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Revista mexicana de ingeniería química

versão impressa ISSN 1665-2738

Rev. Mex. Ing. Quím vol.15 no.1 México Abr. 2016


Ingeniería de alimentos

Papaya (Carica papaya) and tuna (Thunnus albacares) by-products fermentation as biomanufacturing approach towards antioxidant protein hydrolysates

Fermentación de papaya Carica papaya ) y subproductos de atún (Thunnus albacares ) para la biofabricación de hidrolizados de proteínas con actividad antioxidante

M.P. Carballo-Sánchez1 

J.C. Ramírez-Ramírez2 

M. Gimeno3 

G.M. Hall4 

M.G. Ríos-Durán5 

K. Shirai1  * 

1 Universidad Autónoma Metropolitana. Biotechnology Department, Laboratory of Biopolymers and Pilot Plant of Bioprocessing of Agro-Industrial and Food By-Products, Av. San Rafael Atlixco No. 186. Col. Vicentina, C.P. 09340. Iztapalapa, Mexico City, México.

2 Universidad Autónoma de Nayarit, Ciudad de la Cultura Amado Nervo, C.P. 63155 Tepic, Nayarit. Mexico.

3 UNAM, Depto. de Alimentos y Biotecnología, Facultad de Química, (C.P. 04510, Mexico City, México.

4 University of Central Lancashire, Centre for Sustainable Development PR1 2HE United Kingdom.

5 Universidad Michoacana de San Nicolás de Hidalgo, Instituto de Investigaciones Agropecuarias y Forestales. San Juanito Itzícuaro S/N. Unidad San Juanito. CP. 58330) México.


The efficient production of lactic acid, standardization of ingredients, protein hydrolysis, and the presence of an active starter were key factors for development of a controlled lactic acid fermentation of fish wastes and papaya. The maxima buffering capacities, proteases activities, protein and ash contents were considered for the selection of the suitable mixture of bones, fins, viscera, heads and dark meat. In addition, the overproduction of papaya is commonly presented in tropical countries, and herein, papaya surpluses were successfully employed as carbon source for Lactobacillus plantarum fermentation of tuna toward the production of added value protein hydrolysates. The maximum lactic acid produced, acidification rate constant and maximum acid production rate were estimated by Gompertz model with experimental data of 5 d of 1.27 mmol of lactic acid/g, 0.142 1/h and 0.067 mmol LA/gh, respectively. The highest degree of hydrolysis of 87.71% was achieved at 120 h. The radical-scavenging activity of the protein hydrolysates was determined by electron paramagnetic resonance spectroscopy analyses based on the conversion of 2,2-diphenyl-1-picrylhydrazyl free-radical. In addition, the lowest concentration of tuna protein hydrolysates (5.75 (g protein/mg) that gave the half-maximal inhibitory concentration was determined at 72 h of fermentation.

Keywords: Thunnus albacares; radical scavenging; biorefinery; protein hydrolysate; Carica papaya


La producción eficiente de ácido láctico, estandarización de los ingredientes, hidrolisis de proteínas, así como la presencia de un cultivo iniciador activo fueron factores claves para el desarrollo de una fermentación acido láctica controlada. El contenido de proteína, cenizas, enzimas proteolíticas y la capacidad amortiguadora del sustrato fueron empleados como criterios para la selección de la mezcla de cabezas, huesos, vísceras y carne negra de atún (Thunnus albacares). Otro subproducto empleado en este trabajo fue papaya (Carica papaya), la cual comunmente presenta excedentes de producción. La concentración máxima de ácido láctico producido, la tasa de acidificación y la velocidad máxima de producción fueron estimadas mediante modelo de Gompertz en 1.27 mmol de ácido láctico/g, 0.142 1/h y 0.067 mmol de ácido láctico /gh, respectivamente. El grado de hidrolisis más alto determinado a las 120 h fue de 87.71%. La actividad antiradicalaria en los hidrolizados de proteínas fue determinada mediante estudios de espectroscopia de resonancia paramagnética electrónica, basado en la inhibición del radical 2,2-difenil-1-picrilhidrazil. La concentración más baja de hidrolizado proteico que produjo el 50% de actividad antiradicalaria, IC50, fue de 5.75 ?g de proteína/g determinada a las 72 h de fermentación.

Palabras clave: Thunnus albacares; antioxidante; capacidad antiradicalaria; hidrolizados proteicos; Carica papaya

1 Introduction

Tuna species (Thunnus albacares) are extensively processed worldwide giving rise to a large amount of unused by-products. These by-products are composed of viscera, skin, gills, head, bones and dark meat. Despite good nutritional characteristics, dark or red meat have no commercial value due to their unpleasant taste (Graham and Dickson 2004). Several methods have been reported to draw on these by-products; some are based on chemical hydrolysis, regardless of poor final product quality, and others by biological means including hydrolysis by exogenous or endogenous enzymes in mild process conditions (Pihlanto and Korhonen 2003; Chi et al.,2015; Reyes-Mendez et al.,2015). These bio-based methodologies offer ease of control of the reaction and optimal recovery of added-value products without side products (Shirai and Ramirez-Ramirez 2011). Among them, lactic acid fermentation (LAF) proved successful owing to process stability, inhibition of undesirable bacterial growth and the production of biopolymers, valued organic acids and hydrolysates in a GRAS and chemical-free process with low energy requirement (Ramírez-Ramírez et al., 2008; Flores-Albino et al., 2012). These fermentations are widely used to process small pelagic fish or fish by-products, e.g. sauce production in heavily salted conditions. In this regard, some mechanisms have been put forth to describe antioxidant activities of peptides, including ion-chelating capacity, radical scavenging and aldehyde adduction (Mishra et al., 2011). The feed for the lactic acid bacteria (LAB) covers a wide range of substrates; nonetheless the papaya (Carica papaya) is pointed out due to its high protease content and additionally, it is intensively cultivated throughout the year in tropical countries owing to its high demand as fresh fruit and ingredient in food processing (Fabi et al., 2007). On the other hand, the lactic acid (LA) produced in situ has a significant role in fish conservation and antimicrobial effect against undesirable microflora. Generally, the efficacy of LAF is based on the rate of pH drop and its final value, which rely on water activity, temperature and process time, activity of starter and buffer capacity. Practical utilization of fish by-products by biotechnological means requires information on the buffer capacity, endogenous protease activities, and ash and protein contents. In this regard, the bioactive peptide production from tuna wastes by LAF with papaya and molasses as carbon sources has not been reported to the best of our knowledge and the present work establishes the conditions for efficient bioprocess production of antioxidant protein hydrolysates.

2 Materials and methods

2.1 Materials

Lactobacillus plantarum (APG-Eurozym) was cultivated in Man, Rogosa and Sharpe (MRS) agar at 30 °C and kept at 8 °C prior to use. Inoculum was prepared in MRS broth by inoculation from the bacterial slants and incubated at 30 °C for 24 h to reach a cell count of 108 colony-forming units per millilitre (CFU/mL). Yellowfin tuna (Thunnus albacares) wastes were obtained from a canned tuna processing plant in Mazatlan (Sinaloa State, Mexico). The wastes consisted of three factions: i) fresh viscera (V), ii) heads, bones and fins (HBF) and iii) dark meat (DM). The fish were manually gutted and abdominal cavity was washed with water. Eviscerated fish was steam cooked at 100 °C for 3 h. HBF and DM were separated from fish. Each fraction was minced separately through a 5 mm sieve using a meat mincer (Torrey 32-3, Mexico) and stored at -20 °C. Sugar cane molasses were supplied by sugar mill "El Molino S.A. de C.V." in Nayarit state (Mexico). Papaya fruit (Carica papaya L. var Maradol) at commercial maturity (skin yellowed and orange soft pulp) was harvested in the state of Veracruz (Mexico) and milled in a blender prior to use. Soluble sugars in molasses and papaya were determined by the method of phenol sulphuric acid (Dubois et al 1956). Reagents 2,2-diphenyl-1-picrylhydrazyl (DPPH), acrylamide, bisacrylamide, sodium dodecyl sulphate, Tris base, Tricine, glycerol and ultra-low molecular weight marker (1060-26600 Da) were supplied by Sigma-Aldrich (USA). Electrophoresis grade Coomasie Blue G-250, 2-mercaptoethanol and TEMED were supplied by Bio-Rad (USA).

2.2 Determination of tuna wastes proportions in the mixture, starter level and papaya concentration for LAF

Tuna wastes in LAF varied upon the ratio of V and HBF (wt/wt%): 64.9:7.2; 43.8:28.3; 33.3:39.3; 14.4:42.3; 7.2: 64.9. DM was kept at 27.9 wt/wt% considering that this fraction presented the highest protein content. Mixtures were analyzed on their proteases activities (Bougatef et al., 2009) and ash content (AOAC 1997). Five grams of tuna waste mixtures were homogenized in 50 ml of deionized water. Homogenates were equilibrated at 25°C and stirred continuously during titrations with LA (0.054N) employing pH meter equipped with a glass electrode (pH 210 HANNA, Italy). Maximum bufer capacity (?max) was estimated as the slope of pH-neutralization curves in the immediate vicinity of pKa value (pKa of LA=3.86) according to equation 1 (Baicu and Taylor, 2002).


dV is expressed in mmol/l, β max in slykes (mmol of LA required to titrate the pH of 1 g (wet wt) of tuna mixture by one pH unit). The first derivative f (X0) = |a/4b| is the slope of the tangent to the titration curve at pKa location. Limits of linearity are (y 2 - y 1 ) and (x2 - x1) of the pH-LA volume curve. Tangent and linear portion of the curve were overlapped and their slopes coincided.

The starter level was determined using 10 (wt/wt%) sugar cane molasses to the selected tuna wastes mixture and inoculated with 5,10 or 20 (v/wt% wet basis) of L. plantarum at 30 °C. The integrated Gompertz model was applied to analyze the kinetic data of LA production as function of time t according to the equation 2:


where P max is the maximum LA produced (t → ∞), b is a constant related to the initial conditions (when t = 0, then P = Pmax exp(-b)) and k is the acidification rate constant. Kinetic constants P max , b and k were estimated by the non-linear estimation programme STATISTICA (StatSoft, Inc.). Maximum acid production rate (V max ) was calculated from parameters of the Gompertz model as V max = 0.368 kP max.