PN-III-P1-1.2-PCCDI-2017-0473

Use of agro-food residues by feeding solutions which to control the feed contaminants

Context: The recent climate changes and the industrial activity increased the risk of contamination of the cereal grains and forages with various types of contaminants (ranging from heavy metals, pesticides, organofluorine compounds to microorganisms, fungi, mycotoxins, etc.).
The adverse effects of these contaminants can be controlled by many products available on the market, but their high cost increases the total cost of feeding. Hence, the identification of cheaper solutions is a must. The agro-food residues are important sources of bioactive compounds (polyphenols, polyunsaturated fatty acids, fibre, vitamins, minerals, etc.) which might alleviate the effects of contaminants by direct physical adsorption or by other mechanisms (biotransformation/immunostimulation). Several preliminary in vitro studies have substantiated this fact.

Project objective is to develop new formulations and new feed products enriched in bioactive compounds which to alleviate the effects of contaminants in farm animals by recycling the agro-food wastes (RA). Their use can have multiple effects: depress forage contamination, improve farm animals health, avoid pollution with residues and environmental protection, economic growth by decreasing the cost of farming (purchase of decontaminated products) etc.

Foreseen activities. The project consists of 4 stages and16 activities aiming the screening and selection of residues with a high potential of adsorption and control of the contaminants, mycotoxins particularly, by in vitro and in vivo experiments and by substantiating the efficiency of the feed products before their patenting. A feed additive and a compound feed for pigs will be developed, rich in bioactive compounds, which to control the dietary contaminants.

 

Main results of the project

 

Procurement and physic-chemical characterization of agri-food residues with decontaminating potential

 

Vegetable and fruit waste is an important source of antioxidant compounds, organic acids, vitamins, carbohydrates, minerals and fiber. The project selected vegetable and fruit wastes for use as potential decontaminants for mycotoxins in animal feed. The waste tested was shell and pulp residues from three varieties of apple (Delicious Red Apple, Golden Apple, Granny Smith Apple), Carrot, Celery and Beet, as well as Potato Peel, Watermelon, Watermelon and a Mash of grape seeds. The characterization of these residues was done by the component project coordinator 1 (IBNA) and the UBB partner. Freeze-dried samples of apple, carrot, celery, beet, potato peel, melon and grape seed meal were analyzed for the raw chemical composition and a great diversity was observed in terms of protein, fat, mineral and fiber content. The analyzed samples were not contaminated with mycotoxins. In these samples were analyzed bioactive compounds from waste as polyphenols, phenolic acids, vitamin C, organic acids and carbohydrates. Due to the presence of these biologically active compounds, the studied samples represent valuable by-products, with potential application to the decontamination of feed contaminated with mycotoxins. Next, the evaluation of the adsorbent effect of mycotoxins for agri-food residues that were characterized in previous activities in terms of their ability to reduce mycotoxin contamination was performed. Two mycotoxins (aflatoxin and zearalenone), which frequently contaminate cereals in Romania, were chosen for promising residue / residue selection in terms of decontamination capacity. The potato peel demonstrated the highest absorption capacity for both AFB1 and ZEA, both in terms of incubation time and the amount of residue. Residues of apples, carrots, beets and celery had a lower capacity for adsorption of mycotoxins. Regardless of the type of residue there is a small variation of the adsorption regardless of the type of pH in terms of the binding capacity of AFB1. In contrast, the binding capacity of ZEA seems to be more affected by pH variations, the toxin adsorption increasing with pH in the case of potato peels or decreasing as the pH becomes more basic (celery, carrot, grape seeds meal, Golden apple).For docking studies, a three-dimensional structure was generated using CarbBuilder 2.1.17 software which was visually analyzed to identify potential steric conflicts and subjected to a minimization step, using the non-reactive force field UFF, integrated in ArgusLab. Docking studies have shown that AFB1 or ZEA do not appear to have a preferential affinity for certain monosaccharide units or conformations.

 

Development of an in vivo experimental model in pigs to investigate and test the efficiency and functionality of the products obtained: feed additive and feed combined with decontaminating potential enriched in bioactive compounds

 At this stage we analyzed the anti-mycotoxic effect of some residues from the food industry (sea buckthorn seed meal and grape seed meal) in in vivo and in vitro experiments in pigs.  The results obtained in the in vivo experiments showed: - a significant increase in the inflammatory response in the liver as a result of aflatoxin B1 (AFB1) intoxication for inflammatory cytokines: IL-1 beta, IL-6, TNF-α, IL-17A, GM-CSF and chemokine expression: CXCL- 8, CCL-1, CCL-2, CCL-4, CCL-5, CCL-10 and CCL-24 -an increase in gene expression for CYP4A24 and GSTA1 induced in the liver by simultaneous intoxication with AFB1 + OTA (ochratoxin) -grape seed meal caused a significant decrease in aflatoxin-induced inflammatory response -in lymph nodes, AFB1 resulted in a significant decrease in catalase (CAT, superoxide dismutase (SOD) activity of glutathione peroxidase (GPx) and a significant increase in lipid peroxidation -the antioxidant activity affected by AFB1 intoxication was restored in the lymph nodes of AFB1 intoxicated weaned piglets who were given combined fodder containing grape seed meal< -in the spleen, a decrease in catalase activity was observed in the spleen associated with an increase in SOD and TCA and TBARS following AFB1 intoxication -concomitant administration of the rat with AFB1 resulted in a restoration of the parameters affected by AFB1 at  the level of control in the spleen of weaned piglets

 

Viral model development; in vitro testing of the efficacy of residue extracts in case of viral and mycotoxic co-contamination (continuation from 2019) The antiviral effect of grape extract (GE) in the presence or absence of AFB1 on bovine diarrhea virus (BVDV) in the abiotic system or in MDBK cell cultures (Madine Darby Bovine Kidney) was analyzed. GE had a strong antiviral effect (90% inhibition), statistically significant, when administered post-infection, suggesting a post-internalization mechanism of action, most likely in viral replication.In cell viability studies, the results obtained show a possible protective role of GE, against the harmful effect of AFB 1, quite low (15-20%), but statistically significant, regardless of the dilution of GE used, when the extract is administered as a method of prevention (treatment 24 hours before exposure to AFB1). The data also suggest that the molecular mechanisms by which cell toxicity is induced following exposure to AFB may be different from those induced by cytopathic BVDV infection, at least in MDBK cells. 

 

Analytical studies (-omics) to identify hepatotoxic and nephrotoxic effects induced by contaminants and the ability to counteract residues (toxicogenomics; toxicoproteomics, toxicometabolomics)  Our studies in piglets exposed or not to mycotoxins showed changes in protein expression depending on the diet. In the case of the liver there was an increase in protein expression for CYP2E1, CYP3A4 and GSTA1, in the group where the feed was supplemented only with a mixture of mycotoxins (E2) compared to the control group, but for CYP1A2 the effect is completely opposite. At the level of the kidney the most significant changes was observed for piglets that were fed a diet contaminated with mycotoxins and grape seed and consisted in a re-establishment of the expression of CYP1A2, CYP2E1 and GSTA1 towards the control level compared to the group of piglets exposed to mycotoxins. At the same time, as in the case of the liver, there is an increase in protein expression for CYP3A4 in the group of piglets that consumed a diet contaminated with mycotoxins compared to the control group. In addition, GST-specific activity confirms the level of protein expression observed for GSTA1. 

 

Verification of the decontamination potential of the residues in conditions of simultaneous fungal (mycotoxin), viral and microbial co-contamination (in vitro simulation model). 

The results obtained in in vitro experiments showed that:

 - grape seed extract has a strong antiviral effect in vitro in MDBK bovine cell cultures, in the presence and absence of AFB1 contamination

- grape seed extract was most effective in inhibiting BVDV virus (95% inhibition)

- AFB1 induced a moderate inhibition of BVDV virus production of up to 50%

-  the extracts determined the inhibition of the growth of pathogenic bacteria having a similar inhibitory activity for the inhibition of the growth of aureus and E. fecalis;

- grape seed extract has been very effective in inhibiting the combined inflammatory action of toxins (ZEA or OTA) and bacteria (LPS), inhibiting the synthesis of inflammatory cytokines: IL-6, TNF-alpha, IL-1beta

-for sea buckthorn meal the effect was moderately anti-inflammatory; only the grape seed extract caused a significant increase in antioxidant capacity.  

 

Validation of the efficacy of the feed product obtained by using an experimental model of toxicokinetics in an experimental farm The experiment was performed in the IBNA experimental farm on 40 weaned TOPIG hybrid piglets (10 piglets / lot) with an average initial weight of 9.13 ± 0.05 kg, for 30 days. After weaning (at 30 days) the piglets were acclimatized in the experimental hall for one week before being used in the experimental protocol and were treated with colistin (3 days) to prevent weaning stress. The animals were divided into four groups: Control, ochratoxin A (OTA), grinding mixture - sea buckthorn grinding + grape seed grinding (SSC), ochratoxin A + grinding mixture (SSC + OTA). At the end of the experiment (30 days) blood and organ samples (liver and kidney) were collected for analytical determinations.Ingestion of NC contamination with OTA for 30 days resulted in an increase in OTA concentration of 14.81 times compared to the OTA concentration in the liver of control animals. It is important to note that the administration of the feed that contained the mixture of grits led to a reduction in the concentration of OTA by 72% compared to the concentration of OTA determined in the liver of intoxicated piglets. As expected, the concentration of OTA in the liver of piglets in the SSC group had a level similar to that identified in the control group. Administration of OTA-contaminated compound feed for 30 days resulted in a 46-fold increase in OTA from the kidney in control animals. The concentration of OTA in the kidneys of piglets in the SSC group was similar to that identified in the control group. As with the liver, administration of feed containing the mixture of grits resulted in a reduction of the OTA concentration by 91.3% compared to the concentration determined in the kidneys of the piglets intoxicated with OTA.These results suggest that the mixture of grape meal and sea buckthorn meal as well as the combined feed including these by-products is an effective alternative in counteracting the adverse effects of food contaminated with ochratoxin by reducing toxin residues in the liver and kidneys, key organs in metabolic processes of animals. Also the fodder combined with the mixture of grits has the ability to improve the immune status through antibodies. Given that only imported products are used to reduce the effects of mycotoxins on farms, the product resulting from this project is a Romanian product, with high efficiency and low cost price that can be used to feed piglets during weaning. 

 

Activity 4-1-4: Development of a patent application for patenting: feed additive / a new feed recipe based on agri-food residues; partners involved (CO-IBNA, P2-UBB, P3-IBAR); In this activity, a patent application was developed (A / 00689/2020) for a new recipe for compound feed including a mixture of agri-food residues (grape seed meal and sea buckthorn meal) with the effect of reducing the concentration of mycotoxins from organs (liver and kidneys) in pigs. The recipe was transferred to the IBNA development sector and to private farms (AgroLaig Prod Serv SRL).

 

Results dissemination

 ISI Papers

1. Marin D. E., Bulgaru C. V., Anghel C. A., Pistol G. C., Dore M. I., Palade M. L, Taranu I. Grape seed waste counteracts the aflatoxin B1 toxicity in piglets mesenteric lymph nodes. Toxins, 2020 12(12), 800; 10.3390/toxins12120800
2. Taranu I., Hermenean A., Bulgaru C, Pistol G.C, Ciceu A., Grosu I.A, Marin D. E. 2020. Diet containing grape seed meal by-product counteracts AFB1 toxicity in liver of pig after weaning. Ecotoxicology and Environmental Safety 203 (2020) 110899
3. Palade L. M., Dore M. I., Marin D.E , Rotar M. C. Assessment of food by-products potential for simultaneous Aflatoxin B1 and Zearalenone binding. Toxins. 2020 Taranu I. Toxins (Toxins, 2021 13(1), 2; https://doi.org/10.3390/toxins13010002)
4. Marin DE., Pistol G. C., Bulgaru C. V., Taranu I. Cytotoxic and inflammatory effects of individual and combined exposure of HepG2 cells to zearalenone and its metabolites. Naunyn-Schmiedeberg's Archives of Pharmacology. 2019 Aug;392(8):937-947. doi: 10.1007/s00210-019-01644-z. Epub 2019 Mar 27.
5. Grosu IA, Pistol GC, Taranu I, Marin DE. The Impact of Dietary Grape Seed Meal on Healthy and Aflatoxin B1 Afflicted Microbiota of Pigs after Weaning.Toxins (Basel). 2019 Jan 8;11(1). pii: E25. doi: 10.3390/toxins11010025.
6. Marin D.E., Braicu C., Dumitrescu G., Pistol G. C., Cojocneanu R., Berindan Neagoe I., Taranu I. MicroRNA profiling in kidney in pigs fed ochratoxin A contaminated diet. Ecotoxicology and Environmental Safety 184 (2019) 109637 https://doi.org/10.1016/j.ecoenv.2019.109637
7. Popescu R.G., Bulgaru C., Untea A. , Vlassa M., Filip M., Dinischiotu A., Georgescu S.E. Antioxidants The effectiveness of dietary by-product antioxidants on induced CYP genes expression in piglets fed aflatoxin B1 contaminated diet Toxins, Volume: 13, Issue: 2, Article Number: 148, DOI: 10.3390/toxins13020148
8. Marin D. E., Bulgaru C. V., Anghel C. A., Pistol G. C., Dore M. I., Palade M. L.and Taranu I.. Grape seed waste counteracts the aflatoxin B1 toxicity in piglets mesenteric lymph nodes.Toxins (Basel). 2020 Dec; 12(12): 800.Published online 2020 Dec 15. doi: 10.3390/toxins12120800
9. Taranu I., Hermenean A., Bulgaru C., Pistol GC, Ciceu A., Grosu A. I, Marin D.E. Diet containing grape seed meal by-product counteracts AFB1 toxicity in liver of pig after weaning. Ecotoxicology and Environmental Safety. 203 (2020) 110899.
10. Bulgaru CV, Marin DE, Pistol GC, Taranu I. Zearalenone and the Immune Response. Toxins, 2021 13(4):248; https://doi.org/10.3390/toxins13040248

BDI Papers

1. Marin D. E., Pistol G. C., Gras M., Palade M., Taranu,I. 2018. Effect of Cereal Contaminants on the Inflammation and Oxidative Stress in the Gut of Weanling Piglets Scientific Papers: Animal Science and Biotechnologies, Vol 51, No 1, pp38-42
2. Daniela Marin, Gina Pistol, Mihai Gras, Cătălin Rotar, Ionelia Taranu. 2019.Role of probiotics in counteracting the effect of usual feed contaminants in swine. Scientific Papers-Animal Science Series: Lucrări Ştiinţifice - Seria Zootehnie, vol. 72, 155-160.
3. Marin D.E, Pistol G, Taranu I. 2020. Cytotoxic effects of individual and binary combinations of zearalenone and ochratoxin a on liver. Archiva Zootechnica, vol 23, I, 73-80.
4. Palade LM, Pertea AM, Taranu I. Response of antioxidant status in kidney of pigs fed aflatoxin B1 contaminated diets to grape seed meal supplementation. Archiva Zootechnica vol. 24, nr. 1, pp. 17-31, 2021

International Conferences

1. Palade, Laurentiu Mihai; Pertea, Ana-Maria; Marin, Daniela Eliza; Grosu, Iulian Alexandru; Stancu, Mariana; Taranu, Ionelia. Comparative efficacy of agricultural byproducts in sequestering aflatoxin B1 and zearalenone: grape seed and seabuckthorn meals. 13th World Congress on Polyphenols Applications. Malta, September 30 - October 1, 2019.
2.  Marin D.E. Palade M.L; Pistol G.C., Taranu I. Effect of concomitant exposure to zearalenone and soybean of CaCo2 cells. 41th Mycotoxin Workshop 2019, 6-8 Mai, Lisbon, Portugal.
3. Palade L.M., Marin D.E., Grosu I. A., Pistol G.C, Taranu I. Preliminary assessment of fiber and polyphenol rich food-derived residues: aflatoxin B1 and zearalenone binding potential. 12th World Congress on Polyphenols Applications: Bonn Polyphenols University of Bonn, Germany, 25 -28.09.2018
4. Marin D.E, Pistol G.C, Taranu I. Individual and combined cytotoxic effects of co-occurring mycotoxins of zearalenone family on HepG2 cells. 40th Mycotoxin Workshop, Munchen, Germany, 11-13.06.2018
5. Marin DE, Palade M, Pistol GC, Taranu I. Effect Of Concomitant Exposure To Zearalenone And Soybean Of Caco2 Cells. 6-8 May, 41st Mycotoxin Workshop, Lisbon, 2019.
6. Vlassa M, Miuța Filip, Virginia Coman, Daniela Marin, Ionelia Țăranu, HPLC characterization of some bioactive compounds from agro-food residues, 25th International Symposium on Separation Sciences Lodz, Poland, 15-18 September 2019.
7. Vlassa M, Miuța Filip, Perhaița Ioana, Prodan Doina, Borodi Gheorghe, Daniela Marin, Ionelia Țăranu Some freeze dried fibre from agro-food residues characterization by physico-chemical, thermal and spectroscopic methods. 15th International Symposium of Animal Biology and Nutrition organized by the National Research-Development Institute for Animal Biology and Nutrition - IBNA Balotești- September 27, 2019 .
8. Vlassa M, Miuța Filip, Perhaița Ioana, Prodan Doina, Borodi Gheorghe, Daniela Marin, Ionelia Țăranu . Physico-chemical, thermal and spectroscopic characterization of some freeze-dried fruit and vegetable waste, used as feed supplement for the reduction of the toxic effect of aflatoxin B1 in swine. The 33rd EFFoST International Conference: Sustainable Food Systems - Performing by Connecting, Wageningen University & Research , Rotterdam, The Netherlands, 12-14 November 2019.
9. Popescu RG, Marin D, Țăranu I, Georgescu S. E., Dinischiotu A Prevention of combined effects of aflatoxin B1 and ochratoxin A by dietary byproduct antioxidants on protein expression of cytochrome P450 enzymes in piglets liver and kidney.42nd Mycotoxin Workshop, Munchen, 2021

Checks:

In PC1 was used in the second year of the project (2019) a type C mobility check for a young researcher newly hired to learn new microbiology techniques.

In PC1 was used in the third year of the project (2020) a type C mobility check for a young researcher newly hired to learn new microbiology techniques.

 

 

 

Obtaining of feeding products based on unconventional resources (rich in protein) generated of modern technology procedure from food industry and biotechnology.

 

Objective: obtaining and evaluation of an animals feeding product which consist on two components: 1. compound feed formula including vegetable ingredients alternatives to soybean meal; 2. Bacterial additive

Phase’s task:

The following tasks are done in order to realize the objective of the project:

1. Identification of oleo - protein rich resources less known and their nutritional characterization.
2. Formulation and obtaining in lab conditions of a probiotic product (bacterial strains phenotypic selected according to probiotic activity; assessment of stability in the animal diets after micro encapsulated through atomization technique). Testing in vitro and in vivo on animal feeding after incorporated in diets.
3. Obtaining and validation through animal and poultry test of probiotic product after dietary addition
4. Dissemination activity

 

Phase I/2018

Identification of vegetable resources rich oleo-proteaginous less known and their nutritional characterization.

Objective: Providing scientific and technical support as a starting point in the effective implementation of the project development strategy.

ACTIVITIES:

• Activity 1-1-2. The nutritive value of certain local available oleo-protein resources was done: peas, sorghum, soybean, high-protein level sunflower. It was established the level of dietary incorporation.
• Activity 1-2-2. Analytical studies with respect to phenotypic and molecular characterization and genetic confirmation of some bacterial strains.  Realization of a catalogue for bacterial strains isolated from pigs and chickens’ intestinal content (ileum and cecum).

The strains were macroscopic analysed both from solid and liquids medium.  The bacterial strains were analysed macroscopically aiming to determine characters such as morphology, texture, type colony, the colony contour, presence/absence of pigments.

For checking the purity, morphological characters of isolated strains were analysed by Gram staining method. Biochemical characters were performed using catalase test.  It was investigated the carbohydrates fermentation by identification of genus and bacterial species and molecular identification and investigation (qPCR, gene sequence analysis

 16S ARNr, CO I, cpn 60) of probiotics characters of phenotypic characterization strains.  

• Activity 1.3.2. Molecular identification and investigation (qPCR, 16S ARNr, CO I, cpn 60) of probiotics characters of phenotypic characterization strains.

• Activity 1.4.2. Obtaining of a bacterial product based on Bacillus subtilis strain (concentration- 1.6 x 10^9 UFC/ml culture). Establish of experimental protocol Preliminary in vitro studies for probiotic some probiotic character assessment.

RESULTS:

• Technic –scientifically support needs for compound feed formulation / species.

• Catalogue of bacterial strains of Lactobacillus phenotypic characterized and isolated from chicken and pig’s intestinal content.

• Bacterial products based on Bacillus subtilis (concentration 1,6 x 10^9 UFC/ml culture), phenotypic characterized and molecular validated.

• Tables of nutritive values of: peas, sorghum, soybean, high-protein level sunflower.

• Compound feed formulas for piglets.

Communication:

• Lavinia Idriceanu. Researches on in vitro and in vivo digestibility assessment of nutrients on pigs. IBNA Balotești, final internship paper, 2018.

Check used:

In PC2 were used in 2018, 2 mobility checks C type at USV Suceava for training in organoleptic quality of meat.

 

 Phase II/2019

Activity 2.1.2. 

• Obtaining and characterization of bacterial strain-based product with probiotic action.
• Cultivation of bacterial strains selected and microencapsulated by atomization technique.

Activity 2.2.2. Establish effects of feed product on growth performances and nutritional quality of pig’s meat and poultry. 

In phase 2 was obtained, characterized, tested in vitro and validated o complex feeding product based on compound feed (poultry and pigs) plus microbial products based on bacterial strains of Lactobacillus.

Microbial product microencapsulated allows preservation of viable microorganisms in higher concentration for a long period (6 - 12 month).

The studies conducted in this phase led to obtaining of important results from growth parameters point of view and regarding meat quality as well.

2 papers were published, 4 in review process, 1 oral communication and 5 poster presentation and 2 patents applications.

 Dissemination:

BDI papers:

1. Grigore D.M, Ciurescu G., Idriceanu I., Mironeasa S., Iuga M, Băbeanu N Effect of low-fiber sunflower meal and phytase addition on broiler carcass traits, and meat quality. Archiva Zootehnică 22(1), 32-47. 2019
2. Dumitru Mihaela, Sorescu Ionuț, Habeanu Mihaela, Tabuc Cristina, Jurcoane Stefana Preliminary characterization in vitro of Bacillus licheniformis strain for used as a dietary probiotic. Scientific Bulletin. Series F. Biotechnologies, Vol. 23, 164-172. ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

 Conferences:

1. Târnoveanu Emanuel, Lefter Nicoleta A., Gheorghe Anca, Hăbeanu Mihaela Dietary effects of some not conventional feed ingredients on plasma lipoprotein profile in growing fattening pigs The 15th International Symposium of Animal Biology and Nutrition organized by the National Research-Development Institute for Animal Biology and Nutrition 27 sept, 2019, Balotesti, Romania
2. Târnoveanu Emanuel, Dumitru Mihaela, Hăbeanu Mihaela Changes in the gut microbiota of broiler chickens after Lactobacillus sp. probiotic treatments: a mini-review„ International Scientific Symposium „Modern animal husbandry - Food safety and durable development” Faculty of Animal Sciences, University of Agricultural Sciences and Veterinary Medicine, October 17 - 18, 2019, Iasi Romania
3. Dumitru Mihaela, Mihaela Hăbeanu, Ionuț Sorescu, Cristina Tabuc. Effects of Bacillus subtilis use as a dietary probiotic in weaning piglets. European Biotechnology Congress, 11-13 apr 2019, Valencia, Spania.
4. Dumitru Mihaela, Sorescu Ionuț, Ciurescu Georgeta, Tabuc Cristina, Hăbeanu Mihaela, Chelaru Nicoleta-Raluca In vitro probiotic properties of a lactic acid bacteria isolated from a broiler chicken. Symposium " Modern animal husbandry - Food safety and durable development ", Faculty of Animal Sciences, University of Agricultural Sciences and Veterinary Medicine, October 17 - 18, 2019, Iasi Romania
5. Raluca Chelaru, Mihaela Dumitru, Ionut Sorescu, Mihaela Habeanu In vitro evaluation of probiotic properties of a lactic acid bacteria isolated from piglet. International Scientific Symposium „Modern animal husbandry - Food safety and durable development” Faculty of Animal Sciences, University of Agricultural Sciences and Veterinary Medicine, October 17 - 18, 2019, Iasi Romania

 Patents application OSIM:

1. Dumitru M, Hăbeanu M., Lefter N., Sorescu I., Tabuc C., Gheorghe A., Idriceanu L. Nutreț combinat îmbunătățit prin adaos de aditiv zootehnic bacterian pentru purcei în criza de înțărcare OSIM cerere A/00245 /19.04.2019
2. Gheorghe Anca, Dumitru Mihaela, Ciurescu Georgeta, Lefter Nicoleta, Hăbeanu Mihaela, Tabuc Cristina, Sorescu Ionuț. Nutreț combinat îmbunătățit prin adaos de probiotic pentru furajarea puilor de carne. Cerere de brevet nr. A/00770 /20.11.2019

 

Phase 3/2020
The effects of bacterial product on the gut intestinal microbiota in chickens and piglets and sensory quality as well

Objective: Assessment of effects of bacterial products on gut intestinal microbiota in chickens and piglets and sensory quality as well

Obtaining of microencapsulated bacterial product.

1. Incorporation in compound feed.
2. Testing on suckling and weaning piglets.
3. Validation of the complex product.

 Summary

Activity 3.1.2. Evaluation of the meat sensory quality

 # USAMV Cluj – study about piglets and chicken’s microflora characteristics.

The inhibitory effect of a probiotic strain (Lactobacillus plantarum ATCC 8014) on a potentially pathogenic strain (Escherichia coli ATCC 25922) was tested at laboratory level. For this, the probiotic strain of L. plantarum was co-incubated with the enterotoxigenic strain E. coli for 24, 48 and 72h at a temperature of 37oC. Groups of different concentrations of co-cultures were made, thus following the viability of the two strains studied.

Results:

• Group I - 109 CFU / mL plantarum was co-incubated with 107 E. coli: 1) a number of 130x107 CFU / mL for E. coli after 24 h. 2) after incubation for 48 hours, a number of 200x107 CFU / mL;3) after 72 hours a number of 350x107 CFU / mL for E. coli.
• Group II - 109 CFU / mL plantarum with 105 E. coli were co-incubated: 1) a number of 28x105 CFU / mL for E. coli resulted after 24 h. 2) Within the same group, after incubation for 48 hours resulted a number of 30x105 CFU / mL 3) after 72 hours resulted a number of 25x105 CFU / mL for E. coli.
• Group III: after 24h of co-incubation of the two strains were obtained 20x105 CFU / mL, 25x105 CFU / mL after 48h, and 20x105 CFU / mL after 72h.
• In the case of group IV, 1x104 CFU / mL was obtained after 24h, 2x104 CFU / mL after 48h, and 0 CFU / mL after 72h.
• Following the results obtained in the case of the four groups formed, it can be highlighted that the plantarum strain ATCC 8014 with a concentration of 109 CFU / mL has the ability to significantly reduce the development of E. coli strain ATCC 25922 at lower concentrations (107, 105, 104), and after a longer incubation time (48h, 72h).

# INCDBNA Balotesti: in vitro testing for some probiotic properties in Lactobacillus sp. for microencapsulation product; phenotypic and genotypic characteristics were established also (BLAST analysis). A number of 14 bacterial strains of Lactobacillus sp. were isolated and identified from intestinal contents from piglets. Preliminary tests were performed on their viability, pH resistance (2 and 3), bile salts (0.3% ox gall), antibiotics and tolerance to a high temperature level.

The results obtained:

• 14 strains were phenotypically characterized, of which 2 were selected for microencapsulation, molecularly confirmed (BLAST Analysis): Lactobacillus acidophilus, IBNA 76 and Lactobacillus plantarum 1, IBNA 84.

1. Lactobacillus acidophilus, IBNA 76:

Cultural: small, opaque, whitish colonies; very low turbidity, inhomogeneous, with suspended flakes, abundant deposit, flakes.

Morphological: long or medium Gram-positive bacillus, thick or medium, with 1-2 metachromatic granules per cell, grouped in filaments

Biochemical:

Results:

• 14 strains were phenotypic characterized from which 2 were selected for microencapsulation, genetic validated (Analyse BLAST): Lactobacillus acidophilus, IBNA 76 and Lactobacillus plantarum 1, IBNA 84.

1. Lactobacillus acidophilus IBNA 76:

• Cultural: small, opaque, whitish colonies; very low turbidity, inhomogeneous, with suspended flakes, abundant deposit, flakes.
• Morphological: long or medium Gram-positive bacillus, thick or medium, with 1-2 metachromatic granules per cell, grouped in filaments.
• Biochemical:

• API 50 CHL: acidophilus 3, -90,1;
• ABIS online: 3. Taiwanensis - 82,7; 4. L. lindneri, -82,7; Targeted taxon L. acidophilus -75,8 CFU/mL: 2 x 10⁹

1. Lactobacillus plantarum 1, IBNA 84.

• Cultural: large, opaque, whitish colonies, type S; medium / intense turbidity, homogeneous, abundant deposit, powdery.
• Morphological: Gram-positive bacillus short, thin, rarely cocobacillus, arranged in irregular piles.
• Biochemical:

• API 50 CHL: plantarum 1, -85,4;
• ABIS online: 1. nantensis, -92,1; 2. L. plantarum, -90,5 CFU/mL: 9,7 x 10¹⁰

# In order to obtain a microencapsulated probiotic product in powder form, from a total of 14 strains of Lactobacillus sp. isolated, confirmed and evaluated for the degree of survival along the animal gastrointestinal tract, 2 lactic acid strains (Lactobacillus acidophilus IBNA 76 and Lactobacillus plantarum IBNA 84) were selected and subjected to the microencapsulation process, strains that recorded a high accumulation of biomass following the fermentation process (EPPENDORF bioreactor with a capacity of 5L) in compliance with the optimal growth conditions (37 ° C, 24 ± 2 h, 220 rpm, pH 6.4).

# A training stage was done at USAMV Cluj in order to obtain micro-encapsulated bacterial product.

Results:

• It was obtained and optimise 2 bacterial products based on Lactobacillus sp. (Lactobacillus acidophilus IBNA 76, LA and Lactobacillus plantarum IBNA 84, LP).
• Incorporation of LA 1 x 10⁸ UFC/gram in suckling piglets’ diets improved numbers of Lactobacillus sp. In LA group compared to M group:

• At ileum level >6.68%,
• At cecum level > 4.89%.

• A supplement of the suckling diet with additive based on Lactobacillus acidophilus IBNA 76, led to a significant decrease of coliform, clostridia and enterococci, with a positive inhibition on E. coli β-haemolytic colonies, compared to the control group where the degree of multiplication of pathogenic bacteria was very high.

# A biological test was conducted at INCDBNA Balotești for evaluation the effects of products on piglets. The piglets were distributed randomly in  4 groups: 1) control diet (C diet), with by-products associated (mustard meal, hemp meal, hemp oil) + soybean meal 2) experimental LA, where C diet was supplemented with bacterial product microencapsulate based on Lactobacillus acidophilus-1%; 3) experimental LP, where C diet was supplemented with bacterial product microencapsulate based on Lactobacillus plantarum-0,1%; 4) experimental LA:LP, where C diet was supplemented with bacterial product microencapsulate based on mix of Lactobacillus acidophilus: Lactobacillus plantarum-1,1%.

Results:

• The administration of compound feed with additive added to the experimental groups led to an increase of average daily gain and to an average daily feed intake similar to group C. The feed efficiency (kg feed / kg gain), correlated with the average daily feed intake and weight gain recorded comparable values ​​between groups.
• By using micro-encapsulated bacterial product in group LA and group LP, the faecal score grade 2 and 3 were reduced, and the piglets with medium and aggressive diarrhoea as well. The incidence of diarrhoea decreased by 50% compared to the C diet. The microencapsulated bacterial mixture LA: LP led to a grade 2 and 3 faecal score similar to group M. The incidence of diarrhoea in group LA: LP was 37.5% lower compared to group M (insignificant differences; P> 0.05).
• At weaning, a reduction in stomach weight of 2.86 times to 3.96 times and of the small intestine of 1.54 times to 1.80 times, respectively, was observed as an effect of the addition of micro-encapsulated bacterial product with mixture LA: LP.
• At 7 days after weaning, administration of the compound feed added to the experimental diets led to gastrointestinal tract weights similar to C diet.
• At weaning, the use of micro-encapsulated bacterial product LA shows a significant decrease in pH in the stomach by 10.84%, fasting by 6.66%, cecum by 3.33%, in the large intestine with 1.63% compared to C diet. The micro-encapsulated bacterial product with LA: LP mixture contributes to a significant increase in pH in the stomach by 25.00%, in jejunum by 15.49%, in cecum by 7.69 %, in the large intestine by 4.41% compared to C diet. Diets with LP leads to pH values ​​in the digestive tract segment, similar to the C diet.

Activity 3.2.2. Sampling / Molecular analyses packages for intestinal microflora characterization.

# At the end of the experimental test 20 piglets were slaughtered in order to take samples of digestive tract for establishing ileal and cecum microflora, pH evolution in stomach, ileum, cecum gut) as effect of microbial additive microencapsulated added in the diets.

 Results:

• Bacterial strains selected, Lactobacillus acidophilus IBNA 76 and Lactobacillus plantarum IBNA 84, with high probiotic action (high survival percentage to pH and bile salt), had capacity to maintain equilibrium of intestinal microflora (30 ± 3 days, respectively 37 ± 3 days) and to compete with pathogens strains inhibiting their development and multiplication.
• Lactobacillus administrated to as probiotics and added to diets for piglets in intensive systems could stimulate intestinal microflora leading to positive effects that could determine gastrointestinal disorders. 

Activity 3.3.2. Dissemination of research results

• 2 contracts of technological transfer.
• 2 papers ISI and 2 under review process.
• 1 paper BDI, 1 accepted for publication.
• 2 patent application OSIM.
• 2 brochures.

 Indicators of results: 

• Contracts of technological transfer no. 2769/21.04.2020/21.04.2020 - SC AGROPIG Farm SRL
• Contracts of technological transfer nor. 2767/21.04.2020/021.04.2020 - SC Nellmar Transilvania SRL

 

Papers/ communication/ patents application: 

ISI papers

1. Dumitru M., Habeanu M., Lefter N.A., Gheorghe A., 2020 The effect of Bacillus licheniformis as direct-fed microbial product on growth performance, gastrointestinal disorders and microflora population in weaning piglets. Rom Biotechnol Lett.; 25(6): 2060-2069. DOI: 10.25083/rbl/25.6/2060.2069
2. Anca Gheorghe, Nicoleta A. Lefter, Lavinia Idriceanu, Mariana Ropotă and Mihaela Hăbeanu Effects of dietary extruded linseed and Lactobacillus acidophilus on growth performance, carcass traits, plasma lipoprotein response, and cecal bacterial populations in broiler chicks2020. Italian Journal of Animal Science 19(1): 822-832
3. Mihaela Hăbeanu, Anca Gheorghe, Mihaela Dumitru, Nicoleta Aurelia Lefter, Lavinia Idriceanu, Smaranda Mariana Toma, Crina Daniela Neacșu, Gina Cecilia Pistol Assessment of associated effects of the milk and feed composition on suckling piglets’ growth parameters, immune response and digestive disorders. Animals În recenzie
4. Dumitru M., Hăbeanu M., Sorescu I, Effects of Bacillus spp. use as a dietary probiotic in weaning piglets. SASAS Journal. In review.

 BDI Papers

1. Idriceanu Lavinia, Silvia Mironeasa, Anca Gheorghe, Nicoleta Aurelia Lefter, Madalina Iuga, Daniela Mihaela Grigore, Mihaela Habeanu. Effects of the extruded linseed and walnut meal on some quality characteristics of longissimus dorsi and semitendinosus muscle of pigs Scientific Papers. Series D. Animal Science. Vol. LXIII, No. 1, p 128- 134, 2020
2. Dumitru M., Hăbeanu M., Lefter N Effects of direct-feed microbial Bacillus supplementation on piglet’s microbiota. Scientific Bulletin. Series F. Biotechnologies, Acceptat. vol 2 (xx).2020.

  Patents application:

1. Mihaela Habeanu, Gheorghe Anca, Cristina Tabuc Tehnologie nutritionala bazata pe un macro-mix cu proprietati functionale pentru purcei. OSIM A 2309/02.04.2020
2. Dumitru Mihaela, Vodnar Dan, Habeanu Mihaela, Chelaru Nicoleta Raluca, Simon Elemér, Sorescu Ionut, Ciurescu Georgeta, Tabuc Cristina, Gheorghe Anca. Procedeu de izolare, caracterizare si obtinere a unor produse bacteriene de Lactobacillus salivarius. OSIM A/00039/30.01.2020

Communications:

1. Lefter N. A., Dumitru M., Gheorghe A., Idriceanu L., Hăbeanu M The influence of Bacillus licheniformis as feed additive on performances and gastrointestinal disorders of weaning piglets. 7th Annual Conference “Strengthening Food Security and Environmental Sustainability ORAL/ April 28, 2020, Bucharest Romania.
2. Lefter N. A., Dumitru M., Gheorghe A., Idriceanu L., and Hăbeanu M Soluție nutrițională pe bază de sorg, mazăre și Bacillus licheniformis ca aditiv furajer pentru purcei în criza de înțărcare”Abordări sistemice și posibile soluții inovatoare de reducere a emisiilor de gaze cu efect de seră din sectorul zootehnic, în contextul noului pact verde european – «de la fermă în furculiță”, ORAL / INCE/CE-MONT, sept 2020 online
3. Dumitru M., Lefter N., Habeanu M., Cercetări in vitro asupra tulpinii de Bacillus licheniformis în vederea utilizării în hrana purceilor în criza de înțărcare ca sursă de aditiv furajer. Abordări sistemice și posibile soluții inovatoare de reducere a emisiilor de gaze cu efect de seră din sectorul zootehnic, în contextul noului pact verde european – «de la fermă în furculiță”, ORAL / INCE/CE-MONT, sept 2020 online
4. Dumitru M., Lefter N., Habeanu M., Gheorghe A., Idriceanu L New Research on Bacillus licheniformis Bio Feed Additive Effects on Piglet’s Microbiota during Weaning crisis. 7th Annual Conference Strengthening Food Security and Environmental Sustainability. ONLINE 2020/ 27-29 aprilie.
5. Dumitru M., Hăbeanu M., Lefter N. Effects of direct-feed microbial Bacillus supplementation on piglet’s microbiota. Conference Agriculture for life, life for Agriculture. 2020/, 4-6 iunie (online)
6. Lefter N. A., Hăbeanu M., Gheorghe A., Idriceanu L. Growth response and health status of growing pigs fed millet grain as a partial replacement for triticale 22th International Conference on Animal Diseases, Nutrition and Breeding E-Poster / Prague, Czechia, March 19 - 20, 2020.

 Popularization articles:

1. Lefter N.A., Dumitru M., Gheorghe A., Idriceanu L. și Hăbeanu M. Soluții nutriționale pe bază de sorg, mazăre și aditiv furajer microbian pentru purcei Brosura ISBN 978-606-11-7409-6
2. Dumitru M., Lefter N., Habeanu M., Gheorghe A. Obtinerea unui aditiv furajer pe baza de Bacillus licheniformis și efectul acestuia asupra microbiotei purceilor în criza de înțărcare. Brosură ISBN 978-606-11-7410-2

 CHECKS

2 mobility checks at USAMV Cluj.

 

Phase 4/2021

Effects of the bacterial product on gut microbiota in broiler chicken and senzory qualities (pH, Kreiss reaction, hydrogen sulphide assessment).

Objective: Passive and active dissemination of the research results

Summary

Activity 4-2-1. Dissemination of researches results.

• 3 ISI scientific papers were accepted for publication:
• Dumitru M., Hăbeanu M., Sorescu I., 2021. Effects of Bacillus spp. use as a dietary probiotic in weaning piglets. The South African Journal of Animal Science (SAJAS). (IF 0.847). Acceptat.
• Dumitru M., Sorescu I., Ciurescu G., Habeanu M., 2021. Isolation, identification, preservation and determinations of lactic acid bacteria from chicken’s gut content at 26 days old. Rom Biotech Lett.: 26(4), 2765-2772. DOI: 10.25083/rbl/26.4/2765-2772 (IF 0.765).
• Dumitru M., Habeanu M., 2021. Production and evaluation of extracellular enzymes from Bacillus Licheniformis in different raw materials used in animal feed. Scientific Papers. Series D. Animal Science (acceptat). (IF 0). Acceptat
• 1 BDI article was published and 1 accepted:
• Dumitru M., Lefter N., Idriceanu L., Ciurescu G., Habeanu M., 2021. Identification and characterization of Bacillus megaterium as probiotic bacteria in chicken broiler feed. Scientific Papers: Animal Science and Biotechnologies: 54(1) 124-133.
• Gheorghe A., Hăbeanu M., Dumitru M., Lefter N.A., Idriceanu L. Performance and intestinal microflora response of broilers to a probiotic mixture supplementation. Archiva Zootechnica (acceptat)
• Communication:
• Dumitru M., Sorescu I., Vodnar I., Elemer S., Ciurescu G., Habeanu M., 2021. Probiotics potential and microencapsulation of lactic acid bacteria as potential source for poultry. 72nd Annual Meeting of European Federation of animal Science EAAP, Davos (Switzerland) (abstract 35659, Session 99: Free communications) 30 – 03 September.
• Dumitru M., Habeanu M., 2021. Production and evaluation of extracellular enzymes from Bacillus Licheniformis in different raw materials used in animal feed. Conference Agriculture for life, life for Agriculture”, 3-5 juin.
• 1 Collaboration agreement was concluded no. 1976 / 31.03.2021.
• 1 method disseminated of UB partner: Method of molecular diagnosis of various bacterial taxons

 

 

 

 Poultry feeding – a natural way of maintaining gut health, poultry performance and food quality under conditions imposed by the ban of antibiotics

 

Context: Currently, the demand for food of animal origin (both quantity and requirements) is a challenge for the livestock sector with a special focus on nutrition. The qualitative side mainly involves the effects on consumer health, animal welfare and environmental protection. There is also a need for safe food from healthy animals. Ensuring the health of farm animals is another challenge facing the livestock sector, especially after the ban on the antibiotics using in feed as a growth factor as a consequence of the increase in antibiotic resistance of certain strains of bacteria, including the most pathogenic ones such as Salmonella spp., Campylobacter spp., Escherichia

  

The objective of the project is to obtain and validate new feed diets for poultry that contain less known/investigated phytoadditives, produced by local SMEs able to maintain the health of the digestive tract, productive performance and food quality and to remove the need for antibiotics. The partnership formed will ensure the obtaining of in-depth answers, beyond the registration of productive performances.

 

1)            Development and experimental trials of new feeding formula for laying hens to maintain the digestive tract health, the productive performances and egg quality

 

Activity 3-1-1: Local origin phytoadditives identification and characterization that can be used as alternatives to antibiotics in laying hens’ feed; partners involved (CO-IBNA). There were 9 plants identified as vegetable phytoadditives (blueberry leaves, walnut leaves, fennel powder and seeds, mint, marigold, melon peel, sea buckthorn meal, sesame seeds) and a plant mixture consisting of: walnut leaves + blueberry leaves + sea buckthorn meal). The plants were physico-chemically characterized (chemical composition, minerals, polyphenols, antioxidant capacity, vitamins, amino acids, fatty acids) to determine the content of nutrients and bioactive compounds to take into consideration as possible alternatives to the use of antibiotics in poultry feed.

Activity 3-1-2 and 3-1-3: Listing the main parameters to be determined, within "in vivo" experiments, to evaluate the productive performances, digestive tract health and nutritional quality of eggs. Laying hens new feeding formula development to ensure the productive performances, maintaining the health of the digestive tract and the nutritional quality of eggs for consumption. Development of experimental protocol; partners involved (CO-IBNA; P1-UB-DBBM; P4-USV). After their characterization, 4 plants (blueberry leaves, walnut leaves, sea buckthorn meal and watermelon peel) and a plant mixture (25% blueberry leaves, 25% walnut leaves, 50% sea buckthorn meal) were selected for in vivo testing on laying hens. There were 5 new feeding formula developed and manufactured, in accordance with the nutritional requirements of the Tetra SL hybrid, isoproteic and isoenergetic diets (2800 kcal / kg metabolizable energy and 17.8% protein): E1 (blueberry leaves); E2 (walnut leaves); E3 (sea buckthorn meal); E4 (mixture of blueberry leaves, sea buckthorn meal and walnut leaves) and E5 (melon peel). At the same time, the physical and biochemical parameters were determined by measurements from the samples collected during the experiment in order to evaluate their impact on productive performances, health status  and egg nutritional quality.

Activity 3-1-4, 3-1-5 and 3-1-6: Testing the new feeding formula through experiments carried out "in vivo" on laying hens to ensure productive performance, establishing the egg nutritional value o and assessing the digestive tract health (partners involved: CO-IBNA; UB-DBBM; USV). The evaluation of the new feeding formula with phytoadditives was performed by an experiment carried out for 4 weeks on 168 Tetra SL laying hens (age 32 weeks; 6 groups). The effects quantification of using the new feeding formula on  productive performances, egg nutritional value, digestive tract health/health status of laying hens were performed by: (1) monitoring production parameters (daily feed consumption, specific feed consumption, egg weight, laying intensity); (2) physical, rheological and nutritional quality of eggs (internal and external physical parameters, egg compression test, sensory analysis, egg white rheological parameters, yolk chemical composition: fatty acid content, lutein + zeaxanthin, antioxidant capacity, total polyphenols and stability oxidative effects of egg yolk); (3) blood tests and slaughtering process in order to collect samples of intestine and intestinal contents to evaluate the health of the digestive tract by physical determinations (measurements, pH), microbiological, histological and enzymatic.

 

2)            Development and experimental trials of new feeding formula for broilers to maintain the digestive tract health, the productive performances and meat quality

 

Activity 3-2-1:  Local origin phytoadditives identification and characterization that can be used as alternatives to antibiotics in broilers’ feed; partners involved (CO-IBNA; P1-UB-DBBM). The phytoadditives identified as an alternative to antibiotics in broilers’ feed were: flaxseed meal (PUFA source); grape seed oil and grape pomace and citrus peels (sweet orange and red grapefruit). The results obtained for chemical analysis showed that phytoadditives meet the nutritional requirements to use them in broilers’ feed as rich sources of bioactive compounds - sugars, organic acids, flavonoids and vitamins with antioxidant, anticancer, antimicrobial and anti-inflammatory properties.

Activity 3-2-2: Broilers new feed formula development; experimental protocol elaboration; Partners involved (CO-IBNA). For every experiment, several sets of feeding formula were developed for the two rearing phases: experiment 1- a set of 4 feeding fromula based on grape pomace (from white or red grapes); experiment 2- a set of 2 feeding fromula that included grape seed oil; Experiment 3 -  a set of 2 feeding that included citrus peels (orange and red grapefruit); Experiment 4 - a set of 2 feeding fromula that included a natural antioxidant from red grapes. Before preparing the feeding formula, an experimental protocol was elaborated in which the specific characteristics of each experiment were specified.

Activity 3-2-3: Testing new feeding formula through experiments carried out "in vivo" on broilers; (partners involved CO-IBNA). The experiments were carried out on Cobb 500 (40 chickens/lot, growing-finishing phase, 4 weeks length). Parameters followed during the experiments: (1) production parameters (live weight, average daily gain, average daily consumption, specific consumption); (2) microclimate parameters (temperature and humidity; carbon dioxide, ventilation); (3) chemical analysis of feed materials and compound feed; (4) meat nutritional quality; (5) carcass and  organs development (at chickens aged 42 days), (6) birds and their digestive tract health evaluation;

Activity 3-2-4: Broilers’ slaughtering  in order to collect samples of intestine and intestinal contents to evaluate digestive tract health by physical (measurements, pH), microbiological, histological, enzymatic determinations; (partners involved CO-IBNA; P1-UB-DBBM). The results recorded during the experiments highlighted aspects regarding the significant decrease of analyzed pathogenic bacteria and the increase of beneficial bacteria for maintaining a normal microflora. Phenolic compounds can selectively stimulate the growth of beneficial bacteria, such as Lactobacillus and Bifidobacterium, by altering the intestinal microbiota.

Activity 3-2-5: Determination of nutritional value and organoleptic properties from breast and pulp samples collected at slaughter; partners involved (CO-IBNA; USV). The results obtained for the nutritional quality of the meat expressed by the fatty acid content (PUFA) showed significant variations in their content as well as the meat color variation. The analysis of the texture profile (TPA) determined the increase of the parameters: hardness, elasticity and resilience of the chicken leg (Experiments 1, 3 and 4), without influencing the meat texture from experiment 2. Also, the oxidative status of the meat samples have shown a slower lipid degradation process;

 

3)            Evaluation and capitalization of trials results obtained after testing the new feeding formula for laying hens and broilers

 

Activity 3-3-1: Evaluation of the technical, statistical and economic results obtained during the project; partners involved (CO-IBNA). An evaluation of all the results obtained during the whole project period was made in order to allow their capitalization.

Activity 3-3-2 and 3-3-3: Documentation elaboration to fill two patent applications for a new combined feed structure used for laying hens and broilers; partners involved (CO-IBNA). Within the activity, two patent applications were elaborated.

 

4)            Project results capitalization

 

Activity 3-4-1: Dissemination by publishing scientific articles in ISI or BDI journals; partners involved (IBNA-LCF; UB-DBBM; US). Within the activity were elaborated 3 ISI articles, out of which 2 published and 1 in review process; 2 BDI articles published and 3 accepted for publication. The articles included scientific data obtained during the project.

All articles (ISI and BDI), conferences, products and technology transfer obtained in Component Project no. 3 can be found in the Final Technical Report (2018-2021)

Activity 3-4-2: Guide development concerning new poultry nutritional strategies about maintaining the digestive tract health, performance and feed quality under the prohibition of antibiotics; partners involved (IBNA-LCF; UB-DBBM; US). Two guidelines have been developed on new nutritional strategies for poultry in order to maintain the health of the digestive tract, productive performances and food quality under the prohibition of antibiotics.

 

 

 

 

 

  

 Development of feed products based on plants extracts, which to enhance animal performance by manipulation of the rumen environment

  

The objective of the project is to estimate the effects of plant extracts on the ruminal environment, in order to identify some advantages (e.g. improvement of the ruminal protein / energy metabolism) to justify their inclusion in rations / feed products for ruminants.

 

Planned activities. For the evaluation of plant extracts effects, will be used classical methods, already applied by the coordinator (IBNA), but also new “-omics” type methods (metabolomics = analysis of biomarkers / metabolites - UBB, genomics = precise quantification of the ruminal ecosystem - UB-DBBM, proteomics = estimation the effects on the relevant protein fractions in milk - IBAR), using the advanced equipment and expertise of the partners in the respective fields. This collaboration within consortia allow not only a higher potential to generate scientific knowledge and applicative results, with high impact in animal production practice, but will also allow the evolution of the IBNA on research directions toward the confluence of animal nutrition and "-omics" technologies, new collaborations / projects on the mentioned subjects, etc.

 

Stage 1. Identification and nutritional characterization of the studied feedstuffs; ensuring the necessary experimental conditions, methods optimization

This stage included four activities, carried out in collaboration with the four partners of the project (University of Bucharest, Babes-Bolyai University, Institute of Biochemistry - Romanian Academy).

 

Activ. 1.1 - Establishing the experimental design, ensuring the necessary experimental conditions, selecting the studied feedstuffs in accordance with the proposed purpose

Beside ensuring the framework for the tests / experiments provided in the project (ensuring animals, spare parts, analytical methods and procedures, reagents, etc.) this activity led to the development of a bibliographic study and a database, based on which was made the preliminary list of fodder / plant extracts / feed additives to be studied within the project.

For this, the feedstuffs that have an applicative potential for ruminant feeding were also reviewed, in the context of the requirements of the project. An important role in this was played by nutritional characterization of these feeds. In addition, beside classical parameters that define a feed (protein value, energy value, etc.) the nutritional value is also given by the presence of active substances (secondary metabolites of plants) that can influence the ruminal metabolism / productive performances of animals - milk production level, milk protein profile, etc. (positive / negative). If the classical parameters are relatively well known, the effects of the active substances are insufficiently or even not studied (for certain plants / classes of substances). Therefore, the review / nutritional characterization focused on the potential positive effects of these feeds / substances (reduction of ruminal degradability, increased digestibility of cell walls, change in C2/C3 ratio, buffering the acidogenic effect of rations, etc.).

The feeds / plant extracts / feed additives from this list will enter in the study next stage; they will be further selected, based on the results of chemical analyses -> in vitro tests -> ruminal metabolism experiments; so that through successive elimination 2-6 experimental variants will be selected to be assessed through the effects on the productive performance of dairy cows. On the other hand, the list remains open if the analyses provide a special opportunity for feed resources not yet taken into account (e.g. if a certain feeds / feed additives are becoming available in feed / additives market in Romania; if there are express requests from the engaged professional associations in collaborations with the coordinating institute, etc.).

 

Activ. 1.2 - Identification of molecular markers useful for identification of bacterial taxa and in the quantitative characterization of the ruminal microbiota

Within this activity, the markers and the primers that will be used for the characterization of the ruminal microbiota were established. For this, DNA from the ruminal fluid was isolated (by various methods) and the purity and concentration of the DNA samples extracted from the ruminal fluid were determined (by spectrophometry), the integrity of the extracted DNA samples was checked, the amplification of the DNA samples extracted by this method was tested (by temperature gradient PCR), etc.

During the experiments, some working protocols were improved / adapted (e.g. by optimizing the incubation time, optimizing the amplification reactions, optimizing the specific hybridization temperature for the designated primers, etc.).

The development of the activity led to the establishment of the working protocol (extraction method, markers, primers) most suitable for the purpose of the project.

 

Activ. 1.3 - Analysis the feasibility of using metabolites as specific biomarkers for farm animal nutrition

Biomarkers are biological molecules found in the blood, other biological fluids or tissues, which provide information about biological processes in the body - physiological or abnormal. A biomarker can be used to identify / assess the response of organisms to treatments or changes of any kind, the effectiveness of the use of some dietary ingredients, etc.

In this context, the feasibility analysis was focused on taking into account the monitoring potential for metabolic parameters in plasma, ruminal fluid (of high importance in ruminants’ productivity), but also milk (the final product pursued). As a result of the analysis, a short list of potential biomarkers was made, from which the experimental parameters will be selected to be monitored in the next stages of the project (NEFA, BHB, purine derivatives from urine / milk, etc.).

 

Activ. 1.4 - Establishing the methodologies to be used for the assessment of the protein fractions in milk

In this stage of project implementation, two methods for analysing the protein fractions of cow's milk were tested, using the LC-MS / MS technique. 163 protein groups were identified, a number similar to proteomic studies performed on milk samples. No major differences were found regarding the identity of the proteome between the freshly collected milk and the one subjected to the freezing process. TCA precipitation of proteins led to the identification of a larger number of peptides and protein groups, while denaturation with chaotropic agent led to the identification of a slightly increased number of phosphorylated sites.

  

DISSEMINATION ACTIVITIES (STAGE 1):

 Scientific communications:

• Comparative proteomics of fresh and frozen bovine milk. The Annual International Conference of the Romanian Society of Biochemistry and Molecular Biology, Bucharest, 5-7th September, 2018. Cristian V.A. Munteanu, Smaranda Toma, Cătălin Dragomir, Ștefana M. Petrescu

 

Others:

• bibliographic study
• database
• optimized analytical methods and procedures

 Use of the vouchers

In the first stage, a B type voucher was used by the partner UBB Cluj Napoca, on the subject of biomarkers in biological samples (blood / ruminal fluid / milk). The coordinator also used a C type voucher on the subject of FT spectroscopy methods (Fourier transform) and thin layer chromatography.

  

Stage 2/2019 - Carrying out the in vivo experiments in laboratory conditions, completing the optimization of methods, preliminary analyses

This stage was carried out following the seven activities planned and carried out in collaboration with the project partners (University of Bucharest, Babeș-Bolyai University, Institute of Biochemistry of the Romanian Academy).

  Activ. 2.1 - Carrying out the experiment on fistulated animals, determining the ruminal parameters, taking samples (ruminal fluid) for additional determinations

Through this activity, an experimental plan was identified to create the premises for possible effects on the three directions of collaboration with the project partners: biomarkers associated with the feeding condition, the ruminal ecosystem and milk proteomics. Two experiments were performed based on the same typology of ration composition, the replacement of the energy source. Basically, the energy intake of cereal starch was replaced by the energy intake of rapeseed oil, maintaining equal energy and protein levels (isocaloric / isoenergetic) for both diets (control / experimental) fed to the animals. The administration of significant amounts of lipids in the diet of ruminants can lead to ketosis, changes followed by the analysis of specific serum biomarkers (β-hydroxybutyrate - BHB).

After 21 days of adaptation to rations, ruminal fluid samples were taken to study ruminal metabolism (pH, ammonia concentration, volatile fatty acid concentration) and ruminal microbiota (identification and microscopic quantification of protozoa in the rumen + estimation of effects on bacterial and methanogenic populations).

 Activ. 2.2 - Carrying out preliminary tests for optimizing working methods (qPCR, Species Specific PCR) specific to the quantitative and qualitative assessment of the ruminal ecosystem

This activity included isolating the DNA from the ruminal fluid, determining the purity and concentration extracted by spectrophotometric verification and optimizing the method according to the results obtained. Also, optimizations of Species-Specific PCR and qPCR amplification reactions were performed to determine populations of ruminal microorganisms and highlight the effects of nutritional changes.

 Activ. 2.3 - Identification of bacterial taxa in the ruminal ecosystem by analysis of genes encoding rR16S, CO I, cpn 60

The extracted DNA samples were amplified for selected DNA markers using primer pairs. After electrophoretic verification of the amplification products, they were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) kit. The sequences obtained for the analysed samples were compared with those from GenBank, and the BLAST analysis confirmed that they belong to the followed genera and species.

 Activ. 2.4 - Adapting the necessary methods to determine the specific biomarkers for farm animal nutrition

In order to carry out activities 2.4, 2.5 and 2.6 the pre-experimental period related to the experiment on dairy cows planned for the next stage was used, by collecting milk samples that were sent to Babes-Bolyai University and the Institute of Biochemistry for the development / optimization of the methods to be used in the next stages.

For the analysis of purine bases (used as biomarkers of microbial protein synthesis) a calibration curve was constructed using a standard mixture of purines at different dilutions (10 concentrations) between 0.487-200 µg / mL using an HPLC with DAD detector. An HPLC-UV was used for the analysis of milk protein biomarkers, following the optimization being established the characteristics of the working method. The analysis of milk BHB concentration was performed in a transparent microplate, with 96 wells, using a kit based on the reaction of the β-HB Dehydrogenase enzyme on sample BHB (developed by the manufacturer for BHB analysis in blood serum).

 Activ. 2.5 - Optimization the extraction and processing parameters of milk proteins

A series of samples were analysed in order to detect possible changes in the cow's milk proteome, by changing the animals feed and obtaining a comparative proteome, between skimmed milk and whole milk, using the optimized method in the first stage of project implementation. For the comparison of skimmed milk and whole milk proteome, six samples from the control group (three for each condition) were considered. The results showed the identification of a smaller number of both triptych peptides and protein groups in the case of protein extraction from skim milk. Thus, 1074 triptych peptides were identified, corresponding to 150 proteins in the case of skimmed milk and 907 peptides, respectively 127 proteins, in the alternative case.

 Activ. 2.6 - Establishment of optimal experimental parameters specific to the chromatographic system and mass spectrometer for milk proteomic analysis

Samples containing the extracted peptides were subjected to LC-MS / MS analysis, using similar parameters to those described in the previous stage of project implementation. For the analysis of LC-MS / MS data, the SEQUEST algorithm integrated in Proteome Discover v1.4 and the Bos taurus proteome were used. The LC-MS / MS experiments of the 24 samples considered at this stage led to the identification of more than 1500 triptych peptides, corresponding to over 200 protein groups. A high-resolution analyser was used for sample’s analysis, in which both the masses of the identified peptides and their fragments have an accuracy of the ppm order (parts per million), increasing the confidence of the identifications.

The proteome obtained was characterized by a dynamic range of relative protein abundance of about five orders of magnitude, thus reflecting the high variability of protein abundance in cow's milk. The distribution of the number of proteins identified in the analysed samples showed the identification of over 100 proteins in most of the analysed samples (median over 100) and over 600 triptych peptides per sample. The number was variable between samples, which could be an expression of the qualitative variability of the cow's milk proteome (between animals), considering approximately equal amounts of sample. The comparative analysis of milk samples from differentiated fed animals revealed that out of the total 239 identified proteins, 177 were identified under both analysis conditions, while 18 and 44 were specific to group M and E, respectively. The 44 proteins found only in the milk samples taken from the batch with modified feed could indicate an increase in the nutritional protein value of the milk. These results revealed that the developed methods, as well as the established parameters used in the proteomic analysis of milk are adequate to the objective of the project.

 Activ 2.7 - Integration, interpretation and dissemination of partial results

During this stage, the results obtained by the coordinator and the University of Bucharest were integrated (the other partners having planned developments / optimizations of methods) and the dissemination was continued by presenting a poster at an international conference. An article is also being developed based on the results obtained in the experiment performed on fistulated animals.

  

DISSEMINATION ACTIVITIES (STAGE 2):

 International Conferences

Miuța Filip, Mihaela Vlassa , Irina Ciotlăuș , Ioana Boldea , Cătălin Dragomir. Preliminary results on the influence of particular feeding situations on the level of some potential biomarkers in cows' milk. The 33rd EFFoST International Conference: Sustainable Food Systems - Performing by Connecting , Wageningen University & Research , Rotterdam, The Netherlands, 12-14 November 2019.

  

Stage 3/2020 - Carrying out experiments on animals in farm conditions, biochemical determinations

During this stage, all five planned activities were carried out, in collaboration with the partners of the component project: University of Bucharest, Babeș-Bolyai University and the Institute of Biochemistry of the Romanian Academy.

 Activ. 3.1. Organizing and carrying out a test on dairy cows, taking samples for in-depth additional determinations

The experiment was carried out on the IBNA farm, for 58 days, on 30 multiparous dairy cows, in mid-lactation, randomly distributed in two groups, according to a Latin square experimental plan. Two diets were tested, in which the energy intake was provided mainly by starch (control group) or fat (experimental group). The two diets supplied a fat intake of about 1.6% of the diet (the control grpup), respectively about 5.6% of the diet (the experimental ration, in which case the rapeseed was included in a proportion of about 10% of the dietary DM). Preliminary results showed that, in addition to the effects on milk production and its primary composition, the experimental ration led to a change in the profile of milk fatty acids by decreasing the proportion of saturated fatty acids, increasing the proportion of polyunsaturated fatty acids and CLA in the case of rapeseed ration. During this test, the milk samples necessary for carrying out activities 3.4 (determination of biomarkers in milk) and 3.5 were also collected. (milk proteome study).

 Activ. 3.2. Analysis of DNA markers and identification of bacterial taxa in ruminal fluid samples

The analysis of DNA markers and the identification of bacterial taxa was performed on samples that were collected within the experiment on fistulated battles, carried out in the previous stage of the project.

This experiment was based on the same typology of experimental rations used in the experiment on dairy cows: the replacement of the energy source in the combined feed, without substantially changing the energy-protein intake of the ration.

More specifically, part of the energy input from cereal starch has been replaced by energy input from rapeseed oil. Basically, cereals and protein meals have been partially replaced by rapeseed. In this way, the control ration and the experimental ration were isocaloric / isoproteic, but in the experimental ration the fat intake increased abruptly. Even if the allowable limit has not been exceeded, suh a diet has the potential may induce changes in the ruminal ecosystem.

 a) Isolation of DNA from ruminal fluid

It was achieved through the method that was optimized in the previous project stage. The absorbance and concentration values obtained from the spectrophotometric verification showed that the extraction of the samples was not significantly influenced by the experimental design. Thus, the concentration (ng / μL) was 698.18 ± 328.89 in the control group and 724.45 ± 211.82 in the experimental group, and the A260 / A280 ratio was 1.93 ± 0.03 in the control group and 1.86 ± 0.04 in the experimental group.

 b) Identification of bacterial taxa by sequencing

The extracted DNA samples were amplified for the selected DNA markers, the resulting amplicons being subjected to electrophoretic verification. In the next step, they were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) kit. After purification, the DNA fragments were sequenced by the Sanger method, the "dye-terminator" variant. The amplicons obtained during this reaction were subjected to capillary electrophoresis, followed by a fluorescence detection in an automatic genetic analyser. At the end of the reaction has followed the stage of precipitation (obligatory) of the obtained products, which was performed with the XTerminator kit (Life Technologies). The sequences obtained for the analysed samples were compared with those from GenBank, and the BLAST analysis confirmed that they belong to the following classes and genera: total bacteria, methanogenic bacteria, Ruminococcus sp., Prevotella sp.

Activ. 3.3. Quantitative PCR analysis of the number of bacterial populations in ruminal fluid samples and their diversity

The analysis was performed by the method and procedures optimized in the previous stage of the project. Statistical analyses were performed with GraphPad Prism v.6 software, through analysis of variance. It was observed that the change of the diet induced slight changes of the microbiota in the case of the analysed specimens. The administration of the ration based on rapeseed led to a moderate decrease in the relative abundance of methanogenic bacteria and total bacteria. In the case of the Prevotella and Ruminoccocus genera, the decreases recorded are extremely small, statistically insignificant.

 Activ. 3.4. Estimating the effects of feed on nutritionally relevant biomarkers

During the experiment on dairy cows, carried out in activity 3.1., in addition to milk samples used to determine milk quality, samples were collected in order to determine milk biomarkers, analyses performed by the partner UB. A first set of analyses, performed by an HPLC method optimized in the previous stage, aimed to determine the concentrations of purine derivatives in the 60 milk samples taken: 15 animals / batch * 2 periods * 2 repetitions. For each of the 60 samples, two determinations were performed, leading to a total of 120 values. Of the five purine derivatives present in the milk samples, only for uric acid there were significant differences detected between the two rations. The interpretation of these results and their correlation with other determinations will be made, as planned, in the next stage of the project.

Another set of analyses aimed at determining, by gas chromatography, the concentrations of some protein fractions - potential biomarkers, according to the same experimental plan. Preliminary results indicated that there were no significant differences between the two ratios in terms of BSA concentration. On the other hand, there was a decrease in the concentration of α-Lactalbumin, by 11%, a difference that was not detected as significant. There was also a tendency to increase the concentration of β-Lactglobulin B and decrease the concentration of β-Lactglobulin A. These results will be interpreted and correlated with results obtained from other determinations in the next stage, as planned.

 Activ. 3.5. Estimation of the feed effects on milk protein fractions

Effects of the tested rations in Active. 3.1. on protein fractions, it was performed by two methods - a semi-quantitative method (SDS-PAGE), more accessible but less accurate, and a quantitative method, more specific (LC-MS).

 a) Electrophoresis of milk protein fractions using SDS-PAGE

Following the determinations was observed the appearance of the four major caseins αs1 - casein (αs1 - CN), αs2 - casein (αs2 - CN), β - casein (β - CN) and k - casein (k - CN, between approximately 20 and 25 kDa and the two main whey proteins β - lactoglobulin (β –Lg) and α - lactalbumin (α - La), approximately between 14 and 18 kDa. Also, the presence of minority proteins was highlighted, lactoferrin (LF), bovine serum albumin (BSA) and immunoglobulin G (IgG), bands that occur approximately between 50 and 100 kDa.

Following the statistical processing, significant changes were observed in these fractions, which have a role either in milk processing or in consumers health.

 b) Determination of protein fractions by LC / MS

The determinations were performed using methods and procedures optimized in the previous stage of the project. Following the LC-MS / MS analysis were identified 2294 triptych peptides, from 246 proteins, with a median over 150 proteins / sample and ~ 1200 triptych peptides / sample. Qualitatively, peptides from the most abundant protein fractions in cow's milk have been identified, such as alpha caseins, beta-lactoglobulin, lactotransferrin, alpha-lactalbumin, some serotransferins, and some immunoglobulin fractions.

For the relative comparison of the identified peptides and proteins between the two considered conditions, the quantitative values corresponding to the ion currents specific for identified peptides were used, in a first stage excluding chemically modified peptides, and in the second stage using only the values corresponding to the identified phosphopeptides. Thus, the ion current values of the peptides were processed and assembled according to each protein identified for differential quantitative analysis. In order to observe possible changes at the molecular level within the cow's milk proteome, the hierarchical clustering analysis was used, a method based on agglomerative clustering by calculating mathematical distances between the elements of the matrix containing quantitative data. Hierarchical clustering suggests possible quantitative changes in the proteome between the two feeding conditions considered.

 

DISSEMINATION ACTIVITIES (STAGE 3):

 Scientific communications:

• Comparative proteomics of fresh and frozen bovine milk. The Annual International Conference of the Romanian Society of Biochemistry and Molecular Biology, Bucharest, 5-7th September, 2018. Cristian V.A. Munteanu, Smaranda Toma, Cătălin Dragomir, Ștefana M. Petrescu
• Preliminary results on the influence of particular feeding situations on the level of some potential biomarkers in cows' milk. The 33rd EFFoST International Conference: Sustainable Food Systems - Performing by Connecting , Wageningen University & Research , Rotterdam, The Netherlands, 12-14 November 2019. Miuța Filip, Mihaela Vlassa , Irina Ciotlăuș , Ioana Boldea , Cătălin Dragomir.

 Others:

• bibliographic study
• database
• optimized analytical methods and procedures
• technology transfer agreement no. 5899 / 14.09.2020
• Brochure: 1. Dragomir (2020). Brewers grains - nutritional value and particularities

 

Stage 4/2021 - Integration and interpretation of results; completion of their dissemination

During this stage, the two planned activities were carried out, in collaboration with the partners of the component project: University of Bucharest, Babes-Bolyai University and Institute of Biochemistry of the Romanian Academy.

Activity 4.6. Report on the dissemination of results through articles / communications

Within this activity, the publication / communication of the results continued by carrying out the process of re-editing the articles in case of favourable reviews / resubmissions to other journals of the rejected articles. Of the 5 articles that were drafted on December 31, 2020, two more were accepted in January – May, and two more articles are being drafted.

Activity 4.7. Report on the integration, interpretation and dissemination of application results

Within this activity continued the valorisation of experimental data by performing statistical processing, which allowed the validation of the effects of the studied diets on various production parameters (milk production, fat percentage in milk) or other indicators that are relevant in terms of data applicability in practice animal husbandry or food industry (e.g. protein fractions in milk, relevant to consumers or processors). Some of the recipes were sent to the development sector of the institute, in order to include them in the portfolio of feed products.

Articles:

• Simultaneous quantification of four purine derivatives biomarkers in cow’s milk by using high performance liquid chromatography - diode array detection (HPLC-DAD) and solid-phase extraction (SPE). Mihaela Vlassa, Miuta Filip, Catalin Dragomir. 2020. Czech Journal of Food Sciences, 39, 2021 (2): 122–130

• Fatty acids profile of milk altered by the inclusion of the whole rapeseeds and pumpkin seed cakes in the diets of Murciano Granadina goats. 2021. Ioana Boldea, Catalin Dragomir, Mihai Gras, Mariana Ropota. South African Journal of Animal Science (in print)

• The effects of rapeseeds supplementation on bovine milk protein fractions. Oancea, A.-G, Dragomir, C., Pistol, G.-C., Cismileanu, A., Toma, S. 2020. International Food Research Journal (in print)

Communications:

• The effects of rapseeds inclusion in the diets of dairy cows on milk quality. Oancea, A.-G., Dragomir, C., Pistol, G.-C., Yaman S., Cismileanu, A. 3rd International Turkic World Congress on Science and Engineering. June 14-15, 2021 (accepted / included in the congress program)

• The effect of diatery brewers spent yeasts on the quality of sheep milk. Ana Cismileanu, Ioana Boldea, Alexandra-Gabriela Oancea, Catalin Dragomir. 16th International Symposium of Animal Biology and Nutrition, September 30, 2021 (accepted / included in the congress program)

Others:

• nutritional recommendations