Using Steam Treatment to Enhance Storage Stability of Grains from Perennial Intermediate Wheatgrass

Amy Mathiowetz, Catrin Tyl, and Baraem Ismail

Department of Food Science and Nutrition, University of Minnesota, Twin Cities, 1334 Eckles Avenue, St. Paul, MN 55108

MATERIALS AND METHODS

• IWG exhibits a higher protein content than HRW (Table 1).

• IWG has a higher lipase activity than HRW, while HRW exhibits greater lipoxygenaseactivity (Table 2). These enzymes contribute to hydrolytic and oxidative rancidity,respectively, and can be problematic over storage.

• However, IWG boasts superior antioxidant activities (as measured by DPPH andLMB assays), attributed to their greater antioxidant content, specificallyhydroxycinnamic acids (Table 3), which may help to delay oxidation over storage,contributing to longer shelf life.

• DPPH measures radical scavenging activity by electron transfer, whereas LMBmeasures antioxidant capacity by electron transfer and the ability of antioxidantsto reduce hydroperoxides by hydrogen atom transfer 14. Moreover, LMBdemonstrates particularly high correlations (Table 4) with hydroxycinnamic acids,the antioxidants that have been demonstrated as some of the most active in wheatspecies15,16 .

• The following steam treatment conditions were considered optimal in terms ofminimal antioxidant degradation and maximum enzyme inactivation and wereadopted for the remainder of the storage study: 100°C, 90-95%RH, 60 minutes.

• Steamed and not steamed samples of IWG and HRW stored for varying lengths oftime at 45°C and 0.43 aw have been analyzed for peroxide value (an indicator ofprimary oxidation), free fatty acids (an indicator of hydrolytic rancidity) and lipaseactivity up to five weeks.

• HRW that was not steamed reached peak hydroperoxide formation at four weeks,while steamed HRW and IWG (both steamed and not steamed) have considerablyless hydroperoxide formation at four weeks (Figure 2), indicating less oxidation.\

• The steamed grains have less hydroperoxide formation than the grains that werenot steam treated. Additionally, it is to be expected that HRW showed higherperoxide values overall, as HRW has a higher lipoxygenase content, as previouslynoted.

• Aside from steamed IWG, samples have shown minimal change in free fatty acidstatus over time (Figure 3), which may be explained by the higher lipase activity ofIWG (Figure 4).

1. Glover JD, Reganold JP, Bell LW, Borevitz J, Brummer EC, Buckler ES, Cox CM, Cox TS, Crews TE, Culman SW, Dehaan LR, Eriksson D, Gill BS, Holland J, Ho F, Hulke BS, Ibrahim AMH, Jackson W, Jones SS, Murray SC, Paterson AH, Ploschuk E, Sacks EJ, Snapp S, Tao D, Van Tassel DL,

Wade LJ, Wyse DL, Xu Y. 2010. Science. 328(5986):1638-1639.2. Rose DJ, Ogden LV, Dunn ML, Pike OA. 2008. Cereal Chem. 85:218-223.3. De Almeida JL, Pareyt B, Gerits LR, Delcour JA. 2013. Cereal Chem. 91(4): 321-326.4. Meera MS, Bhashyam MK, Ali SZ. 2011. Food Science and Technology. 44(10):2199-2204.5. Ekstrand B, Gangby I, Akesson G, Stollman U, Lingnert H, Dahl S. 1993 Journal of Cereal

Science. 17(3):247-254.6. Guo W, Beta T. 2015. Food Research International 51:518-525.7. Lindenmeier M, Alexander B, Somoza V. 2007. Molecular Nutrition and Food Research. 51(12): 1441-

1446.8. Bright D, Stewart GG, Patino H. 1999. J. Am. Soc. Brew. Chem. 57(4): 133-137.9. Abdel-Aal ESM, Young JC, Rabalski I, Hucl P, Fregeau-Reid J. 2007. Journal of Agricultural and Food

Chemistry 55:787-794.10. Vaidyanathan S and Bunzel M. 2012. Cereal Chem. 89(5): 247-254.11. Li Y and Schwarz PB. 2012. . Am. Soc. Brew. Chem. 70(4): 287-289. 12. Timabud T, Sanitchon J, Pongdontri P. 2013. Food Chemistry 141:2405-2411. 13. Rose DJ and Pike OA. 2006. Journal of the American Oil Chemists’ Society 83:415-419.14. Huang DJ, Ou BX, Prior RI. 2005. Journal of Agricultural and Food Chemistry 53 (6): 1841-1856.15. Verma B, Hucl P, Chibbar RN. 2009. Food Chemistry 116:947-954.16. Tyl CE and Bunzel M. 2012. Journal of Agricultural and Food Chemistry 60:731-739.

REFERENCES

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RESULTS: 45°C Storage Study

This project was supported by grants from the Minnesota Department of Agricultureand the Forever Green Initiative, as well as funds gifted by the Land Institute.Intermediate wheatgrass was supplied by Department of Agronomy and PlantGenetics at the University of Minnesota.

ACKNOWLEDGEMENTS

• Thinopyrum intermedium, or intermediate wheatgrass (IWG), is a novel perennial crop with grains that have a competitive nutritional profile and advantageous environmental attributes.

• With strong agronomic and industrial appeal, IWG has potential to be marketed on a larger scale; however, such an effort requires more information about the grain, including its shelf life.

• Heat treatment has been shown to be an effective means of elongating storage life of grains by inactivating problematic enzymes responsible for hydrolytic and oxidative rancidity, namely lipase and lipoxygenase respectively. Preliminary studies indicated that these enzymes could be more problematic in IWG than in wheat.

• IWG and hard red wheat (HRW) control were subjected to six treatments consisting of two steam treatments (present/absent) and three incubation temperatures (4°C, 23°C, and 45°C). Antioxidant activity pre-and post-steam treatment was analyzed using 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging and leucomethylene blue (LMB) assays. Lipoxygenase activity was measured by the ferrous oxidation-xylenol orange (FOX) assay, and lipase activity was determined spectrophotometrically.

• Throughout storage, samples were analyzed for indicators of hydrolytic and oxidative rancidity, including free fatty acids, hydroperoxides, malondialdehydes, and hexanal.

• Lipoxygenase activity in IWG subjected to 60 min of steam treatment significantly decreased after two weeks of accelerated storage (P<0.05). The antioxidant activity was not significantly affected (P>0.1). Also, compared to our HRW control, IWG has a higher content of ferulic acid, an active antioxidant commonly found in wheat.

• The interruption of enzymatic activity by steam treatment coupled with the preservation of antioxidants could help prolong the shelf life of IWG, ultimately protecting its properties and rendering it marketable.

INTRODUCTION

Samples:

• IWG (Rosemount, August 2015)

• HRW (St. Paul and Lamberton, July 2015)

Steam Treatment

• Half of samples subjected to steam in proofing oven

• 100°C at 95%RH

Storage Conditions

• aw = 0.43

• Groats stored in open petri dishes in dessicators

• Dark environment

• Temp variable: 4, 25, 45°C

Milling

• Samples pulled for analysis at various time points and milled

• UDI Cyclone Sample Mill with 0.5 mm screen size

Moisture

• Moisture content of milled samples analyzed by infrared method

• Ohaus MB45

Chemical Analysis

• Antioxidant activity and content

• Enzymatic activity

• Products of oxidative and hydrolytic rancidity

• Functionality

Figure 4. Comparison of lipase activity over storage at 45°C (aw=0.43) in steamtreated and non-steam treated intermediate wheatgrass (IWG) and hard red wheat(HRW)

Analysis Method

Antioxidant Activity

• DPPH (2,2-diphenyl-1-picryl-hydrazyl) Radical Scavenging Assay6

• Leucomethylene Blue Assay7,8

Antioxidant Content

• Carotenoids by RP-HPLC on a C30 column with PDA detection9

• Hydroxycinnamic acids by RP-HPLC on a C18 column with PDA detection10

Enzymatic Activity • Lipoxygenase activity by ferrous oxidation xylenol orange (FOX)11,12

• Lipase activity per method described by Rose and Pike (2006)13

HydrolyticRancidity

• Free fatty acids (AOAC Method 940.28)

OxidativeRancidity

• Peroxide value (primary oxidation) (AOAC Method 965.33)• Hexanal quantification (secondary oxidation) by GC-FID

ProximateAnalysis

• Fat content by Mojonnier method (Adapted from AOAC 922.06)• Ash content by dry-ashing (AOAC Method 923.03)• Moisture by vacuum oven (AACCI Method 44-40.01)• Protein content by Dumas method (AOAC 990.03)

RESULTS

Constituent HRW IWG

Avg. Percent ± SD*

Protein 13.3 ± 0.172 15.7 ± 0.012

Fat 3.61 ± 0.492 5.07 ± 0.035

Ash 1.54 ± 0.069 2.14 ± 0.064

Moisture 9.66 ± 0.428 7.15 ± 0.049

Carbohydrate (by difference) 71.9 ± 1.161 69.9 ± 0.160

Table 1. Macronutrient composition of intermediate wheatgrass (IWG)and hard red wheat (HRW)

*Standard deviation

Table 3. Comparison of hydroxycinnamic acid content prior to steam treatment inintermediate wheatgrass (IWG) milled using two different methods and ball-milled hardred wheat (HRW)

Sample µg FAᵃ/g flour ± SD* µg pCAᵇ/g flour ± SD* µg SAᵈ/g flour ± SD*

IWG UDI-milled 813 ± 73.7 20.4 ± 3.60 76.4 ± 5.19

IWG Ball-milled 838 ± 83.8 23.5 ± 3.13 71.6 ± 4.42

HRW Ball-milled 506 ± 54.1 7.00 ± 1.72 50.0 ± 5.61

ᵃFerulic acid, ᵇpCoumaric acid, ᵈSinapic acid, *Standard deviation

Measured Variable HRW IWG

Avg. Results ± SD*

Lipoxygenase activity (U/g) 2.83 ± 0.330 1.60 ± 0.155

Lipase activity (U/g) 1.84 ± 0.178 2.79 ± 0.489

Antioxidant activity by DPPHᵃ (acetone extraction) (µmoles TEᵇ/g) 28.1 ± 1.60 61.3 ± 3.69

Antioxidant activity by DPPH (alkaline hydrolysis with ethyl ether extraction) (µmoles TE/g) 34.9 ± 3.82 51.7 ± 2.57

Antioxidant activity by LMBᵉ mmoles (TE/g) 0.589 ± 0.002 0.765 ± 0.002

ᵃ2,2-diphenyl-1-picryl-hydrazyl, ᵇTrolox equivalents, ᵉLeucomethylene blue, *Standard deviation

Table 2. Comparison of enzymatic activities and antioxidant activities in intermediatewheatgrass (IWG) and hard red wheat (HRW) prior to steam treatment

Assay Ferulic acid Sinapic acid pCoumaric acid

Correlation coefficient

DPPHᵃ Radical Scavenging Assay 0.686 0.701 0.703

LMBᵇ Antioxidant Activity Assay 0.981 0.94 0.97

Table 4. Correlations between hydroxycinnamic acid antioxidant content andantioxidant activity by DPPH and LMB antioxidant activity assays

ᵃ2,2-diphenyl-1-picryl-hydrazyl, ᵇLeucomethylene blue

Figure 1. Lipoxygenase activity measured by the FOX assay over time at 60°C in samples steam-treated for various lengths of time

Figure 2. Comparison of primary oxidation as indicated by peroxide value overstorage at 45°C (aw=0.43) in steam treated and non-steam treated intermediatewheatgrass (IWG) and hard red wheat (HRW)

Figure 3. Comparison of hydrolytic rancidity as indicated by percent free fatty acidsover storage at 45°C (aw=0.43) in steam treated and non-steam treatedintermediate wheatgrass (IWG) and hard red wheat (HRW)

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• A steam treatment length of 60 minutes significantly decreased the lipoxygenase activity of IWG after two weeks of accelerated storage (P<0.05), as shown in Figure 1. At this same treatment length, antioxidant activity of steamed-treated IWG as measured by leucomethylene blue and DPPH assays was not significantly decreased as compared to samples that were not steam treated (P>0.1).

• Storage studies are currently underway at 4°C, 25°C, and 45°C. In addition tomeasuring the parameters shown here, samples are also currently beinganalyzed for lipoxygenase activity, antioxidant activity, antioxidant content,and hexanal. As samples are stored at controlled relative humidity (aw=0.43),moisture levels remain constant, at approximately 10%, over storage.

• Future work will focus on storage of flour, as opposed to storage of whole groats,under the same conditions.

• Functionality testing, including loaf volume, starch pasting properties, and doughstrength will be tested over time in stored IWG and HRW samples, steam treatedand not steam treated, as well as in samples stored as flour and whole groats.

• The effect of steam treatment on storage stability and functionality of IWG willinfluence future processing conditions of the grain.

DISCUSSION

FUTURE WORK &

IMPLICATIONS

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