Litter feed additive as source of amino acids and beneficial bacteria

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Abstract


Feed additive was derived from poultry manure by microbiological synthesis. After a 10-day feeding poultry with probiotic strains of bacteria B. subtilis TNP-3 and B. subtilis TNP-5, the litter does not contain potential enteropathogens and can be used as a raw material for feed additive. Based on the results of microbiological and biochemical studies the technology of feed additive (powder) with the use of extrusion was developed. Extrusion at a temperature of up to 120 °C for 5-6 seconds provides presence of beneficial bacteria and significantly high content of essential amino acids. According to the results of biochemical studies, litter feed additive contains 18 free amino acids. The total concentration of free amino acids in the feed additive (powder) is 406.3 mg/kg, which is 1.7 times higher than that in the litter without fermentation and extrusion. The experiments have shown that inclusion of 3.3% feed additive in the diet does not have negative effect on physiological state, viability and productivity of laying hens. Survival of birds in both groups was 100%. Additive application in the experimental group of chickens revealed absence of opportunistic pathogenic microorganisms and microscopic fungi, presence of bifidobacteria and spore-forming aerobic Bacillus bacteria in powder, as well as predominance of beneficial micro flora and lack of potential enteropathogens (compared to control). It allows to conclude that feed additive obtained by microbiological synthesis from bird droppings possess probiotic properties. The results of biochemical study of egg production indicate that the use of food additives (to 3.3% of the basic diet) for laying hens significantly increases content of major micro and macro-elements in eggs, compared to the control. Therefore, the use of feed additive-powder (up to 3.3% of the basic diet) does not reduce egg quality. Thus, based on the results of these studies, it can be concluded that the litter obtained from laying hens, after application of probiotic ‘Nord-Bakt’, further fermentation with strains Bacillus subtilis TNP-3 and Bacillus subtilis TNP-5, followed by extrusion can be used as a feed additive as a source of amino acids and beneficial bacteria.


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Introduction Today, there are many poultry farms in the country, where chickens, geese, turkeys and other bird species are bred in large quantities. Poultry products are meat, eggs, down and feathers. At the same time, the amount of generated waste (bird droppings) can often exceed the volume of the main production, reaching hundreds of tons per year. Therefore, the task of developing safe and waste-free technologies for obtaining poultry products, including organic waste in poultry complexes is urgent [1]. There are several ways to utilize organic poultry waste: they can be used in crop production as fertilizers, raw materials for feed additives, biofuels, as well as an additive for obtaining clean water from wastewater. Feed additives from processed raw materials and animal waste are not inferior to many feeds in nutritional value [2]. In addition, with bird droppings, up to 30...35% of undigested feed is released. Depending on keeping and feeding conditions, bird droppings can serve as a source of nutrients or as an environmental pollution factor [3]. The aim of the study was to develop production of probiotic feed additives from bird droppings processed using Bacillus subtilis bacteria strains. Materials and methods The research work was carried out at Yakutsk Poultry Factory and microbial drug development laboratory of Physics and Technology Center of Siberian Branch of Russian Academy of Sciences. Laying hens of Rodonit-3 cross from industrial herd of No. 18 workshop were studied. Raw poultry manure of chickens was used as material for obtaining a feed additive (powder) after a 10-day use of Nord-Bakt probiotic, 0.01 ml or 5 × 107 CFU per head, daily. For fermentation of fresh litter, Nord-Bakt was also used (equal combination of B. subtilis TNP-3 and B. subtilis TNP-5 bacterial strains containing 5 × 109 CFU/ml) at the rate of 1 ml per 100 g of litter and kept for 2 days at a temperature of 25...28 °C. Then, to obtain powder, litter was treated in a thermal way: passed through Ekorm-1-1600.000 extruder. During the movement, the litter was heated and pressed at a temperature of 100...120 °C for 5-6 seconds, then cooled and crushed. For experiments on testing the obtained feed additive, laying hens at the age of 14 months in the amount of 24 animals were selected. An experimental group of laying hens received 3.3% of the powder from the main diet. The control group received the main diet without powder. The content was similar, corresponding to zootechnical standards, drinking was grooved with free access. Microbiological studies of powdered samples and bird droppings were carried out for presence of bifidobacteria, enterococci, aerobic spore-forming bacteria, mesophilic aerobic facultative anaerobic microorganisms, enterobacteria, staphylococci, microscopic fungi according to the accepted standards [4-7]. Biochemical studies of powder and eggs were carried out on NIR SCANNER model 4250 infrared analyzer. The data were processed using the Snedecor program, Microsoft Excel, and Student's statistical processing. Results and discussion In litter micro biota of the experimental bird groups after a 10-day watering with Nord-Bakt probiotic, only representatives of intestinal normobiosis (lactoand bifidobacteria, enterococci, aerobic spore bacteria) were present, no pathogenic staphylococci, mold and toxigenic fungi were present, unlike litter of the control group of laying hens where the probiotic was not used. The study results confirm the data obtained by A.M. Stepanova [8], when NordBakt probiotic ensured microbiological survival of poultry waste. High-temperature drying allows to effectively neutralizing the litter from opportunistic and pathogenic bacteria, while maintaining useful elements. Upon receipt of the powder, we proceeded from the results of microbiological and biochemical studies. Microbiological studies analyzed normoflora survival: bifidobacteria, enterococci, spore-forming aerobic bacteria and absence of pathogenic microorganisms. As the results (Table 1) showed, the absence of opportunistic microorganisms and microscopic fungi in the powder provided the extruder drying mode at 100...120 °C for 5-6 s. Number of microorganisms in litter feed additive, CFU/g Table 1 Type of microorganisms Number of microorganisms, CFU/g Before heat treatment After heat treatment (extruder) TMC 4 7 ´ 10 4 4.8 ´ 10 Spore bacteria (Bacillus sp.) 4 8 ´ 10 5 1.2 ´ 10 Lactobacillus (Lactobacillus sp.) 5 1.9 ´ 10 - 1 10 3 10 2 1 ´ 10 - 6 10 - - Bifidobacteria (Bifidum sp.) +++ +++ 1 10 3 10 +++ + 6 10 +++ - Enterococci (Enterococcus sp) 5 1.7 ´ 10 3 3.2 ´ 10 Escherichia L + (Escherichia sp) 4 9.2 ´ 10 - Escherichia L- (Escherichia sp) - - Staphylococci (Staphylococcus sp) 4 6 ´ 10 - Yersinia (Yersinia sp) - - Microscopic fingi Yeasts - Designation. L+: Escherichia fermenting lactose; L-: Escherichia not fermenting lactose; -: lack of growth; +: single growth; +++: intensive growth. Litter fermented with B. subtilis strains (1 ml or 5 × 109 CFU per 100 g) and dried for 2 days at 25...28 °C before applying heat treatment contained a significant amount of bifidobacteria, lactobacilli up to 1.9 × 105 CFU/g, enterococcus - 1.7 × 105 CFU/g, spore-forming bacteria - 8.0 × 104 CFU/g, lactose-positive Escherichia - 9.2 × 104 CFU/g, staphylococcus - 6.0 × 104 CFU/g, also sporadic yeasts. The obtained feed additive (powder) after extrusion had sharply decreased TMC, bifidobacteria, enterococci; lactobacilli, lactose-positive escherichiae, staphylococci and yeasts disappeared, and increased the number of spore-forming aerobe Bacillus bacteria (up to 1.2 × 105 CFU/g), which were the basis of Nord-Bakt probiotic, used to obtain powder from bird droppings. Preparations based on B. subtilis are known to withstand heat and granulation [9, 10]. According to the results of biochemical studies, litter feed additive contains 18 free amino acids (table 2). The total concentration of free amino acids in the powder is 406.3 mg/kg, which is 1.7 times higher than in the litter without fermentation and extrusion. Free amino acids, getting into the blood, are involved in protein synthesis. A mixture of amino acids, unbound free proteins in animal feed increases their immune biological status, activates metabolism, improves appetite, digestibility of food and resistance to various diseases [2, 11, 12]. The inclusion of 3.3% of the powder in the diet does not have a negative effect on physiological state, vitality and productivity of laying hens. The survival of birds in both groups was 100%. Amino acid analysis of litter and powder samples Table 2 Amino acid, mg/kg Litter of chickens who took NordBakt probiotic with water (0.01 ml per bird) Litter after B. subtilis fermentation and drying for 2 days at 25...28 °C Powder after heat treatment through Ekorm 1.1600.000 extruder Proportion of the total number of amino acids in the feed additive, % Aspartic acid 22.1 ± 0.1 27.5 ± 1.1 45.1 ± 0.3*** 9.1 Threonine 11.9 ± 0.1 14.8 ± 0.6 24.4 ± 0.2*** 6.0 Serine 10.9 ± 0.1 13.1 ± 0.4 20.3 ± 0.1*** 4.9 Glutamic acid 29.4 ± 0.1 35.0 ± 1.1 53.4 ± 0.3*** 13.1 Proline 9.9 ± 0.1 11.8 ± 0.4 17.8 ± 0.1*** 4.4 Glycine 13.2 ± 0.1 15.7 ± 0.5 23.7 ± 0.1*** 5.8 Alanine 19.2 ± 0.1 21.0 ± 0.3 26.6 ± 0.1*** 6.5 Cysteine 0.4 ± 0.1 0.5 ± 0.0 1.0 ± 0.0*** 0.3 Valine 15.3 ± 0.1 18.5 ± 0.7 28.8 ± 0.2*** 7.0 Methionine 6.0 ± 0.1 7.3 ± 0.2 12.2 ± 0.1*** 3.0 Isoleucine 13.7 ± 0.1 16.7 ± 0.6 26.6 ± 0.2*** 6.5 Leucine 22.3 ± 0.1 25.1 ± 0.6 34.3 ± 0.2*** 8.4 Tyrosine 11.7 ± 0.1 12.9 ± 0.3 16.9 ± 0.22*** 4.2 Phenylalanine 13.4 ± 0.1 14.9 ± 0.3 19.8 ± 0.1*** 4.9 Ornithine 0.6 ± 0.1 0.7 ± 0.1 0.9 ± 0.1*** 0.2 Lysine 17.2 ± 0.1 19.1 ± 0.4 25.3 ± 0.1*** 6.2 Histidine 5.5 ± 0.1 6.6 ± 0.2 10.2 ± 0.1*** 2.5 Arginine - 16.0 ± 0.2 19.0 ± 0.1*** 4.7 Note. *** P > 0.001. Microflora of litter after feed additive application Table 3 Microorganisms The number of microorganisms, CFU/g Experimental group Control group TMC 5 1.2 ± 10 5 1.2 ± 10 Spore bacteria (Bacillus sp.) 5 2.0 ± 10 5 4.3 ± 10 Lactobacillus (Lactobacillus sp.) 5 4.5 ± 10 5 1.2 ± 10 Bifidobacteria (Bifidum sp.) +++ ++ 1 10 3 10 ++ + 6 10 + - Enterococci (Enterococcus sp) 4 8.2 ± 10 4 5.5 ± 10 Escherichia L + (Escherichia sp) 4 9 ± 10 3 5.1 ± 10 Escherichia L- (Escherichia sp) - 3 1.4 ± 10 Staphylococci (Staphylococcus sp) 4 9.1 ± 10 5 1.6 ± 10 Yersinia (Yersinia sp) - - Microscopic fingi - - Designation. L+: Escherichia fermenting lactose; L-: Escherichia not fermenting lactose; -: lack of growth; +: single growth; +++: intensive growth. Biochemical characteristics of chicken eggs after feeding with powder Table 4 Elements Experimental group Control group Yolk White Yolk White Water, % 7.8 ± 0.1* 14.9 ± 0.1* 7.6 ± 0.1 14.5 ± 0.3 Protein, % 38.4 ± 0.1* 81.3 ± 0.4* 38.1 ± 0.2 79.9 ± 0.8 Fat, % 55.7 ± 0.1* 5.4 ± 0.2* 55.6 ± 0.2 4.9 ± 0.3 Carbohydrate, % 8.5 ± 0.1** 9.6 ± 0.1* 8.2 ± 0.1 9.2 ± 0.3 Ash, % 26.9 ± 17.2* 8.3 ± 0.1* 6.7 ± 0.1 7.9 ± 0.3 Sodium, mg % 171.6 ± 1.4* 1071.0 ± 7.3* 168.3 ± 1.5 1048.9 ± 14.0 Potassium, mg % 189.9 ± 1.4 2.2 ± 0.01* 186.7 ± 1.4 2.1 ± 0.1 Calcium, mg % 277.2 ± 0.4* 79.3 ± 0.4* 276.3 ± 0.3 77.9 ± 0.8 Magnesium, mg % 34.9 ± 0.2* 87.5 ± 0.4* 34.4 ± 0.2 86.4 ± 0.7 Phosphorus, g/100 g 1.2 ± 0.01 248.7 ± 2.3** 1.2 ± 0.01 241.6 ± 4.5 Iron, mg % 29.6 ± 0.3 11.7 ± 0.2* 29.0 ± 0.3 11.0 ± 0.4 Vitamin A, mg % 2.6 ± 0.01 - 2.6 ± 0.01 - Vitamin B1, mg % 0.5 ± 0.01 - 0.5 ± 0.01 - Vitamin B2, mg % 0.8 ± 0.01* 6.7 ± 0.1 0.8 ± 0.01 6.3 ± 0.2 Note. *P < 0.05; **P > 0.05. Despite the absence and small number of beneficial microflora in the powder, chickens of the experimental group (Table 3) showed a higher content of lactoand bifidobacteria in the intestinal microbiota, and the absence of lactose-negative escherichia compared to the control chickens, which received the full main feed ration without feed additives. The absence of opportunistic microorganisms, microscopic fungi, the presence of bifidobacteria and Bacillus spore-forming aerobic bacteria in the powder, predominance of beneficial normoflora and the absence of potential enteropathogens in the experimental group of chickens after its use (compared with the control) result in probiotic properties of the feed additive obtained by microbiological synthesis from bird droppings. The data obtained are consistent with the results of studies on the use of feed additives based on B. subtilis, Bac. licheniformis [13-18]. The results of egg biochemical study (Table 4) indicate that the use of powder (up to 3.3% of the main ration for laying hens) significantly increases content of main microand macro-elements in eggs, compared with the control. At the same time, egg quality does not decrease. Thus, the litter obtained by litter extrusion after watering with the Nord-Bakt probiotic and subsequent fermentation with B. subtilis TNP-3 and 1. subtilis TNP-5 is promising as a feed additive containing amino acids and beneficial microorganisms. Conclusions 1. The drying mode for litter feed additive with an extruder at 100...120 °C for 5-6 s ensures destruction of opportunistic pathogenic microorganisms and microscopic fungi. 2. TMC sharply decreased in powder after extrusion, the number of bifidobacteria, enterococci, lactobacilli, lactose-positive escherichia, staphylococcus, and yeast completely disappeared, but the number of spore-forming aerobic Bacillus bacteria increased (1.2 × 105 CFU/g), which are the basis of Nord-Bakt probiotic used to obtain powder from birds litter. 3. The litter contains 18 free amino acids. The total concentration of free amino acids in the powder was 406.3 mg/kg, which was 1.7 times higher than in the litter without fermentation and extrusion. 4. The inclusion of 3.3% of the feed additive in the bird ration did not adversely affect the physiological state, viability and productivity of laying hens, significantly increased the content of the main micro and macro elements in the egg. 5. The powder obtained by extruding bird droppings after watering with the NordBakt probiotic and fermenting with B. subtilis TNP-3 and B. subtilis TNP-5 strains can be added as a source of amino acids and useful bacteria to the ration of laying hens.

About the authors

Anna M Stepanova

Yakut Scientific Research Institute of Agriculture

Author for correspondence.
Email: stepanova_anna1985@mail.ru
Yakutsk, Russian Federation

Candidate of Veterinary Sciences, senior researcher, Laboratory for development of microbial preparations

Nadezhda P Tarabukina

Yakut Scientific Research Institute of Agriculture

Email: hotubact@mail.ru
Yakutsk, Russian Federation

Doctor of Veterinary Sciences, Professor, Head of Laboratory for development of microbial preparations

Marfa P Scryabina

Yakut Scientific Research Institute of Agriculture

Email: hotubact@mail.ru
Yakutsk, Russian Federation

Candidate of Veterinary Sciences, leading research associate, Laboratory for development of microbial preparations

Mikhail P Neustroev

Yakut Scientific Research Institute of Agriculture

Email: mneyc@mail.ru
Yakutsk, Russian Federation

Doctor of Veterinary Sciences, Professor, Head of Laboratory of veterinary biotechnology

Svetlana I Parnikova

Yakut Scientific Research Institute of Agriculture

Email: hotubact@mail.ru
Yakutsk, Russian Federation

candidate of veterinary sciences, senior researcher, Laboratory for development of microbial preparations

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Copyright (c) 2019 Stepanova A.M., Tarabukina N.P., Scryabina M.P., Neustroev M.P., Parnikova S.I.

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