阅读说明：本技术 双直巢螟在虫茶制备方面的应用以及一种虫茶及其制备方法 (Application of eupolyphaga walker in preparation of insect tea, insect tea and preparation method thereof ) 是由 温立香 袁冬寅 陈家献 张芬 彭靖茹 黄寿辉 赵媛 陈燕 冯春梅 于 2021-08-26 设计创作，主要内容包括：本发明涉及虫茶加工制备技术领域,具体是双直巢螟在虫茶制备方面的应用以及一种虫茶及其制备方法。本申请提出了一种新的具有经济价值的产茶昆虫双直巢螟,其具有寄主植物广泛、蚕食周期长、产茶量大、制备虫茶得率高、营养物质丰富等优点,适宜用作虫茶的生产制备。通过本申请的方法制备得到的虫茶产品干茶颗粒饱满,乌润油亮,呈圆柱状,两端平截,直径0.20-0.55mm,高度0.60-1.90mm,干茶显药香,汤色红褐明亮,无沉淀物,滋味醇厚甘甜,茶汤香气浓郁持久,药香明显。营养物质丰富,水浸出物含量32.45-38.67％,茶多酚4-7％,氨基酸5.05-6.66％,必需氨基酸种类尤其丰富,EAA/TAA值达到40.06％,EAA/NEAA值最高达66.83％,达到FAO/WHO推荐的食物蛋白氨基酸理想比例。(The invention relates to the technical field of insect tea processing and preparation, in particular to application of Orthosiphon aristatus in insect tea preparation, insect tea and a preparation method thereof. The application provides a new tea-producing insect dual-direct nestling moth with economic value, which has the advantages of wide host plants, long silkworm feeding period, large tea yield, high insect tea preparation yield, rich nutrient substances and the like, and is suitable for production and preparation of insect tea. The insect tea product prepared by the method has the advantages that the dry tea is full in particles, black, smooth, glossy and cylindrical, the two ends of the insect tea product are truncated, the diameter is 0.20-0.55mm, the height is 0.60-1.90mm, the dry tea has medicinal fragrance, the soup color is red, brown and bright, no precipitate exists, the taste is mellow and sweet, the tea soup fragrance is strong and lasting, and the medicinal fragrance is obvious. The nutrient substances are rich, the water extract content is 32.45-38.67%, the tea polyphenol content is 4-7%, the amino acid content is 5.05-6.66%, the essential amino acid types are particularly rich, the EAA/TAA value reaches 40.06%, the EAA/NEAA value reaches up to 66.83%, and the ideal ratio of food protein amino acid recommended by FAO/WHO is achieved.)

1. The application of the eupolyphaga walker in the preparation of insect tea.

2. The application of the eupolyphaga walker in the preparation of the insect tea according to claim 1, wherein the eupolyphaga walker is applied in the preparation of the insect tea, and particularly is applied to the eupolyphaga walker as an insect for producing the tea by the insect tea.

3. The application of the dirichopus dirichalis in the preparation of the insect tea, according to claim 2, is characterized in that the dirichopus dirichalis is used as an insect for producing the tea, specifically, the dirichopus dirichalis larva is used for eating raw materials, digesting and excreting to obtain a digestion fermentation product of the insect tea, and the insect tea is prepared by using the digestion fermentation product.

4. A preparation method of insect tea is characterized in that the double-straight-nest moth is used in the preparation process.

5. The preparation method of the insect tea according to claim 4, wherein the double straight nest borers are used in the preparation process, specifically, the double straight nest borers are used as tea-producing insects, larvae of the double straight nest borers are used for taking feed raw materials, and digestion fermentation products of the double straight nest borers after being eaten by the tea-producing insects are dried in the air and collected.

6. The preparation method of the insect tea as claimed in claim 4, wherein the preparation method of the insect tea comprises the following steps: (1) selecting a parasitic plant raw material; (2) fermenting raw materials; (3) feeding by tea-producing insects; (4) collecting semi-finished product and removing impurities; (5) removing impurity gas at high temperature; (6) spreading for cooling; (7) drying with full fire; (8) and (7) packaging and storing.

7. The preparation method of the insect tea according to claim 6, wherein the steps are specifically as follows:

(1) selecting a parasitic plant raw material: collecting fresh raw materials of parasitic plant leaves and tender stems according to the feeding characteristics of the tea-producing insect, namely the double-straight-nest borer for later use;

(2) fermenting raw materials: naturally spreading the collected parasitic plant raw materials until no surface water exists, stacking and fermenting for 2-3 days until odor capable of attracting the tea-producing insect, namely the Directus nestling, is generated;

(3) feeding by tea-producing insects: adopting a natural induced or artificial placing mode to make the birectionella sessiliflorus oviposit and hatch, and feeding the larva with raw materials;

(4) collecting semi-finished products and removing impurities: collecting the digested and fermented product after the tea-producing insect, namely the biddulphia nubilalis, takes the food, drying the digested and fermented product in the air, and removing impurities and impurities to obtain a semi-finished product of the insect tea;

(5) removing impurity gas at high temperature: removing miscellaneous gas and part of water from the insect tea semi-finished product collected in the step (4) at high temperature;

(6) spreading for cooling: spreading and cooling the semi-finished product subjected to high-temperature impurity gas removal for 20-30min, and cooling to room temperature to enter the next procedure;

(7) drying with full fire: drying the spread and cooled semi-finished product at 60-90 deg.C for 25-40min until the water content is below 10%;

(8) packaging and storing: the finished product is sealed and packaged and stored under the conditions of light resistance, drying and no peculiar smell.

8. The method for preparing insect tea according to claim 7, wherein the parasitic plant comprises tea tree, wild jujube tree, and uncaria.

9. The preparation method of the insect tea according to claim 8, wherein the parasitic plant raw materials are selected from the following components: tea leaf: ramulus uncariae cum uncis leaf (1: 0: 0) - (3: 1: 3).

10. The insect tea preparation method according to claim 7, wherein during the step (3), the raw material heap temperature is controlled at 25-35 ℃.

Technical Field

The invention relates to the technical field of insect tea processing and preparation, in particular to application of Orthosiphon aristatus in insect tea preparation, insect tea and a preparation method thereof.

Background

The insect tea is called 'tea essence', is a unique forestry insect product in Guizhou mountainous areas in China, is called 'cat excrement coffee' in the tea world, is prepared by taking specific plants by specific insects, and screening, sterilizing, drying and other processes of excrement generated in the bodies of the insects through special physiological and biochemical reactions. With the development of the big health industry and the attention of people on health, the insect tea market gradually gets more and more beautiful.

Currently, the economically valuable insects which can be used for producing the insect tea and are known in China mainly include rice onyx borer (Aglossa dimidata), oriental armyworm (Hydrillodes morrosa), white striped rice borer (Fujima acanicolaralis), striped rice borer (Herculia glaucalis), Arthropoda cunea (Hhdrilides repugnalis), snow looper (Nodaria niphona), yellow spotted webworm borer (Pyralis farinalis), and Grapholitha punctifera (Orthophagia glaucalis); the parasitic plants include Tripterygium hypoglaucum (Malus sieboldii), Syzygium aromaticum (Platycarya strebliacea), Ku Teng (Sageretia), Ku Ding (Rubus henryi), tea tree (Camellia sinensis), wild jujube tree ((Roxb.) Burtt et Hill, and Cinnamomum pantherinum (Litsea coreana), etc.

The physical and chemical properties, efficacy and the like of the insect tea produced by different tea-producing insects and parasitic plants are different, most insect tea needs to be stored for many years and can form higher quality after being fully transformed by substances, and the time cost is high, so that the insect tea products in the current market are few and have higher price. Meanwhile, the production yield of the existing insect tea is generally low, 1 jin of insect tea can be prepared by 30-40 jin of raw materials on average, and the ratio of some parasitic plants such as ampelopsis grossedentata to other raw materials is even up to 100: 1-2, most of the tea-producing insects have single raw materials for feeding parasitic plants, and the nutrient substances of the prepared product are not rich enough. Many researchers continue to explore new tea-producing insects and parasitic plant materials with high yield and rich nutrients.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides an application of the dirachta dichotoma in insect tea preparation, insect tea and a preparation method thereof, and the application comprises the following specific steps:

the application of the eupolyphaga walker in the preparation of insect tea. The nestling is a tea-producing insect collected from a melt region and is determined to be the nestling through morphological, anatomical and molecular biological identification.

Further, the application of the codling moth in the preparation of the insect tea, in particular to the application of the codling moth as an insect for producing the tea.

Further, the biddulphia furnacalis is used as a tea-producing insect of the insect tea, and specifically, the biddulphia furnacalis larva is subjected to raw material feeding, digestion and excretion to obtain a digestion fermentation product and is used for preparing the insect tea.

The inventor of the application discovers a new tea-producing insect dual-direct nestling moth with economic value in the research process, and the insect dual-direct nestling moth has the advantages of wide host plants (including tea trees, wild jujube leaves, uncaria and the like), long silkworm eating period, large tea yield, high insect tea preparation yield, rich nutrient substances and the like.

A method for preparing insect tea uses Orthosiphon aristatus in the preparation process.

Further, the double-straight-nest-borer is used in the preparation process, specifically, the double-straight-nest-borer is used as a tea-producing insect, larvae of the double-straight-nest-borer are used for taking a feed raw material, and digestion fermentation products of the double-straight-nest-borer after eating the tea-producing insect are dried in the air and then collected.

Further, the preparation method of the insect tea comprises the following steps: (1) selecting a parasitic plant raw material; (2) fermenting raw materials; (3) feeding by tea-producing insects; (4) collecting semi-finished product and removing impurities; (5) removing impurity gas at high temperature; (6) spreading for cooling; (7) drying with full fire; (8) and (7) packaging and storing.

Further, the specific operations of the steps are as follows:

(1) selecting a parasitic plant raw material: collecting fresh raw materials of leaves and tender stems of host plants according to the feeding characteristics of the tea-producing insect, namely the double-straight-nest borer for later use;

(2) fermenting raw materials: naturally spreading the collected parasitic plant raw materials until no surface water exists, stacking and fermenting for 2-3 days until odor capable of attracting the tea-producing insect, namely the Directus nestling, is generated;

(3) feeding by tea-producing insects: adopting a natural induced or artificial placing mode to make the birectionella sessiliflorus oviposit and hatch, and feeding the larva with raw materials;

(4) collecting semi-finished products and removing impurities: collecting the digested and fermented product after the tea-producing insect, namely the biddulphia nubilalis, takes the food, drying the digested and fermented product in the air, and removing impurities and impurities to obtain a semi-finished product of the insect tea;

(5) removing impurity gas at high temperature: removing miscellaneous gas and part of water from the insect tea semi-finished product collected in the step (4) at high temperature;

(6) spreading for cooling: spreading and cooling the semi-finished product subjected to high-temperature impurity gas removal for 20-30min, uniformly redistributing the water of the tea body, and cooling to room temperature to enter the next procedure;

(7) drying with full fire: drying the spread and cooled semi-finished product at 60-90 deg.C for 25-40min until the water content is below 10%;

(8) packaging and storing: the finished product is sealed and packaged and stored under the conditions of light resistance, drying and no peculiar smell.

Further, the parasitic plant comprises tea leaves, wild jujube leaves and uncaria. Further, the parasitic plant raw materials are prepared from wild jujube leaves: tea leaf: ramulus uncariae cum uncis leaf (1: 0: 0) - (3: 1: 3). Further, the parasitic plant raw materials are prepared from wild jujube leaves: tea leaf: ramulus Uncariae cum uncis leaf (1: 1: 1) - (3: 1: 3)

Further, in the step (2), the specific stacking height of stacking fermentation is 30-50 cm.

Further, in the step (3), the temperature of the raw material pile is controlled to be 25-35 ℃.

Further, the high-temperature impurity gas removal in the step (5) adopts a hot air method at the temperature of 100 ℃ and 120 ℃ for 20-30 min; or microwave method for 3-4 min; or steaming for 4-8 min.

Compared with the prior art, the invention has the technical effects that:

(1) the application provides a new tea-producing insect with economic value, which is identified as the double-straight nest borer, has the advantages of wide host plants (including tea trees, wild jujube leaves, uncaria and the like), long silkworm eating period, large tea yield, high insect tea preparation yield, rich nutrient substances and the like, and is suitable for being eaten by the tea-producing insect of insect tea.

(2) The insect tea product prepared by the preparation method is subjected to acute oral toxicity, AMES tests, toxicological safety evaluation tests such as mammal erythrocyte micronucleus and mouse spermatogonial chromosome aberration and the like, and the result shows that the tolerance dose of the insect tea to the acute oral toxicity of the mouse is more than 20g/kg BW, and belongs to a non-toxic level; 5 histidine-deficient Salmonella typhimurium strains (TA97a, TA98, TA100, TA102, TA1535) in the Ames test with or without the addition of S_-9The results are negative for the metabolic activation system; compared with a negative control group, 10000, 5000 and 2500mg/kg BW3 dose groups in a mammal erythrocyte micronucleus test and a mouse spermatogonia chromosome aberration test have no significant difference (P)>0.05), which indicates that the insect tea produced by the Orthosiphon aristatus does not causeMutation and aberration, no genetic toxicity, safety and no toxicity. Meanwhile, heavy metals (lead, arsenic and cadmium), pesticide residues (12 cypermethrin, sulfur dioxide, dimethoate and the like), water, ash and the like all meet the requirements of GH/T1091 substituted tea, and staphylococcus aureus, salmonella, shigella and beta-hemolytic streptococcus which are four pathogenic bacteria are not detected, so that the insect tea meets the food safety requirements.

(3) The insect tea product prepared by the preparation method is characterized in that the dry tea is full in particles, black, moist, glossy, cylindrical, truncated at two ends, 0.20-0.55mm in diameter and 0.60-1.90mm in height, the dry tea has medicinal aroma, the soup is red, brown and bright, no precipitate exists, the taste is mellow and sweet, the tea soup aroma is rich and lasting, and the medicinal aroma is obvious. The nutrient substances are rich, the water extract content is 32.45-38.67%, the tea polyphenol content is 4-7%, the amino acid content is 5.05-6.66%, the essential amino acid types are particularly rich, the EAA/TAA value reaches 40.06%, the EAA/NEAA value reaches up to 66.83%, and the ideal ratio of food protein amino acid recommended by FAO/WHO is achieved.

(4) The parasitic plants of the birectionetia longissima which can be eaten by silkworm mainly comprise wild jujube leaves, tea leaves and uncaria, the yield of the insect tea product is high, the average yield can reach more than 27 percent and is 5 to 10 times of other tea-producing insects.

Drawings

FIG. 1 is a graph comparing the effect of insect tea on scavenging DPPH free radicals;

FIG. 2 is a graph comparing the scavenging effect of insect tea on superoxide anions;

FIG. 3 is a graph comparing the effect of insect tea on scavenging hydroxyl radicals.

Detailed Description

The technical solution of the present invention is further defined below with reference to the specific embodiments, but the scope of the claims is not limited to the description.

Examples

Comparison of silkworm feeding rate and yield of tea-producing insect dual-direct nestling borer on raw materials with different proportions

TABLE 1 comparison of the rearing rate and the worm tea yield for different wild jujube leaf and tea leaf ratios

Researches show that parasitic plants which can be eaten by the dual-direct nestling borers through silkworm feeding mainly comprise wild jujube leaves, tea leaves and uncaria, the yield of the insect tea product is high, the average yield can reach over 27 percent and is 5-10 times of that of other tea-producing insects; experiments show that the preference of the dicoryzophragma bilineata walker on the wild jujube leaves and the uncaria is more obvious, the silkworm eating rate is as high as 100%, the silkworm eating rate on pure tea leaves is lower, and the silkworm eating rate can be obviously improved when the tea leaves and the wild jujube leaves or the uncaria raw materials are mixed. Meanwhile, sensory evaluation comparison shows that the insect tea produced by taking pure tea leaves or adding proper tea leaves as raw materials is stronger in medicinal fragrance, and the raw materials of the wild jujube leaves, the tea leaves and the uncaria rhynchophylla are mixed according to the ratio of 1: 1: 1-3: 1: 3 is preferred.

Quality contrast of insect tea obtained by different tea-producing insects

TABLE 2 comparison of insect tea quality for different tea-producing insects

The insect tea produced by different tea-producing insects such as silkworm feeding host plants has different nutritional components, and low caffeine is a common characteristic of insect tea, as shown in table 7, the caffeine produced by the double-straight-nest borer is not detected, which indicates that the degradation effect of the double-straight-nest borer on caffeine in vivo is particularly remarkable. In addition, the insect tea produced by the dual-direct nestling borer is rich in nutrient substances, the water extract is as high as 38.67 percent, the total content of amino acid is 6.66 percent, the types of essential amino acid are particularly rich, the essential amino acid accounts for 40.06 percent of the total amino acid (EAA/TAA value), the EAA/NEAA value is up to 66.83 percent, the ideal proportion of food protein amino acid recommended by FAO/WHO is achieved, and the insect tea produced by the dual-direct nestling borer is a high-quality protein resource.

Research on raw material fermentation stacking height and stacking temperature control

For the dual-direct nestling moth silkworm feeding host plant, the fermentation stacking height of the raw materials is preferably 30-50 cm, the raw materials are slowly dehydrated after being stacked, and a certain moisture condition needs to be ensured in the early stage, so that the fermentation of the raw materials is more facilitated, and a proper dark silkworm feeding environment is also created for tea-producing insects. The stacking is too low (less than 30cm), the water loss is too fast, the fermentation is insufficient, a proper silkworm food dark environment is difficult to create, the insects lack the sense of safety, the silkworm food rate is low, and the production space utilization is not facilitated; the stacking height is 30cm-50cm, the stacking temperature is preferably controlled below 35 ℃.

Tea-producing insect feeding test

The test is carried out in an artificial climate box, 4 temperature gradients of 25 ℃, 30 ℃, 35 ℃ and 40 ℃ are set, the room temperature is taken as a control CK, the relative humidity is 75%, the photoperiod is 14:10(L: D) h, the average room temperature in the test carrying time period is 32 ℃, the result shows that the temperature for living and breeding of the insect-producing tea insect dual-direct-nest borer is 25-35 ℃, and the optimal temperature condition for the dual-direct-nest borer to eat is 25-30 ℃ and 80-85% through further test at the later stage.

Test of influence of high-temperature impurity-removing gas on quality

TABLE 3 Effect of different treatments on insect tea quality

Comparison of antioxidant indexes of insect tea produced by feeding different raw materials to Directus nidulans

The Vc is used as a positive control, and the anti-oxidation index detection and comparison are carried out on insect tea produced by tea-producing insect dual-straight-nest borers such as the juveniles serinae leaf and the tea respectively, the results show that the clearance rates of different raw material insect tea on DPPH free radicals, superoxide anions and hydroxyl free radicals are gradually increased along with the increase of the concentration of tea soup, the clearance rate tends to be stable after the tea soup reaches a certain concentration, the clearance rate on DPPH free radicals is more than 90 percent (shown in figure 1), the clearance rate on superoxide anions is more than 45 percent (shown in figure 2), the clearance rate on hydroxyl free radicals is more than 95 percent (shown in figure 3), and the anti-oxidation effect of the insect tea produced by the juveniles leaf is better than that of the tea integrally.

Toxicological safety evaluation test

1 materials and methods

1.1 sample preparation and pretreatment:

1.1.1 sample preparation

(1) Preparing a parasitic plant material;

(2) naturally spreading until no surface water exists, stacking and fermenting for 2 days, wherein the stacking height is about 40 cm;

(3) adopting a natural priming mode to enable the birectionella furnacalis to lay eggs and hatch on the raw materials, feeding the raw materials by larvae, and controlling the stack temperature of the raw materials to be less than 35 ℃;

(4) collecting the digested and fermented product after silkworm feeding is dried, and removing impurities and impurities by air separation, manual removal and the like to obtain a semi-finished product of the insect tea;

(5) removing impurity gas and part of water by hot air method at 110 deg.C for 25min, wherein the water content is about 15%;

(6) removing impurity gas, and spreading for cooling for 30 min;

(7) drying the spread and cooled semi-finished product at 80 ℃ for 30min until the water content is 9%;

(8) and packaging and storing the mixture in a sealed bag for later use.

1.1.2 sample pretreatment

Weighing 50g of the insect tea sample, adding 500mL of pure water with the temperature of 85 ℃, soaking for 30 minutes, and filtering the soaking solution by using gauze; soaking and extracting repeatedly in the same way. Then combining the two filtrates, concentrating to 50mL by rotary evaporator under reduced pressure to obtain 1.0g/mL (dry basis, the same below) sample extractive solution, and storing in refrigerator.

1.2 Experimental animals and Environment: SPF grade healthy Kunming mouse is bred by Changsha Tianqin biotechnology limited company, and the experimental animal produces license number: SCXK (Xiang) 2019-: 1107261911001645, 430726201100244242. The animal feed is produced by Beijing Ke' ao synergetic feed Co Ltd and Changsha Tianqin biotechnology Co Ltd, and the production license number and the qualification number are respectively as follows: SCXK (Jing) 2014-; SCXK (Xiang) 2019-. Animal laboratories are barrier systems, using license numbers: SYXK 2016-. Animal laboratory temperature: 22-25 ℃, relative humidity: 55 to 70 percent.

1.3 test strains: salmonella typhimurium histidine-deficient strains TA97a, TA98, TA100, TA102, and TA1535, available from MOLTOX, USA.

1.4 mice acute oral toxicity test: the Maximum Tolerated Dose (MTD) test was used. 20 mice were selected, 10 animals were used for females and males, respectively, and animals were fasted for 16 hours before the test without any water. The animals were gavaged with 1.0g/mL of the sample extract at a volume of 0.4mL/20g BW at a dose of 20000mg/kg BW. And observing and recording the poisoning expression of the animals after the gavage. Weigh once a week, observe for a two week period, dissect animals for gross observation at the end of the trial. The test substance is evaluated for acute toxicity according to toxicity grading standards.

1.5 bacterial reverse mutation assay: the test is carried out by five strains of the salmonella typhimurium histidine-deficient strain TA97a, TA98, TA100, TA102 and TA1535 which are identified to meet the requirements. Measuring 5.0mL of sample extracting solution of 1.0g/mL, adding pure water to 100mL, fully and uniformly mixing to prepare 50mg/mL concentration solution, sequentially diluting by 5 times (taking 10mL and adding pure water to 50mL) to prepare 10, 2, 0.4 and 0.08mg/mL concentration solutions respectively, and subjecting the tested solution to autoclaving (0.103Mpa for 20min) for testing. Rat liver microsomal enzyme (S-9) induced by polychlorinated biphenyl was used as an in vitro metabolic activation system. By adopting a plate doping method, 0.1mL of test strain enrichment liquid, 0.1mL of test object solution and 0.5mL of S-9 mixed solution (when metabolic activation is needed) are sequentially added into the heat-preserved top layer culture medium, mixed uniformly and poured onto a bottom layer culture medium plate. The 5 test doses were 5000, 1000, 200, 40, 8 μ g/dish, respectively, with spontaneous regression control, solvent control and positive mutagen control. The spontaneous reversion control was identical to the sample group except that no sample was added. The solvent control was prepared by replacing the sample with sterilized pure water under the same conditions as the sample group. Each strain of each dose group was plated in 3 parallel dishes. The culture was incubated at 37 ℃ for 48 hours, and the number of colonies per dish was counted. The whole set of experiments was repeated twice under the same conditions. If the number of the test object's recurrent colonies increases more than 2 times of the number of the spontaneous recurrent colonies and has a dose-response relationship, the test object is positive for the mutagenesis test.

1.6 micronucleus test of mammalian erythrocytes: the test was performed by oral gavage with two 24 hour intervals. 50 Kunming mice with the weight of 25-30 g are selected and randomly divided into 5 groups, each group comprises 10 mice, and the number of the mice is half of that of the female and the male. 10000, 5000 and 2500mg/kg BW are respectively set for 3 doses of the test group, pure water is used as a negative control, and cyclophosphamide (cp) with 40mg/kg BW dose is used as a positive control. Respectively measuring 20.0 mL, 10.0 mL and 5.0mL of 1.0g/mL sample extracting solution, adding pure water to 40mL, mixing uniformly to prepare 500 mg/mL, 250 mg/mL and 125mg/mL concentration solutions, then irrigating the animals with the volume of 0.4mL/20g BW, irrigating a negative control group with pure water of the same volume, and irrigating a positive control group with cyclophosphamide solution of 2mg/mL of the same volume. Collecting peripheral blood, preparing a sheet, collecting blood from the tail of a mouse 48 hours after 2 nd test object feeding, dripping the blood on a slide, diluting the smear with calf serum, naturally drying the smear, fixing the smear with methanol, staining the smear for 10 minutes with Gimsa application solution, washing the smear with pH6.8 phosphate buffer solution, observing the smear under an oil lens after naturally drying the smear, counting 2000 pleochromogenous erythrocytes (PCE) in each animal, and expressing the micronucleus rate by PCE thousandth rate containing micronucleus; and counting the pleochromophilic erythrocytes (PCE) seen when 1000 Red Blood Cells (RBC) are observed at the same time, and calculating the PCE/RBC ratio. And (4) carrying out statistical treatment by using a Poisson distribution average number comparison method in SPSS statistical software. If the micronucleus rate of the test group is higher than that of the negative control group, and the micronucleus rate has obvious dose-response relation and statistical significance, the result is the positive result.

1.7 mouse spermatogonial chromosome aberration assay: 30 Kunming male mice with the weight of 25-35 g are selected and randomly divided into 5 groups, wherein one group comprises 10 mice, and the other 4 groups comprise 5 mice. 10000, 5000 and 2500mg/kg BW are respectively set for 3 doses of the test group, pure water is used as a negative control, and cyclophosphamide (cp) with 40mg/kg BW dose is used as a positive control. Respectively measuring 20.0 mL, 10.0 mL and 5.0mL of 1.0g/mL sample extracting solution, adding pure water to 40mL, mixing uniformly to prepare 500 mg/mL, 250 mg/mL and 125mg/mL concentration solutions, irrigating the animals with the volume of 0.4mL/20g BW, irrigating the animals with the pure water with the same volume in a negative control group, and irrigating 2mg/mL cyclophosphamide solution with the volume of 0.4mL/20g BW in a positive control group.

The high dose group animals were sacrificed at 24h and 48h after the test subjects, respectively, in 2 batches, and the remaining groups of animals were sacrificed at 24h after the test subjects. 4h before sacrifice, animals in each group were injected intraperitoneally with 0.5mg/mL colchicine (reduced dose 5mg/kg BW) at a volume of 0.2mL/20g BW. Killing animals by cervical dislocation, taking testis from both sides, separating seminiferous tubules by putting the testis into 1% trisodium citrate, fixing in methanol/glacial acetic acid (3:1) fixing solution for 2 times, softening in 60% glacial acetic acid, homogenizing, and centrifuging. And dropping the collected cell suspension on an ice water glass slide to prepare 2-3 pieces of slide, drying, and performing microscopic examination under an optical microscope after Giemsa staining. 100 metaphase cells were counted per animal, spermatogonial cells were observed for chromosomal changes and structural changes, and 1000 spermatogonial cells were observed to determine the mitotic index. The number of cells containing chromosomal structural aberrations and the number of chromosomal aberrations per cell were recorded for each animal, and the number and frequency of chromosomal structural aberrations (chromosomal gaps not counting aberration rates) were counted for each set for different types. SPSS statistical software is applied to carry out statistical treatment on chromosome breakage, fragment, micro-body, translocation, abnormal cell rate and the like of the test group and the negative control group according to the two-term distribution.

If the chromosome aberration rate or the aberrated cell rate of the tested dose group is compared with that of the negative control group, the difference has statistical significance and obvious dose-response relation, and the result is determined to be positive. If the chromosome aberration rate or the aberration cell rate difference in a test dose group is statistically significant but no dose-response relationship is found, the test is repeated and the result is determined to be positive if the result is repeatable.

2 results

2.1 acute oral toxicity test

TABLE 4 acute toxicity test results of insect tea on mice

As can be seen from Table 4, after gavage of the animals with the sample at 20000mg/kg BW, the animals grew well without weight being affected. None of the tested mice had toxic symptoms and no animal death was observed for 14 days. After the experiment is finished, animals are dissected and generally observed, no obvious abnormal change is seen in main organs such as liver, kidney, spleen, heart, lung, stomach, intestine and the like, according to the acute toxicity grading standard of the national food safety standard (GB15193.3-2014), the acute oral toxicity tolerance dose of the sample to mice is more than 20g/kg BW, and the acute oral toxicity belongs to a non-toxic grade.

2.2 bacterial reverse mutation assay

TABLE 5 insect tea first bacterial reverse mutation test results

Note: 1. the results (colony counts) were all mean ± standard deviation of 3 plates.

2. Positive control: TA97a + S-9, TA98+ S-9, and TA100+ S-9 used 2-aminofluorene (dose 10. mu.g/dish); TA 98-S-9 was daunomycin (dose 6. mu.g/dish); TA97 a-S-9 and TA 102-S-9 adopt fenaminosulf (the dosage is 50 mu g/dish); TA 100-S-9, TA 1535-S-9 adopt sodium azide (the dosage is 1.5 mug/dish); TA102+ S-9 was 1, 8-dihydroxyanthraquinone (dose 50. mu.g/dish); TA1535+ S-9 employed cyclophosphamide (dose 200. mu.g/dish). The same applies to Table 6.

TABLE 6 second bacterial back mutation test of Worm teaTest results

In tables 5 and 6, it can be seen that, for five test strains, TA97a, TA98, TA100, TA102 and TA1535, no more than twice the number of spontaneous reversion colonies was observed in each dose group of samples, and no dose-response relationship was observed, indicating that the test result of the mutagenesis test for the test substance was negative.

2.3 micronucleus test for mammalian erythrocytes

TABLE 7 influence of Worm tea on the incidence of micronucleus in peripheral red blood cells of mammals

Note: p <0.01 for negative control group comparison; cp is cyclophosphamide.

As can be seen from Table 7, the peripheral red blood cell micronucleus rates of mice in each dose group of the samples have no significant difference compared with the negative control group (P >0.05), the PCE/RBC values of each dose group are not less than 20% of those of the negative control group, and have no significant difference compared with the negative control group, while the micronucleus rates of cyclophosphamide positive control group have very significant difference (P <0.01) compared with that of the negative control group, which indicates that the samples have no damage and inhibition effect on peripheral red blood cells of the mice.

2.4 mouse spermatogonial chromosome aberration test

TABLE 8 influence of Worm tea on mouse spermatogonial chromosome aberration

Note: the annular change of the structural change comprises a filament-bearing point ring and a non-filament-bearing point ring, and the multi-filament-bearing points comprise more than two filament-bearing points; the abnormal cell rate is not counted in the fissure, and the abnormal cell rate is (number of abnormal cells-number of fissures)/the number of observed cells is multiplied by 100; indicates a very significant difference compared to the negative control group (P < 0.01).

As can be seen from Table 8, the number of chromosome aberrations, the change of each type of structure and the aberration cell rate of the spermatogonium of the mice of each dose group of the sample are compared with those of the negative control group, the difference is not significant (P >0.05), the mitotic index of each dose group is not lower than 50% of that of the negative control group, and the difference is not significant compared with that of the negative control group, while the chromosome aberration cell rate of the cyclophosphamide positive control group is very significant (P <0.01) compared with that of the negative control group, which indicates that the sample has no aberration effect on the spermatogonium chromosome of the mice.

3 results and decisions

3.1 acute oral toxicity test: after the sample with 20000mg/kg BW dosage is administrated to animals for intragastric administration, no toxic symptom is seen in the animals, no animal death occurs, the sample has the tolerance dosage of acute oral toxicity of more than 20g/kg BW to mice, and the acute oral toxicity belongs to non-toxic grade.

3.2 bacterial reverse mutation assay: the 5 histidine-deficient salmonella typhimurium strains (TA97a, TA98, TA100, TA102 and TA1535) are subjected to back mutation tests by using sample solutions with 5 doses of 5000, 1000, 200, 40 and 8 mu g/dish respectively, and the results show that the number of back-mutated colonies of each dose group is not obviously increased and dose-response relationship does not exist no matter whether an S-9 metabolic activation system is added, thereby indicating that the sample mutation test result is negative.

3.3 micronucleus test of mammalian erythrocytes: and respectively performing intragastric administration on the mice for 2 times by using samples with 10000, 5000 and 2500mg/kg BW3 doses, wherein the microkaryocyte rate of peripheral red blood cells of the mice of each dose group is compared with that of a negative control group, the difference is not significant (P is greater than 0.05), the PCE/RBC value of each dose group is not less than 20 percent of that of the negative control group, and the PCE/RBC value is not obviously different from that of the negative control group, so that the condition that the samples have damage and inhibition effects on the peripheral red blood cells of the mice is not shown.

3.4 mouse spermatogonial chromosome aberration assay: the mice are respectively gavaged with 10000, 5000 and 2500mg/kg BW3 doses of samples for 1 time, the chromosome aberration number, the change of each type of structure and the aberration cell rate of the spermatogonium of the mice of each dose group have no significance (P is more than 0.05) when being compared with a negative control group, the mitosis index of each dose group is not less than 50 percent of that of the negative control group, and the difference has no obvious difference when being compared with the negative control group, which indicates that the sample has no aberration effect on the spermatogonium chromosome of the mice.

Example 1

An insect tea is prepared by the following steps:

(1) selecting a parasitic plant raw material: collecting fresh raw materials of host plant leaves and tender stems according to the feeding characteristics of the tea-producing insect dual-straight-nest borer, wherein the parasitic plant raw materials are wild jujube leaves: tea leaf: 1, hooked rattan leaf: 1: 1;

(2) fermenting raw materials: naturally spreading the collected parasitic plant raw materials until no surface water exists, stacking and fermenting for 2.5 days until odor capable of attracting the tea-producing insect Bithonella medicinalis is generated, wherein the specific stacking height of stacking and fermenting is 40 cm;

(3) feeding by tea-producing insects: adopting a natural priming or artificial placing mode to enable the biddulphia webworm to lay eggs and hatch, wherein the larvae eat raw materials, and the temperature of the raw materials is controlled at 30 ℃;

(4) collecting semi-finished products and removing impurities: collecting the digested and fermented product after the tea-producing insect, namely the biddulphia nubilalis, takes the food, drying the digested and fermented product in the air, and removing impurities and impurities to obtain a semi-finished product of the insect tea;

(5) removing impurity gas at high temperature: removing miscellaneous gas and part of water at high temperature from the semi-finished insect tea product collected in the step (4), wherein the miscellaneous gas is removed at the high temperature by adopting a hot air method at 110 ℃ for 25 min;

(6) spreading for cooling: spreading the semi-finished product subjected to high-temperature impurity gas removal for 25min, uniformly redistributing the tea body, and cooling to room temperature to enter the next procedure;

(7) drying with full fire: drying the spread and cooled semi-finished product at 75 ℃ for 30min until the water content is below 10%;

(8) packaging and storing: the finished product is sealed and packaged and stored under the conditions of light resistance, drying and no peculiar smell.

Example 2

An insect tea is prepared by the following steps:

(1) selecting a parasitic plant raw material: collecting fresh raw materials of host plant leaves and tender stems according to the feeding characteristics of the tea-producing insect dual-straight-nest borer, wherein the parasitic plant raw materials are wild jujube leaves: tea leaf: leaf of Uncaria rhynchophylla 3: 1: 3;

(2) fermenting raw materials: naturally spreading the collected parasitic plant raw materials until no surface water exists, stacking and fermenting for 2 days until odor capable of attracting the tea-producing insect, namely the Directionella furcifera, is generated, wherein the specific stacking height of stacking and fermenting is 30 cm;

(3) feeding by tea-producing insects: adopting a natural priming or artificial placing mode for the birectionetia foetida to lay eggs and hatch, wherein the larvae eat raw materials, and the temperature of the raw materials is controlled at 25 ℃;

(4) collecting semi-finished products and removing impurities: collecting the digested and fermented product after the tea-producing insect, namely the biddulphia nubilalis, takes the food, drying the digested and fermented product in the air, and removing impurities and impurities to obtain a semi-finished product of the insect tea;

(5) removing impurity gas at high temperature: removing miscellaneous gas and part of water at high temperature from the semi-finished insect tea product collected in the step (4), wherein the miscellaneous gas is removed at the high temperature by adopting a hot air method at 100 ℃ for 30 min;

(6) spreading for cooling: spreading the semi-finished product subjected to high-temperature impurity gas removal for cooling for 20min, uniformly redistributing the tea body, and cooling to room temperature to enter the next procedure;

(7) drying with full fire: drying the spread and cooled semi-finished product at 60 ℃ for 40min until the water content is below 10%;

(8) packaging and storing: the finished product is sealed and packaged and stored under the conditions of light resistance, drying and no peculiar smell.

Example 3

An insect tea is prepared by the following steps:

(1) selecting a parasitic plant raw material: collecting fresh raw materials of host plant leaves and tender stems according to the feeding characteristics of the tea-producing insect dual-straight-nest borer, wherein the parasitic plant raw materials are wild jujube leaves: tea leaf: 1, hooked rattan leaf: 0: 1;

(2) fermenting raw materials: naturally spreading the collected parasitic plant raw materials until no surface water exists, stacking and fermenting for 3 days until odor capable of attracting the tea-producing insect, namely the Directionella furcifera, is generated, wherein the specific stacking height of stacking and fermenting is 50 cm;

(3) feeding by tea-producing insects: adopting a natural priming or artificial placing mode for the birectionetia foetida to lay eggs and hatch, wherein the larvae eat raw materials, and the temperature of the raw materials is controlled at 35 ℃;

(4) collecting semi-finished products and removing impurities: collecting the digested and fermented product after the tea-producing insect, namely the biddulphia nubilalis, takes the food, drying the digested and fermented product in the air, and removing impurities and impurities to obtain a semi-finished product of the insect tea;

(5) removing impurity gas at high temperature: removing miscellaneous gas and part of water at high temperature from the semi-finished insect tea product collected in the step (4), wherein the miscellaneous gas is removed at high temperature by adopting a hot air method at 120 ℃ for 20 min;

(6) spreading for cooling: spreading the semi-finished product subjected to high-temperature impurity gas removal for cooling for 30min, uniformly redistributing the tea body, and cooling to room temperature to enter the next procedure;

(7) drying with full fire: drying the spread and cooled semi-finished product at 90 ℃ for 25min until the water content is below 10%;

(8) packaging and storing: the finished product is sealed and packaged and stored under the conditions of light resistance, drying and no peculiar smell.

Example 4

An insect tea is prepared by the following steps:

(1) selecting a parasitic plant raw material: collecting fresh raw materials of host plant leaves and tender stems according to the feeding characteristics of the tea-producing insect dual-straight-nest borer, wherein the parasitic plant raw materials are wild jujube leaves;

(2) fermenting raw materials: naturally spreading the collected parasitic plant raw materials until no surface water exists, stacking and fermenting for 2.5 days until odor capable of attracting the tea-producing insect Bithonella medicinalis is generated, wherein the specific stacking height of stacking and fermenting is 40 cm;

(3) feeding by tea-producing insects: adopting a natural priming or artificial placing mode to enable the biddulphia webworm to lay eggs and hatch, wherein the larvae eat raw materials, and the temperature of the raw materials is controlled at 30 ℃;

(4) collecting semi-finished products and removing impurities: collecting the digested and fermented product after the tea-producing insect, namely the biddulphia nubilalis, takes the food, drying the digested and fermented product in the air, and removing impurities and impurities to obtain a semi-finished product of the insect tea;

(5) removing impurity gas at high temperature: removing miscellaneous gas and part of water at high temperature from the semi-finished insect tea product collected in the step (4), wherein the miscellaneous gas is removed at high temperature by adopting a microwave method for 3.5 min;

(6) spreading for cooling: spreading the semi-finished product subjected to high-temperature impurity gas removal for 25min, uniformly redistributing the tea body, and cooling to room temperature to enter the next procedure;

(7) drying with full fire: drying the spread and cooled semi-finished product at 75 ℃ for 35min until the water content is below 10%;

(8) packaging and storing: the finished product is sealed and packaged and stored under the conditions of light resistance, drying and no peculiar smell.

Example 5

An insect tea is prepared by the following steps:

(1) selecting a parasitic plant raw material: collecting fresh raw materials of host plant leaves and tender stems according to the feeding characteristics of the tea-producing insect, namely the double-straight-nest borer, wherein the parasitic plant raw materials are the hooked rattan leaves;

(2) fermenting raw materials: naturally spreading the collected parasitic plant raw materials until no surface water exists, stacking and fermenting for 2.5 days until odor capable of attracting the tea-producing insect Bithonella medicinalis is generated, wherein the specific stacking height of stacking and fermenting is 40 cm;

(3) feeding by tea-producing insects: adopting a natural priming or artificial placing mode to enable the biddulphia webworm to lay eggs and hatch, wherein the larvae eat raw materials, and the temperature of the raw materials is controlled at 30 ℃;

(4) collecting semi-finished products and removing impurities: collecting the digested and fermented product after the tea-producing insect, namely the biddulphia nubilalis, takes the food, drying the digested and fermented product in the air, and removing impurities and impurities to obtain a semi-finished product of the insect tea;

(5) removing impurity gas at high temperature: removing miscellaneous gas and part of water at high temperature from the semi-finished insect tea product collected in the step (4), wherein the miscellaneous gas is removed at high temperature for 4min by adopting a steam method;

(6) spreading for cooling: spreading the semi-finished product subjected to high-temperature impurity gas removal for 25min, uniformly redistributing the tea body, and cooling to room temperature to enter the next procedure;

(7) drying with full fire: drying the spread and cooled semi-finished product at 75 ℃ for 35min until the water content is below 10%;

(8) packaging and storing: the finished product is sealed and packaged and stored under the conditions of light resistance, drying and no peculiar smell.

Example 6

An insect tea is prepared by the following steps:

(1) selecting a parasitic plant raw material: collecting fresh raw materials of host plant leaves and tender stems according to the feeding characteristics of the tea-producing insect dual-straight-nest borer, wherein the parasitic plant raw materials are wild jujube leaves: tea leaf: 1, hooked rattan leaf: 3: 1;

(2) fermenting raw materials: naturally spreading the collected parasitic plant raw materials until no surface water exists, stacking and fermenting for 2.5 days until odor capable of attracting the tea-producing insect Bithonella medicinalis is generated, wherein the specific stacking height of stacking and fermenting is 40 cm;

(3) feeding by tea-producing insects: adopting a natural priming or artificial placing mode to enable the biddulphia webworm to lay eggs and hatch, wherein the larvae eat raw materials, and the temperature of the raw materials is controlled at 30 ℃;

(4) collecting semi-finished products and removing impurities: collecting the digested and fermented product after the tea-producing insect, namely the biddulphia nubilalis, takes the food, drying the digested and fermented product in the air, and removing impurities and impurities to obtain a semi-finished product of the insect tea;

(5) removing impurity gas at high temperature: removing miscellaneous gas and part of water at high temperature from the semi-finished insect tea product collected in the step (4), wherein the miscellaneous gas is removed at high temperature by adopting a steam method for 8 min;

(6) spreading for cooling: spreading the semi-finished product subjected to high-temperature impurity gas removal for 25min, uniformly redistributing the tea body, and cooling to room temperature to enter the next procedure;

(7) drying with full fire: drying the spread and cooled semi-finished product at 75 ℃ for 35min until the water content is below 10%;

(8) packaging and storing: the finished product is sealed and packaged and stored under the conditions of light resistance, drying and no peculiar smell.

Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above-described embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.