KR20080011901A - Probiotic effects of fermented mixture of prepared with milk and soymilk use to lactobacillus salivarius sp. salivarius df20 on piglets - Google Patents

Abstract

A novel strain is provided to decompose effectively high indigestible hydrocarbon such as starchyrose and raffinose with safety to human body and be very useful for preparing a food or a feed. A Lactobacillus salivarius sp. salivarius DF20 having strong acid-resistance and bile juice resistance, inhibiting the growth of pathogenic microorganism, and producing alpha-galactosidase is deposited as a deposition no. KCTC10942BP. A culture material is obtained by fermenting sterilized milk, soybean milk or a mixture thereof using the Lactobacillus salivarius sp. salivarius DF20. A food, medicine, animal drug or feed additive comprises the Lactobacillus salivarius sp. salivarius DF20 and the culture material.

Description

Probiotic effects of fermented mixture of prepared with milk and soymilk use to Lactobacillus salivarius sp. In lactobacillus salivarius sp. Salivavarius die 20 and the fermented milk of soy milk using the same strain. salivarius DF20 on Piglets}

      1 is a procedure showing the selection of lactic acid strain [Lactobacillus salivarius sp. Salivarius DF20 (KCTC10942BP)] and the effect of fermentation using the new strain according to the present invention.

Figure 2 shows the molecular similiarity analysis of the new strain Lactobacillus salivarius sp. Salivarius DF20

Figure 3 shows the weight gain after feeding the probiotics containing the strain Lactobacillus salivarius sp. Salivarius DF20 to weaning pigs

Figure 4 shows the results of measurement of the concentration of ammonia gas in pigs after feeding the probiotics containing the new strain Lactobacillus salivarius sp. Salivarius DF20 to weaning piglets

Fig. 5 shows the change of viable cell counts during refrigeration (7 ° C.) storage of probiotics containing the strain Lactobacillus salivarius sp. Salivarius DF20.

The present invention is to prepare Lactobacillus salivarius sp. Salivarius DF20 (Lactobacillus salivarius sp. Salivarius DF20) and fermented milk and fermented soy milk mixed using the lactic acid bacteria having excellent resolution of the indigestible sugars in carbohydrates of the legumes. More specifically, the present invention relates to a method, more specifically, Lactobacillus salivarius sp. Salivarius, which is a novel microorganism isolated from human feces, which can effectively inhibit the growth of harmful microorganisms while having acid resistance, bile resistance, oxygen resistance and antibiotic resistance. DF20 and its microorganisms are lactic acid bacteria that produce alpha-galactosidase enzymes, and are easily digested and absorbed by polysaccharides such as starchyrose, raffinose, and melobiose, which are indigestible carbohydrates of legumes. Decomposes into glucose monosaccharides, and into other products during fermentation Industrial use as feed additives such as soymilk fermented milk and probiotic activator using lactic acid bacteria for fermentation of soybean cereals such as soybeans by producing organic acids such as lactic acid, acetic acid and citric acid to suppress harmful microorganisms and maintain normal intestinal flora. It is about.

Currently, as a livestock probiotic, Lactic acid bacteria such as Lactobacillus exidophilus, Lactobacillus fermentium, Lactobacillus vulgaris, Lactobacillus cassia, and Bifitus bacterium are mainly predominantly focused on the inhibition of harmful bacteria by strong acid production. Other microorganisms include Aspergillus awamori, Aspergillus arige, Aspergillus tamari, Pichia lilyemondi, Candida zabannica, Bacillus stearomorphils, Cortisium rolfsi, Enterococcus Pesium, Enterococcus pecalis, and Colostridium have been reported and used as livestock probiotics.

Lactobacillus is a bacterium normally loaded in the intestines of humans and animals, and has been shown to suppress the harmful bacteria by acidification in the intestine, improve its function, and improve the immunity and anti-cancer effects of various endocrine and digestive organs. The probiotic properties of lactic acid bacteria make it a healthy food for humans, improve productivity for livestock, and produce high quality fresh meat as a healthy livestock. It has been found to have a large effect as an additive.

However, it has not been clearly identified in relation to the functions and characteristics of Lactobacillus salivarius sp. Salivavarius lactic acid bacteria not only in Korea but also abroad.

In addition, the microorganisms are useful in the legume feed which is added as a major protein source of livestock feed, and in particular, the digestibility of the indigestible carbohydrates of the legume is problematic for livestock as well as for humans. To solve this problem, bacteria such as Bacillus are added as microbial agents, but their utility is extremely low. In addition, the feeding of these microorganisms is a dried microorganism that arrives in the intestinal tract together with the feed and requires significant nutrients and time for the microorganisms to have their original vitality.However, the microorganisms may be properly energized by the effects of gastric acid or bile acid secreted by the digestive system. As it is killed before, the viability of the intestinal microorganisms is extremely low and the microorganisms are difficult to function properly.

Therefore, in the present invention, soybeans that are added to feed are used to decompose polysaccharides, which are indigestible carbohydrates, into monosaccharides to increase the digestibility of feeds, and intestinal harmful microorganisms are produced by organic acids produced by lactic acid bacteria in soy milk and milk mixed fermented milk. It relates to a method for producing a mixed fermented milk that inhibits growth.

The gut microbiota of humans and animals is present and over 100 billion microbiota present. The normal intestinal flora of humans and other animals contains harmful microflora as well as beneficial microorganisms. Among the beneficial microorganisms in the intestinal microflora of the intestinal microorganisms are lactic acid bacteria, which have various probiotic functions along with intestinal intestinal effects. In order for probiotics prepared with these beneficial microorganisms to be listed in the normal intestinal flora, it must first have viability in strong acids of pH 2-4 in the stomach and strong viability in bile acids in the duodenum. Can maintain your gut health.

Conventional pig feed has been developed with the addition of antibiotics for nutrition and disease prevention. Soy milk mixed with soy milk added fermented and fermented feed has never been used domestically and domestically, and when the soymilk fermented milk is fed to pigs, most of the carbohydrates present in the legumes are indigestible polysaccharides by lactic acid bacteria. Since all of the monosaccharides are broken down, all the pigs are absorbed when used as an energy source, and the weight gain can be achieved faster by using the absorbed energy source. In addition, organic acids produced by lactic acid bacteria during soymilk and milk fermentation prevent the growth of harmful microorganisms by adding antibiotics to existing feeds, thereby preventing diarrhea and other diseases.However, by overdose or abuse of antibiotics, the residual concentration and harmful microorganisms in the body The emergence of resistant bacteria against antibiotics eliminates the therapeutic effect of antibiotics when the disease actually occurs. Therefore, the organic acid produced by lactic acid bacteria during fermentation of soy milk has the same inhibitory effect as antibiotics. Soymilk fermented milk has about 2% organic acid, and the acidic acid below pH 4.0 by organic acid is mainly composed of lactic acid and acetic acid. By providing the environment, it has the effect of inhibiting the growth of harmful microorganisms, preventing the growth of intestinal harmful bacteria.

The present invention has been made to solve the problems of the prior art, its main purpose is to effectively decompose the indigestible carbohydrates (starch rose, raffinose, etc.), and is safe for the human body, and is very useful in the field of food and feed manufacturing. To provide useful strains.

Another object of the present invention is to ferment milk and legumes using the novel lactic acid strain to decompose indigestible carbohydrates and to provide the culture products (monosaccharides and various organic acids) as feed additives.

In order to achieve the above object, the present invention provides Lactobacillus salivarius sp. Salivarius DF20 which is fermented in soybeans and has excellent fermentation performance for indigestible carbohydrates.

Hereinafter, the content of the present invention in more detail as follows. The strain according to the present invention is Lactobacillus salivarius sp. Salivarius DF20, which produces alpha galactosidase with excellent resolution for the indigestible carbohydrates of legumes, and particularly the enzymes produced by the same strain are starchyrose, rapi It can decompose nose and melobiose, reducing food processing costs and making it a probiotic feed using soybeans.

As in the procedure shown in FIG. 1, the strain was purely separated from the fecal lactate strain using LBS and MRS agar medium, and a lactic acid strain having good growth in acid and bile-resistant medium and aerobic state was selected. Next, the strains were identified by analyzing the sugar availability test of the selected strains, the fatty acid composition of the cells and the rDNA, and the strains having cholesterol lowering effect and antibiotic resistance were selected, and among them, lactic acid strains having alpha galactosidase activity were selected. It fermented in soy milk to select the cells with excellent fermentation ability.

The strain according to the present invention has the characteristics of typical lactic acid bacteria as Gram-positive bacillus, can grow under acidic conditions, and is also resistant to enteric intestinal tract as bile acid-resistant lactic acid bacteria that are active in bile acids have long intestinal adsorption. City anaerobic and aerobic fermentation are also active, facilitating fermentation. In addition, the organic acid (lactic acid, citric acid, acetic acid, etc.) produced by the fermentation of the strain inhibits the growth of harmful bacteria in the intestine and facilitates the intestinal peristalsis and facilitates digestion and metabolism. In addition, the strain is effective in preventing diseases of the circulatory disorders such as arteriosclerosis caused by excessive cholesterol in the body by reducing the absorption of cholesterol released in the intestine in humans. In addition, the lactic acid bacteria may be resistant to various antibiotics to prevent abnormal growth of the intestinal flora by antibiotics administered in the intestine. That is, the growth of the strain has a high growth effect on various antibiotics can be well grown in the intestine even after treatment with antibiotics.

The lactic acid strain according to the present invention was deposited as KCTC10942BP dated April 27, 2006, at the Biotechnology Research Institute, 52, Eeun-dong, Yuseong-gu, Daejeon. The strain has an excellent digestibility of more than 95% of the indigestible carbohydrates such as starch rose, raffinose, and merobiose, which contains more than 90% of carbohydrates in the carbohydrate, so that the digestion of soybeans used in unit animals is very good. Disability can be effectively responded to

Soybean milk and milk are mixed, sterilized and cooled using the strain to add the strain and fermented soymilk mixed fermented milk can be placed in the intestine of livestock in the form of lactic acid bacteria which is the most vigorous when fed as livestock feed. Since the soy milk mixed fermented milk has an inhibitory effect on the intestinal harmful bacteria in livestock, it can prevent the growth of harmful bacteria and prevent diarrhea and various diseases.

Lactobacillus salivarius sp. Salivarius DF20 of the present invention is a fermentation by-product and a lactic acid bacterium that produces lactic acid as a major organic acid, and starch which is an indigestible carbohydrate in legumes with the effect as a lactic acid bacterium that has been used in dairy products using milk. It is to provide lactic acid strains that can be easily absorbed and used as energy sources of humans and livestock by decomposing rose, raffinose, melobiose, etc. into monosaccharides glucose.

Therefore, when fed to humans and livestock in the state of maintaining the vitality of the microorganisms, the probiotic properties of the lactic acid bacteria are exerted while surviving with resistance from intestinal secretions. The Lactobacillus salivarius sp. Salivarius DF20 is fermented using a large amount of legumes and milk contained in the feed and then fermented as organic acids and the most active probiotic as a feed supplement for livestock.

In order to achieve the above object, the present invention provides a method for producing a mixed fermented soy milk prepared by adding milk to soy milk using soybeans and inoculating the lactic acid bacteria and fermentation.

Example 1 Isolation and Physiological Characteristics of Useful Lactic Acid Strains

Lactobacillus was isolated from human feces using pure LBS agar (BBL, USA) and MRS agar (Difco, USA) and grown in acid-resistant medium adjusted to pH 4.0, pH5.0 and pH 7.0. Primary useful lactic acid strains were isolated by testing as shown in Table 1. In two stages, useful lactic acid strains, which were grown in anaerobic and aerobic conditions and grew well in aerobic conditions, were tested and separated as shown in Table 2. In order to isolate the useful lactic acid strains that can grow well in intestinal mumps acid, the useful lactic acid strains with active growth in the medium containing 0.3% and 1.0% bile acids were selected as shown in Table 3. . The selected strain Lactobacillus salivarius sp. Salivarius DF20 showed the most excellent growth effect compared to other isolated lactic acid strains as shown in Tables 1, 2 and 3. The physiological characteristics of the isolated strain Lactobacillus salivarius sp. Salivarius DF20 showed the characteristics of typical lactic acid bacteria as Gram-positive bacillus as shown in Table 4.

Isolated strain pH 4 pH 5 pH 7 8 h 16 h 24 h 8 h 16 h 24 h 8 h 16 h 24 h Lactobacillus salivarius espi salivarius DF20 0.156 1.123 1.362 0.980 1.589 1.727 1.348 1.567 1.649 Comparison 1 * 0.026 0.048 0.290 0.650 0.916 1.020 1.294 1.354 1.445 Comparison 2 0.030 0.158 0.230 0.645 0.906 0.838 0.264 1.233 1.233 Comparison 3 0.053 0.080 0.122 0.659 0.960 0.872 0.657 1.195 1.278 Compare 4 0.047 0.085 0.120 0.551 0.847 0.882 0.114 1.103 1.189 Compare 5 0.055 0.080 0.122 0.379 0.660 0.624 0.260 1.284 1.362 Comparison 6 0.015 0.445 0.464 0.320 0.998 1.142 0.028 1.303 1.379 Compare 7 0.019 0.084 0.110 0.701 1.408 1.420 0.379 1.505 1.558 Comparison 8 0.035 0.048 0.294 0.594 0.836 1.208 1.120 1.252 1.345 Comparison 9 0.039 0.096 0.584 0.580 0.889 1.327 0.319 1.342 1.398 Comparison 10 0.032 0.069 0.282 0.802 0.972 1.188 0.933 1.261 1.386

* Comparison 1: Lactobacillus ecidophyllose, Comparison 2: Lactobacillus ecidophyllose, Comparison 3: Lactobacillus rhamnose, Comparison 4: Lactobacillus rhamnose, Comparative 5: Lactobacillus rhamnose, Comparative 6: Lactobacillus lactis , Comparison 7: Lactobacillus lactis, comparison 8: Lactavacillus cascai, comparison 9: Lactavacillus cascai, comparison 10: Lactobacillus brevis (hereinafter the same).

Isolated strain % Growth in aerobic state Lactobacillus salivarius espi salivarius DF20 89 Comparison 1 48 Comparison 2 54 Comparison 3 68 Compare 4 78 Compare 5 94 Comparison 6 58 Compare 7 78 Comparison 8 70 Comparison 9 53 Comparison 10 75

Isolated strain Bile acid 0% Bile acid 0.3% Bile acid 1.0% 8 h 16 h 24 h 8 h 16 h 24 h 8 h 16 h 24 h Lactobacillus salivarius espi salivarius DF20 0.556 1.472 1.757 0.359 2.339 2.481 0.147 0.524 2.485 Comparison 1 0.382 1.205 1.239 0.382 2.301 2.459 0.052 0.208 0.268 Comparison 2 0.352 1.209 1.358 0.352 2.256 2.310 0.062 0.080 0.133 Comparison 3 0.107 1.243 1.444 0.107 2.234 2.343 0.130 0.304 1.220 Compare 4 0.237 1.353 1.472 0.237 2.156 2.245 0.082 0.146 0.914 Compare 5 0.300 1.332 1.392 0.300 0.554 1.414 0.108 0.170 2.384 Comparison 6 0.264 1.233 1.233 0.264 0.643 2.049 0.065 0.074 1.502 Compare 7 0.557 1.195 1.278 0.657 1.986 2.016 0.097 0.338 1.687 Comparison 8 0.114 1.103 1.189 0.114 2.340 2.402 0.050 0.899 2.297 Comparison 9 0.260 1.284 1.362 0.260 2.066 2.130 0.066 0.079 0.353 Comparison 10 0.028 1.303 1.379 0.028 0.277 1.321 0.117 0.070 1.235

division Lactobacillus salivarius espi salivarius DF20 Catalase production capacity voice Gram Staining of Cells positivity motility voice shape Bacillus gassing voice Growth temperature 30 ~ 43 ℃

Example 2 Identification of Isolated Lactic Acid Strains

In order to identify and select useful lactic acid strains, cell fatty acids contained in the cytoplasm of lactic acid bacteria were first separated and tested by using a MIDI system (USA). It was shown to be the cell fatty acid of the typical lactic acid bacteria possessing saturated fatty acids 16, 18 and 19.

The carbohydrate availability of the second isolated useful lactic acid bacterium Lactobacillus salivarius sp. Salivarius DF20 was examined as shown in Table 6 using the API CHL50 kit. As a result, it showed the physiological characteristics of typical Lactobacillus lactic acid bacteria that decompose carbohydrates such as galactose, fructose, rhamnose and lactose.

Third, molecular identification was performed by DNA sequence of the isolated useful lactic acid bacteria. Primers were amplified 16S rDNA using a PCR machine using 5'-GCGGCGTGCTTAACACATGCA-3 ', 5'-GGCCTTGTACACACCGCCCG-3'. The amplified 16S rDNA was purified using a PCR product purification kit (Qiagen) and used for sequencing. The sequencing was performed using ABI 377 DNA sequencer applied biosystems (Perkin-Elmer), and sequencing was performed molecularly using CLUSTAL W and PHYLIP programs.

The Lactobacillus strain of the new strain examined rDNA nucleotide sequences of about 1,498 bp, as shown in SEQ ID NO: 1, and analyzed the similarity of molecular system sequences as shown in FIGS. 2 and 7.

Cell Fatty Acid Composition Cell Fatty Acid Content (%) 14: 0 FAME  4.88 14: 1 CIS 7 DMA - 15: 0 FAME  0.77 16: 1 CIS 9 FAME  5.34 16: 0 FAME 16.81 16: 0 DMA - 17: 0 FAME  0.84 16: 0 2OH FAME  1.06 18: 1 CIS 9 FAME 25.98 18: 1 CIS 13 FAME  1.59 18: 0 FAME  7.39 22: 0 NHC  4.97 19 CYC 9,10 /: 1 FAME 21.22 20: 0 FAME - 17: 0 ANTEISO FAME - 17: 0 CYC DMA - 18: 1 CIS 9 FAME - 12: 0 FAME - 9:00 FAME - UN 18. 199 18: Oa DMA - 19 CYC 11,12 /: 1FAME -

Carbohydrate Types Fermentation capacity Glycerol voice(-) Erythritol voice(-) D-arabinose voice(-) L-arabinose Positive (+) Ribose Positive (+) D-Xylose voice(-) L-Xylose voice(-) Adonitol voice(-) Beta methyl-xyloxide voice(-) Galactose Positive (+) D-glucose Positive (+) D-fructose Positive (+) D-Mannose Positive (+) L-Solbo Oz Positive (+) Rhamnose Positive (+) Dulsitol Positive (+) Inositol voice(-) Mannitol Positive (+) Sorbitol Positive (+) Alpha methyl-D-mannoside voice(-) Alpha methyl-D-glucoside voice(-) N Acetyl Glucosamine Positive (+) Amigdalin Positive (+) Arbutin Positive (+) Esklin voice(-) Salinity Positive (+) Cellobioses Positive (+) Maltose Positive (+) Lactose Positive (+) Melibinose Positive (+) Sucrose Positive (+) Trehalozu Positive (+) Inulin voice(-) Melegitozu Positive (+) D-Lappinenose Positive (+) Amidodon False positive (±) Glycogen voice(-) Xylitol voice(-) Beta Genoiobios Positive (+) D-turannose Positive (+) D-Lysoz Positive (+) D-Tagatozu Positive (+) D-fucoose voice(-) L-fucoose voice(-) D-Arabitol Positive (+) L-Arabitol voice(-) Gluconate Positive (+) 2 Keto-Gluconate voice(-) 5 Keto-Gluconate voice(-)

Isolated strain Similarity with standard strain (%) One 2 3 4 5 6 7 8 9 10 11 One Lactobacillus salivarius espi salivarius DF20 2 Lactobacillus salivarius espi salivarius ATCC 11741 ^T 99.7 3 Lactobacillus salivarius es salicinius dsm 20554 ^t 99.7 99.5 4 Lactobacillus avalis DSM 20655 ^T 95.0 94.9 95.5 5 Lactobacillus aguilis DSM 20509 ^T 94.4 94.9 94.8 94.2 6 Lactobacillus animalis DSM 20602 ^T 92.7 93.6 93.7 93.3 94.8 7 Lactobacillus hilgadi DSM 20176 ^T 90.5 91.2 91.4 91.3 91.8 91.7 8 Lactobacillus platinum JCM 1149 ^T 89.6 90.8 90.4 89.9 92.2 91.4 93.2 9 Lactobacillus casei JCM 1134 ^T 90.9 91.7 91.6 91.3 91.6 91.3 93.3 91.9 10 Lactobacillus vipermentans DSM 20003 ^T 88.8 89.7 90.2 89.9 90.6 90.1 92.1 92.2 91.5 11 Lactobacillus sakei DSM 20017 ^T 90.1 91.4 91.2 90.8 90.7 91.8 92.5 93.3 92.7 91.8 12 Lactobacillus rutheri DSM 20016 ^T 89.0 89.9 89.4 90.1 90.0 90.2 91.7 90.9 91.7 90.6 91.3

<Example 3> Harmful microorganism inhibitory ability of the new strain Lactobacillus salivarius sp. Salivarius DF20

In order to investigate the inhibitory ability of the isolated microorganism Lactobacillus salivarius sp. Salivarius DF20 on harmful microorganisms, 8 mm paper disks were attached to LB agar and SPC agar coated with each harmful microorganism and incubated at 37 ° C. for 48 hours. Inhibition of harmful microorganisms was investigated by measuring the size of the inhibitory ring.

As a result of testing the isolated bacterium Lactobacillus salivarius sp. Salivarius DF20 against harmful microorganisms (E. coli and Salonella spp.), The results were as shown in Table 8, and the paper disk tested was twice as large as 8mm. By forming a three-fold inhibitory ring in, it was shown that the ability to suppress the E. coli and Salmonella strains causing food poisoning was excellent.

Harmful microorganisms Lactobacillus salivarius espi salivarius DF20 Comparison 1 Comparison 2 Comparison 3 Comparison 4 Escherichia coli (E. coli) KCTC1039 23 18 19 20 19 Escherichia coli (E. coli) KCTC1021 19 16 15 16 15 Escherichia coli (E. coli) KCTC0115 14 12 14 14 13 Salmonella Typhimurium M-15 25 22 22 22 23 Salmonella typhimurium KCCM40253 23 20 21 21 22 Salmonella Entrepreneur KCCM3313 22 21 20 21 21

* Diameter of ring (unit: mm)

<Example 4> Antibiotic resistance of the new strain Lactobacillus salivarius sp. Salivarius DF20 DF20

In order to investigate the antibiotic resistance of the isolated strain Lactobacillus salivarius sp. Salivarius DF20, antibiotic biodisks (Biomertz, France) contained at each concentration were attached to MRS agar coated with lactic acid bacteria at 37 ° C. Antibiotic resistance was compared by incubating for 48 hours and measuring the size of inhibitory rings.

Antibiotic resistance of the isolated Lactobacillus salivarius sp. Salivarius DF20 lactic acid bacteria showed similar or higher antibiotic growth capacity than the other lactic acid bacteria as shown in Table 9.

Antibiotic Lactobacillus salivarius espi salivarius DF20 Comparison 1 Comparison 2 Comparison 3 Comparison 4 Amikacin (30 µg) 12 11 10 11 11 ampicillin (10 μg) 32 31 28 30 30 Cephaiexin (30 µg) 19 17 15 18 17 Colistin (10 μg) 9 9 9 9 9 Ciprofloxacine (5µg) 9 9 9 9 9 Erythromycin (15㎍) 27 25 24 25 26 Gentamycin (10 μg) 14 12 11 12 11 Neomycin (30 µg) 14 13 11 13 12 Nofloxacin (10µg) 9 9 9 9 9 Penicillin G (10 units) 33 31 30 32 31 Spiramycin (100 µg) 22 20 19 21 20 Tetracycline (30 μg) 24 22 19 22 21

* Diameter of ring (unit: mm)

<Example 5> Enzyme expression characteristics of the new strain Lactobacillus salivarius sp. Salivarius DF20

Enzyme expression of the isolated Lactobacillus salivarius sp. Salivarius DF20 was measured, and 19 enzyme expression abilities were investigated using API-ZYM (bioMerieux SA, France). After culturing in MRS broth, lactic acid strain having good vitality was centrifuged, washed three times with sterile water, diluted, and dropped onto each enzyme plate of API-ZYM for 4 hours at 37 ° C, and each reagent was dropped for discoloration. According to the degree of expression of each enzyme was determined.

As a result of testing the enzyme expression ability of the isolated Lactobacillus salivarius sp. Salivarius DF20, the enzymes of α-glucosidase and β-glucosidase were most actively expressed as shown in Table 10. Therefore, soymilk's indigestible carbohydrates, starchroth and laffinose, have a high decomposition ability. Table 11 shows the degradability of each indigestible carbohydrate using high-performance liquid chromatography, and lactose decomposition is different from other lactic acid bacteria. Similarly, starch rose, raffinose and melobiose showed about 5-20% degradation rate of other lactic acid bacteria, but all of the new strains had high degradation rate of more than 97%. Not only do these strains secrete significant amounts of β-glucosidase enzymes, which break down lactose, a major carbohydrate in milk, but most other lactic acid bacteria contain carbohydrates (starchrose, raffinose, melobees) that are rich in legume crops. The production of α-glucosidase enzyme, which degrades ozone, is low, whereas the new strain produces the largest enzyme, which is a useful lactic acid strain showing high fermentation effect in soymilk.

Constituent enzyme Enzyme activity Alkaline phosphatase usually Esterase (C4) Very many Esterase Lipase (C8) plenty Lipase (C14) usually Leucine arulamidase Very many Valine arylamidase Very many Crystine arylamidase usually Trypsin none α-chymotrypsin usually Acid phospatase Very many Naphtol-AS-B1-phosphohy drolase Very many α-galactosidase Very many β-galactosidase Very many β-glucuronidase none α-glucosidase Very many β-glucosidase a little N-acetyl-β-glucosaminidase none α-mannosidase none α-fucosidase none

Strain Carbohydrate Degradation Rate (%) Lactose Starch Rose Raffinose Melobiose Lactobacillus salivarius espi salivarius DF20 26.53 97.49 98.84 98.79 Comparison 1 24.28 15.49 17.92 20.32 Comparison 2 21.47  5.95  6.92 12.11 Comparison 3 27.72 12.25 16.32 19.22 Compare 4 21.61 22.93 23.94 29.53 Compare 5 25.62 11.45 18.32 21.03

Example 6 Feeding Effect of Mixed Fermented Milk and Soy Milk as Pig Probiotics Using Lactobacillus Salivarius S. Salivarius DF20

Sterilized milk and soy milk mixture as shown in Table 12 for 30 minutes at 85 ℃, cooled to 37 ℃ inoculated and inoculated with the new strain Lactobacillus salivarius S. salivarius DF20 fermentation at pH4.0 and cooled to 4 ℃ It was used in the piglet salary test. Piglets were used for 120 weeks of weaning piglets weaning at 3 weeks of age for 2 weeks. The fermented milk mixed with milk and soy milk was fed 100g / day to investigate the weight gain, feed intake, ammonia gas concentration in pig meal, frequency of diarrhea, and changes in intestinal flora of lactic acid bacilli and Escherichia coli.

Changes in the intestinal flora of test pigs were collected in periods and diluted in anaerobic diluent, and lactic acid bacteria were plated at 37 ° C for 48 hours in BCP agar (Eiken, Japan) containing 0.1% calcium carbonate, and clear and yellow. The colonies of E. coli were cultured in MacConkey agar (DIFCO, USA) and classified according to the type of colonies. EMB agar (BBL, USA) and Brilliant Green Bile Broth (BBL) , US). In general bacteria group, colony counts were measured after plate culture on standard plate agar agar (Difco, USA).

The concentration of ammonia gas in the test piglets was measured by using a Gastec detector tube, and the gas concentration was measured after 1 hour of sealing after adding 500 g of feces.

According to the results of Table 13 and FIG. 4, the feed demand rate was 31% lower in Test A and 31% in Test B than in Control, indicating that pigs fed fermented milk tend to consume relatively small amounts of feed. Indicated. Calculation of the ratio of the feed consumption and the gain gain was performed to increase the weight gain by about 39% compared to the control in Test A and B.

The incidence of weaning piglets in Table 13 was 17.4% in the control group, and there were no piglets that had died. The diarrhea rate of weaning piglets in general was lower than that of 20-47%. As Test A and Test B added fermented milk had no diarrhea rate, the inhibitory effect of diarrhea-inducing microorganisms of probiotic was superior in clinical trials. .

After the fermented milk probiotics were fed to weaning pigs, the concentration of ammonia gas in pig meal was reduced by 5 times or more as compared to the control group. It can be seen that the digestibility of the feed is much higher, and it is showing a good effect on improving the working environment in pigs.

Fecal samples were collected and tested for changes in intestinal flora after 5 days of fermented milk to weaners. In the control group that did not receive fermented milk, E. coli and general bacteria showed 2.29 and 3.79 times more bacteria than the test, respectively, while the number of lactic acid bacteria was 1.70 times higher than that in the control group that fed fermented milk. As a result, the distribution of lactic acid bacteria, which is a beneficial bacterium in the intestinal flora, was significantly present. Therefore, when the fermented milk is fed, the distribution of E. coli and general bacteria is less, so the intestinal intestinal effect by lactic acid bacteria is significantly increased by the feeding of mixed fermented milk.

Raw materials Test Zone A (%) Test Zone B (%) Soybean oil 61 31 Reduced battery milk 31 61 Powdered Glucose 8 8

Control Test Zone A Test B Test head 40 40 40 Starting weight (kg)  7.34  7.03  7.15 Weight (kg) 10.07 10.83 10.95 Daily weight gain (g / day) 195.12 271.79 271.43 Feed Intake (g / day) 401.81 385.77 397.77 Feed rate 2.06 1.42 1.46 Feed demand improvement effect (%) 100 145.1 140.7 % Improvement effect 100 139.3 139.1 Diarrhea incidence (%) 17.4 0 0

Control (mean ± standard deviation) Test sphere (mean ± standard deviation) Remarks Lactobacillus 8.39 ^a ± 0.04 8.62 ^b ± 0.07 Control <Test (1.70 times) Escherichia coli 7.08 ^a ± 0.04 6.72 ^b ± 0.04 Control> Test (2.29 times) General bacteria 8.50 ^a ± 0.24 7.96 ^b ± 0.10 Control> Test (3.79 times)

Unit: viable cell count of log / g

^{a, b} In a row, means followed by a common letter are not significantly different at the 5% level by DMRT.

Example 7 Storage Stability of Mycobacterium Lactobacillus Salivarius Esp Salivarius DF20

Using the isolated Lactobacillus salivarius sp. Salivarius DF20, fermented milk and soy milk in a mixture, incubated pH up to 4.0, and then stored at 5 ° C, 20 ° C, 30 ° C, and 40 ° C for 30 days to measure the survival rate of the new strains. It was. As shown in Table 15, the preservation of the isolated mycobacteria rapidly decreased after 7 days when stored at 20 ℃, and the number of bacteria increased by 3 days after fermentation continued until 1 day when stored at 30 ℃ and 40 ℃. After that, it rapidly decreased. However, when the refrigerated storage at 5 ℃ as shown in the results of Figure 5, the number of viable bacteria does not change significantly until 20 days, so the storage capacity of the viable cells was maintained for at least 20 days when refrigerated storage.

Storage temperature Number of bacteria after storage for each temperature (CFU / ml) 0 days 1 day 3 days 7 days 10 days 20 days 30 days  5 ℃ 5.22 × 10 ⁹ 5.49 × 10 ⁹ 4.36 × 10 ⁹ 2.63 × 10 ⁹ 9.84 × 10 ⁸ 3.52 × 10 ⁸ 4.95 × 10 ⁶ 20 ℃ 6.42 × 10 ⁹ 5.89 × 10 ⁹ 4.58 × 10 ⁸ 3.31 × 10 ⁸ 7.82 × 10 ⁵ 6.39 × 10 ⁴ 3.63 × 10 ³ 30 ℃ 4.87 × 10 ⁹ 3.38 × 10 ¹⁰ 4.73 × 10 ⁷ 6.82 × 10 ³ 3.74 × 10 ² 1.67 × 10 ² 2.00 × 10 ¹ 40 ℃ 5.64 × 10 ⁹ 7.82 × 10 ¹⁰ 2.35 × 10 ⁷ 7.93 × 10 ² 6.29 × 10 ² 5.00 × 10 ¹ 3.00 × 10 ¹

The new strain Lactobacillus salivarius sp. Salivarius DF20 is a lactic acid bacterium with probiotic properties such as acid resistance, bile resistance and oxygen resistance. It is a rare lactic acid bacterium that breaks down. In addition, it was able to adapt to antibiotics and invented a new strain with excellent growth inhibitory ability against pathogenic microorganisms that cause diarrhea and disease. As the new strain grows well in soy milk as well as in milk, it produced the optimal fermentation composition to cultivate the mixture of soy milk and milk.The culture was fed to pig pigs to improve the weight gain, suppressed diarrhea, and mortality was reduced. Its properties were excellent as a supplementary feed (natural probiotic) of piglets.

<110> MIN, Jae Yun          KIM, Kwang Woog          BAE, Hyoung Churl <120> Probiotic effects of fermented mixture of prepared with milk and          soymilk use to Lactobacillus salivarius sp. salivarius DF20 on          Piglets <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1498 <212> DNA <213> Lactobacillus salivarius sp. salivarius DF20 (KCTC10942BP) <400> 1 gtcgcncgaa actttcttac accgaatgct tgcattcacc gtaagaagtt gagtggcgga 60 cgggtgagta acacgtgggt aacctgccta aaagaagggg ataacacttg gaaacaggtg 120 ctaataccgt atatntctaa ggatcgcatg atccttanat gaaagatggt tctgctatcg 180 cttttanatg gacccgcggc gtattaacta gttggngggg taacggccta ccaaggngat 240 gatacgtanc cgaactgaga ggttgatcgg ccacattggg actganacac ggcccaaact 300 cctacgggag gcagcagtag ggaatcttcc acaatggacg caagtctgat ggagcaacnc 360 cgcgtgagtg aanaaggtct tcggatcgta aaactctgtt gttagagaag aacacnagtg 420 agagtaactg ttcattcgat gacggtatct aaccagcaag tcacggctaa ctacgtgcca 480 gcagcccgng gtaatacgta ggtggcaagc gttgtccgga tttatttggg cgtaaaaggn 540 aaccncangc ggtcttttaa attttgttgt gnaaaccttt ggtttnccgg agtantgctt 600 tgnaanctng aaaatttnnt gccnnaaaag aaagngaccn cntntntacg ggaattccta 660 ntttttgaaa acncctngnn naangggttt ttgnctnacc tgccntttgg tnnaacnggg 720 tgncaanana tnanccctnt tnncccctan nanaattnct ngaaatngaa acttagtcaa 780 aaaagnaagg naccncntgg gacggtnaaa ngngaaaaaa aggaanaccc cnnggggaaa 840 anggctnttt tgtttgtaac tacnctttaa ggtttgaaaa ggggggtaac caacangaat 900 tananccctg ggagcccncn ccggtaaacn ataaatgttg gggtggaagg gtttcngccn 960 ttcagngccc aagcnaacgc aataagcatt ccgcctgggg agtacgcccg caaggttgaa 1020 actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca 1080 acgcgaagaa ccttaccagg tcttgacatc ctttgaccac ctaagagatt aggctttccc 1140 ttcggggaca aagtgacagg tggtgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg 1200 gttaagtccc gcaacgagcg caacccttgt tgtcagttgc cagcattaag ttgggcactc 1260 tggcgagact gccggtgaca aaccggagga aggtggggac gacgtcaagt catcatgccc 1320 cttatgacct gggctacaca cgtgctacaa tggacggtac aacgagtcgc nagaccgcga 1380 ggtttagcta atctcttaaa gccgttctca gttcggattg taggctgcaa ctcgcctaca 1440 tgaagtcgga atcgctagta atcgcgaatc agcatgtcgc ggtgaatacg gtcccgaa 1498

Claims (5)

Lactobacillus salivarius sp. Salivarius DF20 (KCTC10942BP) microorganism which has strong adaptability to acid and bile resistance, inhibits the growth of pathogenic microorganisms, and produces alpha-galactoside daisy enzyme The culture according to claim 1, which is sterilized with milk and soy milk or a mixture and fermented with Lactobacillus salivarius sp. Salivarius DF20. The milk and soy milk or mixed culture according to claim 2, wherein the number of bacteria is maintained at an index of 8 or higher of 10 until 30 days upon refrigeration while maintaining the pH of the fermentation culture below 4.0. The composition of claim 2 or 3, wherein the mixed culture is useful for inhibiting enteric pathogenic microorganisms in mammals or for preventing or treating diarrhea caused by pathogenic microorganisms. The food, pharmaceutical, animal drug and feed additive of claim 1 comprising Lactobacillus salivarius sp. Salivarius DF20 (KCTC10942BP) and the culture of claim 2.

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