WO2016046706A1

WO2016046706A1 - Probiotic fermented feed additives

Info

Publication number: WO2016046706A1
Application number: PCT/IB2015/057168
Authority: WO
Inventors: Elena BARTKIENĖ; Gražina JUODEIKIENĖ; Ramūnas GARBARAVIČIUS
Original assignee: Uab "Baltijos Biotechnologijos"
Priority date: 2014-09-24
Filing date: 2015-09-17
Publication date: 2016-03-31
Also published as: LT2014109A; EA201790685A1; EP3198040A1; LT6270B

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 premixtures, 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

Probiotic Fermented Feed Additives

Technical Field

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 premixtures, feed materials and compound feeds.

Background of the Invention

Recently, it is suggested to use probiotic preparations containing lactic acid bacteria (LAB) in feed production. Lactic acid bacteria (LAB) are normally considered to be beneficial (nonpathogenic) microorganisms which live in various environments such as that of soil, plants, insects, animals and human microflora. These microorganisms are antagonistic to decay microflora and excrete a lactic acid as well as the substances which are identical to antibiotics, i.e. bacteriocins or substances similar to bacteriocins. Lactic acid bacteria promote the synthesis of B-group vitamins (biotin, thiamine, riboflavin, nicotinic acid, vitamin B12) and the vitamin K. The growth of lactic acid bacteria reaches the peak when the pH level of the media is 4-5. Their spectrum of activity includes many pathogenic bacteria: Escherichia Coli, Salmonella, Shigella, Pasteurella, Bacillus and Staphylococcus. Probiotics of lactic acid bacteria normalize the livestock gastrointestinal microflora, stimulate digestive processes, help their bodies to assimilate the nutrients contained in a feed, have influence on the livestock resistance to infections and their yield.

One of the most perspective genera of LAB is Pediococcus spp. and, in particular, two closely related group bacteria of this genus Pediococcus pentosaceus and Pediococcus acidilactici. Their brief morphological description is provided in the manual (Bergey's Manual of Systematic Bacteriology, vol.3, Furmicutes, Springer, 2009 p.p. 518, 519). The use of these bacteria in the production of fermented feed is known.

The production method of dry (solid) fermented feeds is known (see the patent WO2013029682). It suggests to use one or several LAB from the group Enterococcus faecium, Lactobacillus rhamnosus, Lactobacillus plantarum, Pediococcus acidililactili and Pediococcus pentosaceus. The method and equipment of drying of fermented feeds is widely described in this invention. The disadvantage of this invention is high temperature necessary for drying fermented feeds: the temperature of the air supplied to the drying chamber exceeds +120°C, and the temperature of the dried fermented feeds exceeds +40°C; this significantly reduces the viability of probiotic bacteria.

The homofermented liquid feeds and the methods of their production are also known (see the patent EP2056681). It suggests to use the same LAB in fermented feed production as the abovementioned invention, however, it additionally indicates specific LAB strains, i.e. Enterococcus faecium MCIMB 30122, Lactobacillus rhamnosus NCIMB 30121, Lactobacillus plantarum LSI (NCIMB 30083), Pediococcus acidililactili NCIMB 30086 and Pediococcus pentosaceus HTS (LMG P- 22549).

The described method of production and formulas are not suitable for dry fermented feed production.

The closest analogue for the proposed invention is described in the patent US20140037786. It is the method of dry fermented feed production in which such LAB strains as Enterococcus faecium MCIMB 30122, Lactobacillus rhamnosus NCIMB 30121, Lactobacillus plantarum LSI (NCIMB 30083), Pediococcus acidililactili NCIMB 30086 and Pediococcus pentosaceus HTS (LMG P-22549) are used.

Essence of the Invention

The objective of the invention is production of the fermented feed additives of high biological activity by using the newly selected LAB strains isolated from the spontaneously fermented cereal material. This objective is achieved by using, together or separately, the LAB Pediococcus acidilactici BaltBioOl and/or Pediococcus pentosaceus BaltBio02 in production of fermented feed additives.

In the environment of the cereal crops grown in Lithuania (rye, wheat, barley, oats), the LAB of genera Pediococcus and Lactobacillus are dominant and convert these cultures into spontaneous starter cultures and grow spontaneously there. The bacterial cultures' isolates from these starter cultures are dominated by the lactic acid bacteria of genera Pediococcus and Lactobacillus: P. pentosaceus— 39 %; P. acidilactici and L. farciminis - 18 % each, L. curvatus - 7 %, L. sakei - 2 % and the unknown species of Lactobacillus spp. - 16 %.

It has been established that 67 % of bacteria in spontaneous cereal starter cultures are antibacterially active and produce different bacteriocins.

Isolation of BaltBio Lactic Acid Bacteria

The BaltBio lactic acid bacteria strains are isolated from the starter cultures of the spontaneously fermented cereal crops grown in Lithuania. The technical works of isolation of the strains have been performed by UAB Bioseka under contracts. The strains have been identified by UAB Biosta.

The starter cultures were produced under the laboratory conditions by changing the moisture content of the medium and the temperature of fermentation. The starter cultures were prepared from rye flour type 1370, oat flour and water in the ratio of 1 : 1 or 1:2, the temperature was cyclically varied within the range from 25 to 35 °C.

10 g of the starter culture were mixed with 90 ml of saline solution (0.9 % NaCl (Balkanpharma- Troyan AD, Troyan, Bulgaria)) in the homogenizer BagMixer® 400 P (Interscience for microbiology, France). The dilutions of 10 -^"2 to 10- 5 were prepared from the obtained suspension. Respectively, the dilutions of 10-⁴ and 10-⁵ were cultured into Petri dishes with MRS agar. The dishes were kept at the temperature of 25 °C, 30 °C or 35 °C under anaerobic conditions. After three days, the number of LAB colony-forming units per gram of the starter culture (CFU/g) was verified and measured. The randomly selected bacterial colonies (30 LAB colonies were isolated from each starter culture (in total, 270)) were purified by sub-culturing them into new dishes. Bacterial cultures were kept at the temperature of - 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, 7.0 g of glucose, fructose and maltose, 2.0 g of sodium glyconate, K2HPO4 and triammonium citrate, 1.0 ml of Tween 80, 0.2 g of MgS0₄-7 H₂0, 0.05 g of MnS0₄-4 H₂0, 0.5 g of cystein chloride, 15.0 g of agar in 1 1 of distilled water. The obtained solution (pH 6.3) was sterilized for 15 min at the temperature of 121 °C.

The MRS agar medium (MRS) was prepared by dissolving 66.2 g of MRS agar in 1 1 of distilled water. The obtained solution was boiled until the agar was completely dissolved and sterilized for 15 min at the temperature of 118 °C.

Identification of Baltbio Lactic Acid Bacteria

The LAB were characterized by identifying their morphology by performing the tests of carbohydrate fermentation and growth at different temperatures.

Having carried out the Gram reaction and catalase tests, it was found that 67 tested LAB selected for identification from the samples of nine spontaneous starter cultures were Gram- positive and did not produce catalase, as it is characteristic for LAB. The results of the microscopic examinations showed that 31 strains were rod-shaped, and 36 strains had a spherical shape (cocci).

Cocci were divided into two groups on the basis of sucrose and maltose fermentation: the first group consisted of 26, and the second group consisted of 10 bacterial strains. All spherical- shaped bacteria hydrolyzed trehalose and glucose. It is known that P. pentosaceus and P. acidilactici differ in fermentation of maltose and trehalose and their potential for growth is also different at the temperature of 50°C, P. pentosaceus ferment both carbohydrates and are unable to grow at the said temperature, while P. acidilactici ferment only trehalose and are able to grow at the temperature of 50 °C. Taking into consideration the fact that 5 bacterial strains fermented maltose and trehalose, but did not grow at the temperature of 50 °C, they were recognized as P. pentosaceus, and 4 bacterial strains were identified as P. acidilactici, since they did not ferment maltose, but grew at the temperature of 50 °C. Amplification by PCR method allowed to identify 16S rDNA gene sequences of these 9 bacterial strains. Comparison of their sequences with the sequences of the Bacteria Bank of the International Center for Biotechnology Information (NCBI) showed and confirmed that these LAC strains belong to P. acidilactici and P. Pentosaceus with at least 95% probability.

They have been given the conditional names: P. acidilactici BaltBioOl, P. acidilactici BaltBio03_s P. acidilactici BaltBio05, P. acidilactici BaltBio07, P. acidilactici BaltBio09 and P. pentosaceus BaltBio02, P. pentosaceus BaltBio04, P. pentosaceus BaltBio06, P. pentosaceus BaltBio08.

Having assessed the activity of bacteriocins of the isolated strains P. acidilactici and P. pentosaceus against pathogenic microorganisms (Bacillus thuringiensis, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Bacillus macerans, Salmonella enteritidis, Micrococcus sp, Yersinia enterocolitica, Listeria sp., Pseudomonas aeroginosa), Pediococcus acidilactici BaltBioOl and Pediococcus pentosaceus BaltBio02 were selected as the most promising microorganisms for ensuring good health of animals by using them for feed additives and other feed products.

LAB Pediococcus acidilactici BaltBioOl and Pediococcus pentosaceus BaltBio02 were deposited in Microbial Strain Collection of Latvia (MSCL) and got identification numbers:

Pediococcus acidilactici BaltBioOl MSCL P1480,

Pediococcus pentosaceus BaltBio02 MSCL P1481.

Further LAB in the text will be used the reduced definition, without indication of the depositing organization and number of registration.

Antimicrobial Effect of LAB Metabolites

The antimicrobial activity of metabolites of the selected lactic acid bacteria (LAB) P. acidilactici BaltBioOl, P. pentosaceus BaltBio02 against pathogenic bacteria (Bacillus thuringiensis, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Bacillus macerans, Salmonella enteritidis, Micrococcus sp, Yersinia enterocolitica, Listeria sp., Pseudomonas aeroginosa) has been found.

The antimicrobial effect of the LAC metabolites against Gram-/+ bacteria (diameter of the zones of inhibition in the agar medium, mm) is presented in Table 1.

It has been established that LAC are able to completely stop the growth of pathogenic bacteria (bactericidal effect) or inhibit or partly inhibit their growth (bacteriostatic effect). The antimicrobial effect of the LAC strains against Gram-/+ bacteria depended on the LAB strain and indicator microorganism. P. acidilactici BaltBioOl demonstrated the bactericidal effect against all examined indicator bacteria (measured diameter of the zones of inhibition in the agar medium varied from 10±0 to 24.5±0.7 mm), while P. pentosaceus BaltBio02 demonstrated the bacteriostatic effect.

Table 1

The greatest antimicrobial effect of LAB was demonstrated by Micrococcus sp. and B. thuringiensis bacteria (diameter of the zones of inhibition in the agar medium varied from 16.2±0.7 to 25±0 mm). The established antimicrobial effect of metabolites of P. acidilactici BaltBioOl on the growth of B. thuringiensis was by 8.4 % higher in comparison with the antimicrobial effect of streptomycin, while P. acidilactici BaltBioOl demonstrated the bactericidal effect on E. coli Ent. faecalis (13±1.4 mm). Bacteriostatic effect was demonstrated by P. pentosaceus BaltBio02 (11±1.4 mm) strain and antibiotics: streptomycin and tetracycline (10 and 29 mm, respectively).

Antimicrobial Effect of the LAB Metabolites on Microscopic Fungi

When assessing the antimicrobial effect of LAB against microscopic fungi, Fusarium poae was selected as the representative of the genus Fusarium, and Aspergillus niger was selected as the representative of the genus Aspergillus. The effect of the LAB metabolites on the fungus Penicillium chrysogenum was additionally assessed. The antimicrobial effect of LAB as well as the effect of antifungal preparations on the microscopic fungi of the genera Fusarium, Aspergillus and Penicillium (diameter of the zones of inhibition in the agar medium, mm) is indicated in Table 2. The LAB metabolites demonstrated fungistatic and fungicidal effect on the growth of Fusarium poae, while the LAB had only fungistatic effect on the growth of Penicillium chrysogenum and Aspergillus niger.

Table 2

Preparation of LAB for Fermentation

Prior to experiment, LAB were kept at the temperature of -70°C (Sanyo, Japan). The thawed LAB were amplified in MRS agar (Sigma Aldrich, Germany) or any other alternative medium by keeping P. pentosaceus BaltBio02 at the temperature of 35 °C and P. acidilactici BaltBioOl - at the temperature of 32 °C for 48 hours, until the level of LAB reached 10⁸-10⁹ CFU/ml.

The pure LAB are amplified in the alternative medium which is made of potato juice by autoclaving them, cooling to a temperature of 30°C and introducing pure LAB cultures (1 ml pure bacteria cultures amplified in MRS bouillon into 0.5 litre of juice). The alternative medium with LAB is kept at the temperature of 30 +/- 2 °C for 24; 48 and 72 hours, until the level of LAB reaches 9.60xl0¹⁰ CFU/ml.

Use of LAB in Fermented Feed Production LAB prepared for fermentation may comprise from 0.5% to 10% of the substratum prepared before fermentation. P. acidilactici BaltBioOl or P. pentosaceus BaltBio02 or their mixture may be used. The recommended duration of fermentation is from 8 hours to 72 hours and the temperature shall be from 20°C to 40°C. Biologically active fermented feed additive shall contain LAB from 10⁶ to 10⁹ CUF/g and pH shall be from 3.0 to 6.0.

Examples of Use of LAB

Example 1. Assessment of the Effect of the Product Fermented by the Mixture of Lactic Acid Bacteria Pediococcus Acidilactici BaltbioOl and Pediococcus Pentosaceus Baltbio02 on the Health Status of Calves.

Intense calf rearing, which is now widely applied, causes gut microflora imbalance, since the appropriate microflora, which is formed from the environment and is obtained with maternal milk in the gastrointestinal tract of the livestock reared under the natural conditions, fails to develop in the gastrointestinal tract of the intensively reared livestock. For various reasons, changes in the composition of microorganisms of small intestine result in growth of coliform baccili which replace the lactic acid bacteria.

Infection caused by pathogenic bacteria results in the damaged mucosa, reduces its defensive functions. The LAB materials are able to normalize the gut microflora, improve the defensive features of the mucosa, promote nutrient assimilation and natural resistance as well as reduce the stress-induced consequences.

It is claimed that LAB contribute to the earlier self-digestion of plant feeds and better assimilation of plant proteins and other nutrients by the bovine offspring. As a result of comparison of the intensively and normally reared livestock, it has been established that the physiological features of the digestive tract change when the normal microflora is ruined. As a result of poorer development of the intestine of calves, the weight of such intestine decreases, too much fluid is accumulated in large intestine and caecum and this may result in enteritis and diarrhoea.

14 calves were selected for experiments: experimental group (n=6) and control group (n=8). Calves were selected by following the principles of analogues according to age, kept under the same conditions, given the cow's milk three times a day and fed with the special compound feed. The experimental group received the probiotic mixture by pouring 50 ml of it into milk and giving it with the morning portion of the cow's milk (approx. at 07:00 a.m.) for 14 days. The following parameters were measured with blood gas analyzer "Epoc" (EPOC, Canada) at the beginning of the experiment (0 day) and at the end of the experiment (on the 14^th day): pH; PC02; P02; Na; K; iCa; Glu; Lactates; Hct; HC03, TC02, cS02, Hb. The blood ferment aspartate aminotransferase (AST) has been analyzed with the blood biochemical analyzer "Hitachi 705" (Hitachi, Japan) by using the reagents of "DiaSys" (Diagnostic Systems GmbH, Germany). The weight of calves was recorded at the beginning and at the end of the experiment. It was found that the changes in the following blood parameters during the examinations were statistically significant (p<0.05): pH; PC02, lactates, AST, leucocytes, lymphocytes and weight gains of calves. The results of the experiments are presented in Table 3.

Table 3

After feeding 50 ml of the mixture of LAB P. acidilactici BaltBioOl and P. pentosaceus BaltBio02 for 14 days:

1 ) the risk of acidosis was reduced (blood pH stabilized, lactates and PC02 concentrations were reduced);

2) the risk of liver damage was reduced (AST concentration was reduced);

3) immunity of the body was strengthened (lymphocyte and leucocyte count in blood increased), 4) other parameters did not change.

Example 2. Assessment of the Effect of the Product Fermented by the Mixture of Lactic Acid Bacteria P. acidilactici BaltBioOl and P. pentosaceus BaltBio02 on the Health Status of Cows.

By following the principle of analogues, 20 experimental and control draught cows at 30 days before the expected calving were selected for the experiment. The cows of the experimental group were fed with the product fermented by the mixture of lactic acid bacteria P. acidilactici BaltBioOl and P. pentosaceus BaltBio02 (quantity per cow 0.3 kilo/day).

The following parameters were recorded prior to dry-off and after calving: milk composition (fat content %, protein content % , lactose %, somatic cell count, (SCC) thousand/ml), urea, (mg/per cent). Following five weeks after calving, the quantity of milk per day (kg/day) was recorded. Blood for biochemical and morphological examinations was taken 30 days prior to calving and 1 day after calving. It was found that the changes occurred in SCC, quantity of milk and concentration of ferment ALT were statistically significant (p<0.05).

Table 4

Results. When giving with the mixture of the LAB strains P. acidilactici BaltBioOl and P. pentosaceus BaltBio02, the fermented product had effect on reduction of subclinical mastitis in the experimental cows as a result of decrease of SCC in the milk of these cows. In addition, the quantity of milk increased and its quality improved.

Example 3. Milk Yield and Chemical Composition when Feeding Cows with the Pediococcus pentosaceus Additive. Methodology of the Experiment of Feeding Lithuanian Black-and- White Cows with the LAB Pediococcus pentosaceus BaltBio02 Amplified and Prepared for Fermentation.

The experiments were carried out with the holsteinized Lithuanian black-and-white cows during the indoor period. Taking into consideration the age, lactation period, milk yield, the cows were divided into two groups: control group (n=10) and experimental group (n=10). All cows were fed with the ration balanced according to requirements of proteins and energy which is in accordance with the physiological norms of cows' feeding.

The experimental cows were fed with the same ration. However, in addition to the feed, these cows were given 100 g of the experimental additive consisting of Pediococcus pentosaceus

BaltBio02 and extruded wheat flour where the level of LAB was 5.0x10 CFU/g each day, in total for 56 days.

During the experimental period, the milk yield was measured three times, i.e. at the beginning of the experiment, in the middle of the experiment (after a half of the experimental period) and at the end of the experiment by carrying out the control milkings. The quantity of milk with natural fat content was recalculated into the quantity of 4 per cent fat (corrected) milk on the basis of the respective ratios. Milk samples were taken from each cow individually in accordance with guidance on milk sampling (LST EN ISO 707: 1999+P:2003 Milk and milk products. Guidance on sampling) at the beginning, in the middle and at the end of the experiment. Milk fat content, milk protein content, lactose and urea were measured by using the device "LactoScope FTIR" (FT1.0. 2001 ; Delta Instruments, Holland) and the somatic cell count (SCC) was measured by using the device "SomaScope" (CA-3A4, 2004; Delta Instruments, Holland) in the samples. The data of experiments was processed by applying the method of statistical analysis (Venables and Smith, 2005). Arithmetic means of the features and their deviations, degree of confidence p<0.05 were calculated.

Milk yield and chemical composition by feeding the cows with the additive Pediococcus pentosaceus BaltBio02 are indicated in Table 5. Table 5

When using the Pediococcus pentosaceus BaltBio02 additive, increase of fat content and lactose in milk was statistically significant. The fat content of the milk of the experimental group at the end of the experiment increased by 0.22 kg/day (p<0.05) in comparison with the milk of the control group and increased by 0.17 kg/day (p<0.05) in comparison with the fat content of the same group at the beginning of the experiment. The quantity of lactose of the milk of the experimental group at the end of the experiment increased by 0.26 kg/day (p<0.05) in comparison with the milk of the control group and increased by 0.2 kg/day (p<0.05) in comparison with the quantity of lactose of the same group at the beginning of the experiment. Protein content, urea and SCC at the beginning and at the end of the experiment differed insignificantly (p>0.05).

The LAB strains P. acidilactici BaltBioOl and P. pentosaceus BaltBio02 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, increase their yield and may be successfully used in fermented feed production.

Claims

1. Use of the lactic acid bacterial strain Pediococcus acidilactici BaltBioOl MSCL P1480 as a probiotic (as the initial fermentative microorganism) in the fermented feed additives, supplements, premixtures, feed materials and compound feeds.

2. Use of the lactic acid bacterial strain Pediococcus pentosaceus BaltBio02 MSCL P1481 as a probiotic (as the initial fermentative microorganism) in the fermented feed additives, supplements, premixtures, feed materials and compound feeds.

3. Use according to claims 1 and 2, charakterized in that the mixtures of the lactic acid bacterial strains Pediococcus acidilactici BaltBioOl MSCL P1480 and Pediococcus pentosaceus BaltBio02 MSCL P1481 are used in production of fermented feed additives.

4. Feed additive containing Pediococcus acidilactici BaltBioOl MSCL P1480 and/or Pediococcus pentosaceus BaltBio02 MSCL P1481 according to claims 1-3, charakterized in that the pure lactic acid bacteria (LAB) are amplified in the alternative medium which is made of potato juice by autoclaving them, cooling to a temperature of 30°C and introducing pure LAB cultures (1 ml pure bacteria cultures amplified in MRS bouillon into 0.5 litre of juice) and by keeping them at the temperature of 32 °C from 24 hours to 72 hours, until the level of LAB reaches 9.60xl0¹⁰ CFU/ml.