TWI782437B - Leuconostoc mesenteroides and composition thereof - Google Patents

Abstract

The invention relates to a Leuconostoc mesenteroidesTEB-8 which has sucrose solution tolerance, acid tolerance and the ability to inhibit the growth of Paenibacillus larvae, and can promote the expression of nutritional genes and immune genes of honeybees. Therefore, the Leuconostoc mesenteroidesTEB-8 can be used as a probiotic for honeybees, improving the health of the colony and increasing the colony’s resistance to environmental changes and the production of bee products.

Description

Leuconostoc enteritidis and compositions thereof

The present invention relates to a lactic acid bacterium and its use, in particular to a Leuconostoc enterica and its use.

Bees ( Apis mellifera ) are important economic insects, which can be applied to crop pollination and bring high agricultural economic benefits; in addition, the bee products produced by bees, such as: honey, royal jelly, pollen, propolis, bee brood, beeswax and bees Natural health products such as poisons also bring considerable economic scale. However, in the past 30 years, the survival of bee colonies has been threatened. Since 1994, a large number of bee colonies have disappeared in Europe and the United States. Until 2006, American scientists officially named this phenomenon of bee colony collapse syndrome (Colony Collapse Disorder) ). In Taiwan, there have also been cases of bee colonies disappearing in recent years. For example, in 2017, nearly 300 colonies of bees disappeared in a short period of time in the Puli area of Nantou and the Dashu area of Kaohsiung.

There are different opinions on the disappearance of bee colonies. Scientists believe that bee colony disintegration syndrome is caused by the interaction of many factors, such as: pathogenic bacteria, viruses, parasitic mites and pesticide chemicals. In terms of pathogens, Spanish and American scholars believe that Nosema ceranae, small RNA-like virus Israel acute paralysis virus ( IAPV ) and deformed wing virus (DWV) are the causes of bee colony disintegration syndrome. They argue that the bee colony disintegration syndrome is caused by the infection of the bee midgut, which destroys the ability of the bee to digest and absorb food, resulting in nutritional deficiency or malnutrition, which leads to the weakness of the bee and the decline in the ability to fly, and even the inability to return to the nest. In terms of chemical agents: Bees exposed to high doses of pesticides will die soon and cannot return to the nest; however, if exposed to low doses of pesticides, adult bees can still bring pesticide-containing food back to the nest for other bees to eat, and even feed it to larvae . Studies have found that eating larvae containing micro-doses of pesticides will damage their neurological development, resulting in impairment of learning and memory abilities, making it possible for them to find no food or a way back to the nest when they go out to collect in the future; our study It was also found that larvae exposed to low doses of pesticides, when developing into queen bees, cannot normally express the royal jelly protein gene. Secretion of royal jelly protein, the development of the hive bees will be greatly hindered, or even die. In terms of parasitic mites: parasitic mites reproduce at a very high rate in the bee colony, and will suck the body fluid and fat body of the larvae, causing the larvae to weaken, and even spread viral diseases. In severe cases, the bees in the nest will soon weaken and die.

However, when exploring the influence of various factors on the physiology of bees, it can be found that many factors that lead to bee colony disintegration syndrome, such as pesticides, habitat destruction, parasitic mites and pathogenic bacteria, etc., mainly interfere with the "nutritional physiology" of individual bees, causing nutritional stress , After the weakening of individual bees, it immediately affects the sound development of the overall bee colony, resulting in the collapse of the bee colony. Therefore, it is an urgent task to develop probiotics that can improve the nutritional and immune physiology of bee colonies and promote the health of bee colonies.

In view of this, one aspect of the present invention is to provide a Leuconostoc mesenteroides TBE-8, which is deposited in the Food Industry Development Research Institute of the Foundation, and the deposit number is BCRC 911036.

According to the aforementioned Leuconostoc enterococcus TBE-8, it may have sucrose solution tolerance, preferably, the sucrose solution tolerance is that the concentration of the sucrose solution is greater than 0% and less than 70%.

According to the aforementioned Leuconostoc enterococcus TBE-8, it may have acid tolerance, preferably, the pH value of the acid tolerance is greater than 4 and less than or equal to 6.5.

According to the aforementioned Leuconostoc enterococcus TBE-8, it may have the ability to inhibit the growth of Bacillus larvae.

According to the aforementioned Leuconostoc enterococcus TBE-8, it has the ability to enhance the expression of one of the bee's nutritional genes and an immune gene, preferably, the nutritional genes include major royal jelly protein 1 ( mrjp1 ) gene and vitellogenin ( vg ) gene, the immune gene comprises hymenoptaecin gene and apidaecin gene.

Another aspect of the present invention is to provide a composition comprising Leuconostoc enteritidis TBE-8 or a derivative of Leuconostoc enteritidis TBE-8.

Thereby, Leuconostoc enterococcus TBE-8 of the present invention has high tolerance to sucrose solution, and also has tolerance to the acidic environment of the gastrointestinal tract of bees, meets the characteristics required by probiotics, and can affect the physiology of bees, By adding Leuconostoc enterococcus TBE-8 into the sucrose solution, it can be fed by bees and enter the intestinal tract to exert its effect, and can effectively stay in the intestinal tract. Therefore, the Leuconostoc enterolis TBE-8 of the present invention, and the composition comprising the Leuconostoc enteritidis TBE-8 or its derivatives of the present invention can be used as probiotics for bees, and have the potential to be applied to the beekeeping industry.

The above summary is intended to provide a simplified summary of the disclosure to provide readers with a basic understanding of the disclosure. This summary is not an extensive overview of the disclosure and it is not intended to identify key/critical elements of the embodiments of the invention or to delineate the scope of the invention.

This case discloses a novel strain of lactic acid bacteria, which was identified as Leuconostoc mesenteroides and named Leuconostoc mesenteroides TBE-8. Leuconostoc enterica belongs to the genus Leuconostoc . It has chemical heterotrophy and is a commonly used heterofermentative lactobacillus. It often exists in dairy products and is a commonly used flavoring agent in many cheese productions. It can also be used for The production of wine, the production of sauerkraut and bread, and the production of sauerkraut as a starter. Observed under the microscope, Leuconostoc enterica grows in pairs and arranged in chains. Leuconostoc enterica has a length of about 0.7-1.2 μm and a diameter of about 0.5-0.7 μm. It is a Gram-positive bacterium and a non-motile, non-porous and spherical facultative anaerobic bacterium.

The following specific test examples are hereby further illustrated to illustrate the present invention, in order to benefit those with ordinary knowledge in the technical field of the present invention, they can fully utilize and practice the present invention without excessive interpretation, and these test examples should not be regarded as To limit the scope of the invention, but to illustrate how to practice the materials and methods of the invention.

[Test example]

1. the cultivation of Leuconostoc enteromenis TBE-8 of the present invention

Leuconostoc enterococci TBE-8 (hereinafter referred to as strain TBE-8) of the present invention was isolated from Taiwan's selected bumblebee ( Bombus eximius ) by the inventor of the present invention, further cultivated, identified and preserved, and deposited in the food industry consortium Development Research Institute, whose deposit number is BCRC 911036. When cultivating bacterial strain TBE-8, MRS liquid medium can be used for cultivation, wherein 1000 mL of MRS liquid medium contains 10 g of peptone, 8 g of beef extract (meat extract), 4 g of yeast extract ( yeast extract), 20 g of glucose (glucose), 2 g of disodium hydrogen phosphate (disodium hydrogen phosphate), 5 g of sodium acetate trihydrate, 2 g of ammonium citrate (triammonium citrate), 0.2 g of seven Magnesium sulfate heptahydrate, 0.05 g of magnesium sulfate tetrahydrate and 1000 mL of sterile water.

In addition, the bacterial strain TBE-8 of the present invention was cultivated on the MRS plate medium, and the growth status of the colony was observed. The MRS plate medium is prepared by adding an additional 12 g of agar gum to the aforementioned MRS liquid medium. Please refer to Figure 1, which is a colony map of strain TBE-8. From the results in Figure 1, it can be seen that the diameter of the colony of strain TBE-8 on the MRS plate medium is about 1 mm, and the surface of the colony is milky white, convex lens-shaped, and the surface is smooth and the edges are flat, which is similar to the known type of Leuconostoc enteritidis. same state.

2. Bee Toxicology Test of Strain TBE-8

Strain TBE-8 is a strain isolated from the intestinal tract of selected bumble bees. Before discussing the characteristics of the strain in depth, the toxicity of this strain to bees is analyzed first. In the experiment, 10 μL of the strain TBE-8 bacteria liquid was fed to the bees, and the experimental group was divided into 3 groups, among which the bacterial content of group A was 6.4×10 ⁸ CFU/mL, and the bacterial content of group B was 6.4×10 ⁷ CFU/mL. mL, the bacterial content of group C was 6.4×10 ⁶ CFU/mL. In the experiment, the bees were fed with 10 μL of 50% sucrose solution as the control group. The number of surviving individuals 7 days after feeding was recorded, and after 7 days of observation, the number of bacteria strain TBE-8 remaining in the intestinal tract of group A honeybees was further analyzed by qPCR.

Please refer to Figure 2A and Figure 2B, which are the analysis results of the bee toxicology test of strain TBE-8, wherein Figure 2A shows the survival rate of bees, and Figure 2B shows the strain TBE remaining in the bee gut after 7 days -8 bacteria count.

The results in Figure 2A show that the survival rate of the bees in the control group, group A, group B and C after 7 days averaged more than 90%, and there was no death at all in the experimental group, showing that the survival rate of the bees was no different among the four groups Significant difference (P > 0.05). It can be seen that the strain TBE-8 is not toxic to honeybees. The results in Figure 2B show that in group A fed with bees with a bacterial content of 6.4×10 ⁸ CFU/mL, the bacterial count of strain TBE-8 in the bee intestines was 8×10 ³ copies/ng DNA after 7 days of feeding , compared with the control group fed bees with 10 μL of 50% sucrose solution, there was a very significant difference (P = 0.000441).

3. Determination of sucrose solution tolerance of bacterial strain TBE-8

Because beekeepers often feed bees sucrose solution in seasons when food sources are scarce to maintain the colony of bees, after confirming that strain TBE-8 is not toxic to bees, this test further confirms the tolerance of strain TBE-8 to sucrose solution . In the experiment, strain TBE-8 was placed in 50%, 60% and 70% sucrose solution respectively, and the number of viable bacteria was measured after 24 hours, 48 hours and 72 hours.

Please refer to Figure 3A and Figure 3B, which are the analysis results of the sucrose solution tolerance test of the strain TBE- ⁸ . ×10 ⁹ CFU/mL, 2.51×10 ⁹ CFU/mL respectively placed in 50%, 60% and 70% sucrose solution, the number of live bacteria after 24 hours; Figure 3B shows the bacterial count of strain TBE-8 The number of live bacteria after 24 hours, 48 hours and 72 hours was 4.33×10 ⁸ CFU/mL placed in 50% sucrose solution.

The results in Figure 3A show that the number of viable bacteria decreased from 2.86×10 ⁹ ± 1.39×10 ⁸ CFU/mL to 2.36×10 ⁹ ± 2.75×10 ⁷ CFU after strain TBE-8 was placed in 50% sucrose solution for 24 hours /mL (survival rate as high as 82.5%). After strain TBE-8 was placed in 60% sucrose solution for 24 hours, the number of viable bacteria decreased from 2.65×10 ⁹ ± 1×10 ⁸ CFU/mL to 9.52×10 ⁸ ± 1.84×10 ⁷ CFU/mL (the survival rate was 35.8 %). After strain TBE-8 was placed in 70% sucrose solution for 24 hours, the number of viable bacteria decreased from 2.51×10 ⁹ ± 1.22×10 ⁸ CFU/mL to 5.73×10 ⁶ ± 3.79×10 ⁵ CFU/mL (the survival rate was only 0.2%). It shows that strain TBE-8 has the highest number of viable bacteria in 50% sucrose solution, which is 412 times that of the group in 70% sucrose solution, so it is known that strain TBE-8 is difficult to survive in 70% sucrose solution , but it is worth mentioning that the viable count of strain TBE-8 is still 5.73×10 ⁶ CFU/mL.

In the experiment, the number of live bacteria of strain TBE-8 after being placed in 50% sucrose solution for 48 hours and 72 hours was further investigated, so as to understand the tolerance of strain TBE-8 in 50% sucrose solution. The results in Figure 3B show that the number of viable bacteria decreased from 4.89×10 ⁸ ± 2.65×10 ⁶ CFU/mL to 2.66×10 ⁸ ± 8.25×10 ⁷ CFU/mL after strain TBE-8 was exposed to 50% sucrose solution for 48 hours (The survival rate was 62.6%). After strain TBE-8 was exposed to 50% sucrose solution for 72 hours, the number of viable bacteria decreased from 4.89×10 ⁸ ± 2.25×10 ⁶ CFU/mL to 1.46×10 ⁸ ± 4.61×10 ⁷ CFU/mL (the survival rate was 36.1%) , showing that strain TBE-8 had the highest number of viable bacteria in 50% sucrose solution within 24 hours, and its number of viable bacteria was 2.5 times that of the number of viable bacteria after 72 hours, and within 72 hours, the number of viable bacteria could be maintained at 10 ⁸ above. From the results in Figure 3A and Figure 3B, it can be seen that the strain TBE-8 of the present invention has extremely high tolerance to sucrose solution.

4. Determination of acid tolerance of bacterial strain TBE-8

The pH value of the honeybee gut is between 5.0 and 5.5. In order to know whether the strain TBE-8 can survive in the acidic environment of the honeybee gut, the acid tolerance of the strain TBE-8 was further tested in this experiment. Replace 3 mL of the strain TBE-8 with an _OD600 value of 1.0 with MRS liquid medium with different pH values (pH 4, 5, 6.5), and measure the viable bacteria after 6, 12 and 24 hours of cultivation number. Please refer to Table 1 below, which is the test results of the acid tolerance of strain TBE-8.

Table I MRS liquid medium Viable bacteria count (1×10 ⁷ CFU/mL) Increase (+) or decrease (-) the number of viable bacteria (1×10 ⁷ CFU/mL) 0 hours 6 hours 12 hours 24 hours pH 6.5 47.6±6.4 127.1 ± 15.4 145.4 ± 21.7 344.9 ± 50.1 +297.3 pH 5 40.6±13.8 96.2 ± 2.6 93.9 ± 1.7 76.3 ± 5.3 +35.7 pH 4 43.7±2.5 136 ± 18.5 49.3 ± 5 8.4 ± 3.3 -35.2

The results in Table 1 show that the number of viable bacteria increased by 297.3×10 ⁷ CFU after the strain TBE-8 was cultured at pH 6.5 for 24 hours, compared with the number of viable bacteria in each group after 24 hours of cultivation and the initial (0 hour) bacterial count /mL; after culturing at pH 5 for 24 hours, the number of viable bacteria increased by 35.7×10 ⁷ CFU/mL; after culturing at pH 4 for 24 hours, the number of viable bacteria decreased by 35.2×10 ⁷ CFU/mL. It was shown that the number of viable bacteria in MRS liquid medium with pH value 6.5 was the highest for strain TBE-8, which was 41 times that of MRS liquid medium with pH value 4. Compared with the environment with pH value 4, the strain TBE-8 is more suitable for growth in the environment with pH value 6.5 and 5, so it can survive in the intestine of bees, and the strains suitable for probiotics are resistant to the acidic environment of the gastrointestinal tract of bees receptive characteristics.

5. Determination of fermentation products of strain TBE-8

Leuconostoc enterolis is a type of lactic acid bacteria, and lactic acid bacteria can produce organic acids during the fermentation process. This test further determined the types and contents of organic acids in the fermentation broth of the strain TBE-8 of the present invention after 24, 48 and 72 hours of fermentation.

In the experiment, the strain TBE-8 was raised in 8 tubes of 3 mL MRS liquid medium, and placed in a shaking incubator at 28 ± 1 ^o C, and one tube was taken at 0, 24, 48, and 72 hours and centrifuged at 5000 rpm for 10 minutes. 2 mL of the supernatant was filtered through a 0.22 μm filter membrane and freeze-dried. Subsequently, 20 μL of the filtrate was used for organic acid component analysis by HPLC.

Please refer to Table 2 for the content of organic acids in the fermentation broth of strain TBE-8. The detected organic acids are Oxalic acid, Tartaric acid, Formic acid, Malic acid, Lactic acid, Acetic acid, Succinic acid and citric acid (Citric acid), each treatment is the mean ± standard deviation of three repetitions.

Table II organic acid 24 hours 48 hours 72 hours oxalic acid 0.03 ± 0.001% ND ND tartaric acid 0.05 ± 0.001% 0.01 ± 0.001% ND formic acid ND ND ND malic acid 0.13 ± 0.001% 0.08 ± 0.001% 0.22 ± 0.001% lactic acid 0.33 ± 0.002% 0.46 ± 0.001% 0.38 ± 0.001% acetic acid 0.58 ± 0.001% 0.83 ± 0.001% 0.81 ± 0.001% Succinic acid ND ND ND citric acid 0.40 ± 0.001% 0.39 ± 0.001% 0.32 ± 0.001%

The results in Table 2 show that after the fermentation of strain TBE-8, formic acid and succinic acid have no production at all, and malic acid, lactic acid, acetic acid and citric acid have an average production of more than 0.1%. The average yield after acetic acid fermentation was the highest, which was 5.2 times that of malic acid, 19 times that of lactic acid, and 2 times that of citric acid. Among them, the acetic acid yield was the highest 24 hours after fermentation, which was 4.5 times the malic acid yield, 1.8 times the lactic acid yield, and 1.5 times the citric acid yield; the acetic acid yield was the highest 48 hours after fermentation, which was 10.4 times the malic acid yield and 1.8 times the lactic acid yield times, 21 times the yield of citric acid; 72 hours after fermentation, the yield of acetic acid was the highest, which was 3.7 times that of malic acid, 2.1 times that of lactic acid, and 2.5 times that of citric acid.

Please refer to Figure 4A, Figure 4B, and Figure 4C, which are the results of the determination and analysis of organic acids in the fermentation broth of the strain TBE-8, which is the calculation of the percentage of the output of each organic acid in the total organic acid production, of which Figure 4A Figure 4B is a graph of analysis results for 24 hours, Figure 4B is a graph of analysis results for 48 hours, and Figure 4C is a graph of analysis results for 72 hours.

The results in Figures 4A to 4C show that after 24 hours of fermentation with strain TBE-8, acetic acid accounted for 38% of the total organic acid production, lactic acid accounted for 22% of the total organic acid production, and citric acid accounted for 26% of the total organic acid production After 48 hours of fermentation, acetic acid accounted for 47% of the total organic acid output, lactic acid accounted for 26% of the total organic acid output, and citric acid accounted for 22% of the total organic acid output; after 72 hours of fermentation, acetic acid accounted for 47% of the total organic acid output %, lactic acid accounts for 22% of the total organic acid production, citric acid accounts for 18% of the total organic acid production, and the proportions of the rest of the organic acids are all lower than 15%. It can be seen that the acetic acid production of strain TBE-8 is the highest after 48 hours of fermentation . In addition, after fermentation, the strain TBE-8 not only produces organic acids, but also produces exopolysaccharide (EPS). 10 mg/mL.

6. Strain TBE-8 persists in the honeybee gut

Leuconostoc enteritidis belongs to lactic acid bacteria, and lactic acid bacteria almost exist in the hindgut of bees. Therefore, after feeding the bee strain TBE-8 in this experiment, the amount of the strain TBE-8 in the bee midgut and hindgut was detected to test the strain TBE-8. Whether it will stay in the bee gut. In the experiment, 10 μL of the strain TBE-8 bacteria solution was fed to the bees, with a bacterial content of 1.46×10 ⁹ CFU/mL, and the midgut and hindgut of 10 bees were collected at 0, 12, 24, 48 and 72 hours respectively. DNA extraction was performed, and the bacterial count of strain TBE-8 in the gut was analyzed by qPCR.

Please refer to Figure 5A and Figure 5B, which are the analysis results of the distribution of strain TBE-8 in the intestines of worker bees, among which Figure 5A is the analysis result of the bacterial count of strain TBE-8 in the midgut of bees, and Figure 5B The picture shows the results of the analysis of the bacterial count of the strain TBE-8 in the hindgut of honeybees, and each treatment is the mean ± standard deviation of three repetitions.

The results in Figure 5A show that the bacterial count of the strain TBE-8 in the honeybee midgut was 2.86×10 ⁴ ± 1.79×10 ³ copies/ng DNA at 12 hours, and the bacterial count at 24 hours was 3.92×10 ² ± 7.95×10 ¹ copy/ng DNA, the bacterial count at 48 hours was 8.85×10 ² ± 2.75×10 ¹ copy/ng DNA, and the bacterial count at 72 hours was 1.58×10 ³ ± 9.22×10 ¹ copy/ng DNA. The results in Figure 5B show that the bacterial count of strain TBE-8 in the honeybee hindgut was 1.02×10 ⁵ ± 6.28×10 ³ copies/ng DNA at 12 hours, and the bacterial count at 24 hours was 2.82×10 ⁵ ± 3.30×10 ⁴ copy/ng DNA, the bacterial count at 48 hours was 1.11×10 ⁵ ± 1.13×10 ⁴ copies/ng DNA, and the bacterial count at 72 hours was 1.07×10 ⁵ ± 1.64×10 ⁴ copies/ng DNA. From the above results, it can be seen that as early as 12 hours ago, the strain TBE-8 had entered the hindgut from the midgut, and the bacterial count reached the peak in the hindgut at 24 hours, although the bacterial count of the strain TBE-8 began to decrease after 24 hours, but There was no difference in the amount of bacteria at 48 and 72 hours, and both remained above 10 ⁵ copies/ng DNA. It can be seen that the strain TBE-8 can reside in the hindgut of bees, which is consistent with the characteristics of probiotic strains that can reside in the intestines of bees.

7. Bacteria distribution of worker bee intestinal strain TBE-8 in bee farm bee colony

Leuconostoc enteritidis was isolated from the intestines of bees in previous studies, so this experiment further tested the presence of strain TBE-8 in the intestines of bees in the apiary of Chung Hsing University. In the experiment, 700 newly emerged bees were marked and returned to the bee colony. On the 1st, 3rd, 8th, 12th, 18th, 21st and 25th day, 10 honeybees were extracted from the midgut and hindgut respectively. And carry out qPCR to analyze the bacteria amount of strain TBE-8 in the bee intestines.

Please refer to Figure 6A and Figure 6B, which are the results of the analysis of the bacterial distribution of the bacterial strain TBE-8 in the intestines of worker bees in the bee colony (untouched/untreated TBE-8 strain), and Figure 6A is Figure 6B is the analysis result of the bacterial count of the strain TBE-8 in the honeybee midgut. Figure 6B is the analysis result of the bacterial strain TBE-8 in the honeybee hindgut. Each treatment is the mean ± standard deviation of four replicates.

The results in Figure 6A show that the strain TBE-8 had the highest bacterial count in the midgut of bees in the bee colony on day 18, with a bacterial count of 9822 ± 1007 copies/ng DNA, and the bacterial count was lower than 50 copies/ng DNA in the rest of the days. The results in Figure 6B show that the highest bacterial count was 3731 ± 22 copies/ng DNA on the 8th day in the hindgut of honeybees, and the bacterial count was lower than 70 copies/ng DNA on the remaining days . From the above results, it can be seen that there are trace amounts of strain TBE-8 in both the midgut and hindgut of honey bees.

8. Physiological effects of strain TBE-8 on bees

Strain TBE-8 is non-toxic to bees, and also conforms to the characteristics of some probiotic strains used in animals. This experiment further tested the effect of strain TBE-8 on the physiology of bees.

In the experiment, they were divided into 4 groups. Group 1 was fed with bees fermented broth of strain TBE-8 with a bacterial content of 5.43×10 ⁸ CFU/mL (hereinafter referred to as group F), and group 1 was fed with 50% sucrose solution of bees and given pollen ( Hereinafter referred to as group P), 1 group was fed with 50% sucrose solution of bees (hereinafter referred to as group S), and 1 group was fed with bee MRS liquid medium (hereinafter referred to as group M). After feeding for 7 days, the total RNA of the head and abdomen of each group of bees was extracted, and the expression levels of nutritional genes in the head, nutritional genes in the abdomen and immune genes in the abdomen were analyzed by qRT-PCR. The nutritional gene analyzed in the bee head is major royal jelly protein 1 ( mrjp1 ) gene, the nutritional gene analyzed in the bee abdomen is vitellogenin ( vg ) gene, and the immune genes analyzed in the bee abdomen are hymenoptaecin gene and apidaecin gene

Please refer to Figure 7A and Figure 7B. Figure 7A is the analysis result of the effect of strain TBE-8 on the bee head mrjp1 gene, and Figure 7B is the analysis result of the effect of strain TBE-8 on the vg gene of the bee abdomen.

The results in Figure 7A show that in the head mrjp1 gene analysis, the gene expression of the S group was 1 times; the gene expression of the P group was 2914.96 times, with a significant difference; the gene expression of the M group was 0.01 times; The gene expression in group F was 1419.61 times, with a significant difference ( F _{3, 12} = 146.94, P < 0.0001). Among them, the expression level of mrjp1 gene in the head of group P was the highest, and the expression level of group F was the second. The results in Figure 7B show that in the analysis of the abdominal vg gene, the gene expression of the S group was 1 times; the gene expression of the P group was 4.07 times, with a significant difference; the gene expression of the M group was 2.06 times; The gene expression of the group was 17.92 times, with a significant difference ( F _{3, 12} = 119.57, P ＜ 0.0001). The results showed that pollen and strain TBE-8 fermented liquid could increase the expression of vg gene in the abdomen of bees, and the expression of vg gene in the abdomen of group F was 4.4 times that of group P, with a significant difference, and the abdomen of group F The expression of vg gene was the highest, followed by that of group P. From the results in Figure 7A and Figure 7B, it can be seen that pollen and the fermentation broth of strain TBE-8 can increase the expression of bee head and abdomen nutritional genes- mrjp1 gene and vg gene, and the expression of mrjp1 gene in group F is the gene in group P 0.5 times the amount of performance. The expression of vg gene in group F was 3 times that of group P; it is worth mentioning that group F was only fed bee TBE-8 fermentation broth without any pollen food.

Please refer to Figure 8A and Figure 8B again. Figure 8A is the analysis result of the effect of the strain TBE-8 on the hymenoptaecin gene in the bee abdomen, and Figure 8B is the analysis result of the effect of the strain TBE-8 on the apidaecin gene in the abdomen of the bee.

As shown in Figure 8A, in the analysis of the hymenoptaecin gene in the honeybee abdomen, the gene expression of the S group was 1 times; the gene expression of the P group was 8.65 times, with a significant difference; the gene expression of the M group was 2.68 times; The gene expression of the group was 17.68 times, with a significant difference ( F _{3, 12} = 160.75, P ＜ 0.0001). Among them, the expression level of hymenoptaecin gene in the abdomen of group F was the highest, and the expression level of group P was the second. As shown in Figure 9B, in the analysis of the apidaecin gene in the honeybee abdomen, the gene expression of the S group was 1 times; the gene expression of the P group was 4.90 times, with a significant difference; the gene expression of the M group was 1.74 times; The gene expression of the group was 7.08 times, with a significant difference ( F _{3, 12} = 16.69, P ＜ 0.0001). Among them, the abdominal apidaecin gene expression level was the highest in the F group, followed by the P group expression level. The above results show that the fermentation broth and pollen of the strain TBE-8 can increase the expression levels of the honeybee abdominal immune genes-hymenoptaecin gene and apidaecin gene, and the expression level of the hymenoptaecin gene in the F group is twice that of the P group, with a significant difference; The gene expression of apidaecin in group F was 1.4 times that of group P, with a significant difference.

The results from Figure 7A to Figure 8B show that the strain TBE-8 has a significant impact on the expression of honeybee head nutritional genes, abdominal nutritional genes and abdominal immune genes.

In order to further understand the amount of bacteria strain TBE-8 in the intestine of bees fed continuously for 7 days, the amount of bacteria strain TBE-8 was divided into 4 groups in the experiment, and the bacteria content of group 1 fed bees was 5.43×10 ⁸ CFU/ mL of strain TBE-8 fermentation broth and 50% sucrose solution (hereinafter referred to as group F), group 1 fed bees with 50% sucrose solution and pollen (hereinafter referred to as group P), group 1 fed bees with 50% sucrose solution (hereinafter referred to as S group), and group 1 was fed with bee MRS liquid medium and 50% sucrose solution (hereinafter referred to as group M). After feeding for 7 days, the intestinal DNA of the above-mentioned groups of bees was extracted, and the bacterial count of the strain TBE-8 in the intestinal tract was determined by qRT-PCR.

Please refer to Figure 9A and Figure 9B, which are the results of the analysis of the total bacterial count in the bee intestine and the bacterial count of the TBE-8 strain after feeding the bee strain TBE-8 fermentation broth, where Figure 9A shows the amount of bacteria in the bee intestine The results of the analysis of the total bacterial count, Figure 9B is the result of the analysis of the bacterial count of the bee intestinal strain TBE.

The results in Figure 9A showed that there was no significant difference in the total bacterial count between Group F and Group S, Group P and Group M ( F _{3, 12} = 1.17, P = 0.3616). The results in Figure 9B show that the strain TBE-8 in Group F had a bacterial count of 1.9×10 ⁶ copies/ng DNA, and the bacterial counts in Group S, Group P, and Group M in the other groups were not detected ( F _{3, 12} = 89.53, P < 0.0001).

9. In vitro test of bacterial strain TBE-8 inhibiting Bacillus larvae

This experiment further tested whether the strain TBE- had the effect of inhibiting Bacillus larvae. In the experiment, the fermentation liquid of strain TBE-8 after 24, 48 and 72 hours of fermentation was used to carry out the in vitro test of inhibiting Bacillus larvae. Please refer to Table 3 below for the results of the in vitro test on Bacillus larvae.

Table three fermentation time 0 hours 24 hours 48 hours 72 hours pH value 5.98 4.60 4.13 4.02 Viable count (Log CFU/mL) - 9.09 ± 7.98 7.54±6.32 6.53 ± 5.00 Antibacterial zone diameter (cm) - 1.2 ± 0.2 1.6±0.4 2.0 ± 0.6

As can be seen from the results in Table 3, the pH values of the strain TBE-8 fermentation broths 24, 48 and 72 hours after fermentation were 4.60, 4.13 and 4.02 respectively, and the diameters of 1.2 ± 0.2 centimeters, 1.6 ± 0.4 centimeters and 2.0 centimeters were produced respectively. The inhibition zone of ± 0.6 cm, so the strain TBE-8 is a probiotic strain that has the ability to resist or inhibit pathogenic bacteria in the intestinal tract.

Because bacterial strain TBE-8 can produce organic acid in the fermentation process, and organic acid is a kind of antibacterial substance, the pH value of the bacterial strain TBE-8 fermented liquid of fermentation 72 hours is 4.02, and the organic acid output that this period produces is highest is acetic acid ( As shown in the results of Fig. 4B), therefore, the in vitro test of acetic acid and strain TBE-8 fermentation broth inhibiting Bacillus larvae was further evaluated.

Please refer to Figure 10A and Figure 10B. Figure 10A is the analysis result of the in vitro test for the inhibition of Bacillus larvae by acetic acid, and Figure 10B is the analysis of the in vitro test for the inhibition of Bacillus larvae by the fermentation broth of strain TBE-8 fermented for 72 hours Result graph. The results in Figure 10A show that acetic acid with a pH value of 2.4 has a large zone of inhibition, and acetic acid with a pH value of 2.9 also has a zone of inhibition, but acetic acid with a pH value of 3.3 and 3.8 has no zone of inhibition. From the results in Figure 10B, it can be seen that the strain TBE-8 fermentation broth with a pH value of 4.02 has a bacteriostatic zone (diameter is 2.0 ± 0.6 cm), and then it is speculated that the TBE-8 fermentation broth may contain inhibitors that can inhibit the growth of Bacillus larvae. Bacterial matter.

10. Mass production and development of strain TBE-8

From the above test results, it can be seen that the strain TBE-8 is non-toxic to bees, which is in line with the characteristics of some probiotic strains used in animals. In addition, the strain TBE-8 can affect the physiological performance of bees and has the ability to inhibit the larval Bacillus. It is considered that the strain TBE-8 has the potential to become a probiotic for bees. In order to commercialize it later, this experiment further amplified the strain TBE-8. A single colony was cultured in 5 mL of MRS liquid medium, and the obtained Add 5 mL of bacterial liquid to 495 mL of MRS liquid medium, and cultivate for another 24 hours to finally obtain 500 mL of bacterial liquid. Then use the freeze-drying preservation method to sublimate the frozen strains to remove water under reduced pressure, so that the strains are stored in the absence of oxygen, moisture, and light, and the number of bacteria after freeze-drying is detected. This test was repeated twice, and the test results are shown in Table 4.

Table four Fermentation broth (L) Dry weight after freeze-drying (g) Bacterial count (CFU/g) 2 40 3×10 ¹² 3 54 4×10 ¹²

Some strains can be stored for up to 30 years after freeze-drying, but some strains may cause bacterial death during the freeze-drying process. The results in Table 4 show that 2 liters of strain TBE-8 fermentation broth can be freeze-dried into 40 grams of freeze-dried powder, and the number of bacteria per gram is 3×10 ¹² CFU; while 3 liters of strain TBE-8 can be freeze-dried 54 grams of freeze-dried powder, the number of bacteria per gram is 4×10 ¹² CFU, so it is known that freeze-drying will not cause the death of strain TBE-8, so the strain TBE-8 can be further amplified and can be stored by freeze-drying Law to save.

In addition, the present invention further provides a composition comprising Leuconostoc enteritidis TBE-8 or a derivative of Leuconostoc enteritidis TBE-8, wherein the Leuconostoc enteritidis TBE-8 derivative comprises Leuconostoc enteritidis TBE -8 bacteriostasis solution or Leuconostoc enterococcus TBE-8 bacteria extract. Based on the above characteristics of Leuconostoc enterococcus TBE-8, the composition of the present invention can be used to improve the nutritional physiology and immune physiology of bee colonies and promote the health of bee colonies. The composition of the present invention can be in any form, for example, it can be in a form selected from the following group: feed composition, nutritional supplement composition and edible composition. For example, Leuconostoc enterica TBE-8 can be added to the feed of bees, or added to the sucrose solution that feeds bees, and any suitable additive can be added to the composition of the present invention as needed, to further strengthen Leuconostoc enterica. The effect of bacteria TBE-8. Alternatively, other nutritional or medicinal ingredients can be added to increase the application flexibility and deployment degree of the composition, as long as the added ingredients have no adverse effect on the benefit of Leuconostoc enterococci TBE-8.

In summary, the Leuconostoc enterococci TBE-8 of the present invention is non-toxic to bees, has high tolerance to sucrose solution, and is also tolerant to the acidic environment of the gastrointestinal tract of bees, and the fermentation product meets the requirements after analysis. Desired properties of probiotics. And Leuconostoc enterococcus TBE-8 can affect the physiology of bees , for example, it can improve the expression of bee nutritional genes and immune genes, and can add Leuconostoc enterococcus TBE-8 to the sucrose solution for the bees to eat the sucrose solution and enter the gastrointestinal tract to exert its effect, and can effectively in retention in the gut. Therefore, the Leuconostoc enterolis TBE-8 of the present invention, and the composition comprising the Leuconostoc enteritidis TBE-8 or its derivatives of the present invention can be used as probiotics for bees, and have the potential to be applied to the beekeeping industry.

Although the present invention has been disclosed as above by way of implementation, it is not intended to limit the present invention. Anyone skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection of the present invention The scope shall be defined by the scope of the appended patent application.

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In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described as follows: Fig. 1 is a colony diagram of Leuconostoc enterolis of the present invention; Fig. 2A and Fig. 2B are the analysis results of the bee toxicology test of Leuconostoc enteromenis TBE-8 of the present invention; Fig. 3A and Fig. 3B are the analysis results of the sucrose solution tolerance of Leuconostoc enteromenis TBE-8 of the present invention; Fig. 4A, Fig. 4B and Fig. 4C are the results of the determination and analysis of organic acids of the Leuconostoc enteromenis TBE-8 fermentation broth of the present invention; Fig. 5A and Fig. 5B are the analysis result diagrams of the distribution of Leuconostoc enterococci TBE-8 in the intestinal tract of worker bees of the present invention; Fig. 6A and Fig. 6B are the results of analysis of the bacterial distribution of Leuconostoc enterica TBE-8 of the present invention in the intestinal tract of worker bees (uncontacted/untreated TBE-8 strain) in bee colonies; Fig. 7A and Fig. 7B are the analysis result diagrams of the effect of Leuconostoc enterococcus TBE-8 of the present invention on the nutritional genes of honeybee head and abdomen; Fig. 8A and Fig. 8B are analysis result diagrams of the impact of Leuconostoc enteromenis TBE-8 of the present invention on the immune gene effect of bee abdomen; Fig. 9A and Fig. 9B are diagrams showing the results of analysis of the total bacterial count in the intestinal tract of bees and the bacterial count of TBE-8 strains after feeding the bees with the Leuconostoc enterolis TBE-8 fermentation broth of the present invention; and Fig. 10A and Fig. 10B are the analysis results of the in vitro test of the inhibition of Bacillus larvae by Leuconostoc enterolis TBE-8 of the present invention.

Domestic deposit information (please note in order of depositor, date, and number) Food Industry Development Institute January 21, 2021 BCRC 911036

Claims (9)

A kind of Leuconostoc mesenteroides ( Leuconostoc mesenteroides ) TBE-8, which is deposited in the Research Institute of Food Industry Development of a consortium legal person, and the deposit number is BCRC 911036, and the Leuconostoc mesenteroides TBE-8 has one of the nutritional genes for promoting bees and Ability to immunize gene expression. The Leuconostoc enterolis TBE-8 as described in Claim 1, which has a sucrose solution tolerance. Leuconostoc enterococci TBE-8 as described in claim 2, wherein the tolerance to the sucrose solution is that the concentration of the sucrose solution is greater than 0% and less than 70%. The Leuconostoc enterococci TBE-8 as described in claim 1, which has an acid tolerance. Leuconostoc enterococci TBE-8 according to claim 4, wherein the pH value of the acid tolerance is greater than 4 and less than or equal to 6.5. The Leuconostoc enterococci TBE-8 as described in Claim 1 has the ability to inhibit the growth of Bacillus larvae. Leuconostoc enteromenis TBE-8 as described in Claim 1, wherein the nutritional gene comprises major royal jelly protein 1 ( mrjp1 ) gene and vitellogenin ( vg ) gene. Leuconostoc enterococci TBE-8 as described in Claim 1, wherein the immunity gene comprises hymenoptaecin gene and apidaecin gene. A composition comprising the Leuconostoc enterica TBE-8 as described in claim 1 or a Leuconostoc enteritidis TBE-8 fermentation broth.

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