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2021.06.01.10
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Fermentation study of Cassava bagasse starch hydrolyzed's using INIAP 650 and INIAP 651 varieties and a strain of lactobacillus leichmannii for the lactic acid production

Shirley Inguillay, Pedro Maldonado-Alvarado
Available from: http://dx.doi.org/10.21931/RB/2021.06.01.10
(registering DOI)
ABSTRACT

Ecuador is an agricultural country, which has an actual production of starch obtained from cassava. Tuber processing residues do not have an economic impact; for example, the cassava bagasse, only used for plant fertilization and animal feeding. This project aimed to study the influence of the fermentation variables (pH and agitation), on the lactic acid production. An enzymatic hydrolysis of cassava bagasse starch, INIAP 650, and INIAP 651 was performed using α-amylase and glucoamylase. Then, glucose was fermented by lactobacillus leichmannii ATCC 7830 strains, varying conditions such as agitation (presence and absence) and pH (4.5, 5, and 5.5). Finally, the determination of lactic acid was performed by potentiometric and FTIR analysis. Conversions of cassava bagasse to reduced sugars were 71.66 and 85.05 % for INIAP 650 and INIAP 651 varieties. The best lactic acid concentrations were 27.62 and 33.48 g/L, obtained at pH 5.5 and agitation, for INIAP 650 and INIAP 651 varieties. Qualitative analysis conducted by FTIR spectrophotometry confirmed the presence of lactic acid in the reacted products. Lactic acid production from cassava bagasse starch could contribute to the Manabí and Esmeraldas provinces of Ecuador's economic development.
Keywords: lactic acid production, fermentation, lactobacillus
INTRODUCTION

In 2016, the global market required 1,220 kton of lactic acid, with growing demand at a rate of 16.2% per year. It is estimated that by 2025 these requirements will reach 2,000 kton; this would represent approximately 9.8 trillion dollars of revenue for the global lactic acid-producing market
Nowadays, green methods that take advantage of residues, or co-products of agroindustry, for their conversion to fuels or industrial interest products is on the rise 1.
Lactobacillus delbrueckii is a homofermentative bacteria that does not develop spores, has tolerance to acidity, and its fermentative metabolic pathway is strictly directed to the production of D-lactic Acid. The fermentation efficiency also depends on the initial concentration of carbohydrates, concentration of nitrogenous material, and the fermentation medium's agitation with ranges of 100-200 rpm 1,2.
Lactic acid or 2-hydroxypropanoic acid is a compound that has alcohol and carboxyl groups in its structure; it is obtained chemically or biotechnologically. There are two optical isomers of this acid: L (+) lactic acid, which is harmful because its consumption generates decalcification in living beings but is used for the production of polylactic acid, and D (-) lactic acid, which is considered a GRAS substance (food additive recognized as safe by the FDA) is used, e.g., as a marker for bacterial infections.
Cassava (Manihot esculenta Crantz) is rich in complex carbohydrates; this tuber grows mainly in tropical climates. In Ecuador, this is grown mainly in the provinces of Manabí, Esmeraldas, 
among others. The root is 80% made up of the tuber and 20% of the cassava bagasse made up of the husk and bark. Cassava bagasse has a starch content of 50 to 60%, cellulose 34%, hemicellulose 
15%, and lignin 7% 3,4. The INIAP 650 variety comes from the MCol 2215 clone; it has a dry matter content of 37% with a yield of 17 to 35 t / ha of fresh roots and a conversion rate from fresh to 
dry matter of 2 - 2.5: 1. The variety INIAP 651 comes from the clone CM 1335-4; it has a dry matter content of 35.5% with a yield of 29 to 40 t / ha, and a conversion rate of 6 - 7.5: 1.

METHODS

Obtaining raw material
The production of cassava bagasse starch was carried out in the "Rallandería Artesanal Bijahual" at the "Las Balsas" sector, located in the province of Manabí - Ecuador. 90 kg of roots of INIAP 650 and INIAP 651 varieties were collected in INIAP's Portoviejo Experimental Station
The cassava root barks were separated and placed on a needle rack for grinding. With a canvas-type cloth, the ground material was washed until the starch was released. The precipitate was collected and sun-dried at room temperature of the extraction site (29 ºC) for 24 h.
Characterization of cassava bagasse starch
A proximal analysis determined the properties of bagasse starch. Standardized norms for these analyses were used, as indicated in Table 1.

Table 1. Standards used in the proximal analysis
Hydrolysis of cassava bagasse starch
10 % of the starch/water (w/v) solution was prepared. The solution was heated at 65 ºC for 1 h at 150 rpm in a thermostatic bath for the gelatinization process. Subsequently, a liquefaction process was carried out with α-amylase from pig pancreas A3176-500KU, SIGMA-ALDRICH (USA) in a proportion of 5 μg of enzyme/mg of starch, dissolved in a phosphate buffer solution 400 mM at pH 7.1. Then, it was added NaN3 (0.02 %), CaCl2 (0.25 %), NaCl (0.013%), and heated in a thermostatic bath for 24 h at 37 ºC and 100 rpm. Finally, a saccharification process was carried out with the amyloglucosidase enzyme from Aspergillus niger A7095-50ML, SIGMA-ALDRICH (USA), in a proportion of 0.025 μg of enzyme/mg of starch in 10 mL of a 0.5 M acetate buffer solution and pH 4.8. It was heated in a thermostatic bath for 48 h at 65 °C and 150 rpm.
After that, the hydrolyzate was stirred at 50 rpm for 12 h and 20 °C. Finally, it was centrifuged at 3000 rpm for 20 min.

Determination of reducing sugars in cassava bagasse starch hydrolyzate
The reducing sugars were determined with the colorimetric method by DNS (3.5-Dinitrosalicylic acid). 0.5 mL of the hydrolyzed sample and 0.5 mL of the DNS reagent were placed in a test tube. It was stirred manually and heated in a boiling thermostatic bath for 5 min. Then, it was cooled at 18 °C, and 3.5 mL of distilled water was added. The absorbance was analyzed in a spectrophotometer (Shimadzu (Brazil) UV-160. A) at a wavelength of 540 nm and the concentration of reducing sugars was calculed.

Inoculum activation and preparation
A Lactobacillus leichmannii ATCC 7830 strain from Microbiologics Company (USA) was used. A pellet of the microorganism was taken and placed in 10 mL of sterilized MRS agar at 121 ºC for 15 min. The agar with the pellet was incubated in a thermostatic bath for 24 h at 37 ºC and 1 mL of this was taken and seeded deeply on MRS agar. The Petri dish with the sowing was incubated for 72 h at 37 ºC. To finish, a colony was taken from the agar, placed in 10 mL of MRS agar, vortexed, and incubated similarly at 37 ºC for 18 h.   

Lactic fermentation
100 mL of cassava bagasse starch hydrolyzate were taken, and enriched with yeast extract 0.5 % (w/v), NH4Cl 0.5 % (w/v) and CaCO3 2.5 % (w/v). The pH was adjusted to 4.5, 5.0 and 5.5 with NaOH 4 % w/v or HCl 5 % w/v. Then, a volume of nitrogen was introduced at a rate of 2 cm3/s for 1 min, and finally, the inoculum at 2 % (v/v) previously prepared was added. The flasks with the substrate to be fermented were placed in an incubator and a thermostatic bath at 37 ºC, with and without shaking, for 120 h.
The input variables were: pH (4.5, 5.0, and 5.5) and agitation (presence or absence), while the output variable was the lactic acid content. The performed treatments are detailed in table 2.


Table 2. Treatments for the production of lactic acid
Recovery of lactic acid
The lactic acid recovery was carried out by a hydrolysis and acidification process. All the fermented was filtered with a canvas. For all fermentation, H2SO4 1 M was added until reaching a pH of 2.0, pH at which CaSO4 precipitates and liberates lactic acid. This mixture was stirred for 30 min and centrifuged at 8000 rpm for 15 min. Lactic acid was contained in the supernatant and CaSO4 in the precipitated.

Determination of lactic acid content by acid-base titration.
Aliquots of 20 mL were taken from the supernatant once the acidification process was finished. The percentage of lactic acid was measured by titration with NaOH 1 N. Finally, the concentration of lactic acid present in the solution was calculated using the equation (1):


Determination of the fermentative efficiency and characterization of lactic acid
The lactic acid solution density was measured with a Mettler Toledo (USA) densimeter and the pH of the final solution containing lactic acid using a Hanna (USA) pH meter. The fermentative yield was determined by the relationship between the lactic acid obtained and the amount of starch used as a substrate. A conversion of 1 glucose molecule to 2 lactic acid molecules was estimated for the theoretical fermentative yield. For processes with homofermentative bacteria, the conversion is 1 to 1 g/L, respectively.
The proximal analysis results, lactic acid concentration, optical density, fermentation efficiency, and pH were statistically analyzed with the StatGraphics Centurion XVIII X64 program using a multifactorial ANOVA followed by a Fisher test with a 95% confidence level to determine statistically significant differences. Three repetitions were made for each treatment.
Furthermore, the comparison between the spectrogram obtained by FTIR of the experimentation with a 98% lactic acid spectrogram was analyzed. The infrared spectrophotometry equipment used was Perkin Elmer (USA) brand, Spectrum One 2370 model with a diamond cell, and three repetitions for each sample.

RESULTS AND DISCUSSION

Characterization of cassava bagasse starch
For this study, the bagasse, a by-product of the cassava grating process, of INIAP 650 and INIAP 651 varieties, was used. Table 3. shows the moisture, ash, fiber, protein, lipids, and amylose contents. The carbohydrates content was calculated as the difference in % with these parameters. It is understood to be made up of the main amylopectin.


Table 3. Physicochemical characteristics of cassava bagasse starch
INIAP 650 and INIAP 651 varieties' moisture content was 11.30 % (w/w) and 14.50 % (w/w), respectively.  A previous study reported the cassava tuber starch oscillates' moisture content in a range of 11-13 % 5. The INIAP 650 variety was found within the indicated range, but the INIAP 651 variety surpasses these values, due to higher hygroscopy, at a rate of 3.30 g of water/g of starch, compared to normal hygroscopy of the tuber.
The ash content in the cassava bagasse starch was 1.40 % and 1.32 % for INIAP 650 and INIAP 651 varieties. Studies showed the range for ash in cassava bagasse starch from 0.9 % to 1.50 % 6. Another work showed points out values of 0.05 % (w/w) for cassava bagasse starch 5, 28 times less ash content concerning the amount obtained in our study. Conversely, an analysis of the literature reported that cassava starch had 7 times less ash content than bagasse 7, due to minerals present in its composition 8.
The fiber present in the cassava bagasse starch was 4.30 and 6% for INIAP 650 and 651 varieties, respectively. No data on the fiber content of bagasse starch was found.
However, the bibliography shows that the amount of fiber present in the cassava tuber's starch is 0.30 %. This is due to the high concentration of cellulose present in cassava bagasse, ranging from 15 to 51 %. It is mainly considered a soluble fiber 6,9.
In lipid content, bagasse starch presented values of 1.90 and 1.50 % for INIAP 650 and INIAP 651 varieties, respectively. These values were similar to the range established for cassava starch's lipid material, which is 0.53 - 1.60 %) 10. The average value of lipids of the tubers presented ranges from 2 to 5 %, and the lipid material of the cassava starch from the experimentation was lower but not far compared to the average value of cassava lipids 4.
The amylose content presented values of 26.50 % for INIAP 651 variety and INIAP variety 650 of 24.34 %. The range of amylose in bagasse starch varies from 15.9 to 22.4 %, as reported by the literature 11. The varieties of the present study had high values compared to those mentioned in the bibliography. These varieties are species improved by the Center for Agricultural Research (INIAP) for starch production.

Hydrolysis of cassava bagasse starch and determination of reducing sugars
Enzymatic hydrolysis using α-amylase and glucoamylase showed conversion levels of cassava bagasse starch to reducing sugars of 71.66 and 85.05 % for INIAP 650 and INIAP 651 varieties, respectively. It should be noted that part of these reducing sugars is glucose chains that were used by lactobacillus. Similar results to this study were reported by analyzing the literature with 81 to 109 % conversion yields from starch to reducing sugars 12. Another study of literature stated 95 % conversions of starch to reducing sugars with α-amylase and glucoamylase 3.
Conversions greater than 100 % are standard because the enzymatic hydrolysis process adds one molecule of water for each broken bond and increases the hydrolyzed starch's weight. Besides, amylose and amylopectin's long chains have n glucose chains; when these chains are hydrolyzed, and they break into n glucose, maltose, and dextrins that increase quantification concentration, reducing sugars 13.

Determination of lactic acid content
Employing the multifactorial ANOVA statistical analysis determined that the agitation and the pH within the fermentation of the hydrolyzed cassava bagasse starch of INIAP 650 and 651 varieties present significant influence response variable, the lactic acid produced.
Table 4. shows the results of the lactic acid concentration, quantified by titration. Statistically significant differences between the studies' treatments were found. Treatment T9 presented higher lactic acid production (33.48 ± 1.87 g/L), which corresponds to the fermentation of the hydrolyzed cassava bagasse starch of INIAP 651 variety pH 5.5 with stirring of 150 rpm.


Table 4. Lactic acid production from cassava bagasse starch varieties INIAP 650 and 651, quantified by titration
In T1, the stoichiometric content of H2SO4 was 6.83 mL, but the real volume used was 5.89 mL. It is then verified that there is a lack of H2SO4, so it reacted with all the calcium lactate, releasing lactic acid and calcium sulfate 14.
Compared to other treatments, the fermentation treatments, T4 to T6 and T10 to T12, presented the lowest lactic acid production values without stirring. The lowest lactic acid production was obtained with T4, with a value of 4.43 ± 0.47 g/L at pH 4.5 conditions and without stirring from the fermentation of a hydrolyzed bagasse starch of cassava INIAP 650 variety. The main reason that would explain this is the lack of agitation of the system, which hinders the transfer of nutrients from the medium to the cells 15.
In this study, the lactic acid production rate increased by 80.65% for the treatments with agitation compared to the treatments without agitation, as seen in T1 and T4, in 120 h of fermentation.
Other studies report an increase in lactic acid production by 42%, when stirred from 0 to 300 rpm was reached in 36 h, with the formation of alcohol at pH between 4.5 and 5.5 at 37 ºC with a Rhizopus oryzae strain 16,17.
It can be noticed that the production of lactic acid increases as the pH increases in the range of 4.5 to 5.5. In values outside this range, there is an inhibition of the microorganisms that act in transforming glucose to lactic acid 15. Besides, there are statistically significant differences between the treatments T3 and T9, the latter with the highest production of lactic acid.
In this work, at pH 5.5, higher lactic acid production was obtained (33.48 ± 1.87 g/L) than at pH 5.0 (27.62 ± 0.69 g/L). This is due to the accumulation of lactic acid within the fermentation process that creates an environment not suitable for developing microorganisms, an acidic medium that reduces microbial growth, and therefore the low transformation of glucose to lactic acid. Furthermore, when the initial pH of the solution approaches the pKa of lactic acid (3.8), the undissociated form of this acid has a high inhibitory effect compared to the dissociated lactate form 2,16.
Studies performed with Lactobacillus strains indicate high lactic acid productions for pH ranges from 5.0 to 6.5. The literature reports lactic acid productions of 74.01 g/L from a hydrolyzed cassava starch with a starch concentration of 120 g/L at pH 5, with a 61.67 % degree of conversion 13. This is a higher production than that obtained in our study (33.48 %), because cassava bagasse starch has non-hydrolyzed lignocellulosic matter that generates interference in fermentation.
A previous study showed lactic acid production from cassava bagasse starch with a Lactobacillus delbrueckii strain reaching 136.8 and 196.4 mg/g of a substrate at pH 4.0 and 5.0, respectively 14. In our study, a yield of 334.80 and 276.20 mg of lactic acid was obtained for each gram of cassava bagasse starch at pH 5.5 and pH 5.0, respectively, so higher contents of lactic acid than those mentioned in the literature. This is because, in the experimentation, cassava bagasse starch was used and not directly the bagasse.

Evaluation of the fermentation efficiency of lactobacillus leichmannii of a starch hydrolyzate from cassava bagasse
The stoichiometric relationship was used under the microorganism's ideal conditions to perform the calculations for lactic acid's theoretical production from glucose (pH 5.0 at 37 ºC) 15.
The conditions of pH, temperature, and agitation are the most important in fermentation. As seen in Figure 1., there is low production of real lactic acid compared to the theoretical one that should reach 71.66 g/L and 85.95 g/L of lactic acid for INIAP 650 and 651 varieties, respectively. Thus, maximum values ​​of 27.62 ± 0.69 g/L and 33.48 ± 1.87 g/L were obtained at pH 5.5 with the agitation of 150 rpm of INIAP 650 and 651 varieties, respectively. This may be because the theoretical production will always be greater than the production that was reached experimentally. Due to the type of microorganisms for fermentation, fermentation times, pH, temperature, and agitation, the medium and substrate are not considered.

Figure 1. Theoretical and real fermentation efficiency of the fermentation at pH 5.5 with stirring of the broth.
Furthermore, the theoretical yield was determined, assuming that reducing sugars corresponds entirely to glucose. The determining factors were pH and substrate. Furthermore, the microorganism used in the fermentation, specifically consumes glucose for the production of lactic acid, and in the hydrolysis of starch, reducing sugars were obtained that include maltose, dextrins, and glucose. Therefore, the conversion of reducing sugars to lactic acid will not reach the total.

Characterization of the final lactic acid solution
The characterization of the purified lactic acid solution in the different treatments is presented below. The parameters to be evaluated were pH and density. Furthermore, the samples were characterized by FTIR with the representative lactic acid spectrograms.

Physicochemical characterization of lactic acid solutions.
The physical properties that were evaluated in the lactic acid solution were density and pH. A comparison was made with 98% lactic acid values at 20 ° C.
In Table 5. the density and final pH of the lactic acid solutions are presented. The density in treatments T7, T8, and T9 reached the highest values because they present the highest concentration of lactic acid. The highest lactic acid production was at pH 5.5, with stirring of the hydrolyzed cassava bagasse starch of INIAP 651 variety. Therefore, the density directly relates to lactic acid concentration with a correlation coefficient of R2 = 0.961. The higher the concentration, the higher the density because the water concentration decreases, and the density takes the value of pure lactic acid.
Within this work, density values were obtained in the range of 1.007 g/mL to 1.133 g/mL. The literature reports density in 98% lactic acid of 1,215 g/mL 15. The value is not within the range obtained in our work because the final lactic acid concentration reached a maximum of 33.48 g/L. The pH of all the treatments decreased due to the presence of lactic acid in the solution. This varies according to the initial pH for fermentation.


Table 5. Physical-chemical characteristics of the final lactic acid solution
Evaluation of the organic compounds present in the final lactic acid samples
FTIR analysis was carried out to check the presence of lactic acid in the samples obtained after fermentation. Figure 2. showed the FTIR spectrogram of the lactic acid sample that presented the best production, T9 of INIAP 651 variety, pH 5.5, and stirring. This spectrogram's organic compound's characteristic bands are shown, and the spectrogram of a commercial lactic acid. All the treatments presented similar characteristics of lactic acid spectrograms.


Figure 2. Spectrogram of lactic acid FTIR obtained from INIAP 651 starch hydrolyzate subjected to fermentation at pH = 5.5 with stirring and commercial lactic acid FTIR spectrogram (Carlo Erba brand, Pharmacopeia grade) 18
The experimental spectrogram shows the OH stretch bands at 3392.2 , alcohol and carboxyl groups at 2940.9 and 2989,1 . For the COOH group: the  bond presents a tension at 1715.4  , typical of the acid group. Groups C  show symmetric flexures at 1375 and 1456 . The C-O stretches of the alcohol and acid groups at 1210.1 , 1119.3 and 1040,4 . The characteristics of the commercial lactic acid spectrogram are linked to OH stretches at 3338 ,  alcohol and carboxyl groups at 2944 and 2995 . For the COOH group: the bond presents a tension at 1716 , typical of the acid group. Groups C show symmetric flexures at 1376 and 1432 . The C-O  stretches of the alcohol and acid groups at 1220 , 1120 , and 1070 18. The resulting spectra showed the characteristic bands of this compound. Therefore, lactic acid was confirmed in all the final samples, with approximately 99% accuracy.
In our research, the characteristic spectrogram presents a band at the level of 2635 , because of the formation of amino compounds, due to the presence of protein in the initial substrate. Besides, the spectrograms of this work do not present peaks own of other substances. It is worth mentioning that the samples do not present the characteristic band of water that ranges from 3600 to 4000 , because the samples were pre-treated by evaporation at 65 ºC and thus there was no interference in the sample scanning with the equipment.

CONCLUSIONS

Lactic acid production from INIAP 650 and 651 varieties of cassava made of bagasse starch hydrolyzate reached relevant yields. The highest production of lactic acid was obtained at pH 5.5 
with stirring thereby;, it could be thought that at higher pH, the microorganism works better in the production of lactic acid. Agitation maximizes the production of lactic acid in the fermentation 
processes of hydrolyzed cassava bagasse starch. So, the pH-agitation system has a significant effect on lactic acid production in the fermentation processes of hydrolyzed cassava bagasse starch. 
The infrared spectrogram showed the characteristic of lactic acid bands for all the treatments analyzed. The quality of lactic acid from cassava by-product was verified and could have significant 
market value for Ecuador's sustainable development.

Acknowledgments
This research was supported economically by EPN through the project PREDU-2016-015. The authors thank F. Jadán and H. Caballero (UTM) for their collaboration in the collection of samples, starch extraction, and physical-chemical analysis of the raw material, and I. Chango (CIAP-EPN) for assistance in recording and interpreting spectra

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Received: 23 October 2021
Accepted: 20 November 2021

Shirley Inguillay, Pedro Maldonado-Alvarado
Department of Food Sciences and Biotechnology. Escuela Politécnica Nacional. Quito-Ecuador
Corresponding author: pedro.maldonado@epn.edu.ec
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