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Cuban Journal of Agricultural Science

Print version ISSN 0864-0408On-line version ISSN 2079-3480

Cuban J. Agric. Sci. vol.54 no.4 Mayabeque Oct.-Dec. 2020  Epub Dec 01, 2020


Animal Science

Effect of calcium carbonate on solid fermentation of Solanum tuberosum post-harvest wastes, inoculated with a microbial preparation

L. M. Borrás1  *

Elaine. C. Valiño2 

A. Elías †2 

J. J. Martínez1

A. M. Sanabria1 

Mónica L. Becerra1

1Pedagogical and Technological University of Colombia, GIBNA research group UPTC. Avenida Central del Norte, Tunja, Boyacá, Colombia

2Instituto de Ciencia Animal (ICA). Carretera Central km 47 y medio, San José de las Lajas, Apartado Postal 24


To evaluate the effect of including calcium carbonate (CaCO3) in solid fermentation of post-harvest wastes of Solanum tuberosum (potato), inoculated with a microbial preparation, a completely randomized design was used, with three repetitions per treatment. Indicators pH, dry matter, crude protein, true protein and concentration of microorganisms were analyzed according to a 2x2x4 factorial arrangement, in which factors were fermentation time (24 and 48 h), incubation temperature (20 and 25ºC) and percentage of inclusion of CaCO3 (0, 0.25, 0.50 and 0.75%). For the concentration of organic acids and ammonia, a 2x2x3 factorial arrangement was used, which differed from the previous one in the percentage of CaCO3 (0.25, 0.50 and 0.75%). It was obtained that pH increased by adding CaCO3 at 0.25 and 0.50% and incubating at 20 or 25 ºC. Meanwhile, pH decreased with fermentation and incubation at 20ºC. Lactic acid production increased with both temperatures and during fermentation, and growth of mesophilic and lactic aerobic bacteria was favored with the inclusion of CaCO3. With fermentation, dry matter and crude and true protein percentages increased. The highest values of these indicators were obtained with 0.50% of CaCO3 at 48 h (40.90, 19.09 and 13.8%, respectively). However, protein synthesis was the same at 20 °C and 25 °C (72.76%). It is concluded that CaCO3 at 0.50% favors the fermentation of post-harvest wastes of S. tuberosum with the microbial preparation, at 20 °C and 24 h.

Keywords: potato; silage; lactobacilli; additive

Potato (Solanum tuberosum) contains nutritional components (energy, macronutrients and micronutrients), as well as non-nutritional components (water, cellulose, hemicellulose, pectin, glycoalkaloids, organic acids, enzymes, among other minority ones). After harvest, tubers contain 80% water and 20% dry matter on average. Of the latter, 60% corresponds to starch (FAO 2015). Organic acids contribute to the characteristic pH of food, which ranges from 5.6-6.2. The most representative are malic and citric acids, as well as the chlorogenic one, which reacts with iron ions. This component and the significant amounts of carbohydrates, mostly starch, and a small percentage of sugars (sucrose, fructose and glucose), propitiate a considerable decrease of pH during its fermentation process.

In post-harvest waste fermentations, pH performance is measured as a quality indicator. If this has a very low value, it limits bacterial growth (Muck et al. 2018), altering the chemical composition of the fermented product and ruminal synthesis in the animal (Elías et al. 1990 and Yang et al. 2015). The pH also depends on the starter culture, which are generally lactic acid bacteria (LAB). With the development of genetics, molecular biology, physiology and biochemistry, and with the discovery of the complete genome sequence of a large number of lactic acid bacteria, new knowledge and applications appeared for these bacteria and a variety of starter and protective cultures, which possess desirable properties (Bintsis 2018).

In the technological process for the development of products for animal feeding, fermentations are essential and must be correctly defined to achieve high productive yields (Sosa et al. 2018). According to Borras (2017), the fermentation of post-harvest wastes of S. tuberosum with a microbial preparation with lactic acid activity affects microbial growth, due to the rapid decrease in pH and high humidity percentage. Therefore, the inclusion of the additive CaCO3 is suggested as acid neutralizer. The stabilizing, thickening and anticaking properties of this compound can cause a technological change and influence on the performance of some fermentative and chemical indicators of the final product.

The objective of this study was to evaluate the inclusion of CaCO3 in the kinetics of solid fermentation of post-harvest wastes of S. tuberosum, inoculated with a microbial preparation with lactic activity.

Materials and Methods

Solid-state fermentation (SSF) experiment was carried out under high tropic conditions (2,860 m.a.s.l.), in the biochemistry and animal nutrition laboratory of the Universidad Pedagógica y Tecnológica of Colombia (UPTC), located on Avenida Central del Norte, via Tunja-Paipa, in Tunja municipality, Boyacá department, Colombia. This region has a mean temperature of 15 °C and mean annual rainfall of 553 mm.

Experimental procedure. A yogurt was made with active strains of Lactobacillus delbrueckiis ssp bulgaricus and Streptococcus thermophilus (commercially freeze-dried, Liofast Y452B, SACCO ®), which was used as inoculum (2% v/v and concentration of 0.99 x 108 CFU/mL) for obtaining the microbial preparation, according to the methodology of Borras (2017). This preparation (2%) was mixed at room temperature (15 ± 2 ºC) with urea (1%), mineral premix (0.50%), sodium sulfate (0.50%), calcium carbonate and potato post-harvest wastes, previously cleaned and chopped. The inclusion percentage of calcium carbonate (CaCO3) varied according to the experimental treatments (0, 0.25, 0.50 and 0.75%).

Table 1 shows the composition of mixtures prepared under the mentioned conditions (0h and 15 ºC). They were distributed in plastic bags, with 1 kg capacity. Each bag was considered an experimental unit, with three repetitions per treatment. They were divided into two groups and incubated at 20 and 25 °C respectively, in individual Memmert® incubators for 48 h. Samples were taken at 24 and 48 h of fermentation to determine chemical and microbiological indicators.

Table 1 Chemical and microbiological composition of mixtures with potato post-harvest wastes, prepared at 15 ºC and without fermenting (n=3) 

Indicator Mixture with inclusion of CaC03, %
0 0.25 0.50 0.75
pH 6.31 7.11 7.12 7.21
Dry matter, % 21.70 21.49 21.22 20.96
Crude protein, % 15.26 15.01 15.32 15.65
True protein, % 10.70 10.58 11.15 11.24
Lactic acid, mmol/L 16.86 16.86 16.85 16.84
Propionic acid*, mmol/L 14.33 14.33 14.32 14.31
NH3, meq/mL 1.79 1.80 1.79 1.78
Mesophilic aerobic bacteria**, CFU/mL 2.1x105 2.5x105 2.0x105 1.1x105
Yeast**, CFU/mL 2.5x104 1.1x104 1.1x104 1.9x104
Lactic acid bacteria**, CFU/mL 7.5x105 2.0x105 1.2x106 1.2x106

*Not significant concentrations of acetic, butyric, isovaleric and isobutyric acid

** Dissolved mixtures at a rate of 1/10 (w/v) in a NaCl solution (0.85%, w/v)

Contents of the bags of each treatment (three repetitions) were collected and homogenized. Then, 5 g of sample were taken and placed in a 100 mL Erlenmeyer flask. Later, 45 mL of sterile distilled water was added. The preparation was shaken for 30 min. on an Adams® electric shaker. Subsequently, the filtrate was obtained to measure pH, concentration of organic acids, ammonia, and also perform a microbiological analysis.

Solids were dried in an oven at 60 °C and ground in a UDY® hammer mill, with a 1 mm sieve, for chemical quantification analysis. Dry matter (DM) and crude protein (CP) were determined according to AOAC (2005), and for true protein (TP), Berstein, cited by Meir (1986), was followed.

pH was measured in an Okaton® automatic potentiometer and ammonia (NH3) was determined by the Berthelot technique (Martínez et al. 2003). The quantification of short chain acids (SCFA) was performed by the method of Dinkci et al. (2007), by means of high pressure liquid chromatography (HPLC). For this, Gemini 5u C18 110A (PHENOMENEX) column was used, with a UV light detector at 214nm, at room temperature (15 °C), with mobile phase of (NH4) 2, PO4 0.5% w/v and acetonitrile 0.4% v/v. The pH was adjusted to 2.24 with H3PO4 (filtered with a 0.22 µm pore membrane, degassed by sonication and bubbling with hydrogen) and a flow of 0.5 mL/min was applied. It was quantified with Claritychrom program, version

The microbiological composition of fermentation samples was determined in a certified microbiological control laboratory, located in Boyacá, Colombia. For this, a 1/10 (w/v) dilution was made and the concentrations were expressed in colony forming units per milliliter (CFU/mL). Mesophilic aerobic bacteria were determined according to AOAC (966.23.C: 2001). The ISO 15213: 2003 was applied for Clostridium spores and reducing sulfite, and ISO 7954: 1987 for fungi and yeasts. Salmonella in 25 g was determined by AS 5013.10: 2009 and lactic acid bacteria by NTC 5034: 2002. For the most probable number (MPN) of total and fecal coliforms, it was proceeded according to ICMSF MPN:2000.

Experimental design and statistical analysis. For indicators pH, dry matter, crude protein, true protein and count of microorganisms, a completely randomized design was used in a 2x2x4 factorial arrangement, in which factors were fermentation time (24 and 48 h), temperature incubation (20 and 25 ºC) and percentage of calcium carbonate inclusion (0, 0.25, 0.50 and 0.75%). In the concentration of organic acids and ammonia, the same design was used with 2x2x3 arrangement, in which factors fermentation time and incubation temperature were maintained with the same levels, while the inclusion percentage of calcium carbonate did not include that of 0%. For microbial counts, the methodology proposed by Herrera et al. (2015) was used and the theoretical assumptions of the analysis of variance were verified. For the normality of wastes, Shapiro-Wilk (1965) test was applied and Levene (1960) was used for the homogeneity of variance. Variables did not meet both assumptions, so logX transformation was used, which improved their fulfillment, so a classic analysis of variance was used. For the comparison of means, Duncan (1955) test was used for P˂0.05. Data was processed in Infostat statistical package, version 2012 (Di Rienzo et al. 2012).

Results and Discussion

Tables 2, 3, 4 and 5 show the results of chemical and microbiological characteristics of solid fermentation of post-harvest wastes of S. tuberosum, inoculated with a microbial preparation. For all indicators, interaction was detected between the factors under study (P <0.0001).

Table 2 demonstrates the effect of CaCO3 on pH during solid fermentation. It could be seen that values increased by incrementing CaCO3 percentage and incubating at 20 or 25 ºC. However, after fermentation and incubation at 20 ºC, a decrease in the indicator was observed, while at 25 ºC and CaCO3 at 0.25 and 0.50%, there were increases. Apparently, the inclusion of calcium carbonate had a positive impact on the fermentation process of potato wastes, and prevented a rapid decline that could be a limitation in growth and performance of present microorganisms, as well as in the stability of pH with its buffer properties.

Table 2 Effect of inclusion of calcium carbonate on pH during solid-state fermentation of post-harvest wastes of S. tuberosum, inoculated with a microbial preparation with lactic acid activity 

Indicator Time, h Temperature, ºC CaCO3, % SE±p-value
0 0.25 0.50 0.75
pH 24 20 5.48k 5.92h 6.56cd 6.66b 0.01 p<0.0001
25 4.93m 5.44l 6.22g 6.59c
48 20 4.96m 5.72i 6.33f 6.48e
25 4.67n 6.53d 6.85a 5.67j

a,b,c,d,e,f,g,h,i,j,l,m,nMeans with different letters differ at p<0.05 (Duncan 1955)

It is considered that pH, temperature, shaking speed and dissolved oxygen are the indicators that most affect microbial growth (Páez et al. 2013) and functional properties (Dong et al. 2014). The optimal values of these factors vary with the species and microbial strain, and must be correctly defined in order to obtain high yields in fermentation. One of the main reasons for the inhibition of microorganism growth is the low pH of the culture medium. Therefore, when this indicator is controlled with a base (carbonates) or an acid (organic acids), higher biomass yields can be obtained (Miranda et al. 2018). The chosen strains should also maintain their viability and activity during manufacturing, transport and storage processes (Anadón et al. 2016). In relation to temperature, most fermentations require between 30 and 37 °C to achieve optimal growth of the microorganism with lactic activity. However, LAB mixture of the microbial preparation, as a starter culture, ranged between 20 and 25 ºC, according to the experimental conditions of this study.

In the treatment without CaCO3 (0%), no appreciable values of SCFA and NH3 were detected (data not shown), so it was necessary to adjust the factorial design to 2x2x3. With respect to SCFA concentration, not significant values were also found, except for the treatment with 0.25 % CaCO3, incubated at 25 ºC for 24 h, in which 11.29 mmol/L of propionic acid was obtained. However, there was an increase of lactic acid concentration with both incubation temperatures and the course of fermentation. This effect indicates an increase of efficiency of the fermentation process, and favors quality and conservation of the final food (Zielinska et al. 2017). NH3 values were low in all treatments, maybe due to this lactic acid production (table 3).

Table 3 Effect of inclusion of CaC03 on organic acid production and NH3 during fermentation of post-harvest wastes of S. tuberosum, with a microbial preparation with lactic acid activity 

Indicator Time, h Temperature, ºC CaCO3, % SE±p-value
0.25 0.50 0.75
Lactic acid, mmol/L 24 20 0.002k 26.02j 35.25h 0.003 P<0.0001
25 42.43f 49.00d 31.31i
48 20 39.36g 52.33c 56.86a
25 52.90b 48.33e 56.86a
NH3 meq/L 24 20 3.74k 3.76k 4.96e 0.02 P<0.0001
25 4.53g 4.80f 6.55a
48 20 5.35c 5.24d 3.82j
25 4.37h 6.14b 4.17i

a,b,c,d,e,f,g,h,i,j,kMeans with different letters differ at p<0.05 (Duncan 1955)

Results indicate that the use of the microbial mixture, of medium and rapid lactic fermentation, in potato post-harvest wastes and CaCO3 additive maintain favorable conditions for the production of organic acids, mainly lactic acid. According to Muck et al. (2018), Lactobacillus buchneri is the dominant species used in silage additives with BAL, obligate heterofermentative. It slowly converts lactic acid to acetic acid and 1,2-propanediol during storage in the silo, improving aerobic stability, without affecting animal productivity. However, these studies are not sufficient to determine the effects it could have on animals.

Table 4 shows the results of the microbiological analysis carried out on the fermentation of potato wastes, inoculated with the microbial preparation. By including calcium carbonate in the mixture, concentration of aerobic bacteria increased with respect to control (0%). The highest concentration was found with 0.25% carbonate and incubation at 20 ºC for 48 h. No defined performance was observed with respect to factors time, temperature and percentage of CaCO3. This result does not appear to be related to the effect of the additive or to the produced lactic acid, but rather to growth inhibition during fermentation and to the humidity of the system. Han et al. (2013) stated that the addition of CaCO3 in fermentations with bacteria increases their growth. In addition, it increases the levels of sugar transporting proteins and of proteins involved in the synthesis, repair, recombination and replication of DNA. Tian et al. (2015) obtained results with the inclusion of CaCO3 in the fermentation of sugarcane bagasse by Clostridium thermocellum and in the degradation of this substrate. They also verified its stimulating effect on biohydrogen production.

LABs, like mesophilic bacteria, increased their concentration compared to control, and a maximum of 1.7x108 CFU/ mL (8.24 log CFU/mL) was obtained with 0.50% CaCO3 in 48 h of fermentation at 25 ºC. However, they maintained concentrations of 107 CFU/mL at the two evaluated temperatures, which indicated that the inoculum, added to the microbial preparation with heterofermentative lactic bacteria, produced lactic acid in the initial period of fermentation with a decrease of pH, regardless of temperature and percentage of inclusion of carbonate as an additive. This condition causes the suppression of enterobacteria, clostridia and other microorganisms, thus reducing DM losses due to proteolysis and fermentation. According to Okubo et al. (2018), in the active fermentation period, it is expected that pH will decrease more quickly, and to a lower value compared to an untreated or not inoculated silage, which improves protein preservation during this process.

In fermented mixtures, yeasts are in a lower proportion in relation to aerobic and lactic bacteria. At 24 h, yeast concentrations decreased with the inclusion of carbonate, without the influence of temperature. However, higher values were found for these populations at 48 h. These results may be associated with the physical-chemical components of the used sources and fermentation conditions. Yeasts appear to take longer to establish and grow in these environments, as well as in synergy with bacteria. According to Miranda et al. (2018), agroindustrial by-products (molasses, whey, soy milk and vinasse) are available sources that can be efficiently used for growth and development of microorganisms with functional activity of their metabolites. However, the combination of Lactobacillus plantarum and molasses has been demonstrated to cause a decrease of yeasts in silage as well as in ruminal fermentation (Zhao et al. 2019). Studies by Marrero et al. (2015) report that yeasts, as efficient microorganisms in the ruminal environment, tolerate a pH range between 3 and 10, but prefer a slightly acidic medium with the addition of C molasses, as a rich source of easily fermented carbohydrates. This corresponds to the values obtained in this study for mixed populations.

Table 4 Microbiological analysis of post-harvest wastes of Solanum tuberosum, fermented in solid state at different temperatures with the inclusion of CaC03 

Indicator, Log 10 CFU/mL (CFU/mL) Time (h) Temperature (0C) Calcium carbonate, % SE± P-value
0 0.25 0.50 0.75
Mesophilic aerobic bacteria 24 20 5.34j (2.1x105) 7.27d (1.9x107) 7.90b (8.0x1.07) 7.05f (1.1x107) 0.02 P<0.0001
25 7.11e (1.2x107) 6.85h (7.0x106) 7.79c (6.2x107)
48 20 8.07a (1.1x108) 7.95b (8.9x107) 7.05f (8.8x107)
25 6.77i (5.9x106) 6.92g (8.3x106) 7.95b (1.2x107)
Yeasts 24 20 4.38c (2.5x104) 3.66d (4.6x103) 3.66d (4.6x103) 2.99e (9.8x102) 0.09 P<0.0001
25 3.16e (1.4x103) 2.69f (4.9x102) 3.61d (4.1x103)
48 20 5.32b (2.0x105) 5.64a (4.3x105) 5.61a (4.1x105)
25 4.40c (5.5x104) 5.65a (5.6x105) 5.74a (4.5x105)
Lactic acid bacteria 24 20 5.87j (7.5x105) 7.90c (8.0x107) 7.50e (3.2x107) 7.04g (1.0x107) 0.01 P<0.0001
25 7.00h (1.0x107) 7.99b 0.97x107) 7.91c (8.1x107)
48 20 7.96b (9.1x107) 7.58d (3.8x107) 7.18f (1.5x107)
25 7.01gh (1.0x107) 8.24a (1.7x108) 6.98i (9.5x106)

a, b, c, d, ….jMeans with different letters indicate differences at p<0.05 (Duncan 1955)

*Data were transformed according to log10 (X) because they do not follow a normal distribution

() means of the colony forming units per milliliters (CFU/mL)

Table 5 shows results of the effect of CaCO3 on protein and DM content during solid-state fermentation of post-harvest wastes of S. tuberosum, inoculated with a microbial preparation with lactic activity. With fermentation, crude protein percentage of mixtures increased in all treatments. The highest values were obtained with the inclusion of 0.50% of CaCO3, which represents a marked difference with protein contents at 25 °C, which were superior to fermentation at 20 °C, at 48 h. During this time, similar performance was found with a tendency to increase in crude protein with the addition of 0.50% of CaCO3 at 25 °C, but lower than those expected according to the results of pH and humidity of the system. However, TP/CPx100 relationship, which expresses microbial protein synthesis, was the same for temperatures of 20 oC and 25 °C with 72.76%. Therefore, under rustic or field solid fermentation conditions, it is more advisable to use temperatures of 20 oC.

Table 5 Effect of CaC03 on protein content and DM during solid state fermentation of post-harvest wastes of S. tuberosum, inoculated with a microbial preparation with lactic acid activity 

Indicator, % Time, h Temperature, ºC Calcium carbonate inclusion, % SE ±p-value
0 0.25 0.50 0.75
Crude protein 24 20 18.48c 18.84b 19.02a 17.72e 0.050 P<0.0001
25 17.31i 16.41j 17.48gh 17.35hi
48 20 17.54fg 18.72b 18.50c 17.66ef
25 17.58efg 18.20d 19.09a 17.74e
True protein 24 20 12.94d 13.28c 13.83a 12.72e 0.050 P<0.0001
25 12.12h 11.57i 12.72e 12.45f
48 20 12.21gh 13.20c 13.46b 12.68e
25 12.31g 12.83de 13.89a 12.73e
DM 24 20 17.49o 17.24p 18.49m 20.72l 0.010 P<0.0001
25 23.51g 21.00j 22.49i 22.73h
48 20 39.90c 40.57b 37.51e 39.66d
25 20.85k 35.18f 40.90a 17.84n

a, b, c, d, e, f, g, h, i,…pMeans with different letters differ at p<0.05 (Duncan 1955)

With respect to DM, at 20 ºC, increases were observed in the different inclusion levels of calcium carbonate during fermentation. Although the highest value was 40.57% in 48 h, it is considered very humid to obtain a final product with the quality indicators for ruminant animals (Borras 2017). However, at 25 °C, the performance was opposite and there was no definite tendency in dry matter values ​​with the different levels of calcium carbonate, finding high and low values, ​​indistinctly. This result is due to the large amount of water still contained in potato post-harvest wastes, which makes their ensiling and preservation difficult (FAO 2015). Thus, a suitable medium is provided for the development of microorganisms, which alter the material and can be pathogenic for animals. However, in the current study, concentration of lactic acid and the maintenance of low pH allowed the removal of undesirable microorganisms.

The performance of chemical indicators of food indicated that when the potato is cut, it quickly begins a leaching process, which leads to significantly wetting food and altering its organoleptic characteristics and conservation. Urea hydrolysis by bacteria within fermentation in its metabolic process of cellular synthesis produces water and ammonia. This could be volatilized depending on final pH of the process and, possibly, on the deamination of peptides and amino acids, on a smaller scale. Part of the water produced during the oxidation of molecules could be evaporated by the metabolic heat generated during the SSF process (Pandey et al. 2001 and Mitchell et al. 2002). However, in the previously mentioned studies, these processes did not significantly influence final dry matter, so the fermentation mixture still maintains high values ​​for the fermentation process to be effective.

True protein demonstrated an increase of 4.98 percentage units with respect to the microbial preparation (8.85%) as a biological accelerator in fermentation, according to Borras (2017) for the temperature of 20 oC during 24 h of fermentation. These values ​​are maintained with differences with respect to calcium carbonate levels at 48 h, so this compound favored the microbial synthesis of concentration of the initial CFU/mL. Siebald et al. (2002) highlighted that around 50% of crude protein belongs to non-protein nitrogenous compounds. One of them is solanidine, an alkaloid that can be present, free or combined in the form of glycoalkaloids, called chaconine and solanine, both toxic to animals. These compounds are eliminated by solid-state fermentation, since most of the protein of this study has microbial origin, and does not come directly from food.

Results of chemical and microbiological indicators evidenced that other raw materials with a high DM proportion should be evaluated, so that they allow to create a mixture for ensiling, with a percentage close to that recommended for animal feed.

It is concluded that the inclusion of 0.50% of CaCO3, at 20 °C during 24 h of fermentation of post-harvest wastes of S. tuberosum with the microbial preparation, maintains favorable conditions for the production of organic acids and the aerobic stability of fermentation. Other plant materials are recommended to increase DM content of the final product.


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Received: February 18, 2020; Accepted: September 10, 2020


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