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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

Solid-state fermentation of post- harvest wastes of Solanum tuberosum and 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

2Animal Science Institute, ICA, Carretera Central km 47 1/2, San José de las Lajas, Mayabeque, Cuba


With the objective of studying the solid -state fermentation of post - harvest wastes of S. tuberosum and a microbial preparation, an experiment with a completely randomized design with factorial arrangement (3x3) and three repetitions was carried out. The factors were temperature (20, 25 and 30oC) and fermentation time (0, 24 and 48 h). Measurements of the indicators pH, ammonia, organic acids, crude fiber, crude protein and true protein, dry matter, ash and microbiological analysis were performed. The results of the microbiological analysis carried out on the fermentation of post-harvest wastes of S. tuberosum shows a high concentration of lactic acid bacteria (LAB) and the low pH found eliminated the pathogens agent(p <0.0001). In the solid fermentation with the microbial preparation, low propionic acid and ammonia values were recorded at 20oC and an increase in lactic acid (p <0.0001). There is a marked effect of temperature on humidity during fermentation. Low fiber and DM values were recorded. True protein increased at 24 h by 2.7 percentage units at 25oC in correspondence with the microbial concentration of mesophilic bacteria (5.5x107 CFU/ mL), LAB (6.1x107 CFU/mL) and yeasts (1.3x104 CFU/mL). It is concluded that the solid state fermentation of post- harvest wastes of Solanum tuberosum and a microbial preparation with lactic activity favors this process and other formulations with drying and fibrous material should be studied for an effective ensiling process of the final product.

Key words: lactic bacteria; agro-industrial wastes; organic acids

The solid-state fermentation is a potential technology for food production, fuel, industrial chemicals products, and pharmaceuticals products (Zhao et al. 2019 and Chohan et al. 2020). Its application offers advantages in bioprocesses such as bioleaching, bio-benefiting, and bioremediation (Romero and Vargas 2017). It is also used for the recycling of agro-industrial wastes and offers an alternative that allows, through simple technologies, to modify or alter the fermentation processes with the addition of microbial preparations rich in lactic bacteria, and with this improve the quality and safety of the process ( Elías and Herrera 2008 and Díaz 2014).

The wastes of potato (Solanum tuberosum) harvest are a raw matter that can be included in animal feeding. According to the Ministry of Agriculture and Rural Development (2015), it has great nutritional value since it is a rich source of protein, carbohydrates, potassium, vitamin C, other vitamins and minerals to a lesser proportion (Waglaya et al. 2019). These characteristics favor the fermentation processes when inoculants are added (Tamasi et al. 2015), so the objective of this research was to study the solid -state fermentation of post -harvest wastes of S. tuberosum with a microbial preparation as inoculant.

Materials and Methods

The experiment of solid-state fermentation (SSF) was carry out under the high tropic conditions (2860 m o.s.l.), in the laboratory of biochemical and animal nutrition from Universidad Pedagógica y Tecnológica de Colombia (UPTC), located in the north central avenue, Tunja-Paipa road, in Tunja municipality, Boyacá department, Colombia. It has an average temperature of 15°C and annual average rainfalls of 553 mm.

Experimental procedure. A yogurt with Lactobacillus delbrueckiis ssp. bulgaricus and Streptococcus thermophilus (LAB) (commercial freeze-dried, Liofast Y452B, SACCO ®) was prepared. This was added at 2 % and a concentration of 0.99x108 CFU/mL to the microbial preparation prepared according to Borrás (2017) methodology.

For the SSF, the post-harvest wastes of the potatoes obtained commercially, clean and chopped (approximate 3x3mm particle size) were used. A 2% of the microbial preparation, 1 % urea, 0.5 % mineral premix and 0.5 % sodium sulfate were added (modification made to the methodology proposed by Elías and Herrera. 2008). These ingredients were mixed until obtaining a homogeneous paste. They were distributed in plastic bags with 1 kg capacity. They were incubated at different temperatures 20 °C, 25 °C and 30 °C, in individual Memmert® incubators, for 48 hours. Each bag represented an experimental unit, with three repetitions each, according to the treatments. Samples at 0, 24 and 48 fermentation hours were taken.

The content of the bags of each treatment was collected in its entirety and homogenized, then 5g of sample were taken and placed in 100 mL Erlenmeyers and 45 mL of sterile distilled water were added with three repetitions. The preparation was shaken for 30 minutes on an Adams® electric shaker and subsequently the filtrate was obtained for pH measurement in an Okaton® automatic potentiometer for microbiological analysis.

The total of solids left in the bags were dried and ground in a UDY®, hammer mill with a 1 mm sieve, for chemical quantification analysis using the following analytical techniques: dry matter (DM), crude protein (CP), by AOAC (2005); true protein (TP) according to Berstein cited by Meir (1986) and crude fiber (CF) according to Van Soest et al. (1991).

The ammonia (NH3) was determined by the Berthelot technique (Martínez et al. 2003). The quantification of short chain acids (SCFA) was carried out by the method reported by Dinkci et al. (2007). By means of high efficiency liquid chromatography HPLC, the Gemini 5u C18 110A column (PHENOMENEX) was used, with a UV vis light detector at 214nm at room temperature (15 °C), with a mobile phase of (NH4)2PO4 0.5 % W/V; acetonitrile 0.4% V/V, pH was fitted 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, quantified with Claritychrom software version

The microbiological composition was determined at 0, 24 and 48h of fermentation, in a certified laboratory for the microbiological control, located in Boyacá, Colombia. For aerobic mesophiles (forming colony units per milliliter, UFC/mL) (AOAC 966.23.C: 2001), total coliforms, Most probably number (MPN) (ICMSF NMP: 2000), total and fecal coliforms (NMP), (ICMSF NMP: 2000), spores of Clostridium sulfite reductor (UFC/mL), (ISO 15213: 2003), fungi and yeasts (UFC/mL)(ISO 7954: 987), Salmonella (AS 5013.10: 2009) and lactic acid bacteria (LAB) (NTC 5034: 2002).

Experimental design and statistical analysis. Analysis of variance was carried out according to a completely randomized design with (3x3) factorial arrangement and three repetitions. The factors were temperature (20, 25 and 30 ºC) and fermentation time (0, 24 and 48 hours) for the following indicators: pH, NH3, SCFA, CF, CP, TP and microbial groups. Duncan's test was applied for P <0.05 when necessary. The statistical package INFOSTAT, version 2012 (Di Rienzo et al. 2012) was used for the analysis. In the case of the microorganism counts, the data did not follow the normal distribution, so they were transformed according to logX.

Results and Discussion

Table 1 shows the interaction of the results achieved in the solid fermentation with the post-harvest wastes of S. tuberosum and the microbial preparation at different temperatures and fermentation times, which was designed to obtain higher protein biomass and metabolite production.

Table 1 Performance of pH, NH3 and SCFA, in relation to temperature during 48 hours of fermentation of post-harvest wastes of Solanum tuberosum with the microbial preparation 

Fermentation time (h) SE±Sig
Indicator Temperature 0C 0 24 48
pH 20 5.70a 5.10b 4.51c 0.040 P<0.0001
25 4.62c 4.44c
30 4.31c 4.42c
Lactic acid mmol/L 20 16.86a 165.64g 160.57f 0.003 P<0.0001
25 54.40e 41.89c
30 50.20d 23.71b
Propionic acid mmol/L 20 14.33d 9.34c 20.65e 0.003 P<0.0001
25 0.002ª 0.002b
30 0.002ª 0.002b
NH3 meq/L 20 1,79a 4.19c 5.47f 0.003 P<0.0001
25 4.57e 4.37d
30 3.88b 6.02g

a, b, c, d,d, e, f, gMeans with different letters show differences to P<0.05 according Duncan (1955)

The performance of the SCFAs studied shows the absence of acetic, butyric, isovaleric and isobutyric acid and only propionic acid values were recorded at 24 and 48 h (9.34 mmol/L and 20.65 mmol/L, respectively) at a 20 °C temperature. These values are not significant to influence on the fermentation deterioration, while at this same temperature high productions of lactic acid during the process were observed. However, a temperature effect was found with notable differences at 25 and 30ºC of fermentation of 118.68 and 136.86 mmol/L respectively at 48h. This could be influenced by the concentration of lactic bacteria, considering that the amount (0.99x108 CFU/mL) as a starter or accelerator culture is adequate to maintain low pH values. This result is considered important in the elaboration of strategy for biological accelerators for the fermentation processes of the post-harvest wastes of Solanum tuberosum and to eliminate undesirable pathogens.

Differences in the performance of pH (P <0.05) with respect to time were also observed and low pH was obtained in the three temperatures evaluated at 48h, where the low ammonia production favors the pH indicator. According to Okubo et al. (2018), the main objective of applying microbial preparations in post-harvest wastes, in addition to accelerating the microbial synthesis processes, is to reduce the pH faster to preserve carbohydrates and proteins (Muck et al. 2018), and inhibit the growth of microorganisms that could deteriorate it. Therefore, the concentration used for fermentation and in the chemical and microbiological composition of the microbial preparation was effective, according to Borrás (2017) methodology.

When there is a low pH, such as that obtained in the treatments at 24 and 48 h of fermentation, the NH3 produced is retained in the substrate by the high humidity of the potato post-harvest wastes, as observed for the three studied temperatures. This performance can be attributed to the use of available sugars by some microorganisms that prevail in the product and to non-protein nitrogen in the mixture of the components to be fermented, for the formation of their cellular protoplasm.

The low NH3 values coincide with that reported by Ramos et al. (2007) with inoculations of 5 % of Vitafert, in Sacchasorgo and Sacchapulido, at a temperature of 25°C. Nout (2014) stated that the inoculation of S. tuberosum wastes in the silage showed a reduction in the concentration of N-ammonia in comparison with the group without the addition of the inoculant. However, Díaz et al. (2014) found a different tendency where the pH initially increases at hour 8 of the process, and then slowly decreases towards the end of the fermentation (96 h) under conditions similar to those described for this preparation, but with other agro-industrial wastes that could influence on the result.

Table 2 shows the results of the performance of the chemical indicators of the post-harvest wastes of S. tuberosum with the microbial preparation (P <0.05) at different incubation temperatures.

Table 2 Performance of the chemical indicators of the post-harvest wastes of S. tuberosum with the microbial preparation 

Indicator (%) Temperature (o C) Fermentation time (h) SE±Sign
0 24 48
DM 20 15.83a 38.13c 19.30b 0.30 P<0.0001
25 39.48c 19.18b
30 38.59c 17.98b
CP 20 15.55ab 21.05c 16.68b 0.420 P<0.0001
25 20.83c 14.85a
30 20.28c 14.84a
TP 20 8.84ab 10.85c 10.31bc 0.480 P=0.0125
25 11.54c 10.31bc
30 7.83a 8.24a
CF 20 5.08a 8.43ab 9.71b 1.170 P=0.0038
25 8.43ab 9.71b
30 9.04b 7.24ab
Ash 20 6.39a 7.89b 16.17e 0.42 P<0.0001
25 8.40b 11.01c
30 13.94d 6.63a

a, b, c, d, eMeans with different letters differ to P<0.05 (Duncan 1955)

The results show interaction between the studied factors (P <0.0001). The DM at 24 h increased to 22.3; 23.65 and 22.76 percentage units with respect to zero hours. This condition is not maintained at 48 h, due to the metabolic processes of the microorganisms, both from the microbial preparation and from the post-harvest wastes of potato, which probably caused a DM reduction in the product due to the use of sugars (sucrose, glucose, and fructose) and starch in their metabolic processes which generate water, CO2 and SCFA. This DM reduction is insufficient for subsequent silage processes, therefore additives such as drying material (calcium carbonate) that do not have an inhibitory effect on microbial synthesis should be considered. Prada (2012) reported humidity values for raw potato storage between 75-76 % attributed to microbial synthesis processes.

Regarding the percentage of ash in the fermentation of post-harvest wastes of S. tuberosum with the microbial preparation, it is considered that they have higher values than the raw potato (Cárdenas et al. 2008). The values found in this study depend on the nature of the soil where the tuber was grown and on the 0.5 % mineral premixture added to the substrate for fermentation (Miranda et al. 2018).

The mineral composition is important for the microbiota metabolism and achieving protein biomass, which is why there is an increase in CP of 5.5; 5.28 and 4.73 percentage units at 20, 25 and 30°C, in 24 h of fermentation, respectively. However, it markedly decreased after 48 fermentation hours in the same proportion and became below the control for the highest temperatures. Fonseca and Borrás (2014) determined a value of 7.73 % of CP in the tuber; value exceeded in this study when fermenting the post-harvest wastes of S. tuberosum and the product fermented in 24 h at 20°C reaches up to 21 %, this condition could be attributed to the microbial preparation with lactic acid activity as a biological accelerator. Okubo et al. (2018) added a raw potato protein concentrate and supplemented the protein of the silage content without inoculum and obtained favorable results.

The true protein indicator increased 2.01 and 2.7 percentage units at temperatures of 20 and 25°C, in 24 h of fermentation. The highest ratio of (TP/CP*100) for these temperatures was 61.2 and 69.42, respectively, at 48 h. fermentation. However, at higher temperatures, microbial synthesis is depressed in 24 h and remains unchanged at 48 h according to the control, so that the increase in 50C for these fermentation conditions limits the microbial growth and therefore could affect protein synthesis.

In this study there was an increase in fiber with respect to the fermentation time, probably due to the increase in the cell walls content, in relation to the starch of potato wastes and the fermentation of sugars by the microorganisms that developed in the system, similar results were found by Aranda et al. (2012). However, the values are still very low, which implies the need to combine this food with other foods high in fiber, in order to produce a better use specifically for ruminants.

Table 3 shows the results of the microbiological analysis carried out on the fermentation of the potato wastes inoculated with the microbial preparation. There is effect of temperature with respect to the fermentation time on the microbial concentrations of mesophilic bacteria, yeasts and lactic acid bacteria (P <0.0001). The presence of fecal coliforms, Salmonella, or Clostridium spores was not detected, a result that supports its quality to use it as a sanitary safe product for animal nutrition.

Table 3 Effect of temperature on the composition of aerobic mesophilic bacteria, yeasts, lactic acid bacteria during the fermentation of post-harvest wastes of S. tuberosum with the microbial preparation 

Microorganism (log 10 UFC/mL) UFC/mL Fermentation Time(h) Temperature (°C) SE±Sign
20 25 30
Aerobic mesophilic bacteria 0 5.34ª (2.1x105) 5.33ª (2.1x105) 5.33ª (2.1x105) 0.02 P<0.0001
24 8.10e (1.2x108) 7.74d (5.5x107 ) 7.80d (6.3x107)
48 7.03b (1.0x107) 7.18c (1.5x107) 8.19f (1.4x108)
Yeats 0 4.40c (2.5x104) 5.33d (2.5x104) 5.33d (2.5x104) 0.01 P<0.0001
24 4.37c (2.1x104) 4.11b (1.3x104) 4.04a (1.1x104)
48 5.75f (5.6x105) 5.74f (5.3x105) 5.61e (4.1x105)
Lactic acid bacteria 0 5.88a (7.5x105) 5.88a (7.5x105) 6.90b (8.0x106) 0.02 P<0.0001
24 7.77f (6.1x107) 7.77f (6.1x107) 7.70e (5.0x107)
48 6.98c (9.5x106) 7.09d (1.0x107) 8.03g (1.0x108)

a, b, c, d, e, f, gMeans with different letters differ to 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-1)

There is an increase in the concentration of aerobic mesophilic bacteria, which increased with fermentation temperatures. This result is related to the decrease in fermentation pH and the organic acids produced at that time as shown in table 1 and probably to bacteriocins produced by lactic acid bacteria that act as antimicrobial substances (Parra 2010 and Nkosi et al. 2019)

Yeasts as part of the fermentation ecosystem markedly increased with the fermentation time. There are differences in its growth with respect to the control and with respect to the studied temperatures (P <0.0001) due to the wide range of growth of this genus and the microbial interactions between the selected strains, which could coexist in stimulation relations and neutralism with LAB, similar studies that are carried out on other substrates with higher acidity (Pardo and Ferrer 2019). While other yeast research suggests that it may be possible to apply a microbial strain of direct-feeding to silage, make it survive silage, and multiply during feeding (Muck et al. 2018).

The concentration of LAB gradually increased until 48 h with the three temperatures under study. These increases in microbial biomass are favorable for the organic acids production which maintain low pH values and eliminate pathogenic microorganisms (Muck et al. 2018), indicating that the humidity of the ecosystem did not influence on the deterioration of the final fermentation.

The lactic acid bacteria used in this study are heterofermentative with medium and rapid acidification, L. delbrueckii ssp bulgaricus and S. thermophilus, were chosen as starter cultures because they were considered sanitary safe and provide aerobic stability (Munk et al. 2018). They are widely distributed in nature and have been isolated from various environments, for their growth they require sugars such as glucose and lactose, in addition to amino acids, vitamins and other factors (Azadnia et al. 2011, Mesa 2016 and Miranda et al. 2018), nutrients that are in the fermentation mixture.

The variables or indicators analyzed in this study (microbial concentration, protein, ammonia, SCFA, pH) as a result of the fermentation of potato post-harvest wastes determine a product with characteristics to be considered in the ruminant's feeding. These indicators are part of the quality control system with biological inoculations, however, the results in the fiber and DM show that other formulations must be studied for their effective process of obtaining the final product. The results show that the microbial preparation with lactic acid activity used acts as a biological accelerator of the solid fermentation process of potato post-harvest wastes under the established experimental conditions.

It is concluded that the solid -state fermentation of post-harvest wastes of Solanum tuberosum and a microbial preparation with lactic activity favors this process and other formulations with drying and fibrous material should be studied for an effective silage process of the final product.


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Received: February 12, 2020; Accepted: September 08, 2020


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