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Fermented products are important for animal production, due to the versatility in their application, either as additives, supplements or food (Xu et al. 2020).
At the Institute of Animal Science, in Cuba, a microbial preparation called Vitafert (Elías and Herrera 2011) was obtained, composed of lactic acid bacteria, yeasts and organic acids that promote a low pH. Several researches describe its use as a zootechnical additive, with positive effects on immunological, productive and health indicators of species of economic interest (Gutiérrez 2005, Gutiérrez et al. 2012 and Beruvides et al. 2018).
For years, different variants and formulations of this biopreparation have been tested, which are related to its form of presentation (liquid/dry), inoculum used (feces/yogurt/ruminal content) and modifications in raw materials (final molasses/raw sugar), used to obtain it (Bustamante et al. 2016, Beruvides 2019 and Savón et al. 2020).
The knowledge of the composition of this preparation is necessary for its application in animal feeding. However, there are few studies on the characterization of these microbial additives.
The objective of this study was to chemically and microbiologically characterize a technological variant of Vitafert, intended for animal production.
Vitafert was prepared in the Food Production Laboratory, belonging to the Central Laboratory Unit of the Institute of Animal Science of the Republic of Cuba. The methodology described by Elías and Herrera (2011) was followed for this purpose. The chemical composition of this preparation is: pH 4.28 ± 0.08, lactic acid 452.0 mMol.L-1, acetic acid 227.36 mMol.L-1, butyric acid 85.32 mMol.L-1, DM 90.9 g/kg ± 0.08 and CP 80.2 g/kg DM ± 0.10. Populations of lactic acid bacteria and yeasts were 1010 and 107 CFU.g -1, respectively. The presence of Escherichia coli was not detected.
The technological variant was prepared from the mixture of Vitafert with corn meal 1:1 (w/v), as proposed by Gutiérrez (2005). Drying was carried out by direct exposure to sun for 72 h, with mean maximum temperature values of 27.3 °C and relative humidity of 76%. Dry Vitafert was stored in double-layer paper bags for five days. Five samples of 1 kg each were taken to perform chemical and microbiological analyzes. Particle size was reduced to 1 mm in a Thomas Wiley mill (Model 4. Thomas Scientific, Swedesboro, NJ).
Chemical analyzes were conducted at the Department of Animal Science at the University of Manitoba, Canada. Standard procedures of the AOAC (2006) were used for calculating DM, CP, ether extract and amino acid profile. Gross energy was determined on an adiabatic bomb calorimeter (model 6400. Parr Instrument Company, Moline, Illinois, USA). Fiber fractioning was performed according to van Soest et al. (1991).
To determine the pH and the concentration of individual short chain fatty acids (iSCFA), 5 g of dry Vitafert were taken and diluted in 45 mL of distilled water 1:10 (w/v) by mechanical stirring for 20 min. The pH was determined with a digital pH meter, WPA brand (CD-70 series of English manufacture). The quantification of iSCFA was carried out in a DANI Master GC gas chromatograph (DANI Instruments S.p.A, Milan, Italy).
A 1 mL aliquot was taken from the aqueous extraction of dry Vitafert to make the serial dilutions in peptone water (0.1% w/v). The 10-5, 10-6 and 10-7 dilutions were cultivated in tubes rolled with Rogosa agar (Rogosa et al. 1951). Tubes were inoculated at 1:10 (v/v) and incubated for 48 h at 37 °C. The number of CFU.mL-1 was quantified by visual counting of colonies. The microbiological quality was carried out in the National Laboratory of Food Hygiene (National Reference Center), belonging to the Central Laboratories Unit for Agricultural Health of the Ministry of Agriculture in Cuba. The current Cuban standards for yeasts and fungi/g (NC-ISO 1004: 2016), Salmonella in 25 g (NC-ISO 6579: 2008) and total coliforms/g (NC-ISO 4832: 2010) were applied.
Five samples were analyzed by triplicate for chemical analysis. In the case of amino acids, a determination was made by duplicate.
For the chemical indicators, the position and dispersion statistics: mean, standard deviation and coefficient of variation (%), were determined. InfoStat statistical package, version 2012 (Di Rienzo et al. 2012), was used.
Table 1 shows the chemical and energy composition of dry Vitafert. DM content was superior to 85%, an aspect that must be considered for the conservation and storage of the product. According to Belén-Camacho et al. (2007), the reduction of humidity content increases the useful life of products, since it causes a decrease of water activity, which delays microbial growth and slows down various deteriorating reactions.
Table 1 Chemical and energy composition of dry Vitafert
| Nutrients, % | Mean | SD | CV (%) |
|---|---|---|---|
| DM | 90.11 | 6.58 | 0.17 |
| CP | 14.58 | 0.02 | 0.11 |
| Ether extract | 2,58 | 0.13 | 1.20 |
| Gross energy* | 18.40 | 0.04 | 0.19 |
| NDF | 11.32 | 0.42 | 0.75 |
| ADF | 3.10 | 0.16 | 1.02 |
| Hemicellulose | 8.22 | 0.30 | 0.28 |
*expressed as MJ
The high content of CP could be associated with biochemical transformations that occur during the absorption time of water present in the system on the solid support and during drying in the sun. This suggests that fermentation process was able to continue. According to Anguita et al. (2006), the biotechnological processing of cereals, rich in carbohydrates, favors an increase of starch hydrolysis and, consequently, an increase of the amount of non-starch polysaccharides. For this reason, microbial populations present in the product were able to use soluble corn starch to increase single-cell biomass. Similarly, the protein contribution of corn meal used as absorbent material is another factor that could have contributed to the increase in protein in the final product.
In general, values of the rest of dry Vitafert indicators can be a result of the concentration of nutrients that takes place during the drying process, together with the previously described aspects.
There are few studies that deepen into the changes that occur during the drying of liquid fermented products. However, it is known about other technological variants which used the same support for this operation.
Gutiérrez (2005) characterized the dry preparation, obtained from the fermentation of fresh poultry manure with final molasse, and observed a rise in protein content from 8.01 to 13.49%. Savón et al. (2017) indicated a value of 8.04% for the dry fermented product, made from rumen content. Variation in results can be related to modifications in the formulation of Vitafert.
Figure 1 shows the amino acid profile of dry Vitafert, with the ten essential amino acids and eight of the non-essential ones. A high content of non-essential amino acids was observed, in the following order: glutamic acid, proline, aspartic acid, alanine, serine, cystine, tyrosine and glycine. However, leucine, arginine, valine, phenylalanine, lysine and threonine predominated in the essentials. The amino acid content of Vitafert is not known, so its availability and concentration in this technological variant could be determined by the factors described above.
The contribution of amino acids is important because they regulate or participate in key metabolic pathways for growth, development, reproduction and health of animals. For this reason, the composition of this fermented product could help meeting the requirements of different monogastric species, due to its contribution of functional amino acids. Wu et al. (2014) stated that functional amino acids contribute to the metabolic needs of the animal for dietary amino acid, beyond the synthesis of tissue protein, in which essential, non-essential and conditionally essential amino acids are included.
The pH of dry Vitafert is acidic, even though the concentration of individual short chain fatty acids is low (table 2). This performance could be related to drying at room temperature, together with the dilution effect of corn meal, which could cause the volatilization of these acids. However, there was a higher concentration of mMol.L-1 of acetic acid, with a reduction of 7.12% with respect to liquid Vitafert. This is associated with the decrease of bacteria populations that produce this acid.
Table 2 Microbiological composition, pH and profile of individual short chain fatty acids of dry Vitafert
| Mean | SD | CV (%) | |
|---|---|---|---|
| pH | 5.41 | 0.02 | 0.45 |
| Individual short chain fatty acids, mMol.L -1 | |||
| Acetic | 16.17 | 0.43 | 2.69 |
| Propionic | 0.40 | 0.01 | 2.58 |
| Butyric | 0.29 | 0.01 | 2.59 |
| Isovaleric | 0.08 | 0.02 | 1.42 |
| Valeric | 0.09 | 0.06 | 1.02 |
| Microorganisms, CFU.g -1 | |||
| Lactic acid bacteria | 15 x 107 | 0.05 | 0.01 |
| Yeasts and fungi | <102 | - | - |
| Salmonella | Absent | - | - |
| Total Coliforms | <102 | - | - |
Yeh et al. (2018) observed that pH of a fermented diet (inoculated with 106 CFU.g-1 food of Bacillus subtilis and Bacillus coagulans) remained acidic during drying with the use of high temperatures. Authors reported that acetic acid content decreased, while butyric and propionic acids were below detection limits. These results indicate that drying does not affect the acidity of the additive, but its concentration of short chain fatty acids does.
The microbiological composition of dry Vitafert showed a high concentration of lactic acid bacteria, a permissible number of yeasts, fungi and an absence of pathogens. However, during the drying process of Vitafert, there was a reduction of 102 CFU.g-1 of lactic acid bacteria and the content of yeast present in the liquid Vitafert was reduced five times. This corresponds to the low concentration of individual short chain fatty acids that was obtained and, probably, with the dilution effect of corn meal as absorbent material.
Even though the concentration of lactic acid bacteria was reduced with this technological variant, it is within the range of 106-107 CFU.g-1, established by FAO/WHO (2002). This indicates that the inclusion of these microorganisms in the diet could have beneficial effects for the animal and express a possible probiotic activity, when ingested in sufficient quantities.
Concentration of viable lactic acid bacteria, acid characteristics and decrease of the number of pathogenic microorganisms contributed to the hygienic-sanitary quality of dry Vitafert.
