Physicochemical Changes and Glycation Reaction in Intermediate

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Physicochemical Changes and Glycation Reaction of Intermediate Moisture Protein-Sugar Foods With and Without Addition of Resveratrol During Storage Zhanwu Sheng, Mantun Gu, Wangjun Hao, Yixiao Shen, Weimin Zhang, Lili Zheng, Binling Ai, Xiaoyan Zheng, and Zhimin Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00877 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on June 5, 2016

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Journal of Agricultural and Food Chemistry

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Physicochemical Changes and Glycation Reaction in Intermediate Moisture

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Protein-Sugar Foods With and Without Addition of Resveratrol During Storage

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Zhanwu Sheng†, ‡, *, Mantun Gu†, Wangjun Hao†, Yixiao Shen†, Weimin Zhang§, Lili

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Zheng†, Binling Ai†, Xiaoyan Zheng† and Zhimin Xu

∥, *

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†

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Academy of Tropical Agricultural Sciences, Haikou, 570101, China;

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‡

Haikou Key Laboratory of Banana Biology, Haikou Experimental Station, Chinese

College of Food Science and Technology, Huazhong Agricultural University, Wuhan

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430070, China;

11

§

College of Food Science and Technology, Hainan University, Haikou 570228, China;

∥

School of Nutrition and Food Sciences, Louisiana State University Agricultural

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Center, Baton Rouge, LA, USA.

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*Corresponding authors:

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Zhanwu Sheng, Haikou Key Laboratory of Banana Biology, Haikou Experimental

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Station, Chinese Academy of Tropical Agricultural Sciences, Haikou 570101, China

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Tel: +86 0898 6670 5612; E-mail address: [email protected]

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Zhimin Xu, School of Nutrition and Food Sciences, Louisiana State University

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Agricultural Center, Baton Rouge, LA, USA

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E-mail address:[email protected]

ACS Paragon Plus Environment


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ABSTRACT

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An intermediate moisture food (IMF) model consisting of whey protein isolate and

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glucose and the IMF model fortified with resveratrol were used to study effect of

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resveratrol on physicochemical changes and glycation of protein-sugar rich foods

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during storage. The water activity (aw) of the storage was controlled at 0.75 or 0.56.

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The browning rate or hardness of fortified IMF was significantly lower than that of

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IMF after 45-day storage. The rate of Maillard reaction in the samples stored at aw

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0.56 was higher than those samples stored at aw 0.75. The fortified IMF had lower

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levels of AGEs (Advanced glycation end products), (Nε-(Carboxymethyl)-l-lysine)

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CML and insoluble protein during storage. The inhibition capability of resveratrol

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against glycation was also confirmed by using sodium dodecyl sulfate polyacrylamide

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gel electrophoresis (SDS-PAGE), liquid chromatography mass spectrometry (LC-MS)

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and Fourier transform infrared spectroscopy (FTIR) analysis to monitor glycated

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proteins and protein aggregation in the samples. The results of this study suggested

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that resveratrol could be used as an inhibitor to reduce the formation of undesirable

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AGEs and other Maillard reaction products in foods during storage.

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KEYWORDS: resveratrol; glycation; advanced glycation end products;

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Maillard reaction; intermediate moisture food


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INTRUCTION

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Advanced glycation end products (AGEs) are a group of compounds which are

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formed by the non-enzymatic reaction between reducing sugars and amino acids.1

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AGEs can lead to the cellular disorders in biological systems through permanently

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modifying the structure and function of proteins and inducing overproduction of the

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reactive oxygen species and inflammatory mediators.2 Some chronic and aging

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diseases, such as diabetes mellitus and complication, cardiovascular diseases,

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neurological disorder and Alzheimer’s disease have been confirmed to associate with

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AGEs.3 A number of AGEs have been identified in our daily food products, especially

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in protein-sugar rich foods.4 Dietary AGEs are usually remained in the body tissues

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and blood circulation before reacted or excreted.2 Therefore, reducing the glycation in

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food products could lower AGEs level in the body after consumption and help prevent

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the risk of development of those chronic diseases. Some AGEs inhibitors from natural

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source, such as grape seed extract, green tea extract and dietary polyphenols

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(naringenin, quercetin, epicatechin, chlorogenic acid, and rosmarinic acid), have

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recently been investigated and proposed as the promising functional food additives

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because of their health-beneficial bioactivities.5 They can prevent the formation of

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AGEs in food systems by scavenging free radical and reactive carbonyl species

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without adverse effects.6

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Intermediate moisture foods, such as snack bars, are very common daily

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confectionary foods with water activity between 0.5 and 0.9.7 Especially, nutrition

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bars which largely contain protein and reducing sugars have been gaining the market

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rapidly and used as meal replacers by the consumers engaged in sports and outdoor

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activities.8 A number of studies found that the protein rich intermediate-moisture

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foods have a high level of AGEs, such as Nε-(Carboxymethyl)-l-lysine (CML) and

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Methylglyoxal (MGO).3, 9, 10 Also, the glycation of protein and sugars in foods during

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storage could result in browning, hardening, and producing of AGEs during storage.8

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Resveratrol is a natural polyphenol present in grape and red wine and has been

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suggested to possess antioxidant activity and prevention capability against diabetes

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and other chronic diseases in in vivo studies.11, 12 Its antioxidant property may play an

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important role in inhibiting production of AGEs by trapping of methylglyoxal.12

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However, the inhibition capability of resveratrol in intermediate moisture food (IMF)

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against glycation has not been well documented. Also, different from most of

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yellowish antioxidant polyphenols, white resveratrol has less effect on the sensory

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quality of food products after added. Therefore, in this study, IMF fortified with

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resveratrol was prepared to evaluate the capability of resveratrol in inhibiting

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production of AGEs during storage.

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FTIR spectra, glycated proteins of IMF and fortified IMF during storage were

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measured and compared. In general, the results of this study could help understand the

The changes of color, texture, insoluble protein,


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inhibition capability of resveratrol against production of AGEs. It would provide a

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way to decrease the level of AGEs in intermediate-moisture food products through

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using as an inhibitor from natural source.

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MATERIALS AND METHODS

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Materials and Chemicals

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Whey protein isolate (WPI) was purchased from Davisco Foods International, Inc.

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(Eden Prairie, Minn., U.S.A.). Alcalase was supplied by Novozymes A/S (Bagsvaerd,

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Denmark). Nε-(Carboxymethyl)-l-lysine (CML) standard was purchased from

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Toronto Research Chemicals Inc. (Toronto, Ontario, Canada). Glucose, glycerol,

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sodium azide, resveratrol (CAS: 501-36-0, ≥99%), sodium dodecyl sulfate (SDS) and

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tris(hydroxymethyl)aminomethane were obtained from Sigma-Aldrich (St. Louis, MO,

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USA). Trichloroacetic acid, acrylamide, methylene bisacrylamide, hydrogen

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phosphate disodium salt, dichloromethane, trifluoroacetic acid anhydrides, odium

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dihydrogen phosphate, Coomassie brilliant blue G-250 and Coomassie brilliant blue

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R-250 were from Sinopharm Chemical Reagent (Guangzhou, China).

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Preparation of Protein-Sugar Rich IMF and Fortified IMF

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The intermediate moisture food (IMF) were prepared according to a previous method

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with minor modification.10 Briefly, a solution of 25 g glucose in 25 g distilled water

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was mixed with 60 g glycerol at room temperature. Then, the mixture was mixed with



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90 g WPI powder and 0.1 g sodium azide which was used to prevent microbial growth

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to form a uniform IMF dough as a control. The fortified IMF dough was prepared as

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same as IMF dough. Resveratrol (0.5 g) was added in the mixture of glucose, water,

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and glycerol and homogenized through agitatedly stirring, before addition of 90 g

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WPI powder and 0.1 g sodium azide. Two of the control IMF doughs and two of the

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fortified IMF doughs were prepared.

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Storage Condition of IMF and fortified IMF

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Each IMF or fortified IMF dough was placed on a Petri dish. Then the dish was laid

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on a rack in an airtight plastic box. In order to maintain a constant water activity

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during storage, a saturated sodium chloride solution (aw 0.75) or sodium bromide

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solution (aw 0.56) was poured into each plastic box until its bottom area was covered.

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Thus, one of the control or fortified dough was stored the box with aw 0.75. Another

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control or fortified dough was stored the box with aw 0.56. All the boxes were capped

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and placed in an incubator (Shanghai Yuanye Bio-Technology, China) which

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temperature was set at 45 °C, based on the study of Zhou.10 After an aliquot

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(approximately 20 g) of each dough was taken rapidly for analysis at each sampling

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time, the box was capped and placed back in the water bath immediately.

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Measurements of Color and Texture

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Color measurement was performed by a colorimeter (CR-400, Konica Minolta,

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Japan). Three chromatic coordinates L*, a*, and b* at four different positions of each


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sample were determined. A white standard plate (L* = 95.26, a* = 0.89, b* = 1.18)

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was used for calibration. Chroma value was calculated as (a*2+ b*2)1/2 and E index

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was calculated as (L*2+ a*2+ b*2)1/2.

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Texture characteristics of each sample was measured by a CT3 10K Texture Analyzer

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(Brookfield, US) after the sample was taken and left at room temperature for 2 h. A

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cylindrical plunger（P / 2.2 mm diameter）was used for compressing samples to 25%

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Cauchy strain at 1 mm/s. The trigger force was 0.05 N. The highest force in the

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process of compression was recorded as the hardness of the sample.

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Measurement of Insoluble Protein

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The formations of insoluble (= 1 - solubility %) protein in IMF and fortified IMF

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during storage were measured based on the solubility test as described in study of

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Zhou et al.13 A dough sample (500 mg) was dissolved in 10 mL double-distilled water.

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The solution was stirred at room temperature for 80 min and centrifuged at 4000 g for

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30 min. Then, 2 mL of the supernatant was collected to carry out the measurement of

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protein concentration. The concentration of soluble protein in the supernatant was

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determined by the absorbance intensity at 595 nm. The amount of soluble protein was

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calculated using a calibration curve constructed by a series of different concentrations

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of WPI solutions. The reduction concentration of soluble protein in the supernatants

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of day 0 sample and the current sample was used to express the formation of insoluble

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protein in the sample at different storage times. The insoluble protein was calculated

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by using the following equation:



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Insoluble protein = (1 - A day n sample/ A day 0 sample) *100

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Where A day n sample is the absorbance of sample after n days storage, A day 0 sample is the

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absorbance of sample at day 0.

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Measurement of Maillard Reaction Rate

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The rate of Maillard reaction in sample was measured according to the method

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described in a previous study.10 The concentration of brown pigments produced by

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Maillard reaction was used to indicate the rate of Maillard reaction. The IMF or

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fortified IMF sample (250 mg) was dissolved in 10 mL of phosphate buffer (pH = 8.0)

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and stirred at room temperature for 60 min. The solution was incubated at 55 °C in a

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water bath for 15 min after 12 µL of alcalase solution was added. One mL of

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trichloroacetic acid (TCA) (80%, w/v) was added to stop the enzyme reaction

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followed by filtration. The absorbance of brown pigments in the filtrate was measured

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at 420 nm and expressed the rate of Maillard reaction in the sample.

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Measurement of Advanced Glycation End Products (AGEs)

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The AGEs content in each sample was analyzed by an F-4500 Luminescence

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Spectrometer (Shimadzu, Japan) based on Lavelli’s method.14 Briefly, 500 mg of

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sample was dissolved in 10 mL double-distilled water and then stirred at room

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temperature for 80 min. After the solution was centrifuged at 4000 g for 30 min, 4 mL

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of the supernatant was collected. The fluorescence intensity of the supernatant was


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measured at excitation wavelength at 370 nm and emission wavelength at 440 nm to

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express the level of AGEs in the sample.

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Measurement of CML by GC-MS

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The CML extraction and determination were performed as described in the study of

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Wang et al.

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adding 20 mL of chloroform/acetone solvent (1:3, v: v). After the sample and solvent

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were completely mixed, the mixture was centrifuged at 4000 g for 15 min. The

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precipitated protein was collected and dried by nitrogen flow. The dried protein was

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hydrolyzed in 8 mL of 6 mol/L of hydrochloric acid solution at 110 °C for 24 h. Fifty

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microliters of the protein hydrolysate was dissolved in 1.0 mL double-distilled water

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and then filtered. The filtrates were dried again by nitrogen flow. The dried sample

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was reacted with 1mL of thionylchloride / methanol (v: v, 1.46: 100) at 110 °C for 30

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min and then dried with nitrogen flow. Then the dried sample was derivatized by 2

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mL dichloromethane and 400 µL trifluoroacetic acid anhydride at room temperature

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for 1 h before analyzed by GC-MS. CML standard was derivatized by the same

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procedure above to prepare the CML standard curve used for quantification.

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Two hundred milligrams of homogenized sample was defatted by

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GC-MS analysis was performed by an Agilent 7890B gas chromatography system

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equipped with a 7693 mass selective detector single quadrupole mass spectrometer

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system and an HP-5MS column（30m x 0.25 mm x 0.25 µm）from Agilent (Palo Alto,



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CA, USA). The GC oven temperature was held at 40 °C for 1 min, and then ramped

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to 70 °C at 20 °C/min followed by ramping to 300 °C at 50 °C/min and holding 2 min

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at 300 °C. The transfer line temperature was 250 °C. High purity helium was used as

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carrier gas at a flow rate of 1.20 mL/min. The mass spectrometer was operated in

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electron ionization (EI) mode under the conditions of ion source temperature 230 °C,

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electron energy 70 eV, and ion scan range of m/z 40-500.

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SDS-PAGE Analysis of Glycated Proteins

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The glycated proteins profile of each sample was determined by the SDS-PAGE

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analysis method reported in a previous study.9 A Mini-PROTEIN unit (Bio-Rad

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Laboratories, USA) was used to carry out the analysis. The soluble protein fraction

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prepared as the measurement of insoluble protein above were diluted 1:1 (v/v) with

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Laemmli sample buffer (Bio-Rad Laboratories, USA). An aliquots (10 µL) of each

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diluted sample and a protein molecular weight maker (14.4 kDa-116 kDa) were

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loaded into a SDS-PAGE analysis gel. After the electrophoresis step was completed,

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the gel was stained by Coomassie Blue R-250 for 4 h and de-stained in a mixture of

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10% acetic acid and 30% methanol solution until the protein bands clearly appeared.

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LC-MS Analysis of Glycated Proteins

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The glycated proteins in each sample was monitored based on the method described

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in a previous study.16 A sample (300 mg) was dissolved in 15 mL double-distilled and


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stirred at room temperature for 80 min. The supernatant was obtained by centrifuge at

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4000 g for 30 min. One hundred µL of the supernatant was diluted 10 times with

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double-distilled water and then filtered for LC-MS analysis. The analysis was carried

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out by a Waters UPLC ZMD 4000 (Waters Co., Milford, U.S.A.) with a TOF mass

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spectrometer. The column was ethylene-bridged hybrid C18 column (2.1 mm ×100

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mm, i.d. Waters Co., Milford, U.S.A.). The mobile phase consisted of A: 100%

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acetonitrile and B: formic acid (0.1%, v/v). The mobile phase gradient program (in

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solvent B) was 80 to 50 %, from 0 to 15 min; 50 to 0 %, from 15 to 20 min; 0 - 80 %,

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from 20 to 21 min; 80% from 21-23 min; flow rate at 0.3 mL/min. The injection

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volume was 3 µL. The mass spectrometer was equipped by an electrospray ion source

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in positive mode at a spray voltage at 4.1 kV. The mass spectra was analyzed using

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Mass Lynx V4.1 (Waters Co., Milford, U.S.A.).

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Determination of FTIR spectra

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The Fourier transform infrared (FTIR) spectra of the samples were analyzed by an

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FTIR spectroscopy (TENSOR27, Bruker, Germany). The test pastilles were prepared

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after the samples were ground and mixed with KBr (sample/KBr: 1/99, w/w). The

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FTIR spectra of each sample was recorded at a spectral region of 400-4000 cm-1 with

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a resolution of 2 cm-1 and 20 scans.

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Statistical analysis

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Each measurement was replicated three times. The experimental data were analyzed

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by ANOVA using the General Linear Model procedure (SAS system, SAS Institute

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Inc., Cary, NC) with significant differences between means at P < 0.05 or 0.01.

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RESULTS AND DISCUSSION

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Changes of Color, Texture and Insoluble Protein in the IMF and Fortified IMF

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During Storage

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The color is an important attribute to food products. It was reported that foods

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fortified by functional polyphenol ingredients, such as quercetin, chlorogenic acid, or

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rosmarinic acid at a level of 0.25% (w/w) would alternate the color of the foods.5, 17, 18

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In this study, the colorimetric parameters of the IMF and fortified IMF, L* (0=

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blackness, 100 = whiteness), a* (-a = greenness, +a = redness), and b* (-b = blueness,

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+b = yellowness) were examined at different storage times. As shown in Table 1,

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browning occurred in both of the IMF and fortified IMF during storage at the two

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different water activities conditions. The whiteness decreased while the redness and

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yellowness increased with extending of storage time. The results were similar to that

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reported in a previous study and indicated the production of various intermediates or

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final products of Maillard reaction.14 However, compared with the IMF at the same

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water activity condition, the a* or b* value of fortified IMF was significantly lower,

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while the L* value of fortified IMF was significantly higher (P

Physicochemical Changes and Glycation Reaction in Intermediate

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