Conflict of interest: The authors declare that there are no conflicts of interests among them
Author´s contribution: A. Beruvides: Design and conducting the experiment, data analysis, manuscript writing. ElÃas,A†. Elaine C. Valiño: Manuscript writing and revision. Grethel Milián: Manuscript writing and revision. Ana J. Rondón: Manuscript writing and revision. Marlen RodrÃguez: Manuscript writing and revision. J. Milián: Manuscript writing and revisión
0103202103202155157651007202024112020This is an open-access article distributed under the terms of the Creative Commons Attribution LicenseAbstract
The objective of this research was to chemically and microbiologically characterize the zootechnical additive VITAFERT and evaluate its stability for 90 days. To analyze the results of the chemical and microbiological characterization, descriptive statistics (mean, standard deviation and coefficient of variation) was applied. One-way analysis of variance was used to study stability. INFOSTAT package, version 2012, was also used. The performance of the chemical composition was similar similar in the five studied batches, with mean for dry matter (9.70%), ashes (10.5%), calcium (1.33%), phosphorus (0.65%), crude protein (7.12%) and pH (4.0). The additive showed high counts of lactic acid bacteria, with values ​​between 3.24 x 1012 CFU.mL-1 and 4.95 x 1012 UFC.mL-1 and yeasts between 7.00 x 107 CFU.mL-1 and 9.66 x 107 CFU.mL-1 respectively, as well as a pH of 4.0. The stability study showed that the product maintains a pH of 4.0 up to 90 days. It is concluded that the zootechnical additive VITAFERT has chemical and microbiological characteristics for its application in animal feed.
In animal production, it is essential to know the bromatological composition of food, such as the levels of protein, fiber, energy and minerals, to establish the nutritional balance in the diets intended for animals. At the same time, it is necessary to determine the microbiological quality of feed to prevent it from affecting animal performance, in terms of intake, digestibility and absorption of nutrients (Lezcano et al. 2014, Caicedo 2015, Brea 2015 and Milián et al. 2019).
For the above reasons, the study of microbiological and chemical characteristics of an additive constitutes a premise to introduce a new product in animal feeding (Caicedo and Valle 2017 and RodrÃguez et al. 2020). In previous studies of ElÃas and Herrera (2008) and Vitaluña (2014), information is provided about the chemical and microbiological characterization of VITAFERT, produced in laboratory-scale fermenters. However, it is unknown whether this composition is maintained under small-scale production conditions. Therefore, the objective of this research was to chemically and microbiologically characterize the zootechnical additive VITAFERT, obtained under small-scale production conditions.
Materials and Methods
Obtaining the inoculum. The inoculum was obtained from natural yogurt, produced in the Empresa Combinado de la Industria Láctea (ECIL) of Matanzas, Cuba. This product was produced with strains from the Instituto de Investigaciones de la Industria de los Alimentos (IIIA) collection: Streptococcus salivarius subspecies thermophilus and Lactobacillus delbrueckii subspecies bulgaricus. The culture was at a concentration of 107 CFU.mL-1, which corresponds to normal values ​​for the production of natural yogurt. The inoculum was stored at 4 °C until its use.
To obtain the microbial preparation, the methodology proposed by ElÃas and Herrera (2008), modified by Beruvides et al. (2018), was used as a guideline. For this research, five batches were elaborated, prepared in 20 L plastic tanks at the same time, in which all the components were weighed and mixed with the substitution of the final molasses, as carbon source, for raw sugar plus the addition of inoculum (natural yogurt). The biopreparation was kept in fermentation for 96 h, at room temperature (24 oC), and was activated every 12 h by shaking with a wooden paddle. Its formulation is presented in table 1, as well as the energy and protein contributions of the used raw materials.
Formulation of the zootechnical additive VITAFERT, obtained under small-scale production conditions
Composition
Inclusion levels, kg
Contribution
Energy, MJ.kg-1
Protein, %
Inoculum (natural yogurt) **
1
3.014
0.3
Corn meal*
4
0.040
0.85
Soy bean meal*
4
0.039
0.83
Urea***
0.5
-
281
Ammonium sulfate*
0.25
-
21
Mineral salt *
0,5
-
-
Raw sugar *
15
0.041
-
Water
100L
-
-
Source: NRC (2012) *, IIIA**, De Blos et al. (2007) ***
Chemical characterization. Three samples were taken from each batch of VITAFERT to determine the content of dry matter (DM), ashes (C), calcium (Ca), phosphorus (P) and crude protein (CP), according to the methodology described by AOAC (2010).
Determination of pH and count of lactic acid bacteria (LAB) and yeasts. To determine pH performance and the presence of these microorganisms during the production of this zootechnical additive, measurements were made every four hours until 96 h in the five studied batches.
To count the LAB and yeasts, serial dilutions of samples (1:10, v/v) were made in peptone water up to 10-11. Of these dilutions, in the first 12 h, 10-7, 10-8 and 10-9 were used for LAB, and 10-9 10-10 and 10-11 for the following hours, for the purpose of deep cultivation on plates with MRS agar (De Mann et al. 1960) (BIOCEN, Cuba). For yeasts, dilutions 10-3, 10-4 and 10-5 were taken in the first 12 h, and later those of 10-5, 10-6 and 10-7 were used. Each of them was repeated three times (1 mL) on Rose Bengal Agar (Rose Bengal 0.05% and chloramphenicol 0.5%) (HISPANLAB, Spain). After incubation at 37 ºC (for 72 h for LAB and 48 h for yeasts), the microbial count was carried out. The number of CFU was determined by visual counting of colonies using a magnifying glass.
Count of contaminating microorganisms. It was carried out in accordance with current standards, described for studies of the microbiological quality of food for human and animal intake NC-ISO (table 2). For this, serial dilutions of samples were carried out (NC ISO 6887-1: 2002) and the techniques for determining the different groups of microorganisms were performed.
Microbiological tests for the determination of contaminating microorganisms in the zootechnical additive VITAFERT
Microbiological tests
References NC- ISO
Count of total and fecal coliforms
4832: 2010
Count of Bacillus cereus
4833-1: 2014
Count of Salmonella in 25 mL
6579: 2008
For the study of VITAFERT stability at room temperature (24 ± 5ºC) for 90 d, an experiment with a completely randomized design was developed. It was carried out in the microbiology laboratory of the Planta de Conservas y Alimentos Libertad, in Colón municipality, Matanzas province, Cuba.
According to the methodology described above, 20 L of the zootechnical additive were prepared and distributed into plastic containers of 1 L capacity that were kept indoors. During days 1, 3, 7, 15, 30, 60 and 90, three samples were taken for analysis. The stability of this biological product was determined from the pH analysis and the counting of LAB and viable yeasts.
Statistical analysis. Dispersion statistics (mean, standard deviation and coefficient of variation) were used for the chemical and microbiological characterization of VITAFERT. The pH values, concentrations of LAB and yeasts were processed by means of a one-way analysis of variance and Duncan (1955) test for P<0.05. Data was processed in INFOSTAT statistical package, version 2012 (Di Rienzo et al. 2012).
Results and Discussion
Tables 3 and 4 show the results of the chemical and microbiological characterization of VITAFERT. Chemical composition values were in correspondence with the determinations reported by ElÃas and Herrera (2008), except DM, which presented figures in the order of 9.72%. Meanwhile, the cited authors obtained values of 15.05%. These results were related to the substitution of molasses for raw sugar.
Chemical composition of the zootechnical additive VITAFERT
Statistics Indicators, %
Mean
SD
CV
Dry matter
9.72
0.05
0.46
Ashes
10.52
0.08
0.80
Calcium
1.33
0.01
0.41
Phosphorous
0.65
0.01
2.01
Crude protein
7.12
0.02
0.26
pH
3.95
0.05
1.29
Results are the average of three determinations SD- standard deviation; CV- coefficient of variation, %
Microbiological composition of the zootechnical additive VITAFERT, after fermentation at 96 h. Microorganism count (CFU mL-1)
It is confirmed that the methodology applied to obtain this biopreparation under small-scale production conditions does not cause considerable variations in its chemical composition, when compared to laboratory-scale fermenters. Data indicate that there is repeatability in the results. This means that when these components are used, under the same conditions, there are no changes in the studied parameters.
Flores-Mancheno et al. (2015) characterized the pH of a biological product, intended for pigs in the pre-fattening and growth-fattening stages, formulated with fresh whey, urea and sugar cane molasses. These authors reported a pH of 3.87, similar to that obtained in the current study. This could be related to the presence of a considerable population of LAB, which produces organic acids (lactic, acetic, propionic and butyric) and lowers the pH (Belkacem-hanfi et al. 2014). Studies carried out by Caicedo and Valle (2017) reported a similar performance to that observed in this study for pH, when they elaborated a microbial biopreparation destined for pigs, which contained natural yogurt, whey, B molasses and taro tubers.
The microbiological analysis did not show contaminating microorganisms in VITAFERT samples (table 4). These results may be caused by the presence of high concentrations of organic acids (mainly lactic and acetic) or bacteriocins provided by LAB (superior to 109 CFU.mL-1), which allow the product to be kept free of contaminants, which makes it viable for the use in animals.
Figure 1 shows the results of LAB and yeast count over time. It was found that during fermentation process, LABs grow to values ​​of 29 natural logarithm (NL) UFC.mL-1 and yeasts, in the order of 20 NL UFC.mL-1.
Growth kinetics of lactic acid bacteria and yeasts and pH performance during the fermentation process of the zootechnical additive VITAFERT
a,b,c,d,e,f,g,h,i,j Means with different letters differ for P<0.05 (Duncan 1955) (LAB: SE=±0.10, P=0.0262; yeasts: SE=±0.03, P=0.0348; pH=±0.01, P=0.0654).
The pH decreased from 8.5 to 4.0 from the beginning of fermentation until 96 h. By studying the growth kinetics of LABs and yeasts in the small-scale fermentation process, it was found that as pH decreases, the growth of both microbial groups progressively increases. This confirms the statements of León (2012), who informed that these microorganisms can grow in a wide pH range (between 4-7), unlike other microbial groups such as coliforms, Salmonella spp. and Bacillus spp., which inhibit their growth under acidic conditions (Pavlović et al. 2016). These values of pH ​​and microbial concentration are in the optimal ranges for the application of this product for feeding pigs. However, it is not known if this composition is maintained over time, so it is necessary to study its stability for a period of 90 d.
Stability performance of the microbiological and chemical indicators in the zootechnical additive VITAFERT under small-scale production conditions for 90 d
a,b,c,d Means with different letters differ for P<0.05 (Duncan 1955) (LAB: SE ± 0.07 P = 0.04568; yeasts: SE ± 0.05, P = 0.0152; pH: SE ± 0.02, P=0.5670)
Figure 2 shows the pH values ​​and LAB and yeast counts of the different batches of VITAFERT. These determinations are within the ranges established by ElÃas and Herrera (2008), and Beruvides (2013).
As demonstrated in figure 2, yeasts from three to 15 d maintained values ​​of 107 CFU.mL-1. Subsequently, there was a decrease of viable cells (106 CFU.mL-1) from 30 d, remaining in this order until the end of the study. LABs gradually increased during the first 15 d to 1012 CFU.mL-1. Then, there was a decrease to 109 CFU.mL-1 at 90 d. The pH showed a value of 4.0 after 72 h, and maintained its stability until 90 d. This result could be associated with the production of organic acids (lactic, acetic, propionic and butyric) by LABs (Vázquez et al. 2009).
The results agree with those obtained by Brizuela (2003), who evaluated some stability parameters of a biopreparation for probiotic purposes, intended for pigs and made with Lactobacillus rhamnosus strains. Similar studies were carried out by Rondón (2009), in which the stability of two biopreparations of Lactobacillus salivarius subspecies salivarius C-7 and C-65 was determined. This author found that, after 30 days, LABs viability is affected, because, under these conditions, cells continue their metabolism and the essential nutrients for their development are consumed.
RamÃrez et al. (2011) and Powthong and Suntornthiticharoen (2015) report that the presence of LAB in biological products guarantees safety and stability for their use in animal feed. These microorganisms have several applications, and one of the most important is food fermentation (milk, meat and vegetables) to obtain products such as yogurt, cheese, pickles, sausages and silage. In this way, it contributes to the biopreservation and quality of food sensorial characteristics.
When carbohydrates ferment, LAB microorganisms produce a mixture of compounds with antimicrobial action, such as lactic acid, acetic acid, butyric acid, hydrogen peroxide, diacetyl and low molecular weight peptides, called bacteriocins, which inhibit the proliferation of other microbial groups that do not tolerate the presence of these compounds (RodrÃguez et al. 2013).
Caicedo and Valle (2017) reported that pH is a very important indicator for fermentation processes. Its decrease is one of the most substantial changes that occur during the production of a biopreparation. Adedeji et al. (2011) defined that pH is directly related to the degradative processes that occur during conservation. In this sense, when a biological product reaches pH values ​​between 3.8 and 4.2, its stability is achieved. This condition causes a restriction of the activity of proteolytic enzymes and the suppression of enterobacteria and Clostridium (López et al. 2013). The results of the current research are in the range of pH values ​​recommended for this type of additive.
According to Rendón et al. (2015), the action mechanisms of biological products with pH values ​​equal or inferior to 4, imply the inhibition of growth of pathogenic bacteria, the production of lactic acid and the decrease of intestinal permeability when diarrhea occurs, as well as the increase of lactase activity and immunity stimulation. In this way, pH decrease in the obtained product is an indicator of the presence of lactic acid bacteria, as resulted in this research.
It is concluded that the methodology used for the preparation of the zootechnical additive VITAFERT, under small-scale production conditions, allowed to obtain a good quality biological product, composed of high levels of LAB and yeasts. These levels of microorganisms within the microbial preparation show stability in their viability up to 15 d, while the pH remains with a value of 4 for 90 d.
En la producción animal es de gran importancia conocer la composición bromatológica de los alimentos, como los niveles de proteÃna, fibra, energÃa y minerales para establecer el balance alimentario en las dietas destinadas a los animales. Al mismo tiempo, es necesario determinar la calidad microbiológica del alimento para evitar que pueda afectar el comportamiento animal, en cuanto al consumo, la digestibilidad y la absorción de nutrientes (Lezcano et al. 2014, Caicedo 2015, Brea 2015 y Milián et al. 2019).
Obtención del inóculo. El inóculo se obtuvo a partir de yogurt natural, producido en la Empresa Combinado de la Industria Láctea (ECIL) de Matanzas, Cuba. Este producto se elaboró con cepas procedentes de la colección del Instituto de Investigaciones de la Industria de los Alimentos (IIIA): Streptococcus salivarius subespecie thermophilus y Lactobacillus delbrueckii subespecie bulgaricus. El cultivo se encontraba a una concentración de 107 UFC.mL-1, lo que se corresponde con los valores normales para la producción de yogurt natural. El inóculo se conservó a 4ºC hasta su utilización.
Fuente: NRC (2012)*, IIIA**, De Blos et al. (2007)***
Caracterización quÃmica. Se tomaron tres muestras de cada lote de VITAFERT para determinar el contenido de materia seca (MS), cenizas (C), calcio (Ca), fósforo (P) y proteÃna bruta (PB), según la metodologÃa descrita por AOAC (2010).
Para el estudio de la estabilidad del VITAFERT a temperatura ambiente (24 ± 5ºC) durante 90 d, se desarrolló un experimento con diseño completamente aleatorizado. Este se realizó en el laboratorio de microbiologÃa de la Planta de Conservas y Alimentos Libertad, del municipio Colón, ubicado en la provincia Matanzas, Cuba.
Análisis estadÃstico. Para la caracterización quÃmica y microbiológica del VITAFERT se utilizaron los estadÃgrafos de dispersión (media, desviación estándar y coeficiente de variación). Los valores de pH, concentraciones de BAL y levaduras se procesaron mediante un análisis de varianza de clasificación simple y la dócima de Duncan (1955) para P < 0.05. Los datos se procesaron en el paquete estadÃstico INFOSTAT, versión 2012 (Di Rienzo et al. 2012).
Resultados y Discusión
En las tablas 3 y 4 se muestran los resultados de la caracterización quÃmica y microbiológica del VITAFERT. Los valores de la composición quÃmica estuvieron en correspondencia con las determinaciones informadas por ElÃas y Herrera (2008), a excepción de la MS, que presentó cifras en el orden de 9.72 %. En tanto, los autores citados obtuvieron valores de 15.05 %. Estos resultados se relacionan con la sustitución de la miel de caña de azúcar por azúcar crudo.
Se confirma que la metodologÃa aplicada para la obtención de este biopreparado en condiciones de producción a pequeña escala no provoca variaciones considerables en su composición quÃmica, si se compara con fermentadores a escala de laboratorio. Los datos indican que existe repetibilidad en los resultados obtenidos. Esto significa que cuando se emplean estos componentes, en las mismas condiciones, no se producen modificaciones en los parámetros estudiados.
En la figura 1 se presentan los resultados del conteo de BAL y levaduras en el tiempo. Se comprobó que durante el proceso de fermentación, las BAL crecen hasta valores de 29 logaritmo neperiano (LN) UFC.mL-1 y las levaduras, en el orden de 20 LN UFC.mL-1.
a,b,c,d,e,f,g,h,i,j Medias con letras diferentes difieren para P < 0.05 (Duncan 1955) (BAL: EE = ± 0.10, P = 0.0262; levaduras: EE = ± 0.03, P = 0.0348; pH = ±0.01, P = 0.0654).
a,b,c,d Medias con letras diferentes difieren para P < 0.05 (Duncan 1955) (BAL: EE ± 0.07 P = 0.04568; Levaduras: EE ± 0.05, P = 0.0152; pH: EE ± 0.02, P=0.5670)
En la figura 2 se presentan los valores de pH y conteos de BAL y levaduras de los diferentes lotes de VITAFERT. Estas determinaciones se encuentran en los rangos establecidos por ElÃas y Herrera (2008) y Beruvides (2013).
RamÃrez et al. (2011) y Powthong y Suntornthiticharoen (2015) refieren que la presencia de las BAL en los productos biológicos garantiza la seguridad y estabilidad para su uso en la alimentación animal. Estos microorganismos tienen diversas aplicaciones, y una de las más importantes lo constituye la fermentación de alimentos (leche, carnes y vegetales) para obtener productos como el yogurt, el queso, los encurtidos, embutidos y ensilajes. De esta forma se contribuye a la biopreservación y a la calidad de las caracterÃsticas sensoriales de los alimentos.
Caicedo y Valle (2017) refirieron que el pH es un indicador de gran importancia en los procesos fermentativos. Su disminución es uno de los cambios más sustanciales que se producen durante la elaboración del biopreparado. Adedeji et al. (2011) definieron que el pH se relaciona directamente con los procesos degradativos que ocurren durante la conservación. En este sentido, cuando un producto biológico alcanza valores de pH entre 3.8 y 4.2, se logra su estabilidad. Esta condición hace que ocurra una restricción de la actividad de las enzimas proteolÃticas y la supresión de enterobacterias y Clostridium (López et al. 2013). Los resultados de esta investigación se encuentran en los Ãndices de pH recomendados para este tipo de aditivo.
Según Rendón et al. (2015), los mecanismos de acción de los productos biológicos con valores de pH iguales o inferiores a 4, implican la inhibición del crecimiento de bacterias patógenas, la producción de ácido láctico y la disminución de la permeabilidad intestinal cuando se producen diarreas, asà como el aumento de la actividad de la lactasa y la estimulación de la inmunidad. De esta manera, la disminución del pH en el producto obtenido es indicador de la presencia de bacterias lácticas, como resultó en esta investigación.