Isolation and Characterization of the Microflora of Nixtamalized Corn Masa

International Journal of Food Microbiology 165 (2013) 209–213

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Short communication Isolation and characterization of the microﬂora of nixtamalized corn masa

Stephen J. Adolphson, Michael L. Dunn, Laura K. Jefferies, Frost M. Steele ⁎ a Department of Nutrition, Dietetics and Food Science, Brigham Young University, S-221 ESC, Provo, UT 84602, United States article info abstract

Article history: Corn tortillas are a staple in the diet among the Mexican population, and are traditionally produced through a Received 19 December 2012 process known as nixtamalization. This traditional process involves steeping whole-kernel corn in an alkaline Received in revised form 29 April 2013 solution overnight and then grinding the corn into dough (masa), which is then baked. While the masa is Accepted 11 May 2013 held before baking, significant microbial change can occur which leads to fermentation and spoilage. The ob- Available online 19 May 2013 jective of this research was to characterize and identify the microflora of nixtamalized corn masa from six different commercial tortilla mills throughout Guadalajara, Mexico. The identification of samples was conducted Keywords: fi Microflora using the microbial identi cation system (MIS), which analyzes cellular fatty acids via gas chromatography to Masa identify bacterial species. Lactic acid bacteria and aerobic mesophiles were the predominant organisms, with Corn both groups having counts ranging from 104 to 107 cfu/g across all mills. Coliform populations were observed Nixtamalization at counts of 102 to 103 cfu/g, while yeast and mold counts were typically less than 101 cfu/g. Some mills Lactic acid bacteria showed no presence of coliforms or yeast or mold. Streptococcus bovis and Lactobacillus oris were isolated from all mills, and were the most prevalent organisms representing 43% and 17% of all lactic acid bacteria isolated, respectively. S. bovis was also isolated on the aerobic tryptic soy plates and was the most prevalent species representing 19% of the total organisms from these aerobic plates. © 2013 Elsevier B.V. All rights reserved.

1. Introduction and baked (Chapman et al., 2010). Due to the availability of nutrients, the high moisture content (approximately 56%) (Ramirezwong et al., Corn tortillas are a common staple in the diets of Mexican and 1994), and warm storage temperature, significant microbial growth many Central American populations. It has been estimated that in cer- can occur while the masa is being held for extended periods. Natural tain areas of Mexico, the corn tortilla provides approximately 50% of fermentation and souring of the masa usually leads to spoilage with- the individual energy intake, constituting 60–90% of cereal grain in- in 4 to 6 h (personal communication: Lorenzo Vital, Todo de Maiz, take (Villalpando, 2004). Traditionally, corn tortillas are prepared by Guadalajara, Mexico). steeping whole-kernel corn in a hot, alkaline solution, containing Published research regarding the microflora of maize products 1.5–2% lime (CaO), and subsequently grinding the drained and rinsed focuses primarily on products such as pozol, sekete, chorote and corn (now referred to as nixtamal) into a fresh dough called masa. other fermented products, and monitors the changes in microbial The ground masa typically exits the grinding stones at a temperature populations throughout the fermentation process (Adegoke et al., near 56 °C (Chapman et al., 2010). Tortillas are produced using this 1995; Diaz-Ruiz et al., 2003; Morales et al., 2005; Sefa-Dedeh et al., process in tens of thousands of very small neighborhood mills 2004; Wacher et al., 1993; Wacher et al., 2000). Since microorgan- scattered throughout towns and cities across Mexico and Central isms are responsible for the fermentation process, it is reasonable America. Many of these mills have limited sanitation capabilities, that most of the research regarding bacterial diversity and character- which can lead to a diverse and abundant microbial population in ization relate to these commonly fermented foods. While tortilla the masa dough. masa is not purposefully fermented, studies of fermented maize Due to the short shelf life of fresh tortillas (Aida et al., 1996), as foods can still offer some knowledge and insight into the microflora well as the daily demand, and the increasing tendency for one mill of nixtamal and other fresh, unfermented maize products. Wacher to transport masa to multiple tortillerias in an expanded distribution et al. (2000) found lactic acid bacteria present at 105–106 cfu/g in area, the fresh masa is often held for a period of time before baking into pozol, a fermented corn dough often consumed as a beverage in tortillas. In commercial tortilla mills, the hot masa is often stacked and Mexico, and identified 25 bacterial isolates to the genus level. They stored in large pails or sacks at ambient temperature for anywhere used selective plating and carbohydrate utilization methods to tenta- from a few minutes to several hours before being formed into tortillas tively identify Leuconostoc and Lactococcus as the primary genera in this fermented product. Escalante et al. (2001) used 16S rDNA sequenc- ⁎ Corresponding author. Tel.: +1 801 422 6670; fax: +1 801 422 0258. ing to identify a variety of Lactobacillus species, including Lactobacillus E-mail address: [email protected] (F.M. Steele). lactis, Lactobacillus alimentarium, Lactobacillus plantarum,aswellas

0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.05.010 210 S.J. Adolphson et al. / International Journal of Food Microbiology 165 (2013) 209–213

Streptococcus suis from fermented pozol dough. Streptococcus bovis was colonies selected for further identification was based on the percent- identified by Diaz-Ruiz et al. (2003) as the predominant microorganism age of each morphology type from the initial spread plates. This gave in samples of pozol. Very little research has been published on the an adequate representation of all the colony types and their approxi- microflora of non-fermented corn products, like tortillas, even mate concentrations. Individual colonies were re-streaked on fresh though,they are a major staple food in Mexico and other countries medium twice to ensure pure cultures. The isolated cultures were (Martinez-Flores et al., 2004). Capparelli and Mata (1975) looked stored in 15% glycerol in combination with their appropriate broth at the microflora, particularly pathogenic bacteria, of both masa (MRS or trypticase soy) at −50 °C until further use. and tortillas in attempts to find sources of bacterial contamination. The masa and tortillas were prepared by housewives in homes in 2.3. Identification of bacteria Guatemala and would probably differ from those produced in commercial mills in Mexico. Rocha and Malcata (2012) examined the micro- Only colonies from the APC and LAB plates were isolated and iden- biological profile of maize and rye flours used to make broa, a traditional tified to the species level due to their substantially higher colony Portuguese sourdough bread. They found bacterial population levels for counts. The identification of samples was conducted with the micro- total plate count, coliforms, yeast and mold, and lactic acid bacteria to bial identification system (MIS), which uses whole cell fatty acid anal- be 107,105,103,and105 cfu/g respectively. Their study looked at the ysis via gas chromatography to identify bacterial species (MIDI Inc., microflora of dry flours instead of hydrated masa dough. Due to the differ- Newark, DE, USA). Extractions of fatty acid methyl esters from sam- ences between the dry flour milling and the wet, alkaline nixtamalization ples were performed according to MIDI instructions. Methanolic process, the microflora of maize flour can significantly differ from that of NaOH, in combination with heat, was added to isolated colonies to fresh nixtamal masa. kill and lyse the cells. Fatty acids were cleaved from the cell lipids, Further investigation of the microflora of commercially prepared converted to their sodium salts, and methylated to increase their vol- masa and tortillas is needed due to the lack of published research atility for the GC analysis. Fatty acid methyl ester extracts were ana- and the widespread production and consumption of tortillas through- lyzed on a 6890 Series Gas Chromatograph (Agilent Technologies, out Mexico. A microbial survey of masa from traditional artisanal Santa Clara, CA, USA) using the MIDI MIS software (MIDI Inc.). Simi- mills would highlight the diversity of microflora present, including larity indices for genus and species were given for the top possible potentially beneficial species, as well as spoilage organisms and matches of each isolate based on the uniqueness of the fatty acid pro- potential pathogens. This knowledge can help tortilla manufacturers file. The species with the highest similarity index for each isolate was implement improved cleaning and sanitation practices, and optimize chosen as the best match and linked to the identity of the given the production process. The objective of this research is to character- isolate. ize and identify the microflora of fresh, nixtamalized corn masa obtained from small, commercial tortilla mills throughout Guadalaja- 3. Results and discussion ra, Mexico. 3.1. Enumeration of microorganisms 2. Materials and methods Bacterial counts from the APC and LAB plates were similar within 2.1. Sample collection each mill, although differences existed between mills. Fig. 1 shows the microbial profiles of the masa samples collected from each of One-kilogram samples of corn masa were collected from six differ- the six mills. Counts for LAB between mills ranged from 2.3 × 104 to ent commercial tortilla mills throughout Guadalajara, Mexico. All 6.0 × 107 cfu/g, while APC populations were between 3.5 × 104 and masa samples were produced through the traditional nixtamalization 8.3 × 107 cfu/g. and wet-milling of whole-kernel corn, and not by using corn masa These counts for LAB and APC are similar to those obtained by flour. Fresh masa samples were collected within a few hours after other researchers. Wacher et al. (2000) found APC and LAB levels of being milled. Samples were collected in plastic bags, placed immediately 105 cfu/g from nixtamal dough used for pozol production, although on ice, and transported on ice to Brigham Young University within 24 h in their study nixtamal dough was purchased from only one manufactur- after collection. er. Sefa-Dedeh (2004) reported LAB levels at 106 cfu/g in nixtamalized corn soaked in both a 0.5 and 1% lime solution. They observed slightly 2.2. Enumeration and isolation of microorganisms higher LAB counts (107 cfu/g) in corn soaked without lime (0% lime solution). In the same study, the level of APC cfu/g was reported at 105 Upon arrival at Brigham Young University, three 20 g replicates in a 1% lime solution, with increasing populations (106 and 107 cfu/g) of each masa sample were aseptically transferred to a Whirl-Pak® as the percent of lime solution dropped to 0.5 and 0%. The percent of stomacher bag (Nasco, Fort Atkinson, WI, USA) along with 180 g of lime used during nixtamalization was not determined for any of the sterile 0.1% peptone water (HiMedia Laboratories, Mumbai, India) mills in our study; however, traditional nixtamalization processes typi- and stomached for 30 s using a Smasher blender (AES Chemunex, cally use a 1 to 2% lime solution. Potential differences in amounts of Bruz, France). Homogenates were serially diluted, and plated on tryp- lime added during nixtamalization could possibly help explain variation tic soy agar (3 M, St. Paul, MN, USA) for aerobic plate count (APC), in microbial populations between the mills, as increasing alkalinity may and de Mann, Rogossa and Sharpe (MRS) agar (Merck, Darmstadt, inhibit total microbial proliferation, or even potentially favor the growth Germany) for lactic acid bacteria (LAB). Coliforms and yeasts and of other organisms. mold were selected for and enumerated using 3M E. coli/coliform Processing and product holding times and temperatures varied and yeast and mold Petrifilm™ (3M), respectively. widely from mill to mill (data not shown) which would contribute APC and LAB plates were incubated at 35 °C for 48 h, with the LAB to any differences observed in microbial populations in each of the plates incubated anaerobically in a nitrogen-flushed incubator. Coli- mills. The levels of microorganisms in each mill might also be related form and yeast and mold Petrifilm™ were incubated at 35 °C for to the extent and type of cleaning done on the equipment and sur- 24 h and 25 °C for 130 h, respectively. Colonies from LAB and APC roundings. We were unable to determine the frequency and depth plates were enumerated and categorized based on their differing of cleaning for each mill, but it was apparent that there were differ- morphologies (colony size, shape, color, and Gram staining). Repre- ences in sanitation, which would affect microbial levels. Mill 6 is sentative colonies of each morphology type were selected for identi- known to chlorinate the water used in the steeping process to help fication to the genus and species level. The number of individual control for microbial growth, and it is highly likely that other mills S.J. Adolphson et al. / International Journal of Food Microbiology 165 (2013) 209–213 211

1E+09

100000000

10000000

1000000

100000 APC LAB 10000 Col. Y/M Counts (cfu/g) Counts

1000

100

1 123456 Mill

Fig. 1. Microbial profile of nixtamalized masa from six tortilla mills in Guadalajara, Mexico. LAB = lactic acid bacteria; APC = aerobic plate count; Col. = coliforms; Y/M = yeast and mold. *No growth is reported as b10 cfu/g. follow this same practice. Although we are uncertain which of the possibly explain differences between the yeast and mold levels other mills chlorinate their water, another miller suggested that Mill found in this and other studies. 5 may also chlorinate their water, which would account for the simi- In this study, coliforms and yeast and mold were plated and enu- lar microbial profiles and general lower levels of APC and LAB popu- merated using 3M Petrifilm™. The use of Petrifilm™ for the enumer- lations between Mills 5 and 6. ation of coliforms and yeasts and mold in foods are approved AOAC Populations of coliforms were insignificant compared to those methods. However, the results by other authors mentioned above from APC and LAB plates, and were lower than those reported in were obtained by plating on conventional agar plates selective for co- other studies. Wacher et al. (2000) found significantly higher coli- liforms and yeast and mold. The differences in plating methods may form counts (104 cfu/g) compared to the levels of 101–102 cfu/g also play a role in the discrepancies between the coliform and yeast reported in this study. Capparelli and Mata (1975) found coliforms and mold populations. present at 106 cfu/g in nixtamalized masa prepared in rural homes from the Guatemalan highlands. In their study, they were looking for sources of contamination of tortillas, and found that soiled hands 3.2. Identification of bacteria and water were a likely cause. The homemade masa and tortillas in their study would likely have a different microbial profile than masa A total of 82 bacterial colonies were isolated for identification from and tortillas produced in a commercial mill, where treated municipal the six mills. These colonies represented 22 different species belonging water was used. Coliforms were expected to be found in all mills, but primarily to the Streptococcus and Lactobacillus genera. Forty-six of the Mill 3 showed no coliforms (b10 cfu/g). This could be due in part to isolates were obtained from the LAB plates, with the remainder from competition from organisms with higher growth rates, although this the APC plates. Tables 1 and 2 show the identification of colonies isolat- is unlikely due to the fact that all other mills show counts around ed from the LAB and APC plates for each mill, respectively. 101–102 cfu/g. The genus Streptococcus was the most common among the iso- Yeasts and mold were also present at insignificant levels com- lates, comprising 56% of the total. Species of Lactobacillus were the pared to the APC and LAB plates. Mills 3 and 6 showed no detectable next largest group, representing 17% of the total. This finding is differ- levels of either yeasts or mold (b10 cfu/g). It would be expected that ent from that of Wacher et al. (2000), who tentatively identified the LAB and APC populations would outcompete the yeast and mold Leuconostoc as the most prevalent genus in nixtamal dough used for in the stored masa due to their higher growth rates; however, the pozol. Escalante et al. (2001) identified a number of Lactobacillus spe- presence of yeast and mold would still be expected based on results cies, with L. lactis and L. alimentarium as the predominant organisms from other researchers. Wacher et al. (2000) reported levels of in pozol dough. In our study, S. bovis was the most prevalent organism yeast and mold at 104 and 102 cfu/g respectively. Yeasts and mold and was isolated from both the LAB and APC plates. S. bovis represent- were present at 104 cfu/g in nixtamal steeped in a 1% lime solution ed 33% of the total 82 isolates, and 43% and 19% of the LAB and APC and increased to 105 cfu/g as the percent lime solution dropped to plates, respectively. This finding agrees with that of Diaz-Ruiz et al. 0.5 and 0% (Sefa-Dedeh et al., 2004). Again, both competition from or- (2003) who also identified S. bovis as the predominant organism ganisms with higher growth rates and chlorination of water could in pozol dough. They noted that the high specific growth rate of 212 S.J. Adolphson et al. / International Journal of Food Microbiology 165 (2013) 209–213

Table 1 Table 2 Bacterial species isolated from nixtamalized masa from six mills in Guadalajara, Mexico Bacterial species isolated from nixtamalized masa from six mills in Guadalajara, Mexico (lactic acid bacteria on MRS agar). (aerobic plate count on tryptic soy agar).

Mill Organism Log #of % of total Similarity Mill Organism Log #of %oftotal Similarity cfu/g isolates population Index (%) cfu/g isolates population Index (%) within mill within mill

1 Streptococcus spp. Lancefield B 5.5 3 63.8 22.6–49.3 1 Lactobacillus pentosus 5.4 1 18.4 77.5 Lactobacillus oris 5.0 1 23.0 56.1 Serratia marcescens GC subgroup A 5.4 2 12.7 13.8–58.6 Streptococcus equinus 5.0 1 3.2 53.2 Enterococcus cecorum 5.4 2 12.0 82.9–89.0 Streptococcus bovis GC subgroup A 4.8 3 8.1 58.2–67.0 Hafnia alvei GC subgroup B 5.1 1 10.8 62.4 2 Enterococcus cecorum 6.5 1 19.2 82.0 Escherichia coli GC group E 5.0 3 10.4 51.9–57.5 Streptococcus bovis GC subgroup A 6.3 3 19.0 26.7–79.2 2 Streptococcus salivarius GC subgroup A 6.5 1 18.0 57.9 Streptococcus anginosus GC 6.2 2 18.2 27.3–71.0 Enterococcus cecorum 6.5 1 16.2 87.9 subgroup A Escherichia coli GC group E 6.3 2 12.8 55.4–57.1 Streptococcus vestibularis 6.1 1 16.4 53.6 Enterococcus gallinarum 6.2 1 12.3 73.6 Lactobacillus oris 6.0 2 14.7 56.7–57.6 Streptococcus bovis GC subgroup A 5.8 1 12.1 60.2 3 Streptococcus bovis GC subgroup A 7.4 3 24.3 26.5–63.5 Rahnella aquaticus 5.7 1 8.7 63.2 Streptococcus vestibularis 7.3 1 27.4 41.2 Streptococcus bovis GC subgroup B 5.0 1 6.6 62.6 Lactobacillus oris 6.5 2 18.4 56.3–59.9 3 Hafnia alvei GC subgroup B 7.9 1 12.5 53.2 Streptococcus equinus 6.3 1 15.0 55.7 Streptococcus intermedius DNA 7.9 1 12.1 51.8 4 Lactobacillus oris 7.1 1 48.4 54.2 homology group II Streptococcus equinus 7.1 1 37.7 55.3 Streptococcus bovis GC subgroup B 7.4 2 10.7 54.4–63.6 Streptococcus bovis GC subgroup A 6.9 2 14.0 46.7–57.8 Streptococcus sanguinis GC subgroup A 7.0 1 10.4 55.1 5 Streptococcus bovis GC subgroup A 4.2 3 25.4 40.4–57.4 Proteus vulgaris GC subgroup B 6.3 1 8.0 52.0 Streptococcus spp. Lancefield D 4.0 1 18.9 74.5 Enterococcus cecorum 6.0 1 7.7 87.1 Lactobacillus pentosus 3.2 2 4.6 57.8–78.1 4 Staphylococcus hominis GC subgroup A 6.9 1 36.4 74.7 Lactobacillus delbruekii-bulgaricus 3.0 1 4.3 48.7 Streptococcus salivarius GC subgroup A 6.8 1 27.2 39.2 Lactobacillus oris 3.0 1 3.6 49.1 Streptococcus bovis GC subgroup B 6.4 2 24.2 66.9–67.9 Streptococcus equinus 3.0 1 3.6 50.9 5 Streptococcus intermedius DNA 4.4 1 18.9 54.8 6 Streptococcus bovis GC subgroup A 4.7 3 43.2 43.0–75.6 homology group II Lactobacillus pentosus 3.6 2 21.4 55.7–74.9 Escherichia coli GC group E 4.3 1 14.9 55.6 Lactobacillus oris 3.3 1 12.2 55.6 Streptococcus bovis GC subgroup B 3.3 2 10.7 63.2–65.8 Hafnia alvei GC subgroup B 3.0 1 8.5 45.5 Streptococcus sanguinis GC subgroup A 3.0 1 8.4 64.0 6 Enterococcus cecorum 4.5 1 58.2 86.3 Rothia dentocariosa 4.0 1 37.4 16.8 S. bovis, along with its efficiency at converting energy to cell biomass, would give it a competitive advantage over other microorganisms. The identification of coliform bacteria such as Escherichia coli, Hafnia other process parameters, all of which may play a role in the micro- alvei, and even Proteus vulgaris as a non-coliform enteric as seen in flora of the masa. Table 2 is of potential concern relating to the sanitation of the mills. Co- As lactic acid bacteria comprise the majority of the microflora, it is liforms were observed in all mills, except Mill 3, as shown in Fig. 1,but understandable that nixtamalized maize would be used in a variety of the higher incidence as calculated from the % within the mill (Table 2) naturally fermented food products. Although desirable in fermented did not correlate with initial counts reported in Fig. 1. This is likely foods, such as pozol, lactic acid and other compounds produced by due to the inherent differences in determining population counts from lactic acid bacteria, are a cause of spoilage and quality deterioration colony morphology groupings and subsequent identifications of a in tortilla masa. Corn tortillas have a very short shelf-life and are typical- small representation of the group (Table 2) versus traditionally accept- ly consumed the same day they are produced, due to spoilage that can ed plating methods such as coliform/E. coli Petrifilm, as reported in occur as a result of high moisture and water activity (Martinez-Flores Fig. 1. The most reliable numbers from a quality assurance perspective et al., 2004). Preservatives such as potassium sorbate, calcium propio- would invariably be the Petrifilm counts due to reproducibility and sim- nate, and others have been added to masa to help extend the shelf-life plicity of use. The presence of coliforms whether it be determined from of the masa and the tortillas, and may be useful in limiting microbial Table 2 or Fig. 1 is still of concern from a food safety perspective and growth in fresh nixtamal masa produced in these small mills (Ortiz should be addressed by the respective mills. and Carrillo, 1997; Tellezgiron et al., 1988). Differences in identification methods may explain discrepancies among predominant genera. Both Sefa-Dedeh et al. (2004) and 4. Conclusion Wacher et al. (2000) used selective media and carbohydrate utilization methods to tentatively identify microorganisms, although Sefa-Dedeh To our knowledge, this is the first study to characterize and iden- identified Lactobacillus as the primary genus, with no Leuconostoc pres- tify bacteria of nixtamalized corn masa from traditional commercial ent. Diaz-Ruiz et al. (2003) and Escalante et al. (2001) used 16S rDNA tortilla mills. In this study, it was demonstrated that corn masa pro- sequencing in their identification methods. This is the first study to duced at selected mills in Guadalajara, Mexico contains a variety of use MIDI fatty acid profile analysis to identify species in nixtamalized bacteria from several different genera. The microbial profile of fresh, corn masa; however, the results generally agree with those of other nixtamalized corn masa is primarily composed of LAB and non-lactic, researchers, particularly with those of Sefa-Dedeh et al. (2004). aerobic, mesophilic bacteria, with smaller amounts of coliforms and Lactobacillus oris and S. bovis were the only organisms to be iso- even fewer numbers of yeasts and mold. This study has the potential lated from all of the six mills studied. Mill 5 was the most diverse of to help commercial producers of nixtamalized masa and tortillas gain all the mills with 11 different species identified between the LAB a better understanding of their products. In addition, it may help man- and APC plates. Mill 3 showed the largest populations with an aver- ufacturers understand how the microflora in the fresh masa can poten- age of 6.0 × 107 LAB/g and 8.3 × 107 APC/g. Although all mills tially lead to spoilage, as well as nutritional and sensory changes in their produced their masa through the traditional nixtamalization pro- products over time. Further study is needed to investigate the effects cess, it is likely that there were differences in the corn supply, the that the microflora may have on the spoilage, nutritional, and sensory lime concentration, the time of steeping in the alkaline solution, or aspects of the masa and tortillas. S.J. Adolphson et al. / International Journal of Food Microbiology 165 (2013) 209–213 213

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