Preparation method of probiotics and lactic acid bacteria, preparation method of saccharomycetes and bee feed

Abstract

The invention discloses probiotics, a preparation method of lactic acid bacteria, a preparation method of saccharomycetes and a bee feed. The probiotics are prepared from the following components in parts by mass: 10-15 parts of lactic acid bacteria, 6-8 parts of saccharomycetes, 25-35 parts of pollen, 28-35 parts of cane sugar, 0.5-1.5 parts of vitamins, 1-5 parts of fructus forsythiae, 1-5 parts of radix isatidis, 1-5 parts of pericarpium citri reticulatae, 1-5 parts of rhizoma atractylodis macrocephalae and 1-5 parts of hawthorn fruit powder. The probiotics can decompose oligosaccharides in the camellia oleifera nectar, so that the digestion burden of camellia oleifera bees can be obviously relieved, the growth of the group potential of the bees is promoted, and the rotten seed rate is reduced. The probiotics and the bee feed can improve the collection enthusiasm of bees to the camellia oleifera nectar and enhance the pollination efficiency, so that the camellia oleifera yield is increased.

Description

Technical Field

[0001] The invention relates to a bee feed formula, in particular to a probiotic formula for improving the pollination efficiency of oil-tea bees, and belongs to the technical field of insect breeding feed. Background Art

[0002] Camellia oleifera is an important woody oil crop in my country, widely cultivated in southern China. Its seeds, which produce tea oil, possess both high economic and nutritional value. The yield and quality of camellia oil are highly dependent on effective pollination, and honeybees are its primary pollinators. However, camellia nectar contains a high proportion of oligosaccharides (such as raffinose and stachyose), which pose a challenge to the bee's digestive system. Oligosaccharides are a class of carbohydrates that are difficult for bees to directly digest and absorb. Bees lack the enzymes required to break down these oligosaccharides. This leads to problems such as indigestion, increased intestinal burden, larval rot, and abdominal swelling in adult workers after collecting camellia nectar. Studies have shown that bees can experience a 30%-50% shortened lifespan and a significant decrease in pollination activity after collecting camellia nectar. This phenomenon not only impacts bee health and colony development but also directly reduces pollination efficiency and yield.

[0003] In order to solve the above problems, the following methods are generally used in the prior art: Supplementing with ordinary syrup: During the camellia oleifera flowering period, beekeepers supplement their bee colonies with sucrose syrup to dilute the concentration of oligosaccharides in the camellia nectar and reduce the digestive burden on the bees. However, this method has limited effectiveness and cannot fundamentally address the negative impact of oligosaccharides on the bees' digestive system.

[0004] Increasing the number of bee colonies: By increasing the number of bee colonies in oil-tea plantations, pollination coverage is attempted to be improved. However, this method is costly and, because the problem of bees digesting oil-tea nectar has not been solved, the improvement in pollination efficiency is not significant.

[0005] Introducing other pollinators: Attempts have been made to introduce other pollinators (such as bumblebees) to replace or supplement honeybee pollination. However, these pollinations are generally less efficient than honeybees and are more difficult to manage, making them difficult to implement on a large scale.

[0006] In recent years, the use of probiotics in agriculture and animal husbandry has gradually gained attention. Probiotics are active microorganisms that are beneficial to the host's health. They can promote the host's growth and health by improving the balance of intestinal flora, enhancing digestion, and boosting immunity. In the field of beekeeping, studies have shown that supplementing with specific probiotics can enhance bees' digestive capacity and increase their efficiency in breaking down complex carbohydrates, thereby improving their health and pollination performance. However, specific probiotic formulas have not yet been developed and applied to address the specificity of oligosaccharides in camellia nectar.

[0007] Furthermore, existing research on the use of probiotics in beekeeping focuses primarily on improving common sugar syrups or preventing and treating bee diseases. There is a lack of specific research on oligosaccharides in camellia nectar, and the negative impact of oligosaccharides on the digestive system of bees during pollination has yet to be effectively addressed. Therefore, providing a method that can help bees efficiently decompose oligosaccharides in camellia nectar has important practical significance and application value. Summary of the Invention

[0008] The present invention aims to provide a probiotic detoxification formula for camellia apis, so as to reduce the digestive burden of camellia apis, improve pollination efficiency and camellia apis yield.

[0009] 1. A probiotic for improving the pollination efficiency of oil-tea bees, characterized in that it is composed of the following components in parts by mass: 10-15 parts of lactic acid bacteria, 6-8 parts of yeast, 25-35 parts of pollen, 28-35 parts of sucrose, 0.5-1.5 parts of vitamins, 1-5 parts of forsythia, 1-5 parts of isatis root, 1-5 parts of tangerine peel, 1-5 parts of atractylodes, and 1-5 parts of hawthorn powder.

[0010] Preferably, the probiotics are composed of the following components in parts by mass: 13 parts of lactic acid bacteria, 7 parts of yeast, 30 parts of pollen, 32 parts of sucrose, 1 part of vitamins, 3 parts of forsythia, 4 parts of isatis root, 4 parts of tangerine peel, 3 parts of atractylodes, and 3 parts of hawthorn powder.

[0011] The vitamins are one or both of vitamin C and vitamin E. The total number of viable lactic acid bacteria and yeasts in each gram of probiotics is 10-30 billion.

[0012] 2. The present invention also provides a method for preparing the lactic acid bacteria in the above-mentioned probiotics, comprising the following steps: Step 1: Add honey bee intestine to 1 ml of PBS and homogenize for 5 minutes; Step 2: gradient dilution of the slurry in step 1, inoculation of the slurry into MRS solid medium, and anaerobically cultured at 30° C. for 48 hours; Step 3: Pick out the dominant lactic acid bacteria colony from step 2, perform three rounds of purification using the plate streak method to obtain a lactic acid bacteria strain, send it to the company for sequencing, perform BLAST analysis and comparison on the NCBI website, and confirm that the lactic acid bacteria strain is Lactobacillus plantarum ( Lactiplantibacillus plantarum ), inoculating the lactic acid bacteria strain into a liquid culture medium for expansion culture to obtain the lactic acid bacteria.

[0013] The lactic acid bacteria culture medium is composed of the following components in parts by weight: 0.1-1.0 parts of peptone, 0.1-0.5 parts of beef extract, 0.1-0.5 parts of yeast extract, 0.1-0.5 parts of sodium chloride, 0.001-0.01 parts of MnSO4·H2O, 0.001-0.01 parts of CaCl2, 0.001-0.1 parts of MgSO4·7H2O, and 90-99 parts of ultrapure water, and is sterilized.

[0014] 3. The present invention also provides a method for preparing the yeast used in the above-mentioned probiotics, comprising the following steps: inoculating 4-6% of the yeast volume into a yeast culture medium, and culturing the culture in a constant temperature environment at a pH of 3.0-7.5 and a temperature of 25°C for 24-72 hours to obtain the yeast. The yeast culture medium is composed of the following components by weight: 0.1-1.0 parts peptone, 0.1-0.5 parts yeast extract, 1-2 parts glucose, and 90-99 parts ultrapure water, and is sterilized.

[0015] 4. The present invention also provides a bee feed using the above-mentioned probiotics. In fact, the above-mentioned probiotics can be directly used as bee feed.

[0016] Due to the adoption of the above technical solution, the present invention has the following advantages: The probiotics of the present invention can decompose oligosaccharides in oil-tea camellia nectar, significantly reduce the digestive burden of oil-tea camellia bees, promote the growth of bee colonies, and reduce the rate of rotten eggs.

[0017] The probiotics and bee feed of the present invention can increase the enthusiasm of bees to collect oil-tea camellia nectar, enhance pollination efficiency, and thus increase oil-tea camellia yield.

[0018] The probiotic formula of the present invention is safe and environmentally friendly, has no side effects on bees and oil-tea camellia, and is suitable for large-scale promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 图1 This is a trend chart showing the effects of different feeds on the colony strength of Italian honey bees. DETAILED DESCRIPTION

[0020] The embodiments of the present application are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended only to explain the present application and are not to be construed as limiting the present application. On the contrary, the embodiments of the present application include all variations, modifications, and equivalents that fall within the spirit and scope of the appended claims.

[0021] Example 1: This example provides a probiotic for improving the pollination efficiency of oil-tea bees, which is composed of the following components in parts by mass: 10-15 parts of lactic acid bacteria, 6-8 parts of yeast, 25-35 parts of pollen, 28-35 parts of sucrose, 0.5-1.5 parts of vitamins, 1-5 parts of forsythia, 1-5 parts of isatis root, 1-5 parts of tangerine peel, 1-5 parts of atractylodes, and 1-5 parts of hawthorn powder. Wherein, the mass fraction of described lactic acid bacteria can be selected but not limited to 10 parts, 13 parts or 15 parts, the mass fraction of described yeast can be selected but not limited to 6 parts, 7 parts or 8 parts, the mass fraction of described pollen can be selected but not limited to 25 parts, 30 parts or 35 parts, the mass fraction of described sucrose can be selected but not limited to 28 parts, 30 parts or 35 parts, the mass fraction of described vitamin can be selected but not limited to 0.5 part, 1 part or 1.5 parts, the mass fraction of described forsythia can be selected but not limited to 1 part, 3 parts or 5 parts, the mass fraction of described isatis can be selected but not limited to 1 part, 3 parts or 5 parts, the mass fraction of described orange peel can be selected but not limited to 1 part, 2 parts or 5 parts, the mass fraction of described hawthorn powder can be selected but not limited to 1 part, 4 parts or 5 parts.Described vitamin is one or both of vitamin C and vitamin E.

[0022] In this embodiment, the probiotics are selected to be composed of the following components in parts by mass: 13 parts of lactic acid bacteria, 7 parts of yeast, 30 parts of pollen, 32 parts of sucrose, 1 part of vitamin C, 3 parts of forsythia, 4 parts of isatis root, 4 parts of tangerine peel, 3 parts of atractylodes and 3 parts of hawthorn powder.

[0023] Wherein, the preparation method of the lactic acid bacteria is: Step 1: Add honey bee intestine to 1 ml of PBS and homogenize for 5 minutes; Step 2: gradient dilution of the slurry in step 1, inoculation of the slurry into MRS solid medium, and anaerobically cultured at 30° C. for 48 hours; Step 3: Pick out the dominant lactic acid bacteria colony from step 2, perform three rounds of purification using the plate streak method to obtain a lactic acid bacteria strain, send it to the company for sequencing, perform BLAST analysis and comparison on the NCBI website, and confirm that the lactic acid bacteria strain is Lactobacillus plantarum ( Lactiplantibacillus plantarum ), inoculating the lactic acid bacteria strain into a liquid culture medium for expansion culture to obtain the lactic acid bacteria.

[0024] The sequence of the lactic acid bacteria is shown below The lactic acid bacteria culture medium consists of the following components in parts by weight: 0.1-1.0 parts of peptone, 0.1-0.5 parts of beef extract, 0.1-0.5 parts of yeast extract, 0.1-0.5 parts of sodium chloride, 0.001-0.01 parts of MnSO4·H2O, 0.001-0.01 parts of CaCl2, 0.001-0.01 parts of MgSO4·7H2O, and 90-99 parts of ultrapure water, and is sterilized.

[0025] The yeast is prepared by inoculating 4-6% of the yeast volume into a yeast culture medium, culturing the culture in a constant temperature environment at a pH of 3.0-7.5 and a temperature of 25°C for 24-72 hours to obtain the yeast. The yeast culture medium comprises the following components by weight: 0.1-1.0 parts peptone, 0.1-0.5 parts yeast extract, 1-2 parts glucose, and 90-99 parts ultrapure water, and is sterilized.

[0026] This embodiment also provides bee feed containing the above-mentioned probiotics, wherein the number of viable probiotic bacteria (i.e., the total number of viable lactic acid bacteria and yeast) contained in each gram of bee feed is 10-30 billion. In fact, the above-mentioned probiotics can be directly used as bee feed.

[0027] Here, bees fed with the bee feed containing the above-mentioned probiotics provided in this embodiment were tested against a control group to detect: Experiment 1: Comparison of bee colony strength and capping capacity Six colonies of Italian honey bees of the same species and comparable strength, without pollen reserves, were randomly divided into two groups: a control group (Group A) without probiotic lactic acid bacteria and yeast, and a probiotic diet group (Group B). Before the experiment began, each group used a pollen remover with a smaller aperture to control pollen collection by field bees. The corresponding diet was then fed to the corresponding bee colonies, and pollen clumps were regularly replaced to ensure a consistently adequate food supply. The experiment lasted 36 days.

[0028] Determination of Colony Strength and Capping: During the experiment, colony strength and capping were observed and recorded four times every 12 days. Colony strength was measured using a "foot-frame bee," and the capping area on each comb was recorded in detail. Total colony strength and capping were calculated for each colony. Designed wooden comb frames (divided into 45 uniformly sized grids by fine lines in the center) were used to record colony strength and capping.

[0029] Here are the results: 1. Effects of different feeds on the colony strength of Italian honey bees Effects of feeding different diets on the development of Italian honey bee colonies 图1As shown in the figure, colony strength developed differently when fed different diets. After 36 days of feeding, the colony strength of the bee group fed with probiotics was significantly higher than that of the control group (P<0.05). Identical letters in the figure indicate no significant difference, while different letters indicate a significant difference (P<0.05).

[0030] 2. Effects of different feeds on the amount of capping by Italian honey bees Table 1 shows the effects of different diets on the number of cappings in Italian honey bees. Significant differences were observed between the different diets. On day 12, there was no significant difference in capping between the two feeding groups (P>0.05). However, on days 24 and 36, the number of cappings in the probiotic group was significantly higher than in the control group (P<0.05).

[0031] Table 1 Effects of different diets on the amount of capping by Italian honey bees

[0032] The data in the table are mean ± standard deviation. Lowercase letters indicate longitudinal comparison. The same letters indicate no significant difference, while different letters indicate significant difference (P < 0.05).

[0033] The above test results fully demonstrate that using the bee feed containing probiotics provided in this embodiment to feed bees can significantly reduce the digestive burden of camellia oleifera bees, promote the growth of bee colonies, and reduce the rate of rotten eggs.

[0034] Experiment 2: Comparison of collection activity Six colonies of Italian honey bees of the same species and comparable strength, without pollen reserves, were randomly divided into two groups: a control group (Group A) without probiotic lactic acid bacteria and yeast, and a probiotic diet group (Group B). Before the experiment began, each experimental group used a pollen remover with a smaller aperture to control pollen collection by field bees. The corresponding diet was then fed to the corresponding bee colonies, and pollen pellets were regularly replaced to ensure a consistently adequate food supply.

[0035] Fifteen days after the start of the experiment, three days of clear and windless weather were selected to observe the collecting activity of worker bees in each bee colony, and the number of worker bees leaving the nest to collect was recorded in five time periods: 9:00-9:05, 11:00-10:05, 13:00-13:05, 15:00-15:05, and 17:00-17:05.

[0036] The results are as follows: The effects of different diets on the foraging activity of Italian honey bees are shown in Table 2. Probiotics significantly promoted the foraging behavior of Italian honey bees (P < 0.05). During the 13:00-13:05 and 15:00-15:05 time periods, the foraging activity of worker bees in the probiotic-fed group was significantly higher than that in the control group (P < 0.05).

[0037] Table 2 Effects of different feeding methods on the foraging activity of Apis mellifera

[0038] The above test results fully demonstrate that using the bee feed containing probiotics provided in this embodiment to feed bees can increase the bees' enthusiasm for collecting camellia nectar, enhance pollination efficiency, and thus increase camellia oleifera yield.

[0039] In addition, this embodiment also provides a method for preparing the probiotics in this embodiment, comprising the following steps: fully crushing the pollen, sucrose, forsythia, isatis root, tangerine peel, atractylodes and hawthorn powder, and fully mixing them with the lactic acid bacteria, yeast and vitamins.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that various changes can be made in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A probiotic for improving the pollination efficiency of oil-tea bees, characterized in that: The invention is composed of the following components in parts by mass: 10-15 parts of lactic acid bacteria, 6-8 parts of yeast, 25-35 parts of pollen, 28-35 parts of sucrose, 0.5-1.5 parts of vitamins, 1-5 parts of forsythia, 1-5 parts of isatis root, 1-5 parts of tangerine peel, 1-5 parts of atractylodes, and 1-5 parts of hawthorn powder.

2. The probiotic for improving the pollination efficiency of oil-tea bees as claimed in claim 1, characterized in that: The invention is composed of the following components in parts by mass: 13 parts of lactic acid bacteria, 7 parts of yeast, 30 parts of pollen, 32 parts of sucrose, 1 part of vitamins, 3 parts of forsythia, 4 parts of isatis root, 4 parts of tangerine peel, 3 parts of atractylodes, and 3 parts of hawthorn powder.

3. The probiotic for improving the pollination efficiency of oil-tea bees according to claim 1 or 2, characterized in that: The vitamins are one or both of vitamin C and vitamin E.

4. The probiotic for improving the pollination efficiency of camellia honey as claimed in claim 3, wherein: The total number of live lactic acid bacteria and yeasts in each gram of probiotics is 10-30 billion.

5. A method for preparing lactic acid bacteria as claimed in claim 1 or 2, characterized in that: The following steps are involved: Step 1: Add honey bee intestine to 1 ml of PBS and homogenize for 5 minutes; Step 2: gradient dilution of the slurry in step 1, inoculation of the slurry into MRS solid medium, and anaerobically cultured at 30° C. for 48 hours; Step 3: Pick out the lactic acid bacteria colonies with the largest number from step 2, perform three rounds of purification using the plate streak method to obtain the lactic acid bacteria strain, and inoculate the lactic acid bacteria strain into a liquid culture medium for expansion culture to obtain the lactic acid bacteria.

6. The method for preparing lactic acid bacteria according to claim 5, wherein: The lactic acid bacteria culture medium consists of the following components in parts by weight: 0.1-1.0 parts of peptone, 0.1-0.5 parts of beef extract, 0.1-0.5 parts of yeast extract, 0.1-0.5 parts of sodium chloride, 0.001-0.01 parts of MnSO4·H2O, 0.001-0.01 parts of CaCl2, 0.001-0.01 parts of MgSO4·7H2O, and 90-99 parts of ultrapure water, and is sterilized.

7. A method for preparing the yeast according to claim 1 or 2, characterized in that: The method comprises the following steps: inoculating yeast into a yeast culture medium at a volume of 4-6% of the culture medium, and culturing the culture medium in a constant temperature environment with a pH value of 3.0-7.5 and a temperature of 25° C. for 24-72 hours to obtain the yeast.

8. The method for preparing yeast according to claim 7, wherein: The yeast culture medium is composed of the following components by weight: 0.1-1.0 parts of peptone, 0.1-0.5 parts of yeast extract, 1-2 parts of glucose, and 90-99 parts of ultrapure water, and is sterilized.

9. A bee feed, characterized in that: The probiotics according to claim 1, 2, 3 or 4 are used.

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