Biochemical and Antioxidative Properties of Unprocessed and Sterilized White and Black Sesame By-product from Northern Thailand

The objective of this research was to determine the effects of sterilization on storage stability of white and black sesame by-products. Results showed that sterilization at 120 0C for 10 min had no effect on proximate compositions and mineral contents of both sesame seed cakes, but the significant reductions of thiamine, riboflavin, sesamin, sesamolin, total phenolic compounds and antioxidant capacity (DPPH and FRAP assays) were observed. During the storage at 37 0C, all bioactive components and antioxidant properties apparently tended to decrease when the storage time rose. At the end of storage, PV (peroxide value) and TBARS (thiobarbituric acid-reactive substances) values of stored black sesame seed cakes were shown to be significantly lower than that in white sesame seed cakes. This study may suggest the application of black and white sesame seeds cakes as functional food ingredients in the future production. Current research in Nutrition and Food Science Journal Website:www.foodandnutritionjournal.org ISSN: 2347-467X, Vol. 5, No. (3) 2017, Pg. 196-205 CoNTACT Pittaya Chaikham pittaya.chaikham@gmail.com Faculty of Science and Technology, Phranakhon Si Ayutthaya Rajabhat University, Phranakhon Si Ayutthaya 13000, Thailand © 2017 The Author(s). Published by Enviro Research Publishers This is an Open Access article licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/ ), which permits unrestricted NonCommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. To link to this article: http://dx.doi.org/10.12944/CRNFSJ.5.3.03 Article history Received: 12 October 2017 Accepted: 29 November 2017

(measured by ABTS assay) in sesame seed cakes were found to range between 129.7 and 355.3 mg GAE/100 g, 3.2 and 25.7 mg/100 g, 208.1 and 537.5 mg/100 g, and 8,460 and 24,311 µM TE, respectively 6 .Moreover, the extracts derived from sesame seed cake were shown to have bioactivities, such as effect on polyunsaturated fatty acid metabolism, hypocholesterolemic, anti-cancer and anti-inflammatory properties 1,7 .These bioactivities were suggested to associate with risk reduction of cardiovascular, atherosclerosis and oxidative stress diseases 8 .Bigoniya et al., 9 found that intake of sesame seed cakes as supplementation could be adopted as a therapeutic strategy for preventing obesity-induced Type 2 hyperglycemia in rats.In addition, Konsoula and Liakopoulou-Kyriakides 10 and Suja et al., 4,11 showed that the extracts of sesame seed cakes were very effective for lowering the lipid peroxidation of various vegetable oils, such as olive, soybean, sunflower and corn oils.
Sterilization is one of the most effective food preservation methods and has been widely employed in food industry since it requires low investment cost.It is used to eliminate pathogenic and spoilage bacteria as well as their spores for extending the foods' shelf-life and ensuring the consumer safety 12 .In this method, high temperature (> 100°C) is applied for an extended period of time, which, however, may reduce the qualities of foods and result in losses of heat-sensitive nutrients and/ or bioactive components [13][14] .Nonetheless, there were limited studies that investigated the effect of this conventional technique on the quality attributes of sesame seed cakes.
In the present study, the biochemical and antioxidative proper ties of Thai white and black sesame seed cakes before and after retort sterilization (120 °C/10 min) were evaluated.The purpose was to determine the feasibility of conventional sterilization to extend the shelf-life of sesame seed cakes during storage at 37 °C for 6 months.

Sterilization of Sesame Seed Cakes
Various cultivars of white and black sesame seed cakes were obtained from six sesame oil production factories in Chiang Mai, Chiang Rai and Mae Hong Son provinces, Thailand.The samples were air-dried at 60 °C for 3 h using a convective hot-air oven.For sterilization, 100 g of dried seed cakes were filled into the retort pouch bags before vacuum sealing and heating at 120 0 C for 10 min.The sterilized samples were kept at 37 0 C, and, after that, all of the stored samples were subjected to quality assessments every 2 months for up to 6 months.

Determination of Proximate Compositions and mineral Contents
All samples were determined for the proximate compositions (%, w/w) viz.moisture content, crude protein, crude fiber, fat, ash and carbohydrate according to AOAC methods 14 .To assess the mineral contents, ash residue was dissolved in nitric acid containing 5% (w/v) lanthanum(III) chloride and then analyzed for the mineral constituents (i.e.Ca, P, K, Mg, Fe, Zn, Se and Mn) using an atomic absorption spectrophotometer (Hitachi Z6100, Tokyo, Japan) 14 .

Determination of B vitamins and lignans
Thiamine (vitamin B 1 ) and riboflavin (vitamin B 2 ) were determined using a spectrophotometric method 14 .To quantify the lignan contents, sesame cakes were ground and 200 mg of their powders were well-mixed with 5 ml of 80% ethanol and then centrifuged at 4,500 rpm for 10 min.The supernatants were filtered through a 0.45 µm nylon membrane before injection (10 µL) into a HPLC system (Agilent 1100, Agilent Technologies, Waldbronn, Germany) that using a reversed phase column (Hypersil BDS C18 5 µm, 150 × 4 mm i.d.; Thermo Electron Co., Southendon-Sea, UK).An absorbance was set at 290 nm for monitoring both compounds.The mobile phase was a mixture of methanol and deionized water at a ratio of 4:1 (v/v) with a flow rate of 0.8 mL/min 15 .

Determination of Total Phenolic Compounds
Total phenolic contents were deter mined following the modified method of Chaikham and Apichartsrangkoon 16 .Accordingly, 1 g of sesame cake powder was extracted with 19 mL of acidified methanol [1% (v/v) HCl in methanol] for 1 hr and then centrifuged at 4,500 rpm for 10 min.The supernatant (0.5 mL) was mixed with 2.5 mL of 10% Folin-Cicalteau reagent and allowed to react for 10 min.Consequently, 7 mL of 20% sodium bicarbonate solution were added to the mixture and incubated at room temperature for 2 hr.The absorbance was measured at 765 nm using a UV-Visible spectrophotometer (Perkin Elmer UVWINLAB, Perkin Elmer, Waltham, MA).The results were reported as mg gallic acid equivalents per gram of sample (mg GAE/g).

Determination of 2,2-diphenyl-1-picrylhydrazyl (DPPh) radical Scavenging Activity
In brief, 2 mL of supernatant (as extracted from above section) or methanol (control) were thoroughly mixed with 0.5 mL of 1.5 µM DPPH radicals in methanol, and allowed to react for 30 min at room temperature.The absorbance was measured at 517 nm using a Perkin Elmer UVWINLAB UV-Visible spectrophotometer.The percentage of DPPH radical inhibition was calculated according to the below formula: where, A 0 = absorbance of the control and A 1 = absorbance of the extracted sample 16 .

Assessment of Ferric ion reducing Antioxidant Power (FrAP) Value
FRAP values of all samples were determined following the method of Zhao et al., 17 with some modifications.Briefly, 7 mL of super natant (as derived from above section) were mixed with 3 mL of FRAP reagent (10:1:1 of 300 mM sodium acetate buffer at pH 3.6, 10 mM 2,4,6-tripyridylstriazine solution and 20 mM FeCl 3 .6H 2 O solution) and incubated at 37 0 C for 30 min.The absorbance was measured at 593 nm.FRAP values were expressed as µmol FeSO 4 per gram sample (µmol FeSO 4 /g).

Determination of rancidity
Levels of peroxide value (PV) and thiobarbituric acid-reactive substances (TBARS) of samples were determined according to the methods of Sakanaka et al., 18 and Buege and Aust 19 , respectively.PV was expressed as mg lipid peroxide/kg sample, while TBARS value was expressed as mg MDA/ kg sample.

microbiological Analysis
Total plate counts, yeast and mold contaminations in unprocessed and sterilized sesame seed cakes were determined following the Bacteriological Analytical Manual (BAM) 20 .

Statistical Analysis
The experiments were performed in triplicates and the results were presented as mean ± standard deviation.The statistical differences were determined at P≤ 0.05.Data were subjected to Duncan's post hoc test and the differences were detected for homogenous subsets.All statistical analyses were performed using IBM SPSS software version 23 .

results and Discussion Proximate Compositions and mineral Contents of Unprocessed and Sterilized Sesame Seed Cakes
In this study, the data showed that sterilization process had no effect on proximate compositions viz.moisture content, carbohydrate, protein, fat, crude fiber and ash of white and black sesame seed cakes (Table 1).Carbohydrate was the major composition of sesame seed cakes, whereby the white one contained higher level of carbohydrate than the black one.In contrast, protein and ash were found in black sesame seed cake at higher levels than that of white sesame seed cake.In addition, fat was the most minute composition in both cakes.These results were comparable to the reports of Kanu 21 and Makinde and Akinoso 1 that showed the high amount of protein in oilseed cakes after extraction.Similarly, Ogunronbi et al., 22 found that flaxseed oil seed cake contained between 38% and 47.3% protein.Rawdkuen et al., 23 reported that sacha inchi seed cakes contained very high amount of protein at approximately 56.61%.According to these findings, oilseed cakes could be good sources for protein and they would be subjected to value-added production.However, future protein configuration is needed to estimate by DSC and CD spectroscopy beside the convectional analytics Similarly, no effect of sterilization was found on mineral contents, including calcium, phosphorus, potassium, magnesium, zinc, iron, manganese and selenium, of both seed cakes (Table 2).In this case, the most predominant mineral found in both seed cakes, particularly the black one, was calcium, followed by phosphorus, potassium and magnesium.The similar results were found by Makinde and Akinoso 1 that reported the contents of calcium, phosphorus and potassium in white and black sesame seeds at 473.6-521.9,466.0-482.8 and 465.7-468.8mg/100 g, respectively.Additionally, Anilakumar et al., 24 revealed that sesame seeds were excellent sources of calcium and they were also rich in phosphorous, iron, magnesium, manganese, zinc and copper.

Changes of Bioactive Components in Sterilized Sesame Seed Cakes During Storage
As shown in Table 3, the levels of bioactive components (i.e.thiamine, riboflavin, sesamin, sesamolin and total phenolic compounds) and antioxidant capacities (i.e.DPPH and FRAP values) of both sesame seed cakes were found to greatly decrease after sterilization and storage at 37 0 C for 6 months (p≤0.05).For untreated seed cakes, the black sesame seed cake was found to contain thiamine, sesamin, total phenolic compounds and FRAP value at the levels higher than white sesame seed cake.At the final stage of storage, the contents of all bioactive compounds and the levels of antioxidant capacities in both sesame seed cakes were shown to reduce by roughly 0.5-1 time when compared to the samples at day 0.
Accordingly, Makinde and Akinoso 1 reported the levels of thiamine and riboflavin in whole white and black sesame seeds at the ranges of 0.71-0.83and 0.36-0.38mg/100 g, respectively.Suja et al., 4 found that the whole white and black sesame seeds contained sesamin and sesamolin at 203.7-399.3 and 205.4-356.3mg/100 g, respectively.
Lieu and Dang25also reported that the amount of total phenols in white and black sesame seed cakes was about 13.18-13.86mg GAE/g.In addition, Shahidi et al., 26 determined the levels of total phenolic compounds and free radical scavenging capacity in whole black and white sesame seed extracts and the results showed the considerable antioxidant activity of tested sesame products, especially the black sesame seed.There was a report that suggested the benefits of consuming sesame seeds to increase plasma ү-tocopherol and vitamin E activity and perhaps to prevent cancer and heart diseases 27 .
Considering the B vitamins, thiamin (vitamin B 1 ) is one of the most unstable B vitamins.Therefore, thermal processes viz.sterilization, pasteurization, baking and boiling can reduce the level of thiamin by up to 50%.Also, the stability of thiamin during storage can greatly depend on the moisture content of the food.The foods which having high moisture content would retain thiamin lesser than that having low moisture content [28][29] .On the contrary, riboflavin (vitamin B 2 ) is very stable during thermal processing, storage and food preparation.However, riboflavin is susceptible to degradation upon exposure to the light [30][31] , and light-proof packaging material is therefore necessary to prevent its deterioration.
Sesames generally comprised of two predominant lignans, including sesamin and sesamolin.The health-promoting effects of these lignans viz.decreasing blood lipids, increasing antioxidative ability, and providing anti-inflammatory function were reported by Hsu et al., 32 and Wu 33 .Gerstenmeyer et al., 34 found that lignan aglycones and glycosides in dr y foods did not degrade by heating at 100 0 C, but high roasting temperature at 250 0 C could degrade them severely.Also, the lignans in sesame seeds with high moisture content were found to apparently degrade.Wu 33 reported that heating at 200 0 C for 20 min caused a significant loss of lignans in commercial sesame oils, particularly sesamolin.
In addition, sesamolin in sesame seeds was also found to be damaged by infrared roasting at the same temperature for 30 min 35 .
On the other hand, Abe et al. 36 and Anilakumar et al., 24 revealed that sesamin and sesamolin were thermostable compounds and remained at 80-90% of the original levels after roasting.Nonetheless, little has been known regarding the effects of storage on both lignans in sesame seeds.Lee et al., 37 found that the levels of sesamin and sesamolin in sesame oil decreased when the storage time increased (25 0 C for 18 months).The storage temperature and processing condition were suggested to be responsible for degradation of sesamin and sesamolin by perhaps conver ting them into sesamol 38 .
Sterilization of sesame seed cakes was found to cause the reduction of total phenolic compounds and antioxidant activities to the contents/levels lower than that in untreated seed cakes.Heating processes, such as roasting, could also degrade the polyphenols and antioxidant capacity (DPPH assay) in sesame seeds and its oil 10 .Similar findings were reported by Abou-Gharbia et al., 39 and Rababah et al., 38 .In fact, sesamin and sesamolin were shown to have very low antioxidative properties 40 .The reduction of antioxidant capacity in sesame cakes might be due to temperature degradation of phenolic compounds.Besides, significant decreases of total phenolic compounds and antioxidant activities in both sesame cakes throughout the storage period were also observed.These antioxidant compounds and properties were also found to decrease significantly in parboiled germinated brown rice 41 , milled rice 42 and other fruits/vegetable products [43][44][45] during their storage.reported that PV and oxidative stability of crude sesame oil were considerably depended on moisture content of the seeds, roasting duration and temperature, and also storage conditions.In this study, the results were comparable to the reports of Elleuch et al., 47 and Lee et al., 48 that investigating the sesame seeds and their by-products.The natural antioxidants found in sesame seeds have been shown to influence the oxidative stability.In this regard, the black sesame seed cake was shown to contain sesamin, sesamolin and total phenolic compounds at the levels higher than that of white seed cake, and therefore it may possess high oxidative stability due to their oxygen-scavenging properties49.In contrast, Lee et al., 37 reported that the roasted sesame oil was oxidized slowly during storage at 25 0 C in the dark, and there was no change of PV up to 9 months of storage.

microbiological Qualities of Sterilized Sesame Seed Cakes During Storage
The colonies of total plate counts (TPC) and yeasts-molds (YM) in untreated and sterilized white and black sesame seed cakes were enumerated using the standard plating methods.The TPC and YM counts in untreated white seed cakes were 6.18±0.62 and 2.50±0.27log CFU/g, respectively; while those in untreated black seed cakes were 5.97±1.01 and 2.61±0.49log CFU/g, respectively.
After sterilization and storage, no detectable levels of both TPC and YM groups were observed in all samples (data not shown).Based on these results, all the indicator microbes in sterilized and stored seed cakes were well complied with the limits of the Thai Community Product Standard (TCPS No. 686/2004) for roasted sesame 50 .

Conclusion
White and black sesame by-products from Northern Thailand can be a good source for functional food ingredients because they contained high amounts of protein, carbohydrate, minerals and various bioactive components.Moreover, the sterilization and storage condition were found to be the key degradation factors of thiamine, riboflavin, sesamin, sesamolin, total phenolic compounds and antioxidant capacity (DPPH and FRAP assays) in these seed cakes.Oxidative stability of both seed cakes during storage was found to depend on the seed types and relate to the amounts of antioxidant compounds.

Table 1 : Proximate composition (%, FW) of untreated and sterilized white and black sesame seed cakes Compositions White sesame seed cake Black sesame seed cake (%, FW) Untreated cake Sterilized cake Untreated cake Sterilized cake
Means in the same rows with the same letters indicate no significant difference(p>0.05).The experiments were performed in triplicates.

Table 2 : mineral contents (mg/100 g, DW) of untreated and sterilized white and black sesame seed cakes mineral contents White sesame seed cake Black sesame seed cake (mg/100 g, DW) Untreated cake Sterilized cake Untreated cake Sterilized cake
Means in the same rows with the same letters indicate no significant difference (p>0.05).The experiments were performed in triplicates.

Table 4 : Changes of bioactive compounds and antioxidant capacity in untreated and sterilized white and black sesame seed cakes during storage at 37 o C for 6 months Bioactive Storage duration of white Storage duration of black compounds sesame seed cake (months) sesame seed cake (months)
bMeans in the same rows with the same letters indicate no significant difference(p>0.05).The experiments were performed in triplicates.PV is Peroxide Value and TBARS is Thio Barbituric Acid Reactive Substance.