduction is in line between sample volume and peak area within this range Sexual intercourse, standard curve are y=2E+10x-2E+06 (y is DHA peak area, and x is DHA concentration), related coefficient 0.9998.
3.3 total DHA contents calculate
It is 50-500mg/100g egg that total DHA content in egg, which can be obtained,.
Table 1：Test the egg measurement that lutein, zeaxanthin and DHA are added in a feed
Table 2：Test the egg measurement that lutein, zeaxanthin and DHA are added in two feeds

Claims (3)

1. a kind of preparation method of the nutritional egg with sight protectio and enhancing, it is characterised in that include the following steps：
One, laying hen several for selecting the health of laying period；
Two, it in the daily feed of above-mentioned laying hen, is added simultaneously according to mass percent in each group in following three groups of constituents It is any, first group：Lutein, marigold or other frequently seen plants rich in lutein；Second group：Zeaxanthin, jade Rice or other frequently seen plants rich in zeaxanthin；Third group：Fish meal, linseed, schizochytrium powder, schizochytrium mud, DHA Fish oil or DHA algal oil；Continuously feed 1-6 weeks acquisition egg；
The three kinds of additives added in above-mentioned egg feedstuff, Lutein additive amount or marigold are other rich in lutein Plant in contained lutein additive amount account for feed for nursing gross mass ratio be 0.0005%~0.01%；Zeaxanthin adds The additive amount of contained zeaxanthin accounts for feed for nursing gross mass in dosage or corn or other plants rich in zeaxanthin Ratio is 0.0003%~0.012%；Fish meal, linseed, schizochytrium powder, schizochytrium mud, DHA fish oil, institute in DHA algal oil Quality containing DHA is the 0.1~1.0% of feed for nursing gross mass.
2. a kind of preparation method of nutritional egg with sight protectio and enhancing according to claim 1, special Sign is that obtaining egg is measured the lutein in egg using high performance liquid chromatography, obtains egg and uses reversed phase high efficiency Liquid chromatography is measured the zeaxanthin in egg, obtains egg using high resolution gas chromatography method to the DHA in egg Content is measured.
3. a kind of nutritional egg rich in lutein, zeaxanthin and DHA simultaneously is for protecting and enhancing human body eyesight.
CN201810639292.1A 2018-06-20 2018-06-20 A kind of preparation method of the nutritional egg with sight protectio and enhancing Pending CN108812538A (en)

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CN111264720A (en) * 2020-03-16 2020-06-12 林大昌 Feed additive and feed for producing health-care eggs capable of preventing myopia and amblyopia and production method of quail health-care eggs
CN114982876A (en) * 2022-02-28 2022-09-02 南京乔康生物科技有限公司 Laying hen feed capable of improving DHA content in eggs based on DHA algae powder and egg production method thereof

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