Wet‐Milling Characteristics of 10 Lines from Germplasm Enhancement of Maize Project Compared with Five Corn Belt Lines

The use of corn (Zea mays L.) hybrids with high grain yield and starch extractability has steadily increased in the processing industry. In light of widespread corn seed industry participation in the Germplasm Enhancement of Maize Project (GEM), which seeks to enhance exotic germplasm, future hybrids may contain more exotic sources in genetic backgrounds. It is necessary to establish and monitor physical, compositional, and milling characteristics of the new exotic breeding materials to determine the processing value. The present study was conducted to determine the wet‐milling characteristics of a set of GEM lines compared with typical Corn Belt lines. Ten GEM lines introgressed with exotic materials from Argentina, Chile, Cuba, Florida, and Uruguay and previously identified as having different starch yields, three commercial inbred lines, and two public inbred lines (B73 and Mo17) were analyzed using both near‐infrared transmittance (NIT) and a 100‐g wet‐milling procedure. There were statistical differences (P < 0.05) in the yield of wet‐milled fractions (starch, fiber, gluten, and germ). The GEM lines AR16035:S19‐227‐1‐B and CUBA117:S1520‐562‐1‐B had similar or better starch yield and starch recovery than B73 and the other adapted inbred lines, indicating that they may be useful in improving the proportion of extractable starch present in kernels of hybrids. Residual protein levels in the starch and gluten fractions were 0.26–0.32% and 38–45%, respectively. The starch yield of GEM lines from wet milling correlated positively with starch content from NIT and was negatively correlated with protein content of the corn kernels. Oil content in the germ varied from 50 to 60%. Our results indicate that incorporating GEM lines in a breeding program can maintain or even improve wet‐milling characteristics of Corn Belt materials if lines with appropriate traits are used.     

Physical,Compositional, and Wet Milling Characteristics of Grain from Crosses of Corn Inbreds with Exotic and Nonexotic Background

Corn breeders have developed hybrids with enhanced compositional characteristics, but exotic germplasm represents little of the germplasm base used to produce these hybrids. Effects of the exotic germplasm on physical, compositional, and wet‐milling properties as well as the proximate composition of recovered fractions need to be determined before these materials are of value to the corn processing industry. Ten lines from the Germplasm Enhancement of Maize (GEM) project with exotic germplasm introgressed from Argentina, Chile, Uruguay, Cuba, and Florida were crossed to three adapted inbred lines (testers) and grain from the resulting 30 hybrids were evaluated for physical, compositional, and wet‐milling characteristics and the expression of heterosis in these variables. The B73xMo17 adapted public hybrid was used as control. Grain obtained by self‐pollination of the hybrid plants was analyzed using near‐infrared transmittance (NIT) technology and a 100‐g wet‐milling procedure. There was great variation among physical, compositional, and wet‐milling characteristics, and some of the experimental hybrids with exotic origin had better starch yield and starch recovery than B73xMo17, which suggests that wet‐milling characteristics of U.S. hybrids can be improved by breeding with exotic germplasm. In particular, GEM breeding crosses AR16035:S19, CH05015:N15, CUBA117:S1520, and FS8B(T):N1802 could be valuable germplasm sources to produce inbreds with good milling properties. Testers varied in ability to produce hybrids with good milling properties, indicating that choice of tester is an important factor when evaluating this end use. Although general trait trends for mid‐ and high‐parent heterosis were revealed, individual variation among hybrids and testers was large for most traits. This demonstrates the importance of analyzing individual hybrids that are intended for the wet‐milling industry when breeding with exotic introgressed lines.     

Laboratory Procedure to Wet‐Mill 100 g of Grain Sorghum into Six Fractions

A small‐scale (100 g of grain) procedure was developed to wet‐mill grain sorghum into six fractions by modifying the procedure of Eckhoff et al (1996). The wet‐milling process was repeated five times on commercial grain sorghum, and the mean yield (69.4%) of starch (≤0.3% protein) varied by 0.3%, whereas the yields of fiber, gluten, and germ plus bran fractions varied by 5–6%. The starch fraction accounted for ≈95% of that in the grain, while the total solids recovered was 99.0%. Four other samples of grain sorghum gave 92–95% recoveries of starches and 98.2–99.8% recoveries of total solids. All grain sorghum starches had lightness (L*) values and pasting curves nearly equal to those of a commercial maize starch.     

Variation Among Physical,Compositional, and Wet‐Milling Characteristics of the F1 Generation of Corn Hybrids of Introgressed Exotic and Adapted Inbred Lines

Hybrids with high grain yield and higher starch, protein, or oil content are available to corn growers; however, they result from crossing adapted Corn Belt inbred lines that rarely include exotic germplasm. This study was conducted to determine whether Corn Belt lines introgressed with exotic materials from Argentina, Chile, Uruguay, Cuba, and Florida have appropriate wet‐milling characteristics in their hybrids. Ten lines from the Germplasm Enhancement of Maize (GEM) project with different starch contents were crossed to three adapted inbred lines used as testers. The B73×Mo17 hybrid was used as a control. The F₁ generation of these 30 experimental hybrids was analyzed using both near‐infrared transmittance (NIT) technology and a 100‐g modified wet‐milling procedure, and measuring test and 1,000‐kernel weight. There was great variation among physical, compositional, and wet‐milling characteristics of the experimental hybrids, suggesting that exotic germplasm can be used to improve wet‐milling characteristics of Corn Belt hybrids.     

Effect of Laboratory Batch Steeping pH on Starch Yield and Pasting Properties of Selected Corn Hybrids

This study evaluated the effect of initial pH on percent of starch yield and pasting characteristics for a laboratory wet‐milling procedure. Four commercial hybrids, selected because they have significantly different starch yield values, were laboratory wet‐milled, and the pasting properties of the starch fractions were evaluated using a Rapid Visco Analyser (RVA). Percent starch yield (db) decreased when initial pH values were >4.0 but was unaffected by any lower initial pH values. The pasting properties of some of the selected hybrids were more sensitive to steepwater pH than others. There was an overall increase in peak, trough, and final viscosity as pH increased.     

Physicochemical Properties of Maize Starch Obtained from Intermittent Milling and Dynamic Steeping (IMDS) Under Various Steeping Conditions

Physicochemical properties of maize starch obtained under different steeping conditions by intermittent milling and dynamic steeping process (IMDS) were studied. Brazilian dent maize (hybrid XL 606) was milled using a 2×2×3 factorial experimental design with two lactic acid levels (0.0 and 0.55%, v/v), two SO₂ levels (0.05 and 0.1%, w/v), and three temperatures (52, 60, and 68°C). Properties of starch obtained by conventional wet‐milling process (36 hr at 52°C, 0.55% lactic acid, and 0.2% SO₂) were used for comparison. Starch protein content and solubility increased with presence of lactic acid, while swelling power decreased. Higher SO₂ concentration (0.1%) had the same effect as lactic acid on some properties. Steeping temperatures of 60 and 68°C increased solubility and most of the thermal properties but reduced swelling power, suggesting stronger starch annealing during IMDS at these temperatures. Some thermal changes on starch granules were visualized by scanning electron microscopy (SEM) at 60 and 68°C. Amylose content as well as pasting properties were affected by steeping factors and interactions. Starches from IMDS and conventional wet‐milling processes were similar in most properties, indicating that IMDS provides starch with quality similar to that from conventional milling.     

Grain and Nutritional Quality Traits of Southwestern U.S. Blue Maize Landraces

Anthocyanin‐rich Southwestern blue maize (Zea mays L.) landraces are receiving interest as functional foods, and commercial production is increasing. We determined variation in kernel color, anthocyanin content, texture, and selected compositional traits of representative varieties. In 2013, eight varieties were grown at four locations in New Mexico. Total kernel anthocyanin content (TAC) and component pigments were measured with spectrophotometry and HPLC, respectively. Oil, protein, starch, and kernel density were determined using NIR spectroscopy and amino acid concentrations using wet chemistry. An average of 49.6 mg/100 g of TAC with a range of 17.6–65.1 mg/100 g was observed. Cyanidin and pelargonidin were major components, and peonidin and succinyl 3‐glucoside were minor components. Low levels of disuccinyl glucoside were detected. Blue kernels were higher in anthocyanin than purple or red kernels. Floury kernels displayed the highest protein and oil contents and the lowest starch content and kernel density. The highest starch and kernel density levels were observed in small flint/dent and pop‐flint/dent kernels. Amino acid content was variable across genotypes and locations.     

Wet Processing of Barley Grains into Concentrates of Proteins, β‐Glucan,and Starch

An improved wet method was developed to process barley into fractions concentrated in protein, (1‐3)(1‐4)‐β‐d ‐glucan (BG), starch, or other carbohydrates (CHO). Alkaline concentration, solvent to barley flour ratio (SFR), and extraction temperature were evaluated for their effects on concentration and recovery of protein, BG, starch, oil, ash, and other CHO in each fraction type. Results show that the three parameters and their interactions all had significant effects, resulting in varying nutrient concentrations and recovery rates in each type of fractions. For protein fractions, protein content varied from 37.7 to 75.2%, protein recovery from 8.5 to 75.7%, and increasing alkaline concentration and SFR improved nutrient recovery. For BG fractions, BG content ranged from 21.5 to 87.0%, BG recovery from 28.6 to 78.0%, and increasing alkaline concentration decreased BG content but increased its recovery significantly. For starch fractions, starch content varied from 76.9 to 93.9%, starch recovery from 33.6 to 63.9%, and all parameters had little effect on the nutrient concentrations, but alkaline concentration and SFR improved recovery of starch, other CHO, and mass. Overall, the improved wet method was effective in concentrating the major nutrients from barley into their respective fractions, but process optimization through manipulating the three parameters is necessary to achieve a maximum concentration or recovery rate of a nutrient of interest in a specific fraction.     

Pasting Properties and Surface Characteristics of Starch Obtained from an Enzymatic Corn Wet‐Milling Process

Recently, we reported the development of an enzymatic corn wet‐milling process that reduces or eliminates sulfur dioxide requirements during steeping, considerably reduces steep time, and produces starch yields comparable to that of conventional corn wet‐milling. The best results so far, using the enzymatic corn wet‐milling procedure, were achieved when a particular protease enzyme (bromelain) was used. In this study, pasting properties and surface characteristics of starch obtained from six different enzyme treatments (three glycosidases [β‐glucanase, cellulase, and xylanase] and three proteases [pepsin, acid protease, and bromelain]) using the enzymatic corn wet‐milling procedure were evaluated and compared with those from starch obtained using the conventional corn wet‐milling procedure. Significant effects from enzymatic milling were observed on all the three starch pasting properties (peak, shear thinning, and setback). The setback viscosities of starch from all enzyme treatments were significantly lower compared with those of the control sample, indicating that starch polymers from enzymatic corn wet‐milling do not reassociate to the same extent as with the control. Comparison between bromelain treatment and the control sample showed that starch samples obtained from bromelain treatment are very similar to control starch in water‐binding capacity, molecular breakdown, and time to swell when cooked in water. Significant effects from enzymatic milling were observed on the surface characteristics of starch granules. The glycosidase treatments, especially the β‐glucanase samples, showed holes in the starch granules. No visual differences were observed in starch granules between bromelain and control samples.     

Viscoelastic Properties of Gelatinized Dispersions of Maize Starch Modified by Solid‐State Milling

Solid‐state milling is an effective physical modification method applied to improve functional properties of starch. In this work the effect of solid‐state milling on the viscoelastic characteristics of maize starch gelatinized dispersions (gels) was investigated by using oscillatory squeeze film rheometry. The relaxation spectrum of the samples under study was calculated from the data obtained. It was found that solid‐state milling resulted in a decrease of the storage (G′) and loss (G″) moduli; meanwhile, loss tangent (tan δ) increased. At a starch milling time more than 10 h, G″ > G′ and the gelatinized starch dispersions behaved as liquid‐like systems. The relaxation maxima shifted to shorter relaxation times, and the heights of the maxima decreased with increasing milling time. The relationship between the complex viscosity η* and steady‐shear viscosity η gradually altered from η* > η to η* < η as the milling time increased. The results can be used to determine the processing conditions of milled starch.     

Comparison of Flour Starch Properties in Half‐Sib Families of Opaque‐2 Maize (Zea mays L.) from Argentina

Since the discovery of the o2 mutation in maize, many studies have reported the characterization of the protein quality of opaque‐2 genotypes. However, few have reported the properties of their starch. The objective of this study was to characterize flour starch properties of 12 half‐sib families of opaque‐2 maize from Argentina. Chemical composition and thermal and pasting properties of whole grain flour were determined. Nonopaque genotypes were used as a control. Starch content of opaque‐2 genotypes did not show significant differences compared with nonopaque genotypes, yet amylose content was significantly lower. A high variability in pasting and thermal properties was observed in genotypes. Opaque samples showed a significantly higher peak viscosity and a lower pasting temperature compared with nonopaque samples, probably owing to larger and less compact starch granules in the floury endosperm. The higher the gelatinization enthalpy of opaque‐2 genotypes was, the lower the amylose content in relation to nonopaque varieties. Two retrogradation endotherms were observed in DSC analysis: one corresponding to amylopectin crystallization and the other to melting of amylose‐lipid complex. Both enthalpies were considered total starch retrogradation (ΔH _RT). A wide range of variation was obtained in ΔH _RT in opaque‐2 genotypes, but no significant differences between opaque and nonopaque genotypes were observed. The differences in starch properties found in this study would make it possible to identify opaque‐2 families with particular characteristics for the development of starchy food items adapted to specific processing traits.     

Study of a Small‐Scale Laboratory Wet‐Milling Procedure for Wheat

The objectives of this research were to study the effects of slurry specific gravity, starch table slope, slurry pumping rate, and their interactions on starch recovery and purity; and to propose a small‐scale laboratory wet‐milling procedure for wheat. First‐order and second‐order response surface regression models were developed to study the effects and interactions of slurry specific gravity, starch table slope, and slurry pumping rate on starch and gluten separation for a 100‐g wheat wet‐milling procedure. The starch and starch protein content data fit the first‐order models (R² = 0.99 and 0.96) better than the second‐order models (R² = 0.98 and 0.93). Regression results from the first‐order models indicated that specific gravity, table slope, pumping rate, and their interactions all had a significant effect on starch yield and purity. However, these effects could be simplified as the effect of the resident time of starch and gluten slurry on the starch table and the specific gravity. Starch yield increased as resident time increased and specific gravity decreased. Protein content in starch decreased as the resident time decreased and the specific gravity increased. The separation condition with specific gravity of 3 Bé, table slope of 1.04 cm/m, and pumping rate of 50 mL/min was recommended. Under this condition, starch recovery was 85.6% and protein content of starch was 0.42%, which was similar to the 1.5‐kg laboratory methods in starch recovery. Total solids recovery was 98.1%, which is similar to that from 1.5‐kg laboratory methods. These results indicated that precision of the 100‐g wheat wet‐milling procedure was similar to that of the 1.5‐kg laboratory methods.     

Relationship Between Wheat (Triticum aestivum L.) Grain Hardness and Wet‐Milling Quality

Grain hardness variation has large effects on many different end‐use properties of wheat (Triticum aestivum). The Hardness (Ha) locus consisting of the Puroindoline a and b genes (Pina and Pinb) controls the majority of grain hardness variation. Starch production is a growing end‐use of wheat. The objective of this study was to estimate the differences in starch yield due to natural and transgenically conditioned grain hardness differences. To accomplish this goal, a small‐scale wet‐milling protocol was used to characterize the wet‐milling properties of two independent groups of isogenic materials varying in grain hardness and in Pin expression level. The first group of lines consisted of hard/soft near‐isogenic lines created in cultivars Falcon or Gamenya in which lines carried either the Pina‐D1a (functional) or the Pina‐D1b (null) alleles of Pina. The second group of lines consisted of Pina, Pinb, or Pina and Pinb overexpressing lines created in Hi‐Line, a hard red spring wheat. Soft near‐isogenic lines had higher starch extractability than the hard Pina null counterparts. This difference in starch extractability was more pronounced between Hi‐Line and its transgenic isolines, with highest levels of extractable starch observed in the transgenic isoline with intermediate grain texture. The results demonstrate that the Ha locus and puroindoline expression are both linked to wet‐milling starch yield and that selection for increased Ha function increases starch yield through the enhanced separation of starch granules and the protein matrix during wet milling.     

Laboratory Wet Milling of Corn: Milling Fraction Correlations and Variations Among Crop Years

Several coproducts result from fractionating corn in the wet‐milling process. Because small changes in product composition and milling characteristics can have a major impact on coproduct yields and values, testing is done to anticipate final product yields. Using small sample size and controlled conditions, a laboratory wet‐milling method proved to be a useful tool for wet milling and genetics industries. A wet‐milling process (100‐g batches) was used for data collection. Data collected during 11 years (1994–2004) were observed for samples used as benchmarks to verify process precision and accuracy and determine correlations among wet‐milling yields. More than 400 milling tests were performed on benchmark samples. Data from benchmark samples also were pooled. Coefficients of variation were low (<6%) for mean yields; year‐to‐year standard deviations of benchmark sample yield means were homogenous and implied precision of the procedure. Some differences were detected in mean yields among years (P ≤ 0.05) for benchmark data due to combined effects of hybrid and environment. A negative correlation (r = –0.58) was observed between starch and gluten yield for pooled benchmark data. Four years (2002–2005) of milling data from commercially available hybrids were analyzed using the milling procedure. For pooled commercial data, the correlation between starch and fiber yield was (r = –0.80); correlation between starch and gluten was (r = –0.76).     

Compositional,Physical, and Wet‐Milling Properties of Accessions Used in Germplasm Enhancement of Maize Project

Forty‐nine accessions used in the Germplasm Enhancement of Maize (GEM) project, two commercial hybrids (Pioneer Brand Hybrids 3394 and 3489), and two Corn Belt inbreds (B73 and Mo17) were evaluated for compositional, physical, and wet‐milling properties. GEM accessions had lower starch contents (65.9–69.1% vs. a mean of 72.2% for the commercial hybrids) and greater protein contents (12.0–14.4% vs. a mean of 8.2% for the commercial hybrids) than did the improved Corn Belt material. Absolute densities were consistently higher for the GEM accessions compared with the commercial hybrids (1.320 vs. 1.265 g/cm³, respectively). The wet‐milling characteristics of the GEM accessions were not nearly as good as for the commercial hybrids. Mean starch yields were only 54.3% for the GEM accessions versus 64.8% for the commercial hybrids. Residual protein levels in the starches recovered from the GEM accessions were much greater (0.45–2.03%) than for commercial corn hybrids (<0.3%).     

Effect of a Cell‐Wall‐Degrading Enzyme Complex on Starch Recovery and Steeping Requirements of Sorghum and Maize

The effect of a commercial cell‐wall‐degrading enzyme (CWDE) complex on the steeping time and starch yields of white regular sorghum (RSOR) compared with yellow maize (YMZ) was determined. An in vitro wet‐milling method standardized to test dosages of 0–120 fungal β‐glucanase units (FBG)/100 mL indicated that starch yields were significantly higher for YMZ than RSOR and increased proportionally as enzyme dosage increased. A factorial experiment with a level of confidence of P < 0.05 was performed to study the effect of CWDE addition to coarsely ground grains for 4 hr after 20 or 44 hr of SO₂ steeping of whole grains. At both regular steep times, YMZ yielded significantly higher amounts of starch than RSOR. When steep times were compared, grains soaked for 48 hr produced 1.7% higher starch yields than counterparts treated for 24 hr. CWDE significantly increased starch yields and recoveries. Enzyme‐treated grains yielded 2.5% more starch than counterparts steeped regularly. For both grains, the best wet‐milling conditions to obtain the highest amount of starch were 48 hr of steeping and CWDE addition. Under these conditions, YMZ and RSOR yielded 66.9 and 66.6% starch, respectively. Starches obtained after the enzyme treatment at both steep times contained higher amounts of residual protein and ash compared with the untreated counterparts. Rapid viscoamylograph properties of YMZ and RSOR starches were not affected by the use of the CWDE nor the steep time. In comparison with RSOR starch, the YMZ starch initiated gelatinization at lower temperature, had less shear thinning and higher viscosity or setback at the end of cooling.     

Effect of Steeping Conditions (Sulfur Dioxide,Lactic Acid,and Temperature) on Starch Yield,Starch Quality,and Germ Quality from the Intermittent Milling and Dynamic Steeping Process (IMDS) for a Brazilian Corn Hybrid

Effect of lactic acid, SO₂, temperature, and their interactions were assessed on the dynamic steeping of a Brazilian dent corn (hybrid XL 606) to determine the ideal relationship among these variables to improve the wet‐milling process for starch and corn by‐products production. A 2×2×3 factorial experimental design was used with SO₂ levels of 0.05 and 0.1% (w/v), lactic acid levels of 0 and 0.5% (v/v), and temperatures of 52, 60, and 68°C. Starch yield was used as deciding factor to choose the best treatment. Lactic acid added in the steep solution improved the starch yield by an average of 5.6 percentage points. SO₂ was more available to break down the structural protein network at 0.1% than at the 0.05% level. Starch‐gluten separation was difficult at 68°C. The lactic acid and SO₂ concentrations and steeping temperatures for better starch recovery were 0.5, 0.1, and 52°C, respectively. The Intermittent Milling and Dynamic Steeping (IMDS) process produced, on average, 1.4% more starch than the conventional 36‐ hr steeping process. Protein in starch, oil content in germ, and germ damage were used as quality factors. Total steep time can be reduced from 36 hr for conventional wet‐milling to 8 hr for the IMDS process.     

Whole Grain Amylose Analysis in Maize Using Near-Infrared Transmittance Spectroscopy

The development of genetically modified starches has relied on the use of maize (Zea mays L.) endosperm mutant alleles that alter starch structural and physical properties. A rapid method for predicting amylose content would benefit breeders and commercial handlers of specialty starch corn. For this reason, a study was conducted to investigate the use of near-infrared transmittance spectroscopy (NITS) as a rapid and nondestructive technique for predicting grain amylose content (GAC) in maize. Many single- and double-mutant inbreds and hybrids were used to create a calibration set for the development of a predictive model using partial least squares analysis. A validation set composed of similar genetic material was used to test the prediction model. A coefficient of correlation (r) of 0.94 was observed between GAC values determined colorimetrically and those predicted by NITS; however, the predicted values were associated with a large standard error of prediction (SEP = 3.5). Overall, NITS discriminated well among high amylose and waxy genotypes. The NITS calibration was used to determine levels of contamination by normal kernels in waxy and high-amylose (Amy VII) grain samples intended for wet milling. In both cases, a 5% contaminated sample could be detected from pure samples according to predicted NITS values.     

Application of Protease and High‐Intensity Ultrasound in Corn Starch Isolation from Degermed Corn Flour

The conventional corn wet‐milling process requires a long steeping time and has environmental and health concerns from the use of SO₂. A recently proposed two‐stage enzymatic milling procedure with the first stage of water soaking and coarse grinding of corn and the second stage of incubating with enzymes has been shown to reduce the soaking time and possibly eliminate the need for SO₂ addition. This current work explored the applications of protease and high‐intensity ultrasound in the second stage of the two‐stage enzymatic milling for corn starch isolation to further shorten the process time without use SO₂. of The starch yield from sonication alone was 55.2–67.8% (starch db) as compared with 53.4% of the water‐only control with stirring for 1 hr and 71.1% of the conventional control with SO₂ and lactic acid steeping for 48 hr. Protease digestion alone for 2 hr was not effective (45.8–63.9% yield) in isolating corn starch, but the starch recovery was increased to 61.2–76.1% when protease was combined with sonication. The preferred combination was neutral protease digestion for 2 hr followed by sonication at 75% amplitude for 30 min. The results demonstrated that combinations of high‐intensity ultrasound and neutral protease could replace SO₂ and shorten the steeping time in the enzymatic wet‐milling process for corn starch isolation.     

Enzymatic Milling of Corn: Optimization of Soaking,Grinding, and Enzyme Incubation Steps

Enzymatic milling is a modified wet‐milling process that uses proteases to significantly reduce the total processing time during corn wet milling and eliminates the need for sulfur dioxide as a processing agent. To optimize the overall enzymatic milling procedure and minimize the amount of enzyme, a series of experiments were done to determine the best first grind parameters and the optimal enzyme additions. The yields for germ, germ quality, and starch recovery were used for evaluation of first grind and enzyme addition, respectively. The specific processing conditions evaluated were the soaking time and first grind parameters. After soaking and first grind optimization, enzyme concentration and pH determinations were evaluated using bromelain as an example. The first grind procedure was optimized by evaluating a combination of different soaking and grinding conditions followed by a fixed enzyme addition and incubation step. The pH profile of bromelain for enzymatic milling was evaluated for pH 3.5–6.5 and the optimum was determined to be pH 5.0. Enzyme addition was then evaluated using the optimized first grind conditions and bromelain additions with 0–1.9 g of enzyme (based on protein)/kg of corn. Results showed that the minimum addition of bromelain to reach starch yields equivalent to conventional yields were ≈0.4 g of protein/kg of corn. This amount is significantly less than what was previously used and reported.