KR20190047756A - A composition comprising lactobacillus probiotics and a functional feed additive comprising the same - Google Patents

Abstract

The present invention relates to probiotics including a complex strain, a lactobacillus probiotics composition, and a functional feed additive including the same. The complex strain of the present invention is selected from the group consisting of an L. plantarum GB-804 strain (accession No: KACC 18613), and an L. plantarum GB-805 strain (accession No: KACC 18614). A lactobacillus strain of the present invention has excellent antibacterial activity, acid resistance, heat resistance, and organic acid producing ability.

Description

[0001] The present invention relates to a lactic acid bacterial prophylactic composition and a functional feed additive comprising the same,

The present invention relates to a complex lactic acid bacteriostatic agent and a functional feed additive comprising the same.

Feed additives are small amounts of feed additives that are used to improve the productivity of livestock. Antibiotics, probiotics, enzymes, organic acids, flavors, sweeteners, antioxidants, various natural substances, and functional substances are classified as feed additives. Antibiotics have been used in the past to inhibit the growth of harmful fungi or microbes, or to destroy the life of the animals and help feed the livestock. Antibiotics have been developed for the control of pathogens, but they have also been shown to be effective in promoting the growth of livestock and improving feed efficiency. However, the issue of abuse of antibiotics is emerging. In 2006, EU prohibited the use of antibiotics as a feed additive, and Korea also prohibited the use of antibiotics in diets since 2011 for the same reason. Currently, the use of therapeutic antibiotics is possible, but the use of antibiotics as feed additives is limited.

The use of antibiotics was forbidden and probiotics were highlighted. The present invention also relates to a kind of probiotic agent. Probiotics, called probiotics, are products that contain living beneficial microorganisms, which act to promote growth by inhibiting the growth of other pathogenic microorganisms and helping digestion and absorption of feedstuffs that are ingested by the animal.

It is preferable that the microorganism for the probiotic agent has an antimicrobial activity against harmful bacteria such as Escherichia coli and can induce a change in intestinal pH by promoting lactic acid production. For example, Korean Patent Registration No. 10-1781211 discloses a strain of Lactobacillus plantarum strain isolated from swine-optimized pigs.

Korean Patent Registration No. 10-1781211

As disclosed in Korean Patent Registration No. 10-1781211, there are various kinds of probiotics introduced in patents and papers which are suitable for livestock such as pigs. However, the activity of E. coli and the like is appropriately suppressed, the growth of livestock is promoted, The development of better strains that can be improved is still insufficient.

In particular, the present inventors have developed a complex probiotic microbicide having excellent antimicrobial activity against Escherichia coli and Salmonella bacteria, and having excellent acid resistance, heat resistance and organic acid production ability.

The present inventors have found that certain lactic acid bacterial strains are excellent in antibacterial activity, heat resistance, acid resistance and organic acid production ability, and in particular, the compound lactic acid probiotic agent comprising two or more of these lactic acid bacterial strains has a synergistic effect on antibacterial activity and organic acid production ability And completed the present invention.

The lactic acid bacteria strain according to the present invention is excellent in antibacterial activity, acid resistance, heat resistance and organic acid production ability. Therefore, the lactobacillus probiotics containing the lactic acid bacteria strain are excellent in storage stability, and when used as a feed additive, the activity of E. coli and the like in the livestock can be appropriately suppressed, the growth of livestock can be promoted, and the disease resistance can be improved.

Figures 1A and 1B show the antimicrobial activity of the selected strain.
FIGS. 2A and 2B show bacterial counts of the strains according to the fermentation method according to the incubation time.
FIGS. 3A and 3B show changes in the number of bacteria produced in the solid fermentation broth and in the liquid fermentation broth. FIG.
4A to 4D show the number of bacteria after heat treatment at various temperatures for strains prepared by solid fermentation or liquid fermentation.
FIGS. 5A to 5F show changes in the number of bacteria and pH of fermentation time depending on the seeding level of various strains.
6 illustrates a number of bacteria fermentation time changes in Lactobacillus Planta room (L. plantarum) GB-804 and Lactobacillus Planta room (L. plantarum) GB-805 complex strains.
7A and 7B show the results of evaluating the heat resistance of the complex-type probiotics of the present invention and commercial products.
FIGS. 8A and 8B show the results of the pH stability evaluation of the complex-type probiotics of the present invention and commercial products.
Fig. 9 shows the results of evaluation of antimicrobial activity against the probiotics of the present invention and commercially available products.

The present invention relates to a Lactobacillus plantarum GB-804 strain (accession number: KACC 18613); Lactobacillus L. plantarum strain GB-805 (accession number: KACC 18614) relates to both strains.

The present invention also relates to a probiotic agent comprising said strain or a culture thereof and a feed additive comprising such a probiotic agent.

The strain is excellent in antibacterial activity, acid resistance, heat resistance and organic acid production ability against Escherichia coli and / or Salmonella.

The present invention also relates to a Lactobacillus plantarum GB-804 strain; And a L. plantarum strain GB-805 or a culture thereof, and a feed additive comprising such a prodrug. The combined lactic acid probiotics containing two strains as described above are more excellent in antibacterial activity, acid resistance, heat resistance and organic acid production ability.

In the present invention, the strain is preferably produced by solid fermentation.

Hereinafter, the present invention will be described in detail based on the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope and spirit of the present invention.

Example 1: Selection of strains according to evaluation of antimicrobial activity

The following 20 strains (including negative control and positive control) were evaluated for their antimicrobial activity against E. coli and Salmonella:

① L. brevis

② L. plantarum (LP2)

③ L. acidophilus

④ L. plantarum

⑤ L. sakei

⑥ L. delbrueckii

⑦ L. plantarum

⑧ L. reuteri

⑨ L. plantarum (LP1)

⑩ P. pentosaceus

⑪ L. plantarum (LP3)

⑫ L. plantarum (GB-805)

⑬ W. paramesenteroides

⑭ L. paracasei

⑮ Negative control (negative control)

 Leuconostocmesenteroides

 L. plantarum

 L. pantosus

 L. plantarum (GB-804)

 Positive control with chloramphenicol (positive control)

The in vitro antimicrobial activity of Escherichia coli and Salmonella strains was evaluated using the above 20 strains (or antibacterial substances). The strains having excellent antimicrobial activity against Escherichia coli and Salmonella strains were firstly selected (evaluation results of antimicrobial activity were shown in Figs. 1A and 1B) through the evaluation, and among them, Two strains of the following strains suitable for production were finally selected:

[Final Selection Strain]

One) Lactobacillus plantarum GB-804 strain (Accession No. KACC 18613);

2) Lactobacillus L. plantarum strain GB-805 (accession number: KACC 18614);

Example 2 Fermentation Characteristics according to Strain Fermentation Method

The two selected strains were cultured by liquid fermentation and solid phase fermentation method, respectively, and the basic fermentation characteristics were confirmed by confirming the number of bacteria.

(One) Liquid fermentation Experimental method: Two strains were cultured in MRS medium for 28 hours in a shaking incubator at 35 ℃, and the number of bacteria was confirmed. The number of bacteria according to the incubation time is shown in Fig.

(2) Solid phase fermentation Experimental method: The culture solution of the gastric juice fermentation experiment was incubated for 28 hours in a 35 ° C incubator using soybean meal as a raw material medium. The number of bacteria according to the incubation time is shown in FIG. 2B.

Comparison of fermentation characteristics of fermentation broth showed that fermentation of fermentation broth was similar to fermentation of fermentation broth in liquid fermentation up to 28 hours.

Example 3: Acid resistance (pH stability) experiment

In order to compare the acid resistance (pH stability) of the fermented product (strain) according to the fermentation method of the selected strain, the following experiment was carried out: the MRS medium was used and the medium was changed to pH 3, 4, 6 and 8 , And cultured for 24 hours.

The changes in the number of bacteria in the solid fermentation broth and in the liquid fermentation broth are shown in FIGS. 3A and 3B, respectively.

Experiments showed that the strain produced through solid fermentation was to have stability at pH 3 ~ 8 range, pH 3 Initial 1.0 × 10 ⁶ after the last 24 hours CFU / mL in maintaining the 8.1 × 10 or more ⁵ CFU / mL bacteria and Thus, it was confirmed that the strain had excellent acid resistance.

Example 4: Evaluation of heat resistance

In order to compare the heat resistance of the fermented product (strain) according to the fermentation method of the selected strain, the following experiment was conducted. In order to increase the thermal conductivity, a 10-fold dilution of each fermented product at 60, 70, After 10 min of heat treatment, bacterial counts were compared.

FIGS. 4A and 4B show the number of bacteria after heat treatment for 5 minutes at each temperature and after heat treatment for 10 minutes, respectively, for strains prepared by solid fermentation. FIG. 4C and FIG. 5 min heat treatment and 10 min heat treatment, respectively.

As a result of the experiment, it was confirmed that the strain produced by the solid fermentation method had better thermal stability than the strain produced by the liquid fermentation method.

Example 5: Evaluation of organic acid (lactic acid) production ability

The contents of lactic acid contained in each fermented product were measured in order to compare the organic acid (lactic acid) production ability of the fermented product (strain) according to the fermentation method of the selected strain. The measurement results are shown in Table 1 below.

As a result of comparing the organic acid production ability in the fermented product according to the fermentation method with the lactic acid content, the lactic acid content in the solid fermented product was at least 6 times higher than the lactic acid content in the liquid fermented product even when fermented for 30 hours Respectively.

Therefore, it was confirmed that the solid fermentation process is more suitable than the liquid fermentation process in consideration of the function and stability of the strain in the mass production of the probiotic microorganism.

Example 6: Evaluation of antibacterial activity of a complex strain

It is possible to economically and effectively produce a probiotic agent by using a solid fermentation process capable of producing an economically viable prophylactic agent without additional steps such as separate formulation in the production process of probiotics.

Particularly, in order to produce an effective microbicide having an excellent effect and to improve the efficiency in the production process, the antimicrobial activity of the product by the single fermentation process and the complex fermentation process in which the selected microbes are mixed is compared Respectively.

As a result of comparison, it was confirmed that Escherichia coli and Salmonella strain exhibited remarkable antimicrobial activity as compared with a single fermentation strain (see FIG. 10).

Example 7: Evaluation of organic acid production ability of a complex strain

The organic acid production ability of the product by the single fermentation process of the selected strains and the complex fermentation process in which the selected strains were mixed was evaluated and compared. In other words, lactic acid and PLA contents in the products were analyzed by complex fermentation in which two different strains were mixed and cultured.

The results of the analysis are shown in Table 2 below:

Organic acid content in product by complex fermentation process Strain Lactic acid (%) PLA (mg / kg) GB-804 2.97 92.1 GB-805 2.96 91.0 GB-804 + GB-805 3.05 175.6

As a result of PLA content analysis of the mixed fermented product of the selected strain using the solid fermentation process, the lactic acid content and the PLA content of the product of the complex fermentation were more increased than those of the product by the single fermentation.

Example 9: Optimum conditions of liquid fermentation process

In order to establish the fermentation process on the pilot scale, based on the medium composition and initial water content established in the fermentation process on the laboratory scale, Fermentation process.

The seeds for solid fermentation were cultured for 24 hours using a 300 L liquid fermenter as a liquid fermentation medium composition established in a laboratory scale fermentation process and used as solid fermentation seeds. Although there was a slight difference, a stable number of bacteria of 5.5 × 10 ⁸ to 1.0 × 10 ⁹ CFU / mL was obtained.

* Result of seed production at 24 hours fermentation by liquid fermentation

For the solid fermentation of the combined strains, solid fermentation was carried out by simultaneously inoculating the two species produced by liquid fermentation.

Example 10 : Optimum Conditions for Solid Fermentation Process

The pilot scale solid fermentation process conditions were established based on the established processes on the laboratory scale to establish the optimal fermentation process by further verification of the level of seedling addition and fermentation time for each combination.

As a result of investigating the changes of bacterial count and pH according to the fermentation time by varying the seeding level of 2 ~ 10%, the rate of bacterial growth during fermentation was faster and the degree of pH reduction was different as the seeding level was increased. However, when the seeding level was more than 6%, it was determined that the increase of the seedling addition level did not cause a significant change in bacterial count and pH. Therefore, the optimal seeding level was determined to be 6% (3% of each strain).

The change in the number of bacteria and the change in pH by fermentation time according to the seeding level are shown in Figs. 5A to 5F.

5a and 5b show changes in pH and number of bacteria according to fermentation time according to the addition level of GB 804, and Figs. 5c and 5d show change in pH and pH with fermentation time according to GB 805 addition level will be.

The optimal fermentation time was determined to minimize the fermentation time considering the mass production process in the future.

For further evaluation of the fermentation time in the pilot scale fermentation process, L. plantarum GB-804 and L. plantarum GB-805 combination was investigated. The results are shown in Fig.

The fermentation time was set to 24 hours since the bacterial count was maintained at 10 ⁹ CFU / g from 15 hours to 24 hours after fermentation.

The pilot scale fermentation process for the production of the combined strains was performed by 5batch of each strain combination. The final pilot scale fermentation conditions were 50% of initial water content, 6% of inoculum size, fermentation temperature of 35 ℃, fermentation time of 24 hours, and bacterial counts after the fermentation /

The results are shown in Table 5 below:

Complex fermentation bacteria Strain combination Batch Throughput (kg) Number of bacteria (CFU / g) pH after drying After grinding L. plantarum GB-804 +
L. plantarum GB-805 One 160 7.80E + 09 4.85E + 09 4.31 2 160 8.50E + 09 2.30E + 09 4.33 3 160 5.50E + 09 1.99E + 09 4.41 4 230 9.50E + 09 5.45E + 09 4.38 5 230 9.80E + 09 5.12E + 09 4.35 Average 188 8.22E + 09 3.94E + 09 4.36

The number of bacteria in the final product was 8.22 × 10 ⁹ CFU / g in the L. plantarum GB-804 + L. plantarum GB-805 strain after fermentation / drying. These strains are used as specimens for in vivo specification testing.

In addition, LC-MS / MS was used to analyze lactic acid and phenyllactic acid (PLA), which are organic acids.

The results of the analysis are shown in Table 6:

The lactic acid content was 4.85% higher than the laboratory fermentation level and the PLA content was found to be 135.3 ppm lower than the laboratory level.

Example 10 : Characterization of Complex Fermentation Strain

The thermostability, pH stability, antimicrobial activity and storage stability of composite strains of GB 804 and GB 805 were evaluated.

(1) Heat resistance

(0, 15, and 30%) at 75, 85, and 95 ° C for 5 minutes, and the heat resistance of the sample of the present invention produced through solid fermentation using a water bath and the commercially available product Respectively.

The sample of the present invention showed a thermal stability of 78% at 95 ° C for 5 minutes after adjusting the moisture content to 30%, and 90% or more of thermal stability at the rest of the treatment (See Fig. 7A).

In the case of commercial products, it was confirmed that the number of bacteria at the level of 80 ~ 100% decreased under all treatment conditions, and there was no thermal stability at all (see FIG.

Therefore, the compound probiotics according to the present invention exhibit thermal stability in a feed processing step such as pellet production in the future, thereby securing differentiation from existing products.

The results of the heat resistance evaluation under actual pellet processing conditions are shown in Table 7:

Analysis of the pelletized samples with pellet processing temperatures of 80, 90, 100, and 108 ℃ showed that the highest pellet stability was achieved (at log value) of 94% or higher even at the highest heat treatment temperature of 108 ℃. This shows the same high thermal stability as the in vitro test results.

(2) pH stability

1% (1 g / 100 mL MRS broth) of 804 and 805 complex-fermented complex strains were added to the MRS broth at pH 3, 4, 6, and 8 using 1N HCl and 1N NaOH. The cultures were incubated at 200 rpm for 24 hours.

The results are shown in Fig.

As a result of the evaluation of the pH stability, it was evaluated that the complex strain of the present invention showed stable bacteria number without decreasing the number of bacteria under all pH conditions, and was evaluated to have acid resistance and biliary cholesterol, which means that it can reach stomach through stomach.

(3) Antimicrobial activity evaluation

804 and 805 strains were evaluated by in vitro agar well diffusion method.

Escherichia coli ( E. coli ) and Salmonella ( S. Enteritidis) strains were used as the strains to be evaluated for antibacterial activity.

The agar medium containing 1% of the indicator strain was mixed with the LB agar medium to prepare an agar medium containing the harmful bacteria. Then, 200 μl of the combined microbial sample and the commercial product extract, which had been filtered with a 0.2 μm syringe filter, zone (low support) formation.

The results of the experiment are shown in Fig.

Unlike the commercial products, the complex strain of the present invention was confirmed to have an inhibition zone formation after 12 hours of incubation, and thus the antibacterial activity was superior to that of the existing commercial products.

Example 11 : Assessment of Efficacy of New Genetic Material Additions for Useful Microorganisms in Feed Intrafauna

(1) Evaluation of efficacy of the combined strains in the breeding pig feed

Average daily gain (ADG) was calculated by weight at the beginning, at 2 weeks and at the end (6 weeks), and feed intake was calculated by subtracting the remaining amount from the feed amount during body weight measurement, Feed efficiency (GF = ADG / ADFI) was calculated by dividing weight gain by feed intake.

Table 8 shows the effect of the breeding stock containing the compound of the invention on the productivity of breeding pigs when given to livestock:

Item Control Treatment -1 Treatment -2 Treatment -3 SEM ² Body weight, kg Initial 24.97 24.96 24.96 24.96 0.01 wk 6 54.69b 55,19ab 55.03ab 55.98a 0.37 Overall ADG, g 708b 720ab 716ab 739a 9 ADFI, g 1615 1639 1624 1645 19 GF 0.438 0.439 0.441 0.449 0.004 ¹ Abbreviation: CON (control), Basal diet; TRT1 (treatment 1), CON + 0.1% GB-LP804; TRT2 (treatment 2), CON + 0.1% GB-LP805; TRT3 (treatment 3), CON + 0.05% GB-LP804 + 0.05% GB-LP805 (total 0.1%).
ADFI: Average Daily Feed Intake ² Standard error of means. ^{a, b} Means in the same row with different superscript differ significantly (P <0.05).

At the end of the study (6 weeks), body weight and body weight gain were significantly higher in treatment group 3 (GB-804 + GB-805) than the control group (P <0.05).

(2) Blood characteristics

At the end of the study (6 weeks), 2 mL of blood was collected from each jugular vein using a Vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) 120, Bayer, USA) were used to examine WBC (white blood cell) and lymphocyte.

In addition, 5 mL of blood was collected using a vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) and serum was centrifuged at 3000 rpm for 15 minutes at 4 ° C for IgG, IgA, IgM Respectively.

The effects of the addition of the fermented probiotics of the present invention on the blood characteristics of the growing pigs are shown in Table 9 below:

Blood characteristics Item Control Treatment -1 Treatment -2 Treatment -3 ^SEM2 wk 6 WBC 13.84 15.14 14.79 15.71 1.24 Lymphocyte 36.68 37.63 37.18 38.28 2.26 IgM 109 124 112 134 20 IgG 191 222 213 246 24 IgA 44 49 46 54 5 ¹ Abbreviation: CON (control), Basal diet; TRT1 (treatment 1), CON + 0.1% GB-LP804; TRT2 (treatment 2), CON + 0.1% GB-LP805; TRT3 (treatment 3), CON + 0.05% GB-LP804 + 0.05% GB-LP805 (total 0.1%). ² Standard error of means.

At the end of the experiment (6 weeks), there were no significant differences in the leucocyte, lymphocyte, IgM, IgG and IgA levels (P> 0.05).

(3) Results of microbial analysis

Signs for Nutrient Digestibility Analysis After collecting the min before the compounding (5 weeks, before Cr compounding) by anal massage method, it was stored frozen at -20 ° C until the experiment, and then suspended in sterilized physiological saline and homogenized The following 10 ³ to 10 ⁷ were used as a sample for the measurement of viable cell count.

To measure the number of E. coli Lactobacillus and Escherichia coli E. coli in the broth obtained by the experimental treatment, the number of viable cells was determined by using MRS agar for Lactobacillus and MacConkey agar (Difco, USA) for E. coli after 38 hours of incubation at 37 ° C Respectively.

The effects of the addition of the fermented probiotics of the present invention on the microorganisms of the breeding stock are shown in Table 10 below:

Item Control Treatment -1 Treatment -2 Treatment -3 ^SEM2 wk 6 Lactobacillus spp. ^{7.28b 7.42a 7.39a 7.47a} 0.03 ¹ Abbreviation: CON (control), Basal diet; TRT1 (treatment 1), CON + 0.1% GB-LP804; TRT2 (treatment 2), CON + 0.1% GB-LP805; TRT3 (treatment 3), CON + 0.05% GB-LP804 + 0.05% GB-LP805 (total 0.1%). ² Standard error of means. ^{a, b} Means in the same row with different superscript differ significantly (P <0.05).

At the end of the experiment (6 weeks), the contents of Lactobacillus in treatments -1, -2 and -3 were significantly higher than those of the control (P <0.05).

The probiotic agent comprising the complex strain according to the present invention can be usefully used as a livestock feed additive.

Depositor Name: Agricultural Biotechnology Research Institute

Accession number: KACC18613

Checked on: 20151030

Depositor Name: Agricultural Biotechnology Research Institute

Accession number: KACC18614

Checked on: 20151030

Claims (7)

Lactobacillus plantarum GB-804 strain (accession number: KACC 18613); And
Lactobacillus strain L. plantarum GB-805 strain (accession number: KACC 18614). A prophylactic agent comprising the strain of claim 1. The probiotics according to claim 2, wherein the strain is a strain produced by solid fermentation. A feed additive containing a probiotic agent according to claim 2 or 3. Lactobacillus plantarum GB-804 strain (accession number: KACC 18613);
Probiotics comprising Lactobacillus L. plantarum strain GB-805 (Accession No: KACC 18614). The probiabant according to claim 5, wherein the two strains are a strain produced by solid fermentation together. A feed additive containing a probiotic agent according to claim 5 or 6.

Images