Technical field

The present invention relates to biotechnologies and is intended for the field of feed production and may be applied for producing fermented feed of plant origin with high antimicrobial activity.

Background of the Invention

Dry fermented feed is becoming more widely used in the modern livestock farming. The so- called fermentation in the solid phase (solid-phase fermentation - SPF) is applied in dry solid feed production. The SPF technology is more cost-effective (simpler, lower volume equipment is used for this fermentation), safer (since the substrate water activity is low during fermentation, the microbial contamination decreases), therefore SPF is the innovative technology in comparison with the usual fermentation in the liquid phase.

It is very important that the fermented feeds with the low moisture content, including dry feeds prepared by applying the SCC technology would contain the sufficient level of lactic acid bacteria (LAB). It is known that the dry fermented feeds prepared by applying the SCC technology are less perishable (they contain the lower number of colony forming units of pathogenic bacteria per gram of the product (CFU/g)) and their nutritional value is better. Various microscopic fungi are the main cause of deterioration of fermented feeds. Some species of microscopic fungi may cause serious diseases in animals since the metabolites produced by them are very toxic. This is particularly the case with the microbial deterioration of the feeds (liquid feeds) with the high moisture content.

The consumption of feeds with the high level of LAB reduces the level of pathogenic microorganisms in the gastrointestinal tract of various domestic animals. Mostly, the feed containing LAB is given to an animal in a liquid form, however, since many animals may not consume liquid feed or liquid feed material may cause the undesirable developments in the main feed mass (undesirable fermentation, etc.), production of thesuch feed in a dry form is perspective.

The traditional drying methods are normally used for production of dry fermented feeds in order to remove moisture from the semi-liquid feeds by heat treatment and obtain dry fermented feed. The latter drying methods (see the patent WO2013029682) work at a very high temperature, therefore they reduce the viability of LAB and other probiotic bacteria in feed. Due to these reasons, in order to ensure efficiency of fermented feeds, it is very important to select the appropriate conditions for their production and to ensure the sufficient concentration of LAB in them. The important thing is not only the sufficient level of LAB (CUF/g) in feeds, but also ensuring of feed safety in the aspect of minimization of pathogenic bacteria.

The dry probiotic feed additive containing the LAB Streptococcus lactis 3186 K 12 VKPM B- 4989, Lactobacilus casei MB VKPM B-4486 and Lactobacilus acidophilus M86 VKPM B-4992 is known and is used to improve the gut microflora of ruminant animals (see the patent RU2063755). The disadvantage of the probiotic feed additive is its insufficient efficiency in destroying pathogenic bacteria of animals for the following reasons: it contains only the LAB of the specific strain; the level of LAB is not sufficient; and the biological activity of LAB is lower.

The method of production of the biologically active feed additive from a plant material, which includes grinding of plant raw material, selection of bacterial cultures, preparation of the culture and fermentation in the solid substrate, is also known (see the patent RU2202224). Bacterial cultures are selected from the different sections of the gastrointestinal tract of grass-feeding animals (mostly, wild animals), starting from larynx cavity, forestomach, gut and ending with excrements.

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. As a plant raw material, it is suggested to use various cereals, i.e. wheat, barley, rye, rice, maize, triticale, oats, soy; vegetables, i.e. potatoes, and, also, dairy products, i.e. whey, curd, skim milk. This invention also suggests dry (solid) feed fermentation method.

The general requirements are raised for dry fermented feeds.

The recommended temperature for production (drying) of fermented feeds is (20-75)°C. The lower the temperature is, the higher level of LAB remains in a feed. Mostly, the moisture content of the fermented feeds is 8-9%, therefore the level of LAB is higher in the smaller feed quantity, because such feed is more concentrated, and, respectively, the effect of LAB on the health status of animals is more significant. The level of LAB in dry fermented feeds shall reach 10 ⁶ - 10 ⁸ CFU/g.

The homofermentative LAB are usually used for feed fermentation. Their main metabolite is lactic acid, and the concentrations of other produced metabolites (acetic acid, ethanol, etc.) are not significant. If heterofermentative bacteria are involved in fermentation, then the main metabolites may be acetic acid, and the quantity of produced lactic acid is insignificant. The LAB used for feed fermentation may be homofermentative and heterofermentative. Homofermentative LAB are the bacteria whose main metabolite is lactic acid, and the lactate produced by heterogermentative LAB is further metabolized to acetate, carbon dioxide and other metabolites.

In order to ensure safety of fermented feeds from pathogenic microorganisms, it is also very important to ensure the sufficiently low pH of fermented feeds. The recommended pH level of fermented feeds is from 3.5 to 5.0.

It is also important to ensure appropriate organoleptic properties and texture of fermented feeds. During fermentation process, LAB produce lactic acid by using substrates containing carbon. The concentration of lactic acid in fermented products may vary depending on the fermentable sugars, LAB species in the medium and other factors.

In case of fermentation of substrates by homofermentative LAB, the concentration of lactic acid in them varies from 50 to 300 mM. In case of fermentation by heterofermentative LAB, the concentration of acetic acid, respectively, may vary from 50 to 300 mM.

Essence of the Invention

The aim of the invention is production of the plant fermented feeds of high biological activity by using the newly selected lactic acid bacteria strains isolated from the fermented cereal material. This objective is achieved by using the substrate of plant origin and the starter culture of LAB dominated by the strains Pediococcus acidilactici BaltBioOl and Pediococcus pentosaceus BaltBio02 in fermented feed production.

The strains Pediococcus acidilactici BaltBioOl and Pediococcus pentosaceus BaltBio02 are isolated from the starter cultures made of the 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. For the purposes of description, the following terms shall have the following meanings:

LAB starter culture shall mean the LAB 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 Pediococcus acidilactici BaltBioOl and/or Pediococcus pentosaceus BaltBio02 (1 ml of pure bacterial cultures, amplified in MRS bouillon, into 0.5 litre of juice).

Substrate shall mean any plant raw material, plant products containing hydrocarbons, including cereal grains and any parts thereof, oily seeds, legumes, hay and grass. In order to reduce the microbial contamination of the substrate and to obtain the fraction of the required size prior to fermentation, the raw material may be ground, treated at high temperatures, with steam, by extrusion and by any other known means.

Fermentation shall mean fermentation of the prepared plant raw material carried out at the temperature of 20-40 °C, in the liquid substrate with moisture content higher than 50%, or in the solid substrate with moisture content lower than 50%. Fermentation is performed under anaerobic conditions or aerobic conditions, depending on the properties of the substrate, i.e. moisture content, composition, etc. The initial content of the LAB starter culture in the substrate may vary from 0.5% to 10%. Duration of fermentation shall vary from 8 hours to 72 hours.

Treatment of the fermented substrate: after termination of fermentation, the fermented mass shall be treated in various ways: ground, sieved, granulated.

Stabilization of the fermented substrate: in order to reduce the moisture content, the substrate is dried, sublimated, pressed-dried, may be frozen, encapsulated, chemically conserved.

Fermented product: depending on the method of stabilization of the fermented substrate, the biologically active solid feed product in the form of paste, powder or granules is obtained. The level of pH of the fermented product shall vary from 3.0 to 6.0, and the level of LAB in it shall vary from 10 ⁶ to 10 ⁹ CFU/g.

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. Isolation, identification, genetics, antimicrobial effect on bacteria, antimicrobial effect on microscopic fungi of the LAB strains Pediococcus acidilactici BaltBioOl and Pediococcus pentosaceus BaltBio02 are described in other patent of these authors (see the patent application LT2014 109).

The strains Pediococcus acidilactici BaltBioOl and/or Pediococcus pentosaceus BaltBio02 distinguish for the particular antimicrobial activity of their metabolites against pathogenic bacteria (Bacillus thuringiensis, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Bacillus macerans, Salmonella enteritidis, Micrococcus sp, Yersinia enterocolitica, Listeria sp., Pseudomonas aeroginosa). The antimicrobial effect of Pediococcus acidilactici BaltBioOl and/or Pediococcus pentosaceus BaltBio02 against microscopic fungi included the fungistatic and fungicidal effect on the growth of Fusarium poae, however only the fungistatic effect on the growth of Penicillium chrysogenum and Aspergillus niger has been established.

In the present invention, the following fermented feed production technology is proposed:

1. Substrate, i.e. barley and wheat (90/10; m/m) barn and water, shall be stirred in slow running stirrer until a homogeneous mass. Temperature of the mixture shall be 29 °C ± 5 °C, moisture content of the mixture shall be 50 % ± 5%.

2. The starter culture of the LAB amplified up to 10 ⁹ CFU/g shall be inserted into the homogenized substrate.

3. Mixture shall be stirred and put into a fermenter. Fermentation shall be performed for 24 hours at the temperature of +30 ± 2 °C and a relative air humidity of 85±2 %.

4. The fermented substrate shall be put into the stirrer again and the feed yeast Saccharomyces cerevisiae (used for livestock feeding) shall be added according to the formula (0.5%). Stirring shall be done for 10 minutes.

5. The fermented product shall be packed and stored under the normal conditions, i.e. at the temperature of (15 - 20)°C and a air humidity of (55-75)%.

As the components of the substrate of fermented products, barley and wheat were selected. After examination carried out with the substrates of various compositions, the substrate of the optimal composition containing 5-20% of wheat and 80-95% of barley barn was selected. The results of the experiments of the fermented product carried out with this substrate are provided below.

The properties of the fermented product depend on the temperature of fermentation. After fermentation performed at the temperature of 37°C and 20°C, the concentration of LAB (CFU/g) in the fermented wheat-barley barn was respectively 60x10 8° CFU/g and 40x 108° CFU/g.

Dependence of LAB CFU in the Dried Fermented Product on a Drying Temperature and Duration (Table 1)

Table 1

It has been found that drying allows to preserve the sufficiently high level of LAB (irrespective of duration of drying), however, if drying is done for 18 and more hours at the temperature of 30°C, mould and yeast start to grow (yeast (5-6)xl0 CFU/g). Therefore the drying process must be as short as possible.

Dependence of Level of pH and Moisture Content of the Dried Fermented Product on Duration of Drying (Table 2).

Table 2

PH Moisture content, %

Duration, h Duration, h

0 72 0 72

5,50 4,06 45,2-46,6 8,2-9,8

Drying temperature - 30°C Dependence of the Fermented Product on Storage Duration.

The fermented product dried to the recommended moisture content (8.82%) was stored at a room temperature in a polyethylene package. The LAB concentration (CFU/g) was measured every 30 days, i.e. after 0, 30, 60 and 90 days (Table 3).

Table 3

L-(+) and D-(-) Lactic Acid Isomers in the Fermented Product.

Pediococcus acidilactici BaltBioOl and Pediococcus pentosaceus BaltBio02 produce:

- 0.111552 μg/100g and 0.892419 μg/100g of D-(-) lactic acid isomers, respectively;

- 0.98g/100g and 0.67 g/lOOg of L (+) lactic acid isomers, respectively.

The barely-wheat substrate is appropriate for production of raw materials of fermented feeds (the obtained LAB concentration in the fermented product with moisture content being 40 % is

10 CFU/g). The barely-wheat substrate is well homogenized, and when it is fermented with the mixture of Pediococcus acidilactici BaltBioOl and Pediococcus pentosaceus BaltBio02, the initial microbial contamination of the raw material with undesirable microorganisms decreases: no yeast and mould fungi have been found in the product dried to the moisture content of 8.82 % (at the temperature of 30 °C for 28 hours). The concentration of LAB in the fermented product is 18xl0 ⁷ CFU/g which decreases to 5.0x10 ⁶ CFU/g after 90 days of storage at a room temperature in a in a polyethylene package. The fermented product is safe with respect to D-(-) lactic acid isomers.

The lactic acid bacteria strains Pediococcus acidilactici BaltBioOl and Pediococcus pentosaceus BaltBio02 are safe to use in feeds, and the products of dry feed materials fermented by them have high biological activity and retain biological activity during storage.

Eamples

Example 1. Assessment of the Effect of the Product Fermented by the Mixture of Lactic Acid Bacteria Pediococcus Acidilactici BaltbioOl and Pediococcus Pentosaceus Baltbio 02 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 lead to growth of coliform baccili which replace the LAB.

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 the 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, received the cow's milk three times a day and were fed with 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 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 occurred 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 4.

Table 4

After feeding 50 ml of the mixture of LAB P. acidilactici BaltBioOl and P. pentosaceus

BaltBio02 per day for 14 days:

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

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

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

Example 2. Milk Yield and Chemical Composition when Feeding Cows with the Pediococcus pentosaceus BaltBio02 Additive. Methodology of the Experiment of Feeding Lithuanian Black-and-White Cows with the amplified Pediococcus pentosaceus BaltBio02.

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 amplified in the substrate of extruded whole-grain wheat flour where the level of LAB was 5.0xl0 ⁸ 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 were 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).

It has been stated on the basis of the obtained results that feeding dairy cows with the fermented feed (90 g / per day, when the moisture content of such feed is 60 %) containing at least 5.0x10 CFU/g of Pediococcus pentosaceus BaltBio02 for the period of at least 56 days resulted in an increase of yield of dairy cows. Isolated LAB strains from the informal way of fermented grain grown in Lithuania materials are safe for use in a safe, dry fermented fodder with high biological activity, loss of biological activity during storage.