LT6270B - Probiotic fermented feed additives - Google Patents

Abstract

The present invention relates to biotechnologies and is intended for the field of feed production and may be applied for producing preparations of high antimicrobial activity, i.e. fermented feed additives, feed supplements, feed premixes, feed materials and compound feeds. It is suggested to use probiotic preparations containing lactic acid bacteria (LAB) in feed production. The LAB strains P. acidilactici BaltBio01 MSCL P1480 and P. pentosaceus BaltBio02 MSCL P1481 which have been isolated from the Lithuanian cereal raw material and tested, are microbiologically safer, have high biological activity and improve the health status of animals.

Description

1

LT 6270 B

TECHNICAL FIELD The present invention relates to biotechnology and is intended for the production of feed and may be used for the preparation of preparations with high antimicrobial activity - fermented feed additives, feed additives, feed premixes, feed materials and compound feed.

Technical Ivan

Recently, it is proposed to use probiotic preparations containing lactic acid bacteria (PRB) in the production of feed. PBRs are generally considered useful (non-pathogenic) micro-organisms that live in different environments, such as soil, plant, insect, animal and human microflora. These microorganisms are antagonistic to rotting microflora and also release lactic acid and antibiotic-like substances - bacteriocins or bacteriocin-like substances. Lactic acid bacteria stimulate the synthesis of B vitamins (biotin, thiamine, riboflavin, nicotinic acid, vitamin B12) and vitamin K. Lactic acid bacteria grow best when the medium is at pH 4-5. They affect many pathogenic bacteria: Escherichia coli, Salmonella, Shigella, Pasteurella, Bacillus and Staphylococcus. The lactic acid bacterial probiotics normalize the microflora of the digestive tract of animals, stimulate digestive processes, help the body to absorb nutrients, and affect the resistance of animals to infections and productivity.

One of the most promising PRBs is Pediococcus spp. bacterial genus and, in particular, two closely related groups of the genus Pediococcus pentosaceus and Pediococcus acidilactici.

A brief description of their morphological characterization is provided in the manual (Bergey's Manual of Systematic Bacteriology, vol. 3, Furmicutes, Springer, 2009, pp. 518,519). The use of these bacteria in the production of fermented fodder is known. Method of producing dry (hard) fermented fodder is known (see patent VVO2013029682).

It proposes the use of one or more PRBs from the Enterococcus faecium, Lactobacillus rhamnosus, Lactobacillus plantarum, 2 in the production of fermented fodder.

LT 6270 B

Pediococcus acidililactili and Pediococcus pentosaceus. The present invention provides a broad description of the method and apparatus for drying fermented feed. The drawback of the present invention is the high drying temperature of the fermented fodder, the temperature of which is fed to the drying chamber by more than + 120 ° C, and the temperature of the dried fermented fodder is more than + 40 ° C, which greatly reduces the viability of the probiotic bacteria.

Also known are homofermented liquid feeds and the method of their production (see patent EP2056681). In the production of fermented fodder, it is proposed to use the same PRBs as in the above-mentioned invention; 22549).

The production method and formulations described are not suitable for the production of dry fermented fodder.

The closest analogue of the proposed invention is described in U.S. Pat. This is a method of producing dry fermented fodder using PRB strains Enterococcus faecium MCIMB 30122, Lactobacillus rhamnosus NCIMB 30121, Lactobacillus plantarum LSI (NCIMB 30083), Pediococcus acidililactili NCIMB 30086 and Pediococcus pentosaceus HTS (LMG P-22549). OBJECT OF THE INVENTION The object of the invention is to obtain high-activity fermented feed additives using newly selected strains of PRB isolated from spontaneously fermented cereal raw material. This objective is achieved by using separately or together in the manufacture of fermented feed additives PRB Pediococcus acidilactici BaltBio 01 MSCL P1480 and / or Pediococcus pentosaceus BaltBio 02 MSCL P1481. The following will be used to abbreviate the PRB without the registration body and registration numbers.

In the environment of grain crops (rye, wheat, barley, oats) grown in Lithuania 3

LT 6270 B is dominated by Pediococcus and Lactobacillus generic PRBs that migrate to these spontaneous yeasts and spontaneously multiply. In bacterial isolates, lactic acid bacteria of the genus Pediococcus and Lactobacillus are predominant in these yeasts: 39% - P. pentosaceus, 18% - P. acidilactici and L pharyngeal, 7% - L. curvatus, 2% - L. sakei and 16% - of unknown species of Lactobacillus spp. In spontaneous rye leakage, 67% of bacteria were found to be antibacterial and produce different bacteriocins.

Isolation of BaltBio lactic acid bacteria

The strains of BaltBio lactic acid bacteria have been extracted (isolated) from the fermented fermented cereal grains in Lithuania. Technical works of strain isolation were contracted by UAB Bioseka. The strains were identified by UAB Biosta.

The yeast is produced under laboratory conditions by changing the humidity of the medium and the fermentation temperature. The yeasts were prepared from 1370 rye flour and oat flour and water at a ratio of 1: 1 or 1: 2, the temperature being cyclically changed (25-35) ° C. 10 g of yeast was mixed with 90 ml of physiological saline (0.9% NaCl (Balkanpharma-Troyan AD, Troyan, Bulgaria)) in a BagMixer® 400 P (Interscience for microbiology, France). The resulting suspension was prepared from 10'2 to 10'5 mortars. Correspondingly, 10 * 4 and 10 * 5 dilutions are seeded in Petri dishes with mMRS agar. Plates were stored at 25 ° C, 30 ° C or 35 ° C under anaerobic conditions. Three days later, the number of grams per gram of yeast (CFU / g) was determined and determined for PRB colonies. Randomly selected bacterial colonies (30 PRB of each yeast sample (270 in total) were purified by transfer to new plates. Bacterial cultures were stored at -40 ° C and -80 ° C.

The modified MRS medium (mMRS) was prepared by dissolving 10.0 g of tryptone peptone and meat extract, 5.0 g of yeast extract and sodium acetate, followed by 7.0 g of glucose, fructose and maltose, 2.0 g of sodium gluconate, K2HPO4 and trisamoniocitrate, 1.0mL tvin 80, 0.2g MgSO4-7 H2O, 0.05g MnSC > 4-4 H2O, 0.5g cysteine chloride, 15.0g agar 1L distilled water. The resulting solution (pH 6.3) was sterilized for 15 min at 121 ° C. MRS Agar Medium (MRS) was prepared by dissolving 66.2 g of MRS agar 1 I distilled 4

EN 6270 B water. The resulting solution was boiled until the agar was completely dissolved and sterilized for 15 min at 118 ° C.

Identification of BaltBi lactic acid bacteria PRB was characterized by morphology, carbohydrate fermentation and growth at different temperatures.

The Gram Reaction and Catalase Assays showed that 67 of the nine investigated PRBs selected for the identification of nine self-test yeast samples were gram-positive and did not produce catalase as characteristic of PRB. The results of the microscopic examination showed that 31 strains were in the form of a rod and 36 strains were spherical (tree).

According to the fermentation of sucrose and maltose, the trees were divided into two groups: 1 group had 26, and the second - 10 bacterial strains. All spherical bacteria hydrolyzed trehalose and glucose. P. pentosaceus and P. acidilactici are known to differentiate between maltose and trehalose and differing in their ability to grow at 50 ° C, P. pentosaceus ferments both carbohydrates and cannot grow at this temperature, P. acidilactici only ferments trehalose and grows at 50 ° C. Due to the fact that 5 bacterial strains fermented maltose and trehalose and did not grow at 50 ° C, they were recognized as P pentosaceus, and 4 bacterial strains were identified as P. acidilactici because they did not ferment maltose but grew at 50 ° C.

The amplification PCR method identified the 16S rDNR gene sequence of these 9 strains. Comparison of these sequences with the sequences of the bacterial bank of the International Biotechnology Information Center (NCBI) showed and confirmed that P. acidilactici and P. pentosaceus belong to at least 95% of the probability of this PRB strain.

They are given conditional names - P. acidilactici BaltBioOI, P. acidilactici BaltBio034 P. acidilactici BaltBio05, P. acidilactici BaltBio07, P. acidilactici BaltBio09 and P. pentosaceus BaltBio02, P. pentosaceus BaltBio04, P. pentosaceus BaltBio06, P. pentosaceus BaltBio08, P. pentosaceus BaltBio08. Evaluation of the bacteriocins activity of isolated P. acidilactici and P. pentosaceus strains in pathogenic microorganisms (Bacillus thuringiensis, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Bacillus macerans, Salmonella 5)

LT 6270 B enteritidis, Micrococcus sp, Yersinia enterocolitica, Listeria sp., Pseudomonas aeroginosa) as the most promising microorganisms for animal health by using feed additives and other feed products, Pediococcus acidilactici BaltBio 01 and Pediococcus pentosaceus BaltBio 02. effect on bacteria

Selected lactic acid bacteria (PRB) P. acidilactici BaltBioOI, P. pentosaceus BaltBio02 metabolism products (MPs) are antimicrobially active against various pathogenic bacteria (Bacillus thuringiensis, Escherichia coli, Enterococcus faecaiis, Staphylococcus aureus, Bacillus macerans, Salmonella enteritidis, Micrococcus sp. Yersinia enterocolitica, Listeria sp., Pseudomonas aeroginosa).

The antimicrobial activity of PRB metabolites against gram - / + bacteria (diameter of inhibition zones on agar medium, mm) is shown in Table 1.

It has been found that PRB can completely stop the growth of pathogenic bacteria (bacteriocidal effect) or inhibit or partially inhibit their growth (bacteriostatic effect). The antimicrobial activity of PRB strains against gram - / + bacteria was dependent on the strain of PRB and the indicator microorganism. P. acidilactici BaltBioOI exhibited bacteriocidal activity against all the indicator bacteria tested (the diameter of the inhibition zone in the agar medium was determined from 10 ± 0 to 24.5 ± 0.7 mm), whereas P. pentosaceus BaltBio 02 had bacteriostatic effect.

Indicator microorganism P. acidilactici BaltBio 01 P. pentosaceus BaltBio 02 Tetracycline 30pg Streptomycin 10pg E. coli 14.5 ± 0.7 14.5 ± 0.7 * 17 13 Ent. faecaiis 13 ± 1.4 11 ± 1.4 * 29 * 10 * B. macerans 13 ± 12 ± 0 * 22 18 * S. enteritidis 10 ± 11 ± 1.4 20 10 Micrococcus sp. 24.5 ± 0.7 25 ± 0 30 18 Listeria sp. 13 ± 1.4 10 ± 1.4 26 0 P. aeroginosa 15 ± 0 15 ± 2.8 11 7 B. thuringiensis 19.3 ± 0.5 17.7 ± 0.5 - 17.8 Where - * bacteriostatic Impact 6

LT 6270 B

The highest antimicrobial activity of PRB was found in Micrococcus sp. and B. thuringiensis bacteria (the diameter of the inhibition zones in the agar medium ranged from 16.2 ± 0.7 to 25 ± 0 mm). Antimicrobial effect of P. acidilactici BaltBioOI MP on growth of B. thuringiensis was found to be 8.4% higher compared to the antimicrobial activity of streptomycin P. acidilactici BaltBioOI exhibited bacteriocidal activity in E. coli Ent. faecalis (13 ± 1.4 mm). Bacteriostatic effect was observed for P. pentosaceus BaltBio02 (11 ± 1.4 mm) strain and antibiotics: streptomycin and tetracycline (10 and 29 mm respectively). Antimicrobial effect of PRB metabolism products on microscopic fungi

In the evaluation of the antimicrobial activity of the PRB against microscopic fungi, as Fusarium's representative was chosen as Fusarium poae, while Aspergian's representative was chosen as Aspergius niger. The effect of PRB metabolites on the fungus of Penicillium chrysogenum was additionally evaluated. The antimicrobial PRB as well as the effect of anti-fungal agents on microscopic fungi of the genus Fusarium, Aspergiiius and Penicillium (diameter of the inhibition zones in the agar medium, mm) are given in Table 2. PRB MP had a fungistatic and fungicidal effect on the growth of Fusarium poae, whereas the growth of Penicillium chrysogenum and Aspergiiius niger was determined only by the fungistatic effect of PRB. Table 2 PRB Indicator microorganism Fusarium poae Penicillium chrysogenum Aspergium niger FS FC FS FC FS FC BaltBioOI 15.3 ± 1.0 10.0 ± 0.8 19.5 ± 1.0 - 11.0 ± 1.8 - BaltBio02 13 , 5 ± 1.3 - 21.0 ± 1.2 - 11.0 ± 1.2 - Antigenic preparations Itraconazole 9.5 ± 0 7.5 ± 0.7 - Nystatin 13.5 ± 2.1 31 ± 1 , 4 16.3 ± 0.4 AFY - - - FS - fungistatic effect, FC - fungicidal effect. 7

Preparation of LT 6270 B PRB for fermentation

Prior to the experiment, the PRB was stored at -70 ° C (Sanyo, Japan). Thawed PRB was agarized in MRS agar (Sigma Aldrich, Germany) or other alternative medium, maintaining P. pentosaceus BaltBioO2 at 35 ° C for 48 hours, P. acidilactici BaltBioOI at 32 ° C to 108-109 PRB CFU / ml.

Pure PRB is propagated in an alternative medium produced from potato juice by autoclaving, cooling to 30 ° C and introducing pure PRB cultures (to 0.5 liter juice / 1 ml pure bacterial cultures grown in MRS broth). An alternative medium with PRB is maintained at 30 +/- 2 ° C 24; 48 and 72 h. to 9.60x1010 PRB CFU / ml. Use of PRB in the production of fermented feed

The pre-fermentation substrate may contain from 0.5% to 10% of ready-to-fermentation PRB. P. acidilactici BaltBioOI or P. pentosaceus BaltBioO2 or a mixture thereof may be used. Duration of fermentation is recommended from 8h to 72h and temperature from 20 ° C to 40 ° C. The biologically active fermented feed additive must contain between 106 and 109 PRB CFU / g and a pH of between 3.0 and 6.0. Examples of using PRB

Example 1. Evaluation of the effect of lactic acid bacteria Pediococcus acidilactici BaltBioOI and Pediococcus pentosaceus BaltBio02 on the health of calves. At present, widespread intensive rearing of calves results in imbalances in the intestinal microflora, as no proper microflora develops in the gastrointestinal tract, which is formed from the environment in the digestive tract of animals reared under natural conditions, and is obtained by breast milk. For a variety of reasons, the change in the composition of the small intestinal microorganisms begins to multiply intestinal sticks, which occupy the place of lactic acid bacteria (PRB). 8

LT 6270 B

Infection with pathogenic bacteria damages the mucous membrane and reduces its protective functions. PRB substances can normalize intestinal microflora, improve mucosal properties, promote nutrient uptake and natural resistance, and reduce the effects of stress.

It is claimed that thanks to the PRB, juveniles of cattle start to self-digest vegetative feeds more quickly, better assimilate vegetable proteins and other nutrients.

Comparison of intensively and normally grown animals shows that the physiological properties of the digestive tract change when normal microflora is killed. The weaker developing intestine reduces its mass, builds up too much fluid in the blind and thick intestines, and calves can get enteritis and diarrhea.

14 calves were selected for the test: the test group (n = 6) and the control (n = 8). The calves were selected by analogy to age, kept under uniform conditions, watered with cow's milk three times a day, fed with a special compound feed. The test group received a probiotic mixture of 50 ml of milk for 14 days during the morning watering (about 07:00 h.). At the beginning of the test (0 days) and at the end (14 days), the following parameters were analyzed for the blood gas analyzer Epoc (EPOC, Canada): pH; PC02; P02; Na; K; iCa; Glu; Lactates; Hct; HCO 3, TCO 2, cSO 2, Hb. Blood biochemical analyzer Hitachi 705 (Hitachi, Japan), using DiaSys (Diagnostic Systems GmbH, Germany) reagents, tested the blood enzyme aspartataminotransferase (AST). At the start and end of the test, calf weight was recorded. The following blood parameters were found to be statistically significant (p < 0.05) during the study: pH; PCO2, lactates, AST, leukocytes, lymphocytes, and calf weight gain. The test results are shown in Table 3. Table 3

Parameter Control group Test group Start of tests End of tests Start of tests End of tests Blood pH 7,36 7,361 7,33 7,28 PCO2 63.95 70,93 63,08 60,71 Lactates 3,95 4,29 3,2 2 , 64 9

LT 6270 B AST, TV / I 50.18 58.9 49.82 33.1 Leukocytes, * 106 10.69 10.32 9.37 11.43 Lymphocytes, * 109 / l 4.57 5.55 4, 57 6.39 Obesity, kg 35.75 49 34.37 45.25 For the PRB P. acidilactici BaltBioOI and P. pentosaceus BaltBio02 mixture 14 days after 50 ml: reduced risk of acidosis (stabilization of blood pH, decrease of lactate, PCO2 concentration) ; decreased risk of liver damage (decreased AST); increased immunity (increased lymphocytes and leukocytes in the blood), other parameters have not changed.

Example 2. Evaluation of the effect of lactic acid bacteria P. acidilactici BaltBioOI and P. pentosaceus BaltBio02 on fermentation of cows' health.

For the test, 20 test and control cows were sampled on the principle of analogues for 30 days before the expected calving. Cows of lactic acid bacteria P. acidilactici BaltBioOI and P. pentosaceus BaltBioO2 were fermented (cow 0.3 kg / day) in the test group cows.

Prior to delay and after calving, the following were recorded: milk composition (% fat content,% protein, lactose%, somatic cell count, (SLS) thousand / ml), urea, (mg /%). Five weeks after calving, the amount of milk per day (kg / d) was recorded. Blood for biochemical and morphological examination was taken 30 days before calving and 1 day after calving. It was found that during the study statistically reliable (p < 0.05) concentrations of other DCS, milk, and enzyme ALT were found. 10

EN 6270 Table B 4

Parameter Control group Test group Start of tests End of tests Start of tests End of tests SLS, 103 / ml 638 602 573 114 Milk, kg 22.28 27.14 19.17 30.79 Enzyme ALT, TV / I 28.2 21.18 24.75 13.21

Results. When feeding PRB P. acidilactici BaltBioOI and P. pentosaceus BaltBio02, the fermented product had an effect on reducing the risk of subclinical mastiffs in experimental cows due to a decrease in cow's milk SLS. The quantity of milk produced has also increased and its quality has improved.

Example 3. Milk yield and chemical composition of cattle fed with Pediococcus pentosaceus.

Methods of Experimentation of Lithuanian Black-and-White Cows Feeding Increased and Prepared for PRB Pediococcus pentosaceus BaltBio02.

The studies were carried out with Holstein-cured Lithuanian black and white cows in the barn period. Depending on age, lactation, milk yield, cows are divided into two groups: control (n = 10) and test (n = 10). All cows were fed on protein and energy needs in a balanced diet, consistent with the physiological standards of cow nutrition.

The test cows were fed the same diet. However, with these feeds, these cows every day 56 d. received 100 g of the test additive consisting of Pediococcus pentosaceus BaltBio02 and extruded wheat flour containing a quantity of PRB of 5.0x108 CFU / g.

Milk yields were determined three times during the investigation period, i.e. at the start of the study, in the middle of the study (half past the time of the study) and at the end of the study, with control cows' milking. The natural fat content of milk was recalculated to 4 percent. the amount of fat (corrected) milk based on the coefficients. Milk samples were 11

EN 6270 B taken from each cow individually according to milk sampling rules (LST EN ISO 707: 1999 + P: 2003 Milk and milk products - Sampling rules) at the beginning, middle and end of the study. Samples contained milk fat, protein, lactose and urea with LactoScope FTIR (FT1.0.2001; Delta Instruments, The Netherlands) and somatic cell count (SLS) with SomaScope (CA-3A4, 2004; Delta Instruments, The Netherlands).

Research data were processed by a statistical analysis method (Venables and Smith, 2005). The arithmetic mean values of the attributes and their errors, the degree of reliability p < 0.05 were calculated.

The milk yield and chemical composition of cow fed Pediococcus pentosaceus with BaltBio02 are shown in Table 5. Table 5

Indicator Control group Test group 1 2 3 1 2 3 Milk, kg / d 15,86 15,08 16,60 18,04 18,87 22,38 4% adjusted milk content, _ 17,22 16,94 17,83 19,17 19,88 23,43 Fat content,% 4,5 4,69 4,56 4,41 4,42 4,40 Fat content, kg / d 0,73 0,73 0,75 0,80 0 , 82 0.97 Lactose content,% 4.44 4.46 4.38 4.46 4.54 4.46 Lactose content, kg / d 0.71 0.70 0.74, 0.80 0.85 1 , 0

Using Pediococcus pentosaceus BaltBio02 additive, milk and lactose increased significantly in milk. The fat content in the milk of the test group cows at the end of the test was 0.22 kg / d higher (p < 0.05) than in the control group milk and 0.17 kg / d higher (p < 0.05) at the start of the same group trial. The lactose content in the milk of the test group cows at the end of the test was 0.26 kg / d higher (p < 0.05) than in the control group milk and 0.2 kg / d higher (p < 0.05) at the start of the same group trial. Protein, urea and SLS varied slightly at the start and end of the test (p > 0.05). Excluded from Lithuanian grain raw material and investigated PRB strains P. acidilactici 12

LT 6270 B

BaltBioOI and P. pentosaceus BaltBio02 are microbiologically safer, have high biological activity and improve animal health, increase their productivity and can be used successfully in the production of fermented feed.

Claims (3)

13 EN 6270 B DEFINITION OF DEFINITION 1. Lactic acid bacteria strain Pediococcus acidilactici BaltBioOI MSCL P1480 as an initial fermentation microorganism in fermented feed additives, supplements, premixtures, feed materials and compound feed. 2. Lactic acid bacteria strain Pediococcus pentosaceus BaltBio02 MSCL P1481 as an initial fermentation microorganism in fermented feed additives, supplements, premixtures, feed materials and compound feed. 3. Use according to claims 1 and 2, characterized in that mixtures of fermented feed additives are used in mixtures of lactic acid strains Pediococcus acidilactici BaltBioOI MSCL P1480 and Pediococcus pentosaceus BaltBio02 MSCL P1481.