Animal Science

Formulation and characterization of a biopreparation with Lactobacillus plantarum CAM-6, from the gastrointestinal tract of Colombian native pigs

0000-0001-8627-2522C. A. Betancur¹  , 0000-0003-3019-1971Ana J. Rondón²  , 0000-0003-2167-4904Y. Martínez^{3  *}  , 0000-0002-6496-4453R. Rodríguez⁴ 

¹Facultad de Medicina Veterinaria y Zootecnia, Universidad de Córdoba, Departamento de Ciencias Pecuarias, Carrera 6 No 76-103, 230002. Montería, Colombia

²Centro de Estudios Biotecnológicos, Universidad de Matanzas, Autopista Varadero km 3 ½. Matanzas, Cuba

³Departamento de Producción Agropecuaria, Escuela Agrícola Panamericana Zamorano. Honduras

⁴Centro de Estudios de Producción Animal, Facultad de Ciencias Agropecuarias, Universidad de Granma. Granma, Cuba

Abstract

The physical-chemical composition of fruit peels was characterized in order to obtain a biopreparation from a culture medium with aqueous extracts of fruit peels (banana, pineapple and papaya) for growing Lactobacillus plantarum CAM-6 and the evaluation of its kinetic parameters and stability over time. Then, an experiment with a completely randomized design and five treatments was developed, in which different mixtures of water with different proportions of fruit peels were evaluated as culture medium. Biomass, concentration of total reducing sugars, lactic acid and pH were determined. Later, growth kinetics was developed in the selected medium for 24 h, and specific growth rate and duplication time were determined. Finally, strain viability was evaluated under refrigeration conditions, at 4 ° C, for 48 days. It was demonstrated that peels presented sufficient soluble solids (12.50-11.67 ºBrix), proteins (0.33-1.31%) and ashes (0.53-1.32%) for bacterial growth. The most suitable medium was the aqueous broth of fruit peel extract, containing 40% fruit peel and 60% water, with a growth of 22.97 LN CFU.mL-1, speed of 0.42 h-1 and duplication time of 1.12 h. Viability was stable for the first 24 days (> 22 LN CFU.mL-1) at 4ºC. It is concluded that the biopreparation with L. plantarum CAM-6 in aqueous extract of papaya, pineapple and banana peels (40:60) guarantees cell growth and viability for 24 days.

Key words: fruit peels; culture medium; growth kinetics; bioprocesses

Papaya (Carica papaya L.), pineapple (Ananas comosus L.) and banana (Musa paradisiaca L.) peels are residues consisting mainly of water, carbohydrates, fiber, proteins, lipids, vitamins and minerals, which could be used as a nutrient source for bacteria, fungi and yeasts. These peels provide the necessary elements for the growth of different species of Lactobacillus spp., because their metabolism is homofermentative, since it mainly uses glucose and fructose, and converts them into lactic acid by means of the Embden-Meyerhof-Parnas glycolytic pathway (^{Jurado-Gámez et al. 2013} and ^{Ravimannan et al. 2014}).

The sustainable use of these agricultural residues allows to reduce pollution and consume what usually is discarded and even generate aggregate value products (^{Cervantes et al. 2016}). Fruit peels are an abundant source of fermentable sugars, which could be used in the production of microbial biomass, enzymes (^{Rojas et al. 2018}), biodiesel (^{Patel et al. 2015}) and animal feed (^{Ospina et al. 2012}, ^{Tijani et al. 2012} and ^{Saheed et al. 2013}). Currently, most developing or economically advanced countries face the problem of disposal and treatment of these residues, which could be reused and reduce their volume, with technical, economic and environmental benefits (^{Ali et al. 2014} and ^{Hamzah et al. 2018}).

In Colombia, large concentrations of solid wastes (peel and bagasse) are generated, as a result of obtaining juices and preserves, mainly of papaya, pineapple and banana, which are the most accepted and highly-produced fruits in the country. These residues could be used in the preparation of products of high biological value, such as probiotics, which would contribute to eliminate a major contamination source (^{Gowe 2015}, ^{DNP 2016} and ^{Anbu et al. 2017}).

Generally, lactic acid bacteria (LAB) are cultivated in media enriched with specific nutrients such as MRS (^{de Mann et al.1960}), which are very expensive and large volumes are required at a laboratory scale. Given these requirements, it is necessary to search for other alternatives to cultivate probiotic lactobacilli in media with national components and high availability.

The objective of this study was to obtain a biopreparation from a culture medium with aqueous fruit extracts (banana, pineapple and papaya) for growing Lactobacillus plantarum CAM-6 and for the evaluation of its kinetic parameters and stability over time.

Materials and Methods

Physicochemical analysis of fruit peels. Three samples were randomly taken of each peel (papaya, banana and pineapple) and then crushed in a mortar. Total sugar content, ºBrix, was determined using a portable refractometer (Toledo Refracto 30P, Spain). In addition, pH (pH meter, OAKLON®, Spain), humidity, protein, ash and acidity (expressed in % of malic acid and citric acid) were also analyzed, according to ^{AOAC (1997)}.

Strain used. To establish the culture medium destined for the growth of L. plantarum CAM-6, Lactobacillus plantarum CAM-6 strain (GenBank accession number: MK523644) was used, which comes from the content of the rectum of Zungo pigs. This strain is deposited in the stock of the Biotechnology Laboratory of the University of Cordoba, Colombia.

Agro-industrial waste treatment. Peels of papaya, banana and pineapple, coming from the processing plant for the handcrafted preparation of fruit salad of Monteria market, Cordoba, Colombia, were transferred to facilities of the Laboratory of Fermentations and Bioprocesses of the University of Cordoba, for obtaining the aqueous extract of fruit peels (CEACF). Peels were washed and mixed in a 1:1:1 proportion. Then, they were crushed in potable water, according to the design, in an industrial blender (Fitmix, Germany). Subsequently, the aqueous extract was filtered three times. The first filtration was carried out through a sieve, the second with a gauze and the third with filter paper.

Design of the culture medium. For the culture medium design, a completely randomized experiment was carried out with five treatments and three repetitions: CEACF-1: 70% of drinking water and 30% of fruit peels, CEACF-2: 60% of drinking water and 40% of fruit peels, CEACF-3: 50% of drinking water and 50% of fruit peels, CEACF-4: 40% of water and 60% of fruit peels and CEACF-5: 30% of water and 70% of fruit peels. For each treatment, 100 mL of the extract obtained were added in Erlenmeyers of 250 mL capacity, which were adjusted to pH 5.6 ± 0.2 (HANNA pHmeter, USA), with calcium citrate or citric acid, at a concentration of 99 and 96% purity, respectively. Then, they were sterilized at 121 °C for 15 min. in an autoclave (Systec VB-55, Germany).

Lactobacillus plantarum CAM-6 strain, cultivated in MRS broth for 18 h, at 37 ºC (1010 CFU.mL-1), was inoculated at 10% (v/v) in each Erlenmeyer. Subsequently, a cap was placed and a venoclysis was adapted as a sample extraction system. Fermentation was carried out at room temperature (30 ± 2 °C), with constant agitation at 100 rpm. on an orbital shaker (SK-o330-Pro LB PRO, USA) for 24 h.

Evaluated indicators. In the 24 h samples, L. plantarum CAM-6 growth was quantified by dry weight (^{Harrigan and McCance 1968}) and the concentration of total reducing sugars (TRS) was also determined according to ^{Miller (1959)} technique, as well as lactic acid (^{AOAC 1997}). The pH was calculated with a digital potentiometer (Metrohm 744, Switzerland).

Evaluated indicators and design. Growth kinetics of Lactobacillus plantarum CAM-6 was evaluated in an experiment with a completely randomized design. For this, the strain was inoculated in MRS broth and incubated at 30ºC for 18 h. This inoculum was cultivated at 10% (v/v) in 27 Erlenmeyers containing the previously selected medium (CEACF-4). The Erlenmeyers were kept in incubation at 30 ºC for 24 h. Every three hours, three repetitions were taken to determine viable count by the method of serial dilutions in peptone water (1%, w/v). For colony counting (^{Harrigan and McCance 1968}), cultivation was performed in dishes with MRS agar for 48 h, at 30 ºC. The MRS broth was used as reference medium, where the strain was cultivated under the same conditions. Dispersion curves were prepared with the use of Microsoft Excel program and data obtained from growth kinetics. By applying the fitting method, the corresponding polynomials and specific growth rate values (µ) were obtained. Duplication time (td) was determined by the formula td=LN 2/µ (^{Madigan et al. 1997}).

Reducing sugars were quantified by the dinitrosalicylic (DNS) method, according to ^{Miller (1959)}. Acidity, expressed in lactic acid, was calculated using titratable acidity, according to ^{AOAC (1997)}. The pH in each sampling was measured with a digital pH meter (Sartorius Meter PP-25, Göttingen, Germany).

Experimental design and viable cell count. For the development of this test, the inoculum was obtained from the cultivation of CAM-6 strain in MRS broth for 18 h, at 37 ºC, under static conditions. It was cultivated at 10% (v/v) in three Erlenmeyers of 5 L capacity, with four liters of the selected medium and pH 5. Erlenmeyers were kept at 37 ºC for 24 h. Subsequently, a test was carried out with the use of a completely randomized design, by distributing the content of each Erlenmeyer into 35 sterile glass flasks, with 50 mL of effective volume and rubber cap. Samples were kept in refrigeration (4 ºC) for 48 d. In each sampling (every eight days), five flasks were taken, from which 1 mL was extracted. They were cultivated (10-7-10-9) in dishes with MRS agar by means of serial dilutions in peptone water (1%, w/v). After incubation at 37 ºC for 48 h, CFU count was performed.

Statistical processing. In the physical-chemical analysis of fruit peels, data was processed using descriptive statistics (standard deviation and coefficient of variation). For determining the best variant of CEACF and viability studies of Lactobacillus plantarum CAM-6 over time, one-way analysis of variance (ANOVA) were performed, with prior determination of normality, data homogeneity and a significance level of P <0.05. ^{Duncan (1955)} test was used for multiple comparison among means, using SPSS statistical package, version 21.0 (^{Pardo and Ruíz 2002}). Results of growth kinetics were processed using Microsoft Excel 2016 program, with which the ^{Monod (1949)} model was run. Viable microorganism counts were transformed to LN to ensure normal conditions.

Results and Discussion

Table 1 shows the results of chemical composition of papaya, banana and pineapple peels. These values are similar to those reported in the literature. ^{FAO (2007)} stated that papaya has approximately 13% sugars, 85% humidity and 0.6% protein. They pointed out that pineapple contains from 12 to 15% sugars, 80 to 85% water and 0.58% protein, and banana has 79.2% humidity, 0.83% protein and 12% sugars.

Table 1 Chemical composition of papaya, banana and pineapple peel, from the fruit processing plant of Montería market, Cordoba, Colombia (n=3) 

Indicators	Papaya peel	SD	CV, %	Banana peel	SD	CV, %	Pineapple peel	SD	CV, %
Soluble solids, ºBrix	11.67	0.251	2.14	11.67	0.208	1.78	12.50	0.264	2.11
pH	6.42	0.059	0.91	5.85	0.005	0.08	4.91	0.551	11.22
Acidity*, %	0.18	0.018	10.00	0.31	0.038	12.25	0.47	0.036	7.65
Humidity, %	87.59	0.722	0.82	88.06	0.114	0.13	82.24	1.18	1.43
Crude protein, %	1.31	0.007	0.53	0.33	0.009	2.72	1.14	0.086	7.54
Ashes, %	0.55	0.039	7.09	1.23	0.068	5.52	0.89	0.075	8.42

*Acidity is expressed according to the abundant acid: malic acid in papaya and banana and citric acid in pineapple. Data of chemical composition is expressed under humid basis.

SD: standard deviation

CV: coefficient of variation

Regarding soluble solids, expressed in degrees Brix, values ​​between 11.6 and 12.5 are referred in fruit peels, levels that are considered admitted as established as the minimum acceptable for commercial growers (^{Zhou et al. 2000}). Low pH is due to the contribution of acids, depending on the type of fruit. The high humidity is given by the high water content of these residues (^{Hamzah et al. 2018}).

Even though protein level of different tested fruit peels is low, the content is sufficient for bacteria growth. Proteins are part of the structure of microbial cells and are necessary for the growth of microorganisms (^{Prescott et al. 2004}). As it is known, fermentation industry uses inorganic nitrogen sources, which tend to be expensive in culture media, and make them a limitation for preparing organic products obtained by fermentation (^{Serna and Torres 2015}).

Ash value, obtained in banana peels (1.23%), demonstrates its high mineral content (Ca, K and Mg). Mineral composition of residues will contribute to the development of some metabolic reactions of bacteria in the formulated culture medium (^{Anbu et al.2017} and ^{Saleem and Saeed 2019}).

Studies carried out by ^{De Oliveira et al. (2015)} and ^{Vargas et al. (2019)} confirm that fruit peels have a higher concentration of mineral elements, total sugars and crude protein than pulp, which indicates that these residues have great potential for their use as raw material for animal feed production. These authors refer that, due to ash content of peels, they are considered as potential mineral sources. Ash composition varies according to the fruit, maturity state, variety and harvest season, as well as cultivation conditions (^{Priego 2007}).

The use of agro-industrial residues as low-cost raw material represents an option to transform waste into compounds with beneficial properties (^{Carota et al. 2016}, ^{Yoong et al. 2017} and ^{Vargas et al. 2019}). It was confirmed that fruit peels contain carbohydrates, proteins, vitamins, and minerals, which could meet the nutritional needs of Lactobacillus plantarum CAM-6.

Table 2 shows the results of Lactobacillus plantarum CAM-6 growth in each of the evaluated treatments to obtain the CEACF culture medium. It was verified that the highest (P <0.05) biomass, lactic acid and TRS production was presented in CEACF-4 and CEACF-5 media. It was also demonstrated that the lowest pH values ​​were found in the last two variants. These results demonstrate that Lactobacillus plantarum CAM-6 is capable of producing acids that lower pH in these media. Probiotic bacteria, especially lactobacilli, transform carbon sources into organic acids that intervene in the inhibition of pathogenic microorganisms and in mineral solubilization, in addition to contribute to the maintenance of the intestinal mucosa integrity (^{Vera et al. 2018}).

Table 2 Characteristics of growing L. plantarum CAM-6 in different variants of CEACF of papaya, banana and pineapple at 30 ºC for 24 h 

Indicators	CEACF-1	CEACF-2	CEACF-3	CEACF-4	CEACF-5	SE±	P
Biomass, g	25.49b	34.37b	44.16ab	55.18a	60.46a	2.977	0.003
pH	5.83a	5.57a	5.28a	4.72b	4.68b	0.066	0.014
Lactic acid, g.L-1	3.49b	5.41b	5.57b	7.01a	7.11a	0.262	0.001
TRS, g.L-1	21.12c	33.27b	44.04a	48.00a	47.00a	0.803	0.001

a,b,c Means with different letters in the same line differ at P < 0.05 (^{Duncan 1955})

TRS: total reducing sugars

Fruit peels constitute a potential raw material for the production of biopreparations, as vehicles for the administration of probiotics, due to their high carbohydrate content (^{Vargas et al. 2019}). TRS concentration was higher in CEACF-3, CEACF-4 and CEACF-5 variants, indicating that after 24 h of fermentation, these media still contain available carbon sources for these bacteria. Specifically, CEACF-4 and CEACF-5 variants presented high biomass production, lactic acid and lower pH values, which could contribute to their conservation over time. On the other hand, with the application of the complete additive (cells + acid), acids will contribute to exerting greater action in animals. CEACF-4 was chosen, since this medium has a lower percentage of fruit peels in its composition, which represents savings in raw material.

Figure 1A shows the results of growth kinetics of Lactobacillus plantarum CAM-6 in CEACF-4 and MRS media for 24 h. It is demonstrated that there are no differences in the count of CFUs during the sampling hours, when both substrates are used (P ˃ 0.05). In each case, the initial population began its growth from the first hours, and once in the exponential phase, cells reproduce at maximum speed without limitation of nutritional substances. Figure 1B shows that the higher the lactic acid production, the lower the TRS concentration and pH in CEACF-4 medium. LABs are known to use fermentable carbohydrates as an energy source mainly to form lactic acid (^{Zamudio and Zavaleta 2003}).

Figure 1A y 1B 1A: Performance of growth kinetics of Lactobacillus plantarum CAM-6 in CEACF-4 and MRS culture media; 1B: Production of lactic acid, TRS concentration and pH for 24 h. Bars represent standard deviation 

CEACF-4 medium was suitable for the growth of Lactobacillus plantarum CAM-6 (table 3). Comparing growth of the strain in this medium with respect to MRS, no differences were observed.

Table 3. Growth of Lactobacillus plantarum CAM-6 in MRS and CEACF-4 media at 30 ºC 

Strain	Medium	LN CFU.mL-1 24 h	SE ±	P	µ (h-1)	td, h	R2
Lactobacillus plantarum CAM-6	MRS	21.97 (3.5X109)	0.572	0.281	0.46 ±0.83	1.05±0.83	0.9977
	CEACF-4	22.97 (9.5X109)			0.42±0.86	1.12±0.86	0.9981

1Growth of bacterial strains are expressed in LN

( ) original data

µ: specific growth rate

td: duplication time

R2: coefficient of determination

Results indicate that CEACF-4 components meet the nutritional requirements of these microorganisms in a similar way to the reference medium, so it could be used as a culture medium for probiotic production. For CEACF-4 medium, a specific growth rate of 0.42 ± 0.86 h-1 and a duplication time of 1.12 ± 0.86 h were obtained, with R2 = 0.9981 data fit. These values ​​are very close to the values ​​obtained by ^{Aguirre et al. (2010)}.

^{Garriga et al. (1998)}report that, for evaluating potentialities of candidate strains for probiotics, it must be verified that they have a high growth rate and a duplication time equal to or approximately one hour. This way, used microorganisms will have greater possibilities of being duplicated quickly in the gastrointestinal tract, to achieve their predominance in this ecosystem. This was demonstrated in this study, in which it was demonstrated that Lactobacillus plantarum CAM-6 strain has these characteristics.

Figure 2 represents performance dynamics of Lactobacillus plantarum CAM-6 viability, grown in CEACF-4, from the first day to 48 d of sampling. Until 24 d, the biopreparation showed count stability under refrigeration conditions (>21 LN CFU.mL-1). It is precisely after that time that the decrease in viable cells in the biopreparation is evident, although cell population that contains at 48 d (19 LN CFU.mL-1) is sufficient to develop probiotic activity (>108 CFU.mL-1). ^{Jin et al. (1998)} defined that probiotic biopreparations should contain 109 CFU.mL-1 to exert their effect.

a,b,c,d Different letters differ at P < 0.05 (^{Duncan 1955})(P < 0.001)SE ± 0.094

Figure 2 Dynamics of Lactobacillus plantarum CAM-6 viability in the biopreparation, under refrigeration conditions, at 4 ºC 

It is concluded that the bioprepared with L. plantarum CAM-6 in aqueous extract broth of papaya, pineapple and banana peels (40:60) has the necessary nutrients to guarantee a high population of cells, with a higher growth rate and duplication time. Furthermore, cell viability was observed for 24 d, under refrigeration conditions, at 4 ° C.

Acknowledgements

The authors would like to thank specialists from the Laboratorio de Ingeniería de Alimentos of the Universidad de Córdoba.

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Received: January 31, 2020; Accepted: June 16, 2020

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Declaración de conflicto de intereses: Los autores declaran no presentar conflicto de intereses

Contribución de los autores: Los autores declaran presentar contribución igualitaria en la concepción de la investigación, obtención y procesamiento de los datos y redacción del documento