Gelling qualities of water soluble carbohydrate from Agave americana L. leaf extracts

Journal Pre-proof Gelling qualities of water soluble carbohydrate from Agave americana L. leaf extracts Mohamed Ali Bouaziz, Abir Mokni, Manel Masmoudi, Brahim Bchir, Hamadi Attia, Souhail Besbes PII:

S2212-4292(18)30449-8

DOI:

https://doi.org/10.1016/j.fbio.2020.100543

Reference:

FBIO 100543

To appear in:

Food Bioscience

Received Date: 24 May 2018 Revised Date:

7 February 2020

Accepted Date: 7 February 2020

Please cite this article as: Bouaziz M.A., Mokni A., Masmoudi M., Bchir B., Attia H. & Besbes S., Gelling qualities of water soluble carbohydrate from Agave americana L. leaf extracts, Food Bioscience (2020), doi: https://doi.org/10.1016/j.fbio.2020.100543. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

AUTHORS’ STATEMENT JOURNAL TITLE: Food Biosciences Manuscript title: Gelling qualities of water soluble carbohydrate from Agave

americana L. leaf extracts All authors state that the article is original, has not been submitted for publication in other journals and has not yet been published either wholly or in part. They state that they are responsible for the research that they have designed and carried out; that they have participated in drafting and revising the manuscript submitted, whose contents they approve. In the case of studies carried out on human beings, the authors confirm that the study was approved by the ethics committee and that the patients gave their informed consent.

Author contributions

Mohamed Ali BOUAZIZ: Conceptualization, Methodology, Writing original draft Abir Mokni Data curation, Writing- Original draft preparation. Manel MASMOUDI: Visualization, Investigation, Formal analysis. Brahim Bchir: Reviewing and Editing, Hamadi ATTIA: Project administration, resources Souhail BESBES: Supervision,

Sfax, 31-12-2019.

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Gelling qualities of water soluble carbohydrate from Agave americana L.

2

leaf extracts

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Mohamed Ali BOUAZIZ1*, Abir MOKNI1, Manel MASMOUDI1, Brahim BCHIR1, Hamadi

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ATTIA1, and Souhail BESBES1

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1

Analytical, Valorization and Food Safety Laboratory; National Engineering School of Sfax,

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Sfax, Tunisia.

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Running Title: A. americana leaf extract: A gelling agent

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* To whom Email should be sent: [email protected]

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Postal address: National Engineering School of Sfax, LAVASA Laboratory, BP 1173 - 3038

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Sfax, Tunisia.

18

Phone number: 00216.74.274.088

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Fax number: 00216.74.275.595

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23 24

Abstract

25

The chemical composition of Agave americana leaves and their water soluble

26

carbohydrate extract (WSCE) were determined. The hydrocolloid extract yield based on dry

27

material was 55% (w/w). The contents of ash, lipid, crude protein, total sugars and total

28

dietary fiber were 1.2, 4.7, 23, 40 and 42.6%, respectively. The WSCE showed the highest

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level of soluble sugars and a lower level of soluble fiber compared to the agave leaves (66

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versus 18.3 and 7 versus 9.3%, respectively). The texture of pectin and WSCE:pectin mixed

31

gels was also investigated. WSCE showed positive effects such as increased gel firmness. For

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the mixed gels, the maximum synergy was observed at 4% pectin concentration. Higher

33

firmness, elasticity and a good general appearance of the mixed gels were observed compared

34

to those of control gel (gel with only pectin at 4%). These results showed the beneficial

35

interaction between pectin and the different hydrocolloids of agave leaves. These

36

hydrocolloids might increase the use of Agave americana and be an alternative gel ingredient

37

for the food industry.

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Keywords: Agave americana L., Pectin, Agave leaves, Dietary fiber, Gel ingredient.

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1. Introduction

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Agave americana is frequently grown in semi-arid regions such as Mexico, Africa, and

51

Australia. This plant is found in many European and African countries mainly Mediterranean

52

countries (Bouaziz et al., 2014). It is a monocotyledon plant that belongs to the Agavaceae

53

family, which is characterized by long and fleshy leaves.

54

In Tunisia, A. americana L. is an abundant variety and Tunisians have recently been

55

interested in its fibers for use in textile applications (Msahli et al., 2015). The extraction of

56

these fibers is done using an immersion in seawater. Such fibers are known for their high

57

levels of insoluble (Bessadok et al., 2008, Msahli et al., 2015) and soluble polysaccharides

58

(Arrizon et al., 2010, Bouaziz et al., 2014). So, it is important to consider developing

59

beneficial uses for the different fractions of A. americana L. for food applications.

60

Many explorations of various plant hydrocolloids sources showed that they can be

61

used in food systems to alter product structure and the introduction of new ingredients into

62

actual food systems can alter these structures and perceived textures. (Carvajal-Millan et al.,

63

2006; Singthong et al., 2005; Vardhanabhuti & Ikeda, 2006; Yamazaki et al., 2008). Limited

64

research on the use of hydrocolloids from plants was found (Lai and Liao, 2002;

65

Vardhanabhuti and Ikeda, 2006; Yamazaki et al., 2008; Corbin et al., 2015). The crystalline

66

structure, morphological and chemical chracterisations of fibers extracted from Agave

67

americana L. have recently been studied (Ben Sghaier et al., 2012, Bouaziz et al., 2014,

68

Corbin et al., 2015). However, few studies have been done on agave gelling properties

69

(Bouaziz et al., 2014).

70

Synergistic effects are cumulative nonlinear effects of two active ingredients with

71

similar or related outcomes of their different activities. (Yechiel, 2005). Interactions of mixed

72

system biopolymers have been studied, such as gelatin:xanthan gum (Wang et al., 2016),

3

73

alginate:pectin (Walkenstrom et al., 2003), starch:pectin (Evageliou et al., 2000) and

74

gelatin:pectin (Al-Ruqaie et al., 1997) solutions. The outcomes of these solutions reflect the

75

positive or negative effects of interactions between pectin and the other hydrocolloids to

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improve or to interfere with the pectin matrix properties.

77

The objective of this study was to characterize the A. americana leaves and their water

78

soluble carbohydrate extract (WSCE) and then to determine its interaction with mixed

79

WSCE:pectin gels as well as to determine the best gel formulation. The effect of pectin to

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WSCE ratio was evaluated by studying the textural properties of the mixed gels.

81

2. Materials and Methods

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2.1. Source of materials

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A. americana L. plants were found wild in M'saken, Tunisia. The agave leaves were

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collected once at the same maturation stage in the winter: the length and width of the leaves

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were about 150 and 20 cm, respectively. The basal A. americana leaves were used. In total, 10

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kg of leaves were cut into large pieces (about 50-60 cm) with a knife and stored at -20 °C

87

until used for the various analyses for a maximum of 6 months.

88

The commercial high methoxyl pectin (HMP, E440) was a clear polysaccharide

89

derived from citrus peel and apple pomace and had an acidic pH (2.5-4). It was supplied by

90

the General Co. of Food Additives and Adjuvants (Sfax, Tunisia).

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2.2. Material preparation

92

One kg of frozen agave leaves was thawed for 24 h at 4 °C and washed with tap water

93

at the start of every assay. The cuticle was removed with a knife and the leaves are cut into

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small pieces and ground using a laboratory blender (M811D10 Perfect Mix, Moulinex,

95

Ecully, France). Finally, the biomass was stored at 4 °C until all analyses were carried out

96

within 7 days.

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2.3. Preparation of the soluble extract from A. americana leaves

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The WSCE was prepared according to the Bouaziz et al. (2014) method. A sample of

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100 g of A. americana leaves were mixed with 600 ml of distilled water using an Ultra Turrax

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homogenizer

101

Germany) with 0.9 g NaCl/l and stirred at 90 °C for 30 min. The WSCE suspension was

102

filtered through a NITEX filtration fabric, 1000 µm pore size mesh (D. Dutscher, Brumath,

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France) and then the supernatant was filtered under vacuum using Whatman paper (Grade 1,

104

Sigma-Aldrich, Taufkirchen, Germany). Finally, the filtered solution was lyophilised (Alpha

105

1-2 LDplus, Martin Christ, Osterode am Harz, Germany) and stored with desiccant until

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analyses were done within 6 months (Figure 1).

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2.4. Characterisation of A. americana leaves

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(T

50,

IKA,

Staufen,

All analytical determinations were done at least in triplicate within 6 months. The values of different parameters are shown as the mean ± standard deviation (S.D.).

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The dry matter, crude protein, fat, and ash contents were determined using standard

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AOAC methods N. 925.40, N. 920.87, N. 922.06 and N. 923.03, (23.1.05), respectively

112

(AOAC, 1995). The nitrogen content of samples was analysed using the Kjeldahl method

113

with a conversion factor of 6.25 (Besbes et al., 2004). The fat content was analysed using a

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Soxhlet continuous hexane extraction on samples previously prepared by drying and grinding.

115

The ash content was determined after incineration at 550 °C for 8 h using a muffle furnace

116

(Nabertherm, Lilienthal, Germany). It was expressed as a percentage of dry weight.

117

Dietary fiber was determined using an enzymatic and gravimetric method developed by

118

Prosky et al. (1988) and adopted in 1995 as AOAC N. 985.29. The A. americana leaves were

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crushed using the laboratory blender to obtain fine particles. Then, the sample was gelatinized

120

using a thermostable α-amylase (A-3306), and treated with a protease (P-3910) and

121

amyloglucosidase (A-3042) to eliminate starch and proteins, respectively. The enzyme kit was

122

also supplied by Sigma (Sigma-Aldrich, Saint Louis, Missouri, USA). After enzymatic 5

123

hydrolysis, the residues were recovered using centrifugation (3000 × g; 30 min; 25 °C, Rotina

124

380R, Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany). After being washed with

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distilled water, 95% ethanol and acetone, residues (R1) were dried overnight in an air oven

126

(Digital drying oven, Raypa Trade, Barcelona, Spain ) at 105 °C and weighed (Jex120, Chyo,

127

Kyoto, Japan).

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The insoluble fiber content was determined using the following formula:

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% Insoluble Fiber = (Residue (R1) – (Ash1 + Protein1)) × 100

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After enzymatic hydrolysis, 95% ethanol was added to the supernatant to precipitate

131

the soluble hydrocolloid. As above, the precipitate was washed successively with 75%

132

ethanol, 95% ethanol and acetone. The dried residue (R2) was weighed.

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The soluble fiber content was calculated using the following formula:

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% Soluble Fiber = (Residue (R2) – (Ash2 + Protein2)) × 100

135

Protein and ash were measured for these specific samples.

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The total dietary fiber is the sum of soluble and insoluble fiber.

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The soluble sugars were extracted with 15 ml of 96% ethanol and then centrifuged at

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10,000 × g for 20 min at 4 °C (Sigma 2-16KL, Sigma Laborzentrifugen GmbH, Osterode am

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Harz, Germany). The residue was washed using 5 ml of 80% ethanol. Then, the supernatants

140

were evaporated to obtain a volume of 1 ml. Finally, it was adjusted to 10 ml with distilled

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water (Ninio et al., 2003). The phenol-sulfuric method was used to analyze the solution. This

142

solution (0.5 ml) was mixed with 1 ml of a phenol solution (80%) followed by the rapid

143

addition of 2.5 ml concentrated sulfuric acid. After 10 min of color development in the dark

144

and an additional 30 min at room temperature (24-25 °C), absorbance was measured at 490

145

nm against distilled water (UV mini 1240, UV/VIS spectrophotometer, Shimdzu, Kyoto,

6

146

Japan). The amount of sugar was determined using a glucose standard (Sigma Aldrich, USA)

147

curve and expressed as glucose equivalents.

148

Polysaccharides were analyzed as follows: the residue obtained from the soluble

149

sugars extraction was stored for 24 h at room temperature to evaporate traces of ethanol. Ten

150

ml HCl (30%) were added and the solution was incubated at 60 °C for 2 h in a water bath and

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then centrifuged at 10,000 × g for 30 min at 4 °C. Then, the supernatant was filtered through a

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Whatman No 1 filter paper (Sigma-Aldrich, Taufkirchen, Germany) and adjusted to 10 ml

153

with distilled water. The solution was analyzed using the phenol-sulfuric method according to

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Bouaziz et al. (2014) to determine polysaccharides. The assay was carried out with 1 ml of the

155

solution with 5% phenol. H2SO4 (5 ml) was added and the solution was heated at 30 °C for 20

156

min in a water bath. Absorbance was measured at 490 nm (Bouaziz et al., 2014).

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The polysaccharide and soluble sugar concentrations were determined using a glucose standard curve. The total sugars were calculated as the sum of the polysaccharides and soluble sugars.

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The pH was measured using a pH-meter (model pH/Ion 510, Eutech Instruments Pte Ltd., Singapore). The amount of soluble solids of the raw material was measured at 20 °C using a refractometer (Mod. DR-101, Coseta S.A., Barcelona, Spain) and expressed as ◦Brix.

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The water activity (aw) was measured at 25 °C using a Novasina aw Sprint TH-500

166

Apparatus ( Pfäffikon, Switzerland) with automatic calibration for up to 8 points between 0.04

167

and 0.98 aw.

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2.5. WSEC:pectin gel preparation

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Pectin gels were used to study the effect of WSCE on gelling properties. Two g of

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lyophilized WSCE was dissolved in distilled water (50 ml) and added to sucrose (≈25g) until

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55 °Brix. Subsequently, the high-methoxyl pectin (HMP) (1, 2, 3, 4 or 5%) was added and

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dissolved using stirring. A 10% citric acid solution (w/v, Ricca Chemical Co., Arlington,

173

Virginia, USA) was used to adjust the pH to 3. The solution was heated to boiling with

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stirring until reaching 65 °Brix. Finally, the preparation was left to set in cylindrical

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containers (3.5 cm in diameter × 3 cm in height). Overnight, the solutions were cooled to 25

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°C (Figure 2). Similarly, pectin gels at 1, 2, 3, 4 or 5% pectin were prepared at pH 3 with

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distilled water to be compared to the mixed WSCE:pectin gels.

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2.6. Texture Profile Analysis

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A texture analyzer (Stable Micro Systems TA-XT Plus Texture Analyzer, Lloyd

180

Instruments, Fareham, UK) interfaced to a personal computer (Windows-based software,

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Nexygen Plot, Lloyd Instruments), was used to analyze the textural profiles of gels. The

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texture profile analysis (TPA) was done using a cylindrical cell and a cylindrical flat probe

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(25 mm in diameter). The samples (4 cm high × 4 cm diameter) were compressed to 50% of

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the original height of the gel at a compression rate of 1.0 mm/sec at room temperature. Five

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sec was used between the 2 cycles of compression. All analyses of texture were done at room

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temperature. The TPA characteristics, firmness (N), adhesiveness (N/mm), cohesiveness and

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elasticity (mm), were obtained.

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The force necessary to achieve a given deformation is the firmness, the maximum

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force required to remove the probe from the sample after applying a compressive force is

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adhesion. Cohesiveness is the ratio of the area under the curve of the second compression

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compared to the area under the first compression curve. Elasticity is the rate at which a

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deformed material goes back to its un-deformed condition after the deforming force is

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removed. It is the ratio of the recovered sample deformation in the second compression to the

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deformation in the first compression (Bouaziz et al., 2014). For each gel, triplicate

195

measurements were carried out.

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2.7 Statistical analysis

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One-way analysis of variance (ANOVA) was used to determine significant differences

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(P < 0.05) between WSCE:pectin gels and pectin gels and Duncan’s test was used. Statistical

199

analyses were done using the statistical analysis package STATISTICA (Release 5.0, Stat

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Soft Inc., Tulsa, Oklahoma, USA).

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3. Results and discussion

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3.1 Chemical composition of leaves and WSCE from A. americana leaves:

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Table 1 shows the proximate composition of leaves from A. americana L. plant.

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Agave is a succulent plant (Deshmukh et al., 2005) so their leaf water content was expected to

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be high (Ayadi et al., 2009). Moreover, it was also high in crude protein and fiber.

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The sugar fraction of agave leaves was mainly soluble sugars and polysaccharides.

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Agave leaves were high in insoluble fiber was consistent with its filamentous flesh

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appearance (Chaabouni et al., 2006, Corbin et al., 2015, Msahli et al., 2015). On the other

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hand, the soluble fiber fraction was lower. The soluble fraction was mainly glucose, sucrose,

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fructose and fructan (Arrison et al., 2010; Bouaziz et al., 2014; Corbin et al., 2015). A.

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americana leaves may be a potential natural source of both soluble and insoluble dietary fiber.

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On the other hand, WSCE of A. americana showed an abundance of soluble sugars

214

and a relatively low content of soluble fiber (Table 1). The soluble sugar content of WSCE

215

was significantly higher when compared to that of the leaves (P < 0.05). These results can be

216

explained if polysaccharides were hydrolyzed to fructose and glucose, which might occur at

217

90 °C, which was used for extraction. The soluble fiber was relatively lower than that of the

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agaves leaves, which led to an extraction yield of 55%. This yield value was similar to those

219

found by Corbin et al. (2015).

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On the other hand, WSCE crude protein content was significantly lower than that in the agave leaves (P < 0.05). These results suggest that many of the proteins may be insoluble.

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Similarly, WSCE has a low amounts of fat compared to the agave leaves (P < 0.05).

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No significant differences were observed between the pH values of WSCE and leaves

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from A. americana. The pH of WSCE was slightly acidic due to the acidity of A. americana

225

leaves (pH = 5.2 - 4.9). These results suggested that the WSCE might be expected to be able

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to resist microbiological alterations.

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3.2. Measure of water activity of gels

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The water activities (aw) of the gels are shown in Table 2. The aw decreased with the

229

increase of pectin concentration both for the control (pectin gels only) and mixed gels. Thus,

230

the nature of the pectin network becomes increasingly tighter (Hua et al., 2018). Besides, the

231

water becomes bound to the gel mesh network as aw decreased in a dose-dependent manner

232

with pectin. For all pectin concentration (from 1 to 5%), mixed gels with WSCE had a

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significantly lower aw than those of the pectin gels (P < 0.05) and the addition of WSCE

234

caused a further decrease of the mixed gel aw.

235

The mixed gels showed a lower aw and would not support microbial growth since their

236

aw was <0.60 (Beuchat et al., 2013).

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3.3. Texture of gels

238

The additional impact of WSCE on texture parameters are shown in Table 3. Pectin

239

gels at pectin concentrations (1, 2, 3, 4 and 5%) were tested and compared to pectin gels

240

without WSCE. Table 3 shows the results. The texture profiles are shown in Figure 3 and the

241

appearance of the gels are shown in Figure 4.

242

Pectin concentration and the WSCE in gels affected the texture profiles (Figure 3) and

243

contributed to the improvement of firmness, elasticity and adhesiveness. However, WSCE

244

affected the cohesiveness regardless of pectin concentration (Table 3).

10

245

For preparations with 1% pectin, no gel could be obtained and the textural parameters

246

of these preparations were not measured but the addition of WSCE led to gel formation

247

(Figure 4 a and b). These results may be related to the associations between different

248

hydrocolloids (polysaccharides and proteins) of WSCE and pectin. The new structure of the

249

WSCE:pectin network may be due to the acidic pH of WSCE. Wang et al. (2016) had

250

confirmed the synergistic gelation of gelatin B and xanthan gum. The optimum gelling

251

properties were obtained at pH 5.5, which may be due to the formation of the densest gelatin

252

B network structure, the strongest association between gelatin B molecules, as well as the

253

strongest additional effect of the xanthan gum.

254

The mixed gels were firmer (P < 0.05). The WSCE:pectin gels firmness increased

255

with increased pectin concentration except at 5% pectin. Indeed, at 2 - 5% pectin, the firmness

256

of WSCE:pectin gels were higher than the pectin gels. For example, firmness of the 4%

257

pectin-WSCE gels was 6.8 versus 1.7 N for the 4% pectin gel and pectin may have reacted

258

with WSCE, which increased gel firmness. The WSCE contained protein that may also

259

contribute to the firm of the mixed gels. This effect is more prominent for gels containing

260

WSCE (Table 3), which confirmed the interactions between these hydrocolloids. Wang et al.

261

(2016) showed that electrostatic forces had an important role in the synergistic effect. The

262

interactions between attractive and repulsive electrostatic interactions between xanthan gum

263

and gelatin B determines the rheological properties at a given pH.

264

For gels at 5% pectin, firmness of WSCE:pectin gels was a little lower than those of

265

WSCE:pectin at 3 - 4% pectin (Figure 3). The firmness decrease could be explained by the

266

possibility that all interactions had occurred, which affects the behavior and the general

267

appearance of the mixed gels (Figure 4 i and j) and, therefore, the excess material interferes

268

with the formation of the gel network. In previous studies, Bouaziz et al. (2014) showed that

269

the firmness of the mixed inulin:pectin gels decreased with the presence of inulin, which

11

270

confirmed the interference with the gelling properties between inulin and pectin. Unlike that

271

work, the firmness increased with the addition of WSCE up to 4% pectin. These results could

272

be due to the nature and structure of WSCE components such as fiber and protein. Beaulieu et

273

al. (2001) reported that increasing the amount of pectin (0.1 up to 1.5%) and the calcium

274

concentration (0, 5, 10 mmolar) made mixed gels firmer. On the other hand, the hardness of

275

gellan:gelatin gels decreased with increasing gelatin proportions (Lee et al., 2003).

276

The adhesiveness of pectin gels was not significantly different whatever the pectin

277

concentration was (P < 0.05). Indeed, adhesion was 0.1 N/mm. On the other hand, it was

278

significantly different when compared with those of mixed gels prepared with WSCE. For

279

example, adhesion of mixed gels at 3% pectin was significantly higher than those of the

280

corresponding pectin gels (P < 0.05). The adhesiveness values of all gels increased with the

281

increase of pectin concentration except for 5% pectin gels. These results could be explained

282

by the hydrocolloid interactions such as inulin:pectin found by Bouaziz et al. (2014).

283

Moreover, the adhesiveness from gels prepared with ‘Golden Delicious’ apple pectin

284

increased significantly with the increase of the pectin concentration (P < 0.05) (Rascón-Chu

285

et al., 2009).

286

No significant differences were measured for the cohesion in both types of gel

287

preparation and the increase of pectin concentration did not change cohesiveness significantly

288

(P ≥ 0.05, Table 3). It was not affected by WSCE. These results were consistent with those of

289

Bouaziz et al. (2014), who showed that cohesiveness of the mixed gels prepared from pectin

290

and A. americana inulin did not change significantly. Lee et al. (2003) characterized

291

gellan:gelatin gels and observed that the cohesiveness increased up to the gellan to gelatin

292

ratio of 40:60 and then decreased.

293

A significant increase of elasticity was observed relative to the concentration of pectin

294

in the control gels (P < 0.05). On the other hand, for the mixed gels, there was a progressive

12

295

increase in this parameter up to 3% pectin. At this concentration, the maximum level of

296

elasticity was obtained. At 5% pectin, the elasticity of mixed gel was slightly decreased. This

297

may probably due to the saturation of links between pectin and hydrocolloid from WSCE.

298

These results were consistent with those found by Bouaziz et al. (2014). They mentioned that

299

the saturation between pectin, protein and inulin affected the mixed gel properties and the

300

pectin matrix had a higher affinity for different compounds such as protein and fiber.

301

Figure 3 shows the texture profile measurements of both preparations: pectin and

302

WSCE:pectin gels. Additive effects were observed for the mixed gels with pectin and WSCE.

303

Namely, the peaks from the first and second compression phases were proportional with the

304

increase of pectin concentration for the mixed gels compared to those of the control gels. The

305

highest peak (7.18N) was observed with 4% pectin (Figure 3 C). Small peaks were observed

306

for the pectin gels (Figure 3: A, B, C and D). These results showed the role of hydrocolloids,

307

probably polysaccharides and proteins, in improving gel textural parameters (Bouaziz et al.,

308

2014). The assumption of a synergy between WSCE hydrocolloids and pectin was observed.

309

At 3 and 4% pectin, the affinity between different compounds (pectin, fiber and protein) was

310

shown. The presence of hydrocolloids probably allowed the disruption of the pectin matrix.

311

Similar results were reported between pectin and inulin from A. americana leaves (Bouaziz et

312

al., 2014) and k-carrageenan and hydrocolloid from the leaves of Corchorus olitorius

313

(Yamazaki et al., 2008).

314

At 5% pectin, texture parameters decreased non-significantly. It meant that the WSCE

315

addition did not improve the gel firmness at higher concentrations (>4% pectin). These results

316

could be explained by the saturation of interaction between WSCE and pectin, which affects

317

the general appearance of the gels such as viscosity, color, clarity (Bouaziz et al., 2014).

318

On the other hand, the WSCE:Pectin gel network interactions, mostly at 4% pectin,

319

was higher than those of pectin gels. The addition of WSCE probably changed the matrix

13

320

properties resulting in a non-polar matrix and led to the formation of new low-energy links

321

and a gel structure with different mesh sizes. Boland et al. (2006) had similar results, the

322

larger the retention of the more hydrophobic compounds the fewer hydrophobic compounds

323

in the strongest gels.

324 325

To understand the mechanism of the interactions between pectin and WSCE, the surface features of WSCE, and the microstructure of the mixed gels might be studied.

326 327

4. Conclusion

328

WSCE from A. americana leaves can form consistent gels with pectin. The

329

physicochemical properties of the WSCE and the textural parameters of different gels made of

330

pectin and WSCE:pectin were reported. A. americana leaves are a potential source of nutritive

331

and functional ingredients, especially fiber and protein confirming the possibility of their use

332

in some food formulations. WSCE showed a gelling effect with pectin in gel preparations.

333

The pectin showed a higher firmness and elasticity due to the new links between the different

334

hydrocolloids, namely protein and polysaccharides from WSCE. With the addition of WSCE,

335

the maximum positive textural effects were observed at 4% pectin with respect to improving

336

the textural properties of WSCE:pectin gels.

337

Acknowledgements

338

We acknowledge the financial support from the Ministry of Higher Education and

339

Scientific Research, Tunisia. Special thanks go to Mr. Salem Makhlouf and Miss. Wissal

340

Charmi (LAVASA, ENIS) for their kind help, all of which enabled us to carry out this study.

341

Conflicts of Interest

342 343

The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript.

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345 346 347 348 349

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18

Table 1: Physico-chemical composition of leaves and WSCE from A. americana L. (g / 100 g of dry matter)

Products Parameters (%) Dry matter Ash Crude protein Lipid Soluble sugars Polysaccharides Total sugars Soluble fibers Insoluble fibers Total fibers pH

A. americana leaves a

7.1±0.4 a 1.2±0.01 a 23±1 a 4.7±0.3 a 18±1 26±0.2 40±1 a 9.3±1 33.3±0.5 42.6±0.4 a 4.9±0.3

--: Not determined Means in the same row with different letters (a-b) are significantly different (P < 0.05).

A. americana WSCE b

4±1 b 1.7±0.1 b 14.4±0.3 b 1.2±0.4 b 66±1 --b 7±1 --a 5.2±0.2

Table 2: Water activity (aw) of prepared gels Products Parameter

aw

1

Pectin concentration (%) 3

2 aA

0.64±0.01

bA

0.52±0.02

Pectin Gels (Control)

0.69±0.02

WSCE:Pectin Gels

0.55±0.03

aB

0.60±0.03

bA

0.49±0.04

All values given are means of three determinations. Means in the same column with different letters (a-b) are significantly different (P < 0.05). Means in the same line with different letters (A-D) are significantly different (P < 0.05).

4

5

aC

0.59±0.04

aC

0.56±0.02

bB

0.41±0.08

aD

bC

0.45±0.01

bB

Table 3: Textural parameters of prepared pectin gels and WSCE:pectin gels

Textural parameters Firmness (N) Pectin Gel Pectin (%)

1 2 3 4 5

-aB 0.2±0.02 bB 0.4±0.2 cC 1.7±0.2 bB 0.4±0.04

Adhesiveness (N/mm)

WSCE:Pectin Gel A

0.3±0.03 dB 2.9±0.0 eC 6.7±0.2 eC 6.8±0.7 jD 4.4±0.1

Pectin Gel -aB 0.1±0.01 aB 0.1±0.02 aB 0.1±0.02 aB 0.1±0.04

Cohesiveness

WSCE:Pectin Gel A

0.1±0.02 aA 0.1±0.02 bC 0.9±0.1 bC 0.8±0.1 cD 0.4±0.1

--: Not determined All values given are means of three determinations. Means in the same line with different letters (a-e) are significantly different (P < 0.05). Means in the same column with different letters (A-D) are significantly different (P < 0.05).

Pectin Gel -aB 0.3±0.03 aB 0.4±0.2 aB 0.4±0.2 aB 0.2±0.01

Elasticity (mm)

WSCE:Pectin Gel B

0.2±0.1 aB 0.2±0.02 aB 0.2±0.00 aB 0.2±0.03 aB 0.2±0.1

Pectin Gel

WSCE:Pectin Gel

-bA 5 ±0.4 cB 9 ±0.5 cC 10 ±0.5 aD 13 ±1

2±0.3 aB 12±1 aB 14±1 aB 13±1 aB 12±0.4

A

Figure legends

Figure 1: Extraction from Agave americana leaves. Figure 2: Diagram of WSCE:pectin gel preparation. Figure 3: Examples of texture profiles of different prepared WSCE:pectin gels. ____: WSCE:pectin gel ____: Control (pectin gel) A: Gel with 2% pectin; B: Gel with 3% pectin; C: Gel with 4% pectin; D: Gel with 5% pectin Note: The different x-axis and y-axis scales between graphs of texture profiles are different.

Figure 4: Visual appearance of WSCE:pectin gels a: Control; b: WSCE:pectin gel (2% pectin) c: Control; d: WSCE:pectin gel (3% pectin) e: Control; f: WSCE:pectin gel (4% pectin) i: Control; j: WSCE:pectin gel (5% pectin)

Agave americana leaves

Removal of chlorophyll cuticle

Cutting and slicing

Grinding

Extraction with distilled water: Agave-water (100 g/600 ml), Temperature (90°C), Time (1.5 h), Salt (0.9 g/l),

Filtration using NITEX filtration fabric (pore size = 1000 µm) Filtration using Whatman No.1 filter paper

Lyophilisation

Water soluble carbohydrate extract (WSCE)

Figure 1

Two g of lyophilised WSCE

Addition of 50 ml of distilled water and sucrose until 55 °Brix (sous agitation) Addition of the high methoxyl pectin (1, 2, 3, 4 or 5%)

Adjustment of pH to 3 using 10% citric acid

Heating to boiling point and stirring until 65 ° Brix was reached Gelation at room temperature overnight

WSCE:pectin gel

Figure 2

A

max≈ 6.65 N

B

max≈ 0.98 N

Time (s)

max≈ 7.18 N

Time (s)

C

max≈ 4.42 N

Time (s)

Time (s)

Figure 3

D

a

b

c

d

e

f

i

j

Figure 4