Effect of microperforation and temperature on quality of modified atmosphere packaged huitlacoche (Ustilago maydis)

Abstract

Huitlacoche (Ustilago maydis) is a food in the rural communities of some Latin American countries, but it is very perishable. For this reason, its postharvest quality was evaluated in refrigeration (3°C) and at ambient thermal conditions (20°C), with modified atmosphere packaging (MAP) implemented in low-density polyethylene bags of 27 × 28 cm, with 0, 2, 5, 10, and 20 microperforations (mp) of 15 μm. Batches of 150 g of huitlacoche galls with firmness of 5.6 N, pH of 4.8, lightness of 52.4, and hue angle of 87.0° were used. The product proved to be resistant to concentrations of O₂ as low as 1.7 kPa. The adjustment of the number of orifices allowed the control of gas concentrations inside bags, and those with 5 mp permitted the best quality characteristics at 20 and 3°C, with weight loss rates of 0.42 and 0.02% d⁻¹, lightness of 43.7 and 40.3, hue angle of 81.4° and 78.0°, respectively, and firmness of 3.4 N a both conditions. Change rates of quality attributes at 3°C were lesser than at 20°C, which proved that with refrigeration the shelf life was extended up to 30 days.

El huitlacoche (Ustilago maydis) se consume como alimento en comunidades rurales de algunos países de Latinoamérica, pero es muy perecedero. Por ello, se evaluó su calidad postcosecha en refrigeración (3°C) y a temperatura ambiente (20°C), con atmósfera modificada (AM) en bolsas de polietileno de baja densidad de 27 × 28 cm, con 0, 2, 5, 10 y 20 microperforaciones (mp) de 15 μm. Se usaron lotes de 150 g de agallas de huitlacoche con 5,6 N de firmeza, 4,8 de pH, 52,4 de luminosidad y 87,0° de ángulo de matiz. El producto mostró resistencia a concentraciones de O₂ tan bajas como 1,7 kPa. El ajuste del número de orificios permitió controlar la concentración de gases en las bolsas, y aquéllas con 5 mp permitieron las mejores características de calidad a 20 y 3°C, con velocidades de pérdida de peso de 0,42 y 0,02 % d⁻¹, luminosidad de 43,7 y 40,3, ángulo de matiz de 81,4 y 78,0°, respectivamente, y firmeza de 3,4 N en ambas condiciones. Las velocidades de cambio de los atributos de calidad a 3°C fueron menores que a 20°C, lo que causó que con refrigeración la vida de anaquel se extendiera hasta 30 d.

Keywords:

• Ustilago maydis
• huitlacoche
• corn smut
• microperforated packaging
• modified atmosphere packaging
• MAP
• postharvest

Palabras clave:

• Ustilago maydis
• huitlacoche
• carbón de maíz
• empaque microperforado
• atmósfera modificada
• AM
• postcosecha

Introduction

Ustilago maydis (D.C.) Corda is a basidiomycete fungus of the Order Ustilaginales and the Class Teliomycetes. U. maydis causes smut of maize (Zea mays) or huitlacoche (Syn. cuitlacoche), which is characterized by the presence of galls on various aerial parts of the plant, including stalks, leaves, tassels, and ears (Valverde, Paredes-López, Pataky, & Guevara-Lara, 1995). During development, basidiospores germinate and produce mycelium that infects the host, followed by dikaryotization by hyphal fusion. Then, mycelium undergoes morphological changes to form diploid teliospores that fill the characteristic galls (Banuett & Herskowitz, 1996).

Although, globally these galls are known as corn smut, a common disease of maize, in some Latin American countries the galls from ears are considered food (Tracy, Vargas, Zepeda, Pataky, & Chandler, 2007; Valverde et al., 1995) that has been part of the rural population diet since the pre-Columbian time. This product is recognized as an exotic dish in countries of the European Community, and also in the USA and Japan (Tracy et al., 2007; Villanueva-Verduzco, Sánchez-Ramírez, & Villanueva-Sánchez, 2007). Besides, huitlacoche has been studied to evaluate its toxicity, but no harmful compounds have been reported (Valdez-Morales et al., 2010; Valverde et al., 1995). The proximate constituents consist of 88–94% dry matter, 3–6% ash (dry basis), 7–12% protein, and 2–5% lipids (Valdez-Morales et al., 2010). Huitlacoche contains almost all the essential and many non-essential amino acids, lysine being the most abundant, corresponding to more than 14% of the total amino acids, followed by leucine (10.4%), glycine (11.3%), and aspartic acid (8.2%) (Juárez-Montiel, Ruiloba de León, Chávez-Camarillo, Hernández-Rodríguez, & Villa-Tanaca, 2011). Free sugar content (dry matter basis) consists mainly of glucose (140–180 mg g⁻¹), fructose (60–100 mg g⁻¹), galactose (0.5–2 mg g⁻¹), and small quantities of arabinose, xylose, and mannose (Valdez-Morales et al., 2010). Also, the fatty acid composition of this food is integrated mainly by the acids oleic (41–46%), linoleic (27–34%), linolenic (1.2–1.8%), palmitic (14–18%), and arachidic (2.4–4.0%) (Juárez-Montiel et al., 2011).

Huitlacoche is a very perishable product; its postharvest life at ambient temperature is characterized by high respiration rate (373 mL [CO₂] kg⁻¹ h⁻¹), no ethylene production, dehydration, modification in color from yellowish-white to black, rupture of periderm, and a shelf life of lesser than three days (Martínez-Flores, Corrales-García, Espinosa-Solares, García-Gatica, & Villanueva-Verduzco, 2008). Consumers prefer fresh huitlacoche with splitted periderm, which occurs around 16–17 days after inoculation, but restaurants do not want it when the periderm has broken, because consistency becomes very soft and makes handling difficult (Tracy et al., 2007). For that, refrigeration at 3°C was recommended by Martínez-Flores et al. (2008) with storage of 11 days, and they achieved to reduce darkening and also respiration rate to 164 mL [CO₂] kg⁻¹ h⁻¹, but in order to offer better commercialization alternatives a longer conservation period is needed. Studies have shown that modified atmosphere packaging (MAP) offers the possibility to preserve the postharvest quality of fresh produces (Ozdemir, Monnet, & Gouble, 2005; Pastorizaa & Bernárdez, 2011). In the case of respiring commodities this technology relies on the interplay of producing respiration and plastic package permeability, causing O₂ concentration to decrease, while CO₂ increases within bags (Mangaraj, Goswami, & Mahajan, 2009). Desirable responses of this include reduction in respiration, reduction in oxidative tissue damage or discoloration, reduction in the rate of chlorophyll degradation, and reduction in ethylene sensitivity, with the concomitant reduction in the rate of ripening and other ethylene-mediated phenomena (Beaudry, 1999). The challenge with this procedure is that O₂ can reach a critical level, which stimulates a fermentative metabolism, whose products can be harmful to tissues and can generate off-flavors and off-odors (Rojas-Graü, Oms-Oliu, Soliva-Fortuny, & Martín-Belloso, 2009).

To improve MAP systems, perforations are created in packages (Mangaraj et al., 2009), which can have several millimeters in diameter (Fonseca, Oliveira, Lino, Brecht, & Chau, 2000; Montanez et al., 2005) or consist of microscopic holes, whose dimensions range up to 200 μm (Makino, Oshita, Kawagoe, & Tanaka, 2008; Ozdemir et al., 2005; Renault, Souty, & Chambroy, 1994). With this strategy, the permeability of plastic walls has been controlled (Allan-Wojtas, Forney, Moyls, & Moreau, 2007; Makino et al., 2008), and products like pre-cut Welsh onions (Ibaraki, Ishii, Ikematsu, Ikeda, & Ohta, 2000) and Charentais-type muskmelons (Rodov et al., 2002) have been handled properly in modified atmosphere, because the excessive O₂ drop inside the package has been avoided. Hence, the objective of the present study was to evaluate the effect of microperforation and temperature on the postharvest shelf life of MA-packaged huitlacoche.

Materials and methods

Plant material and sample preparation

Corncobs of huitlacoche were collected from Ixmiquilpan, Hidalgo, Mexico (20°29′ N, 99°13′ O), and transported by land within a period of 4 h to the experimentation place at Texcoco, Mexico, Mexico (19°30′ N, 98°52′ O), in padded boxes to avoid mechanical damage. Galls were separated from the cobs on the day of harvest to be used as experimental plant material.

Experimental setup

Effect of microperforation on O₂ and CO₂ concentrations

Five groups of six batches of 150 g of huitlacoche galls were placed in transparent low-density polyethylene bags (27 cm × 28 cm; Bol Rol®, Mexico). According to Mangaraj et al. (2009), the permeability (kg m s⁻¹m⁻² Pa⁻¹) of LDPE at 25°C is 2.8 × 10⁻¹⁷ for O₂ and 17.3 × 10⁻¹⁷ for CO₂, and varies with temperature with Q₁₀ values of 1.96 and 1.71, respectively. A pin of 15 μm in diameter was used to punch plastic of four groups to form treatments with 2, 5, 10, and 20 microperforations, which were named M2, M5, M10, and M20, respectively. The units of the remaining group were left without holes and formed the intact bag treatment, named M0. In order to allow thermal conditioning, half of the bags were placed without closing at 20 (±1)°C and the other half at 3 (±1)°C for 12 h. Then, all of them were hermetically sealed, taking this moment as time zero. Sampling of the products' surrounding atmosphere was carried out by placing 25 small (4 mL) open containers (Vacutainer®) with their corresponding stoppers inside each package, which were sequentially closed without opening bags at time intervals of 1 h during 24 h.

The content of the containers was analyzed to determine O₂ and CO₂ concentrations with a gas chromatograph (Varian, model 3400CX, USA), equipped with a Chrompack Poraplot-Q capillary column, a thermal conductivity detector, and a flame ionization detector. The operating conditions were 80°C, 150°C, and 170°C in the column, injector, and detectors, respectively, and nitrogen was used as carrier gas at a column pressure of 158.5 kPa. Concentrations were expressed in partial pressure, considering a standard total pressure of 101.325 kPa. Further, the time–concentration relationship was evaluated through regression routines applied to data, and the steady-state O₂ and CO₂ concentrations (p _∞) were evaluated substituting an infinitive time condition (t _∞) in the resulting models. Later, two treatments in which the O₂ steady state concentration (p _∞) was lesser than 3% were selected and gas concentrations inside their packages were evaluated during 30 days using batches of 150 g of product.

Evaluation of huitlacoche shelf life in a microperforation-based MA

The effect of selected treatments from the previous phase on product postharvest behavior was evaluated in storage of 30 days and compared with a handling under natural air (NA) conditions. Two groups of 135 experimental units with 150 g of product were formed and assigned to 20 (±1) and 3 (±1)°C conditions, with 68 and 92% relative humidity (RH), respectively. In each case 45 batches were packed in LDPE non-perforated bags (treatment M0), 45 in bags with five microperforations (treatment M5), and the remaining 45, named treatment NA, were left unpackaged, exposed to the room NA condition. To sample the internal atmosphere a 4-mL container with its stopper (Vacutainer®) was also placed in each bag. The day when the experiment started (time zero; t = 0) the material was assessed in terms of color, weight, pH, and firmness. At day 2, three experimental units were removed from each treatment and the 4-mL container was closed to obtain a sample of the bag internal atmosphere to evaluate the prevailing O₂ and CO₂ concentrations. After that, bags were opened and the material was evaluated for weight loss, pH, firmness, color, and fermentative metabolites (acetaldehyde and ethanol) content. This was repeated each third day until the 30-day storage was completed.

Quality measurements

Weight loss was evaluated with a digital scale (Ohaus, USA) in relation to the beginning of the storage period. For color measurements a Hunter Lab colorimeter (model Mini Scan XE Plus 45/0-L, USA) was used; values of L*, a*, and b* were determined and hue angle (H* = tan⁻¹ b*/a*) and chroma (C* = [(a*)² + (b*)²]^1/2) were calculated, while L* expressed lightness (McGuire, 1992). Firmness was evaluated in Newton (N) in four individual galls of each experimental unit, using a texture analyzer TA-XT2i (Stable Micro Systems, 2005), where the sample was deformed up to 5 mm at a 5 mm s⁻¹ velocity with a conical element of 2.6 cm at the base, an angle of 75°, and a rounded tip. The pH was measured with a portable potentiometer (Hanna Instruments, USA) in four huitlacoche galls randomly obtained from each experimental unit and macerated with 15 mL of distilled water. The method of Davis and Chase (1969) was adapted to quantify fermentative compounds in 15 g of product that was placed in 37-mL vials. These were incubated at 60°C for 1 h and the composition of their headspace was quantified in μmol 100 mL⁻¹ with the gas chromatography unit operated at 160°C, 170°C, and 170°C in the column, injector, and detector, respectively. In addition, based on the alcoholic fermentation route stoichiometry (Taiz & Zeiger, 2006) the sum of both compounds was obtained to express fermentation metabolites content. Concentrations of O₂ and CO₂ were evaluated with the samples obtained from packages, which were analyzed with the same chromatograph described above.

Statistical analysis

The 24-h evaluation of microperforation effect on steady state O₂ and CO₂ concentrations was based on a completely random 2 × 5 factorial arrangement design, where temperature (20 and 3°C) and package type (M0, M2, M5, M10, and M20) represented the variation sources. On other hand, the 30-day evaluation of product shelf life was based on a completely random 2 × 3 factorial arrangement design, with temperature (20 and 3°C) and product environment (NA, M5, and M0) as variation sources. In both cases analysis of variance and means comparison routines (Tukey, 0.05) were carried out with the SAS statistical software (SAS Institute, 1989). Three replications applied in all cases, and one 150-g bag was considered an experimental unit.

Results and discussion

O₂ and CO₂ concentrations inside packages

The 24-h study of the MA systems implemented with different number of perforations showed that the O₂ concentration diminished and that of CO₂ increased, with a faster change at first, but asymptotic to a constant value at last. In most cases data fitted well to models with the form {p = p ₀ + a[1 – exp (−bt)]} (Figure 1), where p is the package internal gas concentration (kPa) at time t (h), and p ₀ (kPa), a (kPa), and b (h⁻¹) are regression constants. Therefore, from each equation the asymptote was evaluated as [p _∞ = p ₀ + a] to express steady-state O₂ or CO₂ concentration (Table 1).

Table 1. Regression constants related to the model {p = p ₀ + a[1 – exp(-bt)]}.
Tabla 1. Constantes de regresión relacionadas con el modelo {p = p ₀ + a[1 – exp(-bt)]}.

	O2					CO2
Perforations	p 0(kPa)	a(kPa)	b(h^−1)	R ^2	p ∞(kPa)	p 0(kPa)	a(kPa)	b(h^−1)	R ^2	p ∞(kPa)
20°C
0	19.14	−19.86	0.007	0.91	0.00 c^1	0.87	23.53	0.0073	0.95	24.40 a
2	16.67	−16.65	0.012	0.92	0.02 c	0.96	20.55	0.0171	0.94	21.51 a
5	16.60	−15.51	0.006	0.93	1.09 c	2.52	19.53	0.0063	0.93	22.05 a
10	16.77	−10.41	0.009	0.91	6.39 b	2.46	12.64	0.0070	0.90	15.10 b
20	17.83	−8.76	0.017	0.95	9.07 a	2.47	11.94	0.0067	0.90	14.41 b
HSD	–	–	–	–	2.60	–	–	–	–	3.27
3°C
0	18.08	−0.99	0.900	0.91	17.09 a	3.08	7.71	0.1601	0.89	10.79 a
2	20.18	−2.69	0.208	0.92	17.49 a	2.99	7.20	0.1181	0.93	10.19 a
5	19.07	−2.45	0.149	0.88	16.62 a	2.54	3.41	0.2840	0.94	5.95 b
10	16.63	–	0.071	0.81	18.32 a	3.43	2.32	0.0949	0.91	5.75 b
20	16.90	–	0.006	0.83	17.05 a	3.43	–	0.0006	0.95	3.44 c
HSD	–	–	–	–	1.50	–	–	–	–	0.80
Note: The sum [p ∞ = p 0 + a] expresses steady-state O2 or CO2 concentrations. ^1Different letters in p ∞ column indicate significant differences. HSD is honest significant difference (Tukey, 0.05). Values at 3°C condition corresponding to 10 and 20 perforation for O2 and to 20 perforations for CO2 were obtained through linear regression, with a model of the form [p = p 0 + bt]. R ^2 is determination coefficient.
Nota: La suma [p ∞ = p 0 + a] expresa concentración de O2 o CO2 en la condición de equilibrio en régimen estacionario. ^1Letras diferentes en la columna de p ∞ indican diferencias significativas. HSD es diferencia significativa honesta (Tukey, 0,05). Los valores a 3°C que corresponden a 10 y 20 perforaciones para O2 y 20 perforaciones para CO2 fueron obtenidos mediante regresión lineal, con un modelo de la forma [p = p 0 + bt]. R ^2 es coeficiente de determinación.

Figure 1. Concentration change kinetics of O₂ (•) and CO₂ (○) at 20°C (left) and 3°C (right) during the transient phase of modified atmosphere systems in intact bags of low-density polyethylene (a, f), in bags with two microperforations (b, g), in bags with five microperforations (c, h), in bags with 10 microperforations (d, i), and in bags with 20 microperforations (e, j). Punctual symbols (•, ○) represent the mean of three repetitions; bars indicate the corresponding standard error, and lines are the adjustment of data obtained from a non-lineal regression procedure. R _o ² and R _c ² are determination coefficients for O₂ and CO₂, respectively. The fitting data made for O₂ in boxes (i) and (j), and for CO₂ in box (j), corresponded to a linear behavior.

Figura 1. Cinéticas de cambio de concentración de O₂ (•) y CO₂ (○) a 20°C (izquierda) y 3°C (derecha) durante la fase de régimen transitorio de sistemas de atmósfera modificada en bolsas de polietileno de baja densidad intactas (a, f), bolsas con dos microperforaciones (b, g), bolsas con cinco microperforaciones (c, h), bolsas con 10 microperforaciones (d, i) y bolsas con 20 microperforaciones (e, j). Los símbolos (•, ○) representan la media de tres repeticiones, las barras indican error estándar y las líneas corresponden a ajustes obtenidos mediante procedimientos de regresión no lineal. R _o ² and R _c ² son coeficientes de determinación para O₂ y CO₂, respectivamente. Los ajustes de datos hechos para O₂ en los recuadros (i) y (j) y para CO₂ en el recuadro (j) corresponden a comportamiento lineal.

At 20°C, as the punch numbers increased, the O₂ and CO₂ steady-state concentrations were higher and lower, respectively (P ≤ 0.05). In non-perforated bags (M0) and with two perforations (M2) the O₂ was exhausted before 7 h and CO₂ reached concentrations higher than 20 kPa (Figure 1a,b). Thus, neither of these was considered an acceptable alternative for huitlacoche handling, due to the risk of stimulating fermentative metabolism (Taiz & Zeiger, 2006). Meanwhile, treatments with five, 10, and 20 perforations (M5, M10, and M20) showed O₂ steady-state concentrations of 1.1, 6.4, and 9.1 kPa, respectively (p _∞ ; Figure 1c–e). These, being different (honest significant difference (HSD) equal to 2.6 kPa; Table 1), indicated that through variation of the number of microholes, the concentration of gases inside packages can be controlled. In the case of CO₂ concentrations, values of 22.1, 15.1, and 14.4 kPa were found in M5, M10, and M20 treatments, respectively (HSD = 3.3 kPa). This situation was in accordance with the reports of Makino et al. (2008), Allan-Wojtas et al. (2007), and Ozdemir et al. (2005), who demonstrated that the use of microperforation is an effective manner of controlling permeability in container walls and gas concentrations inside packages of modified atmosphere systems. Besides, treatments M0 and M5 were applied during a storage of 30 days and, consistently, the O₂ concentration was different between them (P ≤ 0.05), where those non-perforated bags had values of 0 kPa during all the experimental period, while in bags with five perforations the O₂ ranged between 0.5 and 3.0 kPa, with an average value of 1.7 kPa (Table 2). Meanwhile, the CO₂ had average concentrations of 22.2 and 21.7 kPa in M0 and M5, respectively, during the storage of 30 days, without a difference between them (P > 0.05), and these values were different to the concentration found in the normal air condition, where incipient quantities appeared (0.4 kPa). According to Cliffe-Byrnes and O'Beirne (2007) fungus can support O₂ at very low concentrations without suffering from anaerobic metabolism, and some of them tolerate up to 50 kPa of CO₂, with good sensorial characteristics. In the case of Pleurotus sp., the authors pointed out that O₂ can be present with values as low as 1 kPa and CO₂ as high as 15 kPa. Thus, if a similar behavior is accepted for huitlacoche, handling of this product in bags with five perforations could be adequate for the 20°C condition.

Table 2. Means comparison of concentrations of O₂ and CO₂ and fermentative metabolites production affected by package type (P) and temperature (T), during the shelf life evaluation of huitlacoche galls in postharvest during 30 days.
Tabla 2. Comparación de medias de concentraciones de O₂ y CO₂ y de producción de metabolitos fermentativos afectados por el tipo de empaque (P) y la temperatura (T) durante la evaluación de la vida útil en postcosecha de agallas de huitlacoche durante 30 d

	Package type (P)
Temperature (T)	NA	M5	M0	HSD
	Oxygen concentration (kPa)
20°C	19.8^(A)(a)	1.7^(B)(b)	0^(B)(c)	1.0
3°C	20.6^(A)(a)	16.3^(A)(b)	8.1^(A)(c)	1.2
HSD	0.9	1.1	0.8	–
Interaction T × P	Significant
	Carbon dioxide concentration (kPa)
20°C	0.4^(A)(b)	21.7^(A)(a)	22.2^(A)(a)	2.4
3°C	0.4^(A)(c)	5.7^(B)(b)	12.2^(B)(a)	1.3
HSD	1.1	1.4	1.5	–
Interaction T × P	Significant
	Fermentative metabolites (μmol 100 mL^−1)
20°C	165.3^(A)(b)	178.5^(A)(b)	2331.0^(A)(a)	49.3
3°C	52.5^(B)(c)	107.2^(B)(b)	187.0^(B)(a)	38.7
HSD	103.2	65.8	123.6	–
Interaction T × P	Significant
Note: HSD: honest significant difference (Tukey; 0.05); NA: treatment in normal air; M5: treatment in bag with five perforations; M0: treatment in intact bag without perforations. Different capital letters indicate significant difference between the values of a column, and different lower case letters indicate significant differences between the values of a line (Tukey, 0.05).
Nota: HSD: diferencia significativa honesta (Tukey; 0,05); NA: tratamiento en aire normal; M5: tratamiento en bolsa con cinco perforaciones; M0: tratamiento en bolsa intacta sin perforaciones. Letras mayúsculas diferentes indican diferencias significativas entre los valores de una columna, y letras minúsculas diferentes indican diferencias significativas entre los valores de una misma hilera.

At 3°C the 24-h evaluation did not point out a treatment as the best, since the equilibrium condition was reached with 17.1, 17.5, 16.6, 18.3, and 16.8 kPa of O₂, in systems with 0, 2, 5, 10, and 20 perforations, respectively (Figure 1f–j), without a difference among treatments (HSD = 1.5 kPa). However, when treatments M0 and M5 were tested over a month, the former showed an average O₂ concentration of 8.1 kPa, which was different to the level of 16.3 kPa registered in the latter (P ≤ 0.05; Table 2). In the case of CO₂ a different situation was found: when the M0, M2, M5, M10, and M20 treatments were evaluated during 24 h the concentrations reached were 10.8, 10.2, 5.9, 5.8, and 3.4 kPa, respectively (HSD = 0.8 kPa) (Table 1), but when M0 and M5 were assessed during 30 days, their CO₂ concentrations changed only slightly and averaged 12.2 and 5.7 kPa, respectively, with HSD = 1.3 kPa (Table 2). Renault et al. (1994) showed that the equilibrium concentrations in a perforation-based MA system depend on the number and diameter of perforations, thickness of polymeric film, and temperature. Also, it is documented that a reduction of temperature causes reduction of both produce respiration (Nunes & Emond, 2003) and gas exchange rate through microperforations (Rodov et al., 2007), which explains that in the present work modified atmosphere systems at 3°C may require a period longer than 24 h to reach the equilibrium condition.

Fermentative metabolites production

During the 30-day storage evaluation, fermentation was higher at 20°C than at 3°C (P ≤ 0.05), but a significant interaction was found between factors (Table 2). At 20°C the fermentative metabolites production in non-perforated bags (M0) incremented continuously during the first eight days up to values higher than 3000 μmol 100 mL⁻¹, and then it tended to be constant. On average, such activity was 13.1 and 14.1 times higher (P ≤ 0.05) than that induced by bags with five perforations (M5) and by the NA condition, respectively, where values were approximately constant and no differences (P > 0.05) were found between them. Due to this similar fermentative activity between M5 and NA, a normal aerobic circumstance was inferred in M5, and such treatment was accepted as adequate for handling huitlacoche galls in MAP at 20°C. This also showed that huitlacoche can resist environments with very low O₂ availability, with similar behavior, in that sense, to Pleurotus sp. (Cliffe-Byrnes & O'Beirne, 2007), but it does not resist an anoxic ambient, since the total O₂ exhaustion triggered the fermentation. At 3°C the highest fermentative activity was approximately constant in all treatments during the storage. The highest metabolites production occurred also in treatment M0 (187.0 μmol 100 mL⁻¹), followed by M5 (107.2 μmol 100 mL⁻¹), and finally by the NA condition (52.5 μmol 100 mL⁻¹). Although differences were found (P ≤ 0.05; Table 2), they were considered to have little importance, since the production in bags of M0 at 3°C was similar to those of NA and M5 at 20°C (Table 2). Therefore, experimental units were at similar conditions to that of NA, with the exception of the M0 treatment at 20°C, where fermentation was high.

Weight losses

Weight loss rates were higher at 20 than at 3°C (P ≤ 0.05), which coincided with the report of Martínez-Flores et al. (2008), who found that huitlacoche galls had higher weight losses when handling was applied at moderate to high temperature than when refrigeration was used. In the present work, loss rates at 20 and 3°C were 4.50 and 0.40% d⁻¹ in NA, 0.28 and 0.02% d⁻¹ in M0, and 0.42 and 0.02% d⁻¹ in M5, respectively (Figure 2a,b), the differences between the former and the latter two (P ≤ 0.05) being significant, which was considered normal since the plastic envelopment induces a microenvironment of high RH around the product, thereby reducing the water vapor deficit in relation to the internal product tissue (Ben-Yehoshua & Rodov, 2003).

Figure 2. Variation of weight loss (a, b), firmness (c, d), lightness (e, f), hue angle (g, h), and chroma (i, j) in huitlacoche galls stored in normal air (NA), in perforated bags (M5), and in intact bags (M0), at 20°C (left) and 3°C (right). HSD is honest significant difference (Tukey, 0.05). Each value represents the mean of three repetitions and bars indicate standard error.

Figura 2. Variación de pérdida de peso (a, b), firmeza (c, d), luminosidad (e, f), ángulo de tono (g, h) y cromaticidad (i, j) en agallas de huitlacoche almacenadas en aire normal (NA), en bolsas perforadas (M5) y en bolsas intactas (M0), a 20 (izquierda) y 3°C (derecha). HSD es diferencia significativa honesta (Tukey, 0,05). Cada valor representa la media de tres observaciones y las barras indican error estándar.

pH

At the beginning the product pH was 4.8 (±0.3) and during storage it was affected by both temperature and package type, with interaction between them (P ≤ 0.05). At 20°C, the average values of 3.68, 4.44, and 4.91 were found in materials exposed to treatments M0, M5, and NA, with significant difference among them (HSD = 0.42), while at 3°C values were 3.93, 3.78, and 4.16, respectively (HSD = 0.21). Data showed that huitlacoche is an acidic product, but the behavior found suggested that a handling in modified atmosphere induces reduction in pH, which might have been caused by the higher CO₂ concentrations that appeared in the bags of M0 and M5 treatments. In this regard, Valle-Guadarrama et al. (2013) showed in avocado fruits that as CO₂ concentration increases the pH reduces and they explained that this gas is absorbed as carbonic acid, causing reduction in pH, which may also be occurring in huitlacoche galls.

Firmness

The huitlacoche tissue firmness had a value of 5.6 (±0.3) N at the beginning and during storage it diminished at both thermal conditions, but changes were faster (P ≤ 0.05) at 20°C than at 3°C (Figure 2c,d), which may correspond to a greater metabolic activity at the higher temperature (Nunes & Emond, 2003). At 20°C the fastest firmness changes happened in the NA treatment and the slowest in bagged materials. On average, the packed batches had the highest firmness values (3.4 N for M5 and 2.8 N for M0) and the smallest were found in the NA treatment (1.9 N), being significant the difference (HSD = 0.51; Figure 2c). At 3°C the highest values corresponded also to perforated bags (M5), since they favored an average value of 3.4 N, which was different to materials handled in intact bags (M0; 2.7 N) and to the NA treatment (2.6 N) (HSD = 0.41; Figure 2d). There are no reports about huitlacoche firmness in open literature, but changes observed during storage were considered normal, since it is documented that the horticultural products texture is a time-dependent characteristic that changes with physiology and processing (Smith, Waldron, Maness, & Perkins-Veazie, 2003), and the results of the present work showed that the use of low temperature and MAP systems with microperforations are useful for reducing such changes.

Color

The huitlacoche galls had a pale yellow tonality at the beginning. Lightness (L*) had an average value of 52.4 (±1.9) at that time and during storage it decreased gradually at a rate of − 3.87 (±0.5) d⁻¹ at 20°C and − 0.7 (±0.3) d⁻¹ at 3°C. As a consequence, L* values at 20°C ranged between 13 and 22 at day 12 (Figure 2e), but at 3°C such state was never reached, even after 30 days of storage, when values varied between 25 and 37 (Figure 2f), which emphasized the advantage of using low temperature. On other hand, an interaction was found between temperature and environment (P ≤ 0.05); at 20°C the highest L* was found in treatment with five perforations (M5; 43.7 on average), as it allowed a slower rate of darkening than without perforations (M0) and with a handling in air (NA), which averaged values of 37.5 and 34.0, respectively, without difference between these two (HSD = 3.68). At 3°C average L* values were 42.7, 39.1, and 39.2, for NA, M5, and M0 treatments, respectively, and significant differences were not found (HSD = 2.85).

The hue angle started with an average value of 87.0° (±0.7°) and also diminished during storage. At 20°C the change was similar to a sigmoid curve, where the log phase occurred between days four and six in M0, between days four and 10 in NA, and between days six and 10 in M5. At the end the lowest values were in NA (approximately 65°) and the highest in M0 and M5 (around 70°), without a difference between these two (Figure 2g). If the entire storage period is considered, the highest tonality values were found at 20°C in the treatment with five perforations (M5; 81.4°) and the lowest in treatments of NA and in non-perforated bags (M0), with 77.0° and 77.9°, respectively (HSD = 1.54°), which indicated that at such thermal condition the best treatment was M5. At 3°C there were no significant changes in hue angle during the first eight days, but subsequently the attribute diminished, with a faster rate in NA and M0 treatments than in M5 and, although at the beginning there were no differences between treatments, at the end NA and M0 had an approximate value of 71°, while treatment M5 maintained values above 78° (Figure 2h). On average, the differences were significant (HSD = 1.27°) and the best treatment, in relation to hue angle, was the bag with five perforations. Regarding chroma, this attribute maintained values with certain variation, but without a clear trend of change. No significant differences were found between environments neither between temperatures. At 20°C, the average values were 6.5, 6.8, and 7.2 for material of NA, M5, and M0 (HSD = 1.06; Figure 2i), while at 3°C values were 6.1, 5.7, and 5.6, respectively (HSD = 0.51; Figure 2j).

Martínez-Flores et al. (2008) observed similar lightness, chroma, and hue angle values at the beginning of a storage of huitlacoche galls conducted during 11 days, and also similar behavior in the first two attributes during that period, although they did not find modification in hue angle, which, together with the results of the present work may indicate that product deterioration does not occur immediately after harvest. In other edible fungi, like Agaricus bisporus, the phenomenon of darkening has been associated with the activity of enzymes of the polyphenol oxidase family (Nerya et al., 2006), and Martínez-Flores et al. (2008) proposed that a similar situation occurs in huitlacoche galls. In this regard, Juárez-Montiel et al. (2011) pointed out that the basidiomycete U. maydis produces enzymes like tyrosinase and laccase, which catalyze the polymerization of hydroxy and mono phenols. However, according to Valverde et al. (1995), during development huitlacoche galls are first covered with a glistening, greenish-white to silver-white tissue, and the interior of galls darkens and turns into masses of powdery, dark olive-brown. Huitlacoche is generally consumed with other ingredients such as sauce (Hadley, 1999), and when cooked it may become dark due to manipulation (Valdez-Morales et al., 2010). Thus, the gradual loss of lightness and hue angle reduction found in the present work at both temperatures (3 and 20°C) might also be attributable to the rupture of periderm due to natural aging, which is consistent with the loss of firmness observed. On other hand, although this deteriorative process cannot be stopped, this work showed that the use of a modified atmosphere implemented with a microperforation strategy like that of the M5 treatment is able to reduce rates at which weight loss, softening, and changes in color attributes occur, but when low temperature is applied the shelf life can be extended up to 30 days.

Conclusions

The quality of huitlacoche galls was affected by both temperature and MAP. This product proved to be resistant to low O₂ availability and could be handled in environments with concentrations of that gas as low as 1.7 kPa. Microperforation was an adequate strategy to control gas concentrations inside MAP systems. Bags with five microperforations of 15 μm in diameter allowed the best quality characteristics at ambient conditions (20°C) and under refrigeration (3°C), permitting O₂ concentrations inside MAP systems without significant fermentative activity. These bags caused the lowest weight loss and the highest values in firmness, lightness, and hue angle. The combination of microperforated MAP systems and refrigeration allowed a shelf life of 30 days.

Acknowledgements

The authors wish to acknowledge the financial support received from Consejo Nacional de Ciencia y Tecnología of Mexico (CONACyT; Project SEP-2004-C01–47725) and Universidad Autónoma Chapingo (University Food Research Program and Food Science and Technology Graduate Program).