Effect of storage time on in vitro digestion rate and resistant starch content of tortillas elaborated from commercial corn masas. Tortilla samples were elaborated by four small commercial factories in Mexico, employing masas prepared with the traditional nixtamalization process. Samples were stored at 4 o C for up to 72 hours and their chemical composition and in vitro starch digestibility features were evaluated. Chemical composition did not change with the storage time, but soluble carbohydrates decreased slightly during storage. A significant decrease in available starch content upon storage was observed, concomitant with increased resistant starch RS levels.
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Effect of storage time on in vitro digestion rate and resistant starch content of tortillas elaborated from commercial corn masas. Tortilla samples were elaborated by four small commercial factories in Mexico, employing masas prepared with the traditional nixtamalization process.
Samples were stored at 4 o C for up to 72 hours and their chemical composition and in vitro starch digestibility features were evaluated. Chemical composition did not change with the storage time, but soluble carbohydrates decreased slightly during storage. A significant decrease in available starch content upon storage was observed, concomitant with increased resistant starch RS levels. These changes are possibly due to retrogradation.
The digestion rate DR in the freshly prepared tortillas was similar for the various samples, but after 72 h storage some differences among tortillas were found. Also, when a single tortilla sample was compared throughout the different storage times, lower DRs were determined in samples subjected to prolonged storage, which is related to the concomitant increase in RRS.
The differences found among the various tortilla samples may be due to minor variations in the commercial processing conditions and to the use of different corn varieties. Keywords : Corn, texture, tortillas, resistant starch, starch digestibility. The nixtamalization of corn is an ancient process developed by the Mesoamerican civilizations and is still utilized in the production of "tortillas" and other corn related food products i.
The corn grains are cooked with alkali i. After grinding and washing the "nixtamal" i. The "masa" is a mixture constituted by starch polymers, mixed with partially gelatinized starch granules, intact starch granules, pieces of endosperm, and lipids.
All these components develop a complex heterogeneous network in a continuous water phase 1. Nowadays, table tortillas are highly popular in United States and, to some extent, also in Canada and some European countries 3. The nixtamalization process produces changes that improve the nutritional quality of tortillas.
Many studies have been conducted on nutritional aspects of nixtamalized corn, but very few studies have been carried out on the bioavailability of its carbohydrate constituents 4. Besides, being a major plant metabolite, starch is also the dominating carbohydrate in the human diet 5,6.
Current knowledge on nutritional features of starch indicates that the bioavailability of the polysaccharide in foods may vary widely 7. The resistant starch RS is defined as the sum of starch plus the products of starch degradation not absorbed in the small intestine of healthy individuals 9.
The main classification of RS has been proposed by Englyst et al. RS1 corresponds to physically inaccessible starches, entrapped in a cellular matrix, as in legume seeds RS2 are native uncooked granules of starch, such as raw potato or banana starches, whose crystallinity makes them scarcely susceptible to hydrolysis 11, RS3 are retrograded starches, which may be formed in cooked foods that are kept at low or room temperature It has been proposed that both the rate and extent of starch digestion, and therefore the RS content of foods, will affect a number of physiological functions and thus will have different effects on health, e.
Among the factors affecting the rate and extent of starch digestion, food processing, storage time and botanical origin of the food have a major importance. Starch in raw foods is not easily digested, exhibiting variable levels of RS2 fractions. However, during cooking, starch is gelatinized and rendered available, although a fraction of this available starch retrogrades upon cooling and becomes resistant to enzymatic digestion RS3 5,14,18, Gelatinized starch gels are thermodynamically unstable structures and, on cooling, reassociation of the starch molecules may occur.
The ability of starch chains to form ordered structures in pastes, gels and baked foods during storage, a process often described by the term "retrogradation", greatly influences the texture and shelf-life of these products In a previous study 4 , experimental tortillas were prepared from laboratory-nixtamalized corn, using standardized conditions throughout the whole process. Such materials were evaluated in terms of their in vitro starch digestibility.
The objective of the present study was to evaluate the influence of storage on the "in vitro" digestibility of starch in tortilla made at the traditional commercial level in Mexico, which is more representative of the currently consumed food. Sample preparation : "Masas" were purchased from four small factories A, B, C and D called "tortillerias", in Yautepec, Mexico, and brought immediately to the laboratory, always kept at temperatures between 28 and 32 o C.
These masas were obtained according to the traditional method to produce nixtamal, which shows minor differences among the different "tortillerias"; such variations relate mainly to the length of the steeping step 4.
Once in the laboratory, the masa was quickly molded by pressure and extruded into thin circles to obtain 1mm-thick "tortillas". After baking control or following the corresponding storage period, samples were frozen in liquid nitrogen, freeze-dried and stored at room temperature in sealed plastic containers.
Ash, protein and fat were analyzed according to AACC 21 methods , , and , respectively Total soluble carbohydrates were determined by colorimetry in aliquots of the supernatant Potentially available starch content was assessed following the multienzymatic protocol of Holm et al. Resistant starch was measured by two different protocols: 1 Retrograded resistant starch RRS or RS3 content was measured as starch remnants in dietary fiber residues, according to the so called "Lund method" as modified by Saura-Calixto et al.
The in vitro rate of hydrolysis was measured using hog pancreatic amylase, according to Holm et al. Statistical analysis: A randomized complete design with three replications was used to analyze changes during tortilla storage. The chemical composition of tortillas is presented in Table 1. In general, moisture content in the same kind of tortilla did not change with the storage time; confirming that the poly-ethylene bags where the tortillas were stored restricted water losses.
The highest values were recorded in sample A and the lowest ones in sample C. Ash content in tortillas did not change with the storage time nor with the masa source. Fat content in tortillas A and D had a slight decline with storage time, but samples B and C did not show changes in fat values. Soluble carbohydrates decreased with the storage time; samples A and B showed the highest values whereas C and D exhibited the lowest ones. The values of total starch content TS are presented in Table 2.
Freshly cooked sample B showed the highest values and the A tortillas the lowest ones. The values of available starch AS in the tortillas analyzed ranged between In all samples , AS values decreased with storage time.
Such a tendency was more evident than in the case of TS. In general, total resistant starch RS values in tortilla samples A, B and C increased with storage time ; this in accordance with the fact that tortillas were cold stored, thus favoring the retrogradation phenomenon RRS. However, since RS in D tortillas did not change upon storage Table 2 ; corn varietal differences may again be important for this characteristic.
The in vitro a -amylolysis reaction of tortillas is represented in Figure 1. The control samples 0 h of storage, Fig. Figure 1. Differences were found in moisture level among the different tortilla samples, which may be due to either corn variety or, more likely, to variations in the nixtamalization process, as all tortillas were cooked under the same temperature and time conditions.
However, they found increased moisture levels at the longest storage times. Ash content usually does not vary with the nixtamalization process 29, The ash values ranged between 1. These values are in agreement with the fact that lime concentration used in the nixtamalization process applied to the different samples was rather similar.
The differences determined in fat values may be also attributable to the corn variety and conditions prevailing during nixtamalization process.
There is no clear explanation for the soluble carbohydrate pattern, although it might be due in part to the retrogradation phenomenon, as crystalline structures are formed during storage, resulting in decreased starch solubilization. However, leaching of partly hydrolyzed starch fractions during grain nixtamalization cannot be ruled out.
Variations in TS among the diverse tortilla samples may be explained by the corn variety used for the masa preparation in each "tortilleria". Regarding the storage impact, in general, TS values showed only minor decay with time; the exception was tortilla sample D, the TS level of which remained constant. The reduction in AS content during storage may be explained by the formation of resistant starch due to the retrogradation phenomenon that takes place when a cooked starch product is cold-stored 8.
Thus, when stored, the latter samples seem to contain smaller amounts of indigestible starch fractions. Differences in TS-AS values among the diverse tortillas may reflect differences in corn variety and nixtamalization process, since there is appreciable variation in AS measured in twenty Mexican corn hybrids and varieties unpublished data. It is therefore important to make the appropriate choice of corn variety for obtaining major or minor AS content in tortillas.
The AS value variability recorded among tortillas elaborated with the different masas may also be related to corn variety or nixtamalization conditions, specially in the steeping time or nixtamal washing which, in addition to an important removal of fibrous material 31 , may as well result in RS reduction. Tortillas A and B showed the highest variation in RS with storage time, as the "fresh" control samples contained 1.
For tortilla C, the RS values changed from 1. These data may have interesting implications, as tortillas bought from different "tortillerias" are likely to have different RS levels and, presumably, different structural firmness. In general, the A, C and D tortillas exhibited lower RS values than those determined in laboratory-made tortillas 3. Such a raise in RS during cold storage is consistent with the recorded AS decrease after 72 hours Table 2.
Wet thermal treatment followed by cooling and storage produces retrograded resistant starch RRS , as reported for corn flour 32 and for various starch gels 33, It is noteworthy that RS and RRS values in 72h-stored C tortilla are similar Table 2 ; a fact that suggests most indigestible starch in this sample consists of retrograded fractions.
Appreciable differences between RS and RRS levels were registered for the remaining 72h-stored samples, which indicates that RS2, and perhaps some RS1, are also present in these materials. A RRS value of 2. It has been mentioned that during baking, heat treatment promotes the interaction of starch with other corn components, making it less accessible to enzyme hydrolysis; the tortilla making process appears largely responsible for the recorded changes in indigestible starch levels.
RRS values between 2. Hence, the tortilla-making process may be considered a suitable way to increase resistant starch levels in corn-based products. However, they are lower than those reported by Campas-Baypoli et al. This apparent discrepancy may be consequence of the accuracy differences existing among resistant starch methods of analysis 7 , although the impact of varietal or processing conditions cannot be ruled out. For the 72 h-stored materials Fig. However, at 75 min the A, C and D tortillas behaved similarly.
When the behavior of this tortilla sample was compared along the different storage times Fig. Hence, present results suggest that longer storage times increase RS contents Table 2 and produce a concomitant decrease in hydrolysis rates. No change was observed in moisture content in tortillas stored for different times, which is likely due to the restricted water-losses occurring in the poly-ethylene containers used.
Protein, lipids and ash contents did not change with storage time, but protein and ash contents in tortillas were similar to those reported in raw corn, but lower lipid values were determined, which may be related to solubilization during nixtamalization and nixtamal washing. Decreased available starch and slightly augmented resistant starch RS levels were recorded upon cold-storage.
The differences found in starch bioavailability in tortillas may be mainly due to both corn variety and nixtamalization conditions used in each tortilleria for masa preparation. In general, hydrolysis rate fell as storage time increased, this pattern relates to RRS formation during tortilla storage. Technical assistance of Tech.
Beneficial effects of resistant starch for host health
A recent review by Dr. Anne Nugent from the University College Dublin in Dublin Ireland summarises reported health effects of RS and explores the potential mechanisms of action that underpin them. Dietary fibre is a key nutrient for optimizing gut health and it has been previously documented that a fibre-deprived diet may have a negative impact on the colonic mucus layer and gut microbiota functionality. Resistant starch RS is a form of starch that is not digested in the small intestine and is therefore considered a type of dietary fibre. There are currently 5 types of RS that can be found naturally in plant foods or can be produced or modified commercially and incorporated into food products:.