In animal production, there is an increasing interest in the use of zootechnical additives that would produce beneficial effects on animal health and, at the same time, to be considered as alternatives to antibiotic growth promoters (Kholif et al. 2024Kholif, A.E., Anele, A. & Anele, U.Y. 2024. Microbial feed additives in ruminant feeding. AIMS Microbiology, 10(3): 542-571, ISSN: 2471-1888. https://doi:10.3934/microbiol.2024026. ). These additives have an influence on the digestive and absorptive processes of dietary nutrients, modulate the immune system, improve intestinal and host health, and, therefore, can improve production yields (Anee et al. 2021Anee, I.J., Alam, S., Begum, R.A., Shahjahan, R.Md. & Khandaker, A.M. 2021. The role of probiotics on animal health and nutrition. The Journal of Basic and Applied Zoology, 82: 52, ISSN: 2090-990X. https://doi.org/10.1186/s41936-021-00250-x.). Consequently, the use of zootechnical additives can contribute to increasing the availability and quality of animal products, free of antibiotic residues, intended for humans.
In Ecuador, some educational and research studies address the topic of obtaining and applying additives for animal feeding, as well as their advantages (Flores-Mancheno et al. 2016Flores-Mancheno, L.F., García-Hernández, Y., Usca-Méndez, J.E. & Caicedo-Quinche, W.O. 2016. Estudio comparativo de tres aditivos zootécnicos en el comportamiento productivo y sanitario de cerdos en el período post-destete. Ciencia y Agricultura, 13(2): 95-105, ISSN: 2539-0899. https://doi.org/10.19053/01228420.v13.n2.2016.5557.). However, the available scientific information is scarce. The objective of this research was to determine the microbiological and chemical characteristics of a new zootechnical additive produced in Ecuador for its use in animal feeding.
The study was conducted at an Experimental Poultry Farm of the Faculty of Agricultural Sciences, Technical University of Ambato, located in the Querochaca sector, Cevallos Canton, Tungurahua Province, Republic of Ecuador. The weather features of the site are 2,855 m a. s. l., with a mean annual rainfall of 442.4 mm, and a mean temperature of 16 °C.
The additive production process consisted of: 1) batch submerged fermentation, 2) mixing the culture with a solid support, and 3) natural drying by sun exposure. These steps and the procedure for microbiological and chemical analysis of the additive samples are described below.
Culture medium and inoculum: A medium composed of sugarcane molasses (20%, v/v), unsalted cow milk whey (34%, v/v), corn meal (1%, w/v), soybean cake meal (1%, w/v), urea (1%, w/v), Pecutrin® mineral-vitamin supplement (1%, w/v), and 100% water q.s. was used. Toni natural yogurt (2%, w/v), containing the Lactobacillus GG strain (Industrias Lácteas Toni S.A., Ecuador), was used as the inoculum.
Fermentation and drying process: Two batches of batch fermentation were carried out in duplicate in 50-L plastic tanks. The raw materials were added and weighed on a MOCCO SF-400D technical scale with a precision of ±0.01 units. Later, natural yogurt and drinking water were added to complete the fermentation volume corresponding to 45 L. The components were mixed for 10 min and incubated at room temperature (18 ± 2 °C) for seven days. After this time, the culture in each tank was homogenized, and an aliquot was taken to form a 1 L sample, measure its pH, and check the microbial concentration. The remaining culture was mixed (40:60, v/w) for 20 min with wheat bran and barley meal (50:50 w/w) and, finally, spread on an asphalt and roofing plate, forming a bed approximately 10 cm high. The material was turned every 3 h until dry. Samples of the dried material were taken from the ends and center of the drying plate until a homogeneous 1 kg sample was formed, which was then subjected to microbiological and chemical analyses. The remaining product was packaged in polyethylene bags and stored at room temperature for preservation.
Microbiological and chemical analysis: The additive samples were analyzed in duplicate, according to AOAC procedures (2019)AOAC. 2019. Métodos oficiales de análisis de la Asociación de Químicos Analíticos Oficiales: Métodos oficiales de análisis de la AOAC Internacional. 21.ª edición, AOAC, Washington DC. at the Food Control and Analysis Laboratory of the Faculty of Food Sciences and Engineering and Biotechnology of the Technical University of Ambato (Ecuador), with accreditation No. SAE LEN 10-008. Concentrations of mesophilic aerobes, fungi and yeasts, enterobacteria, Salmonella and Listeria were determined in the samples of the additive, which were previously diluted in a serial and decimal manner, using the 3M Petrifilm plate technique. In addition, total coliforms and Escherichia coli were determined using CompactDry®. Chemical analysis included percentages of humidity, crude protein, ash, fat, crude fiber, total carbohydrates, and pH.
After seven days of fermentation, the microbial culture had a concentration of 107 and 106 cfu/mL of mesophilic aerobic bacteria and yeasts, respectively. Furthermore, it did not contain pathogenic microorganisms, and its pH was 4.10. These results are due to the fact that the microbial population increases its concentration with the fermentation process, metabolizing the nutrients from the substrates used in the mixture and producing organic acids, carbon dioxide, and other metabolites. High concentrations of acids, in turn, contribute to decrease the pH of the medium (García et al. 2020García, Y., Sosa, D., González, L. & Dustet, J.C. 2020. Caracterización química, física y microbiológica de alimentos fermentados para su uso en la producción animal. Livestock Research for Rural Development, 32(7): 105, ISSN: 2521-9952. https://www.lrrd.org/lrrd32/7/Yaneis32105.html. ), which affects the cells of microorganisms such as Salmonella and Escherichia coli, which are sensitive to acidic pH.
Upon mixing the culture with the absorbent material and drying it in the sun, a brown solid with a fermented odor was obtained, the microbiological and chemical composition of which is shown in Tables 1 and 2, respectively. Similar to the culture, the presence of mesophilic aerobic bacteria (107 cfu/g) and yeasts (106 PU/g) were found at concentrations appropriate for their action as an additive and to guarantee its efficacy, as recommended by the FAO/WHO (2002)FAO/WHO (Food and Agriculture Organization of the United Nations/World Health Organization). 2002. Guidelines for the evaluation of probiotics in food. Report of a joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food. April 30 and May 1. London Ontario, Canada. Available at: http://www.who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf. [Consulted: October 25, 2016]. . Likewise, its adequate hygienic and sanitary quality was verified, with no Salmonella or Listeria found, and minimal and harmless concentrations of Enterobacteriaceae and E. coli.
| Indicator/Technique | Method | Unit | Dry additive |
|---|---|---|---|
| Mesophilic aerobic bacteria, Petrifilm | PE-03-7.2-MB 990.12* | cfu/g | 1.4x107 |
| Molds, Petrifilm | PE-02-7.2-MB 997.02* | PU/g | 3.1x106 |
| Yeasts Petrifilm | PE-02-7.2-MB 997.02* | PU/g | 1.0x106 |
| Enterobacteria, Petrifilm | PE-04-5.4-MB 2003.01* | cfu /g | <10 |
| Total Coliforms, CompactDry | PE-01-7.2-MB R.I: 110402* | cfu /g | <10 |
| Escherichia coli, CompactDry | PE-01-7.2-MB R.I: 110402* | cfu /g | <10 |
| Salmonella, Petrifilm | PE-08-7.2-MB 2014.01* | In 25 g | Not found |
| Listeria spp. Petrifilm | R.I: 081203* Petrifilm Listeria Dishes | In 25 g | Not found |
*AOAC (2019)AOAC. 2019. Métodos oficiales de análisis de la Asociación de Químicos Analíticos Oficiales: Métodos oficiales de análisis de la AOAC Internacional. 21.ª edición, AOAC, Washington DC.
| Indicator/Technique | Method | Unit | Dry additive |
|---|---|---|---|
| Humidity, gravimetry | 925.10* | % | 14.2 |
| Protein, Kjendhal | 2001.11* | % (Nx6.25) | 14.2 |
| Ashes, gravimetry | 923.03* | % | 11.8 |
| Fat (with hyrolisis), gravimetry | 2003.06* | % | 0.213 |
| Crude fiber, gravimetry | NTE INEN 522:2013NTE INEN 522. Norma Técnica Ecuatoriana 522:2013 del Instituto Ecuatoriano de Normalización. Harinas de origen vegetal. Determinación de la fibra cruda. Available at: https://www.normalizacion.gob.ec. | % | 14.3 |
| Total carbohydrates, calculation | Calculation | % | 45 |
| pH, potentiometry | 981.12* | pH units | 6.72 |
*AOAC (2019)AOAC. 2019. Métodos oficiales de análisis de la Asociación de Químicos Analíticos Oficiales: Métodos oficiales de análisis de la AOAC Internacional. 21.ª edición, AOAC, Washington DC.
The bran meal, mixed with the microbial culture and naturally dried, had a humidity content of 14.2 %. This value is lower than the maximum established for dehydrated foods, which facilitates their handling and transportation, as well as extends their shelf life by preventing the proliferation of contaminating microorganisms. This is due to the fact that dehydration decreases water content and activity, which, consequently, reduces or inhibits microbial growth and the speed of various spoilage reactions (Toledo 2007Toledo, R.T. 2007. Chapter 12. Dehydration. In: Fundamentals of Food Process Engineering. Third Edition. Springer Science+Business Media, LLC, New York, p. 431-473.). However, Bustamante et al. (2021)Bustamante, D., Savón, L., Elías, A., Caro, Y., Valiño, E.C., Rojas, M., Martin, C. & Mireles, S. 2021. Chemical and microbiological characterization of a technological variant of Vitafert intended for animal production. Technical note. Cuban Journal of Agricultural Science, 55(2): 61-73, ISSN: 2079-3480. http://cjascience.com/index.php/CJAS/article/view/940. suggested that, when products are air-dried and sun-dried, fermentation may continue for a short period and affect protein percentage. This is believed to depend on the proportions in which mixtures are made and the absorption capacity of the material used.
Nutrient concentration also occurs as a result of dehydration. Additionally, these values may increase due to the nutrient content of the material used as absorbent. This study used a mixture of wheat bran and barley meal, which fundamentally influenced on protein and fiber percentage. The aforementioned meals are widely available in Ecuador and contain a maximum of 23.5 % of protein, 16 % of fiber, 8 % of ash, and 14.5 % of humidity (Molinos Miraflores S.A., Ecuador, https://www.molinosmiraflores.com and Prodal S.A., Ecuador, https://www.prodal.com.ec).
Some educational and research work, conducted in Ecuador, addresses the topic of obtaining zootechnical additives. Díaz et al. (2014)Díaz, B.L., Elías, A. & Valiño, E. 2014. Consorcios microbianos con actividad ácido-láctica promisoria aislados desde inoculantes bacterianos nativos para ensilajes. Ciencia y Agricultura, 11(1): 17-25, ISSN: 2539-0899. https://doi.org/10.19053/01228420.3484. obtained a microbial preparation with lactic acid bacteria (106 cfu/mL), total aerobic bacteria (106 cfu/mL), and yeasts (105 cfu/mL), enzymes, organic acids, and a pH of 3.87 through submerged fermentation with agricultural and industrial byproducts. This preparation was applied as an inoculum to post-harvest agricultural residue silages intended for dairy cows, stimulating milk production and increasing milk fat and protein by 11 %. Subsequently, Flores-Mancheno et al. (2016)Flores-Mancheno, L.F., García-Hernández, Y., Usca-Méndez, J.E. & Caicedo-Quinche, W.O. 2016. Estudio comparativo de tres aditivos zootécnicos en el comportamiento productivo y sanitario de cerdos en el período post-destete. Ciencia y Agricultura, 13(2): 95-105, ISSN: 2539-0899. https://doi.org/10.19053/01228420.v13.n2.2016.5557. evaluated the same microbial consortium in growing-fattening pigs and confirmed its beneficial effect on animal health and increased productive yield. However, there was no information in the available scientific literature on additives developed in the country using the procedure proposed in this study.
An example of an additive, which production method is similar to the one used in this research, is Vitafert and its variants, and its beneficial effects have been demonstrated in monogastric animals and ruminants (Valiño et al. 2024Valiño, E., García, Y., Bustamante, D. & Beruvides, A. 2024. Desarrollo y utilización del Vitafert en el sector agropecuario. Cuban Journal of Agricultural Science, 58: e17, ISSN: 2079-3480. http://cjascience.com/index.php/CJAS/article/view/1153.). Dry Vitafert with corn meal showed a similar composition to the product under study, containing 90 % of dry matter, 14.58 % of crude protein, a pH of 5.41, a high concentration of lactic acid bacteria (15x107 cfu/g), an absence of pathogenic microorganisms, and a high content of non-essential amino acids (glutamic acid, proline, aspartic acid, alanine, serine, cystine, tyrosine, and glycine) and essential amino acids (leucine, arginine, valine, phenylalanine, lysine, and threonine).
The chemical and microbiological characteristics of the additive demonstrated that it contains bacteria, yeasts, and nutrients in adequate concentrations, as well as good hygienic and sanitary quality. Therefore, it is considered to have potential for use as a zootechnical additive in animal production. It is recommended that these properties may be considered for future research. It is also suggested to develop future studies directed toward determining the biological response of different categories and species of animals to its use.