CN112741048A - Culture medium for fruit flies and preparation method thereof - Google Patents
Culture medium for fruit flies and preparation method thereof Download PDFInfo
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- CN112741048A CN112741048A CN202011641804.1A CN202011641804A CN112741048A CN 112741048 A CN112741048 A CN 112741048A CN 202011641804 A CN202011641804 A CN 202011641804A CN 112741048 A CN112741048 A CN 112741048A
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- 239000001963 growth medium Substances 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 239000003765 sweetening agent Substances 0.000 claims abstract description 27
- 235000021092 sugar substitutes Nutrition 0.000 claims abstract description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 25
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229920001817 Agar Polymers 0.000 claims abstract description 16
- 239000008272 agar Substances 0.000 claims abstract description 16
- 235000010228 ethyl p-hydroxybenzoate Nutrition 0.000 claims abstract description 13
- 239000004403 ethyl p-hydroxybenzoate Substances 0.000 claims abstract description 13
- 229940043351 ethyl-p-hydroxybenzoate Drugs 0.000 claims abstract description 13
- NUVBSKCKDOMJSU-UHFFFAOYSA-N ethylparaben Chemical compound CCOC(=O)C1=CC=C(O)C=C1 NUVBSKCKDOMJSU-UHFFFAOYSA-N 0.000 claims abstract description 13
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 12
- 235000019260 propionic acid Nutrition 0.000 claims abstract description 12
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 claims abstract description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 11
- 235000021552 granulated sugar Nutrition 0.000 claims abstract description 11
- UDIPTWFVPPPURJ-UHFFFAOYSA-M Cyclamate Chemical compound [Na+].[O-]S(=O)(=O)NC1CCCCC1 UDIPTWFVPPPURJ-UHFFFAOYSA-M 0.000 claims description 26
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical group OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 claims description 25
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 claims description 18
- 239000000625 cyclamic acid and its Na and Ca salt Substances 0.000 claims description 18
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 claims description 18
- 229960001462 sodium cyclamate Drugs 0.000 claims description 18
- 239000000811 xylitol Substances 0.000 claims description 18
- 229960002675 xylitol Drugs 0.000 claims description 18
- 235000010447 xylitol Nutrition 0.000 claims description 18
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- 238000011160 research Methods 0.000 abstract description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 abstract description 4
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- 241000255601 Drosophila melanogaster Species 0.000 description 3
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- 239000002609 medium Substances 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
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- 235000021147 sweet food Nutrition 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
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- 241000894006 Bacteria Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 241001599018 Melanogaster Species 0.000 description 1
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Images
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Preparation Of Fruits And Vegetables (AREA)
Abstract
The invention belongs to the technical field of fruit fly research, and particularly relates to a culture medium for fruit flies and a preparation method thereof. The culture medium for the fruit flies comprises water and also comprises the following components in 1L of water: 10-20 g of agar, 80-120 g of yeast, 40-60 g of white granulated sugar, 5-250 g of sugar substitute, 0.7-0.8 g of anhydrous calcium chloride, 20-40 mL of ethanol, 8-10 g of ethyl p-hydroxybenzoate and 4-6 mL of propionic acid. The culture medium for the fruit flies adopts sugar substitutes to replace cane sugar, so that the service life of the fruit flies is prolonged, the reaction capability to the change of the external environment is promoted, or the reproductive capacity of the fruit flies is promoted to be improved.
Description
Technical Field
The invention belongs to the technical field of fruit fly research, and particularly relates to a culture medium for fruit flies and a preparation method thereof.
Background
More and more people are obese due to the long-term excessive intake of high-sugar foods, and even more, people suffer from diseases such as hypertension, cardiovascular diseases and the like. In order to satisfy the desire of people for sweet taste and simultaneously not intake more sugar, the sugar substitute is produced and widely noticed and used, and the sugar substitute is also widely used as a sweetener in industrial foods nowadays. Although the sugar-substitute substance can bring pleasure to the tongue tip of people and can reduce the intake of sugar, the sugar-substitute substance also brings certain trouble to people to eat, whether the sugar-substitute substance has harm to the body or not, whether the diabetes patients can eat sugar-substitute food or not without considering the consequences or not, therefore, the research on whether various kinds of sugar-substitute substances have certain influence on the human body or not has important practical significance to the masses of sweet food consumption and the patients who love to eat sweet food and the like.
The drosophila melanogaster has the advantages of short life cycle, quick propagation, simple and convenient feeding and the like, and is widely applied in the fields of developmental biology, neuroscience, human disease research and the like. The drosophila genome sequence analysis shows that more than 60 percent of human disease genes have orthologous genes in drosophila, so that the drosophila genome sequence analysis has important research significance through the research based on a drosophila model.
Disclosure of Invention
Based on the defects and shortcomings in the prior art, the invention provides a culture medium for drosophila melanogaster and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a culture medium for fruit flies comprises water and also comprises the following components in 1L of water: 10-20 g of agar, 80-120 g of yeast, 40-60 g of white granulated sugar, 5-250 g of sugar substitute, 0.7-0.8 g of anhydrous calcium chloride, 20-40 mL of ethanol, 8-10 g of ethyl p-hydroxybenzoate and 4-6 mL of propionic acid.
Preferably, the sugar substitute is xylitol or sodium cyclamate.
Preferably, the nutrient solution comprises 15g of agar, 100g of yeast, 50g of white granulated sugar, 250g of xylitol, 0.7126g of anhydrous calcium chloride, 30mL of ethanol, 9g of ethyl p-hydroxybenzoate and 6mL of propionic acid.
Preferably, the nutrient solution comprises 15g of agar, 100g of yeast, 50g of white granulated sugar, 5g of sodium cyclamate, 0.7126g of anhydrous calcium chloride, 30mL of ethanol, 9g of ethyl p-hydroxybenzoate and 6mL of propionic acid.
The invention also provides a preparation method of the culture medium for the fruit flies, which comprises the following steps:
(1) sterilizing the electric food warmer;
(2) mixing part of water with white granulated sugar, sugar substitute and anhydrous calcium chloride, pouring yeast and stirring until the mixture is uniformly stirred;
(3) pouring the rest water into an electric food warmer, electrifying, regulating the rotating speed to a first rotating speed, adding agar, and boiling until the agar is converted into transparent jelly;
(4) pouring the material obtained in the step (2) into an electric food warmer, adjusting the rotating speed to a second rotating speed, heating to boil, and keeping the first target time length; cooling to 60 deg.C, adding propionic acid, adding mixed solution of ethyl p-hydroxybenzoate and alcohol, and stirring; wherein the second rotating speed is greater than the first rotating speed;
(5) and filling into a fruit fly tube or a fruit fly bottle, and cooling and solidifying to obtain the culture medium for fruit flies.
Preferably, the first rotation speed is 100-130 rpm.
Preferably, the second rotating speed is 120-150 rpm.
Preferably, the first target time length is 10-20 min.
As a preferred embodiment, the ratio of the total of the components is as follows.
Compared with the prior art, the invention has the beneficial effects that:
the culture medium for the fruit flies adopts sugar substitutes to replace cane sugar, so that the service life of the fruit flies is prolonged, the reaction capability to the change of the external environment is promoted, or the reproductive capacity of the fruit flies is promoted to be improved.
Drawings
FIG. 1 is a line graph showing the effect of different sugars and sugar substitutes on the life of fruit flies according to an embodiment of the present invention;
FIG. 2 is a graph comparing the average egg laying amount of different experimental groups according to the example of the present invention;
FIG. 3 is a graph comparing the life span differences of drosophila in different experimental groups under starvation conditions in the example of the present invention.
Detailed Description
The technical solution of the present invention is further explained by the following specific examples.
The embodiment of the invention aims at the problem that whether sugar substitutes and saccharides have influence on the life and activity of organisms, the fruit fly melanogaster is used as a model organism, and the influence of the sugar substitutes and the saccharides on the life, the feed intake and the fecundity of the fruit fly is measured on the premise of 6.0 of a sucrose culture medium.
According to the embodiment of the invention, sugar substitutes (two sugar substitutes of xylitol and sodium cyclamate) with the same sweetness are adopted to replace sucrose in the culture medium, so that the influence of the sugar substitutes and the sugar on life activities under the same sweetness can be researched.
Wherein the components of each culture medium are shown in table 1:
TABLE 1 formulation of various media
Specifically, after the culture medium is prepared, the raw materials need to be cooked to be used as the culture medium for feeding the fruit flies. The preparation method of the culture medium comprises the following specific operation steps:
(1) weighing objects according to a formula table, wherein propionic acid is not weighed firstly; sterilizing the electric pan with ethanol;
(2) pouring half of the total amount of water into the basin together with white granulated sugar, other saccharides and anhydrous calcium chloride by using a 1000ml beaker, pouring yeast and stirring for 3-5min by using an egg beater, and continuously rotating the basin in the stirring process until the mixture is uniformly stirred;
(3) pouring the other half water into an electric pan, electrifying, adjusting the rotating speed to 100rpm, adding agar, and boiling until the agar is transparent and gel-like (stirring is required continuously during the coagulation period to prevent the agar from heating and solidifying);
(4) pouring the other raw materials mixed in the step (2) into a pot, adjusting the rotating speed to about 120rpm (higher than the rotating speed in the step (3)), cleaning a basin and a stirrer with additional water, pouring into an electric heating pot, heating to boil (continuous stirring is still needed for preventing food from being burnt and sticking the pot), and then keeping for 15 min; cooling food, weighing propionic acid (antiseptic for preventing growth of bacteria in culture medium from deterioration), pouring into electric pan at about 60 deg.C, pouring mixed solution of ethyl p-hydroxybenzoate and alcohol, and stirring; and finally, filling the food into a fruit fly tube or a fruit fly bottle, and after the food is completely cooled and solidified, obtaining the culture medium which can be used for feeding fruit flies.
The fruit fly will be studied to live in different media for the same time and its life condition will be observed.
In order to obtain the measurement of the influence on the life span, food intake and fecundity of the drosophila, different experimental schemes and test methods need to be established, and the specific experiment is as follows:
experiment one: lifetime determination
The flies emerged within 48 hours were collected from both the Femal and the Male flies, randomly grouped, placed in new medium bottles, and randomly mated for both sexes. After 48 hours, the mated drosophila melanogaster was placed into the experimental group and the control group, respectively. The device is divided into Female and Male, 40 fruit flies are placed in each tube, and each group is provided with 3 repeated groups.
The grouped fruit flies are placed in an incubator with constant temperature and humidity, the experiment is carried out on the culture medium which is changed for the fruit flies at regular intervals of one day (0, 2, 4, 6.. once.), the number of dead fruit flies is counted, and the number of dead fruit flies in each day is recorded.
As shown in fig. 1, it can be seen that there is a great difference in survival time between the xylitol group and the 6.0 and sodium cyclamate group drosophila, and the average life span of the xylitol group drosophila is significantly far lower than that of the 6.0 and sodium cyclamate groups. The P values of females in the xylitol group, the sodium cyclamate group and the 6.0 control group are 6.3621E-06 and 2.85184E-10 respectively through calculation; the P values of males are 9.17783E-08 and 0.15825 (if P <0.01, it indicates very significant difference between the two sets of data; if 0.01< P <0.05, it indicates significant difference between the two sets of data; and if P >0.05, it indicates no significant difference between the two sets of data), respectively, it can be seen that: the xylitol group has a very significant difference from the 6.0 group, and the sodium cyclamate group has a significant difference from the 6.0 group. It can be seen from the figure that the mean life span of males is greater in the 6.0 group than in the females, whereas the mean life span of females is greater in the xylitol group as opposed to the sodium cyclamate group.
Experiment two: food intake measurement
Three diets (i.e. sucrose group 6.0X, xylitol group, sodium cyclamate group) Female and Male were divided, with 4 replicate groups per group, 20 flies per tube. The brilliant blue food was prepared one to two days in advance (i.e., 100 μ L of brilliant blue solution with a concentration of 5.4% was added to the surface of the food) and dried in the air, and replaced on the fourth day (since the brilliant blue solution takes a longer time to soak in the medium and dry in the air, we counted four days as the best). The fruit flies are put into a new culture medium containing brilliant blue and allowed to eat freely for 4 hours, and then are anaesthetized and collected into a 1.5mL centrifuge tube, 20 flies are placed in each tube, the tube is frozen at the temperature of 18 ℃ below zero for one day and then taken out, ground and placed into an enzyme-linked immunosorbent assay instrument, and then the content of the saccharides in the fruit flies can be detected.
The effect of sugar substitutes and sugars on feed intake by Drosophila, P values for the 6.0 control group were compared and calculated with xylitol and cyclamate groups. Wherein the P value of female 6.0 and sodium cyclamate is 0.774087, and the P value of male 6.0 and sodium cyclamate is 0.002642; the P value of female 6.0 and xylitol is 0.290205, and the P value of male 6.0 and xylitol is 0.242261 from these data we can find: there were no significant differences between the male 6.0 group and the sodium cyclamate group, except for the other groups. This demonstrates that different sugars and sugar substitutes do not substantially affect the feed intake of drosophila.
Experiment three: determination of fertility
Three diets (i.e. sucrose group 6.0X, xylitol group, sodium cyclamate group) had only the Female group, 5 replicates per group, 20 flies per tube; adding 100 μ L of 5.4% brilliant blue solution to the surface of food one or two days in advance, and air drying, wherein each tube contains 100 μ L; changing to a brilliant blue food on day four; the mother generation was removed after 24 hours and the amount of eggs laid per tube was counted microscopically.
As shown in FIG. 2, in the experiment of sugar substitution and sugar on the fruit fly fertility, the egg laying amount of female fruit flies on the same days was counted. From the figure it can be seen that the reproductive capacity of xylitol female drosophila is significantly higher than that of the 6.0 and sodium cyclamate groups, while the average oviposition of the other two groups is equal, it can be concluded that: xylitol has a greater effect on female drosophila fertility than 6.0 and sodium cyclamate, and is a promoting effect.
Experiment four: hunger stress test
The preparation method of the culture medium used in the hunger experiment is as follows: adding 7.5g of agar, 500mL of water and 0.365g of anhydrous calcium chloride into a blue-mouth bottle, taking out the mixture by a microwave oven at an interval of 30s, fully shaking the mixture once, cooling the mixture to 60 ℃ after a large amount of bubbles generated in the bottle are gathered, namely the water is boiled, and the temperature is close to 100 ℃, adding 15mL of 95% ethanol solution and 4.5g of ethyl p-hydroxybenzoate, mixing the mixture with 3mL of propionic acid solution after all the solid is dissolved (the process sequence cannot be changed, and the ethyl p-hydroxybenzoate is insoluble in water and only soluble in the ethanol solution), shaking the mixture uniformly, and filling the mixture into a fruit fly tube. Putting four-day-old fruit flies cultured by corresponding xylitol, sodium cyclamate and 6.0 culture medium into the culture medium, separating Female and Male, wherein each group comprises 3 tubes, each tube comprises 20 tubes, and the experimental method is similar to the service life; data were observed and recorded.
As shown in FIG. 3, it was found that the starvation tolerance of Drosophila was also different between different groups and different sexes. The difference is mainly reflected in that: the hunger bearing capacity of female fruit fly is obviously greater than that of male fruit fly. ② in female fruit flies, the hunger bearing capacity of the fruit flies is less than 6.0 and less than sodium cyclamate. ③ in the male fruit flies, the hunger bearing capacity of the fruit flies is that the sodium cyclamate is less than 6.0 and the xylitol. Compared with the P values of the starvation tolerance of the three groups, the P value of the female 6.0 and the P value of the sodium cyclamate is 0.011278318, the P value of the male 6.0 and the P value of the sodium cyclamate is 0.36794981, the starvation tolerance of the female after the female is fed with the sodium cyclamate is obviously increased, and the male does not have obvious difference. The p value of female 6.0 and xylitol is 0.3738391, the p value of male 6.0 and xylitol is 0.20602545, and the xylitol has no great influence on the bearing capacity of the fruit flies.
The results show that: in a life test (under the same sweetness), the sodium cyclamate has a significant difference with the p value of 6.0, so that the life of the fruit fly can be prolonged; xylitol has a very significant difference with 6.0, and the service life of the fruit flies is greatly shortened. The feed intake and the fertility are tested, and the sugar substitute has no great influence on the feed intake of the fruit flies; xylitol can promote the fruit flies to lay eggs. Under starvation conditions, sodium cyclamate can promote the bearing capacity of females, and has no influence on males. Xylitol also has no influence on the hunger bearing capacity of the fruit flies.
In the embodiment and the alternative scheme thereof, the agar can be randomly selected from 10-20 g, the yeast can be randomly selected from 80-120 g, the white granulated sugar can be randomly selected from 40-60 g, the sugar substitute can be randomly selected from 5-250 g, the anhydrous calcium chloride can be randomly selected from 0.7-0.8 g, the ethanol can be randomly selected from 20-40 mL, the ethyl p-hydroxybenzoate can be randomly selected from 8-10 g, and the propionic acid can be randomly selected from 4-6 mL based on 1L of water.
In the above embodiment and its alternatives, the rotation speed of step (3) may also be 110rpm, 115rpm, 120rpm, etc., and the rotation speed of step (4) may also be 125rpm, 130rpm, 140rpm, 150rpm, etc., but it needs to be greater than the rotation speed of step (3).
In the above embodiments and alternatives, the boiling holding time may also be 10min, 12min, 18min, 20min, etc.
The foregoing has outlined rather broadly the preferred embodiments and principles of the present invention and it will be appreciated that those skilled in the art may devise variations of the present invention that are within the spirit and scope of the appended claims.
Claims (8)
1. A culture medium for fruit flies comprises water and is characterized by further comprising the following components in 1L of water: 10-20 g of agar, 80-120 g of yeast, 40-60 g of white granulated sugar, 5-250 g of sugar substitute, 0.7-0.8 g of anhydrous calcium chloride, 20-40 mL of ethanol, 8-10 g of ethyl p-hydroxybenzoate and 4-6 mL of propionic acid.
2. The drosophila culture medium according to claim 1, wherein the sugar substitute is xylitol or sodium cyclamate.
3. The drosophila culture medium according to claim 2, wherein the agar is 15g, the yeast is 100g, the white granulated sugar is 50g, the xylitol is 250g, the anhydrous calcium chloride is 0.7126g, the ethanol is 30mL, the ethyl p-hydroxybenzoate is 9g, and the propionic acid is 6 mL.
4. The culture medium for drosophila according to claim 2, wherein the agar is 15g, the yeast is 100g, the white granulated sugar is 50g, the sodium cyclamate is 5g, the anhydrous calcium chloride is 0.7126g, the ethanol is 30mL, the ethyl p-hydroxybenzoate is 9g, and the propionic acid is 6 mL.
5. The method for preparing a culture medium for drosophila according to any of claims 1 to 4, comprising the steps of:
(1) sterilizing the electric food warmer;
(2) mixing part of water with white granulated sugar, sugar substitute and anhydrous calcium chloride, pouring yeast and stirring until the mixture is uniformly stirred;
(3) pouring the rest water into an electric food warmer, electrifying, regulating the rotating speed to a first rotating speed, adding agar, and boiling until the agar is converted into transparent jelly;
(4) pouring the material obtained in the step (2) into an electric food warmer, adjusting the rotating speed to a second rotating speed, heating to boil, and keeping the first target time length; cooling to 60 deg.C, adding propionic acid, adding mixed solution of ethyl p-hydroxybenzoate and alcohol, and stirring; wherein the second rotating speed is greater than the first rotating speed;
(5) and filling into a fruit fly tube or a fruit fly bottle, and cooling and solidifying to obtain the culture medium for fruit flies.
6. The method according to claim 5, wherein the first rotation speed is 100 to 130 rpm.
7. The method according to claim 6, wherein the second rotation speed is 120 to 150 rpm.
8. The method of claim 5, wherein the first target time period is 10 to 20 min.
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| CN112651658A (en) * | 2020-12-31 | 2021-04-13 | 杭州电子科技大学 | Method for evaluating micro-plastic pollution level based on fruit fly physiological indexes |
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