Patricia Isabel Ramos

Journal of Cereal Science 107 - Contents lists available at ScienceDirect Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs Microbial quality, safety, sensory acceptability, and proximate composition of a fermented nixtamalized maize (Zea mays L.) beverage Patricia Isabel K. Ramos a, b, *, Arvin Paul P. Tuaño c, Clarissa B. Juanico a a Institute of Human Nutrition and Food, College of Human Ecology, University of the Philippines Los Baños, College, Laguna, Philippines Science Education Institute, Department of Science and Technology, Bicutan, Taguig, Philippines c Institute of Chemistry, College of Arts and Sciences, University of the Philippines Los Baños, College, Laguna, Philippines b A R T I C L E I N F O A B S T R A C T Keywords: Ecological nixtamalization Fermented maize beverage Maize extract Maize milk Microbial strains Yogurt Snack Consumption of maize and maize-based snacks in the Philippines is limited despite the high maize production. This study aimed to develop a fermented nixtamalized maize beverage (FNMB) and evaluate the microbial quality, safety, sensory characteristics, acceptability, and proximate composition of the formulated product. Different ratio of maize milk and cow milk were mixed [0:100 (control), 50:50, 70:30, 80:20]. Ecological nix tamalization (using CaCO3) was employed to produce the maize milk and fermentation using L. bulgaricus and S. thermophilus was applied. Results showed that the lactic acid bacteria (LAB) count of the fermented treatments reached the minimum required level for fermented beverages. The most acceptable formulation was that of 50:50 maize milk:cow milk ratio, and was selected for further studies. The FNMB did not exhibit E. coli, Salmonella, and S. aureus. Nixtamalization increased the moisture and protein contents and decreased the carbohydrate and ash levels of the fermented beverage (p < 0.05). Fermentation decreased the amount of moisture and carbohydrate and increased the crude protein and ash contents of the nixtamalized samples (p < 0.05). The FNMB exhibited higher protein, carbohydrate, and lower fat than commercial yogurt. The FNMB is a healthy snack alternative which can promote maize consumption and utilization in the Philippines. 1. Introduction 1.1. Nixtamalization process in maize Maize (Zea mays L.) is a cereal crop with edible grains and is a member of the grass family Poaceae. The kernels may be consumed after subjecting to different cooking methods, or as a processed commodity such as grits, hominy, tamales, tortilla chips, masa or dough. It is considered as a staple food in many countries in Latin America, Africa, and some parts of Asia. The level of human consumption of maize has remained steady despite the increase in production over the years. In the Philippines, the per capita maize production in 2016 was 69.92 kg/year while consumption is only 9.14 kg/year in 2018 (Philippine Statistics Authority, 2017; PSA, 2018). Most of the maize produced is used in livestock and poultry (Salazar et al., 2021). Aside from the high pro duction of maize, it is an affordable crop with nutritional significance. Thus, maize and maize-based products are ideal targets for product development and nutrient enhancement. Nixtamalization is a maize processing method which has been applied as early as 4,500 years ago. It is the process of cooking and soaking maize in alkali solution to be developed into several food products such as flour, tamales, or tortilla chips. The traditional nix tamalization process involves cooking the maize grains in lime solution consisting of water and 1–3% calcium hydroxide at near boiling tem peratures for 35–70 minutes and steeping the grains in the lime solution for 8–16 hours. The lime cooking solution, referred to as nejayote, is discarded after steeping and the cooked maize grains are washed and referred to as the nixtamal. The nixtamal is then subjected to milling which produces the nixtamalized maize dough for making tortillas or may also be dried and processed into flour. Aside from the traditional process, there are two other processes, i.e., classical and ecological nixtamalization. In classical nixtamalization, wood ashes are used as cooking and steeping solution while in ecological nixtamalization, other calcium salts are used such as calcium carbonate, calcium chloride, or calcium sulfate. In ecological nixtamalization, the dissociation of the * Corresponding author. Institute of Human Nutrition and Food, College of Human Ecology, University of the Philippines Los Baños, College, Laguna, Philippines. E-mail address:-(P.I.K. Ramos). https://doi.org/10.1016/j.jcs- Received 10 April 2022; Received in revised form 21 June 2022; Accepted 27 June 2022 Available online 30 June-/© 2022 Elsevier Ltd. All rights reserved. P.I.K. Ramos et al. Journal of Cereal Science 107 - salts does not release hydroxide ions which disrupt the rigid ester bonds of the components supporting the pericarp. According to Campecha no-Carrera et al. (2012), ecological nixtamalization has lesser contam inating effects to the environment due to less residues and near neutral nejayote. Therefore, it would be more ideal to use to improve the nutritional, physicochemical, and sensory characteristics of maize products with less harmful by-products released in the environment. Some of the nutritional advantages reported include improved calcium content, improved bioavailability of niacin, and development of color and flavor compounds making maize products more palatable (Día z-Montes et al., 2016). acceptability, and assess the microbiological quality and safety of the most acceptable FNMB formulation. 2. Materials and methods 2.1. Raw materials and equipment IPB Var 6 maize kernels, a white flint, Philippine quality protein maize (QPM) variety (at 12–14% moisture), were purchased from the Cereals Division of the Institute of Plant Breeding (IPB), College of Agriculture and Food Science (CAFS), University of the Philippines Los Baños (UPLB). Commercial thermophilic yogurt culture (YoFlex YFL812) containing L. bulgaricus and S. thermophilus from Chr. Hansen was used. The commercial cow milk, commercial yogurt, and white sugar were purchased from a local market in Los Baños, Laguna. The incubator used for fermentation was the Kesun 3-in-1 Automatic Yogurt Maker Machine DIY Tool ZCW-S08. Distilled water was used all throughout the study unless otherwise specified. 1.2. Microorganisms involved in fermented beverage production The practice of fermenting food substrates has been used since around 10,000 BC, and it has been one of the oldest forms of food pro cessing and preservation methods. Aside from improving the shelf life, other positive effects also occur upon fermentation of foods such as improving the safety, developing acceptable sensory attributes, and enhancing the nutritional and functional values of foods such as milk, and other protein- and carbohydrate-rich foods. Milk products are commonly fermented using the lactic acid bacteria (LAB) strains Strep tococcus thermophilus (S. thermophilus) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). Both are homofermentative and responsible for metabolizing milk substrates. The growth of L. bulgaricus and S. thermophilus happens in a protocooperative manner. Lactobacillus bulgaricus has high proteolytic activity which help S. thermophilus pro liferate while S. thermophilus produces CO2 which lowers the pH of the milk which is ideal for L. bulgaricus (Mchiouer et al., 2017). In Mexico, different fermented beverages are developed using maize. Atole is a porridge-like product made using dough diluted in water or milk. Maize grains are steeped in water for 4 days then milled, and lactic acid fermentation occurs in a day (Chaudhary et al., 2013). The dough can also be left to ferment to make atole agrio or pozol. Atole agrio is an acidic beverage prepared through solid or liquid state fermentation. In solid state, the nixtamal dough is fermented similar to pozol. In liquid state, the nixtamal dough is mixed with water where a homogenous slurry is made prior to fermentation. In both methods, the resulting product is boiled before consumption. Abundant LAB genera in atole agrio are Weissella, Pediococcus, Lactococcus, and Lactobacillus (Väkeväinen et al., 2017). Pozol is also made using fermented nixtamal dough shaped into balls and wrapped in banana leaves and fermented for several days. The fermented dough balls are then dissolved in water and consumed in an uncooked state (Chaudhary et al., 2013). It is a fermented product ripened by fungal (Aspergillus genus) and LAB strains (L. plantarum, S. suis, Lactococcus lactis. L. delbrueckii, L. casei, L. ali mentarium) (Escalante et al., 2001; Rubio-Castillo et al., 2021; Roble do-Márquez et al., 2021). Supavititpatana et al. (2010) reported that maize milk can be potentially used as a raw material in yogurt pro cessing and showed that maize milk yogurt had a lower fat content but higher protein content without significant differences in the flavor, appearance, and color when compared to cow milk yogurt. In the Philippines, a survey in 2015 showed that there is a significant increase in the purchase of food categories strongly associated with healthier food choices among Filipino households. The sales growth was reported to be 11% and 12% in cereals and yogurt categories, respec tively (Deocareza, 2017). The increasing demand for healthier food products, such as yogurt and yogurt drinks, may lead to an opportunity to utilize maize and develop it into a fermented beverage to provide a nutritious maize snack with added health and nutrition benefits. Moreover, at present, there are limited studies on developing a fer mented beverage made from nixtamalized maize milk. Few attempts have been done in developing maize milk yogurt, but very few have employed nixtamalization for maize milk production. The present study aimed to develop fermented nixtamalized maize beverages (FNMB), evaluate the proximate composition, sensory characteristics and 2.2. Nixtamalization process Sample and product preparation was conducted at the Bio-Assay Laboratory and Analytical Laboratory of the Institute of Human Nutri tion and Food (IHNF), College of Human Ecology (CHE), UPLB. The ecological nixtamalization procedure employed was based on the study of Santiago-Ramos et al. (2017). Maize kernels were cooked in distilled water with 1% (w/v) calcium carbonate (CaCO3) at 90 ◦ C for 30 min and at 1:2 maize-to-alkali solution ratio. Rice cooker was used to nixtamalize the maize kernels to be able to control the cooking temperature. After 30 min, the maize and nejayote were steeped for 16 h at room temper ature (23 ◦ C). Then, the nejayote was discarded and the nixtamal was collected and rinsed using 1:1 ratio of distilled water and finally drained to remove excess CaCO3. 2.3. Preparation of maize milk The preparation of maize milk was adapted from the method of Trikoomdun and Leenanon (2016). The nixtamalized and non-nixtamalized maize kernels were mixed with distilled water at 1:2 ratio. The mixture was then cooked for 10 min at 95 ◦ C and cooled to room temperature. Afterwards, the mixture was homogenized for 3 min and filtered through nylon to obtain nixtamalized maize milk, referred to as maize milk from this point onwards. 2.4. Fermented beverage production The procedure for fermented beverage development was based on the method of Geetha et al. (2018) with some modifications, including addition of 5% sugar (Wang et al., 2017). Different ratios of maize milk and cow milk were mixed [0:100 (control), 50:50, 70:30, 80:20]. Maize milk was first heated at 60 ◦ C for 30 s, then cooled to 45 ◦ C. Commercial cow milk, white sugar, and maize milk were mixed. Using aseptic technique, 2% of the starter culture was added. The fermented beverage was then incubated at 40 ◦ C for 4 h until pH of 4.4–4.6 was reached. The pH values were measured 3 times and 4 batches of fermented beverages were prepared. Each batch was used for microbial safety, LAB count, sensory evaluation, and proximate analyses. Microbial safety was analyzed in duplicates while LAB count and proximate composition were analyzed in triplicates for each formulation. 2.5. Determination of total lactic acid bacteria (LAB) count Lactic acid bacteria (LAB) count analysis was conducted in the Food Safety Laboratory of the Institute of Food Science and Technology (IFST), CAFS, UPLB. Total LAB count was measured using the method of Lestiyani et al. (2014), with slight modifications. Using aseptic 2 P.I.K. Ramos et al. Journal of Cereal Science 107 - technique, the sample of fermented beverage was mixed and 1 mL aliquot was transferred to 9 mL peptone diluents. It was mixed thor oughly and labelled as dilution of 10− 1. Dilutions until 10− 6 were also prepared. Afterwards, appropriate dilutions (1 mL each) were trans ferred to sterile Petri dishes in triplicate. Liquefied de Man Rogosa Sharpe (MRS) agar (15–20 mL) at 44-46 ◦ C was added to each Petri dish containing the 1 mL appropriate dilution. It was allowed to solidify on a level surface and the plates were then incubated at 37 ◦ C for 48 h. Plates with 25–250 colonies were counted. The viable count was calculated using the formula based on US Food and Drug Administration (FDA)- Bacteriological Analytical Manual Procedure: followed by fat extraction. Crude protein was analyzed using Kjeldahl method and gravimetric method was employed to determine total ash content. Total carbohydrate and calories were determined by difference. The carbohydrate, protein, and fat contents of the commercial yogurt was obtained from the product packaging. 2.9. Statistical analysis where: N = Total viable count Σ C = Sum of colonies counted on all plates n1 = Number of plates counted in the lower dilution n2 = Number of plates counted in the higher dilution d = Dilution factor corresponding to the lower dilution. For the sensory evaluation, F-test using one-way repeated measures ANOVA was used to detect differences between average values for a specific variable. Paired t-test with p-values adjusted using Bonferonni correction was done to test significant differences. For the proximate analysis data, homogeneity of variances was checked using Levene’s test and the normality of residuals was checked using Shapiro-Wilk test with assumptions satisfied based on the results. F-test using ANOVA for twofactor factorial design in completely randomized design (CRD) was used followed by Tukey’s Honest Significant Difference (HSD) post-hoc test. The unreplicated data set for the commercial yogurt obtained from its product packaging was not included in the tests of significance. All statistical analyses were done at α = 0.05. 2.6. Sensory evaluation 3. Results and discussion Prior to the actual sensory evaluation of FNMB formulations, a training was conducted in order to level the sensory judgment of the panel on varying degrees and to have a consensus among the panelists on how to rate certain characteristics of the product. For the actual sensory evaluation, ten trained panelists with sufficient sensory evalu ation background assessed 3 ratios of fermented maize beverage with varying proportions of cow milk and maize milk. A 15-point line scale for quality scoring was used to measure the intensity of each sensory attribute (color, consistency, sourness, mouthfeel) and overall accept ability was also rated. The Ethics Review Committee (ERC) of the University of the East Ramon Magsaysay Research Institute for Health Sciences (RIHS) reviewed and approved the implementation of this part of the study involving sensory evaluation panelists and assigned with RIHS ERC Code 0779/E/2020/007. 3.1. Microbial quality N= ΣC [(1xn1 ) + (0.1xn2 ) ]x d All the ratios of the fermented beverages were considered appro priate since pH values ranged from 4.4 to 4.5. Fermentation of the beverage is believed to have caused the decrease in the pH due to con version of sugar sources into organic acids such as lactic acid. The LAB count of the fermented beverage treatments was assessed if the mini mum standard for fermented beverages was met. According to Food and Drug Administration (FDA) standards, yogurt and other fermented milk should have LAB at the minimum viable count of 106 cfu/mL. All the treatments with varying ratios of maize milk to cow milk reached the required minimum level for LAB count (Table 1). Results showed that LAB could survive and propagate in fermented maize beverage with varying ratios of maize milk and cow milk as in the control which contained pure cow milk only similar with results of previous studies (Supavititpatana et al., 2010; Yasni and Maulidya, 2014; Lestiyani et al., 2014). The maize milk contains mainly starch, a polysaccharide con sisting of glucose as monomeric units. The amylase enzyme initiates breakdown of starch producing either glucose or maltose. LAB which can produce amylase enzyme and capable of starch hydrolysis are called amylolytic LAB (ALAB). Some ALAB may be initially present and have flourished in the maize milk during fermentation, providing more sugars for the starter cultures. The 80:20 treatment reached similar pH with the control implying that the starter cultures were still able to induce acidification through production of organic acids even at higher amounts of maize milk. The sugars utilized by the starter cultures in the fermented beverage mix tures were glucose and fructose from the maize milk and lactose from the cow milk. Streptococcus thermophilus was also able to ferment sucrose from the maize milk and from the added white sugar. The 70:30 treat ment reached a lower pH than 50:50 which may explain the higher LAB count in the former. The 70:30 ratio might have been a more ideal environment for the cultures because aside from the sugar sources in the 2.7. Microbial safety analysis Microbial safety was analyzed in the Regional Standards and Testing Laboratory of the Department of Science and Technology (DOST) Region IV-A. The microbial safety of the FNMB treatments was measured based on the procedures described in the Bacteriological Analytical Manual (BAM), Microbiology Manual, 12th ed., Merck. In determining the coliform and E. coli count, pour plate method was employed using Chromocult agar and incubation was at 35 ◦ C for 24 h (Bacteriological Analytical Manual, 2010). Meanwhile, presumptive test was conducted for detecting the presence of Salmonella. Conventional method was done using Hektoen Agar-Bismuth Sulfite Agar-XLD Agar and samples were incubated at 35 ◦ C for 24 h (BAM, 2010). In detecting S. aureus, con ventional method was employed. Baird-Parker Agar was used for isolation and enumeration incubated at 35 ◦ C for 48 h. Coagulase test was done using Brain Heart Infusion Broth and Bactident coagulase with incubation at 37 ◦ C for 24 h (BAM, 2010). Table 1 LAB count of the different fermented beverages at varying maize milk-to-cow milk ratios after three days of incubation at 37 ◦ C. 2.8. Proximate analysis Proximate composition was analyzed in the Chemical Testing Lab oratory of SGS Philippines, Inc. The proximate composition of the most acceptable fermented beverage was measured based on the procedures described in Association of Official Analytical Chemists (AOAC, 2016) Official Methods, 20th ed. Moisture content was determined using air oven method while crude fat was determined through acid hydrolysis Maize milk:cow milk ratio 0:100 (control) 50:50 70:30 80:20 3 Initial pH pH after 4 h - - LAB count (cfu/mL- x 107 x 106 x 107 x 107 P.I.K. Ramos et al. Journal of Cereal Science 107 - cow milk, the cultures were also able to metabolize more sugars from the maize milk. Nevertheless, the 80:20 treatment had a lower LAB count than the 70:30 treatment and the control. Too high saccharification can cause too high sugar content, leading to an increase in the osmotic pressure and the high osmotic stress can hamper growth of LAB (Papa dimitriou et al., 2016). High saccharification and excess soluble sugars from the maize milk could account for the lower LAB count for the 80:20 treatment which contained more starch than the other treatments. 3.2.3. Sourness All the ratios had perceived sourness based on the sensory evaluation results, but the sourness among the fermented beverage treatments was not significantly different (Fig. 1). It is apparent however that the 70:30 ratio had the highest perceived sourness which can be explained by the lower pH than the other ratios as demonstrated previously. The sour taste or the characteristic sharpness and acidity of yogurt is largely caused by the lactic acid and other organic acids produced by the LAB. 3.2.4. Mouthfeel Results showed that all the fermented beverages were slightly creamy (Fig. 1). Creaminess and smoothness perceptions are directly proportional to the fat content and thickness of the product. Although the difference between the fermented beverage treatments was insig nificant, it is noticeable that the 50:50 ratio had the highest mouthfeel rating followed by the 70:30 ratio, and lastly the 80:20 ratio. Cow milk has a higher fat content than maize milk. The 50:50 ratio had the highest cow milk content and therefore had a higher fat level, which also possibly increased the creaminess of this formulation. 3.2. Sensory characteristics and acceptability Sensory evaluation was conducted to determine the sensory attri butes of the treatments and the most acceptable ratio. Sensory evalua tion ratings are shown in Fig. 1. 3.2.1. Color All the ratios were evaluated to have opaque white color which is the most acceptable quality and all ratios had the ideal color. There was no significant difference among the ratios in terms of color even if the nixtamalized maize milk proportion increased (Fig. 1). The kernels used were from a white flint maize variety but with very few yellow streaks due to its minute lutein and zeaxanthin levels. In producing the maize milk used in the fermented beverages, most of the yellow colorcontaining components of the maize were filtered and discarded sub stantially, reducing the yellow color for all the treatments. 3.2.5. Overall acceptability All the fermented beverage treatments were found to be acceptable. Although the results for overall acceptability had no significant differ ences, the 50:50 maize milk to cow milk ratio tended to gain the highest acceptability rating among the treatments. It also had the highest con sistency rating. 3.2.2. Consistency Sensory evaluation results revealed that the 50:50 ratio had a thick consistency while both the 70:30 and 80:20 ratio had a slightly thick consistency with significant difference (p < 0.05) between each. In the preparation of the fermented beverage, the maize milk extracted was heated at 60 ◦ C to eliminate spoilage microorganisms and to gelatinize the starch in the maize milk. Starch gelatinization increased the viscosity of the solutions through formation of gels, thereby improving the con sistency of the fermented beverage. A higher level of milk protein pre sent is associated with higher consistency of yogurt because of the role of protein in coagulum formation. Since the 50:50 ratio had the highest cow milk content, it demonstrated higher gel formation, forming a thicker product than the other two ratios. 3.3. Microbial safety The 50:50 ratio of FNMB (most acceptable formulation based on above sensory evaluation results) was subjected to further studies. For total coliform count, results showed that only the nixtamalized; nonfermented treatment met the standards (Table 2). The other treat ments exceeded the limits and the fermented treatments both had higher coliform counts than their non-fermented counterparts. Coliform contamination of samples may have occurred after fermentation or postprocessing, but most coliforms were possibly destroyed during nixtam alization, but a sufficient number might have survived to return the concentrations to precooking levels within 16 h of room temperature Fig. 1. Mean sensory evaluation ratings for the different treatments (maize milk:cow milk ratios) for each sensory attribute and overall acceptability. Bars with the same letter within a graph are not significantly different (p > 0.05). 4 P.I.K. Ramos et al. Journal of Cereal Science 107 - Table 2 Microbial safety of different beverage formulations containing 50:50 ratio of maize milk and cow milk. NIXTAMALIZED Coliform count (cfu/mL) E. coli count (cfu/mL) Salmonella detection Staphylococcus aureus detection NON-NIXTAMALIZED FDA STANDARD Fermented** Non-fermented Fermented Non-fermented** Microbial limit <250* <10* Negative Negative <10* <10* Negative Negative 2.6 x 103 <10* Negative Negative <250* <10* Negative Negative 102 Negative Negative 102 <10- no growth in 10− 1 dilution. *-estimated plate count. **- all plates in all dilutions yield counts less than 25 colonies. Fermented samples were incubated at 40 ◦ C for 4 h which might have caused evaporation of the water in the mixture lowering the moisture content of the fermented samples. storage. To prevent contamination, pre-treatment of the maize kernels may be employed before nixtamalizing to ensure low coliform counts before processing. Possible pretreatments may be washing the maize kernels with copious clean water and applying sanitizing agents such as 200 ppm aqueous sodium hypochlorite for 5 min. Additionally, food contact surfaces, materials, and containers should also be disinfected prior to use to prevent cross-contamination during processing and postprocessing. Antimicrobial compound solutions which can be used to lower microbial load are hydrogen peroxide for surfaces, 25–200 mg/L sodium hypochlorite, 0.1–0.5 M of citric acid, or 60–80 ppm peroxy acetic acid (Becerra-Sanchez and Taylor, 2021). While coliform counts exceeded the limit for some of the treatments, there had been no growth of E. coli in all the treatments which suggests that there was no fecal contamination. There had also been no Salmonella and S. aureus detected in all the treatments. 3.4.2. Crude fat content In terms of crude fat, nixtamalization did not have a significant effect on the crude fat of the beverages (p-value: 0.4378) which conforms with the study of Vega Rojas et al. (2017). Similarly, fermentation did not have a significant effect on the crude fat of the samples (p-value: 0.9804). Medina et al. (2004) stated that lactic acid bacteria have generally low or weak lipolytic activity, thereby causing insignificant changes in the fat composition in fermented milk as observed in the results of the current study. 3.4.3. Crude protein content Nixtamalization had a significant effect on the average crude protein of the fermented samples (p-value: 0.0099). Nixtamalization increased the average crude protein contents of the samples significantly (p < 0.05). The increase in the protein levels of the nixtamalized samples is in contrast with findings of the majority of previous studies on maize nixtamalization wherein the nixtamalized products had decreased pro tein levels due to its solubility (Escalante-Aburto et al., 2020; De Leon et al., 2022). However, these results were in good agreement with those of Sefa-Dedeh et al. (2004) wherein the nixtamalized samples had higher protein contents than the non-nixtamalized samples. The increase in the protein content has been attributed to the increase in nitrogen content of nixtamal as an effect of lime concentration. Moreover, removal of some soluble starch during nixtamalization consequently increased the pro tein percentage relatively. Exploring the effect of fermentation showed that a significant effect on the average crude protein levels of both the nixtamalized and nonnixtamalized samples (p-value: 0.0000). The fermented treatments had higher average crude protein contents than the non-fermented treat ments. Fermentation significantly increased the average protein con tents of the nixtamalized and non-nixtamalized samples (p < 0.05) similar to the reports of Tangyu et al. (2019) and Ogodo et al. (2017) and the increase in protein content of fermented foods could be attributed to the growth and reproduction of the fermenting cultures since they have a proteinaceous structure from the proteins in the milk as well as due to nitrogen fixation from the air used for growth and reproduction of the fermenting microorganisms. 3.4. Proximate composition Results showed that there was no significant interaction between nixtamalization and fermentation relative to the moisture (p-value: 0.6826) and crude fat (p-value: 0.4128) levels among all the treatments. Thus, the values shown are separately comparing nixtamalized, nonnixtamalized, fermented, and non-fermented treatments (Table 3). However, a significant interaction (p < 0.05) within fermentation and nixtamalization were found for the crude protein (p-value: 0.0021), total carbohydrate (p-value: 0.0490), and total ash (p-value: 0.0074) levels of the beverages. Values for the nixtamalized; fermented, nonnixtamalized; fermented, and nixtamalized; non-fermented samples are shown in Table 4. 3.4.1. Moisture content The average moisture contents of the nixtamalized and nonnixtamalized beverage samples were significantly different (p-value: 0.0474). The nixtamalized samples showed higher average moisture contents than the non-nixtamalized counterparts (p < 0.05). These re sults were consistent with the study of Sefa-Dedeh et al. (2004) reporting that the presence of lime could cause development of osmotic potential in the grain, triggering the kernel to absorb more water until equilibrium is reached. Also, the fermented samples showed significantly lower average moisture content than the non-fermented samples (p < 0.05). Table 3 Effect of nixtamalization and fermentation on the moisture and crude fat con tents of beverages with 50:50 maize milk and cow milk. 3.4.4. Total carbohydrate content Among the fermented treatments, the nixtamalized sample showed significantly lower average total carbohydrate content than the nonnixtamalized sample (p < 0.05). Nixtamalization significantly decreased the average total carbohydrate content in the fermented treatments (p-value: 0.0167). The decrease in the total carbohydrate content of the nixtamalized samples can be attributed to the leaching of the soluble starch granules and sugars into the cooking and steeping solution during nixtamalization. For the effect of fermentation among the nixtamalized treatments, the fermented treatment had lower average total carbohydrate content Mean level (%) Component Moisture Crude fat Moisture Crude fat Nixtamalized 85.75a 0.96a Fermented 85.16a 0.92a Non-nixtamalized 85.64b 0.88a Non-fermented 86.23b 0.92a *Means with the same letter within a row are not significantly different (p > 0.05). 5 P.I.K. Ramos et al. Journal of Cereal Science 107 - Table 4 Effect of nixtamalization and fermentation on the levels of crude protein, total carbohydrate, and total ash of beverages with 50:50 maize milk and cow milk. Mean level (%) Component Crude protein Total carbohydrate Total ash Nixtamalized; fermented 3.36a,a 9.69a,a 0.83a,a Non-nixtamalized; fermented 2.87b 10.30b 0.80a Nixtamalized; non-fermented 1.89b 10.40b 0.80b *Means with the same letter within a row are not significantly different (p > 0.05). First letter in column 2 is compared to letter in column 3 while second letter in column 2 is compared to letter in column 4. than the non-fermented treatment significantly (p-value: 0.0072). Similar to the pattern exhibited by nixtamalization, fermentation also reduced the total carbohydrate of the samples (p < 0.05). The LAB cultures were presumably responsible for the decrease in total carbo hydrates due to their catabolic activities, utilizing the available carbo hydrates for growth. Most of the carbohydrates were converted to organic acids such as lactic acid. commercial yogurt was almost three times higher than that of the developed maize beverage due to its based ingredient, milk. The car bohydrate content of the fermented nixtamalized maize beverage was higher by 1%. The protein content of the fermented nixtamalized maize beverage was also higher by 1% than that of the commercial yogurt. 3.4.5. Total ash content Among the fermented beverage treatments, there was no significant difference between the ash content of the nixtamalized and nonnixtamalized samples. Since ash content indicates the mineral content, it was predicted that the nixtamalized treatments would have higher mineral content as concluded in most of the previous studies in the literature, but results showed otherwise. Nixtamalization did not have a significant effect on the ash content of the fermented samples. However, the results conformed with those of other studies (Sefa-Dedeh et al., 2004; Campechano-Carrera et al., 2012) which reported that inorganic elements from the maize leached into the nejayote. For the effect of fermentation among the nixtamalized treatments, there was a significant difference between the average ash content of fermented and non-fermented treatment (p < 0.05). Fermentation increased the average ash contents in the treatments. The results corroborate with study of Ogodo et al. (2017) wherein fermentation increased the ash content in maize which can be attributed to the metabolic activities of the fermenting cultures. Different food processing techniques such as nixtamalization and fermentation can be applied to cereals such as maize to increase valueadded products in the market and promote maize utilization. In this study, fermented beverage made from nixtamalized maize milk was developed. The product was found to be acceptable and safe for con sumption. Stricter sanitary measures such as using sanitizing agents and proper storage temperature should be observed to avoid increase in coliform counts. The product also had promising macronutrient profile compared to commercial yogurt. Specifically, the FNMB had higher carbohydrate and protein and lower fat contents than the commercial yogurt while still giving the same number of total calories. Ecological method was the nixtamalization employed in the study. The effect of traditional and classical methods on the nutrient content of fermented nixtamalized maize beverage should also be studied since these might have different effects due to difference in calcium source relative to this study. 3.4.6. Comparison between fermented nixtamalized maize beverage and commercial yogurt Comparison between the nutrient profiles of the FNMB and the commercial yogurt is shown in Fig. 2. Values were compared to the Recommended Nutrient Intake (RNI) indicated in the Philippine Dietary Reference Intake (PDRI) (FNRI, 2015) while the total fat and carbohy drate levels were compared to Daily Values (DV). Reference requirement values from RNI were for Filipino male adults aged 19–29 years old and DV was based on a 2,000 calorie diet. The FNMB and commercial yogurt contained similar amount of calories and could both contribute to 3% of the recommended energy intake. However, the calories from fat of the Patricia Isabel Kalacas Ramos: Conceptualization, Formal anal ysis, Investigation, Methodology, Validation, Resources, Writingoriginal draft, Writing-review and editing. Arvin Paul Pavillion Tuaño: Conceptualization, Formal analysis, Funding Acquisition, Resources, Supervision, Validation, Writingoriginal draft, Writing-review and editing. Clarissa Bentillo Juanico: Conceptualization, Formal analysis, Funding Acquisition, Project Administration, Resources, Supervision, Writing-original draft, Writing-review and editing. 4. Conclusion Author statement Fig. 2. Macronutrient profile of fermented nixtamalized maize beverage (left) and commercial yogurt (right). 6 P.I.K. Ramos et al. Journal of Cereal Science 107 - Declaration of competing interest Lestiyani, A.D., Suseno, T.I.P., Srianta, I., 2014. Characteristics of soy corn yogurt. J. Food. Nutri Disorders. 3 (2) https://doi.org/10.4172/-. Medina, R.B., Katz, M.B., González, S., Oliver, G., 2004. Determination of esterolytic and lipolytic activities of lactic acid bacteria. Methods Mol. Biol. 2004 (268), 465–470. https://doi.org/10.1385/-:465.PMID:-. Mchiouer, K., Bennani, S., Meziane, M., 2017. Microbial interactions between Lactobacillus bulgaricus and Streptococcus thermophilus in milk. J. Mater. Environ. Sci. 8 (4),-. 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